British

Pharmacopoeia
2023

Volume V
General Notices
Infrared Reference Spectra
Appendices
Supplementary Chapters
Index

Incorporating the requeriments of the 10th edition of the

European Pharmacopoeia as amended by Supplements 10.1 to 10.8
British Pharmacopoeia 2023

Volume V
British Pharmacopoeia 2023

Volume V
The British Pharmacopoeia Commission has caused this British Pharmacopoeia 2023 to be prepared
under regulation 317(1) of the Human Medicines Regulations 2012 and, in accordance with
regulation 317(4), the Ministers have arranged for it to be published.
The monographs of the Tenth Edition of the European Pharmacopoeia (2019), as amended by
Supplements 10.1 to 10.8, published by the Council of Europe are reproduced either in this edition of
the British Pharmacopoeia or in the associated edition of the British Pharmacopoeia (Veterinary).
See General Notices

Effective date: 1 January 2023

see Notices

London: The Stationery Office

In respect of Great Britain:

THE DEPARTMENT OF HEALTH AND SOCIAL CARE

In respect of Northern Ireland:

THE DEPARTMENT OF HEALTH (NI)

© Crown Copyright 2022

Published by The Stationery Office on behalf of the Medicines and Healthcare products Regulatory
Agency (MHRA) except that:
European Pharmacopoeia monographs are reproduced with the permission of the Council of Europe
and are not Crown Copyright. These are identified in the publication by a chaplet of stars.
This publication is a 'value added' product. If you wish to re-use the Crown Copyright material from
this publication, applications must be made in writing clearly stating the material requested for re-use
and the purpose for which it is required. Applications should be sent to: bpcom@mhra.gov.uk.
First Published 2022
ISBN 978 011 3230 914
British Phannacopoeia Commission Office:
Medicines and Healthcare products Regulatory Agency
10 South Colonnade,
Canary Wharf,
London E14 4PU
E-mail: bpcom@mhra.gov.uk
Web site: http://www.pharmacopoeia.com
Laboratory:
British Pharmacopoeia Commission Laboratory
Queen's Road
Teddington
Middlesex TWI 1 0LY
Telephone: +44 (0)20 8943 8960
E-mail: bpcrs@mhra.gov.uk
Web site: http://www.pharmacopoeia.com
Contents

Contents of Volume I

FOREWORD
NOTICES
PREFACE
BRITISH PHARMACOPOEIA COMMISSION
EXPERT ADVISORY GROUPS, PANELS OF EXPERTS AND WORKING PARTIES
CODE OF PRACTICE
MEMBERSHIP
BP Commission, Expert Advisory Groups, Panels of Experts, Working Parties, Ad-hoe Group
STAFF
British Pharmacopoeia, BP Laboratory, Publisher
INTRODUCTION
Additions, Omissions, Technical Changes, Changes in Title
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances (A - I)
Contents of Volume II
NOTICES
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances G - Z)
Contents of Volume III
NOTICES
GENERAL NOTICES
MONOGRAPHS
Formulated Preparations: General Monographs
Formulated Preparations: Specific Monographs
Contents of Volume IV
NOTICES

V-v
GENERAL NOTICES
MONOGRAPHS
Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products
Materials for use in the Manufacture of Homoeopathic Preparations
Blood-related Products
Immunological Products
Radiopharmaceutical Preparations
Surgical Materials
Contents of Volume V
NOTICES
GENERAL NOTICES
INFRARED REFERENCE SPECTRA
APPENDICES
SUPPLEMENTARY CHAPTERS
INDEX

V-vi
Notices

Monographs of the European Pharmacopoeia are distinguished by a chaplet of stars against the title.
The term European Pharmacopoeia, used without qualification, means the Tenth Edition of the
European Pharmacopoeia comprising, unless otherwise stated, the main volume, published in 201 9, as
amended by any subsequent supplements and revisions.

Patents
In this Pharmacopoeia certain drugs and preparations have been included notwithstanding the
existence of actual or potential patent rights. In so far as such substances are protected by Letters
Patent their inclusion in this Pharmacopoeia neither conveys, nor implies, licence to manufacture.

Effective dates
New and revised monographs of national origin enter into force on 1 January 2023. The monographs
are brought into effect under regulation 320(2) of the Human Medicines Regulations 2012.
Monographs of the European Pharmacopoeia have previously been published by the European
Directorate for the Quality of Medicines & HealthCare, in accordance with the Convention on the
Elaboration of a European Pharmacopoeia, and have been brought into effect under the Human
Medicines Regulations 2012, as amended, and the Veterinary Medicines Regulations 2013, as
amended.

V-vii
2023 General Notices V-1

General Notices
V-2 General Notices 2023

CONTENTS OF THE GENERAL NOTICES

Part I Implementation of Pharmacopoeia! Methods
Italic introduction Conventional Terms
European Pharmacopoeia Interchangeable Methods
References to Regulatory Documents
Part II 1.2 Other Provisions Applying to General Chapters
Italic introduction and Monographs
Official Standards Quantities
Definition of Terms Apparatus and Procedures
Expression of Standards Water-bath
Temperature Drying and Ignition to Constant Mass
Weights and Measures Reagents
Atomic Weights Solvents
Constant Weight Expression of Content
Expression of Concentrations Temperature
Water Bath 1.3 General Chapters
Reagents Containers
Indicators 1.4 Monographs
Caution Statements Titles
Titles Relative Atomic and Molecular Masses
Chemical Formulae rhPmir!'.11 Ah<:tracts Service (CAS) Registry
Definition Number
Production Definition
Manufacture of Formulated Preparations Limits of Content
Freshly and Recently Prepared Herbal Drugs
Methods of Sterilisation Production
Water Choice of Vaccine Strain, Choice of Vaccine
Excipients Composition
Colouring Agents Potential Adulteration
Antimicrobial Preservatives Characters
Characteristics Solubility
Solubility Identification
Identification Scope
Reference Spectra First and Second Identifications
Assays and Tests Powdered Herbal Drugs
Biological Assays and Tests Tests and Assays
Reference Substances and Reference Preparations Scope
Chemical Reference Substances Calculation
Biological Reference Preparations Limits
Storage Indication of Permitted Limit of Impurities
Labelling Herbal Drugs
Action and Use Equivalents
Crude Drugs; Traditional Herbal and Culture Media
Complementary Medicines Storage
Monograph Title Labelling
Definition Warnings
Characteristics Impurities
Control Methods Functionality-related Characteristics of
Homoeopathic Medicines Excipients
Unlicensed Medicines Reference Standards
Part Ill 1.5 Abbreviations and Symbols
Italic introduction Abbreviations used in the Monographs on
General Notices of the European Pharmacopoeia !mmunoglobulins, fo-mmnosera and Vaccines
1.1 n,=,n,:,r~l .~t~tPffiPnt,o;: Collections of Micro-organisms
Quality Systems l .6 Units of the International System (SI) used in
Alternative Methods the Pharmacopoeia and Equivalence with
Demonstration of Compliance with the other Units
Pharmacopoeia International System of Units (SI)
Grade of Materials Notes
General Monographs
Validation of Pharmacopoeia! Methods
2023 General Notices V- 3

Part I

The British Pharmacopoeia comprises the entire text within this publication. The word 'official' is used in the
Pharmacopoeia to signify 'of the Pharmacopoeia'. It applies to any title, substance, preparation, method or
staternent included {n the general notices, monographs and appendices of ihe Pharmacopoeia. The abbreviation
for British Pharmacopoeia is BP.

European Pharmacopoeia
Monographs of the European Pharmacopoeia are reproduced in this edition of the British
Pharmacopoeia by incorporation of the text published under the direction of the Council of Europe
(Partial Agreement) in accordance with the Convention on the Elaboration of a European
Pharmacopoeia (Treaty Series No. 32 (1974) CMND 5763) as amended by the Protocol to the
Convention (Treaty Series No. MISC16 (1990) CMND 1133). They are included for the
convenience of users of the British Pharmacopoeia. In cases of doubt or dispute reference should be
made to the Council of Europe text.
** * ** Monographs of the European Pharmacopoeia are distinguished by a chaplet of stars against
~ : the title and by reference to the European Pharmacopoeia monograph number included
* * * immediately below the title in italics. The beginning and end of text from the European
Pharmacopoeia are denoted by means of horizontal lines with the symbol 'Ph Bur' ranged left and
right, respectively.
The general provisions of the European Pharmacopoeia relating to different types of dosage form
are included in the appropriate general monograph in that section of the British Pharmacopoeia
entitled Monographs: Formulated Preparations. These general provisions apply to all dosage forms of
the type defined, whether or not an individual monograph is included in the British Pharmacopoeia.
In addition, the provisions of the European Pharmacopoeia General Monograph for Pharmaceutical
Preparations apply to all dosage forms, whether or not an individual monograph is included in the
British Pharmacopoeia.
Texts of the European Pharmacopoeia are governed by the General Notices of the European
Pharmacopoeia. These are reproduced as Part III of these notices.
V-4 General Notices 2023

Part II

The following general notices apply to the statements made in the monographs of the British Pharmacopoeia
other than those reproduced from the European Phannacopoeia and to the statements made in the Appendices
of the British Pharmacopoeia other than when a method, test or other matter described in an appendix is
in·voked in a monograph reproduced from the European Pharmacopoeia.

Official Standards
The requirements stated in the monographs of the Pharmacopoeia apply to articles that are intended
for medicinal use but not necessarily to articles that may be sold under the same name for other
purposes. An article intended for medicinal use that is described by means of an official title must
comply with the requirements of the relevant monograph. A formulated preparation must comply
throughout its assigned shelf-life (period of validity). The subject of any other monograph must
comply throughout its period of use.
A monograph is to be construed in accordance with any general monograph or notice or any
appendix, note or other explanatory material that is contained in this edition and that is applicable to
that monograph. All statements contained in the monographs, except where a specific general notice
indicates otherwise and with the exceptions given below, constitute standards for the official articles.
An article is not of pharmacopoeia! quality unless it complies with all of the requirements stated. This
does not imply that a manufacturer is obliged to perform all the tests in a monograph in order to
assess compliance with the Pharmacopoeia before release of a product. The manufacturer may assure
himself that a product is of pharmacopoeial quality by other means, for example, from data derived
from validation studies of the manufacturing process, from in-process controls or from a combination
of the two. Parametric release in appropriate circumstances is thus not precluded by the need to
comply with the Pharmacopoeia. The general notice on Assays and Tests indicates that analytical
methods other than those described in the Pharmacopoeia may be employed for routine purposes.
Requirements in monographs have been framed to provide appropriate limitation of potential
impurities rather than to provide against all possible impurities. Material found to contain an impurity
not detectable by means of the prescribed tests is not of pharmacopoeia! quality if the nature or
amount of the impurity found is incompatible with good pharmaceutical practice.
The status of any statement given under the headings Definition, Production, Characteristics,
Storage, Labelling or Action and use is defined within the general notice relating to the relevant
heading. In addition to any exceptions indicated by one of the general notices referred to above, the
following parts of a monograph do not constitute standards: (a) a graphic or molecular formula given
at the beginning of a monograph; (b) a molecular weight; (c) a Chemical Abstracts Service Registry
Number; (d) any information given at the end of a monograph concerning impurities known to be
limited by that monograph; (e) information in any annex to a monograph. Any statement containing
the word 'should' constitutes non-mandatory advice or recommendation.
The expression 'unless otherwise justified and authorised' means that the requirement in question
has to be met, unless a competent authority authorises a modification or exemption where justified in
a particular case. The term 'competent authority' means the national, supranational or international
body or organisation vested with the authority for making decisions concerning the issue in question.
It may, for example, be a licensing authority or an official control laboratory. For a formulated
preparation that is the subject of monograph in the British Pharmacopoeia any justified and authorised
modification to, or exemption from, the requirements of the relevant general monograph of the
European Pharmacopoeia is stated in the individual monograph. For example, the general monograph
for Tablets requires that Uncoated Tablets, except for chewable tablets, disintegrate within 15
minutes; for Calcium Lactate Tablets a time of 30 minutes is permitted.
2023 General Notices V-5

Many of the general monographs for formulated preparations include statements and requirements
additional to those of the European Pharmacopoeia that are applicable to the individual monographs
of the British Pharmacopoeia. Such statements and requirements apply to all monographs for that
dosage form included in the Pharmacopoeia unless otherwise indicated in the individual monograph.
Where a monograph on a biological substance or preparation refers to a strain, a test, a method, a
substance, etc., using the qualifications 'suitable' or 'appropriate' without further definition in the text,
the choice of such strain, test, method, substance, etc., is made in accordance with any international
agreements or national regulations affecting the subject concerned.

Definition of Terms
Where the term 'about' is included in a monograph or test it should be taken to mean approximately
(fairly correct or accurate; near to the actual value).
Where the term 'corresponds' is included in a monograph or test it should be taken to mean similar
or equivalent in character or quantity.
Where the term 'similar' is included in a monograph or test it should be taken to mean alike though
not necessarily identical.
Further qualifiers (such as numerical acceptance criteria) for the above terms are not included in
the BP. The acceptance criteria for any individual case is set based on the range of results obtained
from known reference samples, the level of precision of the equipment or apparatus used and the level
of accuracy required for the particular application. The user should determine the variability seen in
his/her own laboratory and set in-house acceptance criteria that he/she judges to be appropriate based
on the local operating conditions.

Expression of Standards
Where the standard for the content of a substance described in a monograph is expressed in terms of
the chemical formula for that substance an upper limit exceeding 100% may be stated. Such an upper
limit applies to the result of the assay calculated in terms of the equivalent content of the specified
chemical formula. For example, the statement 'contains not less than 99.0% and not more than
101.0% of C 20H 24N 2 0 2 ,HC1' implies that the result of the assay is not less than 99.0% and not more
than 101.0%, calculated in terms of the equivalent content of C 20H 24N 2 0 2,HCI.
Where the result of an assay or test is required to be calculated with reference to the dried,
anhydrous or ignited substance, the substance free from a specified solvent or to the peptide content,
the determination of loss on drying, water content, loss on ignition, content of the specified solvent or
peptide content is carried out by the method prescribed in the relevant test in the monograph.

Temperature
The Celsius thermometric scale is used in expressing temperatures.

Weights and Measures

The metric system of weights and measures is employed; SI Units have generally been adopted.
Metric measures are required to have been graduated at 20° and all measurements involved in the
analytical operations of the Pharmacopoeia are intended, unless otherwise stated, to be made at that
temperature. Graduated glass apparatus used in analytical operations should comply with Class A
requirements of the appropriate International Standard issued by the International Organization for
Standardization. The abbreviation for litre is 'L' throughout the Pharmacopoeia.

Atomic Weights
The atomic weights adopted are the values given in the Table of Relative Atomic Weights 2001
published by the International Union of Pure and Applied Chemistry (Appendix XXV).
V-6 General Notices 2023

Constant Weight
The term 'constant weight', used in relation to the process of drying or the process of ignition, means
that two consecutive weighings do not differ by more than O. 5 mg, the second weighing being made
after an additional period of drying or ignition under the specified conditions appropriate to the nature
and quantity of the residue (1 hour is usually suitable).

Expression of Concentrations
The term 'per cent' or more usually the symbol '%' is used with one of four different meanings in the
expression of concentrations according to circumstances. In order that the meaning to be attached to
the expression in each instance is clear, the following notation is used:
Per cent w/w (% w/w) (percentage weight in weight) expresses the number of grams of solute in
I 00 g of product.
Per cent w/v (% w/v) (percentage weight in volume) expresses the number of grams of solute in
I 00 mL of product.
Per cent v/v (% v/v) (percentage volume in volume) expresses the number of millilitres of solute in
I 00 mL of product.
Per cent v/w (% v/w) (percentage volume in weight) expresses the number of millilitres of solute in
100 g of product.
Usually the strength of solutions of solids in liquids is expressed as percentage weight in volume, of
liquids in liquids as percentage volume in volume and of gases in liquids as percentage weight in
weight.
When the concentration of a solution is expressed as parts per million (ppm), it means weight in
weight, unless otherwise specified.
When the concentration of a solution is expressed as parts of dissolved substance in parts of the
solution, it means parts by weight (g) of a solid in parts by volume (mL) of the final solution; or parts
by volume (mL) of a liquid in parts by volume (mL) of the final solution; or parts by weight (g) of a
gas in parts by weight (g) of the final solution.
When the concentration of a solution is expressed in molarity designated by the symbol M preceded
by a number, it denotes the number of moles of the stated solute contained in sufficient Purified
Water (unless otherwise stated) to produce 1 litre of solution.

Water Bath
The term 'water bath' means a bath of boiling water, unless water at some other temperature is
indicated in the text. An alternative form of heating may be employed providing that the required
temperature is approximately maintained but not exceeded.

Reagents
The reagents required for the assays and tests of the Pharmacopoeia are defined in appendices. The
descriptions set out in the appendices do not imply that the materials are suitable for use in medicine.

Indicators
Indicators, the colours of which change over approximately the same range of pH, may be substituted
for one another but in the event of doubt or dispute as to the equivalence of indicators for a particular
purpose, the indicator specified in the text is alone authoritative.
The quantit'y of an indicator solution appropriate for use L.11 acid~base titrations described in assays
or tests is 0.1 mL unless otherwise stated in the text.
Any solvent required in an assay or test in which an indicator is specified is previously neutralised to
the indicator, unless a blank test is prescribed.
2023 General Notices V-7

Caution Statements
A number of materials described in the monographs and some of the reagents specified for use in the
assays and tests of the Pharmacopoeia may be injurious to health unless adequate precautions are
taken. The principles of good laboratory practice and the provisions of any appropriate regulations
such as those issued in the United Kingdom in accordance with the Health and Safety at Work etc.
Act 197 4 should be observed at all times in carrying out the assays and tests of the Pharmacopoeia.
Attention is drawn to particular hazards in certain monographs by means of an italicised statement;
the absence of such a statement should not however be taken to mean that no hazard exists.

Titles
Subsidiary titles, where included, have the same significance as the main titles. An abbreviated title
constructed in accordance with the directions given in Appendix XXI A has the same significance as
the main title.
Titles that are derived by the suitable inversion of words of a main or subsidiary title, with the
addition of a preposition if appropriate, are also official titles. Thus, the following are all official titles:
Aspirin Tablets, Tablets of Aspirin; Atropine Injection, Injection of Atropine.
A title of a formulated preparation that includes the full nonproprietary name of the active
ingredient or ingredients, where this is not included in the title of the monograph, is also an official
title. For example, the title Promethazine Hydrochloride Oral Solution has the same significance as
Promethazine Oral Solution and the title Brompheniramine Maleate Tablets has the same significance
as Brornpheniramine Tablets.
Where the English title at the head of a monograph in the European Pharmacopoeia is different
from that at the head of the text incorporated into the British Pharmacopoeia, an Approved Synonym
has been created on the recommendation of the British Pharmacopoeia Commission. Approved
Synonyms have the same significance as the main title and are thus official titles. A cumulative list of
such Approved Synonyms is provided in Appendix XXI B.
'Where the names of pharmacopoeia! substances, preparations and other materials occur in the text
they are printed with capital initial letters and this indicates that materials of Pharmacopoeial quality
must be used. Words in the text that name a reagent or other material, a physical characteristic or a
process that is described or defined in an appendix are printed in italic type, for example, methanol,
absorbance, gas chromatography, and these imply compliance with the requirements specified in the
appropriate appendix.

Chemical Formulae
'When the chemical composition of an official substance is known or generally accepted, the graphic
and molecular formulae, the molecular weight and the Chemical Abstracts Service Registty Number
are normally given at the beginning of the monograph for information. This information refers to the
chemically pure substance and is not to be regarded as an indication of the purity of the official
material. Elsewhere, in statements of standards of purity and strength and in descriptions of processes
of assay, it is evident from the context that the formulae denote the chemically pure substances.
'Where the absolute stereochemical configuration is specified, the International Union of Pure and
Applied Chemistry (IUPAC) RIS and BIZ systems of designation have been used. If the substance is
an enantiomer of unknown absolute stereochemistry the sign of the optical rotation, as determined in
the solvent and under the conditions specified in the monograph, has been attached to the systematic
name. An indication of sign of rotation has also been given where this is incorporated in a trivial name
that appears on an IUPAC preferred list.
All amino acids, except glycine, have the L-configuration unless otherwise indicated. The three-letter
and one-letter symbols used for amino acids in peptide and protein sequences are those recommended
by the Joint Commission on Biochemical Nomenclature of the International Union of Pure and
Applied Chemistry and the International Union of Biochemistry and Molecular Biology.
In the graphic formulae the following abbreviations are used:
V-8 General Notices 2023

Me -CH3 Bus -CH(CH 3)CH2CH3

Et -CH2CH3 Bun -CH2CH2CH 2CH 3
Pi -CH(CH3)2 Bur -C(CH3)3
Pr11 -CH 2 CH2CH3 Ph -C6Hs
-CH 2CH(CH3h Ac -COCH 3

Definition
Statements given under the heading Definition constitute an official definition of the substance,
preparation or other article that is the subject of the monograph. They constitute instructions or
requirements and are mandatory in nature.
Certain medicinal or pharmaceutical substances and other articles are defined by reference to a
particular method of manufacture. A statement that a substance or article i's prepared or obtained by a
certain method constitutes part of the official definition and implies that other methods are not
permitted. A statement that a substance may be prepared or obtained by a certain method, however,
indicates that this is one possible method and does not imply that other methods are proscribed.
Additional statements concerning the definition of formulated preparations are given in the general
notice on Manufacture of Formulated Preparations.

Production
Statements given under the heading Production draw attention to particular aspects of the
manufacturing process but are not necessarily comprehensive. They constitute mandatory instructions
to manufacturers. They may relate, for example, to source materials, to the manufacturing process
itself and its validation and control, to in-process testing or to testing that is to be carried out by the
manufacturer on the final product (bulk material or dosage form) either on selected batches or on
each batch prior to release. These statements cannot necessarily be verified on a sample of the final
product by an independent analyst. The competent authority may establish that the instructions have
been followed, for example, by examination of data received from the manufacturer, by inspection or
by testing appropriate samples.
The absence of a section on Production does not imply that attention to features such as those
referred to above is not required. A substance, preparation or article described in a monograph of the
Pharmacopoeia is to be manufactured in accordance with the principles of good manufacturing
practice and in accordance with relevant international agreements and supranational and national
regulations governing medicinal products.
Where in the section under the heading Production a monograph on a vaccine defines the
characteristics of the vaccine strain to be used, any test methods given for confirming these
characteristics are provided as examples of suitable methods. The use of these methods is not
mandatory.
Additional statements concerning the production of formulated preparations are given in the general
notice on Manufacture of Formulated Preparations.

Manufacture of Formulated Preparations

Attention is drawn to the need to observe adequate hygienic precautions in the preparation and
dispensing of pharmaceutical formulations. The principles of good pharmaceutical manufacturing
practice should be observed.
The Definition in certain monographs for pharmaceutical preparations is given in terms of the
principal ingredients only. Any ingredient, other than those included in the Definition, must comply
with the general notice on Excipients and the product must conform with the Pharmacopoeial
requirements.
The Definition in other monographs for pharmaceutical preparations is presented as a full formula.
No deviation from the stated formula is permitted except those allowed by the general notices on
Colouring Agents and Antimicrobial Preservatives. Where additionally directions are given under the
2023 General Notices V-9

heading Extemporaneous Preparation these are intended for the extemporaneous preparation of
relatively small quantities for short-term supply and use. W'hen so prepared, no deviation from the
stated directions is permitted. If, however, such a pharmaceutical preparation is manufactured on a
larger scale with the intention that it may be stored, deviations from the stated directions are
permitted provided that the final product meets the following criteria:
(1) compliance with all of the requirements stated in the monograph;
(2) retention of the essential characteristics of the preparation made strictly in accordance with the
directions of the Pharmacopoeia.
Monographs for yet other pharmaceutical preparations include both a Definition in terms of the
principal ingredients and, under the side-heading Extemporaneous Preparation, a full formula together
with, in some cases, directions for their preparation. Such full formulae and directions are intended for
the extemporaneous preparation of relatively small quantities for short-term supply and use. When so
prepared, no deviation from the stated formula and directions is permitted. If, however, such a
pharmaceutical preparation is manufactured on a larger scale with the intention that it may be stored,
deviations from the formula and directions stated under the heading Extemporaneous Preparation are
permitted provided that any ingredient, other than those included in the Definition, complies with the
general notice on Excipients and that the final product meets the following criteria:
( 1) accordance with the Definition stated in the monograph;
(2) compliance with all of the requirements stated in the monograph;
(3) retention of the essential characteristics of the preparation made strictly in accordance with the
formula and directions of the Pharmacopoeia.
In the manufacture of any official preparation on a large scale with the intention that it should be
stored, in addition to following any instruction under the heading Production, it is necessary to
ascertain that the product is satisfactory with respect to its physical and chemical stability and its state
of preservation over the claimed shelf-life. This applies irrespective of whether the formula of the
Pharmacopoeia and any instructions given under the heading Extemporaneous Preparation are
followed precisely or modified. Provided that the preparation has been shown to be stable in other
respects, deterioration due to microbial contamination may be inhibited by the incorporation of a
suitable antimicrobial preservative. In such circumstances the label states appropriate storage
conditions, the date after which the product should not be used and the identity and concentration of
the antimicrobial preservative.

Freshly and Recently Prepared

The direction, given under the heading Extemporaneous Preparation, that a preparation must be
freshiy prepared indicates that it must be made not more than 24 hours before it is issued for use. The
direction that a preparation should be recently prepared indicates that deterioration is likely if the
preparation is stored for longer than about 4 weeks at 15° to 25°.

Methods of Sterilisation
The methods of sterilisation used in preparing the sterile materials described in the Pharmacopoeia are
given in Appendix XVIII. For aqueous preparations, steam sterilisation (heating in an autoclave) is the
method of choice wherever it is known to be suitable. Any method of sterilisation must be validated
with respect to both the assurance of sterility and the integrity of the product and to ensure that the
final product complies with the requirements of the monograph.

Water
The term water used without qualification in formulae for formulated preparations means either
potable water freshly drawn direct from the public supply and suitable for drinking or freshly boiled
and cooled Purified Water. The latter should be used if the public supply is from a local storage tank
or if the potable water is unsuitable for a particular preparation.
V-10 General Notices 2023

Excipients
Where an excipient for which there is a pharmacopoeia! monograph is used in preparing an official
preparation it shall comply with that monograph. Any substance added in preparing an official
preparation shall be innocuous, shall have no adverse influence on the therapeutic efficacy of Llie
active ingredients and shall not interfere with the assays and tests of the Pharmacopoeia. Particular
care should be taken to ensure that such substances are free from harmful organisms.

Colouring Agents
If in a monograph for a formulated preparation defined by means of a full formula a specific colouring
agent or agents is prescribed, suitable alternatives approved in the country concerned may be
substituted.

Antimicrobial Preservatives
When the term 'suitable antimicrobial preservative' is used it is implied that the preparation concerned
will be effectively preserved according to the appropriate criteria applied and interpreted as described
in the test for efficacy of antimicrobial preservation (Appendix XVI C). In certain monographs for
formulated preparations defined by means of a full formula, a specific antimicrobial agent or agents
may be prescribed; suitabie aitematives may be substituted provided that their identity and
concentration are stated on the label.

Characteristics
Statements given under the heading Characteristics are not to be interpreted in a strict sense and are
not to be regarded as official requirements. Statements on taste are provided only in cases where this
property is a guide to the acceptability of the material (for example, a material used primarily for
flavouring). The status of statements on solubility is given in the general notice on Solubility.
Solubility Statements on solubility given under the heading Characteristics are intended as
information on the approximate solubility at a temperature between 15° and 25°, unless otherwise
stated, and are not to be considered as official requirements.
Statements given under headings such as Solubility in ethanol express exact requirements and
constitute part of the standards for the substances under which they occur.
The following table indicates the meanings of the terms used in statements of approximate
solubilities.

Descriptive term Approximate volume of solvent in millilitres

per gram of solute
very soluble less than 1
freely soluble from 1 to 10
soluble from 10 to 30
sparingly soluble from 30 to 100
slightly soluble from 100 to 1000
very slightly soluble from 1000 to 10 000
practically insoluble more than 10 000

The term 'pardy soluble' is used to describe a mi..xture of \Vhich only some of the components
dissolve.
2023 General Notices V-11

Iden tifi.cation
The tests described or referred to under the heading Identification are not necessarily sufficient to
establish absolute proof of identity. They provide a means of verifying that the identity of the material
being examined is in accordance with the label on the container.
Unless otherwise prescribed, identification tests are carried out at a temperature between 15° and
25°.
Reference spectra \X'here a monograph refers to an intrared refe1cn1,,;c :spc\.,;trum, Lhi:s :spcurum is
provided in a separate section of the Pharmacopoeia. A sample spectrum is considered to be
concordant with a reference spectrum if the transmission minima (absorption maxima) of the principal
bands in the sample correspond in position, relative intensities and shape to those of the reference.
Instrumentation software may be used to calculate concordance with a previously recorded reference
spectrum.
When tests for infrared absorption are applied to material extracted from formulated preparations,
strict concordance with the specified reference spectrum may not always be possible, but nevertheless
a close resemblance between the spectrum of the extracted material and the specified reference
spectrum should be achieved.

Assays and Tests

The assays and tests described are the official methods upon which the standards of the
Pharmacopoeia depend. The analyst is not precluded from employing alternative methods, including
methods of micro-analysis, in any assay or test if it is known that the method used will give a result of
equivalent accuracy. Local reference materials may be used for routine analysis, provided that these
are calibrated against the official reference materials. In the event of doubt or dispute, the methods of
analysis, the reference materials and the reference spectra of the Pharmacopoeia are alone
authoritative.
Where the solvent used for a solution is not named, the solvent is Purified Water.
Unless otherwise prescribed, the assays and tests are carried out at a temperature between 15° and
25°.
A temperature in a test for Loss on drying, where no temperature range is given, implies a range of
± 2° about the stated value.
Visual comparative tests, unless otherwise prescribed, are carried out using identical tubes of
colourless, transparent, neutral glass with a flat base. The volumes of liquid prescribed are for use with
tubes 16 mm in internal diameter; tubes with a larger internal diameter may be used but the volume
of liquid examined must be increased so that the depth of liquid in the tubes is not less than that
obtained when the prescribed volume of liquid and tubes 16 nun in internal diameter are used. Equal
volumes of the liquids to be compared are examined down the vertical axis of the tubes against a
white background or, if necessary, against a black background. The examination is carried out in
diffuse light.
Where a direction is given that an analytical operation is to be carried out 'in subdued light',
precautions should be taken to avoid exposure to direct sunlight or other strong light. Where a
direction is given that an analytical operation is to be carried out 'protected from light', precautions
should be taken to exclude actinic light by the use of low-actinic glassware, working in a dark room or
similar procedures.
For preparations other than those of fixed strength, the quantity to be taken for an assay or test is
usually expressed in terms of the active ingredient. This means that the quantity of the active
ingredient expected to be present and the quantity of the preparation to be taken are calculated from
the strength stated on the label.
In assays the approximate quantity to be taken for examination is indicated but the quantity actually
used must not deviate by more than 10% from that stated. The quantity taken is accurately weighed
or measured and the result of the assay is calculated from this exact quantity. Reagents are measured
and the procedures are carried out with an accuracy commensurate with the degree of precision
implied by the standard stated for the assay.
V-12 General Notices 2023

In tests the stated quantity to be taken for examination must be used unless any divergence can be
taken into account in conducting the test and calculating the result. The quantity taken is accurately
weighed or measured with the degree of precision implied by the standard or, where the standard is
not stated numerically (for example, in tests for Clarity and colour of solution), with the degree of
precision implied by the number of significant figures stated. Reagents are measured and the
procedures are carried out with an accuracy commensurate with this degree of precision.
The limits stated in monographs are based on data obtained in normal analytical practice; they take
account of normal analytical errors, of acceptable variations in manufacture and of deterioration to an
extent considered acceptable. No further tolerances are to be applied to the limits prescribed to
determine whether the article being examined complies with the requirements of the monograph.
In determining compliance with a numerical limit, the calculated result of a test or assay is first
rounded to the number of significant figures stated, unless otherwise prescribed. The last figure is
increased by 1 when the part rejected is equal to or exceeds one half-unit, whereas it is not modified
when the part rejected is less than a half-unit.
In certain tests, the concentration of impurity is given in parentheses either as a percentage or in
parts per million by weight (ppm). In chromatographic tests such concentrations are stated as a
percentage irrespective of the limit. In other tests they are usually stated in ppm unless the limit
exceeds 500 ppm. In those chromatographic tests in which a secondary spot or peak in a
chromatogram obtained with a solution of the substance being examined is described as corresponding
to a named impurity and is compared with a spot or peak in a chromatogram obtained with a
reference solution of the same impurity, the percentage given in parentheses indicates the limit for that
impurity. In those chromatographic tests in which a spot or peak in a chromatogram obtained with a
solution of the substance being examined is described in terms other than as corresponding to a
named impurity (commonly, for example, as any (other) secondary spot or peak) but is compared with
a spot or peak in a chromatogram obtained with a reference solution of a named impurity, the
percentage given in parentheses indicates an impurity limit expressed in terms of a nominal
concentration of the named impurity. In chromatographic tests in which a comparison is made
between spots or peaks in chromatograms obtained with solutions of different concentrations of the
substance being examined, the percentage given in parentheses indicates an impurity limit expressed in
terms of a nominal concentration of the medicinal substance itself. In some monographs, in particular
those for certain formulated preparations, the impurity limit is expressed in terms of a nominal
concentration of the active moiety rather than of the medicinal substance itself. Where necessary for
clarification the terms in which the limit is expressed are stated within the monograph.
In all cases where an impurity limit is given in parentheses, the figures given are approximations for
information only; conformity with the requirements is determined on the basis of compliance or
otherwise with the stated test.
The use of a proprietary designation to identify a material used in an assay or test does not imply
that another equally suitable material may not be used.

Biological Assays and Tests

Methods of assay described as Suggested methods are not obligatory, but when another method is
used its precision must be not less than that required for the Suggested method.
For those antibiotics for which the monograph specifies a microbiological assay the potency
requirement is expressed in the monograph in International Units (IU) per milligram. The material is
not of pharmacopoeia! quality if the upper fiducial limit of error is less than the stated potency. For
such antibiotics the required precision of the assay is stated in the monograph in terms of the fiducial
limits of error about the estimated potency.
For other substances and preparations for which the monograph specifies a biological assay, unless
otherwise stated, the precision of the assay is such that the fiducial limits of error, expressed as a
percentage of the estimated potency, are within a range not wider than that obtained by multiplying
by a factor of 10 the square roots of the limits given in the monograph for the fiducial limits of error
about the stated potency.
2023 General Notices V-13

In all cases fiducial limits of error are based on a probability of 95% (P = 0.95).
Where the biological assay is being used to ascertain the purity of the material, the stated potency
means the potency stated on the label in terms of International Units (IU) or other Units per gram,
per milligram or per millilitre. When no such statement appears on the label, the stated potency
means the fixed or minimum potency required in the monograph. This interpretation of stated
potency applies in all cases except where the monograph specifically directs otherwise.
Where the biological assay is being used to determine the total activity in the container, the stated
potency means the total number of International Units (IU) or other Units stated on the label or, if
no such statement appears, the total activity calculated in accordance with the instructions in the
monograph.
Wherever possible the primary standard used in an assay or test is the respective International
Standard or Reference Preparation established by the World Health Organization for international use
and the biological activity is expressed in International Units (IU).
In other cases, where Units are referred to in an assay or test, the Unit for a particular substance or
preparation is, for the United Kingdom, the specific biological activity contained in such an amount of
the respective primary standard as the appropriate international or national organisation indicates. The
necessary information is provided with the primary standard.
Unless otherwise directed, animals used in an assay or a test are healthy animals, drawn from a
uniform stock, that have not previously been treated with any material that will interfere with the assay
or test. Unless otherwise stated, guinea-pigs weigh not less than 250 g or, when used in systemic
toxicity tests, not less than 350 g. When used in skin tests they are white or light coloured. Unless
otherwise stated, mice weigh not less than 17 g and not more than 22 g.
Certain of the biological assays and tests of the Pharmacopoeia are such that in the United
Kingdom they may be carried out only in accordance with the Animals (Scientific Procedures) Act
1986. Instructions included in such assays and tests in the Pharmacopoeia, with respect to the
handling of animals, are therefore confined to those concerned with the accuracy and reproducibility
of the assay or test.

Reference Substances and Reference Preparations

Certain monographs require the use of a reference substance, a reference preparation or a reference
spectrum. These are chosen with regard to their intended use as prescribed in the monographs of the
Pharmacopoeia and are not necessarily suitable in other circumstances.
Any information necessary for proper use of the reference substance or reference preparation is
given on the label or in the accompanying leaflet or brochure. Where no drying conditions are stated
in the leaflet or on the label, the substance is to be used as received. No certificate of analysis or other
data not relevant to the prescribed use of the product are provided. The products are guaranteed to be
suitable for use for a period of three months from dispatch when stored under the appropriate
conditions. The stability of the contents of opened containers cannot be guaranteed. The current lot is
listed in the BP Laboratory website catalogue. Additional information is provided in Supplementary
Chapter III E.
Chemical Reference Substances The abbreviation BPCRS indicates a Chemical Reference
Substance established by the British Pharmacopoeia Commission. The abbreviation CRS or EPCRS
indicates a Chemical Reference Substance established by the European Pharmacopoeia Commission.
Some Chemical Reference Substances are used for the microbiological assay of antibiotics and their
activity is stated, in International Units, on the label or on the accompanying leaflet and defined in the
same manner as for Biological Reference Preparations.
Biological Reference Preparations The majority of the primary biological reference
preparations referred to are the appropriate International Standards and Reference Preparations
established by the World Health Organisation. Because these reference materials are usually available
only in limited quantities, the European Pharmacopoeia has established Biological Reference
Preparations (indicated by the abbreviation BRP or EPBRP) where appropriate. Where applicable, the
potency of the Biological Reference Preparations is expressed in International Units. For some
V-14 General Notices 2023

Biological Reference Preparations, where an international standard or reference preparation does not
exist, the potency is expressed in European Pharmacopoeia Units.

Storage
Statements under the side-heading Storage constitute non-mandatory advice. The substances and
preparations described in the Pharmacopoeia are to be stored under conditions that prevent
contamination and, as far as possible, deterioration. Unless otherwise stated in the monograph, the
substances and preparations described in the Pharmacopoeia are kept in well-closed containers and
stored at a temperature not exceeding 25°. Precautions that should be taken in relation to the effects
of the atmosphere, moisture, heat and light are indicated, where appropriate, in the monographs.
Further precautions may be necessary when some materials are stored in tropical climates or under
other severe conditions.
The expression 'protected from moisture' means that the product is to be stored in an airtight
container. Care is to be taken when the container is opened in a damp atmosphere. A low moisture
content may be maintained, if necessary, by the use of a desiccant in the container provided that
direct contact with the product is avoided.
The expression 'protected from light' means that the product is to be stored either in a container
made of a material that absorbs actinic light sufficiently to protect the contents from change induced
by such light or in a container enclosed in an outer cover that provides such protection or stored in a
place from which all such light is excluded.
The expression 'tamper-evident container' means a closed container fitted with a device that reveals
irreversibly whether the container has been opened.

Labelling
The labelling requirements of the Pharmacopoeia are not comprehensive, and the provisions of
regulations issued in accordance with the requirements of the territory in which the medicinal product
is to be used should be met.
Licensed medicines intended for use within the United Kingdom must comply with the
requirements of the Human Medicines Regulations 2012, as amended, in respect of their labelling and
packaging leaflets, together with those regulations for the labelling of hazardous materials.
Best practice guidance on the labelling and packaging of medicines for use in the United Kingdom
advises that certain items of information are deemed critical for the safe use of the medicine (see the
most recent version of the MHRA "Best Practice Guidance on the Labelling and Packaging of
Medicines"). Further information and guidance on the labelling of medicinal products can be found in
Supplementary Chapter I G.
Such matters as the exact form of wording to be used and whether a particular item of information
should appear on the primary label and additionally, or alternatively, on the package or exceptionally
in a leaflet are, in general, outside the scope of the Pharmacopoeia. When the term 'label' is used in
Labelling statements of the Pharmacopoeia, decisions as to where the particular statement should
appear should therefore be made in accordance with relevant legislation.
The label of every official formulated preparation other than those of fixed strength also states the
content of the active ingredient or ingredients expressed in the terms required by the monograph.
Where the content of active ingredient is required to be expressed in terms other than the weight of
the official medicinal substance used in making the formulation, this is specifically stated under the
heading Labelling. Unless othenvise stated in t.11e monograph, the content of the active ingredient is
expressed in terms of the official medicinal substance used in making the formulation.
These requirements do not necessarily apply to unlicensed preparations supplied in accordance with
a prescription. For requirements for unlicensed medicines see the general monograph on Unlicensed
Medicines.
2023 General Notices V-15

Action and Use

The statements given under this heading in monographs are intended only as information on the
principal pharmacological actions or the uses of the materials in medicine or pharmacy. It should not
be assumed that the substance has no other action or use. The statements are not intended to be
binding on prescribers or to limit their discretion.

Crude Drugs; Traditional Herbal and Complementary Medicines

Herbal and complementary medicines are classed as medicines under the Human Medicines Regulations 2012,,
as amended. It is emphasised thatJ although requirements for the quality of the material are provided in the
monograph to assist the registration scheme by the UK Licensing AuthorityJ the Brfrish Pharmacopoeia
Commission has not assessed the safety or efficacy of the material in traditional use.
Monograph Title For traditional herbal medicines, the monograph title is a combination of the
binomial name together with a description of use. Monographs for the material that has not been
processed (the herbal drug) and the processed material (the herbal drug preparation) are published
where possible. To distinguish between the two, the word 'Processed' is included in the relevant
monograph title.
Definition Under the heading Definition, the botanical name together with any synonym is given.
Where appropriate, for material that has not been processed, information on the collection/harvesting
and/or treatment/drying of the whole herbal drug may be given. For processed materials, the method
of processing, where appropriate, will normally be given in a separate section.
Characteristics References to odour are included only where this is highly characteristic.
References to taste are not included.
Control methods Where applicable, the control methods to be used in monographs are:
(a) macroscopical and microscopical descriptions and chemical/chromatographic tests for identification
(b) tests for absence of any related species
(c) microbial test to assure microbial quality
(d) tests for inorganic impurities and non-specific purity tests, including extractive tests, Sulfated ash
and Heavy metals, where appropriate
(e) test for Loss on drying or Water
(f) wherever possible, a method for assaying the active constituent(s) or suitable marker constituent(s).
The macroscopical characteristics include those features that can be seen by the unaided eye or by
the use of a hand lens. When two species/subspecies of the same plant are included in the Definition,
individual differences between the two are indicated where possible.
The description of the microscopical characteristics of the powdered drug includes information on
the dominant or the most specific characters. Where it is considered to be an aid to identification,
illustrations of the powdered drug may be provided.
The following aspects are controlled by the general monograph for Herbal Drugs: they are required
to be free from mouldsJ insects, decay, animal matter and animal excreta. Unless otherwise prescribed
the amount of foreign matter is not more than 2% w/w. Microbial contamination should be minimal.
In determining the content of the active constituents or the suitable marker substances
measurements are made with reference to the dried or anhydrous herbal drug. In the tests for Acid-
insoluble ash, Ash, Extractive soluble in ethanol, Loss on drying, Sulfated ash, Water, Water-soluble
ash and Water-soluble extractive of herbal drugs, the calculations are made with reference to the
herbal drug that has not been specifically dried unless otherwise prescribed in the monograph.

Homoeopathic Medicines
Homoeopathic medicines are classed as medicines under the Human Medicines Regulations 2012, as amended.
It is emphasised that) although requirements for the quality of the material are prozJided in the relei1ant
monograph in order to assist the simplified registration scheme by the UK Licensing Authority, the British
Pharmacopoeia Commission has not assessed the safety or efficacy of the material in use.
V-16 General Notices 2023

All materials used for the production of homoeopathic medicines, including excipients, must
comply with European Pharmacopoeia or British Pharmacopoeia monographs for those materials.
Where such European Pharmacopoeia or British Pharmacopoeia monographs do not exist, each
material used for the production of homoeopathic medicines must comply with an official national
pharmacopoeia of a Member State.
British Pharmacopoeia monographs for homoeopathic medicines apply to homoeopathic stocks and
mother tinctures only, but may be prefaced by a section which details the quality requirements
applicable to the principle component where there is no European Pharmacopoeia or British
Pharmacopoeia monograph for the material. These monographs also include either general statements
on the methods of preparation or refer to specific methods of preparation given in the European
Pharmacopoeia. Homoeopathic stocks and mother tinctures undergo the further process referred to as
potentisation. Potentisation is a term specific to homoeopathic medicine and is a process of dilution of
stocks and mother tinctures to produce the final product.
Identification tests are established for the components in homoeopathic stocks and usually relate to
those applied to the materials used in the production of the homoeopathic stocks. An assay is included
for the principal component(s) where possible. For mother tinctures, an identification test, usually
chromatographic, is established and, where applicable, an assay for the principle component(s); where
appropriate, other tests, related to the solvent, dry matter or known adulterants, are included.
Specifications have not been set for final homoeopathic products due to the high dilution used in
their preparation and the subsequent difficulty in applying analytical methodology.
Statements under Crude Drugs; Traditional Herbal and Complementary Medicines also apply to
homoeopathic stocks and mother tinctures, when appropriate.

Unlicensed Medicines
The General Monograph for Unlicensed Medicines applies to those formulations used in human
medicine that are prepared under a Manufacturer's 'Specials' Licence or prepared extemporaneously
under the supervision of a pharmacist, whether or not there is a published monograph for the specific
dosage form.
An article intended for medicinal use that is described by means of an official title must comply
with the requirements of the relevant monograph. A formulated preparation must comply throughout
its assigned shelf-life (period of validity). The subject of any other monograph must comply
throughout its period of use.
Unlicensed medicines that are prepared under a Manufacturer's 'Specials' Licence comply with the
requirements of the General Monograph for Pharmaceutical Preparations, the requirements of the
General Monograph for Unlicensed Medicines and, where applicable., the requirements of the
individual monograph for the specific dosage form.
Unlicensed medicines prepared extemporaneously under the supervision of a pharmacist comply
with the requirements of the General Monograph for Pharmaceutical Preparations, the requirements
of the General Monograph for Unlicensed Medicines and, where applicable, the requirements of the
individual monograph for the specific dosage form. While it is expected that extemporaneous
preparations will demonstrate pharmacopoeia! compliance when tested, it is recognised that it might
not be practicable to carry out the pharmacopoeia! tests routinely on such formulations. In the event
of doubt or dispute, the methods of analysis, the reference materials and the reference spectra of the
Pharmacopoeia are alone authoritative.
2023 General Notices V-17

Part III

Monographs and other texts of the European Pharmacopoeia that are incorporated in this edition of the British
Phamzacopoeia are governed by the general notices of the European Pharrnacopoeiaj these are reproduced
belmu.

GENERAL NOTICES OF THE EUROPEAN PHARMACOPOEIA

1.1 GENERAL STATEMENTS
1.1.1 General principles
1. 1.1.1 Quality systems
1.1.1.2 Conventional terms
1.1.1.3 References to regulatory documents
1.1.2 Compliance with the Ph. Eur.
1. 1.2.1 Scope
1.1.2.2 Demonstration of compliance with the Ph. Eur.
1.1. 2. 3 Demonstration of suitability of monographs
1.1.2.4 Validation and implementation of Ph. Eur. analytical procedures
1.1.2.5 Alternative analytical procedures
1.1.2.6 Pharmacopoeia! harmonisation
1.2 OTHER PROVISIONS APPLYING TO MONOGRAPHS AND GENERAL CHAPTERS
1. 2 .1 Quantities
1.2.2 Glassware
1.2.3 Temperature
1.2.4 Water-bath
1. 2. 5 Drying and ignition to constant mass
1.2.6 Solutions
1.2.7 Reagents and solvents
1.2.8 Expression of content
1.2. 9 Caution statements
1.3 GENERAL CHAPTERS
1.3.1 Materials for containers and containers
1.4 GENERAL MONOGRAPHS AND GENERAL MONOGRAPHS ON DOSAGE FORMS
1.5 INDIVIDUAL MONOGRAPHS
1.5.1 GENERAL PRINCIPLES
1.5.1.1 Titles
1.5.1.2 Relative atomic and molecular masses, formulae
1. 5 .1. 3 CAS registry number
1. 5 .1. 4 Definition
1. 5. 1.5 Production
1. 5 .1. 6 Potential adulteration
1. 5 .1. 7 Characters
1. 5 .1. 8 Identification
1.:5.1.9 Tests and assays
1.5. 1. 10 Storage
1. 5 .1.11 Labelling
1. 5 .1.12 Impurities
1.5.1.13 Functionality-related characteristics of excipients
1.5.2 MONOGRAPHS ON HERBAL DRUGS
V-18 General Notices 2023

1.5.3 MONOGRAPHS ON MEDICINAL PRODUCTS CONTAINING CHEMICALLY

DEFINED ACTIVE SUBSTANCES
1.5.3.1 Related substances
1.5.3.2 Dissolution / Disintegration
1.5.3.3 Impurities
1.5.3.4 Storage
1.6 REFERENCE STANDARDS
1.7 ABBREVIATIONS AND SYMBOLS
1.8 UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN THE PH. EUR. AND
EQUIVALENCE WITH OTHER UNITS
1.1 GENERAL STATEMENTS
1.1.1 General principles
The General Notices apply to all texts of the European Pharmacopoeia.
The texts of the European Pharmacopoeia are published in English and French. Translations in
other languages may be prepared by the signatory States of the European Pharmacopoeia Convention.
In case of doubt or dispute, the English and French versions published by the EDQM are alone
authoritative.
The date on which texts of the European Pharmacopoeia are to be implemented is fixed by a
resolution of the European Committee on Pharmaceuticals and Pharmaceutical Care (Partial
Agreement) of the Council of Europe, following a recommendation by the Ph. Eur. Commission. This
date is usually 1 year after adoption and about 6 months after publication. Where a text needs to be
implemented at a date earlier than the next publication date of a new edition or supplement of the
European Pharmacopoeia, a resolution of the European Committee on Pharmaceuticals and
Pharmaceutical Care is issued, giving the full text to be implemented. The text is also published in
Pharmeuropa Online for information and posted on the EDQM website as part of the resolution.
In the texts of the European Pharmacopoeia., the word 'Pharmacopoeia' without qualification means
the European Pharmacopoeia. The official abbreviation 'Ph. Bur.' may also be used for this purpose.

1.1.1.1 Quality systems

The quality standards represented by monographs are valid only where the articles in question are
produced within the framework of a suitable quality system. The quality system must assure that the
articles consistently meet the requirements of the Ph. Eur.

1. 1. 1.2 Conventional terms

Medicinal product (a) Any substance or combination of substances presented as having properties
for treating or preventing disease in human beings and/or animals; or (b) any substance or
combination of substances that may be used in or administered to human beings and/or animals with
a view either to restoring, correcting or modifying physiological functions by exerting a
pharmacological, immunological or metabolic action, or to making a medical diagnosis.
Active substance Any substance intended to be used in the manufacture of a medicinal product
and that, when so used, becomes an active ingredient of the medicinal product. Such substances are
intended to have a pharmacological activity or other direct effect in the diagnosis, cure, mitigation,
treatment or prevention of disease, or to affect the structure and function of the body.
Excipieni (auxiliary substance). Any constituent of a medicinai product that is not an active
substance. Adjuvants, stabilisers, antimicrobial preservatives, diluents and antioxidants are examples of
excipients.
Herbal medicinal product Any medicinal product exclusively containing as active ingredients
one or more herbal drugs or one or more herbal drug preparations, or one or more such herbal drugs
in combination with one or more such herbal drug preparations.
2023 General Notices V-1 9

Competent authority The national, supranational or international body or organisation vested

with the authority for making decisions concerning the issue in question. It may, for example, be a
national pharmacopoeia authority (NPA), a licensing authority or an official medicines control
laboratory (OMCL).
'Unless otherwise justified and authorised' This expression means that the requirements
must be met, unless the competent authority authorises a modification (e.g. of an analytical procedure
or limit) or an exemption, if justified by the manufacturer in a particular case.
'Should' Statements containing the word 'should' are informative or advisory.
'Suitable', 'appropriate' In certain texts, the terms 'suitable' and 'appropriate' are used to
describe a reagent, test, micro-organism, etc.; in such cases, if criteria for suitability are not described
in the text, suitability is demonstrated to the satisfaction of the competent authority.

1.1. 1.3 References to regulatory documents

Monographs and general chapters may contain references to documents issued by regulatory
authorities for medicines, for example directives and notes for guidance of the European Union. These
references are provided to users of the Ph. Eur. for information. Inclusion of such a reference does not
modify the status of the documents referred to, unless explicitly stated in the text.

1.1.2 Compliance with the Ph. Eur.

1.1.2.1 Scope
The use of the title or the Latin subtitle of a monograph implies that the article complies with the
requirements of that monograph. Such references to monographs in the texts of the Ph. Eur. are
shown using the monograph title and reference number in italics.
The scope of a monograph is stated in its definition.
Medicinal products whose labels state the modified international nonproprietary name (INNM) of
their active substance (e.g. raltegravir potassium) must comply with the relevant monograph on the
medicinal product, even if the monograph title only refers to the unmodified INN (e.g. Raltegravir
tablets (2938)).
Shelf life and re-test period A medicinal product must comply with the relevant monograph
throughout its shelf life; the shelf life and the time point from which that period is to be calculated are
proposed by the manufacturer in light of experimental results of stability studies and are approved by
the competent authority. A distinct shelf life and/or specifications for opened or broached containers
may be decided by the competent authority.
The subject of any other monograph must comply throughout its re-test period, except for some
substances known to be labile and for certain antibiotics where a shelf life is established rather than a
re-test period.
Monographs on medicinal products provide shelf-life specifications that may differ from release
specifications indicated in marketing authorisations. Monographs on other articles provide
specifications that must be fulfilled until the end of the re-test period.
Human and/or veterinary use The active substances, excipients., medicinal products and other
articles described in monographs are intended for human and veterinary use unless explicitly restricted
to one of these uses in the title or the definition.
Grades Certain articles that are the subject of a monograph may exist in different grades that are
suitable for different purposes. Unless otherwise indicated in the monograph, the requirements apply
to all grades of the article.
In some monographs, particularly those on excipients, a list of functionality-related characteristics
that are relevant to the use of the substance may be appended to the monograph, for information.
Analytical procedures for determination of one or more of these characteristics may be given, also for
information.
V-20 General Notices 2023

1. 1.2.2 Demonstration of compliance with the Ph. Eur.

Unless otherwise indicated in the General Notices or in the monographs, statements in monographs
constitute mandatory requirements.
(1) An article is of Ph. Eur. quality if it complies with all of the requirements stated in the monograph.
This does not imply that a manufacturer must perform all of the tests described in a monograph when
assessing compliance with the Ph. Bur. before release. The manufacturer may obtain assurance that an
article is of Ph. Bur. quality on the basis of its design, together with its control strategy and data
derived, for example, from validation studies of the manufacturing process.
In certain monographs, the sentence 'The following procedure is given as an example' means that the
analytical procedure described has been validated and may be implemented as is or may be replaced
by a suitable, validated procedure (without having to demonstrate its equivalence to the 'example'
procedure), subject to approval by the competent authority.
(2) An enhanced approach to quality control could utilise process analytical technology (PAT) and/or
real-time release testing (including parametric release) strategies as alternatives to end-product testing
alone. Real-time release testing in circumstances deemed appropriate by the competent authority is thus
not precluded by the need to comply with the Ph. Bur.
(3) Reduction of animal testing: the Ph. Eur. is committed to phasing out the use of animals for test
purposes, in accordance with the 3Rs (Replacement, Reduction, Refinement) set out in the European
Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific
Purposes. In demonstrating compliance with the Ph. Bur. as indicated above (1), manufacturers may
consider establishing additional systems to monitor consistency of production. With the agreement of
the competent authority, the choice of tests performed to assess compliance with the Ph. Bur. when
animal tests are prescribed is established in such a way that animal usage is kept to a minimum.

1. 1.2.3 Demonstration of suitability of monographs

The manufacturer must evaluate the suitability of the monograph for the quality control of their
substance or medicinal product, since the choice of analytical procedures may be influenced by the
manufacturing process and/or the composition of the medicinal product. In cases where the
specification described in a monograph is considered to be insufficient to ensure the quality of the
product or substance by a competent authority, the latter may request more-appropriate specifications
from the manufacturer in line with national or regional regulations. In such cases, the competent
authority informs the Ph. Bur. Commission through either the national pharmacopoeia authority or
the Secretariat of the Ph. Eur. Commission (EDQM). The manufacturer is requested to provide the
national pharmacopoeia authority or the EDQM with the details of the alleged insufficiency and the
additional specifications applied, so that the Ph. Eur. Commission can decide on the need to revise
the monograph in question.

1.1.2.4 Validation and implementation of Pb. Eur. analytical procedures

The analytical procedures given in an individual monograph have been validated in accordance with
accepted scientific practice and recommendations on analytical validation. Unless otherwise stated in
the individual monograph or in the corresponding general chapter, validation of these procedures by
the user is not required.
The analytical procedures provided in general chapters may be used for active substances,
excipients, medicinal products and other articles that are not covered by an individual monograph. In
such cases, validation of the procedures is the responsibility of the user.
When implementing a Ph. Eur. analytical procedure, the user must assess whether and to what
extent its suitability under the actual conditions of use needs to be demonstrated according to relevant
monographs, general chapters and quality systems.
2023 General Notices V-21

1. 1.2.5 Alternative analytical procedures

The tests and assays described are the official analytical procedures upon which the standards of the
Ph. Bur. are based. With the agreement of the competent authority, alternative analytical procedures
may be used for control purposes, provided that they enable an unequivocal decision to be made as to
whether compliance with the standards of the monographs would be achieved if the official procedures
were used. In the event of doubt or dispute, the analytical procedures of the Ph. Eur. are alone
l'.l1tthnntl'.l tivP.

1.1.2.6 Pharmacopoeia! harmonisation

The Ph. Eur. is engaged in a process of pharmacopoeia! harmonisation with the Japanese
Pharmacopoeia and the United States Pharmacopeia, within an informal structure referred to as the
Pharmacopoeia! Discussion Group (PDG). More information is available in general chapter 5. 8.
Pharmacopoeial harmonisation.

1.2 OTHER PROVISIONS APPLYING TO MONOGRAPHS AND GENERAL

CHAPTERS
1.2.1 Quantities
In tests with numerical limits and assays, the quantity stated to be taken for examination corresponds
to the quantity used during development of the analytical procedure. The amount actually used may
deviate by not more than 10 per cent from the stated quantity. In all cases, the amount used is
accurately measured and the result of the test is calculated from this exact quantity.
In tests where the limit is not numerical, which usually depend instead upon comparison with the
behaviour of a reference substance in the same conditions, the stated quantity is taken for
examination.
Reagents are used in the prescribed amounts.
The number of significant figures of a quantity value implies specific requirements for the quantities
(masses and volumes) to be measured, as detailed below.
For masses, requirements for balances for analytical purposes are provided in general chapter 2.1. 7
and are applicable to all texts. In addition, during weighing, the indication of a balance must match
the target mass value given in the text when mathematically rounded to the same number of
significant figures. For example, if the target mass value in the text is 50.0 mg, the 'minimum weight
(mmm) of the balance used must be smaller and the weighing is done within ± 5 subunits after the
last figure of the stated mass value (for example, 50.0 mg is to be interpreted as 49.95 mg to
50.04 mg or 49.950 mg to 50.049 mg, depending on the readability of the balance).
For volumes, if the figure after the decimal point is a zero or ends in a zero (for example, 10.0 mL
or 0.50 mL), the target volume is measured using a volumetric pipette, a volumetric flask or a burette,
as appropriate; otherwise, a graduated measuring cylinder or a graduated pipette may be used.
Volumes stated in microlitres are measured using a micropipette or microsyringe.
It is recognised, however, that in certain cases the number of significant figures with which
quantities are stated does not correspond to the number of significant figures stated in a specified
numerical limit. The stated quantities are then measured with a sufficiently improved accuracy.

1.2.2 Glassware
Volumetric glassware complies with Class A requirements of the appropriate International Standard
issued by the International Organisation for Standardisation (ISO).
Unless otherwise prescribed, visual comparative tests are carried out using identical colourless,
transparent, neutral glass tubes with a flat base; the volumes of liquid prescribed are for use with tubes
having an internal diameter of 16 mm, but tubes with a larger internal diameter may be used provided
the voiume of liquid used is adjusted (see general chapter 2.1.5. Tubes for comparative tests). Equai
volumes of the liquids to be compared are examined down the vertical axis of the tubes against a
V-22 General Notices 2023

white background, or if necessary against a black background. The examination is carried out in
diffuse light.

1.2.3 Temperature
Unless otherwise prescribed, analytical procedures are carried out at a temperature between 15 "C and
25 °C.
Where a text describes a temperature without giving a figure, the general terms used have the
following meaning:
in a deep-freeze: below - 15 °C;
in a refrigerator: 2 °c to 8 °C;
cold or cool: 8 °C to 15 °C;
room temperature: 15 °C to 25 °C.

1.2.4 Water-bath
The term 'water-bath' means a bath of boiling water unless water at another temperature is indicated.
Other methods of hearing may be substituted provided the temperature is near to but not higher than
100 °C or the indicated temperature.

1.2.5 Drying and ignition to constant mass

The terms 'dried to constant mass' and 'ignited to constant mass' mean that 2 consecutive weighings do
not differ by more than 0.5 mg, the second weighing carried out after an additional period of drying
or ignition appropriate to the nature and quantity of the residue.
Where drying is prescribed using one of the expressions 'in a desiccator' or 'in vacuo', it is carried
out using the conditions described in general chapter 2.2.32. Loss on drying.

1.2.6 Solutions
'Freshly prepared solution' means that the solution is prepared each time the test/assay is to be carried
out and is used within 24 h.
'Immediately before use' indicates that the stability of the corresponding solution(s) has been found to
be critical during the elaboration of the text. The time between preparation and use must be
minimised.

1.2. 7 Reagents and solvents

The proper conduct of the analytical procedures described in the Ph. Bur. and the reliability of the
results depend, in part, upon the quality of the reagents used. The reagents are described in the
Ph. Eur. in 4. Reagents and subsections. It is assumed that reagents of analytical grade are used; for
some reagents, tests to determine suitability are included in the description.
Any solvent required in a test or assay in which an indicator is to be used is previously neutralised
to the indicator, unless a blank determination is prescribed.
Where the name of the solvent is not stated, the term 'solution' implies a solution in water.
Where the use of water is specified or implied in the analytical procedures described in the Ph. Eur.
or for the preparation of reagents, water complying with the requirements of the monograph Purified
water (0008) is used, except that for many purposes the requirements for bacterial endotoxins
(Purified water in bulk) and microbial contamination (Purified water in containers) are not relevant.
The term 'distilled water' indicates purified water prepared by distillation.
The term 'ethanol' without qualification means anhydrous ethanol. The term 'alcohol' without
qualification means ethanol (96 per cent). Other dilutions of ethanol are indicated by the term
'ethanol' or 'alcohol' followed by a statement of the percentage by volume of ethanol (C 2 H 6 0)
required.
2023 General Notices V-23

1.2.8 Expression of content

In defining content, the expression 'per cent' is used according to circumstances with one of t\vo
meanings:
'per cent m!m' (percentage, mass in mass) expresses the number of grams of substance in 100 g of
final product;
'per cent V/V' (percentage, volume in volume) expresses the number of millilitres of substance in
100 mL of final product.
ThP. P.vprP<::~lcm<:: 'pnrtc pn m17Nnn' (ppm) £tnti 'pnrl~ pPt< hi/Jinn' (pph) rp.fpr rn m!.l<::<:: 1n m~<::<::, 11nlP.<::<;i
otherwise specified.

1.2.9 Caution statements

Articles described in monographs and reagents specified for use in the Ph. Bur. may be injurious to
health unless adequate precautions are taken. The principles of good quality control laboratory
practice and the provisions of any appropriate regulations are to be observed at all times. Attention is
drawn to particular hazards in certain monographs or general chapters by means of a caution
statement; absence of such a statement is not to be taken to mean that no hazard exists.

1.3 GENERAL CHAPTERS

General chapters (see sections 2, 3 and 5 of the Ph. Bur.) become mandatory when referred to in a
monograph, unless the wording clearly indicates that it is not the intention to make the text referred to
mandatory but rather to cite it for information.
When a general chapter is not referred to in any monograph or general chapter, it is given for
information; this is usually indicated in the preamble to the general chapter.
General chapters also become mandatory when referred to in another general chapter that is itself
referred to in a monograph, unless otherwise stated.
Requirements included in general chapters are not repeated in the individual monographs, unless
specific to the article in question (e.g. symmetry factor, signal-to-noise ratio of general chapter 2. 2. 46.
Chromatographic separation techniques).

1.3.1 Materials for containers and containers

Materials used for containers are described in 3.1. Materials used for the manufacture of containers and
subsections. Each subsection covers a defined plastic material with a positive list of accepted additives.
The specifications for each of these materials depend on the formulation and are therefore applicable
only for materials whose formulation is covered by the preamble to the specification. The use of
materials with different formulations, and the corresponding tests and limits applied to them, are
subject to approval by the competent authority.
The specifications for containers in 3. 2. Containers and subsections have been developed for general
application to containers of the stated category, but in view of the wide variety of containers available
and possible new developments, the publication of a specification does not exclude the use, in justified
circumstances, of containers that comply with other specifications, subject to approval by the
competent authority.
Containers for human blood and blood components, transfusion sets and syringes that are not
designed to serve as primary packaging for medicinal products are described in 3.3. Containers for
human blood and blood components> and materials used in their manufacture; transfusion sets and materials
used in their manufacture; syringes and subsections. Most of these texts are published for information
only.
Reference may be made within the monographs of the Ph. Eur. to the definitions and specifications
for containers. The general monographs on dosage forms may, in the Definition/Production section,
require tl1e use of certain types of container, as described in 3. 2. Containers and subsections; certain
other monographs may, in the Storage section, indicate the type of container that is recommended for
use.
V-24 General Notices 2023

1.4 GENERAL MONOGRAPHS AND GENERAL MONOGRAPHS ON DOSAGE

FORMS
General monographs and individual monographs are complementary.
Whenever an individual monograph is used, it is essential to ascertain whether there are one or
more general monographs applicable to the article in question.
Substances and medicinal products that are the subject of an individual monograph are also
required to comply with relevant, applicable general monographs. Cross-references to applicable
general monographs are not given in individual monographs. However, exceptions are possible; for
example, individual monographs on medicinal products containing chemically defined active
substances include a reference to the relevant general monograph on the dosage form.
General monographs give requirements that are applicable to all articles in the given class or, in
some cases, to any article in the given class for which there is an individual monograph in the Ph. Eur.
Where no restriction on scope of a general monograph is given in a preamble, it is applicable to all
articles in the class defined, irrespective of whether there is an individual monograph for the article in
the Ph. Eur.
If the provisions of a general monograph do not apply to a particular article, this is expressly stated
in the individual monograph.
General monographs on dosage forms apply to all medicinal products of the type defined. The
requirements are not necessarily comprehensive for a specific medicinal product and requirements
additional to those prescribed in the general monograph may be imposed by the competent authority.
1.5 INDIVIDUAL MONOGRAPHS
1.5.1 GENERAL PRINCIPLES
1.5.1.1 Titles
Monograph titles are in English or French in the respective versions and there is a Latin subtitle.
Where available, the international nonproprietary name (INN) is used, unless there are justifiable
reasons for not doing so. If needed, it is supplemented by the name of the anion or cation and by the
degree of hydration. Additional qualifiers may limit the applicability of the monograph to certain
classes or forms (e.g. veterinary use., dosage form., route of administration).

1.5. 1.2 Relative atomic and molecular masses, formulae

The relative atomic mass (Ar) or the relative molecular mass (Mr) is shown, where appropriate, at the
beginning of each monograph.
The relative atomic and molecular masses and the molecular and graphic formulae do not
constitute analytical standards for the substances described.

1.5.1.3 CAS registry number

Chemical Abstract Service (CAS) registry numbers are included for information in monographs, where
applicable, to provide convenient access to useful information for users. CAS Registry Number1t is a
registered trademark of the American Chemical Society.

1.5.1.4 Definition
This section provides the official definition of the article that is the subject of the monograph.
Limits of content If prescribed, limits of content are those determined using the analytical
procedure described under Assay.

1.5.1.5 Production
Statements in the Production section draw attention to particular aspects of the manufacturing process
but are not necessarily exhaustive. They constitute mandatory requirements for manufacturers, unless
othenvise stated. They may relate, for example, to source materials, to the manufacturing process itself
2023 General Notices V-25

and its validation and control, to process-related heterogeneity of the article, to in-process testing, or
to tests that are to be carried out by the manufacturer on the final article, either on selected batches or
on each batch prior to release. These requirements cannot necessarily be verified on a sample of the
final article by an independent analyst. The competent authority may establish that the instructions
have been followed, for example, by examining data received from the manufacturer, through
inspection or by testing samples.
The absence of a Production section does not imply that attention to features such as those referred
to above is not required.
Choice of vaccine strain, Choice of vaccine composition The Production section of a vaccine
monograph may define the characteristics of a vaccine strain or vaccine composition. Unless otherwise
stated, analytical procedures given for verification of these characteristics are provided for information
as examples of suitable procedures. Subject to approval by the competent authority, other procedures
may be used without validation against the procedure shown in the monograph.

1.5.1.6 Potential adulteration

Due to the rise in fraudulent activities and cases of adulteration, information may be made available to
users of the Ph. Bur. to help them detect adulterated articles (i.e. active substances, excipients,
intermediate products, bulk products and medicinal products).
To this end, an analytical procedure for the detection of potential adulterants and relevant limits
may be included in this section of monographs on substances for which an incident has occurred or
which are at risk of deliberate contamination. In such cases, a reminder that a suitable quality system
is applied at all stages of production and sourcing is also provided. The frequency of testing by
manufacturers or by users (e.g. manufacturers of intermediate products, bulk products and medicinal
products) depends on a risk assessment, taking into account the level of knowledge of the whole
supply chain and national requirements.
The requirements listed in this section apply to the whole supply chain, from manufacturers to
users. The absence of this section does not imply that attention to features such as those referred to
above is not required.

1.5.1. 7 Characters
The statements in the Characters section do not constitute Ph. Bur. requirements and are given for
information only.
Hygroscopicity, crystallinity, solubility See general chapter 5.11. Characters section in
monographs.
Polymorphism Where a substance shows polymorphism, this is usually stated. Except in rare
cases, no particular crystalline form is required in monographs. However, depending on the function
of a given substance in a medicinal product, it may be necessary for a manufacturer to ensure that a
particular crystalline form is used. The information given in the Characters section is intended to alert
users to the need to evaluate this aspect during the development of a medicinal product. See also
general chapter 5. 9. Polymorphism.

1.5.1.8 Identification
Scope The tests given in the Identification section are not designed to give a full confirmation of the
chemical structure or composition of the article; they are intended to give confirmation, with an
acceptable degree of assurance, that the article conforms to the description on the label.
An identification test may refer to a test in the Tests section of the monograph.
If the monograph lists, for example, identification tests A, B and C, all three tests must be carried
out and must satisfy the requirements.
Certain monographs give two or more sets of identification tests that are equivalent and may be
used independently. They are preceded by a sentence of the type 'Cany out either tests A, B or tests C,
D'. For example, one test determines enantiomeric purity by chromatography, while the other is a test
V-26 General Notices 2023

for specific optical rotation; the intended purpose of the two is the same, i.e. verification that the
correct enantiomer is present.
In some monographs the Identification section is subdivided as follows.
First ident~1ication. The test(s) t11at constitute the first identification may be used in all
circumstances.
Second identification. The test(s) that constitute the second identification may be used in
pharmacies only, provided it can be demonstrated that the article is fully traceable to a batch
certified to comply with all the other requirements of the monograph. The implementation of the
tests under the second identification is subject to national regulation.

1.5.1.9 Tests and assays

Scope The requirements are not designed to take all possible impurities into account. It is not to be
presumed, for example, that an impurity that is not detectable by means of the prescribed tests is
tolerated if common sense and good pharmaceutical practice require that it be absent. See also under
1.5.1.12. Impurities.
Calculation Where the result is to be calculated with reference to the dried or anhydrous
substance or on another specified basis, the determination of loss on drying, water content or another
properv; is carried out bj1 the procedure prescribed in the monograph. The \1.rords 'dr·ied sitbstance' or
'anhydrous substance' etc. appear in parentheses after the result.
'Where a quantitative determination of a residual solvent is carried out and a test for loss on drying
is not carried out, the residual solvent content is taken into account when calculating the assay content
of the substance., the specific optical rotation and the specific absorbance. No further indication is
given in the individual monograph.
Limits The prescribed limits are based on data obtained in routine analytical practice and are
intended to demonstrate that the article being examined complies with the requirements of the
monograph. They take account of normal analytical errors, of acceptable variations in manufacture/
preparation and of deterioration to an extent considered acceptable. No further tolerances are to be
applied to the prescribed limits.
In determining compliance with a numerical limit, the calculated result of an analytical procedure is
first rounded to the number of significant figures stated, unless otherwise prescribed. The limits,
regardless of whether the values are expressed as percentages or as absolute values, are considered
significant to the last digit shown (for example, 0.15 indicates 2 significant figures and 140 indicates 3
significant figures). When rounding, only the digit immediately to the right of the last place in the
limit is to be considered. If this digit is smaller than 5, it is eliminated and the preceding digit is not
changed. If this digit is equal to or greater than 5, it is eliminated and the preceding digit is increased
by L
Indication of permitted limits for impurities The limits for related substances are expressed
either in terms of comparison of peak areas ( comparative method) or as numerical values (quantitative
method). For tests using the comparative method, the approximate tolerated content of the named
impurity, or of the sum of impurities, may be indicated in brackets for information only. Acceptance
or rejection is determined on the basis of compliance or non-compliance with the stated limits.
If the use of a reference standard for the named impurity is not prescribed, the impurity content
may be expressed as a nominal concentration of the substance used to prepare the reference solution
specified in the monograph, unless otherwise described.
Chiral substances Monographs describing a particular enantiomer include a test to confirm
enantiomeric purity, either using specific optical rotation or a chromatographic procedure.
A test for racemic character using optical rotation is included only if there is information on the
specific optical rotation of the enantiomers that indicates that such a test would be discriminating in
terms of enantiomeric purity.
Equivalents Where an equivalent is given, only the figures shown are to be used in applying the
requirements of the monograph. For example, for titrations: l mL of 1 M hydrochloric acid is
equivalent to 50.05 mg of CaCO 3 •
2023 General Notices V-27

Culture media The culture media described in monographs and general chapters have been
found to be satisfactory for the intended purpose. However, the components of media, particularly
those of biological origin, are of variable quality, and for optimal performance it may be necessary to
modulate the concentration of some ingredients, notably:
peptones and meat or yeast extracts, with respect to their nutritive properties;
buffering substances;
bile salts, bile extract, deoxycholate and colouring matter, depending on their selective properties;
antibiotics, depending on their activity.

1.5.1.10 Storage
The information and recommendations given in the Storage section do not constitute a
pharmacopoeial requirement.
The articles described in the Ph. Eur. are stored in such a way as to prevent contamination and, as
far as possible, deterioration. Where special storage conditions are recommended, including the type
of container (see 1.3.1. Materials for containers and containers) and limits of temperature, they are
stated in the monograph.
The following expressions are used in monographs under Storage with the meaning shown below.
'In an airtight container' means that the article is stored in an airtight container (3.2. Containers).
Care is to be taken when the container is opened in a damp atmosphere. A low moisture content may
be maintained, if necessary, by the use of a desiccant in the container provided that direct contact
with the article is avoided.
'Protected from light' means that the article is stored either in a container made of a material that
absorbs actinic light sufficiently to protect the contents from changes induced by such light, or in a
container enclosed in an outer cover that provides similar protection, or that the article is stored in a
place from which all such light is excluded.

1.5.1.11 Labelling
In general, labelling of medicinal products is subject to supranational and national regulation and to
international agreements.
The statements in the Labelling section are not therefore comprehensive. In addition, for the
purposes of the Ph. Eur., only those statements that are necessary to demonstrate compliance or non-
compliance with the monograph are mandatory. Any other labelling statements are included as
recommendations.
When the term 'labeI' is used in the Ph. Eur., the labelling statements may appear on the container,
the package, a leaflet accompanying the package, or a certificate of analysis accompanying the article,
as decided by the competent authority.

1.5.1.12 Impurities
Monographs may include a list of all known and potential impurities that have been shown to be
detected by the tests. See also general chapter 5.10. Control of impurities in substances for pharmaceutical
use. The impurities are designated by a letter or letters of the alphabet. 'Where a letter appears to be
missing, the impurity designated by this letter has been deleted from the list during monograph
development prior to publication or during monograph revision.

1. 5.1.13 Functionality-related characteristics of excipients

This section is included in some monographs on excipients. Its contents do not constitute mandatory
requirements, but the characteristics may be relevant for a particular use of an excipient and are given
for guidance. The decision to control a functionality-related characteristic of an excipient remains \Vith
the manufacturer of the medicinal product and is taken with knowledge of the formulation of the
medicinal product in which it is to be used; the analytical procedures, limits and tolerances are
V-28 General Notices 2023

determined on a contractual basis by the user and the supplier of the excipient (see also Grades under
1.1.2.l. Scope).

L5.2 MONOGRAPHS ON HERBAL DRUGS

This section complements section 1.5. l. General principles.
Definition In monographs on herbal drugs, the definition indicates whether the subject of the
monograph is, for example, the whole drug or the fragmented drug. Where a monograph applies to
the drug in several states, for example both to the whole drug and to the fragmented drug, the
definition clearly indicates this.
Identification Monographs on herbal drugs may contain schematic drawings of the most
diagnostic microscopic botanical structures. These drawings complement the description given in the
relevant identification test.
Tests and Assay The sulfated ash, total ash, water-soluble matter, alcohol-soluble matter, water
content, and content of constituents with known therapeutic activity or markers are calculated with
reference to the drug that has not been specially dried, unless otherwise prescribed in the monograph.

1.5.3 MONOGRAPHS ON MEDICINAL PRODUCTS CONTAINING CHEMICALLY

DEFINED ACTIVE SUBSTANCES
This section complements section 1. 5 .1. General principles.
Monographs on medicinal products are intended for human use only, unless otherwise indicated in
the monograph.
Medicinal products comply with the general monograph Pharmaceutical preparations (2619), the
relevant dosage form monograph, any relevant individual monograph, and any other relevant text.

1.5.3.1 Related substances

Medicinal product monographs limit degradation products arising during the manufacture and the
shelf life of the medicinal product, including any impurities of synthesis that are also degradation
products.
In certain circumstances, it is necessary to identify impurities of synthesis in the medicinal product,
for example when they are detected in the test for related substances at a level greater than the
reporting threshold in the medicinal product. Consequently, the monograph describes how to identify
any such known impurities of synthesis, so that they are disregarded and are not taken into account.
Monographs on medicinal products are not designed to control impurities of synthesis that are not
degradation products. However, tests provided in the monograph could be used to control impurities
of synthesis known to be detected by the monograph, if further validated for that purpose by the user.
It is acknowledged that additional controls may be required to monitor degradation products other
than those controlled by the monograph (e.g. degradation products related to different excipients or
containers used, or from a different manufacturing process).

1. 5. 3.2 Dissolution/Disintegration
The following terms are used hereafter:
monograph dissolution test: analytical procedure and acceptance criteria described in the individual
monograph;
product-spec&4ic dissolution test: analytical procedure and acceptance criteria proposed by the
applicant in a marketing authorisation application (M_A_J\) for a medicinal product;
in-house dissolution test: analytical procedure developed and acceptance criteria defined by the
applicant.
In line with the relevant guidelines applied nationally or regionally (such as the ICH Q6A guideline)
and with the relevant Ph. Eur. dosage form monograph, a suitable product-specific dissolution test has
to be proposed by the applicant for routine quality control to confirm batch-to-batch consistency. This
test must be described in the MAA for submission to the competent authority, unless there is data
2023 General Notices V-29

justifying the replacement of the dissolution test by a disintegration test (see below). The
demonstration of the suitability of the dissolution test has to be made by the applicant to the
satisfaction of the competent authority.
Where appropriate, a dissolution test is described in an individual monograph on a medicinal
product. In such cases, the applicant may either select the monograph dissolution test or develop an
in-house dissolution test as the product-specific dissolution test. In all cases, the applicant has to
demonstrate the suitability of the selected test to the satisfaction of the competent authority.
If an in-house dissolution test is proposed, justification for not selecting the monograph dissolution
test and demonstration of compliance with the monograph dissolution test is normaliy not requested
as part of the MM.
However, when tested, the medicinal product has to comply with the monograph dissolution test,
unless otherwise justified by the applicant.
Where a given medicinal product does not comply with the monograph dissolution test and this
product is approvable by a competent authority, then the competent authority shall bring this to the
attention of the Ph. Eur. Commission so that it can review the monograph and revise it where
appropriate.
As outlined in the ICH Q6A guideline, for rapidly dissolving medicinal products containing active
substances that are highly soluble throughout the physiological range, a disintegration test may be
substituted for a dissolution test. Such a substitution has to be justified by the applicant to the
satisfaction of the competent authority.

1.5.3.3 Impurities
Impurities already listed in the monograph on the active substance, designated by a capital letter (A,
B, C, D, etc.), keep their name. Impurities specific to the medicinal product are designated by 'FP-'
followed by a letter of the alphabet (FP-A, FP-B, etc.).

1.5.3.4 Storage
As for other monographs, the statements included under Storage in a medicinal product monograph
constitute recommendations only; other conditions may be applied depending on the medicinal
product, subject to approval by the competent authority.
1.6 REFERENCE STANDARDS
Certain monographs require the use of reference standards, which can be chemical reference
substances (CRSs), herbal reference standards (HRSs), biological reference preparations (BRPs) or
reference spectra. See also general chapter 5.12. Reference standards. Unless othenvise stated, the
reference standards referred to in texts are alone authoritative in case of arbitration.
V-30 General Notices 2023

1.7. ABBREVIATIONS AND SYMBOLS

A Absorbance mp Melting point

A! per cent
Specific absorbance n20 Refractive index
1 llll D

Ar Relative atomic mass Ph. Bur. U. European Pharmacopoeia Unit

[1]~ Specific optical rotation ppb Parts per billion (micrograms per kilogram)
bp Boiling point ppm Parts per million (milligrams per kilogram)
BRP Biological reference preparation R Substance or solution defined under 4. Reagents
CRS Chemical reference substance RF Retardation factor (see general chapter 2.2.46.
d20 Relative density Chromatographic separation techniques)
20

'A Wavelength Rsr Used in chromatography to indicate the ratio of

the distance travelled by a substance to the
HRS Herbal reference standard distance travelled by a reference substance
IU International Unit RV Substance used as a primary standard in
M Molarity volumetric analysis (see general chapter 4. 2.1.
Mr Relative molecular mass Primary standards for volumetric solutions)

Abbreviations used in the monographs on immunoglobulins, immunosera

CFU C:olony-forming units Lo/10 dose The largest quantity of a toxin that, in the
LDso The statistically determined quantity of a conditions of the test, when mixed with 0.1 IU of
substance that, when administered by the antitoxin and administered by the specified route,
specified route, may be expected to cause the does not cause symptoms of toxicity in the test
death of 50 per cent of the test animals within a animals within a given period
given period U dose The quantity of toxin or toxoid that flocculates in
MLD Minimum lethal dose the shortest time with 1 IU of antitoxin

L+/1O dose The smallest quantity of a toxin that, in the CCID50 The statistically determined quantity of virus that
conditions of the test, when mixed with 0.1 IU of may be expected to infect 50 per cent of the cell
antitoxin and administered by the specified route, cultures to which it is added
causes the death of the test animals within a ED 50 The statistically determined dose of a vaccine
given period that, in the conditions of the test, may be
L+ dose The smallest quantity of a toxin that, in the expected to induce specific antibodies for the
conditions of the test, when mixed with 1 IU of relevant vaccine antigens in 50 per cent of the
antitoxin and administered by the specified route, animals into which it is inoculated
causes the death of the test animals within a EID 50 The statistically determined quantity of virus that
given period may be expected to infect 50 per cent of the
lr/10O dose The smallest quantity of a toxin that, in the fertilised eggs into which it is inoculated
conditions of the test, when mixed ¼ith 0.01 IU ID50 The statistically determined quantity of a virus
of antitoxin and injected intracutaneously causes that may be expected to infect 50 per cent of the
a characteristic reaction at the site of injection animals into which it is inoculated
within a given period PD The statistically determined dose of a vaccine
50
Lp/10 dose The smallest quantity of toxin that, in the that, in the conditions of the test, may be
conditions of the test, when mixed with 0.1 IU of expected to protect 50 per cent of the animals
antitoxin and administered by the specified route, into which it is inoculated against a challenge
causes paralysis in the test animals within a given dose of the micro-organisms or toxins against
period which it is active
PFU Pock-forming units or plaque-forming units
SPF Specified-pathogen-free
2023 General Notices V- 31

Collections of micro-organisms

ATCC American Type Culture Collection NCIMB National Collection of Industrial Food and
CIP Collection des bacteries de l'Institut Pasteur Marine Bacteria Ltd
IMI International Mycological Institute NCPF National Collection of Pathogenic Fungi
IP Institut Pasteur, Collection Nationale de NCTC National Collection of Type Cultures
Cultures de Microorganismes (CNCM) NCYC National Collection of Yeast Cultures
NBRC NITE Biological Resource Center SS! Statens Serum Institut
V-32 General Notices 2023

1.8. UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN THE PH. EUR. AND
EQUIVALENCE WITH OTHER UNITS
INTERNATIONAL SYSTEM OF UNITS (SI)
The International System of Units comprises 2 main classes of units, namely base units and
derived units 1 . The base units are the metre, the kilogram, the second, the ampere, the kelvin, the
mole and the candela.
The derived units are formed as products of powers of the base units according to the algebraic
relationships linking the corresponding quantities. Some of these derived units have special names and
symbols. The derived units used in the Ph. Eur. are shown in Table 1.8.-1.
Some important and widely used units outside the International System are shown in Table 1.8.-2.
The prefixes shown in Table 1.8.-3 are used to form the names and symbols of the decimal
multiples and submultiples of SI units.

Table 1.8.-1. - Derived units used in the Ph. Bur. and equivalence with other units
Quantity Unit
Conversion of other units into SI
Name Symbol Name Symbol Expression Expression in
units
in SI base other SI units
units

Wave number \' one per metre 1/m m-1

Wavelength A, micrometre µm lU- 6 m

nanometre run 10-9 m

Area A,S square metre m2 m2

Volume V cubic metre m3 m3 1 mL::: 1 cm3 ::: 10-6 m 3

Frequency V hertz Hz s-1

Density p kilogram per kg/m3 kg•m-3 1 g/mL =1 g/cm3 ::: 10 3

kg-m~ 3
cubic metre

Velocity, speed V metre per m/s m-s- 1

second

Force F ne\\'ton N m-kg-s- 2 1 dyne= 1 g-cm-s- 2 ::: 10-5 N

I kp::: 9.806 65 N

Pressure, stress p pascal Pa m- 1 -kg·s- 2 N ·m-2 1 dyne/cm2 = 10- 1 Pa::: 10- 1 N-m ~2
1 atm::: 101 325 Pa= 101.325 kPa
1 bar= 105 Pa::: 0.1 MPa
1 mm Hg= 133.322 387 Pa
1 Torr= 133.322 368 Pa
1 psi=6.894 757 kPa
Dynamic lJ pascal second Pa-s m- 1-kg•s- 1 N-s-m- 2 1 P =10- Pa-s::: lff"
1 1
N•s•m~ 2
viscosity 1 cP = 1 mPa·s

Kinematic V square metre m 2/s mz,s-1 Pa·s·m 3 -kg- 1 1 St= 1 cm2-s- 1 = 10-4 m 2 ·s- 1
viscosity per second N-m-s•kg- 1

1
The definitions of the units used in the International System are given in the booklet 'Le Systeme International d'Unitis (SI) 'J published by the
Bureau International des Pouts et Mesures, Pavilion de Breteuil, F-92310 Sevres.
2023 General Notices V-33

Quantity Unit
Conversion of other units into SI
Name Symbol Name Symbol Expression Expression in
units
in SI base other SI units
units

Energy w joule J m 2 •kg•s-2 N·m 1 erg= 1 cm2 ·g·s-2 = 1 dyne•cm =

10-7 J

1 cal = 4.1868 J

Power, radiant p watt w m2·kg•s-3 N·m•s- 1 1 erg/s = 1 dyne-cm-s- i =

flux J ·S -1 10-7 W = 10-7 N-m·s- 1 = 10- 7
J-s- 1

Absorbed dose D gray Gy mz-s-2 J·kg-1 l rad= 10-2 Gy

(of radiant
energy)

Electric u volt V m 2 · kg•s- 3-A- 1 W-A- 1

potential
difference,
voltage

Electric R ohm Q m 2 · kg·s- 3-A-2 V•A- 1

resistance

Electric charge Q coulomb C As

Activity A becquerel Bq s·•l 1 Ci =37•10 9

Bq = 37-109 s- 1
referred to a
radionuclide

Concentration C mole per mol/m3 mol·m-3 1 mol/L = 1 M = 1 mol/dm3 = 10 3

(of amount of cubic metre mol·m- 3

substance),
molar
concentration

Mass p kilogram per kg/m 3 kg·fil-3 1 g/L = 1 gldm3 = 1 kg·m- 3

concentration cubic metre

Catalytic z katal kat mol•s· 1

activity
V-34 General Notices 2023

Table 1.8.-2. - Non-SI units accepted for use with the SI units
Quantity Unit Value in SI units

Name Symbol

Time minute min 1 min = 60 s

hour h 1h =60 min =3600 s

day d 1 d =24 h =86 400 s

1° = (n/180) rad
(J
Plane angle degree

Volume litre L 1 L = l dm3 = 10-3 m 3

Mass tonne t 1t =10 3

kg

dalton Da 1 Da = 1.660539040(20) X 10· 27 kg

Rotational
revolution per minute r/min 1 r/min = (1/60) s- 1
frequency

Em:rgy .L -~--~---1~
t:Jt:UH)UVVU
-'tT
C: V 1 eV =1.602176634 X
111-19
!V
T
J

Table 1.8.-3. - Decimal multiples and sub-multiples of SI units

Factor Prefix Symbol .,.._ -~-
rac1.ur
- ...,. ___ c. __
rreux C'-~-L -1
.;:,y111uu1

1018 exa E 10--1 deci d

1015 peta p 10-2 centi C

1012 tera T 10-3 milli m

109 giga G 10-6 micro µ

106 mega M 10-9 nano n

103 kilo k 10-12 pico p

102 hecto h 10-15 femto f

10' deca da 10- 18 ano a

2023 General Notices V-35

NOTES
1. In the Pharn1acopoeia, the Celsius temperature is used (symbol t). This is defined by the following
equation:
t = T-To
where T0 = 273.15 K by definition. The Celsius or centigrade temperature is expressed in degrees
0
Celsius (symbol C). The unit 'degree Celsius' is equal to the unit 'kelvin'.
2. The radian is the plane angle between two radii of a circle that cut off on the circumference an arc
equal in length to the radius.
3. In the Ph. Eur., conditions of centrifugation are defined by reference to the acceleration of gravity (g):
g = 9.80665m • s2
4. Certain quantities without dimensions are used in the Ph. Eur.: relative density (2.2.5), absorbance
(2.2.25), specific absorbance (2.2.25) and refractive index (2.2. 6).
5. The microkatal is defined as the enzymatic activity that, under defined conditions, produces the
transformation (e.g. hydrolysis) of 1 micromole of the substrate per second.
Infrared Referenee Spectra
V-S2 Infrared Reference Spectra 2023

Preparation of lnfrared Reference Spectra

All spectra presented in this section were recorded using either a Perkin-Elmer model 682 dispersive
infrared spectrophotometer, a Perkin Elmer model 16PC Fourier transform infrared spectrophotometer,
a Perkin Elmer model Spectrum 100 Fourier transform infrared spectrophotometer, or a Perkin Elmer
model Spectrum One Fourier transform infrared spectrophotometer.
Pressed discs, 13 mm in diameter, were prepared using potassium bromide or potassium chloride.
Liquid paraffin mulls and thin films were prepared between potassium bromide plates, and gas and
solution spectra were prepared using cells with potassium bromide windows. Solution spectra were
prepared against a solvent reference and all other spectra were recorded against air. Attenuated Total
Reflectance (ATR) spectra were recorded by pressing the sample against a horizontal diamond ATR
crystal.
For solution spectra the regions of the spectrum within which the solvent shows strong absorption
should be disregarded. Solvent 'cut-offs' in the reference spectra may be recorded as horizontal straight
lines or may appear as blank regions on the spectrum.
 2023 Infrared Reference Spectra V-S3

Polystyrene Instrument: Dispersive Phase: Thin FIim Thickness: 0.038mm

100.0

60

?;'?.

1'3 20
c::

I Cl)
c::
i 0.0
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm-')
100.0

80

C
1'3 20
~
.E
Cl)
c::
i 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S4 Infrared Reference Spectra 2023

Polystyrene Instrument: Fourier Transform Phase: Thin Film Thickness: 0.038mm

100.0

4000.0 3600 3200 2800 2400 2000.0

Wavenumber (cm- 1)

~
e._
Q)
c.:, 20
C:
~
.E
en
C:
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Abacavir RS464 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

60

40

~ 20
C:
CIJ
E
E
"'
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
2023 Infrared Reference Spectra V-SS

Acebutolol Hydrochloride RS380 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

'#-
Q)
u 20
C
CU
;:::,
.E
CJ)
C
CU
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Acenocoumarol RS00l Instrument: Dispersive Phase: Potassium Bromide Disc

'#-
Q)
u 20
C
Jg
.E
CJ)
C
CU
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Acetazolamide RS002 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·')
V-S6 Infrared Reference Spectra 2023

Acetylcysteine RS00J Instrument: Dispersive Phase: Potassium Bromide Disc

100.0. .

60

40

-;fl.
Q)
'-' 20
c::
ro
=
.E
en
c::
~ a.a
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm-1)

~
0

Q)
'-'
20
c::
ro
=
.E
en
c::
~ a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Adrenaline (Epinephrine) RS004 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

1000 800 600 400.0

Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S7

Alfuzosin RS446 Instrument: Fourier Transform Phase: Potassium Chloride Disc

100.0

80

60

40

20

0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Alimemazine RS005 Instrument: Dispersive Phase: 10% w/v Solution in Chloroform Thickness: 0.1mm

;;g-
0

(I)
(.) 20
C
§
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Alverine Citrate RS409 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~ 20
C
§
.E
en
C
ro
i= 0.0

Wavenumber (cm-1)
V-S8 Infrared Reference Spectra 2023

Amantadine RS006 Instrument: Dispersive Phase: Potassium Bromide Disc

80

60

40

~
0

Q)
c., 20
C
CU
:::,
-E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Aminoglutethimide RS403 Instrument: Fourier Transform Phase: Potassium Bromide Disc

'?F.
Q)
c., 20
C
CU
~
E
en
C
CU
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Amiodarone RS008 Instrument: Dispersive Phase: 15% w/v Solution in Dichloromethane Thickness: 0.1 mm

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm- 1)
2023 lnfrared Reference Spectra V-S9

Amisulpride RS462 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100.0

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)

Amitriptytine RSSOl Instrument: Fourier Transform Phase: Attenuated Total Reflectance

40

~
!?...-
2lc:: 20
"'
~,,,
c::
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)

Am1odipine Besilale RS489 Instrument: Fourier Transform Phase: Attenuated Total Reflectance
100

80

60

40

0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)
V-S 10 Infrared Reference Spectra 2023

Amlodipine Malelde RS491 Instrument: Fourier Transform Phase: Attenuated Total Reflectance
100

80

60

40

g 20
Q)

~
E
"'e'!
C

I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)

Amlodipine Mesi.bm, RS490 Instrument: Fourier Transform Phase: Attenuated Total Reflectance
100

80

60

40

lQ)
'-'
C
20
~
.E
,,,
C
e'!
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)

Amoxicillin Sodium RSOlO Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

~ 20
C
ro
=
.E
U)
C
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)
2023 Infrared Reference Spectra V-S 11

Amoxicillin Trihydrate RSOU Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
Q)
0 20
C
co
:::::
.E
CJ)
C
co
,= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Ampicillin Sodium RS366 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
Q)
0 20
C
co
:::::
.E
CJ)
C
co
,= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Ampicillin Trihydrate RS013 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

~ 20
C
co
:::::
E
CJ)
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S 12 Infrared Reference Spectra 2023

Amylmetacresol RS014 Instrument: Dispersive Phase: Thin Film

100.0.. ··················· .....,................................... .

80

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·')

A.nas1ro:mle RS493 Instrument: Fourier Transform Phase: Potassium Bromide Disc

60

40

:,!,!_

Q)
u 20
C:
.l!l
""E
<I)
C:
e!
f-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)

Arginine Hydrochloride RS470 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0 ... . ...................... . ,.....,..............................,....... .... . ................~·-•~•---....................................... ··r .. ·• ....... .

60

40

~ 20
c::
~
E
en
c::
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
 2023 Infrared Reference Spectra V-S 13

Aspirin RS483 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

40

2l 20
ffi

1 0 . .,.......... ······ .........:-• ,...........................

2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)

Atenolol RS015 Instrument: Dispersive Phase: Potassium Bromide Disc

~
Q)
(.) 20
C
Jg
-E
en
C
ro
t-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Azapropazone RS016 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
Jg
E
en
C
ro
t-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S 14 Infrared Reference Spectra 2023

Anhydrous Azapropazone RSOl 7 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

~
0

Q)
(.) 20
C
co
=
.E
CJ)
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Azelastine Hydrochloride RS018 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

40

~ 20
C
co
=
.E
"'
C
co
t-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Azitbromycin RS487 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

j 20
E
"'C:
e
f- 0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)
 2023 Infrared Reference Spectra V-S 15

Beclometasone Di propionate ( 1) RS020 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1mm
100.0 .

60

40

~
0

a,
u 20
C:
:§
.E
CJ)
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Beclometasone Dipropionate (2) RS379 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0

80

60

40

~
0

a,
u 20
C:
:§
.E
CJ)
C:
~
f-- 0.0
2000 0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Beclometasone Dipropionate Monohydrate RS021 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ................................

80

60

C
~ 20
C:
rn
:::::
.E
CJ)
C:
~
t-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S 16 Infrared Reference Spectra 2023

Benorilate RS023 Instrument: Dispersive Phase: Potassium Bromide Disc

?ft
u
Q)
20
C
ro
=
.E
u,
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Benzatropine Mesilate RS026 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

?ft
Q)
u 20
C
ro
=
-E
u,
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Benzoic Acid RS025 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

40

~ 20
C
ro
=
-E
en
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)
2023 Infrared Reference Spectra V-S 17

Benzydamine Hydrochloride RS027 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

~
a,
u 20
C
Jg
-E
Cl)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Benzyl Hydroxybenzoate RS028 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

a,
u 20
C
rn
:i::
.E
Cl)
C
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Betamethasone RS029 Instrument: Dispersive Phase: Potassium Bromide Disc

80

60

40

~ 20
C
rn
::=:
-E
Cl)
C
rn
i= 0.0
2000 0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S 18 Infrared Reference Spectra 2023

Bezafibrate RS419 Instrument: Fourier Transform Phase: Potassium Bromide Disc

80

~
0

2l 20
C
rn
='
.E
en
C
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Bicalutamide RS466 Instrument: Fourier Transform Phase: Potassium Bromide Disc

80

~
0

a.>
0
20
C
rn
='
-E
en
C
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Bleomycin Sulfate RS367 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

2l 20
C
:£l
.E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S 19

Bronopol RSOJl Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

"if!.
a,
0 20
C:
CU
:::::
.E
"'C:
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Buclizine Hydrochloride RS032 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

"if!.
a,
0 20
C:
:§
.E
"'
C:
CU
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Bumetanide RSOJJ Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

~ 20
C:
CU
:::::
.E
"'C:
CU
i= 0.0
2000.0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm- 1)
V-S20 Infrared Reference Spectra 2023

Bupivacaine RS034 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0 ··.····•··················••······················ ·•···•····················. ·. ··.·•······································

BO

40

~
0

Q)
() 20
C
co
=:
.E
U)
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 BOO 600 400.0
Wavenumber (cm-')

Busulfan RS035 Instrument: Dispersive Phase: Potassium Bromide Disc

"#-
Q)
() 20
C

£l
.E
U)
C
co
t= 0.0
2000.0 1800 1600 1400 1200 1000 BOO 600 400.0
Wavenumber (cm-1)

Butyl Hydroxybenzoate RS036 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

40

~ 20
C
co
=:
-E
U)
C
co
~ 0.0
2000.0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm- 1)
2023 Infrared Reference Spectra V-S2 l

Calcium Folinate RS368 Instrument: Fourier Transform Phase: Potassium Bromide Disc

"cf!.
(I)
(..) 20
c::
(IJ
:::::
.E
"'
c::
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Calcium Polystyrene Sulfonate RS037 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~
~ 20
c::
(IJ
:::::
.E
"'
C

~ 0.0.,....... ,,.. , ..., .

2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Candesarlan RS494 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

40

~
a,
g 20
i"'
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)
V-S22 Infrared Reference Spectra 2023

Captopril RS038 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0 .............. • ~•••••• .. T,·.. ·.·.·.·..·.· ........ , ••••e··• ............-........... . .. , .......................................................... .,.,,., . . . . ._ .. ,.,.............. •.•• . . ·••••• . , ....... •-••••••••••••"•' •

40

~
0

CD
u 20
C
:§
.E
"'roC
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Carbamazepine RS406 Instrument Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

CD
u 20
C
ro
=
.E
"'
C
ro
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Carbary! RS039 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
ro
=
.E
U'J
C
ro
.-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
2023 Infrared Reference Spectra V-S23

Carbenoxolone RS041 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

'i?-
a.,
'-' 20
c::
ro
=
-E
en
c::
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Carbimazole RS042 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

:;ii
0

~ 20
C
ro
=
-E
en
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Carvedilol RS476 Instrument: Fourier Transform Phase: Potassium Bromide Disc

80

60

40

~
w 20
'-'
C
Cll
=
"E'
(f)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S24 Infrared Reference Spectra 2023

Cefalexin RS049 Instrument: Fourier Transform Phase: Potassium Bromide Disc

60

~
(I)
(.) 20
C:
ro
:;::
-E
(/)
C:
ro
i-= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Cefotaxime Sodium RS044 Instrument: Dispersive Phase: Potassium Bromide Disc

,R_
0

(I)
(.) 20
C:
ro
:;::
-E
(/)
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Cefoxitin Sodium RS045 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~ 20
C:
ro
:;::
-E
(/)
C:
ro
i-= 0.0 •1•···· •••.. •... •• ..................... .. ... >······ ····--·-··--
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S25

Cefradine RS0S0 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

e::,
(I)
u 20
C
C'O
=
.E
en
C
C'O
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Ceftriaxone Sodium RS046 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~
(I)
u 20
C
ro
=
.E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Cefuroxime Axetil RS047 Instrument: Dispersive Phase: Potassium Bromide Disc

80

60

40

~ 20
C
C'O
=
.E
en
C
C'O
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S26 Infrared Reference Spectra 2023

Cefuroxime Sodium RS048 Instrument: Fourier Transform Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')

Celecoxib RS497 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

40

e_:.
Q)
(.)
C:
20
~
E
"'
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·')

Celiprolol Hydrochloride RS420 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0 - - - - - - - - - ......

80

60

40

~ 20
C
ro
=
-E
<fJ
C
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)
2023 Infrared Reference Spectra V-S27

Cetirizine Hydrochloride RS456 Instrument: Fourier Transform Phase: Potassium Btomide Disc
100.0

80

40

<>'2-
Q)
t) 20
C:
ct1
=
-E
VJ
C:

~ a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Chlorhexidine RS449 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

0~

Q)
t) 20
C:
ct1
=
-E
rJ}
C:
~
t-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Chloroquine RS054 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1 mm
100.0 . -····-··~·······•·.·.·

80

60

40

B 20
C:
ct1
=
-E
en
C:
ct1
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S28 Infrared Reference Spectra 2023

Chloroxylenol RS055 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0. •C" ,,,,.,.,,.,,., .... ,~ .. ,,, . • T"""'""'''''''''''"' ..... , ' ' ' T , """' • , .... ,. ,,

80

~
0

Cl)
(.) 20
C

"'
::::,
.E
en
C
~
f-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Chlorpromazine RS056 Instrument: Dispersive Phase: 5% wlv Solution in Chloroform Thickness: 0.1mm
100.0

80

60

40

~
0

Cl)
(.) 20
C
"'
::::,
.E
en
C
~
f-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Chlorpropamide RS057 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S29

Chlortalidone RS058 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
Q.)
u 20
C
:§
.E
"'
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Choline Salicylate RS059 Instrument: Dispersive Phase: Thin Film

100.0

80

60

40

'#-
Q.)
u 20
C
CU
==
.E
"'
C
CU
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Choline Theophyllinate RS060 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
CU
==
.E
"'
C
~
1- 0.0
2000.0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm-1)
V-S30 Infrared Reference Spectra 2023

Ciclosporin RS445 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

Q)
c.) 20
C
<U
:::::
.E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Cimetidine RS061 Instrument: Dispersive Phase: Potassium Bromide Disc

'if!.
Q)
c.) 20
C
g
.E
en
C
<U
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Cinnamic Acid RS062 Instrument: Dispersive Phase: Potassium Bromide Disc

80

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
2023 Infrared Reference Spectra V-S31

CiDnarizine RS492 Instrument Fourier Transform Phase: Attenuated Total Reflectance

80

60

40

Q) 20
IE
V>

! 0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm 1)

Citalopram RS471 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100.0

80

60

40

~
0

Q)
u 20
C
ro
:::e
.E
C/J
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Clarithromycin (A) RS424 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·')
 V-S32 Infrared Reference Spectra 2023

Clarithromycin (B) RS480 Instrument: Fourier Transform Phase: Attenuated Total Reflectance
100.0 .....·. ·.· . ·. ·. .·. ·.·.·. · . ·. ·. · . · ......,... ·····················.·.·. ··.·.·.·.•·· ............. ...................................................,, .... ____ ......... ,....................................................,................................................................ .

80

60

~
0

Q)
u 20
C
CU
;e
.E
"'CU
C

i= 0. 0
2000 . 0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Clindamycin Hydrochloride RS064 Instrument: Dispersive Phase: Potassium Chloride Disc

80

60

~
Q)
u 20
C
:§
-E
"'C
£ 0. 0
2000.0 1800 1600 1400 1200 1000 800 600 400 . 0
Wavenumber (cm- 1)

Clioquinol RS065 Instrument: Dispersive Phase: Potassium Bromide Disc

100 . 0

80

60

~ 20
C
CU
;e
-E
"'
C
CU
i= 0. 0 + ............................................... ······················..·..····..····.....................................

2000 . 0 1800 1600 1400 1200 1000 800 600 400 . 0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S33

Clobazam RS066 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

~ 20
C
"'
::::
.E
(/]
C:

"'
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Clobetasol Propionate RS067 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

~
Q)
(.) 20
C

"'
::::
.E
(/]
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Clofazimine RS068 Instrument: Dispersive Phase: Liquid Paraffin Mull

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
V-S34 Infrared Reference Spectra 2023

Clomipramine Hydrochloride RS069 Instrument: Dispersive Phase: Potassium Chloride Disc

100 .0 ·····-···-······-······································· ······• . ········. . . ·.·. · . · . . . ·•. · .· .· . · · · . · .. .. . . . . ........ ..... ········ . ......................................... ············----·.. .. ····· ... •·. · . · · •·..... ···•• .

60

"#.
(1)
t) 20
C
:§
.E
"'
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Clonazepam RS432 Instrument: Fourier Transform Phase: Potassium Bromide Disc

"#.
(1)
t) 20
C
ro
=
-E
"'Cro
,= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Clozapine RS444 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 ············································ ····················..····· ..······...................... .................·.·. · . · . ·. . . . . . . ·. · . ·.·. ·.

~ 20 ······························································································"······
C
:§
-E
"'
C
~
f- 0.0 ....................... .,............ ·-··············..·······•· ......................................................................... .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
 2023 Infrared Reference Spectra V-S35

Cocaine RS071 Instrument: Fourier Transform Phase: Potassium Bromide Disc

~
0

(])
(.) 20
C
,gi
.E
"'ro
C

i= 0.0
2000 0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Codeine RS072 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

(])
(.) 20
C
ro
;:::,
.E
U)
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Colestyramine RS369 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

60

40

~ 20
C
ro
;:::,
.E
"'
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S36 Infrared Reference Spectra 2023

Compressible Sugar RS401 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 .... ·······-··· •·· .. . ... ····-~··-····--'"·······--·· ............................

40

;f!.
Q)
u 20
C
Ct)
::::
.E
en
C
Ct)

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Cyclizine RS075 Instrument: Dispersive Phase: Potassium Bromide Disc

1QQ.Q ··• ••••·••••••·····. ·.·. · . · . ·•.··. · . ·.·.·.·.·, •·.·.·.·.·.·.·.·.·.·.·.•·······················r····· -······························•····-···································•······""··......................................................... ,•.•••••.••.

40

;f!.

~ 20
C

£l
.E
en
C
Ct)

i= 0.0
2000.0 1800 1600 1400 1200 1000 BOO 600 400.0
Wavenumber (cm·')

Cyclizine Hydrochloride RS076 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0 '"" ........................................,............................... ········ .. ···········

BO

40

1800 1600 1400 1200 1000 BOO 600 400.0

Wavenumber (cm·')
 2023 Infrared Reference Spectra V-S37

Cyclopenthiazide RS077 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .

80

40

~
0

Cl)
u 20
C
g
.E
"'ro
C

t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Cyclopentolate RS078 Instrument: Dispersive Phase: Thin Film

100.0

80

60

40

-;f!.
Cl)
u 20
C
g
.E
"'
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Cyclophosphamide RS079 Instrument: Dispersive Phase: 10% w/v Solution in Chloroform Thickness: 0.1mm
100.0 _.......,.._,;;,,-.,,..;.;.;;.......,......

80

60

40

2l
C
20
ro
::::::
.E
"'
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S38 Infrared Reference Spectra 2023

Cyproheptadine RS080 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 . ,,....,,., .. .,,.,, ..,..,..... ··--····.· . · . ·. · . ·. · .·.••. ·. ·•.• . ·

60

:ii
0

~ 20
C
Cll
:::::
.E
(J)
C
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Cyproterone Acetate RS395 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ,......... ,,, .... .

80

40

'#.
Q.l
(.) 20
C
g
-E
(J)
C
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Cytarabine RS081 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

~ 20
C
Cll
:::::
.E
(J)
C
Cll
t= 0.0 +····"·"·' ................... ,.. -:,...... ''. ············· ,
2000.0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S39

Dacarbazine RS082 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

Q)
(..) 20
C
ro
=
.E
(f)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Dantrolene Sodium RS422 Instrument: Fourier Transform Phase: Potassium Bromide Disc

80

60

40

~
0

Q)
(..) 20
C
ro
=
.E
(f)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Dantron RS083 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

40

~ 20
C
ro
=
.E
(f)
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S40 Infrared Reference Spectra 2023

Dapsone RS084 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .,,......,..,_,,...,................,........................... ········'······ ••.•. •. •................,................................. ,...- - - - - · , •- - - - - - - - - - - - •- - - - - - - - - , - - - - - - - - - - - - - - - - -•- - - - - - - - ·

?ft
Q)
t.) 20
C
co
:i:::
.E
en
C
co
.,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Desferrioxamine Mesilate RS086 Instrument: Dispersive Phase: Potassium Bromide Disc

~
Q)
t.) 20
C
co
:i:::
.E
en
c::
co
.,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Desipramine Hydrochloride RS087 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

60

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S41

Desogestrel RS088 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

60

40

::ii:
0

a., 20
(.)
C

"'
:::,
.E
(f)
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Dexamethasone RS089 Instrument: Dispersive Phase: Potassium Bromide Disc

of!.
<I)
(.) 20
C

£l
.E
(f)
C

$}. 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Dextromoramide RS090 Instrument: Dispersive Phase: Potassium Bromide Disc

BO

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·1)
V-S42 Infrared Reference Spectra 2023

Dextropropoxyphene RS091 Instrument: Dispersive Phase: Thin Film

100.0 '" ' ' ' " " " " " ' " " " ' ~ - - - · · • " " " ' ' """""············ . • • . , , . , .• , ..

80

60

40

~
0

Q)
(.) 20
C

"'
:::e
.E
"'
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Dextropropoxyphene Napsilate RS092 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0 ...... .

60

40

~
Q)
(.) 20
C

"'
:::e
.E
"'
C

"' 0.0
i=
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Diamorphine Hydrochloride RS093 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S43

Diazoxide RS094 Instrument: Dispersive Phase: Potassium Bromide Disc

~
0

Q)
u 20
C
«:J
:::::
.E
(/)
C
~
I- a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Dibrompropamidine Isetionate RS095 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

e:.
Q)
u 20
C
«:J
:::::
.E
(/)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Diclofenac RS096 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
«:J
:::::
.E
rn
C
«:J
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)
V-S44 Infrared Reference Spectra 2023

Diclofenac Diethylamine RS371 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 '"' '"""""·"'······ '' ...... .

60

40

~
0

CD
(.) 20
C
(ll
:::::
.E
(/)
C:
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Dicycloverine Hydrochloride RS098 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

~
CD
(.) 20
C
:§
.E
(/)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Diethylamine Salicylate RS099 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S45

Diethylcarbamazine RS411 Instrument: Dispersive Phase: Thin Film

100.0

80

60

40

~
0

(I)
(.) 20
C
:§
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Diflucortolone Valerate RSlO0 Instrument: Fourier Transform Phase: 5% w/v Solution in Dichloromethane Thickness: 0.1 mm

~
(I)
(.) 20
C
CU
:::,
-E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Diflunisal (form B) RSlOl Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 '''"""""'""""'

80

40

~ 20
C
:§
.E
en
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S46 Infrared Reference Spectra 2023

Dihydrocodeine RS102 Instrument: Dispersive Phase: Thin Film

100.0 ,.,, .. , '". "'''""'""'"""•'" """""""""'""'" ,,, .... ,, ,, .. ,.,,, "' ,,,,,. . . . . . ,. . . . ,,, . ·. .·. . . , . . . ·•· . ''

40

~
Q)
(.) 20
C
ro
:::::
'E'=
rn
C
ro
,= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Diloxanide Furoate RS103 Instrument: Dispersive Phase: Potassium Bromide Disc

80

40

~
Q)
(.) 20
C
ro
:::::
'E'=
rn
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Dimenhydrinate RS104 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·')
 2023 Infrared Reference Spectra V-S4 7

Dimethyl Phthalate RS105 Instrument: Dispersive Phase: Thin Film

100.0

60

40

'#.
<l>
u 20
C
C1l
E
E
"'C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Dimeticone RS381 Instrument: Fourier Transform Phase: Thin Film

100.0

80

60

40

~
0

<l>
u 20
C
C1l
:::::
-E
"'C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Dinoprostone RS448 Instrument: Fourier Transform Phase: 0.2% w/v solution in chloroform Thickness: 0.5 mm
100. 0 ·,;;:=..__c:;;.;,,,,:,;,;,_.::::;::_:r···········"·"·"·"·"·

80

60

40

~ 20
C
(1J
:::::
.E
"'
C
(1J

~ 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S48 Infrared Reference Spectra 2023

Diphenhydramine Hydrochloride RS450 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

-;!?.
Q)
(.) 20
C

~"'
-E
en
C

t="' 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Diphenylpyraline Hydrochloride RS106 Instrument: Dispersive Phase: Liquid Paraffin Mull

~
0

~ 20
C

"'
~
-E
en
C

t="' 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Dipipanone RS107 Instrument: Dispersive Phase: Thin Film

100.0

80

60

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm· 1)
 2023 Infra red Reference Spectra V-S49

Dipivefrine Hydrochloride RS382 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

~
0

Q)
(.) 20
C
C'il
::::,
.E
(/)
C
C'il
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Dipyridamole RS108 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

40

~
0

Q)
(.) 20
C
fl
.E
(/)
C
C'il
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Disodium Pamidronate RS109 Instrument: Fourier Transform Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
V-SSO Infrared Reference Spectra 2023

Disopyramide RSllO Instrument: Dispersive Phase: 10% w/v Solution in Chlorofrm Thickness: 0.1 mm

'?F.
Q)
0 20
C

="'
-E
en
C
"'
~ 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Disulfiram RS111 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

'?F.
Q)
0 20
C

="'
-E
en
C
"'
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Docusate Sodium RS112 Instrument: Dispersive Phase: 10% w/v Solution in Dichloromethane Thickness: 0.1 mm
100.0

80

60

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
 2023 Infrared Reference Spectra V-S 51

Domiphen Bromide RS383 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100,0

80

60

cf!.
Q)
t.) 20
C
C'O
:i::
.E
en
C
C'O
~ 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Dopamine Hydrochloride RS113 Instrument: Dispersive Phase: Potassium Chloride Disc

cf!.
Q)
t.) 20
C
C'O
:i::
.E
en
C
C'O
~ 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Dosulepin Hydrochloride RS114 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

60

40

~ 20
C
C'O
:i::
.E
en
C
~
r- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S52 Infrared Reference Spectra 2023

Doxapram Hydrochloride RS116 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

60

40

"if!.
Q)
(.) 20
C
ctl
:i::
-E
"'
C
ctl
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Doxepin Hydrochloride RS117 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 T••• .,. ••••••.••.••..•.••.....•........•.... , ••••••.••.••.••.•..•..•..............•....•..•.... , •·•·••·························•··•·••••·•;•···•·······•···········

80

40

"if!.
Q)
(.) 20
C
Jg
-E
"'ctlC
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Droperidol RS384 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 ···-··· ..........................

~ 20
C
Jg
-E
"'
C
~
f- a.a
2000.0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S53

Dydrogesterone RS118 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ...

80

60

40

#-
Q)
0 20
C
ro
=
.E
u,
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Edrophonium Chloride RS120 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

#-
(I)
0 20
C
Jg
.E
u,
C
ro
~ a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Ephedrine RS121 Instrument: Dispersive Phase: Thin Film

100.0

60

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
V-S54 Infrared Reference Spectra 2023

Ephedrine Hydrochloride RS436 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

BO

'?F.
Q)
t.) 20
C

="'
.E
en
C
"' 0.0
i=
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Epinephrine (Adrenaline) RS122 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

'?F.
Q)
t.) 20
C

="'
.E
en
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Erythromycin RS123 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 . . ................ .

60

40

~ 20 ,k.. ,,.·.·.·· .......... .......... ., . . . . . . . . . . . . . . . . . ,

="'
.E
en
C

~ 0.0 +·----~-•.--·•· .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-SSS

Erythromycin Estolate RS124 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

?
0

Q)
u 20
C
,gi
-E
en
C
CU
i-=:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Erythromycin Ethyl Succinate RS125 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0

80

60

?
0

Q)
u 20
C
CU
:::::
.E
en
C
CU
i-=:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)

Erythromycin Lactobionate RS126 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

C
2l 20
CU
:::::
.E
en
C
CU
t-== 0.0
2000 0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm·1)
V-S56 lnfrared Reference Spectra 2023

Erythromycin Stearate RS127 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
Cl)
(.) 20
C
ro
:::::,
.E
en
C
ro
t= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Estramustine Sodium Phosphate RS128 Instrument: Dispersive Phase: Potassium Bromide Disc
100 a

80

60

40

~
0

Cl)
(.) 20
C

~
-E
en
C
ro
t= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Estropipate RS129 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
 2023 Infrared Reference Spectra V-S57

Etamiphylline RSl31 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

:li
0

a.,
u 20
C
(0
:::::
-E
en
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Etamiphylline Camsilate RS132 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

60

40

'2f2.
a.,
u 20
C
(0
:::::
-E
er,
C

~ 0.0 .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Ethambutol Hydrochloride RS134 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

~ 20
C
(0
:::::
-E
er,
C
(0

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S58 Infrared Reference Spectra 2023

Ethyl Cinnamate RS136 Instrument: Dispersive Phase: Thin Film

100 a

80

60

40

?ft
(I)
(.) 20
C
,gJ
-E
en
C
ro
t= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Etidronate Disodium RS440 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~
0

(I)
(.) 20
C
ro
:::::
-E
en
C
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Etodolac RS139 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S59

Etoposide RS396 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100 0

80

60

40

e:.
a,
20
(.)
C
m
=
.E
(J)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Etynodiol Diacetate RS138 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

;ii!.
a, 20
(.)
C
rn
=
.E
(J)
C
rn
~ 0.0
2000.0 1800 1600 1400 1200 1000 BOO 600 400.0
Wavenumber (cm- 1)

Felbinac RS372 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

B
C
20
rn
=
.E
(J)
C
rn
.=: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 V-S60 Infrared Reference Spectra 2023

Fenbufen RS140 Instrument: Dispersive Phase: Potassium Bromide Disc

~
0

Q)
(.) 20
C

"'
:::,
-E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Fenoprofen RS141 Instrument: Dispersive Phase: Thin Film

~
e....-
Q)
(.) 20
C

"'
:::,
-E
en
C

"' 0.0
i=
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Fenoprofen Calcium RS142 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

~ 20
C

"'
:::,
-E
en
C
~
1- 0.0
2000.0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm- 1)
2023 Infrared Reference Spectra V-S61

Fexofenadine Hydrochloride RS459 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

25
C:
20
£l
.E
If)
C:
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm· 1)

Flavoxate Hydrochloride RS143 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

~
(])
u 20
C
m
=
.E
en
C
m
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Plecainide Acetate RS397 Instrument: Fourier Transfonm Phase: Potassium Bromide Disc
100.0

80

60

40

8 20
C:
m
=
.E
en
C
m
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)
V-S62 Infrared Reference Spectra 2023

Flucloxacillin Magnesium RS144 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

~
0

Q)
'-' 20
C

"'
;::::
.E
"'C
I-'"'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Flucloxacillin Sodium RS145 Instrument: Dispersive Phase: Liquid Paraffin Mull

#.
Q)
'-' 20
C

"'
;::::
.E
"'
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Flucytosine RS146 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

~ 20
C
,gi
.E
"'C
~ 0.0 ..,..........................
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
2023 Infrared Reference Spectra V-S63

Fluocinolone Acetonide Dihydrate RS147 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0 ...

60

40

-;F.
Q)
'-' 20
C
rn
~en
C
rn
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Fluocinonide RS148 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
rn
:::::
.E
(/)

ffi
,= 0.0

Wavenumber (cm-')

Fluocortolone Hexanoate RS149 Instrument: Dispersive Phase: Potassium Bromide Disc

BO

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
 V-S64 lnfrared Reference Spectra 2023

Fluorescein Sodium RS151 Instrument: Dispersive Phase: Potassium Bromide Disc

~
(I)
u 20
C
:§
.E
en
C
ro
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Fluorometholone RS152 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

u 20
(I)

C
ro
::::,
.E
en
C
ro
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Fluorouracil RS153 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
25 20
C
ro
::::,
.E
en
C
ro
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
 2023 lnfrared Reference Spectra V-S65

Fluoxetine RS398 Instrument: Fourier Transform Phase: Thin Film

100.0

80

60

40

~
Q)
0 20
C:
ro
:::::
.E
en
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Fluoxetine Hydrochloride RS385 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100 0

80

60

40

~
0

Q)
0 20
C:
ro
:::::
.E
en
C:
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Flupentixol Decanoate RS154 Instrument: Dispersive Phase: Thin Film

100.0

80

60

~ 20
C:
ro
:::::
.E
en
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S66 Infrared Reference Spectra 2023

Flurazepam Monohydrochloride RS155 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1 mm
100.0

80

~
0

~ 20
C
Cl)
;:::,
.E
rn
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Flurbiprofen RS156 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0 .,,,............... •-~···•·••··-·--- .,.. ..........................,,............................ ••----·-··--••·........................... ,....................... - ........... , ......................- ... ,... ,,...... ·--·---•-••·..··.·.··.··.• ,........... . ............... . .,

80

60

40

#-
Q.)
u 20
C
Cl)
;:::,
.E
rn
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Flurbiprofen Sodium RS157 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ·------- ---·- . . . ··- ,------. ·----- .......... •· ..

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 lnfrared Reference Spectra V-S67

Fluticasone Propionate RS158 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
100.0

80

60

40

'#-
(])
u 20
C
ro
::=<
E
"'C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Fluvastatin Sodium RS479 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

~
(])
u 20
C
ro
::=<
.E
en
C
~
t-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Fluvoxamine Maleate RSl59 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

2l
C
20
ro
::=<
.E
en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S68 Infrared Reference Spectra 2023

Foscarnet Sodium RS160 Instrument: Fourier Transform Phase: Potassium Bromide Disc

"ifi.
a,
0 20
C

:£l
.E
en
C
rn
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Fosfestrol Sodium RS161 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

~
0

a,
0 20
C
rn
:;::,
.E
en
C
rn
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Fosinopril RS472 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100. 0 ·-· ...•.... . . ··--···-··· ., ·•. ···-··-·--········ .... •-.. ,......... •-· . ······-·--,-··--·· . ·······-···-·---· ··········-·--······ •·. . . ....... · ..... · ··········---·······,·~·-··-

80

~ 20
C
rn
:;::,
.E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
 2023 Infrared Reference Spectra V-S69

Fumaric Acid RS163 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

<l)
0 20
C

fl
.E
en
C
ro
~ a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Furazolidone RS164 Instrument: Dispersive Phase: Pptassium Bromide Disc

e:-
<l)
0 20
C
ro
::::::
.E
en
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Fusidic Acid RS166 Instrument: Dispersive Phase: 10% w/v Solution in Chloroform Thickness: 0.1mm
100.0

60

40

~ 20
C
ro
::::::
.E
en
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S70 Infrared Reference Spectra 2023

Gemfibrozil RS167 Instrument: Dispersive Phase: Potassium Bromide Disc

~
Q)
(.) 20
C
ro
+:::
.E
Cf)
C
ro
i= 0.0
2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Gliclazide RS168 Instrument: Dispersive Phase: Potassium Bromide Disc

60

40

~
Q)
(.) 20
C
ro
+:::
.E
en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')

Glimepiride RS463 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

1800 1600 1400 1200 1000 BOO 600 400,0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S 71

Glipizide RSI69 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

~
Q)
(.) 20
C
ro
:::,
.E
"'
C

~ 0.0
2000 0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Gliquidone RS170 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

60

40

~
Q)
(.) 20
C
ro
:::,
.E
"'
C
ro
,= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Glycine RSI 71 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ,••·-·-·-•--··•.••,•

80

60

40

~
~ 20
C
ro
:::,
.E
"'roC
,= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S72 Infrared Reference Spectra 2023

Griseofulvin RS172 Instrument: Fourier Transform Phase: 1.5% w/v Solution in Chloroform Thickness: 0.1 mm

#-
<l)
u 20
C
f!
.E
00
C
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Haloperidol RS173 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 .••···-···· ·····--·-····. · . · -. ··-· ......

60

40

~
<l)
u 20
C
ro
='
.E
00
C
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Hexachlorophene RS174 Instrument Dispersive Phase: Potassium Bromide Disc

100.0 ,. ··-···•······--- . . .......,......, •.· .·. •....... ,. . .,. .•. --~---- '"···'·····••. •· ·-· · · --------.,--- _ ····-······· .. -·,··- -·--··. ····- . . .. ······--·· ............. ,.,.,.........-,. .............,.................... -····· .,. ·.·.·.· ·.·. ·.·.... ,

80

40

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·')
 2023 Infrared Reference Spectra V-S73

Homatropine RS175 Instrument: Dispersive Phase: Thin Film

100 0

60

0~
CD
u 20
C
Cll
:::::
.E
Cf)
C
Cll
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Hydralazine RS176 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
CD
uC 20
g
.E
Cf)
C
Cll
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Hydralazine Hydrochloride RS 177 Instrument: Dispersive Phase: Potassium Chloride Disc

100 0

80

60

40

~ 20
C
Cll
:::::
.E
Cf)
C
Cll
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S7 4 Infrared Reference Spectra 2023

Hydrochlorothiazide RS178 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

a,
u 20
C:
CU
~
E
en
C:
CU
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Hydrocortisone Acetate RSI 79 Instrument: Dispersive Phase: Potassium Bromide Disc

~
0

a,
u 20
C:
CU
:::::
.E
en
C
CU
~ 0.0
2000.0 1800 1600
Wavenumber (cm- 1)

Hydrocortisone Sodium Phosphate RS386 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

25C: 20
CU
:::::
.E
en
C
CU
~ 0.0 ·i····•"''·''·'····.·.·.·, ••

2000.0 1800 1600 1400 1200 800 600 400.0

Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S75

Hydrocortisone Sodium Succinate RS180 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0

60

40

~
0

a,
u 20
C:
g
.E
CJ)
C:
co
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Hydroflumethiazide RSI81 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

~
~ 20
C
co
c::,
.E
CJ)
C
co
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Hydroquinone RS495 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

BO

60

40

2l 20
ffi
~
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm-1)
 V-S76 Infrared Reference Spectra 2023

Hydroxycarbamide RS184 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 f······•·· .. ................. .

80

60

~
0

Q)
(.) 20
c:::
ro
:::::
.E
"'
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)

Hydroxychloroquine RS182 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1mm
100.0

80

60

40

~
0

Q)
(.) 20
c:::
ro
:::::
-E
"'c:::
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 BOO 600 400.0
Wavenumber (cm-1)

Hyoscine Butylbromide RS185 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .,.... ... ·••.•---··· ·····~·-" .... -··· ...... -~--·-··············· ...........,...............,.................,........,_.,,. . --··~--·-..·--·· . . . ·····.··········· ... ....................... ·-···-········· .. . ,

80

~ 20
C
(IJ
:::::
-E
"'C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 BOO 600 400.0
Wavenumber (cm- 1)
 2023 Infrared Reference Spectra V-S77

Ibuprofen RS186 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
~
Q)
u 20
C

"'
::::,
.E
CJ)
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Ifosfamide RS428 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
ffi
::::,
-E
(f)
C
"' 0.0
,=:
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

lmipramine Hydrochloride RS509 Instrument: Fourier Transform Phase: Attenuated Total Reflectance
100

80

60

40

1850 1650 1450 1250 1050 850 650

Wavenumber (cm- 1)
V-S78 Infrared Reference Spectra 2023

Indometacin RS187 Instrument: Dispersive Phase: Liquid Paraffin Mull

80

60

40

~
Cl)
(.) 20
C:

~
-E
"'C:
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-1)

Indoramin RS188 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
Cl)
(.) 20
C
ro
:::::
.E
"'C:
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Inositol Nicotinate RS 190 Instrument: Dispersive Phase: Potassium Bromide Disc

80

60

40

~ 20
C:
~
.E
"'
C:
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S79

Iodipamide RS191 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

~
0

a,
(.) 20
C:
g
.E
"'
C:
<tl
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Iopamidol RS441 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 ·:··· . ··········· ..-· .......

60

40

~
0

a,
u 20
C:
<tl
::::
-E
"'
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

lopanoicAcid RS192 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .........................

80

60

40

~ 20
C

~
E
"'C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S80 Infrared Reference Spectra 2023

lrbesartan RS467 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

'if?.
Q)
(.) 20
C
:§
E
"'
C
!}_ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

lsometheptene RS195 Instrument: Dispersive Phase: Thin Film

~
0

Q)
(.) 20
C
ro
::::,
.E
"'
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Isoniazid RS196 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
ro
::::,
.E
"'ro
C

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
 2023 Infrared Reference Spectra V-S81

Isosorbide Dinitrate RS412 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

'#.
Q,)
(.) 20
C
ro
=
.E
"'
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Isradipine RS373 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

60

40

'#.
Q,)
(.) 20
C
ro
:;:::
.E
<n
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Ketoprofen RS198 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 f'"' ......... .

80

60

40

~ 20
C
ro
:;:::
.E
<n
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S82 Infrared Reference Spectra 2023

Ketoprofen RS484 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

a
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)

Labetalol RS199 Instrument: Dispersive Phase: Potassium Bromide Disc

60

~
~
(l)
(.) 20
C
C1J
=
-E
"'
C:
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Lacidipine RS407 Instrument: Fourier Transform Phase: Liquid Paraffin Mull

100.0

60

40

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S83

Lamivudine RS451 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

(I.)
(J 20
C
rn
::::::
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Levobunolol Hydrochloride RS200 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

40

~
0

Cl)
(.) 20
C
rn
::::::
.E
U)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Levodopa RS201 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
V-S84 Infrared Reference Spectra 2023

Levomepromazine RS404 Instrument: Fourier Transform Phase: Liquid Paraffin Mull

100.0

80

60

40

~
e...,
u
Q)
20
C
ro
::=:
.E
"'ro
C

.= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Lidocaine (1) RS202 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .,...............- ............ .

60

40

~
0

Q)
u 20
C
ro
::=:
.E
(/)
C
~
I- a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Lidocaine (2) RS405 Instrument: Fourier Transform Phase: Film on Potassium Bromide Disc
100.0

60

~ 20
C
fl
.E
"'
C
ro
i= 0.0 >······················....... ,............ .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S85

Lincomycin Hydrochloride RS203 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

40

"#-
Q)
(..) 20
C:
<U
:::::
°E'en.
C:
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Lofepramine Hydrochloride (Form A) RS399A Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

60

40

"#-
Cl}
(..) 20
C:
g
.E
C/J
C:
<U
i=
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Lofepramine Hydrochloride (Form B) RS399B Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

40

2l 20
C:
<U
:::::
.E
en
C:
<U
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S86 Infrared Reference Spectra 2023

Lomustine RS204 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

Q)
(.) 20
C
('J
::::,
-E
<J)
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Loprazolam Mesilate RS205 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

40

cfi.
Q)
(.) 20
C
:§
-E
<J)
C
('J

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Loratadine RS435 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
('J
-~
E
<J)
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S87

Lorazepam RS206 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

Q)
<.) 20
C
(U
:::,
.E
en
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Lormetazepam RS207 Instrument: Dispersive Phase: Potassium Bromide Disc

?f.
Q)
<.) 20
C
(U
:::,
.E
en
C
(U

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Losartan RS453 Instrument: Fourier Transform Phase: Liquid Paraffin Mull

100.0

80

~ 20
C
g
.E
en
C
(U

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S88 Infrared Reference Spectra 2023

Mebendazole RS503 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

80

60

40

0 +---- --------- ------·-------+--------------------------------------,-------- ------ - -----------,

4000 3600 3200 2800 2400 2000
Wavenumber (cm-1)

Mebendazole RS503 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100
! i
I (\r,
'' ~A I
80
q ~
-• fiif
60 ---
0 J
( /
II
~ ~~
/ / II ,•••v••nw
r/
••••>•-'•-'•-'
C -
~

40 ------cc-

0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S89

Mebeverine RS208 Instrument: Dispersive Phase: Thin Film

e:.
<J)
20
u
C
:@
-E
en
C
co
i-= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Medroxyprogesterone Acetate RS421 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

;:F.
<J)
u 20
C
:@
-E
en
C
co
i-= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400 0
Wavenumber (cm·')

Mefenamic Acid RS210 Instrument: Dispersive Phase: Liquid Paraffin Mull

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·')
V-S90 Infrared Reference Spectra 2023

Megestrol Acetate RS211 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

?F.
Q)
(.) 20
C
ro
=
.E
rn
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Melatonin RS455 Instrument: Fourier Transform Phase: Potassium Bromide Disc

~
0

Q)
(.) 20
C
ro
=
-E
rn
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Meloxicam RS374 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm- 1)
 2023 Infrared Reference Spectra V-S91

Melphalan RS212 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

Q)
(.) 20
C
ro
-~
E
"'
C
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Menadiol Sodium Phosphate RS213 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0

60

40

~
0

Q)
(.) 20
C
ro
:::::
.E
"'
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Menadione RS214 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

B 20
C
ro
=
.E
"'
C
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S92 Infrared Reference Spectra 2023

Meptazinol Hydrochloride RS215 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 ...

80

40

~
0

Cl)
'-' 20
C
,gi
.E
en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Mepyramine Maleate RS216 Instrument: Dispersive Phase: Liquid Paraffin Mull

'?F-
Cl)
'-' 20
C:
ro
::::,
.E
en
C:
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Mercaptopurine RS426 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
ro
::::,
.E
en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
 2023 Infrared Reference Spectra V-S93

Mesalazine RS454 Instrument: Fourier Transform Phase: Potassium Bromide Disc

~
0

{J
Q)
20
C
(1J

=
-E
en
C
(1J

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Metformin Hydrochloride RS217 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

>!2.
0

Q)
{J 20
C
(1J

=
-E
en
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Methadone RS218 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

40

i15 20
C
(1J

=
-E
en
C
(1J

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S94 Infrared Reference Spectra 2023

Methanol RS387 Instrument: Fourier Transform Phase: Thin Film

4000.0 3600 3200 2800 2400 2000.0

Wavenumber (cm-1)

~
<I)
(.) 20
C
:§
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Methoxamine Hydrochloride RS220 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

~ 20
C

~
E
"'
C
~
1- 0.0
2000 0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
2023 Infrared Reference Spectra V-S95

Methyl Nicotinate RS222 Instrument: Dispersive Phase: Liquid Paraffin Mull

100~ .

80

60

40

#
Q)
u 20
C
ro
=
-E
Cl)
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Methyldopa RS223 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

#
Q)
u 20
C
ro
=
.E
Cl)
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Methyldopate Hydrochloride RS224 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

~ 20
C
ro
~
E
Cl)
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S96 Infrared Reference Spectra 2023

Methylphenidate RS485 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

40

! 20
E
!!!
e!
r o~---··--·---""·.. -·········· ·--·-~··· .,,·.············•··············----·······-0--··-"+······-.. -··---··········'············································
2000 1800 1600 1400 1200 1000 800 400
Wavenumber (cm1)

Methylphenobarbital RS388 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 .,........................... ·········••7••················ ················T'··············

BO

60

~
a.,
(.) 20
C
:§
.E
"'
C

~ a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Methylprednisolone RS225 Instrument: Dispersive Phase: Potassium Bromide Disc

100.Q . .. .... ••· . ·········:· ......................................... :·······. ·······················,······..············-······•·············----······················..·········......,.,-.....................................r···-····· ..························•

60

40

~ 20
C
:§
.E
"'C
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)
 2023 Infrared Reference Spectra V-S97

Methylprednisolone Acetate RS226 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

'#.
(I)
u 20
C
rn
=
.E
U)
C
rn
,= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Methysergide Maleate RS227 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

40

'#.
(I)
u 20
C
rn
=
.E
U)
C
rn
,= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Metipranolol RS375 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

~ 20
C

£:l
.E
U)
C
rn
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S98 Infrared Reference Spectra 2023

Metoprolol RS228 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

Q)
(.) 20
C
:§
.E
en
C
Ct)

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Metronidazole RS229 Instrument: Dispersive Phase: Potassium Bromide Disc

"?F.
Q)
(.) 20
C
Ct)
::::,
.E
en
C
Ct)

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Metronidazole Benzoate RS408 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

60

40

2l 20
C
Ct)
::::,
.E
en
C
Ct)

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S99

Instrument: Dispersive Phase: Liquid Paraffin Mull

80

60

40

~
0

CD
Ll 20
C
ro
:::::
-E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Metyrapone (2) RS231 Instrument: Dispersive Phase: 50% w/v Solution in Macrogol 400 Thickness: 0.1 mm
100.0 , ............................,................... .

80

60

ef!.
CD
Ll 20
C
:§
.E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Mexenone RS232 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

~ 20
C
ro
:::::
.E
en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S 100 Infrared Reference Spectra 2023

Instrument: Dispersive Phase: Potassium Chloride Disc

80

40

~
0

Q)
u 20
C:

=="'
.E
(J)
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Mianserin Hydrochloride RS234 Instrument: Dispersive Phase: Potassium Chloride Disc

100 ,0 T•••~~-••---·--••--••••••••c•••••••••·············•····· ······

~
0

~ 20
C:
Jg
.E
(J)
C:
"'
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Midazolam RS235 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm- 1)
 2023 Infrared Reference Spectra V-S 101

Minocycline Hydrocliloride Dib.ydrale RS502 Instrument: Fourier Transform Phase. Attenuated Total Reflectance
100

80

60

40

"""'g 20
fl
E
"'C
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm-1)

Minoxidil RS376 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
Q)
u 20
C
ro
::::,
.E
en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Mirtazapine RS434 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
ro
::::,
.E
en
C
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)
V-S 102 Infrared Reference Spectra 2023

Mitobronitol RS236 Instrument: Dispersive Phase: Potassium Bromide Disc

80

60

40

~ 20
C
C1l
;:::::
.E
"'
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Modafinil RS505 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

80

60

40

~
a,
'-'
C:
20
~
E
<JI
C:
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm 1)

Morphine RS237 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
2023 lnfrared Reference Spectra V-S103

Moxisylyte Hydrochloride RS238 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

#
Q)
0 20
C
co
=
.E
en
C
co
I-'= 0.0 .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Mucic Acid RS377 lnstnument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

#
©
0 20
C
co
=
.E
en
C
co
I-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Mupirocin RS425 Instrument: Fourier Transform Phase: thin film

100.0

80

60

40

~ 20
C
co
~
E
en
C
~
f- 0.0 .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S 104 Infrared Reference Spectra 2023

Mycophenolate Mofetil RS504 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

60

40

~
e....
Q)
u
C:
20
"'
~
"'
C:
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm-1)

Nabumetone RS239 Instrument: Dispersive Phase: Potassium Bromide Disc

~
~
Cl)
(.) 20
C
rn
::::,
-E
"'
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Naftidrofuryl RS240 Instrument: Fourier Transform Phase: Thin Film

100.0

80

60

40

~ 20
C
rn
::::,
-E
"'rn
C

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
 2023 Infrared Reference Spectra V-S 105

Nalidixic Acid RS241 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

a,
(.) 20
C
:§
.E
en
C
ro
.,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Naproxen RS244 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
ro
:::::
.E
<f)
C
~
1-- a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Nevirapine RS507 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100

80

60

40

g 20
ro
E
E
"'c::
l."
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm·1)
V-S 106 Infrared Reference Spectra 2023

Niclosamide RS245 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

2000.0 1800 1600 1400 1200 800 600 400.0

Wavenumber (cm-')

Nicorandil RS461 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

8
C:
20
£l
.E
"'
C:
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)

Nicotinamide RS246 Instrument: Dispersive Phase: Potassium Bromide Disc

80

60

40

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
 2023 Infrared Reference Spectra V-S 107

Nicotine RS452 Instrument: Fourier Transform Phase: 0.5% w/v Solution in Chloroform
100.0

80

60

40

~
e...-
Q)
0 20
C
ro
+"
.E
<f)
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Nifedipine RS248 Instrument: Dispersive Phase: Potassium Bromide Disc

~
0

Q)
0 20
C
ro
+"
.E
<f)
C
2
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Nikethamide RS249 Instrument: Dispersive Phase: Thin Film

100.0

60

40

8 20
C

-~
E
<f)
C
2
t--- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
 V-S108 Infrared Reference Spectra 2023

Nitrazepam RS482 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100.0 'O"'"'""'"'''''''''""'''''""'"'""" . ''"''''' "''"

80

60

40

~
0

(I)
u 20
C
co
~
-E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Nizatidine RS410 Instrument: Fourier Transform Phase: Potassium Bromide Disc

';J?.
(I)
u 20
C
co
~
.E
en
C
CU
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Olanzapine RS477 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~
0
~ 20
C:
co
;:::,
-E
en
C:

~ 0.0 +·-··"-'"''"'""""""""''''''

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
 2023 Infrared Reference Spectra V-S109

Ondansetron RS418 Instrument: Fourier Transform Phase: Potassium Bromide Disc

"2f2.
Q)
0 20
C
(1J
::ce
-E
(F)
C
(1J
i-= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Ondansetron Hydrochloride RS427 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

cf!.
Q)
<.) 20
C
(1J
-~
E
en
C
(1J

i-= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Oxazepam RS253 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 , ... -------

80

60

40

~ 20
C
(1J
::ce
-E
(F)
C
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S 110 Infrared Reference Spectra 2023

Oxetacaine RS254 Instrument: Dispersive Phase: Potassium Bromide Disc

~
(].)
(.) 20
C
:§
.E
U)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Oxprenolol RS255 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

~
0

Q)
(.) 20
C
ro
:;:::
.E
"' C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Oxybutynin RS442 Instrument: Fourier Transform Phase: Thin Film

100.0

60

~ 20
C
ro
:;:::
.E
en
C
ro
i-= 0.0 , ................................... , .............................
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
2023 Infrared Reference Spectra V-S 111

Oxycodone RS457 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C:
:§
.E
"'C:
l.'?
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)

Oxymetholone RS256 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
CU
=
.E
"'C
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Pantopraz.ole Sodium RS488 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

80

60

40

0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm· 1)
V-S 112 Infrared Reference Spectra 2023

Papaverine Hydrochloride RS415 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

?f?.
(I)
(.) 20
c::
g
.E
en
c::
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Paracetamol RS258 Instrument: Dispersive Phase: Potassium Bromide Disc

?f?.
(I)
(.) 20
c::
ro
;::::
.E
en
c::
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Pentamidine Isetionate RS259 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·1)
2023 Infrared Reference Spectra V-S 113

Pentazocine (Form B) RS261 Instrument: Dispersive Phase: Potassium Bromide Disc

100 0 ·. ·.· . · ·.· ......... .

80

60

40

~
0

Q)
'-' 20
C
Jg
E
Cl)
C
CU
t== 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Pentazocine Lactate RS263 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
Q)
'-' 20
C
CU
=
-E
Cl)
C
CU
t== 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Pentobarbital RS264 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
CU
=
-E
Cl)
C
CU
t== 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S 114 Infrared Reference Spectra 2023

Pethidine RS266 Instrument: Dispersive Phase: Thin Film

100.0

80

60

40

"#-
a, 20
u
C
:§
.E
(J)
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Pethidine Hydrochloride RS267 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

40

"#-
a, 20
u
C
ro
:::::
.E
(J)
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Phenindione RS268 Instrument: Dispersive Phase: Potassium Bromide Disc

60

40

~ 20
C
:§
E
"'
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-Sl 15

Phenoharbital RS270 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

:cf!.
0

(1)
0 20
C
ro
:te
.E
"'
C
ro
..= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Pbenoxybenzamine Hydrochloride RS271 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

cfl.
(1)
0 20
C
ro
:te
E
"'ro
C

..= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Phenytoin RS272 Instrument: Dispersive Phase: Potassium Bromide Disc

80

60

40

8C 20
ro
:te
-E
"'C:
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S 116 Infrared Reference Spectra 2023

Pholcodine RS273 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

a,
u 20
C
rn
::::::
.E
Cl)
C
~
f-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)

Pilocarpine Nitrate RS274 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 . . .,. .·.· .·.· .·.·. ·.·.·. ·.·.. ,.......... ,., .. ,., .,, .... ,.. ,..,,,______,...... ,,,, .., .... ,,_..,.. ,,. ...... ,............ ,.··. ·. ·. ""'"'"'"'"""''"''"""" "·"" .. '"""''·''

60

40

#.
Cl)
u 20
C

~
E
Cl)
C
rn
.,::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Pimozide RS389 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

60

40

2l
C
20
g
.E
Cl)
C
rn
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
2023 lnfrared Reference Spectra V-S 11 7

Pindolol RS275 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
Q)
(.) 20
C
ro
::=<
.E
"'Cro
.,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Piperonyl Butoxide RS478 Instrument: Fourier Transform Phase: Thin Film

'#-
(I.)
(.)
20
C
fl
-E
"'
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Pivmecillinam RS506 Instrument: Fourier Transform Phase: Potassium Chloride Disc

100

80

60

40

8
C:
20
Jll
E
en
C:
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm· 1)
V-S 118 Infrared Reference Spectra 2023

Pizotifen RS276 Instrument: Dispersive Phase: Potassium Bromide Disc

60

40

~
(I)
(.) 20
C:
:§
.E
(J)
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)

Pizotifen Malate RS277 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

~
(I)
(.) 20
C:
(tl
E
E
(J)
C:
(tl
.,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Polythiazide RS280 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

40

~ 20
C:
(tl
:::::
.E
(J)
C:
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)
 2023 Infrared Reference Spectra V-S 119

Prazosin RS281 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
(])
(.) 20
C
ro
:::::
-E
CfJ
C
~
I- a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Prednisolone RS282 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
(])
(.) 20
C
fl
-E
CfJ
C
ro
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Prilocaine RS285 Instrument: Dispersive Phase: Thin Film

100.0

60

40

~ 20
C
ro
:::::
-E
CfJ
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 V-S 120 Infrared Reference Spectra 2023

Primidone RS286 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

'ifl
Q)
u 20
C
ro
=
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Probenecid RS287 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

Q)
u 20
C
ro
=
.E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Procainamide RS288 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1 mm
100.0

80

60

'if!.

~ 20
C
ro
=
.E
en
C
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S 121

Prochlorperazine (1) RS289 Instrument: Dispersive Phase: 10% wlv Solution in Chloroform Thickness: 0.1 mm
100.0

80

60

40

~
<lJ
'-' 20
C
rn
=
-E
(fJ
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)

Prochlorperazine (2) RS390 Instrument: Fourier Transform Phase: Thin Film

100.0

80

60

40

~
0

<lJ
'-' 20
C
,gi
.E
(fJ
C
rn
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Prochlorperazine Mesilate RS290 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

60

40

i1,l 20
C
rn
=
.E
(fJ
C
rn
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S122 Infrared Reference Spectra 2023

Procyclidine RS291 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ,,...................... . .....0 ... • ....

80

60

40

~
Q)
(.) 20
C
:§
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Procyclidine Hydrochloride RS292 Instrument: Dispersive Phase: Potassium Chloride Disc

100,0 T'' , , , , , . ,. ., ,,.,

80

40

,R_
0

Q)
(.) 20
C
co
:i:::
-E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Progesterone RS293 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
C
co
:i:::
-E
en
C
co
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
2023 Infrared Reference Spectra V-S 123

Proguanil Hydrochloride RS294 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

cF-
Q)
(.) 20
C
ro
~
E
VJ
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Promazine RS295 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1 mm
100.0

80

60

40

~
0

Q)
(.) 20
C
ro
=
.E
VJ
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Promethazine RS297 Instrument: Dispersive Phase: 5% wlv Solution in Chloroform Thickness: 0.1 mm

80

60

~ 20
C
:§
.E
VJ
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S 124 Infrared Reference Spectra 2023

Propofol RS416 Instrument: Fourier Transform Phase: Thin Film

100.0

BO

60

~
0

(I)
(.) 20
C
:§
.E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 BOO 600 400.0
Wavenumber (cm-1)

Propylene Glycol RS437 Instrument: Fourier Transform Phase: 40% w/v Solution in water Thickness 0.5 mm
100.0

80

60

~
(I)
(.) 20
C
ro
::::,
.E
en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Propranolol RS298 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ··-····. • . •·. · .· . ·•··•.••.•······.1·· ..••

80

60

40

~
B
C
20, ........ ·"·················->··-···-······ ........- ........ , ............................................ ·fi···· ·I

ro
::::,
.E
en
C
e
1- a.a +....................................,... · · - - - - +.................................+,>--..--····..................... , .....................................--+............. . . ..
2000.0 1800 1600 1400 1200 800 600 400.0
Wavenumber (cm- 1)
 2023 Infrared Reference Spectra V-Sl25

Propylthiouracil RS300 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
CJ)
(.) 20
C
ro
::::::
-E
en
C
ro
..= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Protriptyline RS301 Instrument: Dispersive Phase: Thin Film

100.0

80

60

40

~
0

CJ)
(.) 20
C
ro
::::::
-E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Proxymetacaine RS302 Instrument: Dispersive Phase: Thin Film

100.0

80

60

40

2l
C
20
,g
-E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-Sl26 Infrared Reference Spectra 2023

Proxymetacaine Hydrochloride RS303 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

60

?f!.
Q)
(.) 20
C

="'
.E
en
C
"'
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Pseudoephedrine RS304 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .................... .

60

40

~ 20
C
:§
.E
en
C
"'
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Pyrazinamide (1) RS306 Instrument: Dispersive Phase: Potassium Bromide Disc

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S127

Pyrazinamide (2) RS438 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ,,,,,,,,,,,,,,,,,,,,,,,,

80.

60

40

~
Q)
u 20
C
C\J
:::::
.E
<FJ
C
C\J
i-=: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Pyridoxine Hydrochloride RS308 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

~
Q)
u 20
C
C\J
:::::
.E
<FJ
C
C\J
i-=: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Pyrimethamine RS309 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 '. '""" ,,,,,..,

80

60

40

B 20
C
C\J
:::::
.E
<FJ
C
C\J
t-== 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S128 Infrared Reference Spectra 2023

Quetiapine Fumarate RS496 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

80

60

40

~
Q)
u
C:
20
"'
-~
(/)
C:
(!!
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm 1)

Quinolin-8-ol RS310 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

;fl.
Q)
0 20
C
ro
::=:
.E
(/)
C
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Ramipril RS417 Instrument: Fourier Transfonm Phase: Potassium Bromide Disc

100.0

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S129

Ranitidine Hydrochloride RS311 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

~
a, 20
(.)
C
CU
=
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Ribavirin RS349 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

a,
(.) 20
C
CU
=
.E
(/)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Rifampicin RS312 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
CU
=
.E
en
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-Sl30 Infrared Reference Spectra 2023

Risedronate Sodium RS498 Instrument: Fourier Transfonn Phase: Attenuated Total Reflectance

40

C
Q)

~
20
~
E
"'
~
I-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm-1)

Ritodrine Hydrochloride RS313 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0 ··r···"·"···"·"········"· •······'·····..· •.•

80

40

~
0

Q)
(.) 20
C
co
:i::::
.E
en
C

E. 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Rizatriptan Benzoate RS474 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
100.0

80

2000.0 1800 1600 1400 1200 800 600 400.0

Wavenumber (cm- 1)
 2023 Infrared Reference Spectra V-S 131

Salbutamol RS314 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

(])
u 20
C
"'
:I='
.E
en
C
"' 0.0
i=
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Salbutamol Sulfate RS315 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
(])
u 20
C

"'
:I='
.E
en
C

"' 0.0
i=
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Salicylic Acid RS316 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 C' , • • • • , ....... .

80

60

40

~ 20
C
"'
:I='
.E
en
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S132 Infrared Reference Spectra 2023

SainJelfflll Xinafoale RS499 Instrument: Fourier Transform Phase; Attenuated Total Reflectance

40

~
2:l 20
C
.l!!
'E
"'C
e1
f-
0
2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber (cm-1)

Selegiline RS400 Instrument: Fourier Transform Phase: Thin Film

100.0 ·······················

80

40

~ 20
C
:§
.E
en
C
ro
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Sertraline Hydrochloride (A) RS460 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm· 1)
 2023 Infrared Reference Spectra V-S133

Sertraline Hydrochloride (B) RS443 Instrument: Fourier Transform Phase: 1% w/v solution in dichloromethane Thickness: 0.5 mm
100.0 .

80 ~
60

40

~
QJ
u 20
C
C\J
:::::
-E
<J)
C
C\J
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Simvastatin RS423 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

QJ
u 20
C

fl
-E
<J)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Sodium Amidotrizoate RSJ 17 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~ 20
C
C\J
E
E
<J)
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S 134 Infrared Reference Spectra 2023

Sodium Feredetate RS378 Instrument: Fourier Transform Phase: Potassium Bromide Disc

80

60

40

'#.
a, 20
u
C
g
.E
"'roC
.= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Sodium Polystyrene Sulfonate RS318 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ,·····---·· ...

60

40

~
0

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')

Solifenacin RS508 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

1850 1650 1450 1250 1050 850 650

Wavenumber (cm· 1)
 2023 Infrared Reference Spectra V-S135

Spironolactone RS321 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1 mm
100.0

80

60

40

#,
~ 20
C
co
:::::,
.E
en
C
co
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Stanozolol RS322 Instrument: Dispersive Phase: Potassium Bromide Disc

#,
Q)
(.) 20
C
ro
:::::,
-E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Sulfadiazine RS325 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
fl
E
en
C
co
._'::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S 136 Infrared Reference Spectra 2023

Sulfamethoxazole RS327 Instrument: Dispersive Phase: Potassium Bromide Disc

60

40

~
0

Q)
(.) 20
C:
ctl
;:<
.E
en
C:

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Sulfasalazine RS429 Instrument: Fourier Transform Phase: Potassium Bromide Disc

0~

Q)
(.) 20
C:
ctl
=
.E
en
C:
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Sulindac RS323 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S 13 7

Sulpiride RS391 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 .

80

60

40

?f2.
a,
u 20
C
(1)
:::::
.E
en
C
(1)
t'.:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Sumatriptan RS414 Instrument: Fourier Transform Phase: Liquid Paraffin Mull

100.0

80

60

40

?f2.
a, 20
u
C
(1)
:::::
.E
en
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Sumatriptan Succinate RS413 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
100.0

80

~ 20
C
(1)
:::::
.E
en
C
(1)

.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S 138 Infrared Reference Spectra 2023

Tamoxifen RS328 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

~ 20 ______,,,,,,,.. ,,,,.. ,,,,,,,,, . ,,•,·,,··~--~-··,

C:
:§
.E
"'
C:

~ 0.0 +·······"'"''"'"''''"'"''·"''''""''-"'"'''"'"'''''""''""""""""'''"'"'+····· • '''' , .., , ; .. , , , , , , , , , , , , , ,,,,,,, • ''' ,,+ .

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')

Teicoplanin RS510 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

85

~
C
~
E
<Jl
C

~ 65
1850 1650 1450 1250 1050 850 650
Wavenumber (cm-1)

Telmisartan RS486 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100

0
2000 1800 1600 1400 1200 1000 600 600 400
Wavenumber (cm·1)
2023 Infrared Reference Spectra V-S 139

Temozolomide RSSOO Instrument: Fourier Transform Phase; Potassium Bromide Disc

100

80

60

40

~
a,
'--' 20
C:
2
E
"'~
C:

I-

1800 1600
Wavenumber (cm-1)

Terbinafine Hydrochloride RS475 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 -- _

80

60

40

1800 1600 1400 1200 800 600 400.0

Wavenumber (cm-1)

Testosterone RS329 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1mm
100.0

80

60

40

~ 20
C
:£1
.E
rf)
C
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S 140 Infrared Reference Spectra 2023

Testosterone Decanoate RS330 Instrument: Dispersive Phase: Potassium Bromide Disc

60

~
e...,
Q)
CJ 20
C
C'O
=
-E
(f)
C
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Testosterone Isocaproate RS331 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
Q)
CJ 20
C
C'O
=
-E
"'
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Testosterone Propionate RS332 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
C'O
=
-E
"'C'O
C

t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
 2023 Infrared Reference Spectra V-S 141

Theophylline RS333 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0

80

40

~
0

Q)
(.) 20
C
m
:::::
.E
"'
C
m
i= 0.0 .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Thiopental RS334 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

#
Q)
(.) 20
C
m
:::::
.E
"'
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Thioridazine (1) RS335 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

40

23 20
C
:§
.E
"'
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S 142 Infrared Reference Spectra 2023

Thioridazine (2) RS336 Instrument: Dispersive Phase: Thin Film

100.0 ..,.......... ,.,.....· . ,........... .,...................................,. -,....................... - - ~ - - -.. ·. ·-~--....--................,....- ......................... .,.....................................................,...............................................

60.

#.
Q)
u 20.
C:
(IJ
;::
-E
en
C:

~ a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Thiotepa RS337 Instrument: Dispersive Phase: 2% w/v Solution in Carbon Disulphide Thickness: 0.1 mm
100.0 . .,............................ ,.·.·.·.·.·.·.·.·.·. ·.•· · ·. ·"·' ..........,..................................,................. . ..................................... . ........................................... ,........................................... .. ...................... .

40

~ 20.,....................- ...............,..................- .............., .................................... ;,.................... u .......,...................................... :··············1 .. ,, ..................................... . .

C:
(IJ
;::
-E
en
C:

~ a.a ,........................................ . ,. . . . . .- . . . . . . . . . . . . . . . .,.......................................,...................................... ,....................................................................... > .......................... ,...........

2000.0 1800 1600 1400 1200 800 600 400.0

Wavenumber (cm-1)

Ticarcillin RS458 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

g 20
Jg
.E
"'C:
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·1)
2023 Infrared Reference Spectra V-S 143

Timolol RS339 Instrument: Dispersive Phase: Thin Film

100.0

80

60

40

~
Q)
u 20
C
ro
=
.E
"'
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Tioconazole RS393 Instrument: Fourier Transform Phase: Potassium Bromide Disc

~
Q)
u 20
C
ro
=
.E
"'
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Tioguanine RS340 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

2l 20
C
ro
=
.E
"'
C
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
V-S 144 Infrared Reference Spectra 2023

Tolazamide RS342 Instrument: Dispersive Phase: Potassium Bromide Disc

80

40

e:.
Q)
u 20
C
ro
=
.E
en
C
ro
i= a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Tolbutamide RS343 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

;ii
~
Q)
u 20
C
ro
=
.E
"'
C
~
I- a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Tramadol Hydrochloride RS465 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ·;·--· .................................., ..,____..... _____········ ..................... .

80

60

;ii
~
~ 20 ,.................................,..................................... ,............... ·-tf¥,,. -· , .•.••. ···-··""···-·· ...•. ;...... . .........
C
ro
=
.E
"'C
~ 0.0 ., .........................................................,..........................._ __,____ ......................,...................... ·••.··.·.· .......... .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
 2023 Infrared Reference Spectra V-S 145

Trandolapril RS468 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100.0

80

40

~
(])
'-'
20
C

"'
::::,
-E
rJ>
C

.="' 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Tranexamic Acid RS344 Instrument: Dispersive Phase: Potassium Bromide Disc

~
0

(])
'-' 20
C

"'
::::,
-E
rJ>
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)

Tranylcypromine Sulfate RS345 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

60

40

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
V-S 146 Infrared Reference Spectra 2023

Trazodone Hydrochloride RS346 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

'#.
a, 20
0
C:
:§
.E
en
C:
m
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Triamcinolone Acetonide RS348 Instrument: FourierTransform Phase: Potassium Bromide Disc

'#.
a,
0 20
C:
m
:::::
.E
en
C
m
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Triamcinolone Hexacetonide RS394 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

fl3 20
C:
m
:::::
.E
en
C:
m
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
 2023 Infrared Reference Spectra V-S 14 7

Triclofos Sodium RS350 Instrument: Fourier Transform Phase: Potassium Bromide Disc

~
(])
'--' 20
C
§
"E'=
en
C
ro
,::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Tritluoperazine RS351 Instrument: Dispersive Phase: Thin Film

100.0 .,........ , ,.

80

60

~
(])
'--' 20
C
§
"E'=
en
C
ro
,::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Trifluridine RS469 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
ro
-~
E
en
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S 148 Infrared Reference Spectra 2023

Trimethoprim RS354 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ................................................ ··················· . ······ . . ·.......... .

60

40

?Ji!.
Q)
(.) 20
C
CU
:i::::
.E
rn
C
CU
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Trimipramine Maleate RS355 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ..,........................................... .

40

~
0

~ 20
C
CU
:i::::
.E
rn
C
CU
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Triprolidine Hydrochloride RS356 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm- 1)
2023 Infrared Reference Spectra V-S149

Tropicamide RS357 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~
0

CJ)
c.:, 20
C
ro
::::
.E
"'ro
C

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

lJ rsodeoxycholic Acid RS402 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0

80

60

40

~
CJ)
c.:, 20
C:
ro
::::
.E
"'C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Valaciclovir Hydrochloride RS481 Instrument: Fourier Transform Phase: Attenuated Total Reflectance
100.0

80

60

40

25 20
C:

~
E
en
C:
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S 150 Infrared Reference Spectra 2023

ValproicAcid RS431 Instrument: Fourier Transform Phase: Thin Film

100.0

60

40

~
0

Q)
(.) 20
C

~
.E
en
C
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Valsartan RS473 Instrument: Fourier Transform Phase: Potassium Bromide Disc

~ 20 ·+,.······,.···········,.···············
C
ro
~
E
en
C
ro
i= 0.0 .;. .......................................
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)

Venlafaxine Hydrochloride RS439 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0

80

60

40

~ 20
C
Jg
.E
en
C
e
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·1)
 2023 Infrared Reference Spectra V-S 151

Verapamil RS359 Instrument Dispersive Phase: Thin Film

100.0

80

60

40

sQ)
0 20
C
ro
=
.E
<fJ
C
~
I- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Vigabatrin RS360 Instrument Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

40

~
e.....
Q)
0 20
C
ro
=
.E
<fJ
C
~
I- a.a
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')

Warfarin RS361 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0

80

60

40

~ 20
C
ro
=
.E
<fJ
C
ro
i-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
V-S152 Infrared Reference Spectra 2023

Xylometazoline RS362 Instrument Dispersive Phase: 10% w/v Solution in Dichloromethane Thickness: 0.1 mm
100.0

80

60

40

'#.
a.,
'-' 20
C
:§
.E
"'co
C

i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Zidovudine RS447 Instrument: Fourier Transform Phase: Potassium Bromide Disc

80

40

'#.
a.,
'-' 20
C
co
:::::
.E
"'Cco
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')

Zopiclone RS430 Instrument Fourier Transform Phase: Potassium Bromide Disc

100.0

80

60

~ 20
C
co
:::::
.E
"'
C

~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
2023 Infrared Reference Spectra V-Sl53

Zuclopenthixol Acetate RS363 Instrument: Fourier Transform Phase: Thin Film

100.0

80

60

40

~
Q)
u 20
C
ro
~en
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)

Zuclopenthixol Hydrochloride RS365 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0

80

60

40

~ 20
C
ro
:::::
°El
(/)
C
ro
i= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Appendices
2023 Introduction V-A3

Introduction

When a method, test or other matter described in an appendix is invoked in a monograph reproduced from the
European Pharmacopoeia, Pan III of the General Notices applies. When a method, test or other matter described
in an appendix is invoked in any other monograph, Pan II of General Notices applies.
2023 Contents of the Appendices V-A5

Contents of the Appendices

EUROPEAN PHARMACOPOEIA EQUIVALENT TEXTS A15
APPENDIX I A23
A. General Reagents A23
B. Volumetric Reagents and Solutions A162
C. Standard Solutions A170
D. Buffer Solutions A174
E. Reference Materials A182
F. Polymorphism A183
APPENDIX II A184
A. Infrared Spectrophotometry Al84
B. Ultraviolet and Visible Absorption Spectrophotometry A192
C. Nuclear Magnetic Resonance Spectrometry A196
D. Atomic Spectrophotometry: Emission and Absorption A199
E. Fluorescence Spectrophotometry [Fluorimetry] A205
F. X-Ray Fluorescence Spectrometry A205
G. Mass Spectometry A207
1. Inductively Coupled Plasma-Mass Spectrometry A209
H. Raman Spectrometry A211
J. Flow Cytometry A213
K. Peptide Identification by Nuclear Magnetic Resonance Spectrometry A215
L. Chemical Imaging A216
M. Direct Amperometric and Pulsed Electrochemical Detection A222
N. Process Analytical Technology A223
APPENDIX III A226
Chromatographic Separation Techniques A226
A. Thin-layer Chromatography A232
B. Gas Chromatography A235
c. Size-exclusion Chromatography A237
D. Liquid Chromatography A240
E. Paper Chromatography A242
F. Electrophoresis A242
G. Capillary Electrophoresis A249
H. Supercritical Fluid Chromatography A254
J. Isoelectric Focusing A254
K. Peptide Mapping A256
L. Amino Acid Analysis A260
M. Glycan Analysis of Glycoproteins A267
APPENDIX IV A272
A. Clarity of Solution A272
B. Colour of Solution A274
APPENDIX V A277
Determination of: A277
V-A6 Contents of the Appendices 2023

A. Melting Point A277

B. Freezing Point A280
C. Distillation Range A281
D. Boiling Point A282
E. Refractive Index A282
F. Optical Rotation and Specific Optical Rotation A282
G. Weight per Millilitre, Density, Relative Density and Apparent Density A283
H. Viscosity A284
J. Circular Dichroism A289
K. Approximate pH of Solutions A290
L. Determination of pH Values A290
M. Thermal Analysis A292
N. Osmolality A294
0. Conductivity A295
P. Total Organic Carbon in Water for Pharmaceutical Use A297
Q. Density of Solids A298
R. Detection and Measurement of Radioactivity A299
S. Balances for Analytical Purposes A306
APPENDIX VI A309
Qualitative Reactions and Tests A309
APPENDIX VII A313
Nessler Cylinders A313
Tubes for Comparative Tests A313
Limit Test for: A313
Aluminium A313
Ammonium A313
Arsenic A314
Calcium A314
Chlorides A314
Fluorides A315
Heavy Metals A315
Iron A318
Lead in Sugars A318
Magnesium A319
Magnesium and Alkaline-earth Metals A319
Heavy Metals in Herbal Drugs and Herbal Drug Preparations A319
Nickel in Polyols A321
Phosphates A321
Potassium A321
Sulfates A321
APPENDIX VIII A322
A. Non-aqueous Titration A322
B. Amperometric, Potentiometric and Voltametric Titrations A322
C. Oxygen-flask Combustion A323
2023 Contents of the Appendices V-A 7

D. Complexometric Titrations A323

E. Potentiometric Determination of Ionic Concentration using Ion-selective Electrodes A324
F. Determination of Ethanol A325
G. Determination of Methanol and Propan-2-ol A328
H. Determination of Nitrogen A329
J. Tetrazolium Assay of Steroids A330
K. Ethylene Glycol and Diethylene Glycol in Ethoxylated Substances A330
L. Residual Solvents A330
M. Residual Ethylene Oxide and Dioxan A335
N. N,N-Dimethylaniline A336
0. 2-Ethylhexanoic Acid A337
P. Total Protein A337
Q. Acetic Acid in Synthetic Peptides A341
R. Nickel in Hydrogenated Vegetable Oils A341
Sl. Methyl, Ethyl and Isopropyl Methanesulfonate in Methanesulfonic Acid A341
S2. Methyl, Ethyl and Isopropyl Methanesulfonate in Active Substances A342
S3. Methanesulfonyl Chloride in Methanesulfonic Acid A343
S4. Methyl, Ethyl and Isopropyl Toluenesulfonate in Active Substances A344
SS. Methyl, Ethyl and Isopropyl Benzenesulfonate in Active Substances A345
T. Determination of Elemental Impurities A346
U. Tetrabutylammonium in Radiopharmaceutical Preparations A350
V. N-Nitrosamines in Active Substances A350
APPENDIX IX A354
Determination of: A354
A. Sulfated Ash A354
B. Sulfur Dioxide A354
C. Water A355
D. Loss on Drying A358
E. Limit Test for Carbon Monoxide in Medicinal Gases A358
F. Carbon Dioxide in Medicinal Gases A359
G. Nitrogen Monoxide and Nitrogen Dioxide in Medicinal Gases A360
H. Oxygen in Medicinal Gases A360
J. Water in Medicinal Gases A360
K. Gas Detector Tubes A361
L. Nitrous Oxide in Gases A362
M. Water-Solid Interactions: Determination of Sorption-Desorption Isotherms and of Water
Activity A362
APPENDIXX A366
A. Acetyl Value A366
B. Acid Value A366
C. Ester Value A366
D. Hydroxyl Value A367
E. Iodine Value A367
F. Peroxide Value A367
G. Saponification Value A368
V-A8 Contents of the Appendices 2023

H. Unsaponifiable Matter A369

J. Determination of Cineole A369
K. Determination of Aldehydes A370
L. Oxidising Substances A370
M. Essential Oils A370
N. Fixed Oils A371
0. Anisidine Value A375
P. Oils Rich in Omega-3-acids A375
1. Composition of Fatty Acids A375
2. Total Cholesterol in Oils Rich in Omega-3-Acids A377
Q Sterols in Fatty Oils A378
APPENDIX XI A380
A. Total Solids A380
B 1. Ethanol-soluble Extractive A380
B2. Water-soluble Extractive A380
C. Swelling Index A380
D. Foreign Matter A381
E. Essential Oils in Herbal Drugs A381
F. Continuous Extraction of Drugs A382
G. Complete Extraction of Alkaloids A383
H. Stomata A383
J. Ash A383
K. Acid-insoluble Ash A384
L. Pesticide Residues A384
M. Tannins in Herbal Drugs A385
N. Bitterness Value A386
0. Foam Index A386
P. Dry Residue of Extracts A387
Q. Loss on Drying of Extracts A387
R. Test for Aristolochic Acids in Herbal Drugs A387
1. Test for Aristolochic Acids in Herbal Drugs A387
2. Test for Aristolochic Acids I and II in Herbal Drugs A389
S. Determination of Mycotoxins in Herbal Drugs A390
1. Determination of Aflatoxin B1 in Herbal Drugs A390
2. Determination of Ochratoxin A in Herbal Drugs A391
T. Herbal Drugs: Sampling and Sample Preparation A392
U. Microscopic Examination of Herbal Drugs A394
V. Deoxyribonucleic acid (DNA) Based Identification Techniques for Herbal Drugs A395
W. High-performance Thin-Layer Chromatography of Herbal Drugs and Herbal Drug
Preparations A400
X. Contaminant Pyrrolizidine Alkaloids A401
APPENDIX XII A407
A. Disintegration A407
1. Disintegration Test for Tablets and Capsules A407
2. Disintegration Test for Suppositories and Pessaries A409
2023 Contents of the Appendices V-A9

B. Dissolution A413
1. Dissolution Test for Tablets and Capsules (Dissolution Test for Solid Dosage Forms) A413
2. Dissolution Test for Transdermal Patches A420
3. Dissolution Test for Lipophilic Solid Dosage Forms A423
4. Drug Release from Medicated Chewing Gum A424
5. Intrinsic Dissolution A429
6. Apparent Dissolution A430
C. Consistency of Formulated Preparations A432
1. Uniformity of Weight (Mass) A432
2. Uniformity and Accuracy of Delivered Doses from Multidose Containers A432
3. Uniformity of Content A432
4. Uniformity of Dosage Units A433
5. Extractable Volume of Parenteral Preparations A436
6. Vacant
7. Aerodynamic Assessment of Fine Particles - Fine Particle Dose and Particle Size
Distribution A436
8. Preparations for Nebulisation: Characterisation A448
9. Demonstration of Uniformity of Dosage Units Using Larger Sample Sizes A451
10. Content of active ingredient on actuation of the valve test A452
APPENDIX XIII A455
Particulate Contamination A455
A. Sub-visible Particles A455
B. Visible Particles A458
C. Sub-visible Particles in Non-injectable Liquid Preparations A458
APPENDIX XIV A461
Biological Assays and Tests A461
A. Microbiological Assay of Antibiotics A461
B. Immunochemical Methods A468
C. Test for Bacterial Endotoxins A469
D. Test for Pyrogens A475
E. Vacant
F. Test for Depressor Substances A476
G. Test for Histamine A477
H. Monocyte-Activation Test A477
I. Assay of Pancreatin A483
J. Blood and Related Products A485
Coagulants A485
Al. Assay of Human Coagulation Factor II A485
A2. Assay of Factor VII Fraction A486
A3. Assay of Factor VIII Fraction A487
A4. Assay of Factor IX Fraction A488
AS. Assay of Human Coagulation Factor X A488
A6. Assay of Human Coagulation Factor XI A489
A7. Activated Coagulation Factors A489
AS. Assay of Human von Willebrand factor A489
 V-Al0 Contents of the Appendices 2023

A9. Test for Prekallikrein Activator A490

AlO. Assay of Human Plasmin Inhibitor A491
Anticoagulants A492
Bl. Assay of Heparin in Coagulation Factors A492
B2. Assay of Heparin A492
B3. Assay of Human Antithrombin III A493
B4. Assay of Human Protein C A493
BS. Assay of Human Protein S A495
Immunoglobulins A495
Cl. Test for Fe Function oflmmunoglobulins A495
C2. Test for Anticomplementary Activity of Immunoglobulin A496
C3. Assay of Human anti-D Immunoglobulin A498
C4. Test for anti-D Antibodies in Human Immunoglobulin A501
CS. Anti-A and anti-B Haemagglutinins A501
Other blood-related components A503
Dl. Assay of Human tX-1-Proteinase Inhibitor A503
D2. Assay of Human Cl-Esterase Inhibitor A503
. K. Immunological Products A503
1. Assay of Diphtheria Vaccine (Adsorbed) A503
2. Assay of Pertussis Vaccine (Whole Cell) A508
3. Assay of Tetanus Vaccine (Adsorbed) A508
4. Assay of Hepatitis A Vaccine A513
5. Assay of Hepatitis B Vaccine (rDNA) A513
6. Assay of Pertussis Vaccine (Acellular) A514
7. In viva Assay of Poliomyelitis Vaccine (Inactivated) A516
8. Flocculation Value (Ll) of Diphtheria and Tetanus Toxins and Toxoids (Ramon Assay) A517
9. Residual Pertussis Toxin and Irreversibility of Pertussis A518
L. Nucleic Acid Amplification Techniques A520
M. Assay of Interferons A525
NL Numeration of CD34/CD45+ Cells in Haematopoietic Products A527
N2. Colony-forming Cell Assay for Human Haematopoietic Progenitor Cells A529
N3. Nucleated Cell Count and Viability A530
01. Host-cell Protein Assays A531
02. Quantification and Characterisation of Residual Host-cell DNA A536
P. Determination of Bactericidal, Fungicidal or Yeasticidal Activity of Antiseptic Medicinal
Products A538
APPENDIX XV A540
Production and Testing of Vaccines A540
A. Terminology used in Monographs on Biological Products A540
B. Aluminium in Adsorbed Vaccines A540
C. Calcium in Adsorbed Vaccines A541
D. Free Formaldehyde A541
E. Phenol in Immunosera (Antisera) and Vaccines A541
F. Neurovirulence A541
G. Composition of Polysaccharide Vaccines A542
 2023 Contents of the Appendices V-Al 1

H. Chicken Flocks Free From Specified Pathogens for the Production and Quality Control of
Vaccines A544
J. Cell Substrates for the Production of Vaccines for Human Use A547
K. Carrier Proteins for the Production of Conjugated Polysaccharide Vaccines for Human Use A551
L. Immunonephelometry for Vaccine Component Assay A552
M. Substitution of in Vivo Method(s) by in Vitro Method(s) for the Quality Control of Vaccines A553
APPENDIX XVI A556
A. Test for Sterility A556
B. Microbiological Examination of Non-sterile Products A560
1. Test for Specified Micro-organisms A560
2. Microbial Enumeration Tests A564
3. Test for Absence of Mycoplasmas A568
4. Mycobacteria A573
5. Extraneous Agents in Viral Vaccines A573
C. Efficacy of Antimicrobial Preservation A576
D. Microbiological Quality of Non-sterile Pharmaceutical Preparations and Substances for
Pharmaceutical Use A577
E. Microbiological Examination of Cell-Based Preparations A578
F. Microbiological Examination of Herbal Medicinal Products for Oral Use and Extracts used
in their Preparation A581
G. Microbiological Quality of Herbal Medicinal Products for Oral Use and Extracts used in
their Preparation A583
H. Microbiological Examination of Live Biotherapeutic Products A584
APPENDIX XVII A593
A. Particle Size of Powders A593
1. Particle Size Classification of Powders A593
2. Powder Fineness A593
B. Sieves and Filters A594
1. Sieves A594
2. Filters A595
3. Particle-size Distribution Estimation by Analytical Sieving A595
C. Specific Surface Area by Air Permeability A598
D. Vacant
E. Flowability A600
F. Measurement of Consistency and Texture Analysis A601
1. Measurement of Consistency by Penetrometry A601
2. Texture Analysis of Semi-solids or Gels A603
G. Friability A603
1. Friability of Uncoated Tablets A603
2. Friability of Granules and Spheroids A604
H. Resistance to Crushing of Tablets A606
J. Softening Time Determination of Lipophilic Suppositories A606
K. Pycnometric Density of Solids A607
L. Vacant
M. Specific Surface Area by Gas Adsorption A608
V-A12 Contents of the Appendices 2023

N. Powder Dynamics A611

1. Powder Flow A611
2. Powder Flow Properties by Shear Cell Methods A614
0. Microscopy A617
1. Optical Microscopy A617
2. Scanning Electron Microscopy A619
P. Particle Size Analysis by Laser Light Diffraction A623
Q. Characterisation of Crystalline and Partially Crystalline Solids by X-ray Powder Diffraction
(XRPD) A627
R. Porosity and Pore-size Distribution of Solids by Mercury Porosimetry A633
S. Bulk Density and Tapped Density of Powders A635
T. Wettability of Porous Solids Including Powders A638
U. Crystallinity A641
V. Characterisation of Crystalline Solids by Microcalorimetry and Solution Calorimetry A642
APPENDIX XVIII A645
Methods of Sterilisation A645
APPENDIX XIX A653
Containers A653
A. Introduction A653
B. Glass Containers for Pharmaceutical Use A653
C. Plastic Containers and Closures A660
1. Plastic Containers for Aqueous Solutions for Parenteral Infusions A660
D. Containers for Blood and Blood Components A661
1. Sterile Plastic Containers for Blood and Blood Components A661
2. Empty Sterile Containers of Plasticised Poly(Vinyl Chloride) for Human Blood and
Blood Components A663
3. Sterile Containers of Plasticised Poly(Vinyl Chloride) for Human Blood Containing
Anticoagulant Solution A664
E. Rubber Closures for Containers for Aqueous Parenteral Preparations A665
F. Sets for the Transfusion of Blood and Blood Components A667
G. Sterile Single-use Plastic Syringes A669
APPENDIXXX A671
Materials Used for the Manufacture of Containers A671
A. Materials for Containers for Human Blood and Blood Components A671
1. Materials Based on Plasticised Poly(Vinyl Chloride) for Containers for Human Blood
and Blood Components A671
2. Materials Based on Plasticised Poly(Vinyl Chloride) for Tubing Used in Sets for the
Transfusion of Blood and Blood Components A675
3. Materials Based on Non-plasticised Poly(Vinyl Chloride) for Containers for
Non-injectable, Aqueous Solutions A678
4. Materials Based on Non-plasticised Poly(Vinyl Chloride) for Containers for Solid
Dosage forms for Oral Administration A680
5. Materials Based on Plasticised Poly(Vinyl Chloride) for Containers for Aqueous
Solutions for Intravenous Infusion A682
B. Polyolefins A686
2023 Contents of the Appendices V-A13

C. Polyethylene A690
1. Polyethylene Without Additives for Containers for Parenteral Preparations and for
Ophthalmic Preparations A690
2. Polyethylene With Additives for Containers for Parenteral and for Ophthalmic
Preparations A691
D. Polypropylene for Containers and Closures for Parenteral Preparations and Ophthalmic
Preparations A694
E. Poly(ethylene - vinyl acetate) for Containers and Tubing for Total Parenteral Nutrition
Preparations A698
F. Silicone A700
1. Silicone Oil Used as a Lubricant A700
2. Silicone Elastomer for Closures and Tubing A701
G. Plastic Additives A702
H. Polyethylene Terephthalate for Containers for Preparations not for Parenteral Use A708
APPENDIX XXI A710
A. Abbreviated Titles A710
B. Approved Synonyms A710
C. Eye Drops A723
APPENDIX XXII A724
A. Viral Safety A724
B. Minimising the Risk of Transmitting Animal Spongiform Encephalopathy Agents via
Human and Veterinary Medicinal Products A724
APPENDIX XXIII A738
Weights and Measures A738
B. Conversion Tables for Commonly Used Units A738
APPENDIX XXIV A739
Abbreviations A739
APPENDIX XXV A740
Names, Symbols and Atomic Weights of Elements A740
 2023 European Pharmacopoeia Equivalent Texts V-A15

European Pharmacopoeia Equivalent Texts

In monographs reproduced from the European Pharmacopoeia the analytical methods, tests and other
supporting texts are invoked by means of the reference number of the text in the General Chapters of
the European Pharmacopoeia.
The table below lists the contents of the General Chapters of the European Pharmacopoeia and gives
the British Pharmacopoeia or British Pharmacopoeia (Veterinary) equivalents. It is provided for
information but it is emphasised that, for texts of the European Pharmacopoeia, in cases of doubt or
dispute the text published by the Council of Europe is authoritative. Appendices of the British
Pharmacopoeia (Veterinary) are identified by inclusion of '(Vet)' after the Appendix letter, for example,
Appendix XV J (Vet) 1.

Ph. Eur. Subject of text British Pharmacopoeia reference

1 General Notices
2.1.l Droppers Appendix I A
2.1.2 Sintered-glass Filters Appendix XVII B2
2.1.3 UV lamps Appendix III A
2.1.4 Sieves Appendix XVII B 1
2.1.5 Tubes for Comparative Tests Appendix VII
2.1.6 Gas detector tubes Appendix IX K
2.1.7 Balances for Analytical Purposes Appendix V S
2.2.1 Clarity and Degree of Opalescence of Liquids Appendix NA
2.2.2 Degree of Coloration of Liquids Appendix NB
2.2.3 Potentiometric Determination of pH Appendix VL
2.2.4 Approximate pH of Solutions Appendix V K
2.2.5 Relative Density Appendix VG
2.2.6 Refractive Index Appendix VE
2.2.7 Optical Rotation Appendix VF
2.2.8 Viscosity Appendix V H
2.2.9 Capillary Viscometer Method Appendix V H, Method II
2.2.10 Rotating Viscometer Method Appendix V H, Method III
2.2.11 Distillation Range Appendix V C
2.2.12 Boiling Point Appendix VD
2.2.13 Water by Distillation Range Appendix IX C, Method II
2.2.14 Melting point: Capillary Method Appendix V A, Method I
2.2.15 Melting point: Open Capillary Method Appendix V A, Method N
2.2.16 Melting point: Instantaneous Method Appendix V A, Method V
2.2.17 Drop Point Appendix V A, Method III
2.2.18 Freezing Point Appendix VB
2.2.19 Amperometric Titration Appendix VIII B
2.2.20 Potentiometric Titration Appendix VIII B
2.2.21 Fluorimetry Appendix II E
2.2.22 Atomic Emission Spectrometry Appendix II D
2.2.23 Atomic Absorption Spectrometry Appendix II D
2.2.24 Infrared Spectrophotometry Appendix II A
2.2.25 Visible and Ultraviolet Spectrophotometry Appendix II B
2.2.26 Paper Chromatography Appendix III E
2.2.27 Thin-layer Chromatography Appendix III A
2.2.28 Gas Chromatography Appendix III B
2.2.29 Liquid Chromatography Appendix III D
2.2.30 Size-exclusion Chromatography Appendix III C
2.2.31 Electrophoresis Appendix III F
2.2.32 Loss on Drying Appendix IX D
2.2.33 Nuclear Magnetic Resonance Spectrometry Appendix II C
2.2.34 Thermogravimetry Appendix V M
2.2.35 Osmolality Appendix V N
2.2.36 Ion-selective Potentiometry Appendix VIII E
2.2.37 X-ray Fluorescent Spectrophotometry Appendix II F
2.2.38 Conductivity Appendix V 0
2.2.39 Molecular Mass Distribution in Dextrans Appendix III C
2.2.40 Near-Infrared Spectroscopy Appendix II A
2.2.41 Circular Dichroism Appendix VJ

1 Reproduced in full as Part III of the General Notices of the British Phannacopoeia and British Phannacopoeia (Veterinary).
V-A16 European Pharmacopoeia Equivalent Texts 2023

2.2.42 Density of Solids AppendixV Q

2.2.43 Mass Spectrometry Appendix II G
2.2.44 Total Organic Carbon in Water Appendix VP
2.2.45 Supercritical Fluid Chromatography Appendix III H
2.2.46 Chromatographic Separation Techniques Appendix III
2.2.47 Capillary Electrophoresis Appendix III G
2.2.48 Raman Spectroscopy Appendix II H
2.2.49 Falling Ball and Automatic Rolling Ball Viscometer Methods Appendix V H, Method IV
2.2.54 Isoelectric Focussing Appendix III J
2.2.55 Peptide Mapping Appendix III K
2.2.56 Amino Acid Analysis Appendix III L
2.2.57 Inductively Coupled Plasma-Atomic Emission Spectrometry Appendix II D
2.2.58 Inductively Coupled Plasma-Mass Spectrometry Appendix II G 1
2.2.59 Glycan Analysis of Glycoproteins Appendix III M
2.2.60 Vacant
2.2.61 Characterisation of Crystalline Solids by Microcalorimetry and Appendix XVII V
Solution Calorimetry
2.2.63 Direct Amperometric and Pulsed Electrochemical Detection Appendix II M
2.2.64 Peptide Identification by Nuclear Magnetic Resonance Appendix II K
Spectrometry
2.2.65 Voltametric Titration Appendix VIII B
2.2.66 Detection and Measurement of Radioactivity Appendix V R
2.3.1 Identification Reactions Appendix VI
2.3.2 Identification of Fatty Oils by TLC AppendixX N
2.3.3 Identification of Phenothiazines by TLC Appendix III A
2.3.4 Odour Appendix VI
Limit test for:
2.4.1 -Ammonium Appendix VII
2.4.2 - Arsenic Appendix VII
2.4.3 - Calcium Appendix VII
2.4.4 - Chlorides Appendix VII
2.4.5 - Fluorides Appendix VII
2.4.6 - Magnesium Appendix VII
2.4.7 - Magnesium and Alkaline-earth Metals Appendix VII
2.4.8 - Heavy Metals Appendix VII
2.4.9 - Iron Appendix VII
2.4.10 - Lead in Sugars Appendix VII
2.4.11 - Phosphates Appendix VII
2.4.12 - Potassium Appendix VII
2.4.13 - Sulfate Appendix VII
2.4.14 Sulfated Ash Appendix IX A, Method II
2.4.15 Limit test for Nickel in Polyols Appendix VII
2.4.16 Total Ash Appendix XI J, Method II
2.4.17 Limit test for Aluminium Appendix VII
2.4.18 Free Formaldehyde Appendix XV D
2.4.19 Alkaline Impurities in Fatty Oils AppendixX N
2.4.20 Determination of Elemental Impurities Appendix VIII T
2.4.21 Foreign Oils in Fatty Oils by TLC AppendixX N
2.4.22 Composition of Fatty Acids by Gas Chromatography AppendixX N
2.4.23 Sterols in Fatty Oils AppendixX Q
2.4.24 Residual Solvents Appendix VIII L
2.4.25 Residual Ethylene Oxide and Dioxan Appendix VIII M
2.4.26 N,N-Dimethylaniline Appendix VIII N
2.4.27 Heavy Metals in Herbal Drugs and Fatty Oils Appendix VII
2.4.28 2-Ethylhexanoic acid Appendix VIII 0
2.4.29 Composition of Fatty Acids in Oils Rich in Omega-3-acids Appendix X PI
2.4.30 Ethylene Glycol and Diethylene Glycol in Ethoxylated Appendix VIII K
Substances
2.4.31 Nickel in Hydrogenated Vegetable Oils Appendix VIII R
2.4.32 Total Cholesterol in Oils Rich in Omega-3 Acids Appendix X P2
2.4.33 Tetrabutylammonium in Radiopharmaceutical Preparations Appendix VIII U
2.5.1 Acid Value AppendixX B
2.5.2 Ester Value AppendixX C
2.5.3 Hydroxyl Value AppendixX D
2.5.4 Iodine Value AppendixX E
2.5.5 Peroxide Value AppendixX F
2023 European Pharmacopoeia Equivalent Texts V-Al 7

2.5.6 Saponification Value Appendix X G, Method II

2.5.7 Unsaponifiable Matter Appendix X H, Method II
2.5.8 Assay of Primary Aromatic Amino Nitrogen Appendix VIII B
2.5.9 Semi-micro Determination of Nitrogen by Sulfuric Acid Appendix VIII H
Digestion
2.5.10 Oxygen-flask Method Appendix VIII C
2.5.11 Complexometric Titrations Appendix VIII D
2.5.12 Semi-micro Determination of Water Appendix IX C, Method I
2.5.13 Aluminium in Adsorbed Vaccines Appendix XV B
2.5.14 Calcium in Adsorbed Vaccines Appendix XV C
2.5.15 Phenol in lmmunosera and Vaccines Appendix XV E
Polysaccharide Vaccines:
2.5.16 - Protein Appendix XV G
2.5.17 - Nucleic Acids Appendix XV G
2.5.18 - Phosphorus Appendix XV G
2.5.19 - O-acetyl Appendix XV G
2.5.20 - Hexosamines Appendix XV G
2.5.21 - Methylpentoses Appendix XV G
2.5.22 - Uronic Acids Appendix XV G
2.5.23 - Sialic Acid Appendix XV G
2.5.24 Carbon Dioxide in Medicinal Gases Appendix IX F
2.5.25 Carbon Monoxide in Medicinal Gases Appendix IX E
2.5.26 Nitrogen Monoxide and Nitrogen Dioxide in Medicinal Gases Appendix IX G
2.5.27 Oxygen in Medicinal Gases Appendix IX H
2.5.28 Water in Medicinal Gases Appendix IX J
2.5.29 Sulfur Dioxide Appendix IX B, Method II
2.5.30 Oxidising Substances Appendix XL
2.5.31 Ribose in Polysaccharide Vaccines Appendix XV G
2.5.32 Micro Determination of Water Appendix IX C, Method III
2.5.33 Total Protein Assay Appendix VIII P
2.5.34 Acetic Acid in Synthetic Peptides Appendix VIII Q
2.5.35 Nitrous Oxide in Gases Appendix IX L
2.5.36 Anisidine value Appendix X 0
2.5.37 Methyl, Ethyl and Isopropyl Methanesulfonate in Appendix VIII S1
Methanesulfonic Acid
2.5.38 Methyl, Ethyl and Isopropyl Methanesulfonate in Active Appendix VIII S2
Substances
2.5.39 Methanesulfonyl Chloride in Methanesulfonic Acid Appendix VIII S3
2.5.40 Methyl, Ethyl and Isopropyl Toluenesulfonate in Active Appendix VIII S4
Substances
2.5.41 Methyl, Ethyl and Isopropyl Benzenesulfonate in Active Appendix VIII SS
Substances
2.5.42 N-Nitrosamines in Active Substances Appendix VIII V
2.6.1 Sterility Appendix XVI A
2.6.2 Mycobacteria Appendix XVI B4
2.6.3 Vacant
2.6.4 Vacant
2.6.S Vacant
2.6.6 Vacant
2.6.7 Mycoplasmas Appendix XVI B3 and Appendix XVI B
(Vet)3
2.6.8 Pyrogens Appendix XIV D
2.6.9 Vacant
2.6.10 Histamine Appendix XIV G
2.6.11 Depressor Substances Appendix XN F
2.6.12 Microbiological Examination of Non-sterile Products: Appendix XVI B2
Microbial Enumeration Tests
2.6.13 Microbiological Examination of Non-sterile Products: Test for Appendix XVI Bl
Specified Micro-organisms
2.6.14 Bacterial Endotoxins Appendix XIV C
2.6.15 Prekallikrein Activator Appendix XIV J A9
2.6.16 Extraneous Agents in Viral Vaccines Appendix XVI BS
2.6.17 Anticomplementary Activity of Immunog]obulins Appendix XIV J C2
2.6.18 N eurovirulence of Live Viral Vaccines Appendix XV F
2.6.19 Vacant
2.6.20 Anti-A and Anti-B Haemagglutinins Appendix XN J CS
V-A18 European Pharmacopoeia Equivalent Texts 2023

2.6.21 Nucleic Acid Amplification Appendix XN L

2.6.22 Activated Coagulation Factors Appendix XN J A7
2.6.24 Avian Viral Vaccines: Tests for Extraneous Agents in Seed Appendix XVI B(Vet)4
Lots
2.6.25 Avian Live Virus Vaccines: Tests for Extraneous Agents in Appendix XVI B(Vet)5
Batches of Finished Product
2.6.26 Test for Anti-D Antibodies in Human Immunoglobulin Appendix XN J C4
2.6.27 Microbiological Examination of Cell-based Preparations Appendix XVI E
2.6.30 Monocyte-Activation Test Appendix XN H
2.6.31 Microbiological Examination of Herbal Medicinal Products for Appendix XVI F
Oral Use and Extracts used in their Preparations
2.6.32 Test for bacterial endotoxins using recombinantfactor C Appendix XN C
2.6.33 Residual Pertussis Toxin Appendix XN K9
2.6.34 Host-cell Protein Assays Appendix XN 0
2.6.35 Quantification and Characterisation of Residual Host-cell Appendix XN 0
DNA
2.6.36 Microbiological Examination of Live Biotherapeutic Products: Appendix XVI H
tests for enumeration of microbial contaminants
2.6.38 Microbiological Examination of Live Biotherapeutic Products: Appendix XVI H
tests for specified micro-organisms
2.7.1 Immunochemical Methods Appendix XN B
2.7.2 Microbiological Assay of Antibiotics Appendix XN A
2.7.3 Vacant
2.7.4 Assay of Coagulation Factor VIII Appendix XN J A3
2.7.5 Assay of Heparin Appendix XN J B2
2.7.6 Assay of Diphtheria Vaccine (Adsorbed) Appendix XN Kl
2.7.7 Assay of Pertussis Vaccine (Whole Cell) Appendix XN K2
2.7.8 Assay of Tetanus Vaccine (Adsorbed) Appendix XN K3
2.7.9 Fe Function of Immunog]obulins Appendix XN J C 1
2.7.10 Assay of Human Coagulation Factor VII Appendix XN J A2
2.7.11 Assay of Coagulation Factor IX Appendix XN J A4
2.7.12 Assay of Heparin in Coagulation Factors Appendix XN J Bl
2.7.13 Assay of Anti-D Immunoglobulin Appendix XN J C3
2.7.14 Assay of Hepatitis A Vaccine Appendix XN K4
2.7.15 Assay of Hepatitis B (rDNA) Vaccine Appendix XN KS
2.7.16 Assay of Pertussis Vaccine (Acellular) Appendix XN K6
2.7.17 Assay of Human Antithrombin III Appendix XN J B3
2.7.18 Assay of Human Coagulation Factor II Appendix XN J Al
2.7.19 Assay of Human Coagulation Factor X Appendix XN J AS
2.7.20 In viva Assay of Poliomyelitis Vaccine (Inactivated) Appendix XN K7
2.7.21 Assay of Human von Willebrand factor Appendix XN J AS
2.7.22 Assay of Human Coagulation Factor XI Appendix XN J A6
2.7.23 Numeration of CD34/CD45+ Cells in Haematopoietic Appendix XN N
Products
2.7.24 Flow Cytometry Appendix II J
2.7.25 Assay of Human Plasmin Inhibitor Appendix XN J AlO
2.7.27 Flocculation Value (Lt) of Diphtheria and Tetanus Toxins Appendix XN KS
and Toxoids (Ramon Assay)
2.7.28 Colony-forming Cell Assay for Human Haematopoietic Appendix XN N2
Progenitor Cells
2.7.29 Nucleated Cell Count and Viability Appendix XN N3
2.7.30 Assay of Human Protein C Appendix XN J B4
2.7.31 Assay of Human Protein S Appendix XN J B5
2.7.32 Assay of Human ix-1-Proteinase Inhibitor Appendix XN J D 1
2.7.34 Assay of Human C 1-esterase Inhibitor Appendix XN J D2
2.7.35 Immunonephelometry for Vaccine Component Assay Appendix XV L
2.8.1 Ash Insoluble in Hydrochloric Acid Appendix XI K, Method II
2.8.2 Foreign Matter Appendix XI D
2.8.3 Stomata and Stomata! Index Appendix XI H
2.8.4 Swelling Index Appendix XI C
2.8.5 Water in Essential Oils Appendix X M
2.8.6 Foreign Esters Appendix X M
2.8.7 Fatty Oils and Resinified Volatile Oils AppendixX M
2.8.8 Odour and Taste of Volatile Oils AppendixX M
2.8.9 Residue on Evaporation AppendixX M
2.8.10 Solubility in Ethanol (Volatile Oils) AppendixX M
2023 European Pharmacopoeia Equivalent Texts V-Al 9

2.8.11 Determination of Cineole Appendix X J

2.8.12 Essential Oils in Herbal Drugs Appendix XI E
2.8.13 Pesticide Residues Appendix XI L
2.8.14 Tannins in Herbal Drugs Appendix XI M
2.8.15 Bitterness value Appendix XI N
2.8.16 Dry Residue of Extracts Appendix XI P
2.8.17 Loss on Drying of Extracts Appendix XI Q
2.8.18 Determination of Aflatoxin B 1 in Herbal Drugs Appendix XI S 1
2.8.20 Herbal Drugs: Sampling and Sample Preparation Appendix XI T
2.8.21 Test for Aristolochic Acids in Herbal Drugs Appendix XI Rl
2.8.22 Determination of Ochratoxin A in Herbal Drugs Appendix XI S2
2.8.23 Microscopic Examination of Herbal Drugs Appendix XI U
2.8.24 Foam Index Appendix XI 0
2.8.25 High-performance Thin-Layer Chromatography of Herbal Appendix XI W
Drugs and Herbal Drug Preparations
2.8.26 Contaminant Pyrrolizidine Alkaloids Appendix XI X
2.9.1 Disintegration of Tablets and Capsules Appendix XII Al
2.9.2 Disintegration of Suppositories and Pessaries Appendix XII A2
2.9.3 Dissolution Test for Solid Dosage Forms Appendix XII B 1
2.9.4 Dissolution Test for Transdermal Patches Appendix XII B2
2.9.5 Uniformity of Mass Appendix XII C 1
2.9.6 Uniformity of Content Appendix XII C3
2.9.7 Friability of Uncoated Tablets Appendix XVII G 1
2.9.8 Resistance to Crushing of Tablets Appendix XVII H
2.9.9 Measurement of Consistency by Penetrometry Appendix XVII F
2.9.10 Ethanol Content Appendix VIII F, Method III
2.9.11 Methanol and 2-propanol Appendix VIII G
2.9.12 Particle Size Classification of Powders (Sieve Test) Appendix XVII Al
2.9.13 Vacant
2.9.14 Specific Surface Area by Gas Permeability Appendix XVII C
2.9.15 Vacant
2.9.16 Flowability Appendix XVII E
2.9.17 Extractable Volume of Parenteral Preparations Appendix XII CS
2.9.18 Aerodynamic Assessment of Fine Particles Appendix XII C7
2.9.19 Particulate Contamination: Sub-visible Particles Appendix XIII A
2.9.20 Particulate Contamination: Visible Particles Appendix XIII B
2.9.21 Vacant
2.9.22 Softening Time of Lipophilic Suppositories Appendix XVII J
2.9.23 Pycnometric Density of Solids Appendix XVII K
2.9.24 Vacant
2.9.25 Dissolution Test for Medicated Chewing Gums Appendix XII B4
2.9.26 Specific Surface Area By Gas Adsorption Appendix XVII M
2.9.27 Uniformity and Accuracy of Delivered Doses from Multidose Appendix XII C2
Containers
2.9.28 Vacant
2.9.29 Intrinsic Dissolution Appendix XII ES
2.9.31 Particle Size Analysis by Laser Light Diffraction Appendix XVII P
2.9.32 Porosity and Pore-size Distribution of Solids by Mercury Appendix XVII R
Porosimetry
2.9.33 Characterisation of Crystalline and Partially Crystalline Solids Appendix XVII Q
by X-ray Powder Diffraction (XRPD)
2.9.34 Bulk Density and Tapped Density of Powders Appendix XVII S
2.9.35 Powder Fineness Appendix XVII A2
2.9.36 Powder Flow Appendix XVII N
2.9.37 Optical Microscopy Appendix XVII 0
2.9.38 Particle-size Distribution Estimation by Analytical Sieving Appendix XVII B3
2.9.39 Water-Solid Interactions: Determination of Sorption- Appendix IX M
Desorption Isotherms and of Water Activity
2.9.40 Uniformity of Dosage Units Appendix XII C4
2.9.41 Friability of Granules and Spheroids Appendix XVII G2
2.9.42 Dissolution Test for Lipophilic Solid Dosage Forms Appendix XII B3
2.9.43 Apparent Dissolution Appendix XII B6
2.9.44 Preparations for Nebulisation: Characterisation Appendix XII CS
2.9.45 Wettability of Porous Solids Including Powders Appendix XVII T
2.9.46 Vacant
V-A20 European Pharmacopoeia Equivalent Texts 2023

2.9.47 Demonstration of Uniformity of Dosage Units Using Larger Appendix XII C9

Sample Sizes
2.9.49 Powder Flow Properties by Shear Cell Methods Appendix XVII N
2.9.52 Scanning Electron Microscopy Appendix XVII 0
2.9.53 Particulate Contamination: Sub-visible Particles in Non- Appendix XI X C
injectable Liquid Preparations
3.3.1 Material Used for Containers for Human Blood and Blood Appendix XX A
Components
3.3.2 Materials Based on PVC for Blood and Blood Components Appendix XX Al
3.3.3 Materials Based on PVC for Tubing Used in Sets for the Appendix XX A2
Transfusion of Blood and Blood Components
3.1.3 Polyolefins Appendix XX B
3.1.4 Polyethylene - Without Additives Appendix XX C1
3.1.5 Polyethylene - With Additives Appendix XX C2
3.1.6 Polypropylene Appendix XX D
3.1.7 Poly(ethylene-vinyl acetate) Appendix XX E
3.1.8 Silicone Oil Used as a Lubricant Appendix XX FI
3.1.9 Silicone Elastomer for Closures and Tubing Appendix XX F2
3.1.10 Materials Based on Non-plasticised PVC for Containers for Appendix XX A3
Non-injectable, Aqueous Solutions
3.1.11 Materials Based on Non-plasticised Poly(vinyl chloride) for Appendix XX A4
Containers for Solid Dosage forms for Oral Administration
3.1.12 Vacant
3.1.13 Plastic Additives Appendix XX G
3.1.14 Materials Based on Plasticised Poly(vinyl chloride) for Appendix XX AS
Containers for Aqueous Solutions for Intravenous Infusion
3.1.15 Polyethylene Terephthalate for Containers for Preparations Appendix XX H
Not for Parenteral Use
3.2 Containers Appendix XIX A
3.2.1 Glass Containers for Pharmaceutical Use Appendix XIX B
3.2.2 Plastic Containers and Closures Appendix XIX C
3.2.2.1 Plastic Containers for Aqueous Solutions for Parenteral Appendix XIX C I
Infusions
3.3.4 Sterile Plastic Containers for Blood and Blood Components Appendix XIX D 1
3.3.5 Empty Sterile Plastic Containers for Blood and Blood Appendix XIX D2
Components
3.3.6 Sterile Plastic Containers with Anticoagulant for Blood and Appendix XIX D3
Blood Components
3.3.7 Sets for the Transfusion of Blood and Blood Components Appendix XIX F
3.2.7 Vacant
3.3.8 Sterile Single-Use Plastic Syringes Appendix XIX G
3.2.9 Rubber Closures for Containers for Aqueous Preparations for Appendix XIX E
Parenteral Use

4.1.1 Reagents Appendix IA

4.1.2 Standard Solutions Appendix IC
4.1.3 Buffer Solutions Appendix ID
4.2.1 Reference Substances for Volumetric Solutions Appendix IB
4.2.2 Volumetric Solutions Appendix IB
5.1.1 Methods of Preparation of Sterile Products Appendix XVIII
5.1.2 Biological Indicators (sterilisation) Appendix XVIII
5.1.3 Efficacy of Antimicrobial Preservation Appendix XVI C
5.1.4 Microbiological Quality of Non-sterile Pharmaceutical Appendix XVI D
Preparations and Substances for Pharmaceutical Use
5.1.5 Application of the F Concepts to Heat Sterilisation Processes Appendix XVIII
5.1.6 Alternative Methods for Control of Microbiological Quality Supplementary Chapter IV L
5.1.7 Viral Safety Appendix XXII A
5.1.8 Microbiological Quality of Herbal Medicinal Products for Oral Appendix XVI G
Use and Extracts used in their Preparation
5.1.9 Guidelines for using the Test for Sterility Supplementary Chapter IV P
5.1.10 Guidelines for using the test for bacterial endotoxins Supplementary Chapter I C
5.1.11 Determination of Bactericidal, Fungicidal or Yeasticidal Appendix XIV P
Activity of Antiseptic Medicinal Products
5.1.12 Depyrogenation of items used in the production ofparenteral Supplementary Chapter IV T
preparations
5.2.1 Terminology: Vaccines Appendix XV A and Appendix XV A(Vet)
2023 European Pharmacopoeia Equivalent Texts V-A21

5.2.2 SPF Chicken Flocks Appendix XV H and Appendix XV H

(Vet)
5.2.3 Cell Substrates for the Production of Vaccines for Human Use Appendix XV J
5.2.4 Cell Cultures for the Production of Vaccines for Veterinary Appendix XV J (Vet) I
Use
5.2.5 Management of Extraneous Agents in Immunological Appendix XV J(Vet)2
Veterinary Medicinal Products
5.2.6 Safety: Veterinary Vaccines Appendix XV K(Vet) I
5.2.7 Efficacy: Veterinary Vaccines Appendix XV K(Vet)2
5.2.8 Minimising the Risk of Transmitting Animal Spongiform Appendix XXII B
Encephalopathy Agents via Medicinal Products
5.2.9 Evaluation of Safety of Each Batch of Immunosera for Appendix XV K(Vet)3
Veterinary Use
5.2.11 Carrier Proteins for the Production of Conjugated Appendix XV K
Polysaccharide Vaccines for Human Use
5.2.12 Raw Materials of Biological Origin for the Production of Cell- Supplementary Chapter IV S
based and Gene Therapy Medicinal Products
5.2.13 Healthy Chicken Flocks for the Production of Inactivated Appendix XV(Vet)l
Vaccines for Veterinary Use
5.2.14 Substitution of In Vivo method(s) by In Vitro method(s) for Appendix XV M
the quality control of vaccines
5.3 Statistical Analysis of Biological Assays and Tests Supplementary Chapter IV G
5.4 Residual Solvents Supplementary Chapter IV D
5.5 Alcoholimetric Tables Supplementary Chapter IV E
5.6 Assay of Interferons Appendix XN MI
5.7 Physical Characteristics of Radionuclides Radiopharmaceutical Preparations
5.8 Pharmacopoeia! Harmonisation Supplementary Chapter IV F
5.9 Polymorphism Appendix IF
5.10 Control oflmpurities in Substances for Pharmaceutical Use Supplementary Chapter IV J
5.11 Characters Section in Monographs Supplementary Chapter IV K
5.12 Reference Standards Supplementary Chapter IV M
5.14 Gene Transfer Medicinal Products for Human Use Supplementary Chapter IV N
5.16 Crystallinity Appendix XVII U
5.17.1 Recommendations on dissolution testing Appendix XII B 1
5.17.2 Recommendations on Testing of ParticulateContamination: Supplementary Chapter I N
Visible Particles
5.18 Methods of Pretreatment for Preparing Traditional Chinese Supplementary Chapter VII E
Drugs: General Information
5.19 Extemporaneous Preparation of Radiopharmaceuticals Radiopharmaceutical Preparations
5.20 Elemental Impurities Supplementary Chapter IV Q
5.21 Chemometric Methods Applied to Analytical Data Supplementary Chapter IV R
5.22 Names of Herbal Drugs Used in Traditional Chinese Supplementary Chapter VII B
Medicine
5.23 Monographs on Herbal Drug Extracts (Information Chapter) Supplementary Chapter VII C
5.24 Chemical Imaging Appendix II L
5.25 Process Analytical Technology Appendix II N
5.28 Multivariate Statistical Process Control Supplementary Chapter IV U
2023 Appendix I A V-A23

Appendix I
Additional Information for Reagents
(Ph. Bur. text 4.0)
Additional information for reagents that can only be fully
identified by a trademark or whose availability is limited may be
found in the Knowledge database on the EDQM website. This
information is given only to make it easier to obtain such reagents
and this does not suggest in any way that the mentioned suppliers
are especially recommended or certified by the European
Pharmacopoeia Commission or the Council of Europe. It is
therefore acceptable to use reagents from another source provided
that they comply with the standards of the Pharnzacopoeia.

A. General Reagents 0
I!)

(Ph. Bur. text 4.1)

Where the name of a substance or a solution is followed by
the letter R (the whole in italics), this indicates a reagent 10
included in the following list. The specifications given for
reagents do not necessarily guarantee their quality for use in
medicines.
Within the description of each reagent there is a 7-digit
reference code in italics (for example, 1002501). This
number, which will remain unchanged for a given reagent
during subsequent revisions of the list, is used for
identification purposes by the Secretariat, and users of the
Pharmacopoeia may also find it useful, for example in the
management of reagent stocks. The description may also
include a CAS number (Chemical Abstract Service Registry
Number) recognisable by its typical format, for example
9002-93-1.
Some of the reagents included in the list are toxic and are to
be handled in conformity with good quality control
laboratory practice.
Reagents in aqueous solution are prepared using water R.
j 2::: 1.0
3.00- 3.05
For liquid chromatography, water for chromatography R is
used for the preparation of mobile phases when water, or an Figure 2.1.1.-1. - Standard dropper
aqueous solution, is one of the components. Where a reagent Dimensions in millimetres
solution is described using an expression such as
'hydrochloric acid (10 g/L HCl)', the solution is prepared by Other droppers may be used provided they comply with the
an appropriate dilution with water R of a more concentrated following test.
reagent solution specified in this chapter. Reagent solutions 20 drops of water R at 20 ± 1 °C flowing freely from the
used in the limit tests for barium, calcium and sulfates are dropper held in the vertical position at a constant rate of
prepared using distilled water R. Where the name of the 1 drop per second weighs 1000 ± 50 mg.
solvent is not stated, an aqueous solution is intended. The dropper must be carefully cleaned before use. Carry out
The reagents and reagent solutions are to be stored in well- 3 determinations on any given dropper. No result may
closed containers. The labelling should comply with the deviate by more than 5 per cent from the mean of the
relevant national legislation and international agreements. 3 determinations.
Droppers Acacia See Acacia (0307).
(Ph. Bur. text 2.1.1) Acacia Solution
The term 'drops' means standard drops delivered from a Dissolve 100 g of acacia R in 1000 mL of water R. Stir with
standard dropper as described below. a mechanical stirrer for 2 h. Centrifuge at about 2000 g for
Standard droppers (Figure 2.1.1.-1) are constructed of 30 min to obtain a clear solution.
practically colourless glass. The lower extremity has a circular Storage: in polyethylene containers of about 250 mL capacity
orifice in a flat surface at right angles to the axis. at a temperature of O °C to --20 'C.
Acebutolol Hydrochloride (34381-68-S)
See Acebutolol hydrochloride (0871).
Acetal Acetaldehyde diethyl acetal; 1,1-Diethoxyethane;
C 6 H11O 2 = 118.2 (105-57-7)
V-A24 Appendix I A 2023

Clear, colourless, volatile liquid, miscible with water and with Assay. Dissolve 2.00 gin 50.0 mL of 1 M sodium hydroxide in
ethanol (96 per cent). a ground-glass-stoppered flask and boil under a reflux
dig: about 0.824. condenser for 1 h. Titrate with 1 M hydrochloric acid, using
n~: about 1.382. 0.5 mL of phenolphthalein solution Ras indicator. Calculate
the number of millilitres of 1 M sodium hydroxide required for
bp: about 103 °C.
1 g (n1). Dissolve 2.00 gin 20 mL of cyclohexane Rina
Acetaldehyde Ethanal; C 2H 4 O = 44.1 (75-07-0) ground-glass-stoppered flask, cool in ice and add a cold
Clear, colourless flammable liquid, miscible with water and mixture of 10 mL of aniline Rand 20 mL of cyclohexane R.
with ethanol (96 per cent). Boil the mixture under a reflux condenser for 1 h, add
dig: about 0.788. 50.0 mL of 1 M sodium hydroxide and shake vigorously.
n~: about 1.332. Titrate with 1 M hydrochloric acid, using 0.5 mL of
phenolphthalein solution R as indicator. Calculate the number
bp: about 21 °C.
of millilitres of 1 M sodium hydroxide required for 1 g (n 2).
Acetaldehyde Ammonia Trimer Trihydrate 2,4,6-
Calculate the percentage of C 4H 6O 3 from the following
Trimethylhexahydro-1,3,5-triazine trihydrate;
expression:
C6H1sN3,3H 2O = 183.3 (58052-80-5)
mp: 95 °C to 97 °C. 10.2(n1 - n 2 )
Content: minimum 95.0 per cent.
Acetic Anhydride Solution Rl
Colourless or white or pale yellow crystals or powder
Dissolve 25.0 mL of acetic anhydride R in anhydrous
Assay. Dissolve 0.900 gin water Rand dilute to 50.0 mL pyridine Rand dilute to 100.0 mL with the same solvent.
with the same solvent. Titrate with 1 M hydrochloric acid,
Storage: protected from light and air.
determining the end-point potentiometrically (2.2.20).
Acetic Anhydride-Dioxan Solution
1 mL of 1 M hydrochloric acid is equivalent to 61.08 mg of
C6H1sN3,3H 2O. Add 1 mL of acetic anhydride to 50 mL of 1,4-dioxan.
Acetamide C 2H 5NO = 59.07 (60-35-5) Acetic Anhydride-Sulfuric Acid Solution
mp: about 78°. Carefully mix 5 mL of acetic anhydride R with 5 mL of
sulfuric acid R. Add dropwise and with cooling to 50 mL of
General reagent grade of commerce.
anhydrous ethanol R.
Acetic Acid
Prepare immediately before use.
Content: 290 g/L to 310 g/L of C 2H 4 O 2 (Mr 60.1).
Acetone Propan-2-one; (67-64-1)
Dilute 30 g of glacial acetic acid R to 100 mL with water R.
See Acetone (0872).
Acetic Acid (6 per cent) Acetic acid, dilute Rl
Acetonitrile Methyl cyanide; Ethanenitrile;
Content: 57.5 g/L to 62.5 g/L (Mr 60.1).
C2H3N = 41.05 (75-05-8)
Dilute 6 g of glacial acetic acid R to 100 mL with water R.
Clear, colourless liquid, miscible with water, with acetone
Acetic Acid, Anhydrous Anhydrous glacial acetic acid; and with methanol.
C2H4O2 = 60.1 (64-19-7)
Content: minimum 99.6 per cent m/m of C 2H 4 O 2.
dig: about 0.78.
n~: about 1.344.
Colourless liquid or white or almost white, shining, fem-like
crystals, miscible with or very soluble in water, in ethanol A 100 g/L solution is neutral to litmus paper.
(96 per cent), in glycerol (85 per cent), and in most fatty and Distillation range (2.2. 11). Not less than 95 per cent distils
essential oils. between 80 °C and 82 °C.
dfg: 1.052 to 1.053. Acewnitrile used in spectrophowmetry complies with the following
additional test.
bp: 117 °C to 119 °C.
Absorbance (2.2.25): maximum 0.01 from 255 nm to 420 nm,
A 100 g/L solution is strongly acid (2.2.4).
determined using water R as compensation liquid.
A 5 g/L solution neutralised with dilute ammonia R2 gives
Acetonitrile for Chromatography
reaction (b) of acetates (2.3.1).
Freezing point (2.2.18): minimum 15.8 °C.
See Acetonitrile R.
Acewnitrile used in chromatography complies with the following
Water (2.5.12): maximum 0.4 per cent. If the water content
additional tests.
is more than 0.4 per cent it may be adjusted by adding the
calculated amount of acetic anhydride R. Absorbance (2.2.25): maximum 0.01 at 240 nm and higher
Storage: protected from light. wavelengths, determined using water R as compensation
liquid.
Acetic Acid, Dilute
Content (2.2.28): minimum 99.8 per cent.
Content: 115 g/L to 125 g/L of C 2H 4 O 2 (Mr 60.1).
Acetonitrile Rl
Dilute 12 g of glacial acetic acid R to 100 mL with water R.
Complies with the requirements prescribed for acetonitrile R
Acetic Acid, Glacial C 2H 4 O 2 = 60.1 (64-19-7)
and with the following additional requirements.
See Acetic acid, glacial (0590).
Content: minimum 99.9 per cent.
Solutions of molarity xM should be prepared by diluting 57 x
mL (60x g) of glacial acetic acid to 1000 mL with water. Absorbance (2.2.25): maximum 0.10, determined at 200 nm
using water R as the compensation liquid.
Acetic Anhydride C 4H 6O 3 = 102.1 (108-24-7)
Acetoxyvalerenic Acid (2E)-3-[ ( 1RS,4S, 7R, 7aR)-
Content: minimum 97.0 per cent mlm of C 4 H 6O 3.
1-(Acetyloxy)-3, 7-dimethyl-2,4,5,6, 7,7 a-hexahydro- lH-inden-
Clear, colourless liquid. 4-yl]-2-methylprop-2-enoic acid; C 17 H 24O 4 = 292.4
bp: 136 °C to 142 °C. (81397-67-3)
 2023 Appendix I A V-A25

Colourless or pale yellow viscous oil. Content: minimum 98.0 per cent, calculated by the
Absorbance (2.2.25). A solution in methanol R shows an normalisation procedure.
absorption maximum at about 216 nm. N-Acetylglucosamine 2-(Acetylamino)-2-deoxy-D-
Acetyl Chloride C 2 H 3 ClO = 78.5 (75-36-5) glucopyranose; C 8H 15NO 6 = 221.2 (7512-17-6)
Clear, colourless liquid, flammable, decomposes in contact mp: about 202 °C.
with water and with ethanol (96 per cent), miscible with Acetyl-11-keto-P-boswellic Acid 3rx-(Acetyloxy)-11-
ethylene chloride. oxours-12-en-24-oic acid; (4P)-3rx-(Acetyloxy)-11-oxours-12-
d~g: about 1.10. en-23-oic acid; C 32 H 48 O 5 = 512.7 (67416-61-9)
Distillation range (2.2.11). Not less than 95 per cent distils White or almost white powder, insoluble in water, soluble in
between 49 °C and 53 °C. acetone, in anhydrous ethanol and in methanol.
Acetylacetamide 3-Oxobutanamide; C4 H 7NO 2 = 101.1 mp: 271 °C to 274 °CAcetyl-11-keto-f3-boswellic acid used in
(5977-14-0) liquid chromatography complies with the folwwing additional test.
mp: 53 °C to 56 °C. Assay. Liquid chromatography (2.2.29) as prescribed in the
Acetylacetone 2,4-Pentanedione; C 5H 8O 2 = 100.1 monograph on Indian frankincense (2310).
(123-54-6) Content: minimum 90 per cent, calculated by the
Colourless or slightly yellow, easily flammable liquid, freely normalisation procedure.
soluble in water, miscible with acetone, with ethanol N-(rx)-Acetyl-L-lysine (2S)-2-Acetamido-6-aminohexanoic
(96 per cent) and with glacial acetic acid. acid; C 8H 16N 2O 3 = 188.2 (1946-82-3)
nt 0 : 1.452 to 1.453. N-(s)-Acetyl-L-lysine (2S)-6-Acetamido-2-aminohexanoic
bp: 138 °C to 140 °C. acid; CsH16N2O 3 = 188.2 (692-04-6)
Acetylacetone Reagent Rl N-Acetylneuraminic Acid Sialic acid;
C11H19NO9 = 309.3 (131-48-6)
To 100 mL of ammonium acetate solution R add 0.2 mL of
acetylacetone R. White or almost white acicular crystals, soluble in water and
in methanol, slightly soluble in anhydrous ethanol, practically
Acetylacetone Reagent R2
insoluble in acetone.
Dissolve 0.2 mL of acetylacetone R, 3 mL of glacial acetic
[:x]~: about -36, determined on a 10 g/L solution.
acid Rand 25 g of ammonium acetate R in water Rand dilute
to 100 mL with the same solvent. mp: about 186 °C, with decomposition.
4-Acetylbiphenyl 4-Phenylacetophenone; Acetylsalicylic Acid 2-(Acetyloxy)benzoic acid;
C 9 H 8 O 4 = 180.2 (50-78-2)
C14H1 2 O = 196.2 (51-42-3)
mp: about 117°. White or almost white, crystalline powder or colourless
crystals, slightly soluble in water, freely soluble in ethanol
General reagent grade of commerce.
(96 per cent).
N-Acetyl-s-caprolactam N-Acetylhexane-6-lactam; N-Acetyltryptophan 2-Acetylamino-3-(indol-3-yl)
C 8H 13NO 2 = 155.2 (1888-91-1)
propanoic acid; C 13H 14 N 2 O 3 = 246.3 (1218-34-4)
Colourless liquid, miscible with anhydrous ethanol.
White or almost white powder or colourless crystals, slightly
dJg: about 1.100. soluble in water. It dissolves in dilute solutions of alkali
nt 0 : about 1.489. hydroxides.
bp: about 135 °C. mp: about 205 °C.
Acetylcholine Chloride C 7H 16 CINO 2 = 181.7 (60-31-1) Assay. Liquid chromatography (2.2.29) as prescribed in the
Crystalline powder, very soluble in cold water and in ethanol monograph Tryptophan (1272).
(96 per cent). It decomposes in hot water and in alkalis. Test solution. Dissolve 10.0 mg in a mixture of 10 volumes of
Storage: at -20 °C. acetonitrile R and 90 volumes of water R and dilute to
100.0 mL with the same mixture of solvents.
N-Acetyl-L-cysteine C 5H 9NO 3 S = 163.2 (616-91-1)
[:x]~: about +4.6. Content: minimum 99.0 per cent, calculated by the
normalisation procedure.
mp: about 110°.
Acetyltyrosine Ethyl Ester Ethyl N-acetyl-L-tyrosinate;
General reagent grade of commerce.
C 13 H 17NO 4,H 2 O = 269.3 (36546-50-6)
Acetylene Ethyne; C 2 H 2 = 26.04 (74-86-2) White or almost white, crystalline powder suitable for the
Content: minimum 99.0 per cent V/V. assay of chymotrypsin.
Acetyleugenol 2-Methoxy-4-(2-propenyl)phenylacetate; [,x]~0 : + 21 to+ 25, determined on a 10 g/L solution in
C 12H 14O 3 = 206.2 (93-28-7) ethanol (96 per cent) R.
Yellow coloured, oily liquid, practically insoluble in water, A:~m: 60 to 68, determined at 278 nm in ethanol
freely soluble in ethanol (96 per cent). (96 per cent) R.
nt 0 : about 1.521.
Acetyltyrosine Ethyl Ester, 0.2M 0.2M Ethyl
bp: 281 °C to 282 °C. acetyltyrosinate
Acetyleugenol used in gas chromatography complies with the Dissolve 0.54 g of acetyltyrosine ethyl ester R in ethanol
following additional test. (96 per cent) Rand dilute to 10.0 mL with the same solvent.
Assay. Gas chromatography (2. 2. 28) as prescribed in the rxl-Acid-glycoprotein Silica Gel for Chiral Separation
monograph Clove oil (1091). A very finely divided silica gel for chromatography consisting
Test solution. The substance to be examined. of spherical particles coated with al-acid glycoprotein.
V-A26 Appendix I A 2023

Acid Blue 83 Brilliant blue; Coomassie brilliant blue R dig: about 1.05.
250; C 45 H 4 ,iN 3NaO1S2 = 826 (6104-59-2) ng1: about 1.421.
Colour Index No. 42660 bp: about 141 °C.
Brown powder insoluble in cold water, slightly soluble in mp: 12 °C to 15 °C.
boiling water and in anhydrous ethanol, soluble in sulfuric
Actein (23R,24R,25S,26S)-3~-(~-o-Xylopyranosyloxy)-
acid, glacial acetic acid and in dilute solutions of alkali
l 6 ~,23:23,26:24,25-triepoxy-26-hydroxy-9, 19-cyclolanostan-
hydroxides.
12 ~-yl acetate; C 37 H 56O11 = 677 (18642-44-9)
Acid Blue 90 Coomassie brilliant blue G; Acteoside 2-(3,4-Dihydroxyphenyl)ethyl 3-O-(6-deoxy-lX-L-
C 41H4 8N 3NaO 7S2 = 854 (6104-58-1) mannopyranosyl)-4-O-[ (2E)-3-(3,4-dihydroxyphenyl)prop-2-
Colour Index No. 42655 enoyl]-~-D-glucopyranoside; Verbascoside;
A dark brown powder, with a violet sheen and some particles C 29 H 36O 15 = 624.6 (61276-17-3)
having a metallic lustre, soluble in water and in anhydrous Light yellowish powder, freely soluble in water and in
ethanol. methanol.
Aj~m: greater than 500, determined at 577 nm in a 0.01 g/L mp: about 140 °C, with decomposition.
solution in buffer solution pH 7.0 and calculated with
Adamantane Tricyclo[3.3.l.1 3'7]decane; C10H16 = 136.2
reference to the dried substance. (281-23-2)
Loss on drying (2.2.32): maximum 5.0 per cent, determined mp: about 270 °C.
on 0.500 g by drying in an oven at 105 °C.
Adenine (73-24-5)
Acid Blue 92 Coomassie blue; Anazolene sodium;
Trisodium 8-hydroxy-4 '-(phenylamino )azonaphthalene- See Adenine (0800).
3,5 ',6-trisulfonate; C 26 H 16N 3Na3O1 0S3 = 696 (3861-73-2) Adenosine 6-Amino-9-~-D-ribofuranosyl-9H-purine;
Colour Index No. 13390 C 10 H 13N 5 O 4 = 267.2 (58-61-7)
Dark blue crystals, soluble in water, in acetone and in White or almost white, crystalline powder, slightly soluble in
ethylene glycol monoethylether, slightly soluble in ethanol water, practically insoluble in acetone and in ethanol
(96 per cent). (96 per cent). It dissolves in dilute solutions of acids.
mp: about 234 °C.
Acid Blue 92 Solution Coomassie blue solution
Dissolve 0.5 g of acid blue 92 Rina mixture of 10 mL of Adipic Acid C 6H10O4 = 146.1 (124-04-9)
glacial acetic acid R, 45 mL of ethanol (96 per cent) R and Prisms, freely soluble in methanol, soluble in acetone,
45 mL of water R. practically insoluble in light petroleum.
Acid Blue 93 Methyl blue; Poirrier blue; mp: about 152 °C.
C31H21N3Na 2 O 9S 3 = 800 (28983-56-4) Adrenaline (lR)-1-(3,4-Dihydroxyphenyl)-
Colour Index No. 42780 2-(methylamino )ethanol; 4-[ (lR)- l-hydroxy-2-(methylamino)
Mixture of triphenylrosaniline di- and trisulfonate and of ethyl]benzene-1,2-diol; C 9H 13NO 3 = 183.2 (51-43-4)
triphenylpararosaniline. White or almost white powder, gradually becoming brown on
Dark blue powder. exposure to light and air, very slightly soluble in water and in
ethanol (96 per cent), insoluble in acetone. It dissolves in
Colour change: pH 9.4 to pH 14.0. dilute solutions of mineral acids and alkali hydroxides.
Acid Blue 93 Solution mp: about 215 °C.
Dissolve 0.2 g of acid blue 93 R in water Rand dilute to
Adrenaline Acid Tartrate L-Epinephrine D-hydrogen
100 mL with the same solvent.
tartrate; C 13 H 19NO 9 = 333 (51-42-3)
Acrylamide Propenamide; C 3H 5 NO = 71.1 (79-06-1) General reagent grade of commerce.
Colourless or white flakes or a white or almost white, (±) Adrenaline Hydrochloride DL-adrenaline
crystalline powder, very soluble in water and in methanol,
hydrochloride; DL-epinephrine hydrochloride; C9H13NO3,
freely soluble in anhydrous ethanol. HCl = 220 (329-63-5)
mp: about 84 °C.
General reagent grade of commerce.
Acrylamide/bisacrylamide (29: 1) Solution, Adrenalone Hydrochloride 1-(3,4-Dihydroxyphenyl)-
30 per cent
2-(methylamino)ethanone hydrochloride; 3 ',4' -Dihydroxy-
Prepare a solution containing 290 g of acrylamide R and 10 g 2-(methylamino)acetophenone hydrochloride;
of methylenebisacrylamide R per litre of water R. Filter. C 9H 12 ClNO 3 = 217.7 (62-13-5)
Acrylamide/bisacrylamide (36.5:1) Solution, Pale yellow crystals, freely soluble in water, soluble in ethanol
30 per cent (96 per cent).
Prepare a solution containing 292 g of acrylamide R and 8 g mp: about 244 °C.
of methylenebisacrylamide R per litre of water R. Filter.
Aescin Escin; (6805-41-0)
Acrylic Acid Prop-2-enoic acid; Vinylformic acid; A mixture of related saponins obtained from the seeds of
C 3 H4O 2 = 72.1 (79-10-7) Aesculus hippocastanum L.
Content: minimum 99 per cent. Fine, almost white or slightly reddish or yellowish,
It is stabilised with 0.02 per cent of hydroquinone amorphous powder.
monomethyl ether.
Chromatography. Thin-layer chromatography (2.2.27).
Corrosive liquid, miscible with water and ethanol
Test solution. Dissolve 10 mg of aescin R in ethanol
(96 per cent). It polymerises readily in the presence of
(70 per cent V/V) R and dilute to 10 mL with the same
oxygen.
solvent.
2023 Appendix I A V-A27

Plate: TLC silica gel plate R. Agnuside (1RS,4aSR,5RS,7aRS)-5-Hydroxy-7-[[(4-

Mobile phase: the upper layer of a mixture of 10 volumes of hydroxybenzoyl)oxy]methyl]-l ,4a,5, 7 a-tetrahydrocyclopenta
glacial acetic acid R, 40 volumes of water R and 50 volumes of [c]pyran-1-yl ~-o-glucopyranoside; C 22 H 26 O 11 = 466.4
butanol R. (11027-63-7)
Application: 20 µL of the test solution as bands of 20 mm by White or almost white crystals.
3mm. Air, Hydrocarbon-free
Development: over a path of 12 cm. Complies with the requirements prescribed for the
Drying-. at 100-105 °C. monograph Medicinal air (1238) with the following additional
requirement.
Detection: spray with about 10 mL of anisaldehyde solution R
for a plate 200 mm square and heat again at 100-105 °C. Hydrocarbons: maximum 5 ppm ViV, calculated as CH4 •
Results: the chromatogram shows a principal band with an Rp Alanine L-Alanine; (56-41-7)
of about 0.4. See Alanine (0752).
Cs4Hs4O23,2H2O = 1138 ~-Alanine (107-95-9)
Aflatoxin B 1 (6aR,9aS)-4-Methoxy-2,3,6a,9a- See 3-aminopropionic acid R.
tetrahydrocyclopenta[c] furo [3 ',2 ':4,5] furo [2,3-h] [1] Albumin, Bovine (9048-46-8)
benzopyran-1,11-dione; C 17H 12 O 6 = 312.3 (1162-65-8)
Bovine serum albumin containing about 96 per cent of
White or faint yellow crystals. protein.
Agar (9002-18-0) White to light brownish-yellow powder.
The dried extract from Gelidium sp. and other algae Water (2.5.12): maximum 3.0 per cent, determined on
belonging to the class Rhodophyceae. 0.800 g.
Microbiological reagent grade of commerce. Albumin, Bovine Rl (9048-46-8)
Agarose for Chromatography (9012-36-6) Bovine serum albumin containing about 96 per cent of
Swollen beads 60-140 µm in diameter presented as a protein.
4 per cent suspension in water R. White or light brownish-yellow powder.
Used in size-exclusion chromatography for the separation of Albumin, Human
proteins with relative molecular masses of 6 x 104 to
Human serum albumin containing not less than 96 per cent
20 x 10 6 and of polysaccharides with relative molecular
of albumin.
masses of 3 x 10 3 to 5 x 10 6.
Albumin Solution, Human Albumin; (9048-46-8)
Agarose for Chromatography, Cross-linked
(61970-08-9) See Human albumin solution (0255).
Prepared from agarose by reaction with 2,3-dibromopropanol Albumin Solution Rl, Human
in strongly alkaline conditions. Dilute human albumin solution R with a 9 g/L solution of
It occurs as swollen beads and is presented as a suspension. sodium chloride R to a concentration of 1 g/L of protein.
Adjust the pH to 3.5-4.5 with glacial acetic acid R.
Used in size-exclusion chromatography for the separation of
proteins and of polysaccharides.
Alcohol (64-17-5)
Agarose for Chromatography Rl, Cross-linked See Ethanol (96 per cent) R.
( 65099-79-8) Alcohol, Aldehyde-free Ethanol (96%), aldehyde-free
Prepared for agarose by reaction with 2,3-dibromopropanol Mix 1200 mL of ethanol (96 per cent) R with 5 mL of a
in strongly alkaline conditions. 400 g/L solution of silver nitrate R and 10 mL of a cooled
It occurs as swollen beads 60-140 µm in diameter and is 500 g/L solution of potassium hydroxide R. Shake, allow to
presented as a 4 per cent suspension in water R. stand for a few days and filter. Distil the filtrate immediately
before use.
Used in size-exclusion chromatography for the separation of
proteins with relative molecular masses of 7 x 104 to Alcohol (x% v/v)
40 x 106 and of polysaccharides with relative molecular See Ethanol (x per cent V/V) R.
masses of 1 x 10 5 to 2 x 107 • Aldehyde Dehydrogenase
Agarose for Electrophoresis (9012-36-6) Enzyme obtained from baker's yeast which oxidises
A neutral, linear polysaccharide, the main component of acetaldehyde to acetic acid in the presence of nicotinamide-
which is derived from agar. adenine dinucleotide, potassium salts and thiols, at pH 8.0.
White or almost white powder, practically insoluble in cold Aldehyde Dehydrogenase Solution
water, very slightly soluble in hot water. Dissolve in water R a quantity of aldehyde dehydrogenase R,
Agarose-DEAE for Ion Exchange Chromatography equivalent to 70 units and dilute to 10 mL with the same
(57407-08-6) solvent. This solution is stable for 8 h at 4 °C.
Cross-linked agarose substituted with diethylaminoethyl Aldrin C,2H 8 Cl 6 = 364.9 (309-00-2)
groups, presented as beads. bp: about 145 °C.
Agarose/Cross-linked Polyacrylamide mp: about 104 °C.
Agarose trapped within a cross-linked polyacrylamide A suitable certified reference solution (10 ng/µL in
network; it is used for the separation of globular proteins cyclohexane) may be used.
with relative molecular masses of 2 x 104 to 35 x 104.
V-A28 Appendix I A 2023

Aleuritic Acid (9RS,I0SR)-9,10,16- Aluminium Oxide, Anhydrous (1344-28-1)

Trihydroxyhexadecanoic acid; C 16H 32 O 5 = 304.4 (533-87-9) Aluminium oxide, consisting of y-Al 2O 3, dehydrated and
White or almost white powder, greasy to the touch, soluble activated by heat treatment.
in methanol. Particle size: 75 µm to 150 µm.
mp: about 101 °C. Al 2O 3 = 102.0
Alizarin S Alizarin red S; C 14H 7 NaO 7 S,H 2O = 360.3 Aluminium Oxide, Basic
(130-22-3)
A basic grade of anhydrous aluminium oxide R suitable for
Schultz No. 1145 column chromatography.
Colour Index No. 58005 pH (2.2.3). Shake 1 g with 10 mL of carbon dioxide-free
Orange-yellow powder, freely soluble in water and in ethanol water R for 5 min. The pH of the suspension is 9 to 10.
(96 per cent). When used in monographs other than those of the European
Alizarin S Solution Pharmacopoeia, the following test applies.
A 1 g/L solution of alizarin S R. Activity Pack into a chromatographic tube (25 cm x
Test for sensitivity. If alizarin S solution is used for the 10 mm) sufficient of the basic aluminium oxide to form a
standardisation of 0. 05 M barium perchlorate, it shows a column 50 mm deep. Apply to the column a mixture of
colour change from yellow to orange-red when it is tested 5 mL of sudan yellow solution and 5 mL of sudan red
according to the standardisation of 0. 05 M barium perchlorate. solution and elute with 20 mL of a mixture of 1 volume of
Colour change: pH 3.7 (yellow) to pH 5.2 (violet).
benzene and 4 volumes of petroleum spirit (boiling range,
60° to 80°). Sudan red forms a zone about 10 mm in depth
Aloe Emodin C 15 H 10O 5 = 270.2 (481-72-1) on the upper part of the column separated by a colourless
1,8-Dihydroxy-3-(hydroxymethyl)anthracene-9, 10-dione. zone from a yellow zone of sudan yellow below.
1,8-Dihydroxy-3-(hydroxymethyl)anthraquinone. Aluminium Oxide for Chromatography, Deactivated
Alovudine 1-[ (2R,4S,5R)-4-Fluoro-5-(hydroxymethyl) Aluminium oxide suitably deactivated for the separation and
tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H;-dione; detection of traces of polar hydrocarbons, with porous layer
Fluorodeoxythymidine; 3 '-Deoxy-3 '-fluorothymidine; open tubular (PLOT) design.
C10H13FN 2O4 = 244.2 (25526-93-6) Aluminium Oxide, Deactivated
Content: minimum 95 per cent.
To a suitable basic alumina add 1.5 to 2% of water, mix well
Colourless crystals. and allow to stand overnight in a stoppered bottle.
Aluminium Al= 26.98 (7429-90-5) The product complies with the following test.
White or almost white, malleable, flexible, bluish metal, Prepare a column (20 cm x 10 mm) using the alumina and
available as bars, sheets, powder, strips or wire. In moist air hexane. Add a solution of 0 .2 5 g of calciferol in 10 mL of
an oxide film forms which protects the metal from corrosion. hexane. When the level of the solution falls just to the top of
Analytical grade. the column, begin eluting with a 17.5% v/v solution of ether
Aluminium Chloride Aluminium chloride hexahydrate; in hexane adjusting the rate of flow, if necessary, to between
AICl 3,6H2O = 241.4 (7784-13-6) 1 and 2 mL per minute. Collect 200 mL of eluate;
no calciferol is present. Collect a further 100 mL of eluate;
Content: minimum 98.0 per cent of A!Ch,6H 2O. it contains not less than 95% of the calciferol used in the
White or slightly yellowish, crystalline powder, hygroscopic, test, when determined by the Assay described under
freely soluble in water and in ethanol (96 per cent). Calciferol Oral Solution.
Storage: in an airtight container. Aluminium Oxide G
Aluminium Chloride Reagent A fine, white, homogeneous powder of an average particle
Dissolve 2.0 g of aluminium chloride R in 100 mL of a size between 10 and 40 µm containing about 10% w/w of
5 per cent V/V solution of glacial acetic acid R in methanol R. calcium sulfate hemihydrate.
Aluminium Chloride Solution Content of calcium sulfate Carry out the test described
Dissolve 65.0 g of aluminium chloride R in water R and dilute under silica gel G.
to 100 mL with the same solvent. Add 0.5 g of activated Acidity or alkalinity pH of a suspension prepared by
charcoal R, stir for 10 min, filter and add to the filtrate, with shaking 1 g with 10 mL of carbon dioxide-free water, about
continuous stirring, sufficient of a 10 g/L solution of sodium 7.5, Appendix V L.
hydroxide R (about 60 mL) to adjust the pH to about 1.5. Aluminium Oxide, Neutral See Aluminium oxide,
Aluminium Hydroxide Gel Al(OH) 3+ aq (21645-51-2) hydrated (0311).
A hydrated grade of aluminium hydroxide of commerce. Aluminium Potassium Sulfate Alum; Aluminium
Aluminium Nitrate Aluminium nitrate nonahydrate; potassium sulphate; Aluminium potassium sulfate
Al(NO3)3,9H2O = 375.1 (7784-27-2) dodecahydrate; Aluminium potassium sulphate
dodecahydrate; (7784-24-9)
Crystals, deliquescent, very soluble in water and ethanol
(96 per cent), very slightly soluble in acetone. See Alum (0006).
Storage: in an airtight container. Aluminium Sulfate Aluminium sulfate;
Aluminium Oxide, Activated Acid Al 2O 3 = 102.1 Al2 (SO 4 h,16H2O = 630.4 (10043-01-3)
An almost white, fine, granular powder, very hygroscopic, Analytical reagent grade of commerce.
activated by heating at 200° to 250° for 3 hours. Aluminium Test Strip
Mean particle size, 50 to 200 µm. Commercially available test strip for the determination of
aluminium in aqueous solvents at a level below 5 ppm.
2023 Appendix I A V-A29

Allantoin C 4 H6N4O 3 = 158.1 (97-59-6) (4-Aminobenzoyl)-L-glutamic Acid N-(4-

Allantoin of the British Pharmacopoeia Aminobenzoyl)-L-glutamic acid; C 12 H 14N 2 O5 = 266.3
(4271-30-1)
Amaranth S Cl 16185; acid red 27;
C 20H 11 N 2 Na 3 OIOS 3 = 604 (915-67-3) White or almost white, crystalline powder.
General reagent grade of commerce. mp: about 175 °C, with decomposition.
A deep brown or deep reddish brown, fine powder. 2-Aminobutan-1-ol Aminobutanol; C 4 H 11 NO = 89.1
When used for the titration of iodine and iodides with (5856-63-3)
potassium iodate, the colour changes from orange-red to Oily liquid, miscible with water, soluble in ethanol
yellow. (96 per cent).
Amaranth Solution d~g: about 0.94.
A 0.2% w/v solution of amaranth S. ni 0 : about 1.453.

Americium-243 Spiking Solution bp: about 180 °C.

Contains 50 Bq/L 243Am and a 134 mg/L solution of 4-Amino-N-butyric Acid 4-Aminobutanoic acid;
lanthanum chloride heptahydrate R in a 103 g/L solution of C 4 H 9N0 2 = 103.1 (56-12-2)
hydrochloric acid R. Leaflets from methanol and ether, needles from water and
Amido Black 10B Disodium 5-amino-4-hydroxy-6-[(4- ethanol (96 per cent). Freely soluble in water, practically
nitrophenyl) azo ]-3-(phenylazo )naphthalene-2, 7-disulfonate; insoluble or slightly soluble in other solvents.
C 22H 14N 6Na 2 09S 2 = 617 (1064-48-8) mp: about 202 °C (decreases on rapid heating).
Schultz No. 299 2-Amino-5-chlorobenzophenone
Colour Index No. 20470 Aminochlorobenzophenone; C 13H 10 ClNO = 231.7
Dark-brown to black powder, sparingly soluble in water, (719-59-5)
soluble in ethanol (96 per cent). Yellow, crystalline powder, practically insoluble in water,
Amido Black 10B Solution freely soluble in acetone, soluble in ethanol (96 per cent).
A 5 g/L solution of amido black JOB R in a mixture of mp: about 97 °C.
10 volumes of acetic acid R and 90 volumes of methanol R. Content: minimum 95.0 per cent.
4-Aminoantipyrine 4-Amino-l ,5-dimethyl-2-phenyl-1,2- Storage: protected from light.
dihydro-3H-pyrazol-3-one; C 11 H 13N 3 O = 203.2 (83-07-8) 6-Aminohexanoic Acid 6-Aminocaproic acid;
Light yellow needles or powder, sparingly soluble in water, C 6 H 13NO 2 = 131.2 (60-32-2)
freely soluble in ethanol (96 per cent). Colourless crystals, freely soluble in water, sparingly soluble
mp: about 108 °C. in methanol, practically insoluble in anhydrous ethanol.
4-Aminoantipyrine Solution mp: about 205 'C.
A 1 g/L solution of 4-aminoantipyrine R in buffer solution p-Aminohippuric Acid Aminohippuric acid;
pH9.0R. C 9 H1 0N 2 O 3 = 194.2 (61-78-9)
Aminoazobenzene :x-Aminoazobenzene; White or almost white powder, sparingly soluble in water,
4-Aminoazobenzene; C 12 HllN 3 = 197.2 (60-09-3) soluble in ethanol (96 per cent).
Colour Index No. 11000 mp: about 200 °C.
Brownish-yellow needles with a bluish tinge, slightly soluble Aminohippuric Acid Reagent
in water, freely soluble in ethanol (96 per cent). Dissolve 3 g of phthalic acid Rand 0.3 g of aminohippuric
mp: about 128 °C. acid R in ethanol (96 per cent) R and dilute to 100 mL with
3-Aminobenzoic Acid C 7 H 7NO 2 = 137.1 (99-05-8) the same solvent.
White or almost white crystals. An aqueous solution turns 4-Amino-3-hydroxynaphthalene-1-sulfonic Acid
brown on standing in air. Aminohydroxynaphthalenesulfonic acid;
C 10 H 9 NO 4 S = 239.3 (116-63-2)
mp: about 174 °C.
White or grey needles, turning pink on exposure to light,
Storage: in an airtight container, protected from light.
especially when moist, practically insoluble in water and in
4-Aminobenzoic Acid C 7 H 7 NO 2 = 137.1 (150-13-0) ethanol (96 per cent), soluble in solutions of alkali
White or almost white, crystalline powder, slightly soluble in hydroxides and in hot solutions of sodium metabisulfite.
water, freely soluble in ethanol (96 per cent), practically Storage: protected from light.
insoluble in light petroleum. Aminohydroxynaphthalenesulfonic Acid Solution
mp: about 187 °C. Aminohydroxynaphthalenesulphonic acid solution
Chromatography. Thin-layer chromatography (2.2.27) as Mix 5.0 g of anhydrous sodium sulfite R with 94.3 g of sodium
prescribed in the monograph Procaine hydrochloride (0050); hydrogensulfite Rand 0.7 g of aminohydroxynaphthalenesulfonic
the chromatogram shows only one principal spot. acid R. Dissolve 1.5 g of the mixture in water R and dilute to
Storage: protected from light. 10.0 mL with the same solvent. Prepare the solution daily.
4-Aminobenzoic Acid Solution Aminohydroxynaphthalenesulfonic Acid Solution,
Dissolve 1 g of 4-amiiwbenzoic acid R in a mixture of 18 mL Strong
of anhydrous acetic acid R, 20 mL of water R and 1 mL of Aminohydroxynaphthalenesulphonic acid solution, strong.
phosphoric acid R. Immediately before use, mix 2 volumes of Dissolve O.25 g of 4-amino-3-hydroxynaphthalene-1-sulfonic
the solution with 3 volumes of acetone R. acid in 75 mL of a 15% w/v solution of sodium metabisulfite,
warming to assist solution if necessary. Add 2.5 mL of a
V-A30 Appendix I A 2023

20% w/v solution of sodium sulfi,te, mix and add sufficient of Mix 0.5 g of 8-aminonaphthalene-2-suljonic acid, 30 mL of
the sodium metabisulfite solution to produce 100 mL. glacial acetic acid and 120 mL of water and heat with stirring
5-Aminoimidazole-4-carboxamide Hydrochloride until dissolved. Allow to cool and filter.
C 4 H 6 N 4 O,HC1 = 162.6 (72-40-2) Use the solution within 3 weeks.
mp: about 251 °, with decomposition. 2-Amino-5-nitrobenzophenone
General reagent grade of commerce. Aminonitrobenzophenone; C 13 H 10N 2 O 3 = 242.2 (1775-95-7)
cis-Aminoindanol (1 S,2R)-1-Amino-2,3-dihydro-1H- Yellow, crystalline powder, practically insoluble in water,
inden-2-ol; (-)-cis-1-Aminoindan-2-ol; C9 H 11 NO = 149.2 soluble in tetrahydrofuran, slightly soluble in methanol.
(126456-43-7) mp: about 160 °C.
Content: minimum 98.0 per cent (sum of enantiomers, A:~: 690 to 720, determined at 233 nm using a 0.01 g/L
determined by gas chromatography). solution in methanol R.
[o:]~0 : -69 to -59, determined on a 2 g/L solution in 6-Aminopenicillanic Acid (2S,5R,6R)-6-Amino-3,3-
chloroform R. dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-
mp: 118 °C to 122 °C. carboxylic acid; C 8 H 12N 2O 3 S = 216.3 (551-16-6)
3-Aminomethylalizarin-N,N-diacetic Acid Appearance: white or almost white powder.
Aminomethylalizarindiacetic acid; mp: about 205 °C, with decomposition.
C19H1sNOs,2H2O = 421.4 (3952-78-1) Aminophenazone 4-(Dimethylamino )-1,5-dimethyl-2-
Fine, pale brownish-yellow or orange-brown powder, phenyl- l ,2-dihydro-3H-pyrazol-3-one; C 13H 17N 30 = 231.3
practically insoluble in water, soluble in solutions of alkali (58-15-1)
hydroxides. White or almost white, crystalline powder or colourless
mp: about 185 °C. crystals, soluble in water, freely soluble in ethanol
Loss on drying (2.2.32): maximum 10.0 per cent, determined (96 per cent).
on 1.000 g. mp: about 108 °C.
Aminomethylalizarindiacetic Acid Reagent 2-Aminophenol C 6 H 7 NO = 109.1 (95-55-6)
Solution A. Dissolve 0.36 g of cerous nitrate R in water Rand Pale yellowish-brown crystals which rapidly become brown,
dilute to 50 mL with the same solvent. sparingly soluble in water, soluble in ethanol (96 per cent).
Solution B. Suspend 0.7 g of aminomethylalizarindiacetic mp: about 172 °C.
acid R in 50 mL of water R. Dissolve with the aid of about Storage: in an airtight container, protected from light.
0.25 mL of concentrated ammonia R, add 0.25 mL of glacial
3-Aminophenol C 6 H 7NO = 109.1 (591-27-5)
acetic acid R and dilute to 100 mL with water R.
Pale yellowish-brown crystals, sparingly soluble in water.
Solution C. Dissolve 6 g of sodium acetate R in 50 mL of
mp: about 122 °C.
water R, add 11. 5 mL of glacial acetic acid R and dilute to
100 mL with water R. 4-Aminophenol C 6H 7NO = 109.1 (123-30-8)
To 33 mL of acetone R add 6.8 mL of solution C, 1.0 mL of Content: minimum 95 per cent.
solution B and 1.0 mL of solution A and dilute to 50 mL White or slightly coloured, crystalline powder, becoming
with water R. coloured on exposure to air and light, sparingly soluble in
Test for sensitivity. To 1.0 mL of fluoride standard solution water, soluble in anhydrous ethanol.
(10 ppm F) R add 19.0 mL of water Rand 5.0 mL of the mp: about 186 °C, with decomposition.
aminomethylalizarindiacetic acid reagent. After 20 min, the Storage: protected from light.
solution assumes a blue colour.
Aminopolyether 4,7 ,13, 16,21,24-hexaoxa-1,1 O-
Storage: use within 5 days. diazabicyclo[8,8,8]hexacosane; C 18H 3 ~ 2 O 6 = 376.5
Aminomethylalizarindiacetic Acid Solution (23978-09-8)
Dissolve 0.192 g of aminomethylalizarindiacetic acid R in 6 mL mp: 70 °C to 73 °C.
of freshly prepared 1 M sodium hydroxide. Add 750 mL of 4-( 4-Aminobenzene-1-sulfonyl)phenol
water R, 25 mL of succinate buffer solution pH 4. 6 R and, C 12H 11 NO 3 S = 249.3 (25963-47-7)
dropwise, 0.5 M hydrochloric acid until the colour changes
Grey or light brown powder, hygroscopic, slightly soluble in
from violet-red to yellow (pH 4.5 to 5). Add 100 mL of
methanol.
acetone Rand dilute to 1000 mL with water R.
mp: about 138 °C.
4-Aminomethylbenzoic Acid C 8H 9NO 2 = 151.2
(56-91-7) 4-( 4-Aminophenoxy)-N-methylpicolinamide 4-(4-
Aminophenoxy)-N-methylpyridine-2-carboxamide;
3-(Aminomethyl)pyridine (3-Pyridylmethyl)amine;
C 13 H 13N 3 O 2 = 243.3 (284462-37-9)
3-picolylamine; C 6 H 8N 2 = 108.1 (3731-52-0)
Content: minimum 99.0 per cent.
General reagent grade of commerce.
Light brown powder.
8-Aminonaphthalene-2-sulfonic Acid 8-Amino-2-
naphthalenesulfonic acid; 1-naphthylamine-7-sulfonic acid; mp: 110 °C to 112 °C.
8-aminonaphthalene-2-sulfonic acid; C 10 H 9NO 3 S = 223.2 3-Aminopropionic Acid 3-Amino-1-propanol;
(119-28-8) C 3 H 7NO 2 = 89.1 (107-95-9)
General reagent grade of commerce. Content: minimum 99 per cent.
Aminonaphthalenesulfonic Acid Solution White or almost white, crystalline powder, freely soluble in
Aminonaphthalenesulphonic acid solution. water, slightly soluble in ethanol (96 per cent), practically
insoluble in acetone.
2023 Appendix I A V-A31

mp: about 200 °C, with decomposition. Ammonia, Methanolic

Aminopterine 4-Aminofolic acid; C 19 H 20N 8 O 5 = 440.4 Solutions of the requisite molarity may be obtained by
(54-62-6) diluting 13.5M ammonia or 18M ammonia with methanol as
Yellowish powder. directed under ammonia.
mp: about 230 °C. Ammonia Rl, Concentrated
Aminopyrazolone 4-Aminophenazone; (83-07-8) Content: minimum 30.0 per cent m/m of NH3 (Mr 17.03).
See 4-aminoantipyrine R. A clear, colourless liquid.
Aminopyrazolone Solution See 4-aminoantipyrine d~g: less than 0.892.
solution R. Assay. Weigh accurately a ground-glass-stoppered flask
3-Aminosalicylic Acid 3-Amino-2-hydroxybenzoic acid; containing 50.0 mL of 1 M hydrochloric acid. Introduce 2 mL
C 7 H 7 NO 3 = 153.1 (570-23-0) of concentrated ammonia Rl and weigh again. Titrate the
mp: about 240 °C. solution with 1 M sodium hydroxide, using 0.5 mL of methyl
red mixed solutwn R as indicator.
Slightly soluble in water.
1 mL of 1 M hydrochloric acid is equivalent to 17.03 mg of
4-Aminosalicylic Acid 4-Amino-2-hydroxybenzoic acid;
NH3.
C7H 7 NO 3 = 153.1 (65-49-6)
Storage: protected from atmospheric carbon dioxide, at a
White or almost white, bulky powder, slightly soluble in temperature below 20 °C.
water, soluble in ethanol (96 per cent), in dilute nitric acid
and in sodium hydroxide. It darkens on exposure to air and Ammonia Rl, Dilute
light. Content: 100 g/L to 104 g/L ofNH3 (Mr 17.03).
mp: 135 °C to 145 °C. Dilute 41 g of concentrated ammonia R to 100 mL with
Storage: at a temperature not exceeding 30 °C, in an airtight water R.
container, protected from light. Ammonia R2, Dilute
Ammonia Content: 33 g/L to 35 g/L of NH 3 (Mr 17.03).
Content: 170 g/L to 180 g/L of NH3 (Mr 17 .03). Dilute 14 g of concentrated ammonia R to 100 mL with
Dilute 67 g of concentrated ammonia R to 100 mL with water R.
water R. Ammonia R3, Dilute
dig: 0.931 to 0.934. Content: 1.6 g/L to 1.8 g/L of NH 3 (Mr 17 .03).
When used in the test for iron, ammonia R complies with the Dilute 0.7 g of concentrated ammonia R to 100 mL with
following additional requirement. Evaporate 5 mL of water R.
ammonia to dryness on a water-bath, add 10 mL of water R, Ammonia R4, Dilute
2 mL of a 200 g/L solution of citric acid monohydrate R and Content: 8.4 g/L to 8.6 g/L of NH3 (Mr 17.03).
0.1 mL of thioglycollic acid R. Make alkaline by adding Dilute 3.5 g of concentrated ammonia R to 100 mL with
ammonia R and dilute to 20 mL with water R. No pink water R.
colour develops.
Ammonium Acetate C 2 H 7NO 2 = 77.1 (631-61-8)
Storage: protected from atmospheric carbon dioxide, at a
temperature below 20 °C. Colourless crystals, very deliquescent, very soluble in water
and in ethanol (96 per cent).
For 18M and 13.5M ammonia use analytical reagent grade
solutions of commerce containing 35% and 25% w/w of NH3 Storage: in an airtight container.
and weighing 0.88 g and 0.91 g per mL, respectively. Ammonium Acetate C2 H 7 NO 2 = 77.1 (631-61-8)
Solutions of molarity xM should be prepared by diluting 75x Content: minimum 99.0 per cent (anhydrous substance).
mL of 13.5M ammonia or 56x mL of 18M ammonia to Colourless crystals, very deliquescent, very soluble in water
1000 mL with water. and in ethanol (96 per cent).
Ammonia, Chloride-free Water (2.5.12): maximum 1 per cent.
13.5M ammonia that complies with the following test. Su/fated ash (2.4.14): maximum 0.01 per cent.
Chloride Evaporate 54 mL on a water bath almost to Storage: in an airtight container.
dryness and dilute to 15 mL with water. The solution Ammonium Acetate Solution
complies with the limit test for chlorides, Appendix VII Dissolve 150 g of ammonium acetate R in water R. Add 3 mL
(1 ppm). of glacial acetic acid R and dilute to 1000 mL with water R.
Ammonia, Concentrated See Concentrated ammonia Storage: use within 1 week.
solutwn (0877).
(lR)-(-)-Ammonium 10-Camphorsulfonate
Ammonia, Lead-free 10-Camphorsulfonic acid; 10-camphorsulphonic acid;
Complies with the requirements prescribed for dilute Ammonium salt; (lR)-(-)-Ammonium 10-Camphorsulfonate;
ammonia Rl with the following additional test: to 20 mL of C10H19NO4S = 249.3
lead-free ammonia, add 1 mL of lead-free potassium cyanide Content: minimum 97.0 per cent of (lR)-(-)-ammonium
solutwn R, dilute to 50 mL with water Rand add 0.10 mL of 10-camphorsulfonate.
sodium sulfide solution R. The solution is not more intensely
coloured than a reference solution prepared without sodium [o:]~0 : -18 ± 2, determined on a 50 g/L solution.
sulfide. Ammonium Carbamate Carbamic acid ammonium salt;
CH 6N 2 O 2 = 78.1 (1111-78-0)
V-A32 Appendix I A 2023

Ammonium Carbonate A mixture of varying proportions Ammonium Hexafluorogermanate(1v)

of ammonium hydrogen carbonate (NH 4 HCO 3, Mr 79.1) (NH 4) 2 GeF 6 = 222.7 (J 6962-47-3)
and ammonium carbamate (NH 2 COONH4 , Mr 78.1); White or almost white crystals, freely soluble in water.
(506-87-6)
Ammonium Hydrogen Carbonate Ammonium
White or almost white translucent mass, slowly soluble in bicarbonate; NH 4HCO 3 = 79.1 (1066-33-7)
about 4 parts of water. It is decomposed by boiling water.
Content: minimum 99 per cent.
Ammonium carbonate liberates not less than
30 per cent mlm ofNH 3 (Mr 17.03). Ammonium lron(m) Citrate Ferric ammonium citrate;
(1185-57-5)
Assay. Dissolve 2.00 gin 25 mL of water R. Slowly add
50.0 mL of 1 M hydrochloric acid, titrate with 1 M sodium General reagent grade (brown) of commerce.
hydroxide, using 0.1 mL of methyl orange solution Ras Ammonium Iron(n) Sulfate Ferrous ammonium sulfate;
indicator. Fe(NH 4 )z(SO 4 )z,6H 2 O = 392.2 (7783-85-9)
1 mL of 1 M hydrochloric acid is equivalent to 17.03 mg of Pale bluish-green crystals or granules, freely soluble in water,
NH3. practically insoluble in ethanol (96 per cent).
Storage: at a temperature below 20 °C. Storage: protected from light.
Ammonium Carbonate Solution Ammonium Iron(m) Sulfate Ferric ammonium sulfate;
A 158 g/L solution of ammonium carbonate R. FeNH4 (SO 4 )z,12H 2 O = 482.2 (7783-83-7)
Ammonium Carbonate Solution Rl Pale-violet crystals, efflorescent, very soluble in water,
practically insoluble in ethanol (96 per cent).
Dissolve 20 g of ammonium carbonate R in 20 mL of dilute
ammonia Rl and dilute to 100 mL with water R. Ammonium Iron(m) Sulfate Solution Rl
Ammonium Carbonate Solution, Dilute Ammonium iron(m) sulphate solution Rl
Dissolve 5 g of ammonium carbonate in a mixture of 7.5 mL Dissolve 0.2 g of ammonium iron(III) sulfate in 50 mL of
of 5M ammonia and 50 mL of water, dilute to 100 mL with water, add 5 mL of nitric acid and dilute to 100 mL with
water and filter, if necessary. water.
Ammonium Cerium(1v) Nitrate Ammonium and cerium Ammonium Iron(m) Sulfate Solution R2 Ferric
nitrate; (NH 4) 2 Ce(NO 3) 6 = 548.2 (16774-21-3) ammonium sulfate solution R2
Orange-yellow, crystalline powder, or orange transparent A 100 g/L solution of ferric ammonium sulfate R. If necessary
crystals, soluble in water. filter before use.
Ammonium Cerium(1v) Sulfate Ammonium and cerium Ammonium Iron(m) Sulfate Solution R5 Ferric
sulfate; (NH 4 ) 4 Ce(SO 4 ) 4 ,2H 2 O = 633 (10378-47-9) ammonium sulfate solution R5
Orange-yellow, crystalline powder or crystals, slowly soluble Shake 30.0 g of ferric ammonium sulfate R with 40 mL of
in water. nitric acid R and dilute to 100 mL with water R. If the
solution is turbid, centrifuge or filter it.
Ammonium Chloride (12125-02-9)
Storage: protected from light.
See Ammonium chloride (0007).
Ammonium Iron(m) Sulfate Solution R6 Ferric
Ammonium Chloride Solution
ammonium sulfate solution R6
A 107 g/L solution of ammonium chloride R.
Dissolve 20 g of ferric ammonium sulfate R in 75 mL of
Ammonium Citrate Diammonium hydrogen citrate; water R, add 10 mL of a 2.8 per cent V/V solution of suljuric
C6H 14N 2 O 7 = 226.2 (3012-65-5) acid R and dilute to 100 mL with water R.
White or almost white, crystalline powder or colourless Ammonium Mercaptoacetate Solution
crystals, freely soluble in water, slightly soluble in ethanol
Add 300 mL of water to 50 mL of mercaptoacetic acid,
(96 per cent).
neutralise with about 40 mL of 13.5M ammonia and dilute to
pH (2.2.3): about 4.3 for a 22.6 giL solution. 500 mL with water.
Ammonium Citrate Solution Ammonium Mercurithiocyanate Reagent
Dissolve, with cooling, 500 g of citric acid in a mixture of Dissolve 30 g of ammonium thwcyanate and 27 g of
200 mL of water and 200 mL of 13.5M ammonia. Filter and mercury(II) chloride in sufficient water to produce 1000 mL.
dilute to 1000 mL with water. Ammonium Metavanadate Ammonium vanadate;
Ammonium Cobaltothiocyanate Solution NH4VO 3 = 117.0 (7803-55-6)
Dissolve 37.5 g of cobalt(II) nitrate and 150 g of ammonium White or slightly yellowish, crystalline powder, slightly
thiocyanate in sufficient water to produce 1000 mL. soluble in water, soluble in dilute ammonia Rl.
Use within 1 day of preparation. Ammonium Metavanadate Solution
Ammonium Dihydrogen Orthophosphate Ammonium Ammonium vanadate solution
dihydrogen phosphate; (NH4 )H 2PO 4 = 115.0 (7722-76-1) Dissolve 1.2 g of ammonium vanadate R in 95 mL of water R
White or almost white, crystalline powder or colourless and dilute to 100 mL with sulfuric acid R.
crystals, freely soluble in water. Ammonium Molybdate (NH 4 ) 6 Mo 7 O 24,4H2 O = 1236
pH (2.2.3): about 4.2 for a 23 g/L solution. (12054-85-2)
Ammonium Formate CH5 NO 2 = 63.1 (540-69-2) Colourless or slightly yellow or greenish crystals, soluble in
Deliquescent crystals or granules, very soluble in water, water, practically insoluble in ethanol (96 per cent).
soluble in ethanol (96 per cent). Ammonium Molybdate Reagent
mp: 119 °C to 121 °C. Mix, in the given order, 1 volume of a 25 g/L solution of
Storage: in an airtight container. ammonium molybdate R, I volume of a 100 g/L solution of
2023 Appendix I A V-A33

ascorbic acid R and 1 volume of suljuric acid R (294.5 g/L Ammonium Oxalate C 2H 8 N 2O 4,H2O = 142.1
H 2SO 4). Add 2 volumes of water R. (6009-70-7)
Storage: use within 1 day. Colourless crystals, soluble in water.
Ammonium Molybdate Reagent Rl Ammonium Oxalate Solution
Mix 10 mL of a 60 g/L solution of disodium arsenate R, A 40 g/L solution of ammonium oxalate R.
50 mL of ammonium molybdate solution R, 90 mL of dilute Ammonium Persulfate Ammonium Peroxodisulfate;
sulfuric acid R and dilute to 200 mL in water R. Ammonium Peroxodisulphate; Ammonium Persulphate;
Storage: in amber flasks at 37 °C for 24 h. (NH4)2S 2 O 8 = 228.2 (7727-54-0)
Ammonium Molybdate Reagent R2 White or almost white, crystalline powder or granular
Dissolve 50 g of ammonium molybdate R in 600 mL of crystals, freely soluble in water.
water R. To 250 mL of cold water R add 150 mL of suljuric Ammonium Polysulfide Solution Ammonium
acid R and cool. Mix the 2 solutions together. Storage: use polysulphide solution
within 1 day. Dissolve a sufficient quantity of precipitated suljur to produce
Ammonium Molybdate Solution a deep orange solution in a solution prepared in the following
A 100 g/L solution of ammonium molybdate R. manner. Immediately before use saturate 120 mL of
Ammonium Molybdate Solution R2 6M ammonia with hydrogen suljide and add 80 mL of
6M ammonia.
Dissolve 5.0 g of ammonium molybdate R with heating in
30 mL of water R. Cool, adjust the pH to 7.0 with dilute Ammonium Pyrrolidinedithiocarbamate Ammonium
ammonia R2 and dilute to 50 mL with water R.
tetramethylenedithiocarbamate; C 5 H 12N 2S2 = 164.3
(5108-96-3)
Ammonium Molybdate Solution R3
White or pale yellow, crystalline powder, sparingly soluble in
Solution A. Dissolve 5 g of ammonium molybdate R in 20 mL
water, very slightly soluble in ethanol (96 per cent).
of water R with heating.
Storage: in a bottle containing a piece of ammonium
Solution B. Mix 150 mL of ethanol (96 per cent) R with
carbonate in a muslin bag.
150 mL of water R. Add with cooling 100 mL of suljuric
acid R.
Ammonium Pyrrolidinedithiocarbamate Solution
Immediately before use add 80 volumes of solution B to A 1.0% w/v solution of ammonium pyrrolidinedithiocarbamate
20 volumes of solution A. that has been washed immediately before use with three
25-mL quantities of 4-methylpentan-2-one.
Ammonium Molybdate Solution R4
Ammonium Reineckate Ammonium
Dissolve 1.0 g of ammonium molybdate R in water R and
tetrathiocyanatodiamminochromate(rn) monohydrate;
dilute to 40 mL with the same solvent. Add 3 mL of
NH4[Cr(NCSMNH 3) 2],H2 O = 354.4 (13573-16-5)
hydrochloric acid R and 5 mL of perchloric acid R and dilute to
100 mL with acetone R. Red powder or crystals, sparingly soluble in cold water,
soluble in hot water and in ethanol (96 per cent).
Storage: protected from light; use within 1 month.
Ammonium Reineckate Solution
Ammonium Molybdate Solution R5
A 10 g/L solution of ammonium reineckate R. Prepare
Dissolve 1.0 g of ammonium molybdate R in 40.0 mL of a immediately before use.
15 per cent VIV solution of sulfuric acid R. Prepare the
solution daily. Ammonium Sulfamate Ammonium sulphamate;
NH2 SO 3 NH4 = 114.1 (7773-06-0)
Ammonium Molybdate Solution R6
White or almost white, crystalline powder or colourless
Slowly add 10 mL of sulfuric acid R to about 40 mL of crystals, hygroscopic, very soluble in water, slightly soluble in
water R. Mix and allow to cool. Dilute to 100 mL with ethanol (96 per cent).
water Rand mix. Add 2.5 g of ammonium molybdate Rand
1 g of cerium sulfate R, and shake for 15 min to dissolve. mp: about 130 °C.
Ammonium Molybdate-Sulfuric Acid Solution Storage: in an airtight container.

Dissolve 10 g of ammonium molybdate in sufficient water to

Ammonium Sulfate Ammonium sulphate;
produce 100 mL and add the solution slowly to 250 mL of (NH4)2SO4 = 132.1 (7783-20-2)
cold 1OM suljuric acid. Colourless crystals or white or almost white granules, very
Store in a plastic bottle protected from light. soluble in water, practically insoluble in acetone and in
ethanol (96 per cent).
Ammonium Nitrate NH4NO 3 = 80.0 (6484-52-2)
pH (2.2.3): 4.5 to 6.0 for a 50 g/L solution in carbon dioxide-
White or almost white, crystalline powder or colourless
free water R.
crystals, hygroscopic, very soluble in water, freely soluble in
methanol, soluble in ethanol (96 per cent). Sulfated ash (2.4. 14): maximum 0.1 per cent.

Storage: in an airtight container. Ammonium Sulfide Solution Ammonium sulphide

solution
Ammonium Nitrate Rl
Saturate 120 mL of dilute ammonia Rl with hydrogen suljide R
Complies with the requirements prescribed for ammonium
and add 80 mL of dilute ammonia Rl. Prepare immediately
nitrate R with the following additional requirements.
before use.
Acidity. The solution of the substance is slightly acid (2. 2. 4).
Ammonium Thiocyanate NH 4 SCN = 76.1 (1762-95-4')
Chlorides (2.4.4): maximum 100 ppm, determined on 0.50 g.
Colourless crystals, deliquescent, very soluble in water,
Sulfates (2.4.13): maximum 150 ppm, determined on 1.0 g. soluble in ethanol (96 per cent).
Suljated ash (2.4.14'): maximum 0.05 per cent, determined on Storage: in an airtight container.
1.0 g.
V-A34 Appendix I A 2023

Ammonium Thiocyanate Solution The area of the principal peak is not less than 92.0% of the
A 7 6 g/L solution of ammonium thiocyanate R. total area of the peaks.
Amoxicillin Trihydrate See Amoxicillin trihydrate (0260). Anhydrous Colloidal Silica (7631-86-9)
Amygdalin (R)-[ (6-O-P-o-Glucopyranosyl-P-o- See Anhydrous colloidal silica (0434).
glucopyranosyl)oxy] (phenyl)acetonitrile; Anhydrous Trisodium Orthophosphate Tribasic
C20H21NO11 = 457.4 (29883-15-6) Sodium Phosphate; Na 3PO 4 = 163.94 (7601-54-9)
Amyl Acetate C 7 H 14O 2 = 130.2 mp: about 75°
Consists principally of 3-methylbutyl acetate with a small General reagent grade of commerce.
proportion of 2-methylbutyl acetate. Aniline Benzeneamine; C 6 H 7 N = 93.1 (62-53-3)
bp: about 140°. Colourless or slightly yellowish liquid, soluble in water,
Analytical reagent grade of commerce. miscible with ethanol (96 per cent).
A colourless liquid; weight per mL, about 0.87 g. d~g: about 1.02.
Amyl Alcohol Isoamyl alcohol; C 5 H 12 O = 88.1 (123-51-3) bp: 183 °C to 186 °C.
Colourless liquid, slightly soluble in water, miscible with Storage: protected from light.
ethanol (96 per cent). Aniline Hydrochloride Benzenamine hydrochloride;
bp: about 130 °c. C 6 H 8 CIN = 129.6 (142-04-1)
:x-Amylase 1,4-:x-o-glucane-glucanohydrolase (EC 3.2.1.1) Crystals. It darkens on exposure to air and light.
White or light brown powder. mp: about 198 °C.
:x-Amylase Solution Storage: protected from light.
A solution of a-amylase R with an activity of 800 FAU/g. Content: minimum 97.0 per cent.
P-Amyrin Olean-12-en-3P-ol; C 30H 50 O = 426.7 Aniline Hydrochloride Solution
(559-70-6) Dissolve 2 g of aniline hydrochloride in a mixture of 65 mL of
White or almost white powder. ethanol (96%) and 35 mL of water and add 2 mL of
mp: 187 °C to 190 °C. hydrochloric acid.
L-Analyl-L-proline C 8 H 14N 2 O 3 = 186.2 (13485-59-1) Use within one day of preparation.
General reagent grade of commerce. Anion Exchange Resin
Andrographolide (3E,4S)-3-[2-[(1R,4aS,5R,6R,8aS)-6- Resin in chlorinated form with a quaternary ammonium
Hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2- funtionalised latex cross-linked with divinylbenzene.
methylenedecahydronaphthalen-1-yl]ethylidene]-4- Wash the resin with 1 M sodium hydroxide on a sintered-glass
hydroxydihydrofuran-2 (3H)-one; C 20 H 30 O 5 = 350.4 filter (40) (2.1. 2) until the washings are free from chloride,
(5508-58-7) then wash with water R until the washings are neutral.
Anethole 1-Methoxy-4-(propen-1-yl)benzene; Suspend in freshly prepared ammonium-free water R and
C1 0 H 12 O = 148.2 (4180-23-8) protect from atmospheric carbon dioxide.
White or almost white, crystalline mass up to 20 °C to Anion Exchange Resin, Strongly Basic
21 °C, liquid above 23 °C, practically insoluble in water, Gel-type resin in hydroxide form containing quaternary
freely soluble in anhydrous ethanol, soluble in ethyl acetate ammonium groups [CH 2 N+(CH 3 ) 3 , type l] attached to a
and in light petroleum. polymer lattice consisting of polystyrene cross-linked with
nf}: about 1.56. 8 per cent of divinylbenzene.
bp: about 230 °C. Brown transparent beads.
Anethole used in gas chromatography complies with the following Particle size: 0.2 mm to 1.0 mm.
additional test. Moisture content: about 50 per cent.
Assay. Gas chromatography (2.2.28) as prescribed in the Total exchange capacity: minimum 1.2 meq/mL.
monograph Anise oil (0804). Anion Exchange Resin for Chromatography, Strongly
Test solution. The substance to be examined. Basic
Content: minimum 99.0 per cent of trans-anethole (retention Resin with a quaternary ammonium functionalised latex
time: about 41 min), calculated by the normalisation cross-linked with divinylbenzene.
procedure. Anion-exchange Resin for Chromatography, Strongly
cis-Anethole (Z)-1-Methoxy-4-prop-1-enylbenzene; Basic Rl
C10H1 2O = 148.2 Non-porous resin agglomerated with a 100 nm alkyl
nf}: about 1.56. quaternary ammonium functionalised latex.
bp: about 230°. Anion-exchange Resin for Chromatography, Strongly
General reagent grade of commerce. Basic R2
A white crystalline mass at 20°; liquid above 23°. Resin agglomerated with a quaternary ammonium
Cis-anethole used in gas chromatography complies with the
functionalised latex cross-linked with ethylvinylbenzene-
following test. divinylbenzene.

Assay Examine by gas chromatography, Appendix III B, Anion Exchange Resin Rl

under the conditions described in the test for Resin containing quaternary ammonium groups
Chromatographic profile in the monograph for Anise Oil [CH2 N+(CH 3 h] attached to a lattice consisting of
using the reagent being examined as solution (1). methacrylate.
2023 Appendix I A V-A35

Anion Exchange Resin R2 mp: about 218 °C.

Conjugate of homogeneous 10 µm hydrophilic polyether Anthranilic Acid 2-Aminobenzoic acid;
particles, and a quaternary ammonium salt, providing a C 7H 7N0 2 = 137.1 (118-92-3)
matrix suitable for strong anion-exchange chromatography of A white or pale-yellow, crystalline powder, sparingly soluble
proteins. in cold water, freely soluble in hot water, in ethanol
Anion Exchange Resin R3 (96 per cent) and in glycerol. Solutions in ethanol
Resin with quaternary ammonium groups attached to a (96 per cent) or in ether and, particularly, in glycerol show a
lattice of ethylvinylbenzene crosslinked with 55 per cent of violet fluorescence.
divinylbenzene. mp: about 145 'C.
Anion Exchange Resin, Weak Anthrone 9(10H)-Anthracenone; C 14H 10 0 = 194.2
Resin with diethylaminoethyl groups attached to a lattice (90-44-8)
consisting of poly(methyl methacrylate). Pale yellow, crystalline powder.
Anisaldehyde 4-Methoxybenzaldehyde; C 8H 8 0 2 = 136.1 mp: about 155 °C. When used in an assay for glucose or
(123-11-5) dextrans, complies with the following test.
Oily liquid, very slightly soluble in water, miscible with Sensitivity to glucose Add, carefully, 6 mL of a 0.2% w/v
ethanol (96 per cent). solution in a mixture of 19 mL of sulfuric acid and 1 mL of
bp: about 248 °C. water to 3 mL of a solution containing 5 µg of D-glucose per
Anisaldehyde used in gas chromatography complies with the mL and heat in a water bath for 5 minutes. The solution is a
fallowing additional test.
darker green than a solution prepared in the same manner
but omitting the glucose.
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Anise oil (0804). Anthrone Reagent
Test solutwn. The substance to be examined. A 0.2% w/v solution of anthrone in nitrogen-free sulfuric acid.
The solution should be allowed to stand for 4 hours before
Content: minimum 99.0 per cent, calculated by the
use and should be discarded after 7 days.
normalisation procedure.
Antimony Potassium Tartrate Antimony potassium
Anisaldehyde Solution oxide (+)-tartrate; C 8 H 4 K 2 0 12 Sb2 ,3H 2 0 = 668 (28300-74-5)
Mix in the following order, 0.5 mL of anisaldehyde R, 10 mL White or almost white, granular powder or colourless,
of glacial acetic acid R, 85 mL of methanol R and 5 mL of transparent crystals, soluble in water and in glycerol, freely
sulfuric acid R.
soluble in boiling water, practically insoluble in ethanol
Anisaldehyde Solution Rl (96 per cent). The aqueous solution is slightly acid.
To 10 mL of anisaldehyde R add 90 mL of ethanol Antimony Trichloride SbC13 = 228.1 (10025-91-9)
(96 per cent) R, mix, add 10 mL of sulfuric acid R and mix
Colourless crystals or a transparent crystalline mass,
again. hygroscopic, freely soluble in anhydrous ethanol. .1\ntimony
Anisaldehyde Solution R2 trichloride is hydrolysed by water.
To 170 mL of cold methanol R add 20 mL of glacial acetic Storage: in an airtight container, protected from moisture.
acid R and 10 mL of sulfuric acid R. Mix well. Cool to room
Antimony Trichloride in Dichloroethane Solution
temperature and add 1.0 mL of anisaldehyde R.
Prepare a 22.0% w/v solution of antimony trichloride in dry
Anise Ketone 1-(4-Methoxyphenyl)propan-2-one; 1,2-dichloroethane that has been purified by passing it down a
C1 0 H 12 02 = 164.2 (122-84-9) column of silica gel and allow the solution to stand for
p-Anisidine 4-Methoxyaniline; C 7 H 9 NO = 123.2 24 hours. To 100 mL add 2.5 mL of aceryl chloride and allow
(104-94-9) to stand for a further 24 hours before use.
White or almost white crystals, sparingly soluble in water, Antimony Trichloride Solution
soluble in anhydrous ethanol. Rapidly wash 30 g of antimony trichloride R with two
Content: minimum 97.0 per cent. quantities, each of 15 mL, of ethanol-free chloroform R, drain
Caution: skin irritant, sensitiser. off the washings, and dissolve the washed crystals
Storage: protected from light, at O °C to 4 °C. immediately in 100 mL of ethanol-free chloroform R, warming
On storage, p-anisidine tends to darken as a result of slightly.
oxidation. A discoloured reagent can be reduced and Storage: over a few grams of anhydrous sodium sidfate R.
decolorised in the following way: dissolve 20 g of p- Antithrombin III ATIII; (90170-80-2)
anisidine R in 500 mL of water R at 75 °C. Add 1 g of sodium Antithrombin III is purified from human plasma by heparin
sulfi,te heptahydrate R and 10 g of activated charcoal R and stir agarose chromatography and should have a specific activity of
for 5 min. Filter, cool the filtrate to about 0 °C and allow to at least 6 IU/mg.
stand at this temperature for at least 4 h. Filter, wash the Antithrombin III Solution Rl
crystals with a small quantity of water R at about 0 °C and
dry the crystals in vacua (2.2.32). Reconstitute antithrombin III R as directed by the
manufacturer and dilute with tris(hydroxymethyVaminomethane
Anolyte for Isoelectric Focusing pH 3 to 5 sodium chloride buffer solution pH 7.4 R to 1 IU/mL.
0.lM glutamic acid, 0.5M phosphoric acid. Dissolve 14.71 g Antithrombin III Solution R2
of glutamic acid in water, add 33 mL of orthophosphoric acid
and dilute to 1000 mL with water. Reconstitute antithrombin III R as directed by the
manufacturer and dilute with tris(hydroxyrnethyVaminomethane
Anthracene C 14Hrn = 178.2 (120-12-7) sodium chloride buffer solutwn pH 7.4 R to 0.5 IU/mL.
White or almost white, crystalline powder, practically
insoluble in water, slightly soluble in chloroform.
V-A36 Appendix I A 2023

Antithrombin III Solution R3 Content: minimum 96 per cent of C 20H 42 O.

Reconstitute antithrombin III R as directed by the Arachis Oil
manufacturer and dilute to 0.3 IU/mL with phosphate buffer Of the British Pharmacopoeia.
solution pH 6. 5 R.
Arbutin Arbutoside; 4-Hydroxyphenyl-~-D-
Antithrombin III Solution R4 glucopyranoside; C 12 H 16 O 7 = 272.3 (497-76-7)
Reconstitute antithrombin III R as directed by the Fine, white or almost white, shiny needles, freely soluble in
manufacturer and dilute to 0.1 IU/mL with water, very soluble in hot water, soluble in ethanol
tris(hydroxymethyl) aminomethane-EDTA buffer solution (96 per cent).
pH 8.4 R. Chromatography. Thin-layer chromatography (2.2.27) as
Antithrombin III Solution R5 prescribed in the monograph Bearbeny leaf (1054); the
Reconstitute antithrombin III R as directed by the chromatogram shows only one principal spot.
manufacturer and dilute to 0.125 IU/mL with Arginine L-Arginine; (74-79-3)
tris(hydroxymethyl)aminomethane-EDTA buffer solution See Arginine (0806).
pH 8.4 Rl.
Argon Ar= 39.95 (7440-37-1)
Antithrombin III Solution R6
Content: minimum 99.995 per cent V/V.
Reconstitute antithrombin III R as directed by the
Carbon monoxide (2.5.25, Method I): maximum 0.6 ppm V/V;
manufacturer and dilute to 1.0 IU/mL with
tris(hydroxymethyl) aminomethane-EDTA buffer solution
after passage of 10 L of argon R at a flow rate of 4 L/h, not
more than 0.05 mL of 0.002 M sodium thiosulfate is required
pH 8.4 Rl.
for the titration.
Apigenin 4',5,7-Trihydroxyflavone; C 15 H 10 O 5 = 270.2
(520-36-5)
Argon Rl Ar= 39.95 (7440-37-1)
Content: minimum 99.99990 per cent V/V.
Light yellowish powder, practically insoluble in water,
sparingly soluble in ethanol (96 per cent). Argon for Chromatography Ar= 39.95 (7440-37-1)
mp: about 310 °C, with decomposition. Content: minimum 99.95 per cent V/V.
Chromatography. Thin-layer chromatography (2.2.27) as Aromadendrene (IR,2S,4R,8R,11R)-3,3,11-Trimethyl-7-
prescribed in the monograph Roman chamomile flower (0380): methylenetricyclo- [6.3.0.0 2' 4]undecane; C 15H 24 = 204.4
apply 10 µL of a 0.25 g/L solution in methanol R; the (489-39-4)
chromatogram shows in the upper third a principal zone of Clear, almost colourless liquid.
yellowish-green fluorescence. df 0 : about 0.911.
Apigenin-7-glucoside Apigetrin; 7-(~-D- ng1: about 1.497.
Glucopyranosyloxy)-5-hydroxy-2-( 4-hydroxyphenyl)-4H-1-
[a]~: about+ 12.
benzopyran-4-one; C 21 H 200 10 = 432.4 (578-74-5)
bp: about 263 °C.
Light yellowish powder, practically insoluble in water,
sparingly soluble in ethanol (96 per cent). Aromadendrene used in gas chromatography complies with the
following additional test.
mp: 198 °C to 201 °C.
Assay. Gas chromatography (2.2.28) as prescribed in the
Chromatography. Thin-layer chromatography (2.2.27) as
monograph on Tea tree oil (1837).
prescribed in the monograph Roman chamomile flower (0380):
apply 10 µL of a 0.25 g/L solution in methanol R; the Content: minimum 92 per cent, calculated by the
chromatogram shows in the middle third a principal zone of normalisation procedure.
yellowish fluorescence. Arsenazo III 3,6-Bis[(2-arsonophenyl)diazenyl]-4,5-
Apigenin-7-glucoside used in liquid chromatography complies with
dihydroxynaphthalene-2, 7-disulfonic acid;
the following additional test. C22H1sAs2N4O14S2 = 776 (1668-00-4)
Assay. Liquid chromatography (2.2.29) as prescribed in the Brown powder.
monograph Matricaria flower (0404). Arsenious Trioxide Arsenic trioxide; As 2 O 3 = 197 .8
(1327-53-3)
Test solution. Dissolve 10.0 mg in methanol R and dilute to
100.0 mL with the same solvent. Crystalline powder or a white or almost white mass, slightly
Content: minimum 95.0 per cent, calculated by the soluble in water, soluble in boiling water.
normalisation procedure. Arsenite Solution
Apiole Apiol; apioline; parsley apiole; 5-allyl-4, 7- Dissolve 0.50 g of arsenious trioxide in 5 mL of 2M sodium
dimethoxy-1,3-benzodioxol; C 12H 14O 4 = 222.24 (484-31-1) hydroxide solution, add 2.0 g of sodium hydrogen carbonate and
General reagent grade of commerce. dilute to 100 mL with water.
Aprotinin (9087-70-1) L-Ascorbic Acid Ascorbic acid; (50-81-7)
See Aprotinin (0580). See Ascorbic acid (0253).
L-Arabinose Arabinose; C 5H 10 O 5 = 150.1 (87-72-9) Ascorbic Acid Solution
White or almost white, crystalline powder, freely soluble in Dissolve 50 mg in 0.5 mL of water R and dilute to 50 mL
water. with dimethylformamide R.
[a]t0 : + 103 to+ 105, determined on a 50 g/L solution in Asiaticoside O-6-Deoxy-rt-L-mannopyranosyl-( 1 ➔ 4)-O-~­
water R containing about 0.05 per cent ofNH 3 • D-glucopyranosyl-( 1 ➔ 6 )-~-D-glucopyranosyl 2rt,3 ~,23-
trihydroxy-4rt-urs-12-en-28-oate; C 48 H 78 O 19 = 959
Arachidic Alcohol Eicosan-1-ol; Arachidyl alcohol; (16830-15-2)
Arachidyl alcohol; C 20H 42 O = 298.5 (629-96-9)
mp: about 65 °C.
2023 Appendix I A V-A37

White or almost white powder, hygroscopic, soluble in Chromatography. Thin-layer chromatography (2.2.27) as
methanol, slightly soluble in anhydrous ethanol, insoluble in prescribed in the monograph Frangula bark (0025); the
acetonitrile. chromatogram shows only one principal spot.
mp: about 232 °C, with decomposition. Barbitone Sodium Barbital sodium;
Water (2.5.12): 6.0 per cent. C8H 11 N 2 NaO 3 = 206.2 (144-02-5)
Asiaticoside used in liquid chromatography complies with the Content: minimum 98.0 per cent.
following additional test. A white or almost white, crystalline powder or colourless
Assay. Liquid chromatography (2.2.29) as prescribed in the crystals, freely soluble in water, slightly soluble in ethanol
monograph Centella (1498). (96 per cent).
Content: minimum 97.0 per cent, calculated by the Barbitone Barbital; C 8H 12 N 2O 3 = 184.2 (57-44-3)
normalisation procedure. 5,5-Diethyl-2,4,6(1H,3H,5H)-pyrirnidinetrione.
Storage: protected from humidity. Content: minimum 98.0 per cent.
Asparagine C 4H 8N 2 O 3 = 132.12 (70-47-3) mp: 188-192 °C.
Aspartic Acid (56-84-8) A white or almost white, crystalline powder, freely soluble in
See Aspartic acid (0797). water, soluble in ethanol (96 per cent).
o-Aspartic Acid C 4 H 7 NO 4 = 133.1 (1783-96-6) Barbituric Acid 2,4,6-Trihydroxypyrirnidine;
L-Aspartyl-L-phenylalanine Aspartame; C4H4N2 O 3 = 128.1 (67-52-7)
C13H16N2Os = 280.3 (13433-09-5) White or almost white powder, slightly soluble in water,
White or almost white powder. freely soluble in boiling water and in dilute acids.
mp: about 210 °C, with decomposition. mp: about 253 °C.
Astragaloside IV (20R,24S)-20,24-Epoxy-16P,25- Barium Acetate Barium diacetate; C 4H 6BaO 4 = 255.4
dihydroxy-3P-(P-o-xylopyranosyloxy)-9, 19-cyclolanostan-6cr- (543-80-6)
yl P-o-glucopyranoside; C 41 H 68 O 14 = 785 (84687-43-4) White or almost white powder, soluble in water.
Atenolol C14H 22 N2O 3 = 266.3 (56715-13-0) dig: 2.47.
General reagent grade of commerce. Barium Carbonate BaCO 3 = 197.3 (513-77-9)
Atropine Sulfate Atropine sulphate; (5908-99-6) White or almost white powder or friable masses, practically
See Atropine sulfate (0068). insoluble in water.
Aucubin (1 S,4aR,5S, 7aS)-5-Hydroxy-7-(hydroxymethyl)- Barium Chloride Barium dichloride;
1,4a,5, 7a-tetrahydrocyclopenta [c]pyran-1-yl P-o- BaC12 ,2H 2O = 244.3 (10326-27-9)
glucopyranoside; C 15H 22 O9 = 346.3 (479-98-1) Colourless crystals, freely soluble in water, slightly soluble in
Crystals, soluble in water, in ethanol (96 per cent) and in ethanol (96 per cent).
methanol, practically insoluble in light petroleum. Barium Chloride Solution
mp: about 181 °C. A 10.0% w/v solution of barium chloride.
Azadirachtin C35H 44 0 16 = 720.7 (11141-17-6) Barium Chloride Solution Rl
mp: about 165°. A 61 g/L solution of barium chloride R.
General reagent grade of commerce. Barium Chloride Solution R2
Azobenzene C 12H 10N 2 = 182.2 (103-33-3) A 36.5 g/L solution of barium chloride R.
mp: about 69°. Barium Hydroxide Barium dihydroxide;
Use a grade of commerce suitable for chromatographic Ba(OH)z,8H 2 O = 315.5 (12230-71-6)
standardisations. Colourless crystals, soluble in water.
Azomethine H Monosodium salt hydrate; Barium Hydroxide Solution
C 17H1 2NNaO 8 S2 = 445.4 (5941-07-1) A 4 7.3 g/L solution of barium hydroxide R.
Azomethine H Solution Barium Nitrate Ba(NO 3)z = 261.3 (10022-31-8)
Dissolve 0.45 g of azomethine H R and 1 g of ascorbic acid R Crystals or crystalline powder, freely soluble in water, very
with gentle heating in water R and dilute to 100 mL with the slightly soluble in ethanol (96 per cent) and in acetone.
same solvent. mp: about 590 °C.
Baicalin C 21 H 18 O 11 = 446.4 (21967-41-9) Barium Sulfate (7727-43-7)
5,6-Dihydroxy-4-oxo-2-phenyl-4H-1-benzopyran-7-yl-P-o- See Barium sulfate (0010).
glucopyranosiduronic acid. Benzaldehyde C 7 H 6 O = 106.1 (100-52-7)
Barbaloin Aloin; 1,8-Dihydroxy-3-hydroxymethyl-1 0-P-o- Colourless or slightly yellow liquid, slightly soluble in water,
glucopyranosyl-1 0H-anthracen-9-one; C21 H 22 O9 , miscible with ethanol (96 per cent).
H 2 0 = 436.4 (1415-73-2)
Yellow to dark-yellow, crystalline powder, or yellow needles,
d~g: about 1.05.
darkening on exposure to air and light, sparingly soluble in nEJ0 : about 1.545.
water and in ethanol (96 per cent), soluble in acetone, in Distillation range (2.2.11). Not less than 95 per cent distils
ammonia and in solutions of alkali hydroxides. between 177 °C and 180 °C.
A\'1i111 : about 192 at 269 nm, about 226 at 296.5 nm, about Storage: protected from light.
259 at 354 nm, determined on a solution in methanol R and Benzalkonium Chloride
calculated with reference to the anhydrous substance. Of the British Pharmacopoeia.
V-A38 Appendix I A 2023

Benzalkonium Chloride Solution Prismatic crystals, practically insoluble in water, freely soluble
Of the British Pharmacopoeia. in ethanol (96 per cent).
Benzalphthalide 3-Benzylidenephthalide; mp: about 48 °C.
C1sH 10 O2 == 222.24 (575-61-1) 1,4-Benzoquinone Cyclohexa-2,5-diene- l ,4-dione;
light yellow powder or crystals. C6H4O 2 == 108.1 (106-51-4)
Analytical reagent grade of commerce. Content: minimum 98.0 per cent.
Benzene C 6 H 6 == 78.1 (71-43-2) Benzoylarginine Ethyl Ester Hydrochloride N-Benzoyl-
L-arginine ethyl ester hydrochloride; Ethyl (S)-2-benzamido-
Clear, colourless, flammable liquid, practically insoluble in
water, miscible with ethanol (96 per cent). 5-guanidinovalerate hydrochloride; C 15H 23 ClN4 O 3 == 342.8
(2645-08-1)
bp: about 80 °C.
White or almost white, crystalline powder, very soluble in
Where benzene is used to prepare a reference solution, for water and in anhydrous ethanol.
safety reasons, the pure reagent may be replaced by a
commercially available reference material containing a [1X]t0: -15 to -18, determined on a 10 g/L solution.
certified amount of benzene. mp: about 129 °C.
Where benzene is used to prepare a reference solution, for A\~m: 310 to 340, determined at 227 nm using a 0.01 g/L
safety reasons, the pure reagent may be replaced by a solution.
commercially available reference material containing a Benzoyl Chloride C 7 H 5 ClO == 140.6 (98-88-4)
certified amount of benzene. Colourless, lachrymatory liquid, decomposed by water and
4-(Benzenesulfonyl)aniline C 12HuNO 2S == 233.3 by ethanol (96 per cent).
(7019-01-4) d?g: about 1.21.
light brown powder. bp: about 197 °C.
mp: about 176 °C. Benzoylecgonine Hydrate C 16H 19NO 4,H 2O == 307.3
Benzene-1,2,4-triol Hydroxyhydroquinone; (519-09-5)
Hydroxyquinol; C 6H 6O 3 == 126.1 (533-73-3) General reagent grade of commerce.
Freely soluble in water, in ethanol (96 per cent) and in ethyl Benzoyl Peroxide C 14H 10O 4 == 242.2 (94-36-0)
acetate. mp: after drying, about 104°.
mp: about 140 °C. White or almost white granules.
Benzethonium Chloride Benzyldimethyl[2-[2-[4-(l,l,3,3- General reagent grade of commerce.
tetramethylbutyl)phenoxy]ethoxy] ethyl] ammonium chloride;
For safety Benzoyl Peroxide should be kept moistened with
C21H42ClNO2 == 448.1 (121-54-0)
about 23% w/w of water.
Fine, white or almost white powder or colourless crystals,
soluble in water and in ethanol (96 per cent). N-Benzoyl-L-prolyl-L-phenylalanyl-L-arginine
4-Nitroanilide Acetate C 35 H 42N 8 O 8 == 703
mp: about 163 °C.
3-Benzoylpropionic Acid 4-Oxo-4-phenylbutanoic acid;
Storage: protected from light.
C10H 10 O3 == 178.2 (2051-95-8)
Benzidine Biphenyl-4,4'-diamine; C 12 H 12N 2 == 184.2 mp: about 118 °C.
(92-87-5)
2-Benzoylpyridine Benzoylpyridine; C 12H 9NO == 183.2
Content: minimum 95 per cent. (91-02-1)
White or slightly yellowish or reddish powder, darkening on Colourless crystals, soluble in ethanol (96 per cent).
exposure to air and light.
mp: about 43 °C.
mp: about 120 °C.
Benzyl Alcohol (100-51-6)
Storage: protected from light.
See Benzyl alcohol (0256).
Benzil Diphenylethanedione; C 14H 10O 2 == 210.2
(134-81-6) Benzyl Benzoate (120-51-4)
Yellow, crystalline powder, practically insoluble in water, See Benzyl benzoate (0705).
soluble in ethanol (96 per cent), ethyl acetate and toluene. Chromatography. Thin-layer chromatography (2.2.27) as
mp: 95 °C. prescribed in the monograph Peru balsam (0754): apply
20 µL of a 0.3 per cent V/V solution in ethyl acetate R; after
Benzocaine C 9 HuNO 2 == 165.2 (94-09-7) spraying and heating, the chromatogram shows a principal
See Benzocaine (0011). band with an RF of about 0.8.
Benzohydrazide Benzoyldiazane; C 7 H 8 N 2O == 136.2 Benzyl Cinnamate Benzyl 3-phenylprop-2-enoate;
(613-94-5) C16H 14O 2 == 238.3 (103-41-3)
Benzoic Acid (65-85-0) Colourless or yellowish crystals, practically insoluble in water,
See Benzoic acid (0066). soluble in ethanol (96 per cent).
Benzoin et,-Hydroxy-cx-phenylacetophenone; mp: about 39 °C.
C14H12O2 == 212.3 (579-44-2) Chromatography. Thin-layer chromatography (2.2.27) as
Slightly yellowish crystals, very slightly soluble in water, freely prescribed in the monograph Peru balsam (0754): apply
soluble in acetone, soluble in hot ethanol (96 per cent). 20 µL of a 3 g/L solution in ethyl acetate R; after spraying and
mp: about 137 °C. heating, the chromatogram shows a principal band with
Benzophenone Diphenylmethanone; C 13H 10O == 182.2 an RF of about 0.6.
(119-61-9)
2023 Appendix I A V-A39

Benzyl Cyanide Phenylacetonitrile; C 8 H 7N = 117.2 Betulin Lup-20(39)-ene-3~,28-diol; C 30H 50 O 2 = 442.7

(140-29-4) ( 473-98-3)
Content: minimum 95.0 per cent. White or almost white, crystalline powder.
Clear, colourless or light yellow liquid. mp: 248 °C to 251 °C.
nt0 : about 1.523. Bibenzyl 1,2-Diphenylethane; C 14H 14 = 182.3 (103-29-7)
bp: about 233 °C. White or almost white, crystalline powder, practically
Benzyl Ether Dibenzyl ether; C 14 H 14O = 198.3 insoluble in water, very soluble in methylene chloride, freely
(103-50-4) soluble in acetone, soluble in ethanol (96 per cent).
Clear, colourless liquid, practically insoluble in water, mp: 50 °C to 53 °C.
miscible with acetone and with anhydrous ethanol. Biphenyl C 12 H 10 = 154.2 (92-52-4)
dig: about 1.043. mp: 68 °C to 70 °C.
nt0: about l.562. Biphenyl-4-ol 4-Phenylphenol; C 12 H 10 O = 170.2
bp: about 296 °C, with decomposition. (90-43-7)
Benzylpenicillin Sodium (69-57-8) mp: 164° to 167°.
See Benzylpenicillin sodium (0114). White or almost white, crystalline powder, practically
2-Benzylpyridine C 12 H 11 N = 169.2 (101-82-6) insoluble in water.
Content: minimum 98.0 per cent. (-)-a-Bisabolol Levomenol; C 15H 26 O = 222.4
(23089-26-1)
Yellow liquid.
Colourless, viscous liquid with a slight, characteristic odour,
mp: 13 °C to 16 °C.
practically insoluble in water, freely soluble in ethanol
4-Benzylpyridine C 12H 11 N = 169.2 (2116-65-6) (96 per cent), in methanol, in toluene, in fatty oils and in
Content: minimum 98.0 per cent. essential oils.
Yellow liquid. dig: 0.925 to 0.935.
mp: 72 °C to 78 °C. nt 0 : 1.492 to 1.500.

Benzyltrimethylammonium Chloride [:x]t0 : -54.5 to -58.0, determined on a 50 g/L solution in

Trimethylphenylmethanaminium chloride; ethanol (96 per cent) R.
C 10H 16CIN = 185.7 (56-93-9) (-)-:x-Bisabolol used for gas chromatography complies with the
White or almost white powder, soluble in water. following additional test.
mp: about 230 °C, with decomposition. Assay. Gas chromatography (2. 2. 28) as prescribed in the
Berberine Chloride 9,10-Dimethoxy-5,6-dihydrobenzo monograph Matricaria 017 (1836).
[g]-1,3-benzodioxolo [5, 6-a] quinolizinium chloride; Test solution. A 4 g/L solution in cyclohexane R.
C 20 H1sCINO4,2H2O = 407.8 (5956-60-5) Content: minimum 95.0 per cent, calculated by the
Yellow crystals, slightly soluble in water, practically insoluble normalisation procedure.
in ethanol (96 per cent). 1,3-bis(2-Acetyl-3-hydroxyphenoxy)-2-propanol
mp: 204 °C to 206 °C. C19H2001 = 360.4 g/mol (16150-44-0)
Berberine chloride used in l¼uid chromatography complies with the Analytical reagent grade of commerce.
following additional test. Bisbenzimide 4-[5-[5-( 4-Methylpiperazin-1-yl)
Assay. Liquid chromatography (2.2.29) as prescribed in the benzimidazol-2-yl]benzimidazol-2-yl]phenol trihydrochloride
monograph Goldenseal rhizome (1831). pentahydrate; C 25 H 27 Cl3N 6O,5H 2 O = 624 (23491-44-3)
Content: minimum 95 per cent, calculated by the Bisbenzimide Stock Solution
normalisation procedure. Dissolve 5 mg of bisbenzimide R in water R and dilute to
Bergapten 5-Methoxypsoralen; C 12H 8 O 4 = 216.2 100 mL with the same solvent.
(484-20-8) Storage: in the dark.
Colourless crystals, practically insoluble in water, sparingly Bisbenzimide Working Solution
soluble in ethanol (96 per cent) and slightly soluble in glacial
Immediately before use, dilute 100 µL of bisbenzimide stock
acetic acid. solution R to 100 mL with phosphate buffered saline pH 7. 4 R.
mp: about 188 °C.
Bis(diphenylmethyl) Ether C 26 H 22 O = 350.5 (574-42-5)
Beta-cyclodextrin Derivative of Silica Gel for Chiral [Oxybis(methanetriyl)]tetrakisbenzene. 1,1 ',1 '', 1'' '-(Oxy-
Separation methylidyne) tetrakis benzene.
Substituted beta-cyclodextrin coated on a very finely divided
Bismuth Nitrate Pentahydrate Bi(NO 3h,5H2 O = 485.1
silica gel for chiral separation. (10035-06-0)
~-Cyclodextrin Derivative of Silica Gel for Chiral mp: about 30 °C.
Separation See Beta-cyclodextrin derivative of silica gel for
chiral separation R. Bismuth Oxycarbonate Bismuth subcarbonate;
(5892-10-4)
Betamethasone 9a-Fluoro-11 ~' 17a,21-trihydroxy-16~-
methylpregna-1,4-diene-3,20-dione; C 22 H 29 FO 5 = 392.5 A basic salt corresponding approximately to the formula
(378-44-9)
(BiO) 2 CO 3 ,V2H2O, containing the equivalent of about 91 %
of Bi 2 O 3 •
mp: about 236°.
Bismuth Oxynitrate Bismuth subnitrate; 4BiNO 3 (OH) 2 ,
General reagent grade of commerce.
BiO(OH) = 1462 (1304-85-4)
V-A40 Appendix I A 2023

White or almost white powder, practically insoluble in water. Boric Acid (10043-35-3)
Bismuth Oxynitrate Rl Bismuth subnitrate Rl See Boric acid (0001).
Content: 71.5 per cent to 74.0 per cent of bismuth (Bi), and Boric Acid Solution
14.5 per cent to 16.5 per cent of nitrate, calculated as Dissolve 5 g of boric acid in a mixture of 20 mL of water and
nitrogen pentoxide (N2 O 5). 20 mL of absolute ethanol and dilute to 250 mL with absolute
Bismuth Oxynitrate Solution ethanol.
Bismuth subnitrate solution Boric Acid Solution, Cold Saturated
Dissolve 5 g of bismuth subnitrate Rl in a mixture of 8.4 mL To 3 g of boric acid R add 50 mL of water Rand shake for
of nitric acid R and 50 mL of water R and dilute to 250 mL 10 min. Place the solution for 2 h in the refrigerator.
with water R. Filter if necessary. o-Borneol Borneo!; C 10H 18 O = 154.3 (507-70-0)
Acidity. To 10 mL add 0.05 mL of methyl orange solution R.
Colourless crystals, readily sublimes, practically insoluble in
5.0 mL to 6.25 mL of 1 M sodium hydroxide is required to
water, freely soluble in ethanol (96 per cent) and in light
change the colour of the indicator. petroleum.
Bismuth Subcarbonate See Bismuth oxycarbonate. mp: about 208 °C.
N,0-Bis(trimethylsilyl)acetamide C 8H 21 NOSi2 = 203.4 Chromatography. Thin-layer chromatography (2.2.27), using
(10416-59-8)
silica gel G R as the coating substance. Apply to the plate
Colourless liquid. 10 µL of a 1 g/L solution in toluene R. Develop over a path
dig: about 0.83. of 10 cm using chloroform R. Allow the plate to dry in air,
Bis(trimethylsilyl)trifluoroacetamide spray with anisaldehyde solution R, using 10 mL for a plate
N,O-bis(Trimethylsilyl)trifluoroacetamide; 200 mm square, and heat at 100-105 °C for 10 min.
CsH1sF3NOSi2 = 257.4 (25561-30-2) The chromatogram obtained shows only one principal spot.
Colourless liquid. o-Bornyl Acetate Bornyl acetate; C 12H 20 O 2 = 196.3
(5655-61-8)
dfg: about 0.97.
Colourless crystals or a colourless liquid, very slightly soluble
ngi: about 1.38.
in water, soluble in ethanol (96 per cent).
bp12mm: about 40 °C
mp: about 28 °C.
Biuret C 2H 5N 3 O 2 = 103.1 (108-19-0)
Chromatography. Thin-layer chromatography (2.2.27), using
White or almost white crystals, hygroscopic, soluble in water, silica gel G R as the coating substance. Apply to the plate
sparingly soluble in ethanol (96 per cent). 10 µL of a 2 g/L solution in toluene R. Develop over a path
mp: 188 °C to 190 °C, with decomposition. of 10 cm using chloroform R. Allow the plate to dry in air,
Storage: in an airtight container. spray with anisaldehyde solution R, using 10 mL for a plate
Biuret Reagent 200 mm square, and heat at 100-105 °C for 10 min.
The chromatogram obtained shows only one principal spot.
Dissolve 1.5 g of copper sulfate pentahydrate Rand 6.0 g of
sodium potassium tartrate R in 500 mL of water R. Boron Trichloride BCh = 117.2 (10294-34-5)
Add 300 mL of a carbonate-free 100 g/L solution of sodium Colourless gas. Reacts violently with water. Available as
hydroxide R, dilute to 1000 mL with the same solution and solutions in suitable solvents (2-chloroethanol, methylene
m!X. chloride, hexane, heptane, methanol).
Blocking Solution ngi: about 1.420.
A 10 per cent VIV solution of acetic acid R. bp: about 12.6 °C.
Blue Dextran 2000 (9049-32-5) Caution: toxic and corrosive.
Prepared from dextran having an average relative molecular Boron Trichloride-Methanol Solution
mass of 2 x 106 by introduction of a polycyclic A 12 per cent mlm solution of boron trichloride R in
chromophore that colours the substance blue. The degree of methanol R.
substitution is 0.017. Storage: protected from light at -20 °C, preferably in sealed
It is freeze-dried and dissolves rapidly and completely in tubes.
water and aqueous saline solutions. Boron Trifluoride BF3 = 67.8 (7637-07-2)
Absorbance (2.2.25). A 1 g/L solution in a phosphate buffer
Colourless gas.
solution pH 7. 0 R shows an absorption maximum at 280 nm.
Boron Trifluoride Solution Boron trifluoride-
Boldine 1, 10-Dimethoxy-6ao:-aporphine-2, 9-diol; methanol solution
C19H 21 NO 4 = 327.3 (476-70-0)
A 140 g/L solution of boron trifluoride R in methanol R.
White or almost white crystalline powder, very slightly
soluble in water, soluble in ethanol (96 per cent) and in
Bovine Coagulation Factor Xa (9002-05-5)
dilute solutions of acids. An enzyme which converts prothrombin to thrombin.
The semi-purified preparation is obtained from liquid bovine
[a]~: about+ 127, determined on a 1 g/L solution in
plasma and it may be prepared by activation of the zymogen
anhydrous ethanol R.
factor X with a suitable activator such as Russell's viper
mp: about 163 °C.
venom.
Borate Solution Storage: freeze-dried preparation at -20 °C and frozen
Dissolve 9.55 g of disodium tetraborate R in suljuric acid R, solution at a temperature lower than -20 °C.
heating on a water-bath, and dilute to 1 L with the same
acid.
2023 Appendix I A V-A41

Bovine Factor Xa Solution Bromobenzene C 6H 5 Br = 157.0 (108-86-1)

Reconstitute as directed by the manufacturer and dilute with ni0 : about 1.56.

tris(hydroxymethyl)aminomethane sodium chloride buffer solution bp: about 156°.

pH 7.4 R.
General reagent grade of commerce.
Any change in the absorbance of the solution, measured at
A colourless to pale yellow liquid; weight per mL, about
405 nm (2.2.25) against tris(hydroxymethyl)aminomethane
1.50 g.
sodium chloride buffer solution pH 7. 4 R and from which the
blank absorbance has been substracted, is not more than Bromocresol Green 3',3 11 ,5',5 11 -Tetrabromo-m-cresol-
0.20 per minute. sulfonphthalein; 4,4 '-(3H-2, l-Benzoxathiol-3-ylidene)bis(2,6-
dibromo-3-methylphenol)-S,S-dioxide; C 21 H 14Br4 O 5 S = 698
Bovine Factor Xa Solution Rl
(76-60-8)
Reconstitute as directed by the manufacturer and dilute to Brownish-white powder, slightly soluble in water, soluble in
1.4 nkat/mL with tris(hydroxymethyl)aminomethane-EDTA
ethanol (96 per cent) and in dilute solutions of alkali
buffer solution pH 8. 4 R. hydroxides.
Bovine Factor Xa Solution R2
Bromocresol Green-Methyl Red Solution
Reconstitute as directed by the manufacturer and dilute with
Dissolve 0.15 g of bromocresol green Rand 0.1 g of methyl
tris(hydroxymethyl) aminomethane-EDTA buffer solution
red R in 180 mL of anhydrous ethanol Rand dilute to 200 mL
pH 8.4 Rl to obtain a solution that gives an absorbance
with water R.
between 0.65 and 1.25 at 405 nm when determining the
blank amidolytic activity according to general chapter 2. 7.5 Bromocresol Green Solution
using the end-point method. Dissolve 50 mg of bromocresol green R in 0.72 mL of 0.1 M
Brilliant Blue (6104-59-2) sodium hydroxide and 20 mL of ethanol (96 per cent) R and
dilute to 100 mL with water R.
See acid blue 83 R.
Test for sensitivity. To 0.2 mL of the bromocresol green
Brilliant Green CI 42040; basic green 1;
solution add 100 mL of carbon dioxide-free water R.
C27H3 4 N 2O 4 S = 482.6 (633-03-4')
The solution is blue. Not more than 0.2 mL of 0.02 M
Technical grade of commerce. hydrochloric acid is required to change the colour to green.
Small, glistening golden crystals. Colour change: pH 3.6 (yellow) to pH 5.2 (blue).
Bromelains (37189-34-7) Bromocresol Purple 3',3"-Dibromo-o-
Concentrate of proteolytic enzymes derived from Ananas cresolsulfonphthalein; 4,4' -(3H-2, 1-Benzoxathiol-3-ylidene)
comosus Merr. bis(2-bromo-6-methylphenol)-S,S-dioxide;
Dull-yellow powder. C21H15Br2OsS = 540.2 (J 15-40-2)
Activity. 1 g liberates about 1.2 g of amino-nitrogen from a Pinkish powder, practically insoluble in water, soluble in
solution of gelatin R in 20 min at 45 °C and pH 4.5. ethanol (96 per cent) and in dilute solutions of alkali
Bromelains Solution hydroxides.
A 10 g/L solution of bromelains R in a mixture of 1 volume of Bromocresol Purple Solution
phosphate buffer solution pH 5.5 Rand 9 volumes of a 9 g/L Dissolve 50 mg of bromocresol purple R in 0.92 mL of 0.1 M
solution of sodium chloride R. sodium hydroxide and 20 mL of ethanol (96 per cent) R and
Bromine Br2 = 159.8 (7726-95-6) dilute to 100 mL with water R.
Brownish-red fuming liquid, slightly soluble in water, soluble Test for sensitivity. To 0.2 mL of the bromocresol purple
in ethanol (96 per cent). solution add 100 mL of carbon dioxide-free water R and
0.05 mL of 0.02 M sodium hydroxide. The solution is bluish-
dfg: about 3.1. violet. Not more than 0.2 mL of 0.02 M hydrochloric acid is
To prepare 0.05M bromine dissolve 3 g of potassium required to change the colour to yellow.
bromate and 5 g of potassium bromide in sufficient water to Colour change: pH 5.2 (yellow) to pH 6.8 (bluish-violet).
produce 1000 mL. Weaker solutions should be prepared
using proportionately lesser amounts of reagents or by 5-Bromo-2' -deoxyuridine 5-Bromo-1-(2-deoxy-~-d-
appropriate dilution. erythro-pentofuranosyl)-1 H,3H-pyrimidine-2,4-dione;
C 9 H 11 BrN 2O 5 = 307.1 (59-14-3)
Bromine Solution
mp: about 194 °C.
Dissolve 30 g of bromine R and 30 g of potassium bromide R in
water R and dilute to 100 mL with the same solvent. Chromatography. Thin-layer chromatography (2.2.27) as
prescribed in the monograph Idoxuridine (0669): apply 5 µL
Bromine Solution, Acetic of a 0.25 g/L solution; the chromatogram shows only one
Dissolve 100 g of potassium acetate in glacial acetic acid and principal spot.
add 4 mL of bromine and sufficient glacial acetic acid to Bromomethoxynaphthalene 2-Bromo-6-
produce 1000 mL. methoxynaphthalene; C 11 H 9 BrO = 237.1 (5111-65-9)
Bromine Water mp: about 109 'C.
Shake 3 mL of bromine R with 100 mL of water R to Bromophenol Blue 3',3",5',5"-
saturation. Tetrabromophenolsulfonphthalein; 4,4 '-(3H-2, 1-
Storage: over an excess of bromine R, protected from light. Benzoxathiol-3-ylidene) bis(2, 6-dibromophenol) S,S-dioxide;
Bromine Water Rl C19HJOBr4 O 5 S = 670 (115-39-9)
Shake 0.5 mL of bromine R with 100 mL of water R. Light orange-yellow powder, very slightly soluble in water,
Storage: protected from light; use within 1 week. slightly soluble in ethanol (96 per cent), freely soluble in
solutions of alkali hydroxides.
V-A42 Appendix I A 2023

Bromophenol Blue Solution Colourless crystals, slightly soluble in water, freely soluble in
Dissolve O.1 g of bromophenol blue R in I. 5 mL of 0.1 M ethanol (96 per cent).
sodium hydroxide and 20 mL of ethanol (96 per cent) R and mp: about 178 °C.
dilute to 100 mL with water R. Butanal Butyraldehyde; C 4H 8O = 72.1 (123-72-8)
Test for sensitivity. To 0.05 mL of the bromophenol blue dig: 0.806.
solution add 20 mL of carbon dioxide-free water R and
ngi: 1.380.
0.05 mL of 0.1 M hydrochloric acid. The solution is yellow.
Not more than 0.1 mL of 0.1 M sodium hydroxide is required bp: 75 °C.
to change the colour to bluish-violet. i-Butane Isobutane; 2-Methylpropane; C 4 H 10 = 58.12
(75-28-5)
Colour change: pH 2.8 (yellow) to pH 4.4 (bluish-violet).
Content: minimum 99.0 per cent V/V.
Bromophenol Blue Solution Rl
Dissolve 50 mg of bromophenol blue R with gentle heating in
N-Butane Butane; C 4 H 10 = 58.12 (106-97-8)
3.73 mL of 0.02 M sodium hydroxide and dilute to 100 mL Content: minimum 99.0 per cent V/V.
with water R. Butane-1,3-diol C 4 H 10O 2 = 90.1 (6290-03-5)
Bromophenol Blue Solution R2 nt0 : about 1.440.
Dissolve with heating 0.2 g of bromophenol blue R in 3 mL of bp: about 203°.
0.1 M sodium hydroxide and I O mL of ethanol (96 per cent) R. General reagent grade of commerce.
After solution is effected, allow to cool and dilute to 100 mL
Butane-1,4-diol HO(CH 2 ) 4 OH = 90.12 (110-63-4)
with ethanol (96 per cent) R.
Butan-1-ol 1-Butanol; N-Butanol; N-Butyl alcohol;
Bromophos C 8 H 8 BrC12 O 3 PS = 366.0 (2104-96-3) Butanol; C 4 H 10O = 74.1 (71-36-3)
A suitable certified reference solution ( 10 nglµL in iso- Clear, colourless liquid, miscible with ethanol (96 per cent).
octane) may be used.
Bromophos-ethyl C 10H 12 BrC12 O 3 PS = 394.0 (4824-78-6)
d?g: about 0.81.
bp: 116 °C to 119 °C.
A suitable certified reference solution (10 nglµL in iso-
octane) may be used. Butan-2-ol sec-Butyl alcohol; 2-butanol; C 4 H 10 O = 74.12
(78-92-2)
Bromothymol Blue 3 ',3 "-Dibromothymolsulfonphthalein;
4,4 '-(3H-2, 1-Benzoxathiol-3-ylidene)bis(2-bromo-6- d?g: about 0.81.
isopropyl-3-methylphenol) S,S-dioxide; C 27 H 28Br2 O 5 S = 624 bp: about 99°.
(76-59-5) Analytical reagent grade of commerce.
Reddish-pink or brownish powder, practically insoluble in A colourless liquid.
water, soluble in ethanol (96 per cent) and in dilute solutions Butan-2-ol Rl sec-Butyl alcohol; 2-Butanol Rl;
of alkali hydroxides. C4H 10O = 74.1 (78-92-2)
Bromothymol Blue Solution Rl Content: minimum 99.0 per cent.
Dissolve 50 mg of bromothymol blue R in a mixture of 4 mL Clear, colourless liquid, soluble in water, miscible with
of 0. 02 M sodium hydroxide and 20 mL of ethanol ethanol (96 per cent).
(96 per cent) R and dilute to 100 mL with water R.
dig: about 0.81.
Test for sensitivity. To 0.3 mL of bromothymol blue solution Rl
Distillatwn range (2.2.11). Not less than 95 per cent distils
add 100 mL of carbon dioxide-free water R. The solution is
between 99 °C and 100 °C.
yellow. Not more than 0.1 mL of 0.02 M sodium hydroxide is
required to change the colour to blue. Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Isopropyl alcohol (0970).
Colour change: pH 5.8 (yellow) to pH 7.4 (blue).
Butan-2-one Methyl ethyl ketone; Ethyl methyl ketone;
Bromothymol Blue Solution R2 (78-93-3)
A 10 glL solution of bromothymol blue R in
See methyl ethyl ketone R.
dimethylformamide R.
Butyl Acetate C 6 H 12 0 2 = 116.2 (123-86-4)
Bromothymol Blue Solution R3
Clear, colourless liquid, flammable, slightly soluble in water,
Warm 0.1 g of bromothymol blue R with 3.2 mL of 0.05 M miscible with ethanol (96 per cent).
sodium hydroxide and 5 mL of ethanol (90 per cent V/V) R.
After solution is effected, dilute to 250 mL with ethanol dig: about 0.88.
(90 per cent Vlv; R. nt0 : about 1.395.
Bromothymol Blue Solution R4 Distillatwn range (2.2.11). Not less than 95 per cent distils
Dissolve 100 mg of bromothymol blue R in a mixture of equal between 123 °C and 126 °C.
volumes of ethanol (96 per cent) R and water R and dilute to Butyl Acetate Rl
100 mL with the same mixture of solvents. Filter if Content: minimum 99.5 per cent, determined by gas
necessary. chromatography.
BRP Indicator Solution Clear, colourless liquid, flammable, slightly soluble in water,
Dissolve 0.1 g of bromothymol blue R, 20 mg of methyl red R miscible with ethanol (96 per cent).
and 0.2 g of phenolphthalein R in ethanol (96 per cent) Rand dig: about 0.883.
dilute to 100 mL with the same solvent. Filter. ngi: about 1.395.
Brucine 10, 11-Dimethoxystrychnine; Butanol: maximum 0.2 per cent, determined by gas
C23H 26N 2 O 4 = 394.5 (357-57-3) chromatography.
2023 Appendix I A V-A43

n-Butylfom1ate: maximum 0.1 per cent, determined by gas bp: about 163 °C.
chromatography. Butyrolactone Dihydro-2(3H)-furanone; y-Butyrolactone;
n-Butyl propionate: maximum 0.1 per cent, determined by gas C 4 H 6 O 2 = 86.1 (96-48-0)
chromatography. Oily liquid, miscible with water, soluble in methanol.
Water: maximum 0.1 per cent. n~: about 1.435.
N-Butylamine 1-Butanamine; Butylamine; bp: about 204 °C.
C 4 HllN = 73.1 (109-73-9) Cadmium Cd = 112.4 (7440-43-9)
Distil and use within one month. Silvery-white, lustrous metal, practically insoluble in water,
Colourless liquid, miscible with water, with ethanol freely soluble in nitric acid and in hot hydrochloric acid.
(96 per cent). Cadmium Acetate C4H6 O 4 Cd,2H 2 O = 266.5
nt 0 : about 1.401.
(5743-04-4)
bp: about 78 °C. Analytical reagent grade of commerce.
tert-Butylamine (75-64-9) Cadmium and Ninhydrin Solution
See 1, 1-dimethylethylamine R. Dissolve 50 mg of cadmium acetate in a mixture of 5 mL of
4-(Butylamino)benzoic Acid C 11 H 15NO 2 = 193.2 water and 1 mL of glacial acetic acid and dilute with butan-2-
(4740-24-3) one to 50 mL. Immediately before use add and dissolve
White or almost white powder. sufficient ninhydrin to produce a solution containing
Content: 96.5 per cent to 103.5 per cent.
0.2% w/v.
Butylated Hydroxyanisole 2-tert-Butyl-4-methoxyphenol; Cadmium Iodide CdI 2 = 366.2 (7790-80-9)
C1 1H1 6 O 2 = 180.2 (25013-16-5) Analytical reagent grade of commerce.
mp: about 61°. Cadmium Iodide Solution
General reagent grade of commerce. A 5.0% w/v solution of cadmium iodide.
A white or almost white, crystalline powder. Cadmium Nitrate Tetrahydrate
Butylated Hydroxytoluene (128-37-0) Cd(NO 3 )z,4H 2 O = 308.5 (10022-68-1)
See Butylhydroxytoluene R. Hygroscopic orthorhombic crystals, very soluble in water,
soluble in acetone and in ethanol (96 per cent).
Butylboronic Acid C 4H 11 BO2 = 101.9 (4426-47-5)
mp: about 59.5 °C.
Content: minimum 98 per cent.
Caesium Chloride CsCI = 168.4 (7647-17-8)
mp: 90 °C to 92 °C.
White or almost white powder, very soluble in water, freely
Butyl Chloride See 1-Chlorobutane. soluble in methanol, practically insoluble in acetone.
tert-Butylhydroperoxide Caffeic Acid 3,4-Dihydroxycinnamic acid;
1,1-Dimethylethylhydroperoxide; C 4H 10O 2 = 90.1 (75-91-2) C 9 H 8 O 4 = 180.2 (331-39-5)
Flammable liquid, soluble in organic solvents. White or almost white crystals or plates, freely soluble in hot
dig: 0.898. water and in ethanol (96 per cent), sparingly soluble in cold
ngi: 1.401. water.
bp: 35 °C. Absorbance (2.2.25). A freshly prepared solution at pH 7.6
Butyl 4-hydroxybenzoate (94-26-8) shows 2 absorption maxima at about 288 nm and about
See Butyl parahydroxybenzoate R. 313 nm.
Butylhydroxytoluene (128-37-0) Caffeine (58-08-2)
See Caffeine (0267).
See Butylhydroxytoluene (0581).
2-Butyloctanol (2.3')-2-Butyloctan-1-ol; C 12 H 26 O = 186.3 Calciferol Ergocalciferol; vitamin D 2 ; C 28H 44 O = 396.7
(50-14-6)
(3913-02-8)
Butyl Parahydroxybenzoate (94-26-8) [ci]~: about +105 (4% w/v in ethanol).
mp: about 117°.
See Butyl parahydroxybenzoate (0881).
tert-Butyl Methyl Ether (1634-04-4) Crystalline reagent grade of commerce.
Colourless crystals or a white, crystalline powder.
See 1, 1-dimethylethyl methyl ether R.
Butyl Methacrylate Butyl 2-methylpropenoate; Calcium Acetate Calcium diacetate; See Calcium acetate
(2128); C 4 H 6 CaO 4 = 158.2 (62-54-4)
CsH14O 2 = 142.2 (97-88-1)
Clear, colourless solution. Calcium Acetate, Dried C 4 H 6 O4 Ca = 158.2 (62-54-4)
General reagent grade of commerce.
dI 0 : about 0.894.
Calcium bis(formyl homotaurine) Calcium bis(3-
nt0 : about 1.424.
formamidopropane-1-sulfonate); C 8 H 16 CaN2 O 8 S2 = 372.4
bp: about 163 °C.
White or almost white powder.
Butyric Acid N-Butyric acid; C 4H 8 O 2 = 88.1 (107-92-6)
Content: minimum 80.0 per cent.
Content: minimum 99.0 per cent.
Calcium Carbonate (471-34-1)
Oily liquid, miscible with water and with ethanol
See Calcium carbonate (0014).
(96 per cent).
Calcium Carbonate Rl
dig: about 0.96.
Complies with the requirements prescribed for calcium
ngi: about 1.398.
carbonate R with the following additional requirement.
V-A44 Appendix I A 2023

Chlorides (2.4.4): maximum 50 ppm. Calconcarboxylic Acid Triturate

Calcium Chloride Calcium Chloride Dihydrate; Calconecarboxylic acid triturate
(10035-04-8) Mix 1 part of calconecarboxylic acid R with 99 parts of sodium
See Calcium chloride (0015). chloride R.
Calcium Chloride, Anhydrous CaC12 = 111.0 Test for sensitivity. Dissolve 50 mg of calconecarboxylic acid
(10043-52-4) triturate in a mixture of 2 mL of strong sodium hydroxide
Content: minimum 98.0 per cent (dried substance). solution R and I 00 mL of water R. The solution is blue but
becomes violet on addition of 1 mL of a 10 g/L solution of
White or almost white granules, deliquescent, very soluble in magnesium sulfate Rand 0.1 mL of a 1.5 g/L solution of
water, freely soluble in ethanol (96 per cent) and in calcium chloride R and turns pure blue on addition of
methanol. 0.15 mL of 0.01 M sodium edetate.
Loss on drying (2.2.32): maximum 5.0 per cent, determined Campesterol C 28 H 48 O = 400.7 (474-62-4)
by drying in an oven at 200 ± 10 °C.
(24R)-Ergost-5-en-3~-ol.
Storage: in an airtight container, protected from moisture.
White or almost white crystalline solid.
Calcium Chloride Rl Calcium chloride tetrahydrate;
CaCl2,4H2O = 183.1 Camphene 2,2-Dimethyl-3-methylenebicyclo [2.2.1]
heptane; C 10 H 16 = 136.2 (79-92-5)
Iron: maximum 0.05 ppm.
Camphene used in gas chromatography comphes with the following
Calcium Chloride Solution additional test.
A 73.5 g/L solution of calcium chloride R. Assay. Gas chromatography (2.2.28) as prescribed in the
Calcium Chloride Solution, 0.01M monograph Rosemary Oil (1846).
Dissolve 0.147 g of calcium chloride R in water Rand dilute to Content: minimum 90 per cent, calculated by the
100.0 mL with the same solvent. normalisation procedure.
Calcium Chloride Solution, 0.02M Camphor (76-22-2)
Dissolve 2.94 g of calcium chloride R in 900 mL of water R, See Camphor, racemic (0655).
adjust to pH 6.0 to 6.2 and dilute to 1000.0 mL with Camphor used in gas chromatography compli,es with the following
water R. additional test.
Storage: at 2 °C to 8 °C.
Assay. Gas chromatography (2.2.28) as prescribed in the
Calcium Chloride Solution, 0.025M monograph Lavender oil (1338).
Dissolve 0.368 g of calcium chloride R in water Rand dilute to Test solutwn. A l O g/L solution of the substance to be
100.0 mL with the same solvent. examined in hexane R.
Calcium Hydroxide Calcium dihydroxide; Content: minimum 95.0 per cent, calculated by the
Ca(OH) 2 = 7 4.1 (1305-62-0) normalisation procedure.
White or almost white powder, almost completely soluble in (1S)-(+)-10-Camphorsulfonic Acid (lS)-(+)-10-
600 parts of water. Camphorsulphonic Acid; C 10 H 16O 4 S = 232.3 (3144-16-9)
Calcium Hydroxide Solution Prismatic crystals, hygroscopic, soluble in water.
A freshly prepared saturated solution. Content: minimum 99.0 per cent of (lS)-(+)-10-
Calcium Lactate Calcium lactate pentahydrate; camphorsulfonic acid.
(41372-22-9) [()(]~: + 20 ± 1, determined on a 43 g/L solution.
See Calcium lactate pentahydrate (0468). mp: about 194 °C, with decomposition.
Calcium Phosphate Monobasic Monohydrate Calcium M (2.2.41): 10.2 x 10 3 determined at 290.5 nm on a
tetrahydrogen bisphosphate monohydrate; Phosphoric acid 1.0 g/L solution.
calcium salt (2:1) monohydrate; CaH4OsP2,H 2O = 252.1 Capric Acid Decanoic acid; C 10H 20O 2 = 172.3 (334-48-5)
(10031-30-8)
Crystalline solid, very slightly soluble in water, soluble in
White or almost white, crystalline powder, soluble in water. anhydrous etl!anol.
Calcium Sulfate Calcium sulphate hemihydrate; Calcium bp; about 270 °C.
sulphate; Plaster of Paris; CaSO4 ,1/2 H2O = 145.1
(10034-76-1) mp: about 31.4 °C.
Capric acid used in the assay of total fatty acids in Saw palmetto
White or almost white powder, soluble in about 1500 parts
of water, practically insoluble in ethanol (96 per cent). When fruit (1848) complies with the following additional test.
mixed with half its mass of water it rapidly solidifies to a Assay. Gas chromatography (2.2.28) as prescribed in the
hard and porous mass. monograph Saw palmetto fruit (1848).
Calcium Sulfate Solution Calcium sulphate solution Content: minimum 98 per cent, calculated by the
Shake 5 g of calcium sulfate R with I 00 mL of water R for 1 h normalisation procedure.
and filter. Caproic Acid Hexanoic acid; C6H12O 2 = 116.2
(142-62-1)
Calconcarboxylic Acid Panon and Reeder's reagent;
2-Hydroxy-1-(2-hydroxy-4-sulfo- l-naphthylazo)naphthalene- Oily liquid, sparingly soluble in water.
3-carboxylic acid; Calconecarboxylic acid; dJ 0 : about 0.926.
C21 H 14N 2O7S = 438.4 (3737-95-9) ngi: about 1.417.
Brownish-black powder, slightly soluble in water, very slightly bp: about 205 °C.
soluble in acetone and in ethanol (96 per cent), sparingly Caproic acid used in the assay of total fatty acids in Saw palmetto
soluble in dilute solutions of sodium hydroxide. fruit (1848) complies with the following additional test.
2023 Appendix I A V-A45

Assay. Gas chromatography (2.2.28) as prescribed in the bp: 76 °C to 77 °C.

monograph Saw palmetto fruit (1848). Carbophenothion 0, O-Diethyl S-[[ (4-chlorophenyl)thio]
Content: minimum 98 per cent, calculated by the methyl]-phosphorodithioate; C 11 H 16ClO 2 PS 3 = 342.9
normalisation procedure. (786-19-6)
e-Caprolactam Hexane-6-lactam; C 6 H 11 NO = 113.2 Yellowish liquid, practically insoluble in water, miscible with
(105-60-2) organic solvents.
Hygroscopic flakes, freely soluble in water, in anhydrous df 5 : about 1.27.
ethanol and in methanol. For the monograph Wool Fat (0134), a suitable certified
mp: about 70 °C. reference solution (10 ng/µL in iso-octane) may be used.
Capryl Alcohol Decan-1-ol; Decanol; Capric alcohol; 5-Carboxyuracil 2,4-Dioxo-1,2,3,4-tetrahydropyrimidine-
See Decanol R. 5-carboxylic acid; Uracil-5-carboxylic acid;
Capsaicin (E)-N-[ (4-Hydroxy-3-methoxyphenyl)methyl]-8- C 5H 4 N 2 O 4 = 156.1 (23945-44-0)
methylnon-6-enamide; C 18H 27NO 3 = 305.4 (404-86-4) mp: about 283 °C.
White or almost white, crystalline powder, practically Car-3-ene 3, 7, 7-Trimethylbicyclo[4.1. O]hept-3-ene;
insoluble in water, freely soluble in anhydrous ethanol. 4,7,7-Trimethyl-3-norcarene; C 10H 16 = 136.2 (498-15-7)
mp: about 65 °C. Liquid with a pungent odour, slightly soluble in water,
Capsaicin used in the assay in Capsicum (1859) complies with soluble in organic solvents.
the following additwnal test. dig: about 0.864.
Assay. Liquid chromatography (2.2.29) as prescribed in the nt0 : 1.473 to 1.474.

monograph Capsicum (1859). [a]~:+ 15 to+ 17.

Content: minimum 95.0 per cent, calculated by the bp: 170 °C to 172 °C.
normalisation procedure. Car-3-ene used in gas chromatography complies with the following
Carbazole Dibenzopyrrole; C 12 H 9N = 167.2 (86-74-8) additional test.
Crystals, practically insoluble in water, freely soluble in Assay. Gas chromatography (2.2.28) as prescribed in the
acetone, slightly soluble in anhydrous ethanol. monograph Nutmeg oil (1552).
mp: about 245 °C. Content: minimum 95.0 per cent, calculated by the
Carbomer (9007-20-9) normalisation procedure.
A cross-linked polymer of acrylic acid; it contains a large Carminic Acid 7-rx-o-Glucopyranosyl-3,5,6,8-
proportion (56 per cent to 68 per cent) of carboxylic acid tetrahydroxy-1-methyl-9, 1O-dioxo-9, 10-dihydroanthracene-2-
(CO 2 H) groups after drying at 80 °C for 1 h. Average carboxylic acid; C 22 H 20O 13 = 492.4 (1260-17-9)
relative molecular mass about 3 x 106 • Dark red powder, very slightly soluble in water, soluble in
pH (2.2.3): about 3 for a 10 g/L suspension. dimethyl sulfoxide, very slightly soluble in ethanol
Carbon Dioxide (124-38-9) (96 per cent).
See Carbon dwxide (0375). Carob Bean Gum Locust bean gum
Carbon Dioxide Rl CO 2 = 44.01 (124-38-9) The ground endosperm of the fruit kernels of Ceratonia
siliqua L. Taub.
Content: minimum 99.995 per cent V/V.
Carbon monoxide: less than 5 ppm.
White or almost white powder containing 70 per cent to
80 per cent of a water-soluble gum consisting mainly of
Oxygen: less than 25 ppm. galactomannoglycone.
Nitric oxide: less than 1 ppm. Carvacrol 5-Isopropyl-2-methylphenol; C 10H 14 O = 150.2
Carbon Dioxide R2 CO 2 = 44.01 (124-38-9) (499-75-2)
Content: minimum 99 per cent V/V. Brownish liquid, practically insoluble in water, very soluble in
Carbon Disulfide Carbon disulphide; CS 2 = 76.1 ethanol (96 per cent).
(75-15-0) dig: about 0.975.
Colourless or yellowish, flammable liquid, practically
insoluble in water, miscible with anhydrous ethanol.
nt0 : about 1.523.

bp: about 237 °C.

dfg: about 1.26. Carvacrol used in gas chromatography complies with the following
bp: 46 °C to 47 °C. additional test.
Carbon for Chromatography, Graphitised Assay. Gas chromatography (2.2.28) as prescribed in the
Porous spherical carbon particles comprised of flat sheets of monograph Peppermint oil (0405).
hexagonally arranged carbon atoms. Test solutwn. Dissolve 0.1 gin about 10 mL of acetone R.
Carbon Monoxide CO = 28.01 (630-08-0) Content: minimum 95.0 per cent, calculated by the
Content: minimum 99.97 per cent VIV. normalisation procedure.
Carbon Monoxide Rl CO= 28.01 (630-08-0) Carveol p-Mentha-1 (6 ),8-dien-2-ol; 2-Methyl-5-( l-
Content: minimum 99 per cent V/V. methylethenyl)cyclohex-2-enol; C 10H 160 = 152.2 (99-48-9)
Carbon Tetrachloride Tetrachloromethane; The substance contains a variable content of trans- and cis-
CC1 4 = 153.8 (56-23-5) carveol.
Clear, colourless liquid, practically insoluble in water, Carveol used in gas chromatography complies with the following
miscible with ethanol (96 per cent). additional test.
dig: 1.595 to 1.598.
V-A46 Appendix I A 2023

Assay. Gas chromatography (2.2.28) as prescribed in the test Content: minimum 99.0 per cent, calculated by the
for chromatographic profile in the monograph Caraway normalisation procedure.
oil (1817). Casein (9000-71-9)
Content: minimum 97 per cent, calculated by the Mixture of related phosphoproteins obtained from milk.
normalisation procedure. White or almost white, amorphous powder or granules, very
Carvone (+ )-p-Mentha-6,8-dien-2-one; (5S)-2-Methyl- slightly soluble in water and in non-polar organic solvents.
5-( l-methylethenyl)-cyclohex-2-enone; C 10H 14 O = 150.2 It dissolves in concentrated hydrochloric acid giving a pale-
(2244-16-8) violet solution. It forms salts with acids and bases.
Liquid, practically insoluble in water, miscible with ethanol Its isoelectric point is at about pH 4. 7. Alkaline solutions are
(96 per cent). laevorotatory.
d?g: about 0.965 Casein Substrate, Concentrated
ni7: about 1.500. Suspend a quantity of casein EPBRP equivalent to 2.5 g in
[ix]~: about+ 61. 5 mL of water, add 18 mL of 0 .1 M sodium hydroxide and stir
bp: about 230 °C. for 1 minute. Add 60 mL of water and stir with a magnetic
stirrer until the solution is practically clear. Adjust the pH of
Carvone used in gas chromatography complies with the following the solution to 8.0 with either 0.lM sodium hydroxide or
additional test. 0.lM hydrochloric acid and add sufficient water to produce
Assay. Gas chromatography (2.2.28) as prescribed in the 100 mL.
monograph Peppennint oil (0405) using the substance to be Use on the day of preparation.
examined as the test solution.
Casticin 5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-3,6,7-
Content: minimum 98.0 per cent, calculated by the trimethoxy-4H-1-benzopyran-4-one; C 19H 18O 8 = 374.3
normalisation procedure. (479-91-4)
Carvone Rl Yellow crystals.
Complies with the requirements prescribed for carvone R Catalpol (laS, 1bS,2S,5aR,6S,6aS)-6-Hydroxy-
with the following additional requirement. 1a-(hydroxymethyl)-la, 1b,2,5a,6,6a-hexahydrooxireno [4,5]
Assay. Gas chromatography (2.2.28) as prescribed in the test cyclopenta[l,2-c]pyran-2-yl ~-D-glucopyranoside;
for chiral purity in the monograph Caraway oil (1817). C1sH22O1o = 362.3 (2415-24-9)
Content: minimum 98 per cent. mp: 203 °C to 205 °C.
(-)-Carvone (-)-p-Mentha-1(6),8-dien-2-one; (5R)-2- Catechin (+ )-(2R,3S)-2-(3,4-Dihydroxyphenyl)-3,4-
Methyl-5-(l-methylethenyl)cyclohex-2-enone; dihydro-2H-chromene-3,5, 7-triol; Catechol; Cianidanol;
C 10H 14O = 150.2 (6485-40-1) Cyanidol; C 15 H 14O6'xH 2O = 290.3 for the anhydrous
Liquid. substance (154-23-4)
d~g: about 0.965. Catechol Pyrocatechol; C 6H 6O 2 = 110.1 (120-80-9)
ni7: about 1.4988. Colourless or slightly yellow crystals, soluble in water, in
[ix];0 : about -62. acetone and in ethanol (96 per cent).
bp: about 230 °C. mp: about 102 °C.
Assay. Gas chromatography (2.2.28) as prescribed in the test Storage: protected from light.
for chiral purity in the monograph Caraway oil (1817). Cathine Hydrochloride (1S,2S)-2-Amino-l-
Content: minimum 99 per cent. phenylpropan-1-ol hydrochloride; Norpseudoephedrine
~-Caryophyllene (E)-(1R,9S)-4, 11, 11-Trimethyl-8- hydrochloride; C 9 H 14CINO = 187. 7 (2153-98-2)
methylenebicyclo[7 .2.0]undec-4-ene; C 15H 24 = 204.4 White or almost white solid.
(87-44-5) Content: minimum 95.0 per cent.
Oily liquid, practically insoluble in water, miscible with Catholyte for Isoelectric Focusing pH 3 to S
ethanol (96 per cent). (0. lM {]-Alanine) Dissolve 8.9 g of alanine in sufficient water
f]-Caryophyllene used in gas chromatography complies with the to produce 1000 mL.
following additumal test. Cation Exchange Resin
Assay. Gas chromatography (2.2.28) as prescribed in the A resin in protonated form with sulfonic acid groups attached
monograph Clove oil (1091). to a polymer lattice consisting of polystyrene cross-linked
Test solutwn. The substance to be examined. with 8 per cent of divinylbenzene. It is available as spherical
Content: minimum 90.0 per cent, calculated by the beads.
normalisation procedure. Cation Exchange Resin Rl
Caryophyllene Oxide (-)-~-Caryophyllene epoxide; A resin in protonated form with sulfonic acid groups attached
( IR,4R,6R, 10S)-4, 12, 12-Trimethyl-9-methylene-5- to a polymer lattice consisting of polystyrene cross-linked
oxatricyclo [8.2. 0. 04'6] dodecane; C 15H 240 = 220.4 with 4 per cent of divinylbenzene. It is available as spherical
(1139-30-6) beads.
Colourless, fine crystals with lumps. Cation Exchange Resin R2
mp: 62 °C to 63 °C. Resin containing strongly acidic propylenesulfonic acid
Caryophyllene oxide used in gas chromatography complies with the groups.
following additwnal test.
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Turpentine oil, Pinus pinaster type (1627).
2023 Appendix I A V-A4 7

Cation Exchange Resin, Strong pressure and extract the dried material with 20 mL of
Strong cation-exchange resin in protonated form with chloroform for 2 hours, shaking the mixture frequently. After
sulfonic acid groups attached to a polymer lattice consisting removal of the solid material by filtration or centrifugation,
of polystyrene cross-linked with divinylbenzene. evaporate the chloroform from the extract under reduced
Cation-exchange Resin, Weak pressure. Suspend the residue in 5 to 10 mL of saline solution.
This stock emulsion may be stored frozen or freeze-dried for
Resin with a carboxylate functionalised latex cross-linked
3 months.
with ethylvinylbenzene-divinylbenzene.
Cerium(m) Nitrate Cerium trinitrate hexahydrate; Cerous
Cation Exchange Resin (Calcium Form), Strong nitrate; Ce(NO 3),,6H 2 O = 434.3 (10294-41-4)
Resin in calcium form with sulfonic acid groups attached to a Colourless or pale yellow, crystalline powder, freely soluble in
polymer lattice consisting of polystyrene cross-linked with water and in ethanol (96 per cent).
8 per cent of divinylbenzene.
Cerium(m) Nitrate Solution
Cation Exchange Resin (Sodium Form), Strong
Dissolve 0.22 g of cerium(III) nitrate in 50 mL of water, add
Resin in sodium form with sulfonic acid groups attached to a 0 .1 mL of nitric acid and 50 mg of hydroxylamine hydrochloride
polymer lattice consisting of polystyrene cross-linked with and dilute to 1000 mL with water.
divinylbenzene.
Cerium(1v) Sulfate Ceric sulfate; Ceric sulphate;
Cationic Resin, Weak Cerium(1v) sulfate tetrahydrate; Cerium sulfate;
Polymethacrylic resin, slightly acid, with carboxyl groups Ce(SO 4)z,4H 2 O = 404.3 (10294-42-5)
present in a protonated form. Yellow or orange-yellow, crystalline powder or crystals, very
Particle size: 75 µm to 160 µm. slightly soluble in water, slowly soluble in dilute acids.
pH limits of use: 5 to 14. Cetostearyl Alcohol (67762-27-0)
Maximum temperature of use: 120 °C. See Cetostearyl akohol (0702).
Cedarwood Oil Cetrimide (8044-71-1)
A grade of commerce for microscopy thickened as necessary See Cetrimide (0378).
in temperate or tropical climates. Cetirizine N-Oxide C 21 H 25 CIN2 O 4 = 404.9
Cellulose Cellulose for chromatography; (9004-34-6) (1076199-80-8)
Fine, white or almost white, homogeneous powder with an Cetyl Alcohol Hexadecan-1-ol; C 16H 34 O = 242.4
average particle size less than 30 µm. (36653-82-4)
Preparation of a thin layer. Suspend 15 g in 100 mL of Content: minimum 95.0 per cent.
water R and homogenise in an electric mixer for 60 s. Coat mp: about 48 °C.
carefully cleaned plates with a layer 0.1 mm thick using a
spreading device. Allow to dry in air. Cetylpyridinium Chloride Monohydrate
1-Hexadecylpyridinium chloride monohydrate; C 21 H 38 CIN,
Cellulose F 254 Cellulose for chromatography F 254 H 2O = 358.0 (6004-24-6)
Microcrystalline cellulose F 254 . A fine, white or almost white, White or almost white powder, freely soluble in water and in
homogeneous powder with an average particle size less than ethanol (96 per cent).
30 µm, containing a fluorescent indicator having an optimal
intensity at 254 nm. mp: 80 °C to 83 °C.
Preparation of a thin layer. Suspend 25 gin 100 mL of Cetylt:rimethylammonium Bromide Cetrimonium
water R and homogenise using an electric mixer for 60 s. bromide; N-Hexadecyl-N,N,N-trimethylammonium bromide;
Coat carefully cleaned plates with a layer 0.1 mm thick using C19H42BrN = 364.5 (57-09-0)
a spreading device. Allow to dry in air. White or almost white, crystalline powder, soluble in water,
Cellulose, Microcrystalline Cellulose for freely soluble in ethanol (96 per cent).
chromatography Rl mp: about 240 °C.
Microcrystalline cellulose. Chamazulene 7-Ethyl-1,4-dimethylazulene;
Preparation of a thin layer. Suspend 25 g in 90 mL of water R C 14H 16 = 184.3 (529-05-5)
and homogenise in an electric mixer for 60 s. Coat carefully Blue liquid, very slightly soluble in water, soluble in ethanol
cleaned plates with a layer 0.1 mm thick using a spreading (96 per cent), miscible with fatty oils, with essential oils and
device. Allow to dry in air. with liquid paraffin, soluble with discolouration in phosphoric
Cephaeline Dihydrochloride (R)-1-[ (2S,3R, 11 bS)-3- acid (85 per cent m/m) and sulfuric acid (50 per cent V/V).
Ethyl-1,3,4,6, 7, l l b-hexahydro-9 ,l 0-dimethoxy-2H- benzo[a] Appearance of solution. 50 mg is soluble in 2.5 mL of
quinolizinylmethyl]-1,2,3,4-tetrahydro-7- methoxyisoquinolin- hexane R. The blue solution is clear in a thin-layer obtained
6-ol dihydrochloride heptahydrate; by tilting the test-tube.
CzsH40Cl2N2O4, 7H2O = 666 (5884-43-5) Chamazulene used for gas chromatography complies with the
[:z]t0: about +25 (2% w/v in water). following additi.onal test.
General reagent grade of commerce. Assay. Gas chromatography (2. 2. 28) as prescribed in the
Cephalin Reagent monograph Matricaria oil (1836).
Solvents used to prepare this reagent should contain a Test soluti.on: a 4 g/L solution in cyclohexane R.
suitable antioxidant such as butylated hydroxyanisole at a Content: minimum 95.0 per cent, calculated by the
concentration of 0.002% w/v. normalisation procedure.
To 0.5 to 1 g of acetone-dried ox brain add 20 mL of acetone Charcoal, Activated (64365-11-3)
and leave for 2 hours. Centrifuge for 2 minutes at 500 g and See Activated charcoal (0313).
decant the supernatant liquid. Dry the residue under reduced
V-A48 Appendix I A 2023

Chelerythrine Chloride l,2-Dimethoxy-12-methyl[l,3] Chloroauric Acid Gold chloride; hydrogen

benzodioxolo [5 ,6-c] phenanthridin-12-ium chloride; tetrachloroaurate; HAuC14 + aq (27988-77-8)
C 21 H1 8 ClNO4 = 383.8 (3895-92-9) General reagent grade of commerce.
mp: 200-206 °C (about 199 °C). Brown, deliquescent masses.
Orange-yellow crystalline powder, soluble in methanol. Chloroauric Acid Solution
Storage: protected from light and moisture. General reagent grade of commerce containing about 2% w/v
Chloral Hydrate (302-17-0) of HAuC14,3H 2 O, or a 2.0% w/v solution of chloroauric acid
See Choral hydrate (0265). in water.
Chloral Hydrate Solution Chlorobenzene C 6 H 5 Cl = 112.6 (108-90-7)
A solution of 80 g in 20 mL of water R. bp: about 131°.
Chloramine Solution Analytical reagent grade of commerce.
A 20 g/L solution of chloramine R. Prepare immediately A colourless liquid.
before use. 4-Chlorobenzenesulfonamide
Chloramine Solution Rl 4-Chlorobenzenesulphonamide; C 6H 6 ClNO 2 S = 191.6
(98-64-6)
A 0.1 g/L solution of chloramine R. Prepare immediately
before use. White or almost white powder.
Chloramine Solution R2 mp: about 145 °C.
A 0.2 g/L solution of chloramine R. Prepare immediately 2-Chlorobenzoic Acid C 7 H 5 ClO 2 = 156.6 (118-91-2)
before use. Slightly soluble in water, soluble in hot water, very soluble in
Chloramine T The sodium salt of N-chlorotoluene-p- anhydrous ethanol.
sulfonamide; Chloramine; (7080-50-4) bp: about 285 °C.
See Tosylchloramide sodium (0381). mp: about 140 °C.
Chlordane C 10 H 6 Cl 8 = 409.8 (12789-03-6) 4-Chlorobenzoic Acid C 7 H 5 ClO 2 = 156.57 (74-11-3)
bp: about 175 °C. mp: about 240°.
mp: about 106 °C. General reagent grade of commerce.
A suitable certified reference solution of technical grade 3-(4-Chlorobenzoyl)propionic Acid
(10 ng/µL in iso-octane) may be used. C 10H 9 ClNO 3 = 212.6 (3984-34-7)
Chlordiazepoxide (58-25-3) mp: about 129°.
See Chlordiazepoxide (0656). General reagent grade of commerce.
Chlorfenvinphos C 12H 14Cl3 O 4 P = 359.6 (470-90-6) 4-Chlorobenzophenone C 13H 9 ClO = 216.66 (134-85-0)
A suitable certified reference solution (10 ng/µL in bp: about 195°.
cyclohexane) may be used. mp: about 75°.
Chlorhexidine Acetate Chlorhexidine diacetate; p-Chlorobenzyhydrylpiperazine 1-(4-Chlorobenzhydryl)
C 22 H 3 0Cl2N10,2C2H4O 2 = 625.6 (56-95-1) piperazine; C 17H 19 ClN2 = 286.80 (303-26-4)
mp: about 154°. bp: about 180°.
General reagent grade of commerce. mp: about 67°.
Chlorhexidine Hydrochloride Chlorhexidine General reagent grade of commerce.
dihydrochloride; C 22 H 30 Cl2 N 10,2HC1 (3697-42-5) 1-Chlorobutane Butyl chloride; C 4 H 9 Cl = 92.57
General reagent grade of commerce. (109-69-3)
4' -Chloroacetanilide Chloroacetanilide; n~: about 1.402.
C 8 H 8 ClNO = 169.6 (539-03-7) bp: about 78°.
Content: minimum 95 per cent. General reagent grade of commerce.
Crystalline powder, practically insoluble in water, soluble in Weight per mL, about 0.886 g.
ethanol (96 per cent).
Chlorobutanol Anhydrous chlorbutol; 1,1,1-trichloro-2-
mp: about 178 °C. methylpropan-2-ol; (57-15-8)
Chloroacetic Acid C 2 H 3 ClO 2 = 94.5 (79-11-8) See Chlorobutanol (0382).
Colourless or white or almost white crystals, deliquescent, 4-Chloro-o-cresol 4-Chloro-2-methylphenol;
very soluble in water, soluble in ethanol (96 per cent). C 7 H 7 ClO = 142.6 (1570-64-5)
Storage: in an airtight container. mp: about 144°.
3-Chloroaniline C 6 H 6 ClN = 127.6 (108-42-9) General reagent grade of commerce.
n~: about 1.594. 2-Chloro-2-deoxy-o-glucose C 6 H 11 ClO 5 = 198.6
General reagent grade of commerce. (14685-79-1)
4-Chloroaniline Chloroaniline; C 6H 6 ClN = 127.6 White or almost white crystalline, very hygroscopic powder,
(106-47-8) soluble in water and in dimethyl sulfoxide, practically
Crystals, soluble in hot water, freely soluble in ethanol insoluble in ethanol (96 per cent).
(96 per cent). 1-Chloro-2,4-dinitrobenzene C 6 H 3 ClN 2 O 4 = 202.6
mp: about 71 °C. (97-00-7)
mp: about 51°.
2023 Appendix I A V-A49

Analytical reagent grade of commerce. White or almost white, crystalline powder or needles, freely
Pale yellow crystals or crystalline powder. soluble in boiling water, in acetone and in ethanol
2-Chloroethanol Ethylene chlorohydrin; C 2H 5 ClO = 80.5 (96 per cent).
(107-07-3) [o:]~6: about -35.2.
Colourless liquid, soluble in ethanol (96 per cent). mp: about 208 °C.
d?g: about 1.1 97. Chromatography. Thin-layer chromatography (2.2.27) as
nt 0 : about 1.442. prescribed on Identification A in the monograph Belladonna
leaf dry extract, standardised (1294); the chromatogram shows
bp: about 130 °C. only one principal zone.
mp: about -89 °C.
Chlorogenic acid used in li,quid chromatography complies with the
2-Chloroethanol Solution fallowing additional test.
Dissolve 125 mg of 2-chloroethanol R in 2-propanol Rand Assay. Liquid chromatography (2.2.29) as prescribed in the
dilute to 50 mL with the same solvent. Dilute 5 mL of the monograph Artichoke Leaf (1866).
solution to 50 mL with 2-propanol R. Content: minimum 97.0 per cent.
Chloroethylamine Hydrochloride 2-Chloroethanamine 5-Chloro-8-hydroxyquinoline 5-Chloroquinolin-8-ol;
hydrochloride; C 2 H 7 Cl 2N = 116.0 (870-24-6) C 9 H 6CINO = 179.6 (130-16-5)
mp: about 145 °C.
Sparingly soluble in cold dilute hydrochloric acid.
(2-Chloroethyl)diethylamine Hydrochloride mp: about 123 °C.
2-Diethylaminoethyl chloride hydrochloride;
C 6 H 15 Cl 2 N = 172.1 (869-24-9) Content: minimum 95.0 per cent.

White or almost white, crystalline powder, very soluble in 3-Chloro-2-methylaniline 6-Chloro-2-toluidine;

water and in methanol, freely soluble in methylene chloride, C 7 H 8CIN = 141.6 (87-60-5)
practically insoluble in hexane. Not miscible with water, slightly soluble in anhydrous
mp: about 211 °C. ethanol.
Chloroform Trichloromethane; CHCh = 119.4 (67-66-3) d?g: about 1.1 71.
Clear, colourless liquid, slightly soluble in water, miscible nt0 : about 1.587.

with ethanol (96 per cent). bp: about 115 °C.

dig: 1.475 to 1.481. mp: about 2 °C.
bp: about 60 °C. 2-Chloro-N-(2,6-dimethylphenyl)acetamide
Ethanol: 0.4 per cent mlm to 1.0 per cent mlm.
C 10H 12 CINO = 197.7 (1131-01-7)
Chloroform, Acidified 2-Chloronicotinic Acid 2-Chloropyridine-3-carboxylic
acid; C 6H 4 CINO 2 = 157.6 (2942-59-8)
To 100 mL of chloroform R add 10 mL of hydrochloric acid R.
Shake, allow to stand and separate the 2 layers. White or almost white powder.
Chloroform, Ethanol-free mp: about 177 °C.
Shake 200 mL of chloroform R with four quantities, each of Content: minimum 95 per cent.
100 mL, of water R. Dry over 20 g of anhydrous sodium 2-Chloro-4-nitroaniline C 6H 5 CIN2 O 2 = 172.6
sulfate R for 24 h. Distil the filtrate over 10 g of anhydrous (121-87-9)
sodium sulfate R. Discard the first 20 mL of distillate. Prepare Yellow, crystalline powder, freely soluble in methanol.
immediately before use. mp: about 107 °C.
Chloroform IR Storage: protected from light.
Spectroscopic reagent grade of commerce. 2-Chloro-5-Nitrobenzoioc Acid C 7 H 4 ClNO4 = 201.6
Chloroform Stabilised with Amylene CHC1 3 = 119 .4 (2516-96-3)
Clear, colourless liquid, slightly soluble in water, miscible mp: 165 °C to 168 °C.
with ethanol (96%). 4-Chlorophenol Chlorophenol; C 6H 5 ClO = 128.6
Water maximum 0.05%. (106-48-9)
Residue on evaporation maximum 0.001 %. Colourless or almost colourless crystals, slightly soluble in
Minimum transmittance (2.2.25) using water as water, very soluble in ethanol (96 per cent) and in solutions
compensation liquid : 50% at 255 nm, 80% at 260 nm, 98% of alkali hydroxides.
at 300 nm. mp: about 42 °C.
Content, minimum 99.8% of CHC13, determined by gas 2-[2-( 4-Chlorophenyl)acetyl]benzoic Acid
chromatography. C1 5H11ClO 3 = 274.7 (53242-76-5)
Chloroform Water 1-Chlorophthalazine C 8 H 5 CIN2 = 164.6 (5784-45-2)
Shake 2.5 mL of chloroform with 900 mL of water until Yellow powder.
dissolved and dilute to 1000 mL with water. Chloroplatinic(1v) Acid Platinic chloride;
Chlorogenic Acid (1S,3R,4R,5R)-3-[(3,4- Chloroplatinic acid; H 2 Cl6Pt,6H 2 O = 517.9 (18497-13-7)
Dihydroxycinnamoyl)oxy]-1,4,5- Content: minimum 37.0 per cent mlm of platinum (Ar 195.1).
trihydroxycyclohexanecarboxylic acid; C 16H 18 O 9 = 354.3
Brownish-red crystals or a crystalline mass, very soluble in
(327-97-9)
water, soluble in ethanol (96 per cent).
Assay. Ignite 0.200 g to constant mass at 900 ± 50 °C and
weigh the residue (platinum).
V-ASO Appendix I A 2023

Storage: protected from light. Choline Chloride (2-Hydroxyethyl)trimethylammonium

Chloroplatinic Acid Solution chloride; C 5 H 14CINO = 139.6 (67-48-1)
A solution of chloroplatinic(IV) acid in water, containing the Deliquescent crystals, very soluble in water and in ethanol
equivalent of 5.0% w/v of H 2 PtCl6 ,6H2 O. (96 per cent).
3-Chloropropane-1,2-diol C 3 H 7 ClO 2 = 110.5 (96-24-2) Chromatography. Thin-layer chromatography (2.2.27) as
Colourless liquid, soluble in water and ethanol (96 per cent). prescribed in the monograph Suxamethonium chloride (0248):
apply 5 µL of a 0.2 g/L solution in methanol R; the
dig: about 1.322. chromatogram shows one principal spot.
nb0 : about 1.480.
Storage: in an airtight container.
bp: about 213 °C. Chondroitinase ABC
1-Chloropropyl (dimethylamine) Hydrochloride Pectin lyase-like enzyme secreted by Flavobacterium
3-Dimethylaminopropyl chloride hydrochloride; heparinum. Available in vials containing 5-10 units. It cleaves
C 5 H 12 CIN = 158.1 (5407-04-5) both glucuronate-containing disaccharides, e.g. chondroitin
mp: about 142°. sulfate, and iduronate-containing disaccharides, e.g. dermatan
General reagent grade of commerce. sulfate.
4-Chlororesorcinol 4-Chlorobenzene-1,3-diol; Chondroitinase AC
1,3-Dihydroxy-4-chlorobenzene; C 6 H 5 ClO 2 = 144.6 Pectin lyase-like enzyme secreted by Flavobacterium
(95-88-5) heparinum. Available in vials containing 5-10 units. It cleaves
mp: 106 °C to 108 °C. only glucuronate-containing disaccharides, e.g. chondroitin
5-Chlorosalicylic Acid C 7 H 5ClO3 = 172.6 (321-14-2) sulfate.
White or almost white, crystalline powder, soluble in Chrome Azurol S Trisodium 5-((3-carboxylato- 5-methyl-
methanol. 4-oxocyclohexa-2, 5-dien-1-ylidene) (2,6-dichloro-3-
sulfonatophenyl)methyl]-2-hydroxy-3-methylbenzoate;
mp: about 173 °C.
Chromazurol S; C 23 H 13 Cl2Na 3 O 9 S = 605 (1667-99-8)
4-Chlorosulfamoylbenzoic Acid C 7H 6 CINO 4 S = 235.7
(1205-30-7)
Schultz No. 841
Colour Index No. 43825
General reagent grade of commerce.
Chlorothiazide 6-Chloro-2H-1,2,4-benzothiadiazine-7- Brownish-black powder, soluble in water, slightly soluble in
ethanol (96 per cent).
sulfonamide 1,1-dioxide; C 7 H 6 CIN3 O4 S2 = 295.7 (58-94-6)
Chromic Acid Cleansing Mixture
Content: minimum 98.0 per cent.
White or almost white, crystalline powder, very slightly See Chromic-sulfuric acid mixture.
soluble in water, sparingly soluble in acetone, slightly soluble Chromic-Sulfuric Acid Mixture
in ethanol (96 per cent). It dissolves in dilute solutions of Chromic-sulphuric acid mixture
alkali hydroxides. A saturated solution of chromium(VI) oxide in sulfuric acid.
Chlorotriphenylmethane Triphenylchloromethane; Chromium(m) Acetylacetonate (OC-6-11 )-Tris(2,4-
triphenylmethyl chloride; C 19 H 15 Cl = 278.8 (76-83-5) pentanedionato-KO,KO')chromium; C 15 H 21 Cr0 6 = 349.3
mp: about 112°. (21679-31-2)
General reagent grade of commerce. Chromium(v1) Oxide Chromium trioxide; CrO 3 = 100.0
A pale yellow or buff, crystalline solid. (1333-82-0)
Chlorpyriphos C 9 H 11 Cl3NO 3 PS = 350.6 (2921-88-2) Dark brownish-red needles or granules, deliquescent, very
bp: about 200 °C. soluble in water.
Storage: in an airtight glass container.
mp: 42 °C to 44 °C.
Chromium(m) Potassium Sulfate Chrome alum;
A suitable certified reference solution (10 ng/µL in
Chromic potassium sulphate; Chromium(m) potassium
cyclohexane) may be used.
sulphate; Chromic potassium sulfate;
Chlorpyriphos-methyl C 7H 7 Cl3 NO 3PS = 322.5 CrK(SO 4 ) 2,12H2 O = 499.4 (7788-99-0)
(5598-13-0)
Large, violet-red or black crystals, freely soluble in water,
mp: 45 °C to 47 °C. practically insoluble in ethanol (96 per cent).
A suitable certified reference solution (10 ng/µL in Chromium(m) Trichloride Hexahydrate
cyclohexane) may be used. [Cr(H2 O) 4 Cl2]Cl,2H 2 O = 266.5 (10060-12-5)
Chlortetracycline Hydrochloride Dark green crystalline powder, hygroscopic.
See Chlortetracycline hydrochloride (0173). Storage: protected from humidity and oxidising agents.
(5a)-Cholestane C 27 H 48 = 372.7 (481-21-0) Chromogenic Substrate R4
Slightly soluble in anhydrous ethanol. Dissolve D-phenylalanyl-L-pipecolyl-L-arginine-4-nitroanilide
mp: about 81 °C. dihydrochloride in water R to give a 0.008 M solution. Dilute
Cholesterol (57-88-5) to 0.0025 M with phosphate buffer solution pH 8.5 R before
See Cholesterol (0993). use.
Cholesteryl Benzoate 5-Cholesten-3~-ol-3-benzoate; Chromogenic Substrate R5
C34HsoO2 = 490.8 (604-32-0) Dissolve N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-4-
mp: about 150°. nitroanilide hydrochloride in water R to give a 0.003 M
solution.
General reagent grade of commerce.
2023 Appendix I A V-A5 l

Chromophore Substrate Rl Chromogenic substrate Rl Cinchonidine (R)-(Quinol-4-yl) [(2S,4S,5R)-5-

Dissolve N-':1-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine- vinylquinuclidin-2-yl]methanol; C 19 H 22 N 2 O = 294.4
4-nitroanilide dihydrochloride in water R to give a 0.003 M (485-71-2)
solution. Dilute in tris(hydroxymethyl)aminomethane-EDTA White or almost white, crystalline powder, very slightly
buffer solution pH 8.4 R to 0.0005 M before use. soluble in water and in light petroleum, soluble in ethanol
Chromophore Substrate R2 Chromogenic substrate R2 (96 per cent).
Dissolve D-phenylalanyl-L-pipecolyl-L-arginine-4-nitroanilide [o:]~0 : -105 to - 110, determined on a 50 gJL solution in
dihydrochloride in water R to give a 0.003 M solution. Dilute ethanol (96 per cent) R.
before use in titrating in tns(hydroxymethyl)aminomethane- mp: about 208 °C, with decomposition.
EDTA buffer solution pH 8.4 R to give a 0.0005 M solution. Storage: protected from light.
Chromophore Substrate R3 Chromogenic substrate R3 Cinchonine (S)-(Quinol-4-yl) [(2R,4S,5R)-5-
Dissolve o-valyl-leucyl-lysyl-4-nitroanilide dihydrochloride in vinylquinuclidin-2-yl]methanol; C 19H 22N 2 0 = 294.4
water R to give a 0.003 M solution. (118-10-5)
Chromotrope 11B Chromotrope 2B; White or almost white, crystalline powder, very slightly
C 16 H9N3Na2O10S 2 = 513.4 (548-80-1) soluble in water, sparingly soluble in ethanol (96 per cent)
Schultz No. 67 and in methanol.
Colour Index No. 16575 [o:]~: + 225 to+ 230, determined on a 50 gJL solution in
Reddish-brown powder, soluble in water giving a yellowish- ethanol (96 per cent) R.
red colour, practically insoluble in ethanol (96 per cent). mp: about 263 °C.
Chromotrope IIB Solution Chromotrope 2B solution Storage: protected from light.
A 0.05 gJL solution of chromotrope II BR in sulfuric acid R. Cineole 1,8-Cineole; Eucalyptol; 1,8-Epoxy-p-menthane;
Chromotropic Acid 4,5-Dihydroxy-2,7- C 10H 18 O = 154.3 (470-82-6)
naphthalenedisulfonic acid; C 10H 8 O 8 S2 = 320.3 (148-25-4) Colourless liquid, practically insoluble in water, miscible with
anhydrous ethanol.
mp: about 300°.
General reagent grade of commerce. d~g: 0.922 to 0.927.
Chromotropic Acid Sodium Salt Disodium nt 0 : 1.456 to 1.459.

1,8-dihydroxynaphthalene-3,6-disulfonate dihydrate; Freezing point (2.2.18): 0 °C to 1 °C.

C 10H 6Na2O 8 S2,2H2O = 400.3 (5808-22-0) Distillation range (2.2.11): 174 °C to 177 °C.
Schultz No. 1136 Phenol. Shake 1 g with 20 mL of water R. Allow to separate
A yellowish-white powder, soluble in water, practically and add to 10 mL of the aqueous layer O.1 mL offerric
insoluble in ethanol (96 per cent). chloride solution Rl. No violet colour develops.
Chromotropic Acid, Sodium Salt Solution Turpentine oil. Dissolve 1 g in 5 mL of ethanol
(90 per cent V/V) R. Add dropwise freshly prepared bromine
Dissolve 0.60 g of chromotropic acid, sodium salt R in about
80 mL of water R and dilute to 100 mL with the same water R. Not more than 0.5 mL is required to give a yellow
solvent. Use this solution within 24 h. colour lasting for 30 min.
Residue on evaporation: maximum 0.05 per cent.
Chromotropic Acid Solution
Dissolve 0.5 g of chromotropu acid in sufficient water to To 10.0 mL add 25 mL of water R, evaporate on a water-
bath and dry the residue to constant mass at 100-105 °C.
produce 100 mL.
Use the solution within 24 hours. Cineole used in gas chromatography complies with the folwwing
additional test.
Chromotropic Acid-Sulfuric Acid Solution
Chromotropic Acid-Sulphuric Acid Solution Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Peppermint oil (0405).
Dissolve 5 mg of chromotropic acid, sodium salt R in 10 mL of
a mixture of 9 mL of suljuric acid R and 4 mL of water R. Test solution. The substance to be examined.
Chrysanthemin Cyanidin 3-O-glucoside chloride; Content: minimum 98.0 per cent, calculated by the
Kuromanin chloride; 2-(3,4-Dihydroxyphenyl)-3-(~-D- normalisation procedure.
glucopyranosyl)oxy-5, 7-dihydroxy-1-benzopyrylium chloride; 1,4-Cineole l-Methyl-4-(1-methylethyl)-7-oxabicyclo
C21H21ClO 11 = 485.8 (7084-24-4) [2.2. l]heptane; l-Isopropyl-4-methyl-7-oxabicyclo[2.2. l]
Reddish-brown crystalline powder, soluble in water and in heptane; C 10H 18 O = 154.3 (470-67-7)
ethanol (96 per cent). Colourless liquid.
Absorbance (2.2.25). A 0.01 gJL solution in a mixture of di 0 : about 0.900.
1 volume of hydrochloric acid R and 999 volumes of nt 0 : about 1.445.

methanol R shows an absorption maximum at 528 nm. bp: about 173 °C.
':1-Chymotrypsin for Peptide Mapping Cinnamaldehyde 3-Phenylpropenal; Cinnamic aldehyde;
':1-Chymotrypsin of high purity, treated to eliminate tryptic C 9 H 8 O = 132.2 (104-55-2)
activity. Yellowish or greenish-yellow, oily liquid, slightly soluble in
Cimifugin (2S)-7-(Hydroxymethyl)-2-(l-hydroxy-1- water, very soluble in ethanol (96 per cent).
methylethyl)-4-methoxy-2,3-dihydro-5H-furo (3,2-g) [1) n~?: about 1.620.
benzopyran-5-one; C 16 H1 8 O 6 = 306.3 (37921-38-3) Storage: protected from light.
Cinnamamide (E)-3-Phenylprop-2-enamide;
C 9 H 9 NO = 147.2 (621-79-4)
V-A52 Appendix I A 2023

White or almost white powder. Dilute to 20 mL with water R. No pink colour appears in the
mp: about 149 °C. solution.
Cinnamic Acid trans-3-Phenylacrylic acid; (2E)-3- Citric Acid, Anhydrous (77-92-9)
Phenylprop-2-enoic acid; trans-Cinnamic acid; See Citric acid (0455).
C 9 H 8 O 2 = 148.2 (140-10-3) Citric-Molybdic Acid Solution
Colourless crystals, very slightly soluble in water, freely Mix 54 g of molybdenum(v1) oxide with 200 mL of water, add
soluble in ethanol (96 per cent). 11 g of sodium hydroxide and heat, with stirring, until almost
mp: 133 °C. complete solution has been obtained. Dissolve 60 g of citric
trans-Cinnamic Aldehyde (E)-3-Phenylprop-2-enal; acid in 250 mL of water and add 140 mL of hydrochloric acid.
C9 H 8 0 = 132.2 (14371-10-9) Add the first solution to the second, stirring continuously,
trans-Cinnamic aldehyde used in gas chromatography complies cool, filter if necessary, dilute to 1000 mL with water and
with the following additional test. add, dropwise, sufficient of a 1% w/v solution of potassium
bromate to discharge the green colour.
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Cassia oil (1496). Store in a well-closed container, protected from light.
Content: minimum 99.0 per cent, calculated by the Citronella! 3,7-Dimethyl-6-octenal; C 10H 18 O = 154.3
(106-23-0)
normalisation procedure.
Cinnamyl Acetate 3-Phenylprop-2-en-l-yl acetate; Very slightly soluble in water, soluble in ethanol
(96 per cent).
C 11 H12O2 = 176.2 (103-54-8)
nfr about 1.542.
dig: 0.848 to 0.856.
bp: about 262 °C. nt 0 : about 1.446.

Cinnamyl acetate used in gas chromatography complies with the Citronella! used in gas chromatography complies with the following
following additional test. additional test.
Assay. Gas chromatography (2.2.28) as prescribed in the Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Cassia oil (1496). monograph Citronella oil (1609).
Content: minimum 99.0 per cent, calculated by the Content: minimum 95.0 per cent, calculated by the
normalisation procedure. normalisation procedure.
Citral 3,7-Dimethylocta-2,6-dienal; C 10H 16O = 152.2 Citronellol 3,7-Dimethyloct-6-en-1-ol; C 10H 20 O = 156.3
(106-22-9)
(5392-40-5)
Light yellow liquid, practically insoluble in water, miscible Clear, colourless liquid, practically insoluble in water,
with ethanol (96 per cent) and with propylene glycol. miscible with ethanol (96 per cent).
Chromatography. Thin-layer chromatography (2.2.27), using dfg: o.857.
silica gel GF254 R as the coating substance: apply to the plate n~: 1.456.
10 µL of a 1 g/L solution in toluene R. Develop over a path bp: 220 °C to 222 °C.
of 15 cm using a mixture of 15 volumes of ethyl acetate R and Citronellol used in gas chromatography complies with the following
85 volumes of toluene R. Allow the plate to dry in air and additional test.
examine in ultraviolet light at 254 nm. The chromatogram Assay. Gas chromatography (2.2.28) as prescribed in the
shows only one principal spot. monograph Citronella oil (1609).
Citral used in gas chromatography complies with the following Content: minimum 95.0 per cent, calculated by the
additional test. normalisation procedure.
Assay. Gas chromatography (2.2.28) as prescribed in the Storage: in an airtight container, protected from light.
monograph Citronella oil (1609).
Citronellyl Acetate 3,7-Dimethyl-6-octen-1-yl acetate;
Content of citral (neral + geranial): minimum 95.0 per cent, C1 2 H 22 O2 = 198.3 (150-84-5)
calculated by the normalisation procedure.
Citrate Buffered Saline
dig: o.890.
Dissolve 11. 76 g of sodium citrate in 1800 mL of water,
nt 0 : 1.443.

adjust to pH 7.0 with lM hydrochloric acid and add suffient bp: 229 °C.
water to produce 2000 mL. Citronellyl acetate used in gas chromatography complies with the
Citrated Rabbit Plasma following additional test.
Collect blood by intracardiac puncture from a rabbit kept Assay. Gas chromatography (2.2.28) as prescribed in the
fasting for 12 h, using a plastic syringe with a No. 1 needle monograph Citronella oil (1609).
containing a suitable volume of 38 glL solution of sodium Content: minimum 95.0 per cent, calculated by the
citrate R so that the final volume ratio of citrate solution to normalisation procedure.
blood is 1: 9. Separate the plasma by centrifugation at Storage: in an airtight container, protected from light.
1500 g to 1800 g at 15 °C to 20 °C for 30 min. Citropten 5,7-Dimethoxycoumarin; C 11 H 10O 4 = 206.2
Storage: at 0 °C to 6 °C; use within 4 h of collection. (487-06-9)
Citric Acid Citric acid monohydrate; (5949-29-1) Needle-shaped crystals, practically insoluble in water and in
See Citric acid monohydrate (0456). light petroleum, freely soluble in acetone and in ethanol
W7zen used in the test for iron, it complies with the fallowing (96 per cent).
additional requirement. mp: about 145 °C.
Dissolve 0.5 gin 10 mL of water R, add 0.1 mL of Chromatography. Thin-layer chromatography (2.2.27), using
thioglycollic acid R, mix and make alkaline with ammonia R. silica gel GF254 R as the coating substance: apply to the plate
2023 Appendix I A V-A53

10 µL of a 1 g/L solution in toluene R. Develop over a path Codeine (6059-47-8)

of 15 cm using a mixture of 15 volumes of ethyl acetate R and See Codeine monohydrate (0076).
85 volumes of toluene R. Allow the plate to dry in air and Codeine Phosphate (52-28-8)
examine in ultraviolet light at 254 nm. The chromatogram
obtained shows only one principal spot. See Codeine phosphate hemihydrate (0074).
Clobetasol Propionate 21-Chloro-9-fluoro- l l ~, 17- 2,4,6-Collidine 2,4,6-Trimethylpyridine; C 8 H 11 N = 121.2
dihydroxy-16 ~-methylpregna-1,4-diene-3,20-dione (108-75-8)
17-propionate; C 25 H 32 CIFO 5 = 467 .0 (25122-46-7) bp: about 170°.
White or almost white crystalline powder, insoluble in water, General reagent grade of commerce.
soluble in ethanol (96 per cent) and in acetone. A colourless to pale yellow liquid; weight per mL, about
[a]t0 : about+ 104 (in dioxan). 0.92 g.
mp: about 196 °C. It may be stabilised by the addition of aluminium oxide.
Coagulation Factor V Solution Clotting factor V Columbin ( 1R,4R,4aR,6aR, 9S, 10aS, 10bS)-9-(3-Furanyl)-
solution 1,4,4a,5,6,6a,9, 10, 10a, 10b-decahydro-4-hydroxy-4a, 10a-
Coagulation factor V solution may be prepared by the dimethyl-1,4-etheno-3H, 7H-benzo[l,2-c:3,4-c']dipyran-3, 7-
following method or by any other method which excludes dione; C 20H 22 O 6 = 358.4 (546-97-4)
factor VIII. Reagent grade of commerce.
Prepare the factor V reagent from fresh oxalated bovine Crystalline solid.
plasma, by fractionation at 4 °C with a saturated solution of Congo Red Disodium (biphenyl-4,4 '-diyl-bis-2,2 '-azo)
ammonium sulfate R prepared at 4 °C. Separate the fraction bis(l-aminonaphthalene-4-sulfonate );
which precipitates between 38 per cent and 50 per cent of C 32H22N 6Na2O 6 S2 = 697 (573-58-0)
saturation, which contains factor V without significant Schultz No. 360
contamination with factor VIII. Remove the ammonium
Colour Index No. 22120
sulfate by dialysis and dilute the solution with a 9 g/L
solution of sodium chloride R to give a solution containing Brownish-red powder, soluble in water.
between 10 per cent and 20 per cent of the quantity of Congo Red Fibrin Fibrin congo red
factor V present in fresh human normal plasma. Take 1.5 g of fibrin and leave overnight in 50 mL of a
Assay of factor V. Prepare two dilutions of the preparation of 20 g/L solution of congo red R in ethanol (90 per cent Vlv'.) R.
factor V in imidazole buffer solution pH 7.3 R containing Filter, rinse the fibrin with water R and store under ether R.
1 volume of the preparation in 10 volumes and in Congo Red Paper
20 volumes of the buffer solution respectively. Test each Immerse strips of filter paper for a few minutes in congo red
dilution as follows: mix 0.1 mL of plasma substrate deficient in solutwn R. Allow to dry.
factor V R, 0.1 mL of the solution to be examined, 0.1 mL of
Congo Red Solution
thromboplastin R and 0.1 mL of a 3.5 g/L solution of calcium
chloride R and measure the coagulation times, i.e. the interval Dissolve 0.1 g of congo red Rina mixture of 20 mL of
between the moment at which the calcium chloride solution ethanol (96 per cent) R and water R and dilute to 100 mL
is added and the first indication of the formation of fibrin, with water R.
which may be observed visually or by means of a suitable Test for sensitivity. To 0.2 mL of the congo red solution add
apparatus. 100 mL of carbon dioxide1ree water Rand 0.3 mL of 0.1 M
In the same manner, determine the coagulation time (in hydrochloric acid. The solution is blue. Not more than 0.3 mL
duplicate) of four dilutions of human normal plasma in of 0.1 M sodium hydroxide is required to change the colour to
imidazole buffer solution pH 7.3 R, containing respectively, pink.
1 volume in 10 (equivalent to 100 per cent of factor V), Colour change: pH 3.0 (blue) to pH 5.0 (pink).
1 volume in 50 (20 per cent), 1 volume in 100 (10 per cent), Convallatoxin 3~-((6-Deoxy-'Y.-L-mannopyranosyl)oxy]-
and 1 volume in 1000 (1 per cent). Using two-way 5, 14-dihydroxy- l 9-oxo-5~-card-20(22)-enolide;
logarithmic paper plot the average coagulation times for each 5, 14-Dihydroxy-19-oxo-3~-[ (a-L-rhamnopyranosyl)oxy]-5 ~-
dilution of human plasma against the equivalent percentage card-20(22)-enolide; C 29 H 42 O 10 = 550.6 (508-75-8)
of factor V and read the percentage of factor V for the two White or slightly yellow, crystalline powder, slightly soluble in
dilutions of the factor V solution by interpolation. The mean water, soluble in ethanol, and in acetone, slightly soluble in
of the two results gives the percentage of factor V in the ethyl acetate.
solution to be examined.
mp: 235-242 °C.
Storage; in the frozen state at a temperature not higher than
Coomassie Blue (3861-73-2)
-20 °c.
See acid blue 92 R.
Cobalt(n) Acetate Cobaltous acetate;
(CH 3CO 2 hCo,4H 2 O = 249.1 (6147-53-1) Coomassie Blue Solution See acid blue 92 solution R.
General reagent grade of commerce. Coomassie Staining Solution
Cobalt(n) Chloride Cobaltous chloride; Cobalt chloride; A 1.25 g/L solution of acid blue 83 R in a mixture consisting
CoC12,6H 2O = 237.9 (7791-13-1) of 1 volume of glacial acetic acid R, 4 volumes of methanol R
and 5 volumes of water R. Filter.
Red, crystalline powder or deep-red crystals, very soluble in
water, soluble in ethanol (96 per cent). Coomassie Staining Solution Rl
Cobalt(u) Nitrate Cobaltous nitrate; Cobalt nitrate; Dissolve 0.275 g of acid blue 83 R in 200 mL of methanol R.
Co(NO 3 )z,6H 2 O = 291.0 (10026-22-9) Stir until complete dissolution of the crystals (for about 2 h).
Add 750 mL of water Rand 50 mL of glacial acetic acid R.
Small garnet-red crystals, very soluble in water.
Stir overnight (for at least 16 h); filter.
V-A54 Appendix I A 2023

Copper Cu = 63.55 (7440-50-8) ammonia R until the precipitate which forms dissolves
Cleaned foil, turnings, wire or powder of the pure metal of completely. Keeping the temperature below 20 °C, add
electrolytic grade. dropwise with continuous shaking 30 mL of strong sodium
Copper(n) Acetate Cupric acetate; Copper acetate; hydroxide solution R. Filter through a sintered-glass filter (40)
(2.1.2), wash with water R until the filtrate is clear and take
C 4 H 6 CuO 4,H2 O = 199.7 (6046-93-1)
up the precipitate with 200 mL of concentrated ammonia R.
Blue-green crystals or powder, freely soluble in boiling water, Filter through a sintered-glass filter (2.1. 2) and repeat the
soluble in water and in ethanol (96 per cent), slightly soluble filtration to reduce the residue to a minimum.
in glycerol (85 per cent).
Corallin Sodium salt of rosolic acid; ( 603-45-2)
Copper Carbonate Approximately CuCO 3,Cu(OHh,H2 O
(12069-69-1)
Colour Index No. 43811
General reagent grade of commerce. General reagent grade of commerce.
Copper(n) Chloride Cupric chloride; Hard, dull red masses.
CuC12,2H2 O = 170.5 (10125-13-0) Corallin Solution, Alkaline
Greenish-blue powder or crystals, deliquescent in moist air, Dissolve 5 g of corallin in 100 mL of ethanol (90%).
efflorescent in dry air, freely soluble in water, in ethanol Immediately before use add 1 mL of the solution to 20 mL
(96 per cent) and in methanol, sparingly soluble in acetone. of a 20% w/v solution of sodium carbonate.
Swrage: in an airtight container. Cortisone C 21 H 28 O 5 = 360.4 (53-06-5)
Copper Chloride-Pyridine Reagent Content: minimum 95.0 per cent.
Dissolve 40 mg of copper(II) chloride in pyridine, warming until mp: 223-228 °C.
complete dissolution is effected, and cool. Add 1 mL of Cortisone Acetate (50-04-4)
carbon disulfide and sufficient pyridine to produce 100 mL. See Cortisone acetate (0321).
Copper Edetate Solution Corydaline (13S,13aR)-5,8,13,13a-Tetrahydro-2,3,9,10-
To 2 mL of a 20 g/L solution of copper acetate R add 2 mL tetramethoxy-13-methyl-6H-dibenzo [a,g] quinolizine;
of 0.1 M sodium edetate and dilute to 50 mL with water R. C22H21NO4 = 369.4 (518-69-4)
Copper(n) Nitrate Chloride dinitrate trihydrate; Cupric Costunolide (3aS,6E, 1OE, 11 aR)-6, 10-Dimethyl-3-
nitrate; Copper nitrate; Cu(NO 3 ) 2 ,3H2 O = 241.6 methylene-3a,4,5,8, 9, 11 a-hexahydrocyclodeca[b] furan-2(3H)-
(10031-43-3) one; C 15 H 20 O 2 = 232.3 (553-21-9)
Dark blue crystals, hygroscopic, very soluble in water giving a Coumaphos C 14H 16 Cl0 5 PS = 362.8 (56-72-4)
strongly acid reaction, freely soluble in ethanol (96 per cent) mp: 91 °C to 92 °C.
and in dilute nitric acid. A suitable certified reference solution (10 ng/µL in iso-
Swrage: in an airtight container. octane) may be used.
Copper Oxide Solution, Ammoniacal o-Coumaric Acid (E)-2-Hydroxycinnamic acid; (2E)-
Triturate 0.5 g of copper carbonate with 10 mL of water and 3-(2-Hydroxyphenyl)prop-2-enoic acid; C 9 H 8 O 3 = 164.2
gradually add 10 mL of 13.5M ammonia. (614-60-8)
Copper(n) Sulfate Copper Sulfate; Copper sulphate; White or almost white powder.
Copper(u) sulphate; Cupric sulfate; Cupric sulphate; Copper mp: about 217 °C.
sulfate pentahydrate; CuSO 4 ,5H 2 O = 249.7 (7758-99-8)
Coumarin 2H-Chromen-2-one; 2H-1-Benzopyran-2-one;
Blue powder or deep-blue crystals, slowly efflorescent, very C9H 6 O2 = 146.1 (91-64-5)
soluble in water, slightly soluble in ethanol (96 per cent).
Colourless, crystalline powder or orthorhombic or rectangular
Copper Sulfate, Anhydrous CuSO 4 = 159.6 (7758-98-7) crystals, very soluble in boiling water, soluble in ethanol
Greenish-grey powder, hygroscopic, freely soluble in water, (96 per cent). It dissolves in solutions of alkali hydroxides.
slightly soluble in methanol and practically insoluble in mp: 68 °C to 70 °C.
ethanol (96 per cent).
Coumarin used in gas chromatography complies with the following
Copper Sulfate-Pyridine Reagent additional test.
Copper sulphate-pyridine reagent Assay. Gas chromatography (2.2.28) as prescribed in the
Dissolve 4 g of copper(n) sulfate in 90 mL of water and add monograph Cassia oil (1496).
30 mL of pyridine. Content: minimum 98.0 per cent, calculated by the
Prepare immediately before use. normalisation procedure.
Copper Sulfate Solution Copper sulphate solution Coumestrol C 15 H 8 O 5 = 268.22 (479-13-0)
A 125 g/L solution of copper sulfate pentahydrate R. General reagent grade of commerce.
Copper Sulfate Solution Rl m-Cresol (108-39-4)
To 600 mL of water R slowly add 80 mL of phosphoric See metacresol (2077).
acid R. Dissolve with stirring 100 g of anhydrous copper o-Cresol Cresol; C 7H 8 O = 108.1 (95-48-7)
sulfate R and dilute to 1 L with water R. Crystals or a super-cooled liquid becoming dark on exposure
Copper Sulfate Solution, Weak to light and air, miscible with anhydrous ethanol, soluble in
Copper sulphate solution, weak about 50 parts of water and soluble in solutions of alkali
A 10% w/v solution of copper(II) sulfate. hydroxides.
Copper Tetrammine, Ammoniacal Solution of dig: about 1.05.
Dissolve 34.5 g of copper sulfate pentahydrate R in 100 mL of ngi: 1.540 to 1.550.
water R and, whilst stirring, add dropwise concentrated bp: about 190 °C.
2023 Appendix I A V-A55

Freezing point (2.2.18): minimum 30.5 °C. a) To 25.0 mL add 3 g of potassium iodide R. Add 25 mL of
Residue on evaporation: maximum 0.1 per cent mlm, a 25 per cent mlm solution of sulfuric acid R with precaution
determined by evaporating on a water-bath and drying in an and in small quantities. Titrate with 0.1 M sodium thiosulfate
oven at 100-105 °C. using 0.5 mL of starch solution R, added towards the end of
Storage: protected from light, moisture and oxygen. the titration, as indicator.
Distil before use. 24.5 mL to 25.5 mL of 0.1 M sodium thiosulfate is used in the
titration.
p-Cresol 4-Methylphenol; C 7 H 8 O = 108.1 (106-44-5)
b) Dilute 10.0 mL to 100.0 mL with water Rand mix.
Colourless or white or almost white crystals or crystalline
To 10.0 mL of the solution, add 25.0 mL of 0.1 M
mass. hydrochloric acid and heat for 1 h on a water-bath. Cool,
dig: about 1.02. adjust with water R to the initial volume and titrate with
bp: about 202 °C. 0.1 M sodium hydroxide, using 0.1 mL of phenolphthalein
m-Cresol Purple m-Cresolsulfonphthalein; solution Rl as indicator.
C 21 H 18 O 5 S = 382.44 (2303-01-7) 5.7 mL to 6.3 mL of 0.1 M sodium hydroxide is used in the
Olive-green, crystalline powder, slightly soluble in water, titration.
soluble in ethanol (96 per cent), in glacial acetic acid and in c) Dilute 10.0 mL to 100.0 mL with water Rand mix.
methanol. Titrate 10.0 mL of the solution with 0.1 M hydrochloric acid,
m-Cresol Purple Solution using 0.1 mL of phenolphthalein solution Rl as indicator.
Dissolve 0.1 g of m-cresol purple R in 13 mL of 0.01 M 6.0 mL to 7.5 mL of 0.1 M hydrochloric acid is used in the
sodium hydroxide, dilute to 100 mL with water Rand mix. titration.
Colour change: pH 1.2 (red) to pH 2.8 (yellow); pH 7.4 Cupriethylenediamine Hydroxide Solution
(yellow) to pH 9.0 (purple). (14552-35-3)
Cresol Red Cresolsulfonphthalein; 4,4 1-(3H-2,l- The molar ratio of ethylenediamine to copper is
Benzoxathiol-3-ylidene)bis-(2-methylphenol) S,S-dioxide; 2.00 ± 0.04.
C21H1sO 5 S = 382.4 (1733-12-6) This solution is commercially available.
A reddish-brown crystalline powder, slightly soluble in water, Cupri-tartaric Solution
soluble in ethanol (96 per cent) and in dilute solutions of Solution A. Dissolve 34.6 g of copper sulfate pentahydrate R in
alkali hydroxides. water R and dilute to 500 mL with the same solvent.
Cresol Red Solution Solution B. Dissolve 173 g of sodium potassium tartrate R and
Dissolve 0.1 g of cresol red Rina mixture of 2.65 mL of 50 g of sodium hydroxide R in 400 mL of water R. Heat to
0.1 M sodium hydroxide and 20 mL of ethanol (96 per cent) R boiling, allow to cool and dilute to 500 mL with carbon
and dilute to 100 mL with water R. dioxide-free water R.
Test for sensitivity. A mixture of 0 .1 mL of the cresol red Mix equal volumes of the 2 solutions immediately before use.
solution and 100 mL of carbon dioxide-free water R to which Cupri-tartaric Solution Rl
0.15 mL of 0.02 M sodium hydroxide has been added is
Fehling's solution
purple-red. Not more than 0.15 mL of 0.02 M hydrochloric
acid is required to change the colour to yellow. Solution A Dissolve 34.6 g of copper(II) sulfate in a mixture
of 0.5 mL of sulfuric acid and sufficient water to produce
Colour change: pH 7.0 (yellow) to pH 8.6 (red).
500 mL.
Crystal Violet Basic violet 3; C 25H 30 ClN 3 = 408.0 Solution B Dissolve 176 g of potassium sodium ( +)-tartrate
(548-62-9)
and 77 g of sodium hydroxide in sufficient water to produce
Schultz No. 78 500 mL.
Colour Index No. 42555 Mix equal volumes of solutions A and B immediately before
Dark-green powder or crystals, soluble in water and in use.
ethanol (96 per cent). Cupri-tartaric Solution R2 Dilute potassium cupri-
Crystal Violet Solution tartrate solution
Dissolve 0.5 g of crystal violet R in anhydrous acetic acid Rand Add 1 mL of a solution containing 5 g/L of copper sulfate
dilute to 100 mL with the same solvent. pentahydrate R and 10 g/L of potassium tartrate R to 50 mL of
Test for sensitivity. To 50 mL of anhydrous acetic acid R add sodium carbonate solution Rl. Prepare immediately before use.
0.1 mL of the crystal violet solution. On addition of0.l mL Cupri-tartaric Solution R3
of 0.1 M perchloric acid the bluish-purple solution turns Prepare a solution containing 10 g/L of copper sulfate
bluish-green. pentahydrate R and 20 g/L of sodium tartrate R. To 1.0 mL of
Cupri-citric Solution the solution add 50 mL of sodium carbonate solution R2.
Dissolve 25 g of copper sulfate pentahydrate R, 50 g of citric Prepare immediately before use.
acid monohydrate R and 144 g of anhydrous sodium carbonate R Cupri-tartaric Solution R4
in water R and dilute to 1000 mL with the same solvent. Solution A. 150 g/L copper sulfate pentahydrate R.
Cupri-citric Solution Rl Solution B. Dissolve 2.5 g of anhydrous sodium carbonate R,
Dissolve 25 g of copper sulfate pentahydrate R, 50 g of citric 2.5 g of sodium potassium tartrate R, 2.0 g of sodium hydrogen
acid monohydrate R and 144 g of anhydrous sodium carbonate R carbonate R, and 20.0 g of anhydrous sodium sulfate R in
in water R and dilute to 1000 mL with the same solvent. water R and dilute to 100 mL with the same solvent.
Adjust the solution so that it complies with the following Mix 1 part of solution A with 25 parts of solution B
requirements. immediately before use.
V-A56 Appendix I A 2023

Curcumin 1, 7-Bis(4-hydroxy-3-methoxyphenyl)hepta-1,6- ~-Cyclodextrin for Chiral Chromatography, Modified

diene-3,5-dione; C 21 H 20 O 6 = 368.4 ( 458-37-7) 30 per cent of 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl-~-
Orange-brown, crystalline powder, practically insoluble in cyclodextrin dissolved in polysiloxane substituted with
water, soluble in glacial acetic acid. 15 per cent of phenyl groups and 85 per cent of methyl
mp: about 183 °c. groups.
Curcuminoids ~-Cyclodextrin for Chiral Chromatography,
A mixture of curcumin (C 21 H 20O 6; Mr 368.4), Modified Rl
demethoxycurcumin (C 20H 18 O 5 ; Mr 338.4) and bis- 30 per cent of 2,3-di-O-acetyl-6-O-tert-butylsilyl-~-
demethoxycurcumin (C 19H 16 O4 ; Mr 308.3). cyclodextrin dissolved in polysiloxane substituted with
Cyanoacetic Acid Malonic acid mononitrile; 15 per cent of phenyl groups and 85 per cent of methyl
C3H3NO2 = 85.1 (372-09-8) groups.
White or yellowish-white, hygroscopic crystals, very soluble in ~-Cyclodextrin Hydroxypropyl Ether Derivative for
water. Chiral Chromatography
Storage: in an airtight container.
Beta cyclodextrin, R,S-hydroxypropyl ether derivative,
bonded to porous silica particles, 3 to I O µm in diameter.
Cyanocobalamin Coix-[ix-(5,6-Dimethylbenzimidazolyl)]-
Co~-cyanocobamide; Vitamin B 12; (68-19-9) Cyclohexane C 6 H 12 = 84.2 (110-82-7)
See Cyanocobalamin (0547). Clear, colourless, flammable liquid, practically insoluble in
water, miscible with organic solvents.
Cyanogen Bromide Solution (506-68-3)
Add dropwise, with cooling 0.1 M ammonium thiocyanate to
d~g: about 0.78.
bromine water R until the yellow colour disappears. Prepare
bp: about 80.5 °C.
immediately before use. Cyclohexane used in spectrophotometry complies with the following
1-Cyanoguanidine Dicyandiamide; Cyanoguanidine; additional test.
C2H4N 4 = 84.1 (461-58-5) Absorbance (2.2.25): maximum 0.35 at 220 nm, 0.16 at
White or almost white, crystalline powder, sparingly soluble 235 nm, 0.05 at 240 nm, 0.01 at 250 nm, determined using
water R as compensation liquid.
in water and in ethanol (96 per cent), practically insoluble in
methylene chloride. Cyclohexane Rl
mp: about 210 °C. Complies with the requirements prescribed for cyclohexane R
Cyanopropylphenylene(6)methyl(94)polysiloxane with the following additional requirement.
Polysiloxane substituted with 6 per cent of cyanopropyl and The fluorescence, measured at 460 nm, under illumination
phenylene groups and 94 per cent of methyl groups. with an excitant light beam at 365 nm, is not more intense
than that of a solution containing 0.002 ppm of quinine R in
Cyanopropyl(3)phenyl(3)methyl(94)polysiloxane dilute sulfuric acid Rl.
Polysiloxane substituted with 3 per cent of cyanopropyl Cyclohexylamine Cyclohexanamine; C 6 H 13 N = 99.2
groups, 3 per cent of phenyl groups and 94 per cent of (108-91-8)
methyl groups.
Colourless liquid, soluble in water, miscible with usual
Cyanopropyl(7)phenyl(7)methyl(86)polysiloxane organic solvents.
Polysiloxane substituted with 7 per cent of cyanopropyl n~: about 1.460.
groups, 7 per cent of phenyl groups and 86 per cent of
methyl groups. bp: 134 °C to 135 °C.
Cyanopropyl(25)phenyl(25)methyl(S0)polysiloxane Cyclohexylenedinitrilotetra-acetic Acid ( ± )-trans-1,2-
Diaminocyclohexane-N,N,N 1,N '-tetra-acetic acid;
Polysiloxane substituted with 25 per cent of cyanopropyl
C14H22N2Os,H2O = 364.4
groups, 25 per cent of phenyl groups and 50 per cent of
methyl groups. White or almost white, crystalline powder.
Cyanopropylpolysiloxane mp: about 204 °C.
Polysiloxane substituted with 100 per cent of cyanopropyl Cyclohexylmethanol Cyclohexylcarbinol;
groups. C7H1 4 O = 114.2 (100-49-2)
Cyasterone (2~,3~,5~,22R,24S,24 1R,25S)-24 1,26-Epoxy- Liquid with a slight odour of camphor, soluble in ethanol
2,3, 14,20,22-pentahydroxystigmast-7-ene-6,26-dione; (96 per cent).
C29H44Os = 520.7 (17086-76-9) n;']: about 1.464.
Cyclobutane-1,1-dicarboxylic Acid C 6H 10 O 4 = 144.1 bp: about 185 °C.
(5445-51-2) 3-Cyclohexylpropionic Acid C 9 H 16 O 2 = 156.2
mp: about 160°. (701-97-3)
General reagent grade of commerce. Clear liquid.
ix-Cyclodextrin Cyclohexakis-(1--+4)-(ix-o-glucopyranosyl); dig: about 0.998.
Cyclomaltohexaose; Alfadex; C 36H 60 O 30 = 972 (10016-20-3) n~: about 1.4648.
~-Cyclodextrin (7585-39-9) bp: about 130 °C.
See Betadex (1070). Cyhalothrin C21H 19 ClF3NO 3 = 449.9 (91465-08-6)
y-Cyclodextrin Gamma-cyclodextrin bp: 187 °C to 190 °C.
C4sHsoO40 = 1297 (17465-86-0) mp: about 49 °C.
General reagent grade of commerce. A suitable certified reference solution (10 ng/µL in
cyclohexane) may be used.
2023 Appendix I A V-A57

Cymarin 3 ~-[ (2, 6-Dideoxy-3-O-methyl-~-D-ribo- o,p'-DDE C 14 H 8Cl 4 = 318.0 (3424-82-6)

hexopyranosyl) oxy)-5~, 14-dihydroxy-19-oxocard-20(22)- 1-(2-Chlorophenyl)-l-( 4-chlorophenyl)-2,2-dichloroethylene.
enolide; C 30H 44 O 9 = 548.7 (508-77-0) A suitable certified reference solution (10 ng/µL in
White or slightly yellow powder, slightly soluble in water, cyclohexane) may be used.
soluble in methanol. p,p'-DDE l,l-Bis(4-chlorophenyl)-2,2-dichloroethylene;
mp: about 148 °C. C1 4H 8 Cl4 = 318.0 (72-55-9)
p-Cymene 4-Isopropyltoluene; C 10H 14 = 134.2 (99-87-6) bp: 316 °C to 317 °C.
Colourless liquid, practically insoluble in water, soluble in mp: 88 °C to 89 °C.
ethanol (96 per cent). A suitable certified reference solution (10 ng/µL in
dig: about 0.858. cyclohexane) may be used.
nt0 : about 1.4895. o,p' -DDT 1-(2-Chlorophenyl)-l-(4-chlorophenyl)-2,2,2-
bp: 175 °C to 178 °C. trichloroethane; C 14H 9 Cl5 = 354.5 (789-02-6)
p-Cymene used in gas chromatography complies with the following A suitable certified reference solution (10 ng/µL in
additional test. cyclohexane) may be used.
Assay. Gas chromatography (2.2.28) as prescribed in the p,p' -DDT 1, 1-Bis(4-chlorophenyl)-2,2,2-trichloroethane;
monograph Peppemzint oil (0405). C 14H 9 Cl5 = 354.5 (50-29-3)
Test solution. The substance to be examined. bp: about 260 °C.
Content: minimum 96.0 per cent, calculated by the mp: 108 °C to 109 °C.
normalisation procedure. A suitable certified reference solution (10 ng/µL in
Cynarin (1R,3R,4S,5R)-1,3-Bis[[3-(3,4- dihydroxyphenyl) cyclohexane) may be used.
propenoyl] oxy)-4, 5- dihydroxycyclohexanecarboxylic acid; Decanal Decyl aldehyde; C 10H 20 O = 156.3 (112-31-2)
C2 5 H24O 12 = 516.4 (30964-13-7) Oily, colourless liquid, practically insoluble in water.
White or almost white amorphous mass, odourless. Decanal used in gas chromatography compli,es with the following
Cypermethrin C 22 H 19 Cl 2 NO 3 = 416.3 (52315-07-8) additional test.
bp: 170 °C to 195 °C. Assay. Gas chromatography (2.2.28) as prescribed in the
mp: 60 cc to 80 °C. monograph Sweet orange oil (1811).
A suitable certified reference solution (10 ng/µL in Content: minimum 97 per cent, calculated by the
cyclohexane) may be used. normalisation procedure.
L-Cysteine C 3 H 7 NO 2 S = 121.1 (52-90-4) N-Decane Decane; C 10H 22 = 142.3 (124-18-5)
Powder, freely soluble in water, in ethanol (96 per cent) and Colourless liquid, practically insoluble in water.
in acetic acid, practically insoluble in acetone. n~: about 1.411.
Cysteine Hydrochloride (7048-04-6) bp: about 174 °C.
See Cysteine hydrochloride monohydrate (0895). Decan-1-ol Capryl Alcohol; Capric alcohol; Decanol;
L-Cystine C 6 H 12N2O 4S2 = 240.3 (56-89-3) C10H22 O = 158.3 (112-30-1)
White or almost white, crystalline powder, practically Viscous liquid, solidifying at about 6 °C, practically insoluble
insoluble in water and in ethanol (96 per cent). It dissolves in in water, soluble in ethanol (96 per cent).
dilute solutions of alkali hydroxides. nt0 : about 1.436.

[a]~: -218 to -224, determined in 1 M hydrochloric acid. bp: about 230 °C.
mp: 250 °C, with decomposition. Decloxizine Hydrochloride 2-[2-( 4-benzhydryl-1-
Cytochrome c Ferricytochrome c (oxidized state); piperazinyl)ethoxy] ethanol; C 21 H 28N 2O2.2HC1 = 413.39
(9007-43-6) (3733-63-9)
Purity, minimum 95%. Analytical reagent grade of commerce.
Cytosine C4 H 5N 3 0 = 111.1 (71-30-7) Defluorohydroxy-PSMA-1007 (3S,10S,14S)-l-[4-[[(2S)-
4-Carboxy-2-[ (2S)-4-carboxy-2-( 6-hydroxypyridin-3-amido)
Content: minimum 95.0 per cent.
butanamido)butanamido) methyl)phenyl]-3-[ (naphthalen-2-yl)
Daidzein 7-Hydroxy-3-( 4-hydroxyphenyl)-4H- l- methyl)-1,4, 12-trioxo-2,5, 11,13-tetraazahexadecane-
benzopyran-4-one; C 15H 10 O4 = 254.2 (486-66-8) 10, 14, l 6-tricarboxylic acid; C 49H 56N 8O 17 = 1029
Daidzin Daidzein-7-O-glucoside; 7-(~-D- White or almost white powder.
Glucopyranosyloxy)-3-(4-hydroxyphenyl)-4H- l -benzopyran-
4-one; C 21 H 20 O 9 = 416.4 (552-66-9) Defluorotrimethylaminium-PSMA-1007
Trifluoroacetate 5-[[(2S)-4-Carboxy-l-[[(2S)-4-carboxy-
o,p' -DDD 1-(2-Chlorophenyl)-l-(4-chlorophenyl)-2,2- l-[[[4-[(3S,10S,14S)-l 0, 14-dicarboxy-17-hydroxy-3-
dichloroethane; C 14H 10Cl4 = 320.0 (53-19-0) [(naphthalen-2-yl)methyl)-l,4, 12, 17-tetraoxo-2,5, 11, 13-
A suitable certified reference solution (10 ng/µL in tetraazaheptadecan- l-yl)phenyl]methyl) amino )-1-oxobutan-2-
cyclohexane) may be used. yl) amino)-1-oxobutan-2-yl] carbamoyl]-N,N,N-
p,p 1-DOD 1, 1-Bis(4-chlorophenyl)-2,2-dichloroethane; trimethylpyridin-2-aminium trifluoroacetate;
C1 4H10Cl 4 = 320.0 (72-54-8) Cs4H 64F3N9O 18 = 1184 (2226894-58-0)
bp: about 193 °C. White or almost white powder.
mp: about 109 °C. Dehydrocostus Lactone (3aS,6aR, 9aR, 9bS)-3,6, 9-
A suitable certified reference solution (10 ng/µL in Trismethylenedecahydroazuleno [4,5-b) furan-2(3H)-one;
cyclohexane) may be used. C1 5H1 8O2 = 230.3 (477-43-0)
V-A58 Appendix I A 2023

Deltamethrin C 22 H 19Br2 NO 3 = 505.2 (52918-63-5) Deuterated Dimethyl Sulfoxide C 2D 6 OS; Deuterated

bp: about 300 °C. dimethyl sulphoxide; (2H 6)-dimethyl sulphoxide;
mp: about 98 °C. C/H 6 OS = 84.2 (2206-27-1)
Degree of deuteration: minimum 99.8 per cent.
A suitable certified reference solution (10 nglµL in
cyclohexane) may be used. Very hygroscopic liquid, practically colourless, viscous,
soluble in water, in acetone and in anhydrous ethanol.
Demeclocycline Hydrochloride
See Demeclocycline hydrochloride (0176). d~g: about 1.18.
mp: about 20 °C.
Demethylflumazenil Ethyl 8-fluoro-6-oxo-5,6-dihydro-
4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate; Water and deuterium oxide: maximum 0.1 per cent.
C 14H 12FN3O 3 = 289.3 (79089-72-8) Storage: in an airtight container.
Colourless needles, soluble in dimethyl sulfoxide and in hot Deuterated Methanol CD4O; C 2H 4 O = 36.1 (811-98-3)
methanol. Degree of deuteration: minimum 99.8 per cent.
mp: about 288 °C. Clear, colourless liquid miscible with water, with ethanol
14-Demcy-11,12-didehydroandrographolide 3-[(lE)-2- (96 per cent) and with methylene chloride.
[(1R,4aS,5R,6R,8aR)-6-Hydroxy-5-(hydroxymethyl)-5,8a- dig: about 0.888.
dimethyl-2-methylenedecahydronaphthalen- l-yl] ethenyl]
furan-2(5H)-one; C 20 H 28O 4 = 332.4 (42895-58-9)
ni0 : about 1.326.

bp: 65.4 °C.

2 '-Deoxyuridine l-(2-Deoxy-~-d-erythro-pentofuranosyl)-
1H,3H-pyrimidine-2,4-dione; C 9 H 12N 2 O 5 = 228.2
Deuterated N-Nitroso-diethylamine N,N-Bis[(2H 5 )
(951-78-0)
ethyl]nitrous amide; NDEA-d10; C/H 10N 2 O = 112.2
(1219794-54-3)
mp: about 165 °C.
Degree of deuteratwn: minimum 98 per cent.
Chromatography. Thin-layer chromatography (2.2.27) as
prescribed in the monograph Idoxuridine (0669): apply 5 µL
Deuterated Sodium T rimethylsilylpropionate
of a 0.25 glL solution; the chromatogram shows only one C6H9D4NaO 2 Si; C 6 H/H4 NaO 2 Si = 172.3 (24493-21-8)
principal spot. Degree of deuteratwn: minimum 98 per cent.
4-Desoxypyridoxine Hydrochloride 5-(Hydroxymethyl)- White or almost white powder.
2,4-dimethylpyridin-3-ol; 4-Deoxypyridoxine hydrochloride; Deuterium Chloride DC!; 2 HCI = 37.47 (7698-05-7)
C 8 H 12NO2Cl = 189.6 (148-51-6) Gas.
Destaining Solution Degree of deuteratwn: minimum 99 per cent.
A mixture consisting of 1 volume of glacial acetic acid R, Caution: toxic.
4 volumes of methanol R and 5 volumes of water R.
Deuterium Chloride Solution
Desmethylmisonidazole (2RS)-3-(2-Nitro-1H-imidazol- Dilute 1 mL of deuterium chloride R (38 per cent m/m) with
1-yl)propane-1,2-diol; C 6H 9 N 3O 4 = 187.2 (13551-92-3)
5 mL of deuterium oxide R.
Content: minimum 95 per cent. Deuterium Oxide D2O; 2 H 2 O = 20.03 (7789-20-0)
Yellow powder.
Degree of deuteration: minimum 99.7 per cent.
Deuterated Acetic Acid C 2D 4 O 2 ; C/H4 O 2 = 64.1 d~g: about 1.11.
(1186-52-3)
n~: about 1.328.
Degree of deuteration: minimum 99. 7 per cent.
bp: about 101 °C.
dJg: about 1.12. Deuterium Oxide, Isotopically Pure
n~t about 1.368. Deuterium oxide Rl; 2 H 2 O = 20.03 (7789-20-0)
bp: about 115 °C.
Degree of deuteration: minimum 99.95 per cent.
mp: about 16 °C.
Deuterochloroform (2H)-Chloroform; Chloroform-d;
Deuterated Acetone C 3D 6 O; Deuteroacetone; CDC13; Deuterated chloroform; C 2 HCl3 = 120.4 (865-49-6)
C/H6 0 = 64.1 (666-52-4') Degree of deuteration: minimum 99. 7 per cent.
Degree of deuteration: minimum 99.5 per cent.
Clear, colourless liquid, practically insoluble in water,
Clear, colourless liquid, miscible with water, with miscible with acetone and with ethanol (96 per cent). It may
dimethylformamide, with anhydrous ethanol and with be stabilised over silver foil.
methanol.
d~g: about 1.51.
d~g: about 0.87. nt0 : about 1.445.
nf;1: about 1.357. bp: about 60 °C.
bp: about 55 °C.
Water and deuterium oxide: maximum 0.05 per cent.
Water and deuterium oxide. Not more than 0.1 per cent. Devarda's Alloy (8049-11-4')
Deuterated Acetonitrile C/H 3N = 44.1 (2206-26-0) Copper, 50 parts; aluminium, 45 parts; zinc, 5 parts.
Degree of deuteration: minimum 99.8 per cent.
General reagent grade of commerce containing not more
Clear, colourless liquid, miscible with water, with acetone than 20 ppm of nitrogen as NH4 •
and with methanol.
Developer Solution
dig: about 0.78. Dilute 2.5 mL of a 20 g/L solution of citric acid
nf;1: about 1.344. monohydrate Rand 0.27 mL of formaldehyde R to 500.0 mL
with water R.
2023 Appendix I A V-A59

Dexamethasone C22H 29FO 5 = 392.5 (50-02-2) siliceous frustules of fossil diatoms or of debris of fossil
mp: about 263°. diatoms. The substance may be identified by microscopic
General reagent grade of commerce. examination with a magnification of x 500. The substance is
acid-washed, then water-washed until neutral.
Dextran for Chromatography R2, Cross-linked
Diatomaceous Support, Alkali-washed
Bead-form dextran with a fraction range suitable for the
separation of peptides and proteins with relative molecular Diatomaceous support that has been treated with potassium
masses of 15 x 102 to 30 x 103 . When dry, the beads have hydroxide solution to reduce peak-tailing of basic
a diameter of 20-80 µm. compounds.
Dextran for Chromatography R3, Cross-linked Diatomaceous Support, Silanised Diatomaceous earth
for gas chromatography, silanised
Bead-form dextran with a fraction range suitable for the
separation of peptides and proteins with relative molecular Diatomaceous earth for gas chromatography R silanised with
masses of 4 x 103 to 15 x 104 . When dry, the beads have a dimethyldichlorosilane or other suitable silanising agents.
diameter of 40-120 µm. Diazinon C 12H 21 N 2 O 3 PS = 304.3 (333-41-5)
3,3'-Diaminobenzidine Tetrahydrochloride 3,3',4,4'- bp: about 306 °C.
Biphenyl-tetramine; C 12H 18 Cl4 N 4 , 2H2 O = 396.1 A suitable certified reference solution (10 ng/µL in iso-
(7411-49-6) octane) may be used.
Almost white or slightly pink powder, soluble in water. Diazobenzenesulfonic Acid Solution
mp: about 280 °C, with decomposition. Diazobenzenesulphonic acid solution
1,2-Diamino-4,5-methylenedioxybenzene Heat 0.2 g of sulfanilic acid with 20 mL of lM hydrochloric
Dihydrochloride 2H-l,3-Benzodioxole-5,6-diamine acid until dissolved, cool to about 4" and add, dropwise,
dihydrochloride; C 7 H1 0 Cl 2 N2O 2 = 225.1 (81864-15-5) 2.2 mL of a 4% w/v solution of sodium nitrite, swirling
Content: minimum 99 per cent (HPLC). continuously. Allow to stand in ice for 10 minutes and add
1,3-Diaminopropan-2-one Dihydrochloride 1 mL of a 5% w/v solution of sulfamic acid.
Monohydrate C 3 H 10 Cl2N 2 O,H2O = 179.0 (207226-24-2) Diazobenzenesulfonic Acid Solution Rl
mp: 179 °C, with decomposition. Diazobenzenesulphonic acid solution Rl
Diammonium 2,2 '-azinobis(3-ethylbenzothiazoline-6- Dissolve 0.9 g of sulfanilic acid R in a mixture of 30 mL of
sulfonate) Diammonium 2,2'-azinobis(3- di1ute hydrochloric acid Rand 70 mL of water R. To 3 mL of
ethylbenzothiazoline-6-sulphonate); C 18H 24N 6 O 6 S4 = 548.7 the solution add 3 mL of a 50 g/L solution of sodium
(30931-67-0) nitrite R. Cool in an ice-bath for 5 min, add 12 mL of the
sodium nitrite solution and cool again. Dilute to 100 mL
Chromogenic substrate suitable for use in ELISA procedures. with water R and keep the reagent in an ice-bath. Prepare
Green tablets, freely soluble in water. extemporaneously but allow to stand for 15 min before use.
pH (2.2.3): 4.2 to 5.8 for a 0.1 g/L solution. Dibenzosuberone Dibenzo [a,d] cyclohepta-1,4-dien-3-
Diammonium Hydrogen Orthophosphate Ammonium one; H-dibenzo[a,d]cyclohepten-5-one; C 15 H 12 O = 208.3
phosphate; (NH 4 ) 2 HP0 4 = 132.1 (7783-28-0) (1210-35-1)
White or almost white crystals or granules, hygroscopic, very mp: about 34°.
soluble in water, practically insoluble in ethanol General reagent grade of commerce.
(96 per cent). Dibromomethane CH2Br2 = 173.8 (74-95-3)
pH (2.2.3): about 8 for a 200 g/L solution. Colourless liquid, slightly soluble in water.
Storage: in an airtight container. bp: about 96 °C.
Diatomaceous Filter-aid, Washed, Flux-calcined Di-N-butylamine N-Butylbutan-1-amine; Dibutylamine;
To 500 g of flux-calcined, diatomaceous filter-aid (Celite 545 CsH 19N = 129.3 (111-92-2)
is suitable), add 2000 mL of hydrochloric acid, mix, allow to Colourless liquid.
stand with occasional stirring for 12 hours, filter and wash
the residue with water until the washings are neutral to litmus n~r about 1.417.
paper. Continue washing the residue on the filter paper, using bp: about 159 °C.
500 mL of methanol followed by 1000 mL of a mixture of Dibutylanunonium Phosphate for Ion-pairing
equal volumes of methanol and ether. Finally dry the washed A colourless solution of 10 per cent to 15 per cent V/V of
residue at 100° until the odour of solvent is no longer di-n-butylamine and 12 per cent to 17 per cent V/V of
detectable. It should be stored in an airtight container. phosphoric acid in water, suitable for ion-pairing in liquid
Diatomaceous Support Diatomaceous earth; chromatography.
(91053-39-3) Dibutyl Ether C 8 H 18 O = 130.2 (142-96-1)
White or almost white, fine granular powder, made up of Colourless, flammable liquid, practically insoluble in water,
siliceous frustules of fossil diatoms or of debris of fossil miscible with anhydrous ethanol.
diatoms, practically insoluble in water and in ethanol
(96 per cent).
dfg: about 0.77.
The substance may be identified by microscopic examination
ni0 : about 1.399.

with a magnification of x 500. Do not distil if the dibutyl ether does not comp~v with the test for
peroxides.
Diatomaceous Support, Acid-washed Diatomaceous
earth for gas chromatography Peroxides. Place 8 mL of potassium iodide and starch solution R
in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in
White or almost white, fine granular powder, practically
diameter. Fill completely with the substance to be examined,
insoluble in water and in ethanol (96 per cent), made up of
V-A60 Appendix I A 2023

shake vigorously and allow to stand protected from light for dig: about 1.25.
30 min. No colour is produced. Distillation range (2.2.11). Not less than 95 per cent distils
The name and concentration of any added stabiliser are between 82 °C and 84 °C.
stated on the label. 2, 7-Dichlorofluorescein 2-(2, 7-dichloro-6-hydroxy-3-oxo-
Dibutyl Phthalate Di-N-butyl phthalate; 3H-xanthen-9-yl)benzoic acid; Dichlorofluorescein;
C 16H22O4 = 278.3 (84-74-2) C20H 10 Cl2O 5 = 401.2 (76-54-0)
Clear, colourless or faintly coloured, oily liquid, very slightly Yellowish-brown or yellow-orange powder, slightly soluble in
soluble in water, miscible with acetone and with ethanol water, freely soluble in ethanol (96 per cent) and in dilute
(96 per cent). solutions of alkali hydroxides giving a solution showing a
dfg: 1.043 to 1.048. yellowish-green fluorescence.
ngi: 1.490 to 1.495. 5, 7-Dichloro-8-hydroxyquinoline 5, 7-Dichlorooxine;
5,7-Dichloroquinolin-8-ol; C 9H 5 Cl2 NO = 214.1 (773-76-2)
Dicarboxidine Hydrochloride 4,4'-[(4,4'-
Diaminobiphenyl-3,3'-diyl)dioxy]dibutanoic acid Yellow, crystalline powder, soluble in acetone, slightly soluble
dihydrochloride; C 20H 26 Cl 2 N 2 O 6 = 461.3 (56455-90-4') in ethanol (96 per cent).
Dichlofenthion C 10H 13 Cl 2 O 3 PS = 315.2 (97-17-6) mp: about 179 °C.
A suitable certified reference solution (10 nglµL in Content: minimum 95.0 per cent.
cyclohexane) may be used. Dichloromethane Methylene chloride; CH2 C]z = 84.9
Dichloroacetic Acid C 2H 2 Cl 2 O 2 = 128.9 (79-43-6) (75-09-2)
Colourless liquid, miscible with water and ethanol Colourless liquid, sparingly soluble in water, miscible with
(96 per cent). ethanol (96 per cent).
d~g: about 1.566. bp: 39 °C to 42 °C.
ngi: about 1.466. Methylene chloride used in fluorimetry complies with the
following additional test.
bp: about 193 °C.
Fluorescence. Under irradiation at 365 nm, the fluorescence
Dichloroacetic Acid Solution
(2.2.21) measured at 460 nm in a 1 cm cell is not more
Dilute 67 mL of dichloroacetic acid R to 300 mL with water R intense than that of a solution containing 0.002 ppm of
and neutralise to blue litmus paper R using ammonia R. Cool, quinine R in 0. 5 M suljuric acid measured in the same
add 33 mL of dichloroacetic acid Rand dilute to 600 mL with conditions.
water R.
Dichloromethane, Acidified Methylene chloride,
3,5-Dichloroaniline 3,5-dichlorophenylamine; acidified
C 6H 5 Cl2N = 162.0 (626-43-7)
To 100 mL of methylene chloride R add 10 mL of hydrochloric
mp: 46 °C to 52 °C. acid R, shake, allow to stand and separate the two layers.
1,2-Dichlorobenzene Dichlorobenzene; C 6H 4 Cl2 == 147.0 Use the lower layer.
(95-50-1) Dichloromethane IR
Colourless, oily liquid, practically insoluble in water, soluble Spectroscopic reagent grade of commerce.
in anhydrous ethanol.
Dichloromethane Rl Methylene Chloride Rl
g:
df about 1.31. Content (2.2.28): minimum 99.8 per cent.
bp: about 180 °C.
Dichloromethane Reagent
2,4-Dichlorobenzoic Acid C 7 H 4 Cl 2 O 2 = 191.0 (50-84-0)
Add 50 g of sodium hydrogen carbonate to 1000 mL of
Faintly beige powder. dichloromethane, allow to stand overnight and filter.
mp: about 160 °C. 2,4-Dichloro-1-naphthol C 10H 6Cl 2 O = 213.1
2,3-Dichloro-5,6-dicyanobenzoquinone 4,5-Dichloro- (2050-76-2)
3,6-dioxo-cyclohexa- l ,4-diene-1,2-dicarbonitrile; mp: about 107°.
CsC12N 2O 2 = 227.0 (84-58-2)
General reagent grade of commerce.
Yellow or orange crystals, soluble in dioxan and in acetic
acid, slightly soluble in methylene chloride. It decomposes in
2,6-Dichlorophenol C 6H 4 Cl2O = 163.0 (87-65-0)
water. mp: 64 °C to 66 °C.
mp: about 214 °C. 2,6-Dichlorophenolindophenol Sodium Salt The
sodium derivative of 2,6-dichloro-N-(4-hydroxyphenyl)-1,4-
Storage: at a temperature of 2 °C to 8 °C.
benzoquinone monoimine; Tillman's reagent;
(S)-3 ,5-Dichloro-2,6-dihydroxy-N-[ (1-ethylpyrrolidin- Dichlorophenolindophenol, sodium salt;
2-yl)methyl]benzamide Hydrobromide C,2H6Cl2NNaO2,2H2O = 326.1 (620-45-1)
C1 4 H 19 BrC12N 2 O 3 = 414.1 (113310-88-6)
Dark-green powder, freely soluble in water and in anhydrous
White or almost white, crystalline powder. ethanol. The aqueous solution is dark blue; when acidified it
[o:]f;: + 11.4, determined on a 15.0 glL solution in anhydrous becomes pink.
ethanol R. 2,6-Dichlorophenolindophenol Solution
mp: about 212 °C. Warm O.1 g of 2, 6-dichlorophenolindophenol sodium salt with
1,2-Dichloroethane Ethylene chloride; C 2 H 4 Cl 2 = 99.0 100 mL of water and filter.
(107-06-2) Use within 3 days of preparation.
Clear, colourless liquid, soluble in about 120 parts of water
and in 2 parts of ethanol (96 per cent).
2023 Appendix I A V-A61

2 ,6-Dichlorophenolindophenol Solution, Double- Didodecyl 3,3'-thiodipropionate C 30H 58 O 4S = 514.8

strength Standard (123-28-4)
Dissolve 0.1 g of 2,6-dichlorophenolindnphenol sodium salt in White or almost white, crystalline powder, practically
100 mL of water, filter, standardise by the method described insoluble in water, freely soluble in acetone and in light
under standard dichlorophenolindophenol solution and dilute the petroleum, slightly soluble in ethanol (96 per cent).
solution with water so that 1 mL is equivalent to 0.2 mg of mp: about 39 °C.
ascorbic acid. Dieldrin C 12 H 8 Cl6 O = 380.9 (60-57-1)
Use within 3 days of preparation and standardise bp: about 385 °C.
immediately before use.
mp: about 176 °C.
Dichlorophenolindophenol Solution, Standard
A suitable certified reference solution (10 nglµL in
Dissolve 50.0 mg of dichlorvphenolindnphenol, sodium salt R in cyclohexane) may be used.
100.0 mL of water Rand filter.
Diethanolamine 2,2 '-Iminobisethanol;
Assay. Dissolve 20.0 mg of ascorbic acid R in 10 mL of a C4HllNO2 = 105.1 (111-42-2)
freshly prepared 200 g/L solution of metaphosphoric acid R
and dilute to 250.0 mL with water R. Titrate 5.0 mL rapidly Viscous, clear, slightly yellow liquid or deliquescent crystals
with the dichloro-phenolindophenol standard solution, added melting at about 28 °C, very soluble in water, in acetone and
from a microburette graduated in 0.01 mL, until the pink in methanol.
colour persists for 10 s, the titration occupying not more than dig: about 1.09.
2 min. Dilute the dichlorophenolindophenol solution with pH (2.2.3): 10.0 to 11.5 for a 50 glL solution.
water R to make 1 mL of the solution equivalent to 0.1 mg Diethanolamine used in the test for alkaline phosphatase complies
of ascorbic acid (C 6 HsO6)- with the following additional test.
Storage: use within 3 days. Ethanolamine. Gas chromatography (2.2.28).
Standardise immediately before use. Internal standard solution. Dissolve 1.00 g of
2,6-Dichloroquinone-4-chloroimide 3-aminoprvpanol R in acetone Rand dilute to 10.0 mL with
Dichloroquinonechlorimide; C 6 H 2 Cl3 NO = 210.4 (101-38-2) the same solvent.
Pale yellow or greenish-yellow crystalline powder, practically Test solution (a). Dissolve 5.00 g of the substance to be
insoluble in water, soluble in ethanol (96 per cent) and in examined in acetone Rand dilute to 10.0 mL with the same
dilute alkaline solutions. solvent.
mp: about 66 °C. Test solution (b). Dissolve 5.00 g of the substance to be
Dichlorvos 2,2-Dichlorovinyl dimethyl phosphate; examined in acetone R, add 1.0 mL of the internal standard
C 4 H7Cl 2 O 4 P = 221 (62-73-7) solution and dilute to 10.0 mL with the same solvent.
Colourless or brownish-yellow liquid, soluble in water, Reference solutions. Dissolve 0.50 g of ethanolamine R in
miscible with most organic solvents. acetone Rand dilute to 10.0 mL with the same solvent.
n;}: about 1.452. To 0.5 mL, 1.0 mL and 2.0 mL of this solution, add 1.0 mL
of the internal standard solution and dilute to 10.0 mL with
Di-2-cyanoethyl Ether (2-Cyanoethyl) ether; acetone R.
C 6H 8 N 2O = 124.1 (1656-48-0)
Column:
nfr 1.4400. - size: l = l m, 0 = 4 mm;
bp: about 111 °. - stationary phase: diphenylphenylene oxide polymer R
Chromatographic grade of commerce. (180-250 µm).
Dicyclohexyl Bicyclohexyl; C 12 H 22 = 166.3 (92-51-3) Carrier gas: nitrogen for chromatography R.
diS: about 0.864. Flow rate: 40 mLJmin.
bp: about 227 °C. Temperature:
mp: about 4 °C.
Time Temperature
Dicyclohexylamine N,N-Dicyclohexylamine; (min) (C)
C 12 H 23N = 181.3 (101-83-7)
Column 0-, 3 125
Colourless liquid, sparingly soluble in water, miscible with 3-> 17.6 125 ➔ 300
the usual organic solvents. Injection port 250
nt 0 : about 1.484.
Detector 280
bp: about 256 °C.
Freezing point (2.2.18): 0 °C to 1 °C. Detection: flame-ionisation.
1,3-Dicyclohexylurea Dicyclohexylurea; Injection: 1.0 µL.
C13H2 4N 2O = 224.4 (2387-23-7) Limit:
White or almost white, crystalline powder. - ethanolamine: maximum 1.0 per cent.
mp: about 232 °C. 2,5-Diethoxytetrahydrofuran Diethoxytetrahydrofuran, a
Didocosahexaenoin Diglyceride of docosahexaenoic acid mixture of the cis and trans Isomers; C 8 H 16O 3 = 160.2
(C22:6); Glycerol didocosahexaenoate; (all-Z)- (3320-90-9)
Docosahexaenoic acid, diester with propane-1,2,3-triol; Clear, colourless or slightly yellowish liquid, practically
C47H6 8 O 5 = 713.0 (88315-12-2) insoluble in water, soluble in ethanol (96 per cent) and in
The reagent from Nu-Chek Prep, Inc. has been found most other organic solvents.
suitable dig: about 0.98.
V-A62 Appendix I A 2023

nt 0:about 1.418. Diethylphenylenediamine Sulfate Solution

Diethylamine C 4H 11 N = 73.1 (109-89-7) Diethylphenylenediamine sulphate solution
Clear, colourless, flammable liquid, strongly alkaline, miscible To 250 mL of water R add 2 mL of sulfuric acid Rand
with water and with ethanol (96 per cent). 25 mL of 0. 02 M sodium edetate. Dissolve in this solution
1.1 g of diethylphenylenediamine sulfate R and dilute to
d~g: about 0.71. 1000 mL with water R.
bp: about 55 °C.
Do not use if the solution is not colourless.
Diethylamine Rl N-Ethylethanamine; C 4H 11 N = 73.1
Storage: protected from light and heat for 1 month.
(109-89-7)
Diethyl Sulfone 1-(Ethylsulfonyl)ethane;
Content: minimum 99.5 per cent.
1-(Ethanesulfonyl)ethane; C 4 H 10 O 2S = 122.2 (597-35-3)
Clear, colourless, flammable liquid, strongly alkaline, miscible
Content: minimum 97 per cent.
with water and with ethanol (96 per cent).
Crystalline powder.
d~g: about 0.71.
mp: about 73 °C.
bp: about 55 °C.
Diflubenzuron 1-( 4-Chlorophenyl)-3-(2,6-
Diethylaminoethyldextran
difluorobenzoyl)urea; C 14H 9 ClF2 N 2 O 2 = 310.7 (35367-38-5)
Anion-exchange resin presented as the hydrochloride.
Colourless or white or almost white crystals, practically
Powder forming gels with water. insoluble in water, freely soluble in dimethyl sulfoxide,
N,N-Diethylaniline C 10H 15N = 149.2 (91-66-7) slightly soluble in acetone.
dig: about 0.938. mp: 230 to 232 °C.
bp: about 217 'C. Digitonin 3 B-[ O-B-D-Glucopyranosyl-(1 ➔ 3)-O-B-D-
mp: about -38 °C. galactopyranosyl-(l-+ 2)-O-[B-D-xylopyranosyl-(1-+ 3)]-O-B-D-
Diethylene Glycol Digol; C 4 H 10O 3 = 106.1 (111-46-6) galactopyranosyl-(l -+4)-O-B-D-galactopyranosyloxy]-(25R)-
51X-spirostan-21X,15B-diol; C 56 H 92 O 29 = 1229 (11024-24-1)
Content: minimum 99.5 per cent mlm.
Crystals, practically insoluble in water, sparingly soluble in
Clear, colourless liquid, hygroscopic, miscible with water,
anhydrous ethanol, slightly soluble in ethanol (96 per cent).
with acetone and with ethanol (96 per cent).
Digitoxin (71-63-6)
dJg: about 1.118.
See Digitoxin (0078).
n;!7:about 1.447.
Diglycine 2-[(2-Aminoacetyl)amino]acetic acid;
bp: 244 °C to 246 °C.
Glycylglycine; C 4H 8 N 2O3 = 132.1 (556-50-3)
Storage: in an airtight container.
Digoxin See Digoxin (0079).
N,N-Diethylethane-1,2-diamine
Digoxin Reagent
N,N-Diethylethylenediamine; C 6 H 16N 2 = 116.2 (100-36-7)
Add 98 mL of glacial acetic acid to 2 mL of sulfuric acid and
Content: minimum 98.0 per cent.
add 0.1 mL of a 5% w/v solution of anhydrous iron(III)
Slightly oily liquid, colourless or slightly yellow, strong odour chloride in glacial acetic acid.
of ammonia, irritant to the skin, eyes and mucous
Dihydrocapsaicin N-[ (4-Hydroxy-3-methoxyphenyl)
membranes.
methyl]-8-methylnonanamide; C 18 H 29NO 3 = 307.4
dig: 0.827. (I 9408-84-5)
bp: 145 °C to 147 cc. White or almost white, crystalline powder, practically
Water (2.5.12): maximum 1.0 per cent, determined on insoluble in cold water, freely soluble in anhydrous ethanol.
0.500 g. 10, 11-Dihydrocarbamazepine 10, l 1-Dihydro-5H-
Di(2-ethylhexyl)Phthalate Dioctyl phthalate; dibenzo [b.J] azepine-5-carboxamide; C 15H 14N 2 O = 238.3
C24H3sO4 = 390.5 (3564-73-6)
Colourless, oily liquid, practically insoluble in water, soluble mp: 205 °C to 210 °C.
in organic solvents. Dihydrocarvone p-Menth-8-en-2-one; 2-Methyl-5-(1-
dig: about 0.98. methylethenyl)cyclohexanone; C 10H 16O = 152.2 (7764-50-3)
n~7: about 1.486. Dihydrocarvone used in gas chromatography complies with the
Viscosiry (2.2.9): about 80 mPa-s. fallowing additional test.
Diethyl Phthalate C 12 H 14O4 = 222.2 (84-66-2) Assay. Gas chromatography (2.2.28) as prescribed in the test
bp: about 298°. for chromatographic profile in the monograph Caraway
oil (1817).
General reagent grade of commerce.
Content calculated by the normalisation procedure:
N,N-Diethyl-p-phenylenediamine Sulfate N,N-Diethyl-
- major component (trans-dihydrocarvone): minimum
p-phenylenediamine sulphate; Diethylphenylenediamine
70 per cent;
sulphate; Diethylphenylenediamine sulfate;
- sum of cis- and trans-dihydrocarvone: minimum 98 per cent.
C10H1sN2O4S = 262.3 (6283-63-2)
1,8-Dihydroxyanthraquinone Danthron;
White or slightly yellow powder, soluble in water.
Dihydroxyanthraquinone; 1,8-Dihydroxyanthracene-9, 10-
mp: about 185 °C, with decomposition. dione; Dantron; C 14H 8 O4 = 240.2 (117-10-2)
Storage: protected from light. Crystalline orange powder, practically insoluble in water,
slightly soluble in ethanol (96 per cent), soluble in solutions
of alkali hydroxides.
mp: about 195 °C.
2023 Appendix I A V-A63

2 ,4-Dihydroxybenzaldehyde P-Resorcylaldehyde; Clear, colourless or light yellow liquid.

C 7 H 6 O 3 = 138.1 (95-01-2) bp: 127 °C.
2,5-Dihydroxybenzoic Acid Gentisic acid; N,N' -Di-isopropylethylened.iamine N,N' -Bis( l-
C 7 H 6 O 4 = 154.1 (490-79-9) methylethyl)-1,2-ethanediamine; C 8 H 20N 2 = 144.3
Light yellow crystals. ( 4013-94-9)
mp: about 200 °C. Colourless or yellowish, corrosive, flammable, hygroscopic
5, 7-Dihydroxy-4-methylcoumarin 5, 7-Dihydroxy-4- liquid.
methyl-2H- l-benzopyran-2-one; C 10H 8 O 4 = 192.2 d~g: about 0.798.
(2107-76-8) n5°: about 1.429.
Light yellowish powder, practically insoluble in water, bp: about 170 °C.
sparingly soluble in ethanol (96 per cent).
Dillapiole 6-Allyl-4,5-dimethoxy-1,3-benzodioxole;
mp: 295 °C to 303 °C. C 12H14O4 = 222.24 (523-80-8)
1,3-Dihydroxynaphthalene N aphthalene-1,3-diol; General reagent grade of commerce.
C 10H 8 O 2 = 160.2 (132-86-5)
2,5-Dimethoxybenzaldehyde C 8H 10 O 3 = 166.2 (93-02-7)
Crystalline, generally brownish-violet powder, freely soluble
mp: about 50°.
in water and in ethanol (96 per cent).
General reagent grade of commerce.
mp: about 125 °C.
4,4 '-Dimethoxybenzophenone Bis (4-methoxyphenyl)
5, 7-Di-iodo-8-hydroxyquinoline 5, 7-Diiodoquinolin-8-
methanone; C 15 H 14O 3 = 242.3 (90-96-0)
ol; C 9 H 512 NO = 397.0 (83-73-8)
White or almost white powder, practically insoluble in water
Yellowish-brown powder, sparingly soluble in acetone and in
and slightly soluble in ethanol (96 per cent).
ethanol (96 per cent).
mp: about 142 °C.
Content: minimum 95.0 per cent.
3,4-Dimethoxyphenethylamine C 10H1 5NO 2 = 181.2
1,5-Di-iodopentane C 5H 1012 = 323.9 (628-77-3) (120-20-7)
bp: about 101 °.
n~: about 1.546.
General reagent grade of commerce.
An oily liquid; weight per mL, about 1.07 g.
A colourless liquid.
General reagent grade of commerce.
Di-isobutyl Ketone 2,6-Dimethyl-4-heptanone;
3,4-Dimethoxy-L-phenylalanine (2S)-2-Amino-3-(3,4-
C 9 H 18 O = 142.2 (108-83-8)
dimethoxyphenyl)propanoic acid; C 11 H 15NO 4 = 225.2
Clear, colourless liquid, slightly soluble in water, miscible (32161-30-1)
with most organic solvents.
Content: minimum 95 per cent.
n5°: about 1.414 White or almost white powder.
bp: about 168 °C.
2,2-Dimethoxypropane Dimethoxypropane;
Di-isopropyl Ether lsopropyl ether; C 6 H 14 O = 102.2 C 5H 12O 2 = 104.1 (77-76-9)
(108-20-3)
Colourless liquid, decomposing on exposure to moist air or
Clear, colourless liquid, very slightly soluble in water, water.
miscible with ethanol (96 per cent).
d?g: about 0.847.
dig: 0.723 to 0.728. 115°: about 1.378.
bp: 67 °C to 69 °C.
bp: about 83 °C.
Do not distil if the di-isopropyl ether does not comply with the test
Dimethylacetamide N,N-Dimethylacetamide;
for peroxides.
C 4H 9NO = 87.1 (127-19-5)
Peroxides. Place 8 mL of potassium iodide and starch solution R
Content: minimum 99.5 per cent.
in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in
diameter. Fill completely with the substance to be examined, Colourless liquid, miscible with water and with many organic
shake vigorously and allow to stand protected from light for solvents.
30 min. No colour is produced. d?g: about 0.94.
The name and concentration of any added stabiliser are 115°: about 1.437.
stated on the label. bp: about 165 °C.
Storage: protected from light. Dimethylamine N-Methylmethanamine; C 2 H 7 N = 45.09
Di-isopropyl Ether, Stabiliser-free (124-40-3)
Di-isopropyl ether that is free from stabiliser. Content: minimum 98.0 per cent.
Reagent grade of commerce. Colourless, flammable gas.
Di-isopropylethylamine Ethyldi-isopropylamine; Dimethylamine Hydrochloride C 2 H 8CIN = 81.6
C 8 H 20N 2 = 144.3 (7087-68-5) (506-59-2)
n~: about 1.414. mp: about 172°.
bp: about 127°. General reagent grade of commerce.
General reagent grade of commerce. Dimethylamine Solution
N,N-Diisopropylethylamine N-Ethyl-N-(propan-2-yl) A 400 g/L solution of dimethylamine R.
propan-2-amine; N-Ethyldiisopropylamine; C 8 H 19N = 129.2 Clear, colourless solution.
(7087-68-5) Density: about 0.89.
V-A64 Appendix I A 2023

bp: about 54 °C. Dimethylaminoethanol 2-(Dirnethylamino )ethan-1-ol;

mp: about -37 °C. C4H 11 NO = 89.1 (108-01-0)
4-Dimethylaminobenzaldehyde Colourless or slightly yellow liquid, miscible with water.
Dimethylaminobenzaldehyde; C 9 H 11 NO = 149.2 (100-10-7) bp: about 135 °C.
White or yellowish-white crystals, soluble in ethanol 2-(Dimethylamino)ethyl Methacrylate
(96 per cent) and in dilute acids. 2-(Dimethylamino)ethyl 2-methylpropenoate;
mp: about 74 °C. CsH 15 NO2 = 157.2 (2867-47-2)
Dimethylaminobenzaldehyde Reagent di 0 : about 0.930.

Dissolve 0.5 g of 4-dimethylaminobenzaldehyde in a cooled bp: about 187 °C.

mixture of 53 mL of sulfuric acid and 50 mL of water and Dimethylaminonaphthalenesulfonyl Chloride
add 0.5 mL of iron(m) chloride solutwn Rl. Dirnethylaminonaphthalenesulphonyl chloride;
Allow to stand for 2 hours before use. 5-Dirnethylaminonaphthalene-1-sulphonyl chloride; Dansyl
chloride; C 12H 12 ClNO 2 S = 269.8 (605-65-2)
Dimethylaminobenzaldehyde Solution, Alcoholic
Dissolve 1 g of 4-dimethylaminobenzaldehyde in 30 mL of Yellow, crystalline powder, slightly soluble in water, soluble
in methanol.
ethanol (96%) and add 180 mL of butan-1-ol and 30 mL of
hydrochloric acid. mp: about 70 °C.
Prepare immediately before use and discard if a pink colour 3-Dimethylaminophenol 3-(Dimethylamino)phenol;
develops. CsH 11 NO = 137.2 (99-07-0)
Dimethylaminobenzaldehyde Solution Rl Grey powder, slightly soluble in water.
Dissolve 0.2 g of dimethylaminobenzaldehyde R in 20 mL of mp: about 80 °C.
ethanol (96 per cent) Rand add 0.5 mL of hydrochloric acid R. 2-(Dimethylamino)thioacetamide Hydrochloride
Shake the solution with activated charcoal Rand filter. C 4 H11ClN 2 S = 154.7 (27366-72-9)
The colour of the reagent is less intense than that of iodine Dimethylaniline N,N-Dirnethylaniline; (J 21-69-7)
solutwn R3. Prepare immediately before use.
See N,N-Dimethylanuine R.
Dimethylaminobenzaldehyde Solution R2 N,N-Dimethylaniline C 8 H 11 N = 121.2 (121-69-7)
Dissolve 0.2 g of dimethylaminobenzaldehyde R, without Clear, oily liquid, almost colourless when freshly distilled,
heating, in a mixture of 4.5 mL of water R and 5.5 mL of darkening on storage to reddish-brown, practically insoluble
hydrochloric acid R. Prepare immediately before use.
in water, freely soluble in ethanol (96 per cent).
Dimethylaminobenzaldehyde Solution R6 nt0 : about 1.558.
Dissolve 0.125 g of dimethylaminobenzaldehyde Rina cooled Distillatwn range (2.2.11). Not less than 95 per cent distils
mixture of 35 mL of water Rand 65 mL of suljuric acid R. between 192 °C and 194 °C.
Add 0.1 mL of a 50 g/L solution of ferric chloride R. Before
use allow to stand for 24 h, protected from light. 2,3-Dimethylaniline 2,3-Xylidine; C 8 H 11N = 121.2
(87-59-2)
Storage: when stored at room temperature, use within
Yellowish liquid, sparingly soluble in water, soluble in
1 week; when stored in a refrigerator use within several
ethanol (96 per cent).
months.
Dimethylaminobenzaldehyde Solution R7 dig: 0.993 to 0.995.
Dissolve 1.0 g of dimethylaminobenzaldehyde R in 50 mL of ng1: about 1.569.
hydrochloric acid R and add 50 mL of ethanol (96 per cent) R. bp: about 224 °C.
Storage: protected from light; use within 4 weeks. 2,4-Dimethylaniline 2,4-Xylidene; C 8 H 11 N = 121.2
(95-68-1)
Dimethylaminobenzaldehyde Solution R8
Dissolve 0.25 g of dimethylaminobenzaldehyde Rina mixture General reagent grade of commerce.
of 5 g of phosphoric acid R, 45 g of water R and 50 g of 2,6-Dimethylaniline 2,6-Xylidine; C 8 H 11 N = 121.2
anhydrous acetic acid R. Prepare immediately before use. (87-62-7)
Dimethylaminobenzaldehyde Solution R9 Colourless liquid, sparingly soluble in water, soluble in
Dissolve 1.0 g of dimethylaminobenzaldehyde R in 3.5 mL of ethanol (96 per cent).
perchloric acid (600 g/L HCI04 ) and slowly add 6.5 mL of d~g: about 0.98.
2-propanol R. Prepare immediately before use. 2,6-Dimethylaniline Hydrochloride
4-Dimethylaminocinnamaldehyde 3-( 4- 2,6-Dirnethylbenzenamide hydrochloride; 2,6-Xylidine
Dirnethylaminophenyl)prop-2-enal; C 11 H 13NO = 175.2 hydrochloride; C 8 H 12 ClN = 157.6 (21436-98-6)
(6203-18-5) Content: minimum 98.0 per cent.
Orange or orange-brown crystals or powder. Sensitive to 2,4-Dimethyl-6-tert-butylphenol 6-tert-Butyl-2,4-
light. dimethylphenol; C 12H 18 O = 178.3 (1879-09-0)
mp: about 138 °C. Dimethyl Carbonate Carbonic acid dimethyl ester;
4-Dimethylaminocinnamaldehyde Solution C3H5O3 = 90.1 (616-38-6)
Dissolve 2 g of 4-dimethylaminocinnamaldehyde R in a mixture Liquid, insoluble in water, miscible with ethanol
of 100 mL of hydrochloric acid Rl and 100 mL of anhydrous (96 per cent).
ethanol R. Dilute the solution to four times its volume with df: 1.065.
anhydrous ethanol R immediately before use. ng1: 1.368.
bp: about 90 °C.
2023 Appendix I A V-A65

Dimethyl-~-cyclodextrin Heptakis(2,6-di-O-methyl) White or almost white, crystalline powder or colourless

cyclomaltoheptaose; Cycloheptakis-(1->4)-(2,6-di-O-methyl- crystals, practically insoluble in cold water, very slightly
cr-o-glucopyranosyl); soluble in boiling water, soluble in ethanol (96 per cent).
2A 28 2c 2° 2E 2F 2G 6A 68 6c 6° 6E 6F 6G-Tetradeca-O- mp: about 240 'C, with decomposition.
H0
m~th;l-~~cy~lodex~rin: c'56 98 35 , = '1331 (51166-71-3)
Sulfated ash (2.4.14): maximum 0.05 per cent.
White or almost white powder.
1,3-Dimethyl-2-imidazolidinone N,N' -Dimethylethylene
Dimethyldecylamine N,N-dimethyldecylamine; urea; 1,3-Dimethyl-2-imidazolidone; C 5H10N2O = 114.2
C 12H 27N = 185.4 (1120-24-7) (80-73-9)
Content: minimum 98.0 per cent mlm. nt 0:
1.4720.
bp: about 234 cc. bp: about 224 °C.
1, 7-dimethyl-3, 7-dihydro-lH-purine-2,6-dione N,N-Dimethyl-p-nitrosoaniline C 8 H 10N2O = 150.2
paraxanthine; 1,7-dimethylxanthine; C 7 H 8N 4 O2 = 180.16 (138-89-6)
(611-59-6)
mp: about 86°.
mp: about 298c
General reagent grade of commerce.
General reagent grade of commerce.
Green crystals or a green, crystalline powder.
N,N-Dimethyldodecylam.ine N-oxide N,N-Dimethyloctylam.ine Octyldimethylamine;
C 14H 31 NO = 229.4 (1643-20-5)
C 10H 23N = 157.3 (7378-99-6)
mp: about 132°.
Colourless liquid.
General reagent grade of commerce.
d~S: about 0.765.
1,1-Dimethylethylam.ine 2-Amino-2-methylpropane;
tert-Butylamine; C 4HllN = 73.1 (75-64-9)
nb0: about 1.424.
bp: about 195 °C.
Liquid, miscible with ethanol (96 per cent).
2,5-Dimethylphenol p-Xylenol; CsH1oO = 122.2
d~g: about 0.694. (95-87-4)
nb 0 : about 1.378.
White or almost white crystals.
bp: about 46 °C.
2,6-Dimethylphenol C 8 H 10 O = 122.2 (576-26-1)
1,1-Dimethylethyl Methyl Ether tert-Butyl methyl ether; Colourless needles, slightly soluble in water, very soluble in
2-methoxy-2-methylpropane; CsH 12 O = 88.1 (1634-04-4)
ethanol (96 per cent).
Colourless, clear, flammable liquid.
bp: about 203 °C.
nt 0 : about 1.376.
mp: 46 °C to 48 °C.
Absorbance (2.2.25): maximum 0.30 at 240 nm, 0.10 at 3,4-Dimethylphenol C 8 H 10 O = 122.2 (95-65-8)
255 nm, 0.01 at 280 nm, determined using water Ras
compensation liquid. White or almost white crystals, slightly soluble in water,
freely soluble in ethanol (96 per cent).
1,1-Dimethylethyl Methyl Ether Rl 2-Methoxy-2-
bp: about 226 °C.
methylpropane; tert-Butyl methyl ether; C 5H12O = 88.1
(1634-04-4) mp: 25 °C to 27 °C.
Content: minimum 99.5 per cent. N,N-Dimethyl-L-phenylalanine (2S)-
2-(Dimethylamino)-3-phenylpropanoic acid;
d~g: about 0.741. C 11 H 15NO 2 = 193.2 (17469-89-5)
nb 0 : about 1.369.
mp: about 226 °C.
bp: about 55 °C.
N,N-Dimethyl-p-phenylenediam.ine Dihydrochloride
Dimethylformamide C 3H 7 NO = 73.1 (68-12-2) C 8H 12N 2,2HC1 = 209.1 (536-46-9)
Clear, colourless neutral liquid, miscible with water and with
General reagent grade of commerce.
ethanol (96 per cent).
Darkens readily on exposure to air.
dfg: 0.949 to 0.952. Dimethyl Phthalate C 10 H 10 O 4 = 194.2 (131-11-3)
bp: about 153 °C.
General reagent grade of commerce.
Water (2.5.12): maximum 0.1 per cent.
A colourless or faintly coloured liquid; weight per mL, about
Dimethylformamide Diethylacetal 1.19 g.
N,N-Dimethylformamide diethylacetal; C 7 H 17NO2 = 147.2
(1188-33-6)
N,N' -Dimethylpiperazine 1,4-Dimethylpiperazine;
Dimethylpiperazine; C 6 H 14N 2 = 114.2 (106-58-1)
ngi: about 1.40.
A colourless liquid, miscible with water and with ethanol
bp: 128 °C to 130 °C. (96 per cent).
N,N-Dimethylformamide Dimethylacetal dig: about 0.85.
1,1-Dimethoxytrimethylamine; C 5H 13NO 2 = 119.2
(4637-24-5) nt0 : about 1.446.

Clear, colourless liquid. bp: about 131 °C.

dfg: about 0.896. Dimethylstearamide Dimethylstearylamide;
N,N-dimethyloctadecanamide; C 20 H41NO = 311.6
n~: about 1.396.
White or almost white solid mass, soluble in many organic
bp: about 103 °C.
solvents, including acetone.
Dimethylglyoxime 2,3-Butanedione dioxime; mp: about 51 C•c.
C 4 H 8N 2 O 2 = 116. l (95-45-4)
V-A66 Appendix I A 2023

Dimethylstearylamide See dimethylstearamide R. Dimeticone (9006-65-9)

Dimethyl Sulfone Dimethyl sulphone; C 2H 6O 2 S = 94.1 See Dimeticone (0138).
(67-71-0) Dimidium Bromide 3,8-Diamino-5-methyl-6-
White or almost white, crystalline powder, freely soluble in phenylphenanthridinium bromide; C 20H 18BrN3 = 380.3
water, soluble in acetone and ethanol (96 per cent). (518-67-2)
mp: 108 °C to 110 °C. Dark-red crystals, slightly soluble in water at 20 °C, sparingly
Dimethyl Sulfoxide Dimethyl sulphoxide; (67-68-5) soluble in water at 60 °C and in ethanol (96 per cent).
See Dimethyl sulfoxide (0763). Dimidium Bromide-Sulfan Blue Mixed Solution
Dimethyl sulfoxide used in spectrophotometry complies with the Dimidium bromide-sulphan blue mixed solution
following additional test. Dissolve separately 0.5 g of dimidium bromide Rand 0.25 g of
Absorbance (2.2.25): maximum 1.00 at 262 nm, 0.46 at sulfan blue R in 30 mL of a hot mixture of 1 volume of
270 nm, 0.16 at 290 nm, 0.01 at 340 nm and higher anhydrous ethanol R and 9 volumes of water R, stir, mix the
wavelengths, determined using water R as compensation two solutions, and dilute to 250 mL with the same mixture
liquid. of solvents. Mix 20 mL of this solution with 20 mL of a
14.0 per cent V/V solution of sulfuric acid R previously
Dimethyl Sulfoxide Rl Dimethyl sulphoxide Rl diluted with about 250 mL of water R and dilute to 500 mL
Content: minimum 99.7 per cent, determined by gas with water R.
chromatography. Storage: protected from light.
Dimethyl Sulfoxide R2 Dimethyl sulphoxide R2 1,3-Dinitrobenzene Dinitrobenzene; C 6H 4 N 2 O 4 = 168.1
Content: minimum 99.9 per cent, determined by gas (99-65-0)
chromatography. Yellowish crystalline powder or crystals, practically insoluble
Residue on evaporation: maximum 0.0005 per cent. in water, slightly soluble in ethanol (96 per cent).
Water (2.5.32): maximum 0.005 per cent. mp: about 90 °C.
N,N-Dimethyltetradecylamine C 16H 35N = 241.5 Dinitrobenzene Solution
dig: about 0.80. A 10 g/L solution of dinitrobenzene R in ethanol
bp: about 260°. (96 per cent) R.
General reagent grade of commerce containing 98.0 to 3,5-Dinitrobenzoic Acid Dinitrobenzoic acid;
101.0% w/w of C16H3sN. C1H4N2O6 = 212.1 (99-34-3)
A clear or almost clear, colourless or slightly yellow liquid. Almost colourless crystals, slightly soluble in water, very
Complies with the following tests. soluble in ethanol (96 per cent).
Water Not more than 0.3%, Appendix IX C, Method I A. mp: about 206 °C.
Assay Dissolve 0.2 gin 10 mL of ethanol (96%) and titrate Dinitrobenzoic Acid Solution
with 0.lM hydrochloric acid VS using methyl red solution as A 20 g/L solution of dinitrobenzoic acid R in ethanol
indicator until a red colour is produced. Each mL of 0.lM (96 per cent) R.
hydrochloric acid VS is equivalent to 24.15 mg of C 16H 35N. Dinitrobenzoyl Chloride 3,5-Dinitrobenzoyl chloride;
Dimethyl Yellow Cl 11020; 4-dimethylaminoazobenzene; C 7 H 3CIN2 O 5 = 230.6 (99-33-2)
C14H1sN3 = 225.3 (60-11-7) Translucent, yellow or greenish-yellow powder or yellowish
Produces a red colour in moderately acidic alcoholic crystals, soluble in acetone and in toluene.
solutions and a yellow colour in weakly acidic and alkaline mp: about 68 °C.
solutions (pH range, 2.8 to 4.6). Suitability test. To 1 mL of anhydrous ethanol Rand 0.1 g of
Complies with the following test. dinitrobenzoyl chloride R add 0.05 mL of dilute sulfuric acid R
Homogeneity Carry out the method for thin-layer and boil under a reflux condenser for 30 min. After
chromatography, Appendix III A, using silica gel G as the evaporation on a water-bath add 5 mL of heptane R to the
coating substance and dichloromethane as the mobile phase. residue and heat to boiling. Filter the hot solution. Wash the
Apply to the plate 10 µL of a 0.01 % w/v solution in crystals formed on cooling to room temperature with a small
dichloromethane. The chromatogram shows only one spot. quantity of heptane R and dry in a desiccator. The crystals
Dimethyl Yellow and Oracet Blue Solution melt (2.2.14) between 92 °C and 95 °C.
Dissolve 10 mg of dimethyl yellow and 10 mg of oracet blue B 2,4-Dinitrophenylhydrazine Dinitrophenylhydrazine;
in 300 mL of dichloromethane. C 6 H 6N 4 0 4 = 198.1 (119-26-6)
Dimethyl Yellow and Oracet Blue 2R Solution Reddish-orange crystals, very slightly soluble in water, slightly
soluble in ethanol (96 per cent).
Dissolve 10 mg of dimethyl yellow and 10 mg of oracet blue 2R
in 300 mL of dichloromethane. mp: about 203 °C (instantaneous method).
Dimethyl Yellow Solution Dinitrophenylhydrazine-aceto-hydrochloric Solution
A 0.2% w/v solution of dimethyl yellow in ethanol (90%). Dissolve 0.2 g of dinitrophenylhydrazine R in 20 mL of
methanol R and add 80 mL of a mixture of equal volumes of
Complies with the following test.
acetic acid R and hydrochloric acid Rl. Prepare immediately
Sensitivity A solution containing 2 g of ammonium chloride before use.
in 25 mL of carbon dioxide-free water to which is added
Dinitrophenylhydrazine-hydrochloric Solution
0.1 mL of the dimethyl yellow solution is yellow. Not more
than O.10 mL of 0.1 M hydrochloric acid VS is required to Dissolve by heating 0.50 g of dinitrophenylhydrazine R in dilute
change the colour of the solution to red (pH range, 2.8 to hydrochloric acid Rand dilute to 100 mL with the same
4.6).
2023 Appendix I A V-A67

solvent. Allow to cool and filter. Prepare immediately before Dwxan used for liquid scintillatwn is of a suitable analytical
use. grade.
Dinitrophenylhydrazine-sulfuric Acid Solution Dioxan Solution
Dinitrophenylhydrazine-sulphuric acid solution Dissolve 1.00 g of dioxan R in water R and dilute to
Dissolve 1.5 g of dinitrophenylhydrazine R in 50 mL of a 100.0 mL with the same solvent. Dilute 5.0 mL of this
20 per cent V/V solution of sulfuric acid R. Prepare solution to 100.0 mL with water R (0.5 mg/mL of dioxan).
immediately before use. Dioxan Solution Rl
Dinonyl Phthalate C 26 H 42 O4 = 418.6 (28553-12-0) Dilute 10.0 mL of dioxan solution R to 50.0 mL with water R.
Colourless to pale yellow, viscous liquid. (0.1 mg/mL of dioxan).
dig: 0.97 to 0.98. Dioxan Solution R2
nt0 : 1.482 to 1.489. Dilute 2.0 mL of dwxan solutwn R to 50.0 mL with water R
Acidity. Shake 5.0 g with 25 mL of water R for 1 min. Allow (0.02 mg/mL of dioxan).
to stand, filter the separated aqueous layer and add 0.1 mL Diphenylamine C 12 H 11N = 169.2 (122-39-4)
of phenolphthalein solutwn R. Not more than 0.3 mL of 0.1 M White or almost white crystals, slightly soluble in water,
sodium hydroxide is required to change the colour of the soluble in ethanol (96 per cent).
solution (0.05 per cent, calculated as phthalic acid). mp: about 55 °C.
Water (2.5.12): maximum 0.1 per cent. Storage: protected from light.
Dioctadecyl Disulfide Dioctadecyl disulphide; Diphenylamine Solution
C36 H, 4 S2 = 571.1 (2500-88-1)
A 1 g/L solution of diphenylamine R in suljuric acid R.
White or almost white powder, practically insoluble in water.
Storage: protected from light.
mp: 53 °C to 58 °C.
Diphenylamine Solution Rl
2,2 '-Di(octadecyloxy)-5,5 '-spirobi(l,3,2-
dioxaphosphorinane) 2,2 '-Di(octadecyloxy)-5 ,5 ' - A 10 g/L solution of diphenylamine R in sulfuric acid R.
spirobi(l,3,2-dioxaphosphorin-ane); C 41 H 82O 6P 2 = 733 The solution is colourless.
White or almost white, waxy solid, practically insoluble in Diphenylamine Solution R2
water, soluble in hydrocarbons. Dissolve 1 g of diphenylamine R in 100 mL of glacial acetic
mp: 40 °C to 70 °C. acid Rand add 2.75 mL of szdfunc acid R. Use immediately.
Dioctadecyl 3,3'-Thiodipropionate C 42H 82 O 4S = 683 9 ,10-Diphenylanthracene Diphenylanthracene;
(693-36-7) C26H1 8 = 330.4 (1499-10-1)
White or almost white, crystalline powder, practically Yellowish or yellow, crystalline powder, practically insoluble
insoluble in water, freely soluble in methylene chloride, in water.
sparingly soluble in acetone, in ethanol (96 per cent) and in mp: about 248 °C.
light petroleum. N,N' -Diphenylbenzidine N,N' -Diphenylbiphenyl-4,4 ' -
mp: 58 °C to 67 °C. diamine; Diphenylbenzidine; C 24H 20 N 2 = 336.4 (531-91-9)
Di-n-octyl Phthalate Dioctyl benzene-1,2-dicarboxylate; White or faintly grey, crystalline powder, practically insoluble
C24H3sO4 = 390.6 (117-84-0) in water, slightly soluble in acetone and in ethanol
Colourless viscous liquid, insoluble in water. (96 per cent).
Density: about 0.98 g/mL (20 °C). mp: about 248 °C.
Dioctyl Sodium Sulfosuccinate Docusate sodium; Nitrates. Dissolve 8 mg in a cooled mixture of 5 mL of
Sodium 1,4-bis [(2-ethylhexyl)oxy)-1,4-dioxobutane-2- water R and 45 mL of nitrogen-free sulfuric acid R.
sulfonate; l,4-bis(2-ethylhexyl) sulfobutanedioate sodium The solution is colourless or very pale blue.
salt; Sodium dioctyl sulfosuccinate; C 20 H 37NaO 7 S = 444.6 Szdfated ash (2.4.14): maximum 0.1 per cent.
(577-11-7) Storage: protected from light.
White or almost white, waxy solid. Diphenylboric Acid Aminoethyl Ester
Diosgenin (25R)-Spirost-5-en-3~-ol; C 27H 42 O 3 = 414.6 C 14H 16 BNO = 225.1 (524-95-8)
(512-04-9) White or slightly yellow, crystalline powder, practically
1,4-Dioxan Dioxan; C 4H 8 O2 = 88.1 (123-91-1) insoluble in water, soluble in ethanol (96 per cent).
Clear, colourless liquid, miscible with water and with most mp: about 193 °C.
organic solvents. 1,5-Diphenylcarbazide 1,5-diphenylcarbonodihydrazide;
dig: about 1.03. Diphenylcarbazide; C 13H 14N 4O = 242.3 (140-22-7)
Freezing point (2.2.18): minimum 11.0 °C. White or almost white, crystalline powder which gradually
Water (2.5.12): maximum 0.5 per cent. becomes pink on exposure to air, very slightly soluble in
water, soluble in acetone, in ethanol (96 per cent) and in
Do not distil if the dwxan does not comply with the test for
glacial acetic acid.
peroxides.
mp: about 170 °C.
Peroxides. Place 8 mL of potassium iodide and starch solution R
in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in Sulfated ash (2.4.14): maximum 0.1 per cent.
diameter. Fill completely with the substance to be examined, Storage: protected from light.
shake vigorously and allow to stand in the dark for 30 min.
No colour is produced.
V-A68 Appendix I A 2023

Diphenylcarbazide Solution 2,2 '-Dipyridylamine N-(Pyridin-2-yl)pyridin-2-amine;

Dissolve 0.2 g of diphenylcarbazide R in 10 mL of glacial C 10H 9 N 3 = 171.2 (1202-34-2)
acetic acid R and dilute to 100 mL with anhydrous ethanol R. mp: about 95 °C.
Prepare immediately before use. Disodium Arsenate Sodium arsenate heptahydrate;
1,5-Diphenylcarbazone Diphenylcarbazone; Na2HAsO 4 ,7H 2O = 312.0 (10048-95-0)
C 13H 12N 4O = 240.3 (538-62-5) Crystals, efflorescent in warm air, freely soluble in water,
Orange-yellow, crystalline powder, practically insoluble in soluble in glycerol, slightly soluble in ethanol (96 per cent).
water, freely soluble in ethanol (96 per cent). The aqueous solution is alcaline to litmus.
mp: about 157 °C, with decomposition. d?g: about 1.87.
2,2-Diphenylglycine Amino(diphenyl)acetic acid; mp: about 57 °C when rapidly heated.
C 14H 13NO 2 = 227.26 (3060-50-2) Disodium Bicinchoninate 2,2'-Biquinoline-4-4'-
1,2-Diphenylhydrazine Hydrazobenzene; dicarboxylate disodium salt; C 20 H 10N2Na2O4 = 388.3
1,2-Diphenyldiazane; C 12H 12N 2 = 184.3 (122-66-7) (979-88-4)
Orange powder. Disodium Catechol-3,5-disulfonate 4,5-Dihydroxy-1,3-
mp: about 125 °C. benzene disulfonic acid disodium salt monohydrate,
4,5-dihydroxy-1,3-benzene disulphonic acid disodium salt
Diphenylmethanol Benzhydrol; C 13H 12 O = 184.2
(91-01-0)
monohydrate, disodium catechol-3,5- disulphonate;
C 6H 4 O 8Na,H2O = 332 (149-45-1)
White or almost white, crystalline powder.
General reagent grade of commerce.
mp: about 66 °C.
Disodium Edetate Disodium dihydrogen
Diphenyloxazole 2,5-Diphenyloxazole; ethylenediaminetetra-acetate, dihydrate; Sodium edetate;
C 15H 11NO = 221.3 (92-71-7) (6381-92-6)
White or almost white powder, practically insoluble in water, See Disodium edetate (0232).
soluble in methanol, sparingly soluble in dioxan and in glacial
acetic acid. Disodium Ethanedisulfonate Disodium
ethanedisulphonate; 1,2-ethanedisulfonic acid disodium salt;
mp: about 70 °C. 1,2-ethanedisulphonic acid disodium salt;
A]:~: about 1260 determined at 305 nm in methanol R. C 2H 4Na 2 O6S2 = 234.2 (5325-43-9)
Diphenykixazole used for liquid scintillation is of a suitable General reagent grade of commerce.
analytical grade.
Disodium Hydrogen Citrate Sodium acid citrate;
Diphenylphenylene Oxide Polymer 2,6-Diphenyl-p- Disodium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate
phenylene oxide polymer sesquihydrate; C 6H 6Na2 O 7,l 1/2H 2O = 263.1 (144-33-2)
White or almost white, porous beads. The size range of the White or almost white powder, soluble in less than 2 parts of
beads is specified after the name of the reagent in the tests water, practically insoluble in ethanol (96 per cent).
where it is used. Disodium Hydrogen Orthophosphate Disodium
Dipotassium Edetate Dipotassium dihydrogen hydrogen phosphate dodecahydrate; (10039-32-4)
ethylenediaminetetra-acetate; C 10H 14N2K2Os,2H2O = 404.5 See Disodium phosphate dodecahydrate (0118).
(25102-12-9)
Disodium Hydrogen Orthophosphate, Anhydrous
General reagent grade of commerce. Disodium hydrogen phosphate, anhydrous;
Dipotassium Hydrogen Orthophosphate Na2HPO 4 = 142.0 (7558-79-4)
Dipotassium hydrogen phosphate; K2HPO 4 = 174.2 Disodium Hydrogen Orthophosphate Dihydrate
(7758-11-4)
Disodium hydrogen phosphate dihydrate; (10028-24-7)
White or almost white, crystalline powder, hygroscopic, very See Disodium phosphate dihydrate (0602).
soluble in water, slightly soluble in ethanol (96 per cent).
Disodium Hydrogen Orthophosphate Heptahydrate
Storage: in an airtight container. Disodium hydrogen phosphate heptahydrate;
Dipotassium Hydrogen Phosphate Trihydrate Na2HPO 4,7H2 O = 268.1 (7782-85-6)
K 2HPO4 ,3H2O = 228.2 (16788-57-1) Disodium Hydrogen Phosphate Heptahydrate
Colourless or white or almost white powder or crystals, freely Na 2 HPO 4,7H 2O = 268.1 (7782-85-6)
soluble in water. Disodium Hydrogen Phosphate Solution
Dipotassium Sulfate Potassium sulfate; Dipotassium A 90 g/L solution of disodium hydrogen phosphate
sulphate; Potassium sulphate; K 2SO 4 = 174.3 (7778-80-5) dodecahydrate R.
Colourless crystals, soluble in water. Ditalimphos 0, 0-Diethyl ( 1,3-dihydro-l ,3-dioxo-2H-
Dipotassium (+)-Tartrate Potassium tartrate; isoindol-2-yl)phosphonothioate; C 12H 14NO 4 PS = 299.3
C 4 H 4 K 2O6,1/2H2O = 235.3 (921-53-9) (5131-24-8)
White or almost white, granular powder or crystals, very Very slightly soluble in water, in ethyl acetate and in
soluble in water, very slightly soluble in ethanol anhydrous ethanol.
(96 per cent). A suitable certified reference solution may be used.
2,2'-Dipyridyl 2,2'-Bipyridine; C 10H 8 N 2 = 156.2 5,5'-Dithiobis(2-nitrobenzoic) Acid 3-Carboxy-4-
(366-18-7) nitrophenyldisulphide; C 14H 8N 2O 8 S2 = 396.4 (69-78-3)
mp: about 72°. Yellow powder sparingly soluble in ethanol (96 per cent).
General reagent grade of commerce. mp: about 242 °C.
 2023 Appendix I A V-A69

Dithioerythritol C 4H 10O 2 S2 = 154.3 (6892-68-8) indicator, titrate the solution immediately with 0.1 M ferrous
(2R,3S)- l ,4-Disulfanylbutane-2,3-diol. DTE. sulfate until a greenish-red colour is obtained.
mp: about 83 °C. 1 mL of 0.1 M ferrous sulfate is equivalent to 9.095 mg of
Dithiol Toluene-3,4-dithiol; 4-Methylbenzene-1,2-dithiol; V2Os.
C7HsS2 = 156.3 (496-74-2) Divanadium Pentoxide Solution in Sulfuric Acid
White or almost white crystals, hygroscopic, soluble in Divanadium Pentoxide Solution in Sulphuric Acid
methanol and in solutions of alkali hydroxides. Dissolve 0.2 g of divanadium pentoxide R in 4 mL of sulfuric
mp: about 30 °C. acid R and dilute to 100 mL with water R.

Storage: in an airtight container. Divinylbenzene and Vinylpyrrolidone Copolymer for

Chromatography
Dithiol Reagent Toluenedithiol reagent
Chromatographic reagent grade of commerce.
To 1 g of dithiol R add 2 mL of thioglycollic acid R and dilute
to 250 mL with a 20 g/L solution of sodium hydroxide R. Spherical particles (30 µm) of an m-divinylbenzene and
Prepare immediately before use. N-vinylpyrrolidone copolymer.
Dithiothreitol threo-l,4-Dimercaptobutane-2,3-diol; Docosahexaenoic Acid Methyl Ester DHA methyl ester;
C4H10O2S 2 = 154.2 (27565-41-9) Cervonic acid methyl ester; (all-Z)-Docosa-4,7,10,13,16,19-
hexaenoic acid methyl ester; C 23 H 34O 2 = 342.5 (301-01-9)
Slightly hygroscopic needles, freely soluble in water, in
acetone and in anhydrous ethanol. Content: minimum 90.0 per cent, determined by gas
chromatography.
Storage: in an airtight container.
Docusate Sodium (577-11-7)
Dithizone 1,5-Diphenylthiocarbazone;
C13H12N4S = 256.3 (60-10-6) See Docusate sodium (1418).
A bluish-black, brownish-black or black powder, practically Dodecan-1-ol Laury! alcohol; C 12 H 26O = 186.3
(112-53-8)
insoluble in water, soluble in ethanol (96 per cent).
Storage: protected from light. d~g: about 0.820.
Dithizone Rl 1,5-Diphenylthiocarbazone; mp: 24 °C to 27 °C.
C13H 12N 4S = 256.3 (60-10-6) Content: minimum 98.0 per cent, determined by gas
Content: minimum 98.0 per cent.
chromatography.
Bluish-black, brownish-black or black powder, practically 4-Dodecylresorcinol CH3(CH2) 11 C 6 Hr
insoluble in water, soluble in ethanol (96 per cent). l,3-(OH) 2 = 278.43 (24305-56-4)
Storage: protected from light. General reagent grade of commerce.
Dithizone Solution Dodecyltrimethylammonium Bromide N,N,N-
Trimethyldodecan-1-aminium bromide; C 15 H 34BrN = 308.4
A 0.5 g/L solution of dithizone R in chloroform R. Prepare (1119- 94-4)
immediately before use.
White or almost white crystals.
Divanadium Pentoxide Vanadium(v) oxide;
V2 O 5 = 181.9 (1314-62-1) mp: about 246 °C.
Content: minimum 98.5 per cent. Domiphen Bromide Dodecyldimethyl-2-
phenoxyethylammonium bromide; C 22 H 40 BrNO = 414.5
Yellow-brown or rust-brown powder, slightly soluble in (538-71-6)
water, soluble in strong mineral acids and in solutions of
alkali hydroxides with formation of salts. General reagent grade of commerce.
Appearance of solution. Heat 1 g for 30 min with 10 mL of D-Dopa (2R)-2-Amino-3-(3,4-dihydroxyphenyl)propanoic
sulfuric acid R. Allow to cool and dilute to 10 mL with the acid; 3-Hydroxy-D-tyrosine; 3,4-Dihydroxy-o-phenylalanine;
same acid. The solution is clear (2.2.1). C9HllNO4 = 197.2 (5796-17-8)
Sensitivity to hydrogen peroxide. Dilute 1.0 mL of the solution [a]t0 : + 9.5 to+ 11.5, determined on a 10 g/L solution in
1 M hydrochloric acid.
prepared for the test for appearance of solution cautiously to
50.0 mL with water R. To 0.5 mL of the solution add mp: about 277 °C.
0.1 mL of a solution of hydrogen peroxide (0.1 g/L of H 2 O 2) Dotriacontane n-Dotriacontane; C 32H 66 = 450.9
prepared from dilute hydrogen peroxide solution R. The solution (544-85-4)
has a distinct orange colour compared with a blank prepared White or almost white plates, practically insoluble in water,
from 0.5 mL of the solution to be examined and 0.1 mL of sparingly soluble in hexane.
water R. After the addition of 0.4 mL of a solution of
mp: about 69 °C.
hydrogen peroxide (0.1 g/L of H 2 O 2) prepared from dilute
hydrogen peroxide solution R, the orange solution becomes Impurities. Not more than 0.1 per cent of impurities with the
orange-yellow. same tR value as o:-tocopherol acetate, determined by the gas
chromatographic method prescribed in the monograph
Loss on ignition: maximum 1.0 per cent, determined on
a-Tocopherol acetate (0439).
1.00 g at 700 ± 50 °C.
Doxycycline
Assay. Dissolve 0.200 g with heating in 20 mL of a
70 per cent mlm solution of sulfuric acid R. Add 100 mL of See Doxycycline monohydrate (0820).
water R and 0. 02 M potassium permanganate until a reddish ~-Ecdysterone (2~,3~,5~,22R)-2,3,14,20,22,25-
colour is obtained. Decolorise the excess of potassium Hexahydroxycholest-7-en-6-one; C 27 H 44O 7 = 480.6
permanganate by the addition of a 30 g/L solution of sodium (5289-74-7)
nitrite R. Add 5 g of urea R and 80 mL of a 70 per cent mlm
solution of sulfuric acid R. Cool. Using 0.1 mL offerroin Ras
V-A70 Appendix I A 2023

Echimidine ( lR, 7 aR)-7-([[ (2R,3S)-2,3-Dihydroxy-2-(2- Eosin Cl 45380; acid red 87; C 20H 6 Br4 Na2O 5 = 691.9
hydroxypropan-2-yl)butanoyl] oxy] methyl]-2,3,5, 7a- (17372-87-1)
tetrahydro-1 H-pyrrolizin-1-yl (2Z)-2-methylbut-2-enoate; General reagent grade of commerce.
C20H3 1NO1 = 397.5 (520-68-3) A red powder.
Viscous liquid, miscible in methanol. (-)-Epicatechin (2R,3R)-2-(3,4-Dihydroxyphenyl)-3,4-
Echimidine N-Oxide (1R,7aR)-7-[[[(2R,3S)-2,3- dihydro-2H-1-benzopyran-3,5, 7-triol; C 15 H14O6 = 290.3
Dihydroxy-2-(2-hydroxypropan-2-yl)butanoyl] oxy] methyl]-1- (490-46-0)
[ [(2Z)-2-methylbut-2-enoyl] oxy]-2,3,5,7 a-tetrahydro- lH-
4-epichlortetracycline hydrochloride C22H23ClN2Os,
pyrrolizine 4-oxide; C 20 H 31 NO8 = 413.5 (41093-89-4) HCl = 515.34 (101342-45-4)
Light brown powder, soluble in methanol. General reagent grade of commerce.
Echinacoside P-(3 ',4 '-Dihydroxyphenyl)-ethyl-O-cx-L- (-)-Epigallocatechin-3-0-gallate (2R,3R)-5,7-
rhamnopyranosyl (1 ➔ 3)-O-P-o-[P-o- Dihydroxy-2-(3,4,5-trihydroxyphenyl)-3, 4-dihydro-2H- l-
glucopyranosyl( 1➔ 6)]-( 4-O-caffeoyl)-glucopyranoside; benzopyran-3-yl 3,4,5-trihydroxybenzoate;
C 35H 46 O 20 = 787 (82854-37-3) C 22H 18O 11 = 458.4 (989-51-5)
Pale yellow powder, odourless. Epilactose C 12H 22 O 11 = 342.3 (20869-27-6)
Edotreotide N-[ [4, 7, 10-Tris(carboxymethyl)-1,4, 7, 10- 4-O-P-o-Galactopyranosyl-o-mannopyranose.
tetraazacyclododecan-1-yl] acetyl]-D-phenylalanyl-L-cysteinyl-
L-tyrosyl-D-tryptophyl-L-lysyl-L-threonyl-N-[ ( 1R,2R)-2- Content: minimum 98 per cent.
hydroxy- l -(hydroxymethyl)propyl]-L-cysteinamide cyclic 4-Epioxytetracycline C 22 H 24N 2O 9 = 460.4 (14206-58-7)
(2 ➔ 7)-disulfide; DOTATOC; DOTA-[Tyr3]-octreotide; Analytical reagent grade of commerce.
C 65H 92N 14O 18 S2 = 1422 (204318-14-9) Erucamide (Z)-Docos-13-enoamide; C22H43NO = 337.6
White or almost white powder. (112-84-5)
Content: minimum 95.0 per cent. Yellowish or white powder or granules, practically insoluble
n-Eicosane C 20 H 42 = 282.6 (112-95-8) in water, very soluble in methylene chloride, soluble in
mp: about 37°. anhydrous ethanol.
General reagent grade of commerce. mp: about 70 °C.
Electrolyte Reagent for the Determination of Water Erucifoline (5R, 7R,9Z,12R, 18R)-9-Ethylidene-
Electrolyte reagent for the micro determination of water 7-(hydroxymethyl)-5-methyl-3,6, l 1-trioxa-15-azatetracyclo
[10.5.1.0 5 ' 7.0 15' 18] octadec-1 ( 17)-ene-4, 10-dione;
Commercially available anhydrous reagent or a combination C 18H 23NO 6 = 349.4 ( 40158-95-0)
of anhydrous reagents for the coulometric titration of water,
containing suitable organic bases, sulfur dioxide and iodide White or almost white powder, soluble in methanol.
dissolved in a suitable solvent. Erucifoline N-Oxide (5R,7R,9Z,12R,18R)-9-Ethylidene-
Embonic Acid C 23 H1 6O 6 = 388.4 (130-85-8) 7-(hydroxymethyl)-5-methyl-4,1 0-dioxo-3,6, 11-trioxa-15-
azatetracyclo [10.5. l .05'7 .015'18]octadec-1 (17)-ene 15-oxide;
General reagent grade of commerce. C 18H 23NO 7 = 365.4 (123864-94-8)
Emodin 1,3,8-Trihydroxy-6-methylanthraquinone; White or almost white powder, soluble in water and in
C 15H 10O 5 = 270.2 (518-82-1)
methanol.
Orange-red needles, practically insoluble in water, soluble in Erythritol (149-32-6)
ethanol (96 per cent) and in solutions of alkali hydroxides.
See Erythritol (1803).
Chromatography. Thin-layer chromatography (2.2.27) as
prescribed in the monograph Rhubarb (0291); the Esculetin 6,7-Dihydroxy-2H-1-benzopyran-2-one;
chromatogram shows only one principal spot. Aesculetin; C 9 H 6O 4 = 178.1 (305-01-1)
Endoprotease LysC Esculin 6-(P-o-Glucopyranosyloxy)-7-hydroxy-2H-
chromen-2-one; C 15H 16O 9 ,l 1/2H 2O = 367.3 (531-75-9)
Microbial extracellular proteolytic enzyme secreted by
Achromobactcr lyticus. A lyophilised powder, free of salts. White or almost white powder or colourless crystals,
sparingly soluble in water and in ethanol (96 per cent), freely
cx-Endosulfan cx-Endosulphan; C 9 H 6Cl6O3S = 406.9 soluble in hot water and in hot ethanol (96 per cent).
(959-98-8)
Chromatography (2.2.27). Thin-layer chromatography (2.2.27)
bp: about 200 °C. as prescribed in the monograph Eleutherococcus (1419); the
mp: about 108 °C. chromatogram shows only one principal spot.
A suitable certified reference solution (10 nglµL in Estradiol Estra-1,3,5 (1 0)-triene-3, 17P-diol; P-Estradiol;
cyclohexane) may be used. C1 8H24O2 = 272.4 (50-28-2)
P-Endosulfan P-Endosulphan; C 9 H 6Cl6O3S = 406.9 Prisms stable in air, practically insoluble in water, freely
(33213-65-9) soluble in ethanol (96 per cent), soluble in acetone and in
bp: about 390 °C. dioxan, sparingly soluble in vegetable oils.
mp: about 207 °C. mp: 173 °C to 179 °C.
A suitable certified reference solution (10 nglµL in 17cx-Estradiol C 18 H 24 O 2 = 272.4 (57-91-0)
cyclohexane) may be used. White or almost white, crystalline powder or colourless
Endrin C 12H 8Cl6O = 380.9 (72-20-8) crystals.
A suitable certified reference solution (10 nglµL in mp: 220 °C to 223 °C.
cyclohexane) may be used. Estragole 4-Allylanisole; C 10 H 12 O = 148.2 (140-67-0)
Liquid, miscible with ethanol (96 per cent).
 2023 Appendix I A V-A71

nft about 1.52. Injection: 1 µL of the test solution and 1 µL of the reference
bp: about 216 °C. solution, alternately, three times.
Estragole used in gas chromatography complies with the following After each chromatography, heat the column to 230 °C for
test. 8 min. Integrate the methanol peak. Calculate the percentage
Assay. Gas chromatography (2.2.28) as prescribed in the
methanol content from the following expression:
monograph Anise oil (0804). axb
Test solution. The substance to be examined. c-b
Content: minimum 98.0 per cent, calculated by the
normalisation procedure. a percentage V/V content of methanol in the reference solution,
b area of the methanol peak in the chromatogram obtained with
Ethane C 2 H 6 = 30.07 (74-84-0) the test solution,
Content: minimum 99.0 per cent V/V. area of the methanol peak in the chromatogram obtained with
the reference solution.
Ethane-1,2-diol Ethylene glycol; C 2H 6 O2 = 62.1
(107-21-1) Limit:
Content: minimum 99.0 per cent. - methanol: maximum 0.005 per cent VIV:
Colourless, slightly viscous liquid, hygroscopic, miscible with Ethanol (96 per cent) (64-17-5)
water and with ethanol (96 per cent). See Ethanol (96 per cent) (1317).
dig: 1.113 to 1.115. Ethanol (96%)
njs0 : about 1.432. Alcohol
bp: about 198 °C. Analytical reagent grade ethanol of commerce containing not
mp: about -12 °C. less than 95.l % v/v and not more than 96.9% v/v of C 2 H 6 O.
Acidity. To 10 mL add 20 mL of water R and 1 mL of A colourless liquid; weight per mL, about 0.81 g.
phenolphthalein solution R. Not more than 0.15 mL of 0.02 M Diluted ethanols may be prepared by diluting the volumes of
sodium hydroxide is required to change the colour of the ethanol (96%) indicated in the following table to 1000 mL
indicator to pink. with water.
Water (2.5.12): maximum 0.2 per cent
Ethanol Absolute ethanol; (64-17-5)
Strength Volume of ethanol Weight per
See Ethanol, anhydrous R. (96%J(approx) ml
Ethanol, Absolute Ethanol; C 2H 6 O = 46.07 (64-17-5) % v/v ml g

dig: 0.791 to 0.794. 90 934 0.83

85 885 0.85
bp: 78° to 79°.
80 831 0.86
Analytical reagent grade of commerce containing not less
70 727 0.89
than 99.5% v/v of C2H5O.
65 676 0.90
A colourless, hygroscopic liquid. 60 623 0.91
Store protected from light at a temperature not exceeding 50 519 0.93
30°. 45 468 0.94
Ethanol, Anhydrous (64-17-5) 25 259 0.97
See Ethanol, anhydrous (1318). 20 207 0.975
10 104 0.986
Ethanol Rl, Absolute Ethanol Rl
Complies with the requirements prescribed for the
monograph Ethanol, anhydrous (1318) with the following Ethanol (96%), Aldehyde-free
additional requirement. Aldehyde-free alcohol
Methanol. Gas chromatography (2.2.28).
Mix 1200 mL of ethanol (96%) with 5 mL of a 40% w/v
Test solution. The substance to be examined. solution of silver nitrate and 10 mL of a cooled 50% w/v
Reference solution. Dilute 0.50 mL of anhydrous methanol R to solution of potassium hydroxide. Shake, allow to stand for a
100.0 mL with the substance to be examined. Dilute 1.0 mL few days and filter. Distil the filtrate immediately before use.
of this solution to 100.0 mL with the substance to be Ethanol (x% v/v)
examined. Mix appropriate volumes of water R and ethanol
Column: (96 per cent) R, allowing for the effects of warming and
- material: glass; volume contraction inherent to the preparation of such a
=
- size: l 2 m, 0 = 2 mm; mixture, to obtain a solution whose final content of ethanol
- stationary phase: ethylvinylbenzene-divinylbenzene corresponds to the value of x.
copolymer R (75-100 µm).
Ethanolamine 2-Aminoethanol; C 2 H 7 NO = 61.1
Cam·er gas: nitrogen for chromatography R. (141-43-5)
Flow rate: 30 mUmin. Clear, colourless, viscous, hygroscopic liquid, miscible with
Temperature: water and with methanol.
- column: 130 °C; dig: about 1.014.
- injection port: 150 'C;
njs0 : about 1.454.
- detector. 200 °C.
mp: about 11 °C.
Detection: flame-ionisation.
Storage: in an airtight container.
V-A72 Appendix I A 2023

Ether Diethyl ether; C 4H 10 O = 74.1 (60-29-7) Ethyl Acetate Rl C 4 H 8 O2 = 88.1 (141-78-6)

Clear, colourless, volatile and very mobile liquid, very Suitable for gas chromatography BCD and FID.
flammable, hygroscopic, soluble in water, miscible with Liquid, miscible in water (i.e. 85.3 g/L).
ethanol (96 per cent).
d~g: 0.90.
dig: 0.713 to 0.715. Content: minimum 99.8 per cent.
bp: 34 °C to 35 °C. Evaporation residue: maximum 3.0 mg/L.
Do not distil if the ether does not comply with the test for Water: maximum 0.02 per cent.
peroxides.
Ethyl Acrylate Ethyl prop-2-enoate; C 5H 8 O 2 = 100.1
Peroxides. Place 8 mL of potassium iodide and starch solution R (140-88-5)
in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in
diameter. Fill completely with the substance to be examined, Colourless liquid.
shake vigorously and allow to stand in the dark for 30 min. dig: about 0.924.
No colour is produced. nl 0: about 1.406.
The name and concentration of any added stabilisers are bp: about 99 °C.
stated on the label. mp: about -71 °C.
Storage: in an airtight container, protected from light, at a Ethyl Benzoate C 9 H 10 O 2 = 150.2 (93-89-0)
temperature not exceeding 15 °C. A clear, colourless, refractive liquid, practically insoluble in
Ether, Peroxide-free See Anaesthetic ether (0367). water, miscible with ethanol (96 per cent) and with light
Shake 1000 mL of ether with 20 mL of a solution of 30 g of petroleum.
iron(II) sulfate in 55 mL of water and 3 mL of sulfuric acid. di 5 : about 1.050.
Continue shaking until a small sample no longer produces a
njl;>: about 1.506.
blue colour when shaken with an equal volume of a 2% w/v
solution of potassium iodide and 0.1 mL of starch mucilage. bp: 211 °C to 213 °C.
Ethion C 9 H 22O 4P 2S4 = 384.5 (563-12-2) Ethyl 5-Bromovalerate Ethyl 5-bromopentanoate;
C 7H 13 Br0 2 = 209.1 (14660-52-7)
mp: -24 °C to -25 °C.
Clear, colourless liquid.
A suitable certified reference solution (10 ng/µL in
cyclohexane) may be used. d~g: about 1.321.
Ethoxychrysoidine Hydrochloride 4-p-Ethoxyphenylazo- bp: 104 °C to 109 °C.
m-phenylenediamine hydrochloride; C 14H 17 ClN4O = 292.8 Ethyl Cinnamate C 11 H 12 O 2 = 176.2 (103-36-6)
(2313-87-3) General reagent grade of commerce.
Reddish powder, soluble in ethanol (96 per cent). A colourless or very pale yellow liquid; weight per mL, about
Ethoxychrysoidine Solution 1.05 g.
A 1 g/L solution of ethoxychrysoidine hydrochloride R in ethanol Ethyl Clorazepate Ethyl (3RS)- 7-chloro-2-oxo-5-phenyl-
(96 per cent) R. 2,3-dihydro-lH-l,4-benzodiazepine-3-carboxylate;
Test for sensitivity. To a mixture of 5 mL of dilute hydrochloric C1sH1 5 ClN2O3 = 342.8 (5606-55-3)
acid R and 0.05 mL of the ethoxy-chrysoidine solution add Ethyl Cyanoacetate C 5 H 7 NO 2 = 113.1 (105-56-6)
0.05 mL of 0.0167 M bromide-bromate. The colour changes Colourless or pale yellow liquid, slightly soluble in water,
from red to light yellow within 2 min. miscible with ethanol (96 per cent).
2-Ethoxyethanol Ethylene glycol monoethyl ether; bp: 205 °C to 209 °C, with decomposition.
C4H1oO2 = 90.1 (110-80-5) Ethyl Formate Ethyl methanoate; C 3H 6 O 2 = 74.1
Content: minimum 99.0 per cent. (109-94-4)
Clear, colourless liquid, miscible with water, with acetone Clear, colourless, flammable liquid, freely soluble in water,
and with ethanol (96 per cent). miscible with ethanol (96 per cent).
dig: about 0.93. dig: about 0.919.
nii: about 1.406. nl0: about 1.36.
bp: about 135 °C. bp: about 54 °C.
(2-ethoxyphenoxy) acetic acid C 10H 12O 4 = 196.2 g/mol Ethyl 4-Hydroxybenzoate (120-47-8)
(3251-30-7) See Ethyl parahydroxybenzoate R.
General reagent grade of commerce Ethyl Methanesulfonate C 3H 8 O 3S = 124.2 (62-50-0)
Ethyl Acetate C 4 H 8 O 2 = 88.1 (141-78-6) Clear, colourless liquid.
Clear, colourless liquid, soluble in water, miscible with Content: minimum 99.0 per cent.
ethanol (96 per cent).
Density: about 1.206 g/cm3 (20 °C).
dig: 0.901 to 0.904.
nl0 : about 1.418.
bp: 76 °C to 78 °C.
bp: about 213 °C.
Ethyl Acetate, Treated
Ethyl Parahydroxybenzoate Ethyl 4-hydroxybenzoate;
Disperse 200 g of sulfamic acid R in ethyl acetate R and make (120-47-8)
up to 1000 mL with the same solvent. Stir the suspension
See Ethyl parahydroxybenzoate (0900).
obtained for three days and filter through a filter paper.
4-[(Ethylamino)methyl]pyridine C 8 H 12N 2 = 136.2
Storage: use within 1 month.
(33403-97-3)
2023 Appendix I A V-A73

Pale yellow liquid. Ethylene Oxide Solution R2

dfg: about 0.98. Weigh 1.00 g of cold ethylene oxide stock solution R (equivalent
nb0 : about 1.516. to 2.5 mg of ethylene oxide) into a cold flask containing
bp: about 98 °C. 40.0 g of cold macrogol 200 Rl. Mix and determine the exact
mass and dilute to a calculated mass to obtain a solution
Ethylbenzene C 8 H 10 = 106.2 (100-41-4) containing 50 µg of ethylene oxide per gram of solution.
Content: minimum 99.5 per cent mlm, determined by gas Weigh 10.00 g into a flask containing about 30 mL of
chromatography. water R, mix and dilute to 50.0 mL with water R (10 µg/mL
Clear, colourless liquid, practically insoluble in water, soluble of ethylene oxide). Prepare immediately before use.
in acetone, and in ethanol (96 per cent). The solution can be prepared using commercially available
d~g: about 0.87. reagents instead of ethylene oxide stock solution R, making
nb0 : about 1.496. appropriate dilutions.
bp: about 135 °C. Ethylene Oxide Solution R3
Ethyl Benzenesulfonate C 8 H 10O 3 S = 186.2 (515-46-8) Dilute 10.0 mL of ethylene oxide solutwn R2 to 50.0 mL with
water R (2 µg/mL of ethylene oxide). Prepare immediately
Content: minimum 97.0 per cent.
before use.
Colourless or slightly yellow liquid, slightly soluble in water,
miscible with ethanol (96 per cent). Ethylene Oxide Solution R4
Dilute 1.0 mL of ethylene oxide stock solution Rl to 100.0 mL
Densiry: about 1.22 g/mL (25 °C).
with water R. Dilute 1.0 mL of this solution to 25.0 mL with
4-Ethylcatechol 3,4-Dihydroxyethylbenzene; water R.
CsH10O 2 = 138.2
Ethylene Oxide Solution R5
General reagent grade of commerce.
A 50 g/L solution of ethylene oxide R in methylene chloride R.
Ethylene Bis [3,3-di(3-(1, I-dimethyl) ethyl-4-
Either use a commercially available reagent or prepare the
hydroxyphenyl) butyrate] Ethylene bis[3,3-di(3-tert-butyl-
4-hydroxyphenyl)butyrate); C 50 H 66 O8 = 795 (32509-66-3) solution corresponding to the above-mentioned composition.
Crystalline powder, practically insoluble in water and in light
Ethylene Oxide Stock Solution
petroleum, very soluble in acetone and in methanol. All operations carried out in the preparation of these solutwns must
be conducted in a fume cupboard. The operator must protect both
mp: about 165 °C.
hands and face by wean·ng polyethylene protective gloves and an
Ethylene Bis[3,3-di(3-tert-butyl-4-hydroxyphenyl) appropriate face mask.
butyrate] (32509-66-3)
Store all solutwns in an airtight container in a refrigerator at 4 °C
See ethylene bis[3,3-di (3-(1, 1-dimethylethyl)-4-hydroxyphenyl) to 8 °C. Cany out all determinatwns three times.
butyrate] R.
Into a dry, clean test-tube, cooled in a mixture of 1 part of
Ethylene Glycol Monododecyl Ether 2-(Dodecyloxy) sodium chloride R and 3 parts of crushed ice, introduce a slow
ethan-1-ol; C 14 H 30 O 2 = 230.4 (4536-30-5) current of ethylene oxide R gas, allowing condensation onto
Colourless or faintly green liquid. the inner wall of the test-tube. Using a glass syringe,
Ethylene Oxide Oxirane; C 2H 4 O = 44.05 (75-21-8) previously cooled to -10 °C, inject about 300 µL
Colourless, flammable gas, very soluble in water and in (corresponding to about 0.25 g) of liquid ethylene oxide R into
anhydrous ethanol. 50 mL of macrogol 200 Rl. Determine the absorbed quantity
of ethylene oxide by weighing before and after absorption
Liquefaction point: about 12 °C.
(Me 0 ). Dilute to 100.0 mL with macrogol 200 Rl. Mix well
Ethylene Oxide Solution before use.
Weigh a quantity of cool ethylene oxide stock solution R Assay. To 10 mL of a 500 g/L suspension of magnesium
equivalent to 2.5 mg of ethylene oxide into a cool flask and chloride R in anhydrous ethanol R add 20.0 mL of 0.1 M
dilute to 50.0 g with macrogol 200 Rl. Mix well and dilute alcoholic hydrochloric acid R in a flask. Stopper and shake to
2.5 g of this solution to 25.0 mL with macrogol 200 Rl (5 µg obtain a saturated solution and allow to stand overnight to
of ethylene oxide per gram of solution). Prepare immediately equilibrate. Weigh 5.00 g of ethylene oxide stock solution
before use. (2.5 g/L) into the flask and allow to stand for 30 min. Titrate
The solution can be prepared using commercially available with 0.1 M alcoholic potassium hydroxide R determining the
reagents instead of ethylene oxide stock solutwn R, making end-point potentiometrically (2.2.20).
appropriate dilutions. Carry out a blank titration, replacing the substance to be
Ethylene Oxide Solution Rl examined with the same quantity of macrogol 200 Rl.
Dilute 1.0 mL of cooled ethylene oxide stock solution R ( check Ethylene oxide content in milligrams per gram is given by:
the exact volume by weighing) to 50.0 mL with macrogol
200 Rl. Mix well and dilute 2.5 g of this solution to 25.0 mL (Vo - Vi) Xf X 4.404
with macrogol 200 Rl. Calculate the exact amount of ethylene m
oxide in parts per million from the volume determined by
weighing and taking the relative density of macrogol 200 Rl as volumes of 0.1 M a/.coholic potassium hydroxide used
respectively for the blank titration and the assay,
1.127. Prepare immediately before use.
I factor of the alcoholic potassium hydroxide solution,
The solution can be prepared using commercially available 111 mass of the sample taken, in grams.
reagents instead of ethylene oxide stock solution R, making
appropriate dilutions. Ethylene Oxide Stock Solution Rl
A 50 g/L solution of ethylene oxide R in methanol R.
V-A74 Appendix I A 2023

Either use a commercially available reagent or prepare the dig: about 0.939.
solution corresponding to the aforementioned composition. nt0 : about 1.496.

Ethylene Oxide Stock Solution R2 bp: about 149 °C.

A 50 g/L solution of ethylene oxide R in methylene chloride R. 1-Ethylquinaldinium Iodide l-Ethyl-2-
Either use a commercially available reagent or prepare the methylquinolinium iodide; C 12H 141N = 299.2 (606-55-3)
solution corresponding to the aforementioned composition. General reagent grade of commerce.
Ethylenediamine 1,2-Diaminoethane; C 2H 8N 2 = 60.1 Ethyl Toluenesulfonate Ethyl 4-methylbenzenesulfonate;
(107-15-3) Ethyl tosilate; C 9 H 12O 3 S = 200.3 (80-40-0)
Clear, colourless, fuming liquid, strongly alkaline, miscible Content: minimum 97.0 per cent.
with water and with ethanol (96 per cent). Density: about 1.17 g/mL (25 °C).
bp: about 116 °C. bp: about 160 °C.
Ethylenediaminetetra-acetic Acid (Ethylenedinitrilo) mp: about 33 °C.
tetra-acetic acid; C 10H 16N 2O 8 = 292.2 (60-00-4)
Ethylvinylbenzene-Divinylbenzene Copolymer
White or almost white crystalline powder, very slightly
soluble in water. Porous, rigid, cross-linked polymer beads. Several grades are
available with different sizes of bead. The size range of the
mp: about 250 °C, with decomposition. beads is specified after the name of the reagent in the tests
2-Ethylhexane-1,3-diol C 8H 18O 2 = 146.2 (94-96-2) where it is used.
Slightly oily liquid, soluble in anhydrous ethanol, 2-propanol, Eugenol 4-Allyl-2-methoxyphenol; C 10 H 12 O 2 = 164.2
propylene glycol and castor oil. (97-53-0)
dig: about 0.942. Colourless or pale yellow, oily liquid, darkening on exposure
n~: about 1.451. to air and light and becoming more viscous, practically
bp: about 244 °C. insoluble in water, miscible with ethanol (96 per cent) and
2-Ethylhexanoic Acid 2-Ethylhexoic acid; with fatty and essential oils.
CsH16O2 = 144.2 (149-57-5) dig: about 1.07.
Colourless liquid. bp: about 250 °C.
dig: about 0.91. Eugenol used in gas chromatography complies with the fallowing
additional test.
n~: about 1.425.
Related substances. Gas chromatography (2.2.28). Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Clove ozl (1091).
Injection: 1 µL of the test solution.
Test solution. The substance to be exainined.
Test solution: suspend 0.2 g of the 2-ethylhexanoic acid in
Content: minimum 98.0 per cent, calculated by the
5 mL of water R, add 3 mL of dilute hydrochloric acid R and
5 mL of hexane R, shake for 1 min, allow the layers to normalisation procedure.
separate and use the upper layer. Carry out the Storage: protected from light.
chromatographic procedure as prescribed in the test for Euglobulins, Bovine
2-ethylhexanoic acid in the monograph on Amoxicillin Use fresh bovine blood collected into an anticoagulant
sodium (0577). solution (for example, sodium citrate solution). Discard any
Limir. the sum of the area of any peaks, apart from the haemolysed blood. Centrifuge at 1500-1800 g at 15-20 °C to
principal peak and the peak due to the solvent, is not greater obtain a supernatant plasma poor in platelets.
than 2.5 per cent of the area of the principal peak. To 1 L of bovine plasma add 75 g of barium sulfate R and
1,1'-Ethylidenebis(tryptophan) C 24 H 26N 4O 4 = 434.5 shake for 30 min. Centrifuge at not less than 1500-1800 g at
(132685-02-0) 15-20 °C and draw off the clear supernatant. Add 10 mL of
mp: about 223°, with decomposition. a 0.2 mg/mL solution of aprotinin R and shake to ensure
General reagent grade of commerce containing not less than mixing. In a container with a minimum capacity of 30 L in a
chamber at 4 °C introduce 25 L of distilled water R at 4 °C
98.0% of C24H25N4O4.
and add about 500 g of solid carbon dioxide. Immediately
Assay Proceed as described in the test for 1, 1' - add, while stirring, the supernatant obtained from the
Ethylidenebis(tryptophan) and other related substances in the plasma. A white precipitate is formed. Allow to settle at 4 °C
monograph for Tryptophan. The area of the principal peak for 10-15 h. Remove the clear supernatant solution by
in the chromatogram obtained with reference solution (a) is siphoning. Collect the precipitate by centrifuging at 4 °C.
not less than 98.0% of the area of all the peaks. Suspend the precipitate by dispersing mechanically in
N-Ethyhnaleimide l-Ethyl-lH-pyrrole-2,5-dione; 500 mL of distilled water Rat 4 °C, shake for 5 min and
C 6H 7NO 2 = 125.1 (128-53-0) collect the precipitate by centrifuging at 4 °C. Disperse the
Colourless crystals, sparingly soluble in water, freely soluble precipitate mechanically in 60 mL of a solution containing
in ethanol (96 per cent). 9 g/L of sodium chloride Rand 0.9 g/L sodium citrate Rand
mp: 41 °C to 45 °C. adjust to pH 7 .2-7.4 by adding a 10 g/L solution of sodium
hydroxide R. Filter through a sintered glass filter (2.1.2);
Storage: at a temperature of 2 °C to 8 °C.
to facilitate the dissolution of the precipitate crush the
2-Ethyl-2-methylsuccinic Acid 2-Ethyl-2- particles of the precipitate with a suitable instrument. Wash
methylbutanedioic acid; C 7 H 12O 4 = 160.2 (631-31-2) the filter and the instrument with 40 mL of the chloride-
mp: 104 °C to 107 °C. citrate solution described above and dilute to 100 mL with
2-Ethylpyridine C 7 H 9N = 107.2 (100-71-0) the same solution. Freeze-dry the solution. The yields are
Colourless or brownish liquid. generally 6 g to 8 g of euglobulins per litre of bovine plasma.
2023 Appendix I A V-A75

Test for suitability. For this test, prepare the solutions using Europine N-Oxide (1S,7aR)-l-Hydroxy-7-[[[(2R,3S)-2-
phosphate buffer solution pH 7. 4 R containing 30 g/L of bovine hydroxy-2-(2-hydroxypropan-2-yl)-3-methoxybutanoyl]oxy]
albumin R. methyl]-2,3,5, 7a-tetrahydro-1H-pyrrolizine 4-oxide;
Into a test-tube 8 mm in diameter placed in a water-bath at C 16H 27 NO 7 = 345.4 (65582-53-8)
37 °C introduce 0.2 mL of a solution of a reference White or almost white powder, soluble in ethanol
preparation of urokinase containing 100 IU/mL and 0.1 mL (96 per cent).
of a solution of human thrombin R containing 20 IU/mL. Evodiamine (13bS)-14-Methyl-8,13, 13b,14-
Add rapidly O. 5 mL of a solution containing 10 mg of bovine tetrahydroindolo [2 ',3 ':3,4]pyrido [2, 1-b] quinazolin-5(7 H)-
euglobulins per millilitre. A firm clot forms in less than 10 s. one; C 19H 17 N 3 O = 303.4 (518-17-2)
Note the time that elapses between the addition of the
Extraction Resin
solution of bovine euglobulins and the lysis of the clot.
The lysis time does not exceed 15 min. Solid phase extraction resin containing 2,2'-oxybis(N,N-
dioctylacetamide) (N,N,N' ,N' -tetra-n-octyldiglycolamide).
Storage: protected from moisture at 4 °C; use within 1 year.
Fargesin C 21 H 22 O 6 = 370.4 (31008-19-2)
Euglobulins, Human
5-[ (3SR,3aRS,6RS, 6aRS)-6-(3,4-Dimethoxyphenyl)-
For the preparation, use fresh human blood collected into an 1,3,3a,4, 6,6a-hexahydrofuro [3,4-c] furan-3-yl]-1,3-
anticoagulant solution (for example sodium citrate solution)
benzodioxole.
or human blood for transfusion that has been collected in
plastic blood bags and which has just reached its expiry date. (E,E)-Farnesol trans,trans-Farnesol; (2E,6E)-3,7,11-
Discard any haemolysed blood. Centrifuge at 1500-1800 g at Trimethyldodeca-2,6,10-trien-1-ol; C 15 H 26 O = 222.4
(106-28-5)
15 °C to obtain a supernatant plasma poor in platelets. Isa-
group plasmas may be mixed. Fast Blue B Salt 3,3'-Dimethoxy(biphenyl)-4,4'-
To 1 L of the plasma add 75 g of barium sulfate R and shake bisdiazonium dichloride; C 14 H 12 Cl2 N 4O 2 = 339.2
for 30 min. Centrifuge at not less than 15 000 g at 15 °C (84633-94-3)
and draw off the clear supernatant. Add 10 mL of a solution Schultz No. 490
of aprotinin R containing 0.2 mg/mL and shake to ensure Colour Index No. 37235
mixing. In a container with a minimum capacity of 30 L in a Dark green powder, soluble in water. It is stabilised by
chamber at 4 °C introduce 25 L of distilled water R at 4 °C addition of zinc chloride.
and add about 500 g of solid carbon dioxide. Immediately
Storage: in an airtight container, at a temperature between
add while stirring the supernatant obtained from the plasma. 2 °C and 8 °C.
A white precipitate is formed. Allow to settle at 4 °C for
10-15 h. Remove the clear supernatant solution by Fast Blue B Salt Solution
siphoning. Collect the precipitate by centrifuging at 4 °C. Dissolve 140 mg offast blue B salt R in 10 mL of water R
Suspend the precipitate by dispersing mechanically in and mix with 50 mL of methylene chloride R and 140 mL of
500 mL of distilled water R at 4 °C, shake for 5 min and methanol R.
collect the precipitate by centrifuging at 4 °C. Disperse the Storage: protected from light at a temperature of 4 °C; use
precipitate mechanically in 60 mL of a solution containing within 1 week.
9 g/L of sodium chloride Rand 0.9 g/L of sodium citrate R, and Fast Red B Salt 2-Methoxy-4-nitrobenzenediazonium
adjust the pH to 7.2-7.4 by adding a 10 g/L solution of hydrogen naphthalene-1,5-disulfonate;
sodium hydroxide R. Filter through a sintered-glass filter C 17 H 13N 3 O 9 S2 = 467.4 (49735-71-9)
(2. 1. 2); to facilitate the dissolution of the precipitate crush
Schultz No. 155
the particles of the precipitate with a suitable instrument.
Wash the filter and the instrument with 40 mL of the Colour Index No. 37125
chloride-citrate solution described above and dilute to Orange-yellow powder, soluble in water, slightly soluble in
100 mL with the same solution. Freeze-dry the solution. ethanol (96 per cent).
The yields are generally 6 g to 8 g of euglobulins per litre of Storage: in an airtight container, protected from light, at 2 °C
human plasma. to 8 °C.
Test for suitability. For this test, prepare the solutions using Fenbufen C 16H 14O 3 = 254.3 (36330-85-5)
phosphate buffer solution pH 7.2 R containing 30 g/L of bovine General reagent grade of commerce.
albumin R. Into a test-tube 8 mm in diameter placed in a
Fenchlorphos C 8 H 8 Cl 3 O 3 PS = 321.5 (299-84-3)
water-bath at 37 °C introduce 0.1 mL of a solution of a
reference preparation of streptokinase containing 10 IU of mp: about 35 °C.
streptokinase activity per millilitre and 0.1 mL of a solution A suitable certified reference solution (10 ng/µL in
of human thrombin R containing 20 IU/mL. Add rapidly cyclohexane) may be used.
1 mL of a solution containing 10 mg of human euglobulins Fenchone (1R)-1,3,3-Trimethylbicyclo[2.2. l]heptan-2-
per millilitre. A firm clot forms in less than 10 s. Note the one; C 10H 16O = 152.2 (7787-20-4)
time that elapses between the addition of the solution of Oily liquid, miscible with ethanol (96 per cent), practically
human euglobulins and the lysis of the clot. The lysis time insoluble in water.
does not exceed 15 min.
Storage: in an airtight container at 4 °C; use within 1 year.
ni0 : about 1.46.

bP,smm: 192 "C to 194 °C.

Europine Hydrochloride [(1S,7aR)-1-Hydroxy-2,3,5,7a-
Fenchone used in gas chromatography complies with the following
tetrahydro-1H-pyrrolizin-7-yl]methyl (2R,3S)-2-hydroxy-2-(2-
test.
hydroxypropan-2-yl)-3-methoxybutanoate hydrochloride;
C,6H2sClNO 6 = 365.9 ((Free base: 570-19-4)) Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Bitter fennel (0824).
White or almost white powder, soluble in ethanol
(96 per cent). Test solution. The substance to be examined.
V-A76 Appendix I A 2023

Content: minimum 98.0 per cent, calculated by the Fixing Solution

normalisation procedure. To 250 mL of methanol R, add 0.27 mL of formaldehyde R
Fenvalerate C 25 H 22 ClNO3 = 419.9 (51630-58-1) and dilute to 500.0 mL with water R.
bp: about 300 °C. Fixing Solution for Isoelectric Focusing in
A suitable certified reference solution (10 ng,'µL in Polyacrylamide Gel
cyclohexane) may be used. A solution containing 35 g of sulfosalicylic acid Rand 100 g of
Ferric Chloride-ferricyanide-arsenite Reagent trichloroacetic acid R per litre of water R.
Immediately before use mix 10 mL of a 27 g,'L solution of Flufenamic Acid 2-[[3-(Trifluoromethyl)phenyl]amino]
ferric chloride R in dilute hydrochloric acid R, 7 mL of potassium benzoic acid; C 14H 10 F 3 NO 2 = 281.2 (530-78-9)
ferricyanide soluti.on R, 3 mL of water R and I O mL of sodium Pale yellow, crystalline powder or needles, practically
arsenite solution R. insoluble in water, freely soluble in ethanol (96 per cent).
Ferric Chloride Solution R3 mp: 132 °C to 135 °C.
Dissolve 2.0 g of ferric chloride R in anhydrous ethanol R and Flumazenil (78755-81-4)
dilute to 100.0 mL with the same solvent. See Flumazenil (1326).
Ferric Chloride Solution, Radiolabelled [59Fe], Flunitrazepam (1622-62-4)
Concentrated See Flunitrazepam (0717).
A commercially available solution of [59Fe]ferric chloride Fluoranthene C 16H 10 = 202.3 (206-44-0)
(approximate specific activity: 100-1000 MBq/mg of Fe).
bp: about 384°.
Ferric Chloride Solution, Radiolabelled [59Fe]
mp: 105° to 110°.
Dilute the concentrated radio labelled f 9Fe]fernc chloride solution
in sodium citrate buffer soluti.on pH 7.8 to obtain a solution General reagent grade of commerce.
with an activity of 3.7 x 104 Bq/mL Fluorene Diphenylenemethane; C 13 H 10 = 166.2 (86-73-7)
F errocyphen Dicyanobis( 1, 10-phenanthroline)iron(n); White or almost white crystals, freely soluble in anhydrous
Ferrocyphene; C 26 H 16FeN 6 = 468.3 (14768-11-7) acetic acid, soluble in hot ethanol (96 per cent).
Violet-bronze, crystalline powder, practically insoluble in mp: 113 °C to 115 °C.
water and in ethanol (96 per cent). 9-Fluorenone C 13 H 18 O = 180.2 (486-25-9)
Storage: protected from light and moisture. mp: about 83°.
Ferrocyphen Solution General reagent grade of commerce.
Dissolve, without warming, 0.5 g of ferrocyphen in 50 mL of Fluorenone Solution
suljuric acid. Dissolve 50 mg of 9-fiuorenone in 10 mL of warm methanol,
F erroin Solution F erroin; (1463 4-91-4) transfer to a 500-mL graduated flask with the aid of 190 mL
Dissolve 0.7 g of ferrous sulfate Rand 1.76 g of phenanthroline of methanol and add sufficient water to produce 500 mL.
hydrochloride R in 70 mL of water Rand dilute to 100 mL The solution should be clear. Immediately before use dilute
with the same solvent. 10 mL of this solution to 100 mL with methanol.
Test for sensitivity. To 50 mL of dilute sulfuric acid R add (9-Fluorenyl)methyl Chloroformate Fluoren-9-ylmethyl
0.1 mL offerroin R. After the addition of 0.1 mL of 0.1 M chloromethanoate; C 15 H 11 ClO 2 = 258.7 (28920-43-6)
ammonium and cerium nitrate the colour changes from red to mp: about 63 °C.
light blue. Fluorescamine 4-Phenylspiro[furan-2(3H),1 '(3' H)-
Ferulic Acid 4-Hydroxy-3-methoxycinnamic acid; 3-(4- isobenzofuran]-3,3'-dione; C 17 H 10O 4 = 278.3 (38183-12-9)
Hydroxy-3-methoxyphenyl)propenoic acid; mp: 154 °C to 155 °C.
C 10H1 0 O4 = 194.2 (1135-24-6) Fluorescein 3 ',6 '-Dihydroxyspiro [isobenzofurane-
Faint yellow powder, freely soluble in methanol. 1(3H), 9 '-[9H] xanthen]-3-one; C 20H 12O 5 = 332.3
mp: 172.9 °C to 173.9 °C. (2321-07-5)
Ferulic acid used in the assay of eleutherosides in Orange-red powder, practically insoluble in water, soluble in
Eleutherococcus (1419) complies with the following additional test. warm ethanol (96 per cent), soluble in alkaline solutions.
Assay. Liquid chromatography (2.2.29) as prescribed in the In solution, fluorescein displays a green fluorescence.
monograph Eleutherococcus (1419). mp: about 315 °C.
Content: minimum 99 per cent, calculated by the Fluorescein Sodium Disodium 2-(3-oxo-6-oxido-3H-
normalisation procedure. xanthen-9-yl)benzoate; Sodium fluoresceinate;
Fibrin Blue C20H 10Na 2 O 5 = 376.3 (518-47-8)
Mix 1.5 g of fibrin with 30 mL of a 5 g,'L solution of indigo Schultz No. 880
carmine R in 1 per cent V/V dilute hydrochloric acid R. Heat Colour Index No. 45350
the mixture to 80 °C and maintain at this temperature whilst Orange-red powder, freely soluble in water. Aqueous
stirring for about 30 min. Allow to cool. Filter. Wash solutions display an intense yellowish-green fluorescence.
extensively by resuspension in 1 per cent VIV dilute Fluorocholine Chloride N-(Fluoromethyl)-2-hydroxy-N,
hydrochloric acid R and mixing for about 30 min; filter.
N-dimethylethan-1-aminium chloride; C 5 H 13 ClFNO = 157.6
Repeat the washing operation three times. Dry at 50 °C. (459424-38-5)
Grind.
Colourless, hygroscopic crystals.
Fibrinogen (9001-32-5)
mp: about 184 °C.
See Human fibrinogen, freeze-dried (0024).
2023 Appendix I A V-A77

2-Fluoro-2-deoxy-D-glucose C 6H 11 FO 5 = 182.2 Formaldehyde Solution Rl

( 86 783-82-6) Complies with the requirements prescribed in the monograph
White or almost white crystalline powder. Formaldehyde solution (35 per cent) (0826) with the following
mp: 174 °C to 176 °C. modification.
2-Fluoro-2-deoxy-o-mannose C 6 H 11 FO 5 = 182.1 Content: 36.5 per cent mlm to 38.0 per cent mlm of
(38440-79-8) formaldehyde (CH 2 0; Mr 30.03).
Colourless semi-solid. Formaldehyde Solution See Formaldehyde solution
(35 per cent) (0826).
1-Fluoro-2,4-dinitrobenzene 2,4-Dinitrofluorobenzene;
Fluorodinitrobenzene; C 6H 3FN 2O 4 = 186.1 (70-34-8) Formamide CH3NO = 45.0 (75-12-7)
Pale yellow liquid or crystals, soluble in propylene glycol. Clear, colourless, oily liquid, hygroscopic, miscible with water
mp: about 29 °C. and with ethanol (96 per cent). It is hydrolysed by water.
Content: minimum 99.0 per cent, determined by gas dig: about 1.134.
chromatography. bp: about 210 °C.
1-Fluoro-2,4-dinitrophenyl-5-L-alaninamide N'--(5- Content: minimum 99.5 per cent.
Fluoro-2,4-dinitrophenyl)-L-alaninamide; Marfey's reagent; Storage: in an airtight container.
FDAA; C9H9FN4 O 5 = 272.2 (95713-52-3) Formamide Rl
Yellow or orange powder. Complies with the requirements prescribed for formamide R
mp: about 228 °C. with the following additional requirement.
Enantiomeric purity: minimum 99.5 per cent. Water (2.5.12): maximum 0.1 per cent determined with an
DL-6-Fluorodopa Hydrochloride (2RS)-2-Amino-3-(2- equal volume of anhydrous methanol R.
fluoro-4,5-dihydroxyphenyl)propanoic acid hydrochloride; Formamide, Treated
2-Fluoro-5-hydroxy-DL-tyrosine hydrochloride; Disperse 1.0 g of suifamic acid R in 20.0 mL offormamide R
C 9 HuClFNO 4 = 251.6 containing 5 per cent V/V of water R.
White or almost white powder. Formic Acid CH2O 2 = 46.03 (64-18-6)
Fluoroethyl(2-hydroxyethyl)dimethylammonium Content: minimum 98.0 per cent mlm.
Chloride N-(2-Fluoroethyl)-2-hydroxy-N,N-dimethylethan- Colourless liquid, corrosive, miscible with water and with
l-aminium chloride; C 6H 15 ClFNO = 171.6 (479407-08-4) ethanol (96 per cent).
Slightly yellow powder.
dig: about 1.22.
Fluoroethyl-o-tyrosine Hydrochloride (2R)-2-Amino-3- Assay. Weigh accurately a conical flask containing 10 mL of
[4-(2-fluoroethoxy)phenyl]propanoic acid hydrochloride;
water R, quickly add about 1 mL of the acid and weigh
CuH15FNO3Cl = 263.7 again. Add 50 mL of water R and titrate with 1 M sodium
Content: minimum 95 per cent. hydroxide, determining the end-point potentiometrically
Colourless or almost colourless crystals. (2.2.20) or using 0.5 mL of phenolphthalein solution R as
Fluoroethyl-L-tyrosine Hydrochloride (2S)-2-Amino-3- indicator.
[4-(2-fluoroethoxy)phenyl]propanoic acid hydrochloride; 1 mL of 1 M sodium hydroxide is equivalent to 46.03 mg of
C11H1sFNO3Cl = 263.7 CH2O2.
Content: minimum 95 per cent. When used for mass spectrometry detection, a special grade may be
Colourless or almost colourless crystals. needed.
6-Fluorolevodopa Hydrochloride (2S)-2-Amino-3-(2- Formic Acid, Anhydrous Formic Acid; (64-18-6)
fluoro-4,5-dihydroxyphenyl)propanoic acid hydrochloride; See formic acid R.
2-Fluoro-5-hydroxy-L-tyrosine hydrochloride; Formononetin 7-Hydroxy-3-( 4-methoxyphenyl)-4-
C 9 HuClFNO4 = 251.6 (144334-59-8) benzopyrone; 7-Hydroxy-3-(4-methoxyphenyl)chromone;
Colourless or almost colourless solid, soluble in water. 7-Hydroxy-4'-methoxyisoflavone; C 16H 12 O4 = 268.26
Fluoromisonidazole (2RS)-1-Fluoro-3-(2-nitro-1H- (485-72-3)
imidazol-1-yl)propan-2-ol; FMISO; C 6H 8 FN3 O 3 = 189.1 General reagent grade of commerce.
(13551-89-8) Forsythoside A (2R,3S,4R,5R,6R)-6-(2-(3,4-
Content: minimum 95 per cent. Dihydroxyphenyl)ethoxy]-4,5-dihydroxy-2-
Yellow crystals. [[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy]
1-Fluoro-2-nitro-4-trifluoromethylbenzene 4-Fluoro-3- methyl]oxan-3-yl (2£)-3-(3,4-dihydroxyphenyl)prop-2-
nitrobenzotrifluoride; l-Fluoro-2-nitro-4-(trifluoromethyl) enoate; 2-(3,4-Dihydroxyphenyl)ethyl 6-O-(6-deoxy-11-L-
benzene; C 7 H 3 F 4 NO 2 = 209.1 (367-86-2) mannopyranosyl)-4-O-[(2£)-3-(3,4-dihydroxyphenyl)prop-2-
enoyl]-~-D-glucopyranoside; C 29H 360 15 = 625 (79916-77-1)
mp: about 197 °C.
D-Fructose Laevulose; Fructose; (57-48-7)
Folic Acid (75708-92-8)
See Fmctose (0188).
See Folic acid hydrate (0067).
Fuchsin, Basic (632-99-5)
Formaldehyde (50-00-0)
A mixture of rosaniline hydrochloride (C 20 H 20ClN3 ;
See Formaldehyde solution R. Mr 337.9; Colour Index No. 42510; Schultz No. 780) and
para-rosaniline hydrochloride (C 19H 18 CIN3; Mr 323.8;
Colour Index No. 42500; Schultz No. 779).
V-A78 Appendix I A 2023

If necessary, purify in the following manner. Dissolve 1 g in o-Galactose Galactose; C 6H 12O 6 = 180.2 (59-23-4)
250 mL of dilute hydrochloric acid R. Allow to stand for 2 h at White or almost white, crystalline powder, freely soluble in
room temperature, filter and neutralise with dilute sodium water.
hydroxide solution R and add 1 mL to 2 mL in excess. Filter [1JC]t0 : + 79 to+ 81, determined on a 100 g/L solution in
the precipitate through a sintered-glass filter (40) (2.1. 2) and water R containing about 0.05 per cent ofNH3 •
wash with water R. Dissolve the precipitate in 70 mL of
methanol R, previously heated to boiling, and add 300 mL of 1,6-Galactosylgalactose 6-O-~-o-Galactopyranosyl-o-
water R at 80 °C. Allow to cool to room temperature, filter galactopyranose; C 12H 22 O 11 = 342.3 (5077-31-6)
and dry the crystals in vacuo. White or almost white powder.
Crystals with a greenish-bronze sheen, soluble in water and Galacturonic Acid o-(+ )-galacturonic acid;
in ethanol (96 per cent). (2S,3R,4S,5R)-2,3,4,5-Tetrahydroxy-6-oxo-hexanoic acid;
Storage: protected from light. C6H 10 O 7 = 194.1 (685-73-4)
Fuchsin Solution, Basic [1JC]g': about+ 53°, determined on a 100 giL solution.
Dissolve 0.1 g of basic fuchsin in 3 mL of methanol, dilute to Gallic Acid 3,4,5-Trihydroxybenzoic acid monohydrate;
100 mL with water, mix and filter. C 7 H 6 O 5,H2O = 188.1 (5995-86-8)
Fuchsin Solution, Decolorised Crystalline powder or long needles, colourless or slightly
yellow, soluble in water, freely soluble in hot water, in
Dissolve 0 .1 g of basic juchsin R in 60 mL of water R. Add a ethanol (96 per cent) and in glycerol.
solution containing 1 g of anhydrous sodium sulfite R or 2 g of
sodium sulfite heptahydrate R in 10 mL of water R. Slowly and It loses its water of crystallisation at 120 °C.
with continuous shaking add 2 mL of hydrochloric acid R. mp: about 260 °C, with decomposition.
Dilute to 100 mL with water R. Allow to stand protected Chromatography. Thin-layer chromatography (2.2.27) as
from light for at least 12 h, decolorise with activated prescribed in the monograph Bearbeny leaf (1054); the
charcoal R and filter. If the solution becomes cloudy, filter chromatogram shows only one principal spot.
before use. If on standing the solution becomes violet, Gallium (68 Ga) Chloride Solution 68 GaC13 = 174.3
decolorise again by adding activated charcoal R.
See Gallium { 8 Ga) chloride solution for radiolabelling (2464) or
Test for sensitivity. To 1.0 mL add 1.0 mL of water R and Gallium { 8 Ga) chloride (accelerator-produced) solution for
0.1 mL of aldehyde-free alcohol R. Add 0.2 mL of a solution radio labelling (3109).
containing 0.1 giL of formaldehyde (CH 2 O, Mr 30.03). Gallium Edotreotide Complex of gallium with
A pale-pink colour develops within 5 min.
N-[[4, 7, 10-tris(carboxymethyl)-1,4, 7,1 0-
Storage: protected from light. tetraazacyclododecan-1-yl] acetyl]-o-phenylalanyl-L-cysteinyl-
Fuchsin Solution Rl, Decolorised L-tyrosyl-D-tryptophyl-L-lysyl-L-threonyl-L-cysteinyl-L-
To 1 g of basic juchsin R add 100 mL of water R. Heat to threoninol cyclic (2 ➔ 7)-disulfide; Gallium DOTATOC;
50 °C and allow to cool with occasional shaking. Allow to C6sH 89GaN14O1 8 S2 = 1488 (293295-70-2)
stand for 48 h, shake and filter. To 4 mL of the filtrate add White or almost white powder.
6 mL of hydrochloric acid R, mix and dilute to 100 mL with Gallium PSMA-11 C 44H 59GaN 6 O 17 = 1014
water R. Allow to stand for at least 1 h before use. Complex of gallium with (3S,7S)-22-[3-[[[2-[[[5-(2-
L-Fucose Fucose; C 6H 12 O5 = 164.2 (6696-41-9) carboxyethyl)-2-hydroxyphenyl]
White or almost white powder, soluble in water and in methyl] (carboxymethyl)amino]ethyl] (carboxymethyl)amino]
ethanol (96 per cent). methyl]-4-hydroxyphenyl]-5, 13,20-trioxo-4,6, 12, 19-
[1JC]t0 : about -76, determined on a 90 g/L solution 24 h after tetraazadocosane-1,3, 7-tricarboxylic acid (PS1\.1A-1 l).
dissolution. Colourless or almost white powder.
mp: about 140 °C. Content: minimum 95.0 per cent (anhydrous and
Fumaric Acid (E)-Butenedioic acid; C 4 H 4 O4 = 116.1 trifluoroacetic acid-free substance).
(110-17-8) Ganoderic Acid A (25R)-7~,15a-Dihydroxy-3,ll,23-
White or almost white crystals, slightly soluble in water, trioxolanost-8-en-26-oic acid; C 30H 44O 7 = 516.7
soluble in ethanol (96 per cent), slightly soluble in acetone. (81907-62-2)
mp: about 300 °C. Gastric Juice, Artificial
Furfuraldehyde Furan-2-aldehyde; Furlural; Dissolve 2.0 g of sodium chloride Rand 3.2 g of pepsin
CsH 4 O 2 = 96.1 (98-01-1) powder R in water R. Add 80 mL of 1 M hydrochloric acid and
Clear, colourless to brownish-yellow, oily liquid, miscible in dilute to 1000 mL with water R.
11 parts of water, miscible with ethanol (96 per cent). Gastrodin 4-(Hydroxymethyl)phenyl a-o-glucopyranoside;
d~g: 1.155 to 1.161. (2R,3S,4S,5R,6S)-2-(Hydroxymethyl)-6-
[4-(hydroxymethyl)phenoxy]oxane-3,4,5-triol;
Distillation range (2.2.11). Not less than 95 per cent distils
C13H 18 O 7 = 286.3 (62499-27-8)
between 159 °C and 163 °C.
GC Concentrical Column
Storage: in a dark place.
A commercially available system consisting of 2
Gadolinium Chloride Hexahydrate Gadolinium concentrically arranged tubes. The outer tube is packed with
trichloride hexahydrate; GdCl3 ,6H2 O = 371.7 (13450-84-5) molecular sieves and the inner tube is packed with a porous
Content: minimum 99.9 per cent. polymer mixture. The main application is the separation of
Gadolinium Sulfate Octahydrate gases.
Gd2 (SO 4h,8H2 O = 747 (13450-87-8') Gelatin (9000-70-8)
Colourless, crystalline powder. See Gelatin (0330).
2023 Appendix I A V-A79

Gelatin, Hydrolysed Ginsenoside Rgl (20S)-6P-D-Glucopyranosyl-D-

Dissolve 50 g of gelatin R in 1000 mL of water R. Autoclave glucopyranosylprotopanaxatriol; (20S)-6a,20-Bis(~-D-
in saturated steam at 121 °C for 90 min and freeze dry. glucopyranosyloxy)-5a-dammar-24-ene-3~, 12~-diol; (20S)-
Geniposide Methyl (1S,4aS,7aS)-1-(P-D- fo,20-Bis (~-D-glucopyranosyloxy)-4 ,4,8, 14-tetramethyl-18-
glucopyranosyloxy)-7-(hydroxymethyl)-1,4a,5, 7a- nor-5a-cholest-24-ene-3 P, 12 ~-diol; C 42 H 72 O 14,2H2O = 837
tetrahydrocyclopenta[c] pyran-4-carboxylate; (22427-39-0)
C 17H 24O 10 = 388.4 (24512-63-8) A colourless solid, soluble in water, in anhydrous ethanol and
Geraniol (E)-3, 7-Dimethylocta-2,6-dien-1-ol; in methanol.
C 10H 18O = 154.2 (106-24-1) [algi: + 31.2 determined on a 10 giL solution in methanol R.
Oily liquid, slight odour of rose, practically insoluble in mp: 188 °C to 191 °C.
water, miscible with ethanol (96 per cent). Water (2.5.12): maximum 4.8 per cent.
Geraniol used in gas chromatography complies with the following Assay. Liquid chromatography (2.2.29) as prescribed in the
additional test. monograph Ginseng (1523).
Assay. Gas chromatography (2.2.28) as prescribed in the Test solution. Dissolve 3.0 mg, accurately weighed, of
monograph Citronella oil (1609). ginsenoside Rgl in 10 mL of methanol R.
Content: minimum 98.5 per cent, calculated by the Content: minimum 95.0 per cent, calculated by the
normalisation procedure. normalisation procedure.
Storage: in an airtight container, protected from light Ginsenoside Rg2 W, 12~,20-Trihydroxydammar-24-en-fo-
Geranyl Acetate (E)-3, 7-Dimethylocta-2,6-dien- l-yl yl 2-O-(6-deoxy-a-L-mannopyranosyl)-P-D-glucopyranoside;
acetate; C 12 H 20 O 2 = 196.3 (105-87-3) C42H12O 13 = 785 (52286-74-5)
Colourless or slightly yellow liquid, slight odour of rose and Ginsenoside Ro (3P)-28-(P-D-Glucopyranosyloxy)-28-
lavender. oxoolean-12-en-3-yl 2-O-~-D-glucopyranosyl-~-D-
glucopyranosiduronic acid; C 48 H 76 O 19 = 957 (34367-04-9)
Geranyl acetate used in gas chromatography complies with the
following additional test. Gitoxin Glycoside of Digitalis purpurea L; 3P-(O-2,6-
Assay. Gas chromatography (2.2.28) as prescribed in the
Dideoxy-~-d-ribo-hexopyranosyl-(1-+4)-O-2,6-dideoxy-p-d-
monograph Bitter-orange-flower oil (1175). ribo-hexopyranosyl-(1-+4)-2,6-dideoxy-P-d-ribo-
hexopyranosyloxy)-l 4, 16~-dihydroxy-5~, 14P-card-20(22)-
Test solution. The substance to be examined. enolide; C 41 H 64 O 14 = 781 (4562-36-1)
Content: minimum 98.0 per cent, calculated by the
A white or almost white, crystalline powder, practically
normalisation procedure.
insoluble in water and in most common organic solvents,
Ginsenoside Rbl (20S)-W-Di-D-glucopyranosyl-20-di-D- soluble in pyridine.
glucopyranosylprotopanaxadiol; (20S)-3~-[ (2-O-P-D-
[alt0 : + 20 to + 24, determined on a 5 g/L solution in a
Glucopyranosyl-~-D-glucopyranosyl) oxy]-20-[ (6-O-P-D-
mixture of equal volumes of chloroform R and methanol R.
glucopyranosyl-P-D-glucopyranosyl)oxy]-5a-dammar-24-en-
Chromatography. Thin-layer chromatography (2.2.27) as
12~-ol; (20S)-3P-[(2-O-P-v-Glucopyranosyl-P-D-
glucopyranosyl) oxy]-20-[ (6-O-~-D-glucopyranosyl-~-D- prescribed in the monograph Digitalis leaf (0117); the
chromatogram shows only one principal spot.
glucopyranosyl) oxy]-4,4,8, l 4-tetramethyl- l 8-nor-5a-cholest-
24-en- l 2 ~-ol; C 54 H 92 O 23 ,3H2O = 1163 (41753-43-9) Glucosamine Hydrochloride D-Glucosamine
hydrochloride; C 6H 14ClNO 5 = 215.6 (66-84-2)
A colourless solid, soluble in water, in anhydrous ethanol and
in methanol. Crystals, soluble in water.
[al;r + 11.3 determined on a 10 giL solution in methanol R. [algi: + 100, decreasing to+ 47.5 after 30 min, determined
mp: about 199 °C. on a 100 giL solution.
Water (2.5.12): maximum 6.8 per cent.
Glucose (50-99-7)
Assay. Liquid chromatography (2.2.29) as prescribed in the See Glucose (0177).
monograph Ginseng (1523). o-Glucose Dextrose; (50-99-7)
Test solution. Dissolve 3.0 mg, accurately weighed, of See glucose R.
ginsenoside Rb 1 in 10 mL of methanol R. o-Glucose Monohydrate C 6H 12 O 6,H2O = 198.2
Content: minimum 95.0 per cent, calculated by the (5996-10-1)
normalisation procedure. [algi: about +52.5 (anhydrous) (10% w/v in water containing
Ginsenoside Re (3~,6a, 12~)-20-(~-D-Glucopyranosyloxy)- about 0.2% ofNH 3).
3, l 2-dihydroxydammar-24-en-6-yl 2-0-( 6-deoxy-a-L- General reagent grade of commerce.
mannopyranosyl)-~-D-glucopyranoside; C 48H 82 O 18 = 947.2 Colourless crystals or a white to cream, crystalline powder.
(52286-59-6)
o-Glucuronic Acid C 6 H 10 O 7 = 194.1 (6556-12-3)
Colourless solid, soluble in water, in ethanol (96 per cent)
Content: minimum 96.0 per cent, calculated with reference to
and in methanol.
the substance dried in vacua (2.2.32).
Ginsenoside Rf (20S)-6-O-W-D-Glucopyranosyl-(1-+ 2)-~- Soluble in water and in ethanol (96 per cent).
o-glycopyranoside]-dammar-24-ene-3 ~' 6a, 12 P,20-tetrol;
C 12H 72 O 14,2H2O = 837 (52286-58-5) Shows mutarotation: [alf)4 : + 11. 7 -+ + 36.3.
A colourless solid, soluble in water, in anhydrous ethanol and Assay. Dissolve 0.150 gin 50 mL of anhydrous methanol R
while stirring under nitrogen. Titrate with 0.1 M
in methanol.
tetrabwylammonium hydroxide, protecting the solution from
[alt0 : + 12.8 determined on a 10 giL solution in methanol R. atmospheric carbon dioxide throughout solubilisation and
mp: about 198 °C.
V-A80 Appendix I A 2023

titration. Determine the end-point potentiometrically 181'.l-Glycyrrhetinic Acid (2OB)-3B-Hydroxy-11-oxo-181'.l-

(2.2.20). olean-12-en-29-oic acid; C 30H 46 0 4 = 470.7 (1449-05-4)
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to White or almost white powder, practically insoluble in water,
19.41 mg of C6H1001. soluble in anhydrous ethanol, sparingly soluble in methylene
Glutamic Acid (56-86-0) chloride.
See Glutamic acid (0750). ~-Glycyrrhetinic Acid Glycyrrhetinic Acid; Glycyrrhetic
L-Glutamine (S)-2,5-Diamino-5-oxopentanoic acid; acid; C 30H 460 4 = 470.7 (471-53-4)
CsH 10N 20 3 = 146.2 (56-85-9) A mixture of l'.l- and B-glycyrrhetic acids in which the
White crystalline powder. B-isomer is predominant.
mp: about 185 °C, with decomposition. White or yellowish-brown powder, practically insoluble in
water, soluble in anhydrous ethanol and in glacial acetic acid.
Glutamyl Endopeptidase for Peptide Mapping
Endoproteinase Glu-C of high purity from Staphylococcus [ix];;°: + 145 to+ 155, determined on a 10.0 g/L solution in
aureus strain VB (EC 3.4.21.19); (137010-42-S) anhydrous ethanol R.
L-y-Glutamyl-L-cysteine C 8 H 14N 20 5 S = 250.3 Chromatography. Thin-layer chromatography (2.2.27) using
(636-58-8)
silica gel GF254 R as the coating substance; prepare the slurry
using a 0.25 per cent V/V solution of phosphoric acid R. Apply
Glutaraldehyde C 5 H 8 0 2 = 100.1 (111-30-8) to the plate 5 µL of a 5 g/L solution of the glycyrrhetic acid
Oily liquid, soluble in water. in a mixture of equal volumes of chloroform R and
n'f]: about 1.434. methanol R. Develop over a path of 10 cm using a mixture of
bp: about 188 °C. 5 volumes of methanol R and 95 volumes of chloroform R.
Examine the chromatogram in ultraviolet light at 254 nm.
Glutaric Acid Pentanedioic acid; C 5 H 8 0 4 = 132.1
(110-94-1)
The chromatogram shows a dark spot (Rp about 0.3)
corresponding to B-glycyrrhetic acid and a smaller spot (Rp
White or almost white, crystalline powder. about 0.5) corresponding to l'.l-glycyrrhetic acid. Spray with
L-Glutathione, Oxidised Bis(L-y-glutamyl-L- anisaldehyde solutwn Rand heat at 100-105 °C for 10 min.
cysteinylglycine) disulfide; C 20H 3 2N6 012S 2 = 612.6 Both spots are coloured bluish-violet. Between them a
(27025-41-8) smaller bluish-violet spot may be present.
Glycerol Propane-1,2,3-triol; (56-81-5) Glycyrrhizic Acid C 42H 62 0 16 = 822.93 (1405-86-3)
See Glycerol (0496). Analytical reagent grade of commerce.
Glycerol Rl Glyoxal Bis(2-hydroxyanil) Bis(2-hydroxyphenylimino)
Complies with the requirements prescribed for the ethane; Glyoxalhydroxyanil; C 14H 12N 20 2 = 240.3
monograph Glycerol (0496) and free from diethylene glycol (1149-16-2)
when examined as prescribed in the test for impurity A and White or almost white crystals, soluble in hot ethanol
related substances in that monograph. (96 per cent).
Glycerol (85%) See Glycerol (85 per cent) (0497). mp: about 200 °C.
Glycerol (85 per cent) Rl Glyoxal Sodium Bisulfite Glyoxal sodium bisulphite;
Complies with the requirements prescribed for the C2H4Na20sS2,H20 = 284.2
monograph Glycerol 85 per cent (0497) and free from General reagent grade of commerce.
diethylene glycol when examined as prescribed in the test for A white or cream powder.
impurity A and related substances in that monograph.
Glyoxal Solution (107-22-2)
Glycerol 1-decanoate (2RS)-2,3-Dihydroxypropyl
Contains about 40 per cent ( mlm) glyoxal.
decanoate; l'.l-Monocaprin; 1-Monodecanoyl-rac-glycerol;
C13H 26 0 4 = 246.3 (2277-23-8) Assay. In a ground-glass stoppered flask place 1.000 g of
glyoxal solution, 20 mL of a 70 g/L solution of hydroxylamine
Content: about 99 per cent.
hydrochloride R and 50 mL of water R. Allow to stand for
Glycerol 1-octanoate (2RS)-2,3-Dihydroxypropyl 30 min and add 1 mL of methyl red mixed solution R and
octanoate; l'.l-Monocaprylin; 1-Monooctanoyl-rac-glycerol; titrate with 1 M sodium hydroxide until the colour changes
CllH 22 0 4 = 218.3 (502-54-5) from red to green. Carry out a blank titration.
Content: about 99 per cent. 1 mL of 1 M sodium hydroxide is equivalent to 29.02 mg of
Glycidol C 3 H 60 2 = 74.1 (556-52-5) glyoxal (C2H20z).
Slightly viscous liquid, miscible with water. Gonadotrophin, Chorionic (9002-61-3)
df 0 : about 1.115. See Chorwnic gonadotrophin (0498).
nt 0: about 1.432. Gonadotrophin, Serum
Glycine Aminoacetic acid; (56-40-6) Dry preparation of a glycoprotein fraction obtained from the
See Glycine (0614). serum or plasma of pregnant mares. It has follicle-stimulating
Glycine Anhydride Piperazine-2,5-dione (2,5-DKP); and luteinising activities. The potency is not less than
C4H6N 2 0 2 = 114.1 (106-57-0) 1000 IU of serum gonadotrophin per milligram, calculated
with reference to the anhydrous substance.
Glycolic Acid 2-Hydroxyacetic acid; C 2H 40 3 = 76.0
(79-14-1) Gramine 1-(lH-Indol-3-yl)-N,N-dimethylmethanamine;
CllH 14N 2 = 174.2 (87-52-5)
Crystals, soluble in water, in acetone, in ethanol
(96 per cent) and in methanol. Flakes, practically insoluble in water, soluble in ethanol
(96 per cent), slightly soluble in acetone.
mp: about 80 °C.
2023 Appendix I A V-AS 1

mp: 132 °C to 134 °C. Hederacoside C O-6-Deoxy-C(-L-mannopyranosyl-(l -->4)-

Guaiacol 2-Methoxyphenol; 1-Hydroxy-2- O-~-D-glucopyranosyl-( 1--> 6)-~-D-glucopyranosyl (4R)-3~-
methoxybenzene; C 7H 8O 2 = 124.1 (90-05-1) [ [2-O(-6-deoxy-C(-L-mannopyranosyl)-C(-L-arabinopyranosyl]
Crystalline mass or colourless or yellowish liquid, oxy]-23-hydroxyolean-12-en-28-oate; C59 H 96O26 = 1221
(14216-03-6)
hygroscopic, slightly soluble in water, very soluble in
methylene chloride, freely soluble in ethanol (96 per cent). Colourless crystals or white or almost white powder.
bp: about 205 °C. mp: about 220 °C.
mp: about 28 °C. Hederacoside C used in liquid chromatography complies with the
Guaiacum Resin Gum guaiac following additional test.
Resin obtained from the heartwood of Guaiacum officinale L. Assay. Liquid chromatography (2.2.29) as prescribed in the
and Guaiacum sanctum L. monograph Ivy leaf (2148).
Reddish-brown or greenish-brown, hard, glassy fragments; Test solution. Dissolve 5.0 mg of hederacoside C in 5 .0 mL of
fracture shiny. methanol R.
Guaiazulene 1,4-Dimethyl-7-isopropylazulene; Content: minimum 95 per cent, calculated by the
C1 5H 18 = 198.3 (489-84-9) normalisation procedure.
Dark-blue crystals or blue liquid, very slightly soluble in Hederagenin Astrantiagenin E; Caulosapogenin; 3~,23-
water, miscible with fatty and essential oils and with liquid Dihydroxy-4C(-olean-12-en-28-oic acid; C 30H 48 O4 = 472.7
paraffin, sparingly soluble in ethanol (96 per cent), soluble in (465-99-6)
500 g/L sulfuric acid and 80 per cent mlm phosphoric acid, C(-Hederin (+ )-( 4R)-3~-[[2-O-(6-Deoxy-a-L-
giving a colourless solution. mannopyranosyl)-C(-L-arabinopyranosyl] oxy]-23-
mp: about 30 °C. hydroxyolean- l 2-en-28-oic acid; C 41 H 66 O 12 = 751.0
(27013-91-8)
Storage: protected from light and air.
White or almost white powder.
Guaiphenesin Guaifenesin; guaiacol glyceryl ether;
C 10H 14 O4 = 198.2 (93-14-1) mp: about 256 °C.
mp: about 79°. Heliotrine [(lS, 7aR)-1-Hydroxy-2,3,5, 7a-tetrahydro-1H-
pyrrolizin-7-yl]methyl (2S,3R)-2-hydroxy-3-methoxy-
General reagent grade of commerce.
2-(propan-2-yl)butanoate; C 16H 27NO 5 = 313.4 (303-33-3)
Guanidine Hydrochloride CH 5N 3HC1 = 95.5 (50-01-1)
White or almost white powder, soluble in ethanol
Crystalline powder, freely soluble in water and in ethanol (96 per cent).
(96 per cent).
Heliotrine N-Oxide (lS, 7aR)-1-Hydroxy-7-[[((2S,3R)-2-
Guanine 2-Aminopurin-6-one; C 5H 5N 5O = 151.1 hydroxy-3-methoxy-2-(propan-2-yl)butanoyl]oxy]methyl]-
(73-40-5) 2,3,5, 7 a-tetrahydro- lH-pyrrolizine 4-oxide;
Amorphous white or almost white powder, practically C16H27NO6 = 329.4 (6209-65-0)
insoluble in water, slightly soluble in ethanol (96 per cent). White or almost white powder, soluble in ethanol
It dissolves in ammonia and in dilute solutions of alkali (96 per cent).
hydroxides.
Helium Helium for chromatography; He= 4.003
Haemoglobin (9008-02-0) (7440-59-7)
Nitrogen: 15 per cent to 16 per cent. Content: minimum 99.995 per cent V/V of He.
Iron: 0.2 per cent to 0.3 per cent. Heparin (9041-08-1)
Loss on drying (2.2.32): maximum 2 per cent. See Heparin sodium (0333).
Sulfated ash (2.4.14): maximum 1.5 per cent. Heparinase I Heparin lyase (EC 4.2.2.7); (9025-39-2)
Haemoglobin Solution Enzyme from Flavobacterium heparinum that performs
Transfer 2 g of haemoglobin R to a 250 mL beaker and add eliminative cleavage of polysaccharides containing (1-->4)-
75 mL of dilute hydrochloric acid R2. Stir until solution is linked D-glucuronate or L-iduronate residues and (l-->4)-a-
complete. Adjust the pH to 1.6 ± 0.1 using 1 M hydrochloric linked 2-sulfoamino-2-deoxy-6-sulfo-n-glucose residues to
acid. Transfer to a 100 mL flask with the aid of dilute give oligosaccharides with terminal 4-deoxy-a-D-gluc-4-
hydrochloric acid R2. Add 25 mg of thiomersal R. Prepare enuronosyl groups at their non-reducing ends.
daily, store at 5 ± 3 °C and readjust to pH 1.6 before use. Heparinase II (149371-12-0)
Storage: at 2 °C to 8 °C. Enzyme from Flavobacterium heparinum that depolymerises
Hamamelitannin (2R,3R,4R)-2-Formyl-2,3,4- sulfated polysaccharide chains containing 1-->4 linkages
trihydroxypentane-1,5-diyl bis(3,4,5-trihydroxybenzoate); between hexosarnines and uronic acid residues (both iduronic
2-C-[ (Galloyloxy)methyl]-D-ribose 5-gallate; and glucuronic acid residues). The reaction yields
C 20H 20O 14 = 484.4 (469-32-9) oligosaccharide products (mainly disaccharides) containing
Harpagide (1S,4aS,5R, 7S, 7aR)-4a,5, 7-Trihydroxy-7- unsaturated uronic acids.
methyl-1,4a,5,6, 7, 7a-hexahydrocyclopenta[c]pyran-1-yl ~-D- Heparinase III Heparin-sulfate Iyase (EC 4.2.2.8);
glucopyranoside; C 15 H 24 O 10 = 364.3 (6926-08-5) (37290-86-1)
Harpagoside C24H 30 O 11 = 494.5 Enzyme from Flavobacterium heparinum that depolymerises
White or almost white, crystalline powder, very hygroscopic, selectively sulfated polysaccharide chains containing 1-->4
soluble in water and in ethanol (96 per cent). linkages between hexosamines and glucuronic acid residues
Storage: in an airtight container. to give oligosaccharide products (mainly disaccharides)
containing unsaturated uronic acids.
V-A82 Appendix I A 2023

Heptachlor C 10 H 5Cl7 = 373.3 (76-44-8) A suitable certified reference solution (10 ng/µL in
bp: about 135 °C. cyclohexane) may be used.
mp: about 95 °C. ~-Hexachlorocyclohexane C 6H 6Cl6 = 290.8 (319-85-7)
A suitable certified reference solution (10 ng/µL in A suitable certified reference solution (10 ng/µL in
cyclohexane) may be used. cyclohexane) may be used.
Heptachlor Epoxide C 10 H 5 Ch0 = 389.3 (1024-57-3) 6-Hexachlorocyclohexane C 6H 6Cl 6 = 290.8 (319-86-8)
bp: about 200 °C. A suitable certified reference solution (10 ng/µL in
mp: about 160 °C. cyclohexane) may be used.
A suitable certified reference solution (10 ng/µL in Hexacosane C 26 H 54 = 366.7 (630-01-3)
cyclohexane) may be used. Colourless or white or almost white flakes.
Heptafluorobutyric Acid HFBA; C 4HF7 0 2 = 214.0 mp: about 57 °C.
(375-22-4) Hexadimethrine Bromide l,5-Dimethyl-1,5-
Clear, colourless liquid. Corrosive. diazaundecamethylene polymethobromide; Poly(l,l,5,5-
dig: about 1.645. tetramethyl-1,5-azonia-undecamethylene dibromide);
n~r about 1.300.
(C13H30Br2N2)n (28728-55-4)
White or almost white, amorphous powder, hygroscopic,
bp: about 120 °C. soluble in water.
Content: minimum 99.5 per cent.
Storage: in an airtight container.
Heptafluorobutyric Anhydride C 8 F 14 0 3 = 410.1 2,2' ,2' ',6,6' ,6' '-Hexa-(1,1-dimethylethyl)-4,4' ,4' '-[2,4,6-
(336-59-4)
trimethyl-1,3,5-benzenetriyltrismethylene]triphenol
n1°: about 1.287. 2,2 ',2 ",6,6 ',6 "-Hexa-tert-butyl-4,4' ,4 " -[ (2,4,6-trimethyl-
bp: about 108°. 1,3,5-benzenetriyl)trismethylene]triphenol; C 54H 78 0 3 = 775
Use a grade of commerce suitable for derivatisation. Crystalline powder, practically insoluble in water, soluble in
Heptafluoro-N-methyl-N-(trimethylsilyl)butanamide acetone, slightly soluble in ethanol (96 per cent).
2,2,3,3,4,4,4-Heptafluoro-N-methyl- mp: about 244 °C.
N-(trimethylsilyl)butyramide; C 8H 12F 7NOSi = 299.3 1,1,1,3,3,3-Hexafluoropropan-2-ol C 3H 2 F 60 = 168.0
(53296-64-3) (920-66-1)
Clear, colourless liquid, flammable. Content: minimum 99.0 per cent, determined by gas
nf~: about 1.351. chromatography.
bp: about 148 °C. Clear, colourless liquid, miscible with water and with
N-Heptane Heptane; C 7H 16 = 100.2 (142-82-5) anhydrous ethanol.
Colourless, flammable liquid, practically insoluble in water, d~g: about 1.596.
miscible with anhydrous ethanol. bp: about 59 °C.
d~g: 0.683 to 0.686. Hexamethyldisilazane C 6 H 19NSi2 = 161.4 (999-97-3)
n~: 1.387 to 1.388. Clear, colourless liquid.
Distillation range (2.2.11). Not less than 95 per cent distils d~g: about 0. 78.
between 97 °C and 98 °C. n~: about 1.408.
n-Heptylamine C 7 H 17N = 115.22 (111-68-2) bp: about 125 °C.
General reagent grade of commerce. Storage: in an airtight container.
Boiling point, about 156 °C. Hexamine 1,3,5, 7-tetra-azatricyclo [3 .3 .1.13, 7] decane;
2-Heptylamine 2-Aminoheptane; C 7 H 17N = 115.2 Hexamethylenetetramine; C 6H 12N 4 = 140.2 (100-97-0)
bp: about 143°. Colourless, crystalline powder, very soluble in water.
General reagent grade of commerce. n-Hexane Hexane; C 6H 14 = 86.2 (110-54-3)
Hesperidin (S)-7-[ [6-0-( 6-Deoxy-o:-L-mannopyranosyl)-~- Colourless, flammable liquid, practically insoluble in water,
D-glucopyranosyl] oxy]-5-hydroxy-2-(3-hydroxy-4- miscible with anhydrous ethanol.
methoxyphenyl)-2,3-dihydro-4H- l-benzopyran-4-one; dig: 0.659 to 0.663.
C28H340 15 = 611 (520-26-3) n1°: 1.375 to 1.376.
Hygroscopic powder, slightly soluble in water and in Distillation range (2.2.11). Not less than 95 per cent distils
methanol. between 67 °C and 69 °C.
mp: 258 °C to 262 °C. Hexane used in spectrophotometry complies with the following
Hexachlorobenzene C 6Cl 6 = 284.8 (118-74-1) additional test.
bp: about 332 °C. Absorbance (2.2.25): maximum 0.01 from 260 nm to 420 nm,
mp: about 230 °C. determined using water R as compensation liquid.
A suitable certified reference solution (10 ng/µL in Hexane, Purified
cyclohexane) may be used. A grade of hexane containing not more than 0.002% w/v of
o:-Hexachlorocyclohexane C 6H 6Cl6 = 290.8 (319-84-6) non-volatile matter.
bp: about 288 °C. Hexylamine Hexan-1-amine; C 6H 15N = 101.2 (111-26-2)
mp: about 158 °C. Colourless liquid, slightly soluble in water, soluble in ethanol
(96 per cent).
2023 Appendix I A V-A83

dig: about 0.766. phospholipids and calcium buffers. Suitable stabilisers may
nt 0: about 1.418. be added.
bp: 127 °C to 131 °C. Hyaluronate Solution
Hibifolin 2-(3,4-Dihydroxyphenyl)-3,5, 7-trihydroxy-4-oxo- Dilute potassium hyaluronate stock solution with an equal
4H-1-benzopyran-8-yl P-o-glucopyranosiduronic acid; volume of phosphate-buffered saline pH 6. 4.
Gossypetin 8-O-glucuronide; Gossypetin 8-O-P-D- Use on the day of preparation.
glucuropyranoside; C 21 H 18 O 14 = 494.4 (55366-56-8) Hyaluronidase Diluent
Storage: protected from light, at a temperature not exceeding Mix 100 mL of phosphate buffer solution pH 6. 4 R with
8 °C, in a dry place. 100 mL of water R. Dissolve 0.140 g of hydrolysed gelatin R in
Hippuric Acid Benzamidoacetic acid; the solution at 37 °C.
Benzoylaminoethanoic acid; N-Benzoylglycine; Storage: use within 2 h.
C 9H 9NO 3 = 179.2 (495-69-2)
Hydrastine Hydrochloride (3S)-6,7-Dimethoxy-3-[(5R)-
White or almost white crystalline powder. 6-methyl-5,6, 7,8-tetrahydro-1,3-dioxolo [4,5-g]isoquinolin-5-
mp: 187-191 °C. yl]isobenzofuran-1 (3H)-one hydrochloride;
Histamine Dihydrochloride (56-92-8) C 21 H 22 ClNO 6 = 419.9 (5936-28-7)
See Histamine dihydrochloride (0143). White or almost white powder, hygroscopic, very soluble in
Histamine Phosphate Histamine acid phosphate; water and in ethanol (96 per cent).
CsH9N3,2H3PO4 = 307.1 (23297-93-0) [a]!-;: about+ 127.
Of the British Pharmacopoeia. mp: about 116 °C.
Histamine Solution Hydrastine hydrochloride used in liquid chromatography complies
A 9 g/L solution of sodium chloride R containing 0.1 µg per with the following additional test.
millilitre of histamine base (as the phosphate or Assay. Liquid chromatography (2.2.29) as prescribed in the
dihydrochloride). monograph Goldenseal rhizome (1831).
Histidine (2S)-2-Amino-3-(1H-imidazol-4-yl)propanoic Content: minimum 98 per cent, calculated by the
acid; (71-00-1) normalisation procedure.
Histidine Monohydrochloride (RS)-2-Amino- Hydrazine Diazane; H 4 N 2 = 32.05 (302-01-2)
3-(imidazol-4-yl)propionic acid hydrochloride monohydrate; Slightly oily liquid, colourless, with a strong odour of
C 6 H 10 CIN3O 2 ,H2 O = 209.6 (123333-71-1) ammonia, miscible with water. Dilute solutions in water are
Crystalline powder or colourless crystals, soluble in water. commercially available.
mp: about 250 °C, with decomposition. nt0 : about 1.470.

Chromatography. Thin-layer chromatography (2.2.27) as bp: about 113 °C.

prescribed in the monograph Histamine mp: about 1.5 °C.
dihydrochloride (0143); the chromatogram shows only one Caution: toxic and corrosive.
principal spot.
Hydrazine Hydrate N 2 H 4 ,H2 O = 50.06 (10217-52-4)
Holmium Oxide Diholmium trioxide; Ho 2 O 3 = 377.9
Analytical reagent grade of commerce.
(12055-62-8)
A colourless liquid; weight per mL, about 1.03 g.
Yellowish powder, practically insoluble in water.
Hydrazine Dihydrochloride Cl 2H 6N 2 = 105.0
Holmium Perchlorate Solution (5341-61-7)
A 40 g/L solution of holmium oxide R in a solution of
White or almost white powder.
perchloric acid R containing 141 g/L ofHC1O 4 •
Content: minimum 97.5 per cent.
DL-Homocysteine (2RS)-2-Amino-4-sulfanylbutanoic
acid; C 4 H 9NO 2 S = 135.2 (454-29-5) Hydrazine Sulfate Hydrazine sulphate; H 6N 2 O 4 S = 130.1
(10034-93-2)
White or almost white, crystalline powder.
Colourless crystals, sparingly soluble in cold water, soluble in
mp: about 232 °C.
hot water (50 °C) and freely soluble in boiling water,
L-Homocysteine Thiolactone Hydrochloride (3S)-3- practically insoluble in ethanol (96 per cent).
Aminodihydrothiophen-2(3H)-one hydrochloride;
Content: minimum 99 per cent.
C 4 H 8CINOS = 153.6 (31828-68-9)
Hydrindantin 2,2 '-Dihydroxy-2,2 '-bi-indan-1, 1 ',3,3 ' -
White or almost white, crystalline powder.
tetraone dihydrate; C 18H 10 O 6 ,2H 2 O = 358.3 (5950-69-6)
mp: about 202 °C.
mp: about 258°.
Homoorientin 2-(3,4-Dihydroxyphenyl)-6-P-o-
General reagent grade of commerce.
glucopyranosyl-5, 7-dihydroxy-4H-1-benzopyran-4-one;
Isoorientin; Luteolin-6-C-glucoside; C 21 H 20 O" = 448.4 Hydriodic Acid HI= 127.9 (10034-85-2)
(4261-42-1) Prepare by distilling hydriodic acid over red phosphorus,
Honokiol 3' ,5-Di(prop-2-enyl)biphenyl-2,4'-diol; 3' ,5- passing carbon dioxide R or nitrogen R through the apparatus
Diallyl-2,4'-dihydroxybiphenyl; 3 ',5-Di-2-propenyl-[1,1 ' - during the distillation. Use the colourless or almost
biphenyl]-2,4'-diol; C1 8H 18 O 2 = 266.3 (35354-74-6) colourless, constant-boiling mixture (55 per cent to
58 per cent of HI) distilling between 126 °C and 127 °C.
Human Tissue Factor Solution
Place the acid in small, amber, glass-stoppered bottles
Solution containing human tissue factor, which may be
previously flushed with carbon dioxide R or nitrogen R, seal
produced by recombinant DNA technology, combined with with paraffin.
V-A84 Appendix I A 2023

Storage: in a dark place. Solutions of the requisite molarity may be obtained by

Hydrobromic Acid, 30 per cent (10035-10-6) diluting hydrochloric acid with ethanol (96%) in place of water
A 30 per cent solution of hydrobromic acid in glacial acetic as directed under hydrochloric acid.
acid R. Hydrochloric Acid, Heavy Metal-free
Degas with caution the contents before opening. Complies with the requirements prescribed for hydrochloric
Hydrobromic Acid, 47 per cent acid R with the following maximum contents of heavy metals.
A 47 per cent mlm solution of hydrobromic acid. As: 0.005 ppm.
Cd: 0.003 ppm.
Hydrobromic Acid, Dilute
Place 5.0 mL of 30 per cent hydrobromic acid R in amber vials Cu: 0.003 ppm.
equipped with polyethylene stoppers. Seal under argon R and Fe: 0.05 ppm.
store in the dark. Add 5.0 mL of glacial acetic acid R Hg: 0.005 ppm.
immediately before use. Shake. Ni: 0.004 ppm.
Storage: in the dark. Pb: 0.001 ppm.
Hydrobromic Acid Rl, Dilute Zn: 0.005 ppm.
Contains 7,9 g/L of HBr. Hydrochloric Acid, Lead-free
Dissolve 16.81 g of 47 per cent hydrobromic acid R in water R Complies with the requirements prescribed for hydrochloric
and dilute to 1000 mL with the same solvent. acid R with the following additional requirement.
Hydrocarbons (Type L), Low-vapour-pressure Lead: maximum 20 ppb.
Unctuous mass, soluble in benzene and in toluene. Atomic emission spectrometry (2.2.22, Method l).
Hydrochloric Acid (7647-01-0) Test solution. In a quartz crucible evaporate 200 g of the acid
See Concentrated hydrochloric acid (0002). to be examined almost to dryness. Take up the residue in
Solutions of molarity xM should be prepared by diluting 85x 5 mL of nitric acid prepared by sub-boiling distillation of
mL of hydrochloric acid to 1000 mL with water. nitric acid R and evaporate to dryness. Take up the residue in
5 mL of nitric acid prepared by sub-boiling distillation of
0.1M Hydrochloric Acid, Alcoholic
nitric acid R.
Dilute 9.0 mL of hydrochloric acid R to 1000.0 mL with
aldehyde-free alcohol R. Reference solutions. Prepare the reference solutions using lead
standard solution (0.1 ppm Pb) R diluted with nitric acid
2M Hydrochloric Acid
prepared by sub-boiling distillation of nitric acid R.
Dilute 206.0 g of hydrochloric acid R to 1000.0 mL with Wavelength: 220.35 nm.
water R.
Hydrochloric Acid, Methanolic
3M Hydrochloric Acid
Dilute 4.0 mL of hydrochloric acid R to 1000.0 mL with
Dilute 309.0 g of hydrochloric acid R to 1000.0 mL with
methanol R2.
water R.
Hydrochloric Acid Rl
6M Hydrochloric Acid
Contains 250 g/L of HCI.
Dilute 618.0 g of hydrochloric acid R to 1000.0 mL with
Dilute 70 g of hydrochloric acid R to 100 mL with water R.
water R.
Hydrochloric Acid, Brominated Hydrochloric Acid Rl, Dilute
Contains 0.37 g/L of HCI.
To 1 mL of bromine solution R add 100 mL of hydrochloric
acid R. Dilute 1.0 mL of dilute hydrochloric acid R to 200.0 mL with
Hydrochloric Acid, Dilute water R.
Contains 73 g/L of HCI.
Hydrochloric Acid R2, Dilute
Dilute 20 g of hydrochloric acid R to 100 mL with water R. Dilute 30 mL of 1 M hydrochloric acid to 1000 mL with
water R; adjust to pH 1.6 ± 0.1.
Hydrochloric Acid, Dilute, Heavy Metal-free
Hydrochloric Acid, Dilute R3
Complies with the requirements prescribed for dilute
Contains 3. 7 g/L of HCI.
hydrochloric acid R with the following maximum contents of
heavy metals. Dilute 10.0 mL of dilute hydrochloric acid R to 200.0 mL with
As: 0.005 ppm. water R.
Cd: 0.003 ppm.
Hydrochloric Acid, Stannated
Cu: 0.003 ppm. Use stannated hydrochloric acid low in arsenic, grade of
commerce, or prepare by adding 1 mL of tin(II) chloride
Fe: 0.05 ppm. solution to 100 mL of hydrochloric acid.
Hg: 0.005 ppm.
Hydrocortisone C21 H 30 0 5 = 362.5 (50-23-7)
Ni: 0.004 ppm. mp: about 214°, with decomposition.
Pb: 0.001 ppm. General reagent grade of commerce.
Zn: 0.005 ppm. Hydrocortisone Acetate (50-03-3)
Hydrochloric Acid, Ethanolic See Hydrocortisone acetate (0334).
Dilute 5.0 mL of 1 M hydrochloric acid to 500.0 mL with Hydrofluoric Acid HF= 20.01 (7664-39-3)
ethanol (96 per cent) R.
Content: minimum 40.0 per cent m/m.
Clear, colourless liquid.
2023 Appendix I A V-ASS

Loss on ignition: not more than 0.05 per cent mlm; evaporate 4-Hydroxybenzohydrazide p-Hydroxybenzohydrazide;
the hydrofluoric acid in a platinum crucible and gently ignite C 7 H 8N 2 O 2 = 152.2 (5351-23-5)
the residue to constant mass. 4-Hydroxybenzoic Acid Parahydroxybenzoic acid;
Assay. Weigh accurately a glass-stoppered flask containing C 7H 6 O 3 = 138.1 (99-96-7)
50.0 mL of 1 M sodium hydroxide. Introduce 2 g of the Crystals, slightly soluble in water, very soluble in ethanol
hydrofluoric acid and weigh again. Titrate the solution with (96 per cent), soluble in acetone.
0.5 M sulfuric acid, using 0.5 mL of phenolphthalein solution R
mp: 214 °C to 215 °C.
as indicator.
4-Hydroxybiphenyl See Biphenyl-4-ol
1 mL of 1 M sodium hydroxide is equivalent to 20.01 mg of
HF. 4-Hydroxycoumarin 4-Hydroxy-2H- l-benzopyran-2-one;
C 9 H 6 0 3 = 162.2 (1076-38-6)
Storage: in a polyethylene container.
White or almost white powder, freely soluble in methanol.
Hydrogen Hydrogen for chromatography; H 2 = 2.016
(1333-74-0) Content: minimum 98.0 per cent.

Content: minimum 99.95 per cent VIV. 6-Hydroxydopa (2RS)-2-Amino-3-(2,4,5-

trihydroxyphenyl)propanoic acid; 2,5-Dihydroxy-DL-tyrosine;
Hydrogen Peroxide Solution (200 vol) H 2 0 2 = 34.02 C 9 H 11 NO 5 = 213.2 (21373-30-8)
(7724-84-1)
mp: about 257 °C.
General reagent grade of commerce containing about
2-[4-(2-Hydroxyethyl)piperazin-t-yl]ethane-t-sulfonic
60% w/v of H2O2.
Acid 2-[ 4-(2-hydroxyethyl)piperazin- l-yl] ethanesulphonic
A colourless liquid; weight per mL, about 1.18 g.
acid; HEPES; C 8 H 18N 2 O 4 S = 238.3 (7365-45-9)
Hydrogen Peroxide Solution (100 vol) See Strong
White or almost white powder.
hydrogen peroxide solution
mp: about 236 °C, with decomposition
Hydrogen Peroxide Solution (20 vol)
4-Hydroxyisophthalic Acid 4-Hydroxybenzene-1,3-
Analytical reagent grade of commerce containing about dicarboxylic acid; C 8 H 6 O 5 = 182.1 (636-46-4)
6% w/v of H 2 0 2 or hydrogen peroxide solution (100 vol)
diluted with 4 volumes of water. Needles or platelets, very slightly soluble in water, freely
soluble in ethanol (96 per cent).
A colourless liquid; weight per mL, about 1.02 g.
mp: about 314 °C, with decomposition.
Hydrogen Peroxide Solution (10 vol) See Dilute hydrogen
peroxide solution Hydroxylamine Hydrochloride Hydroxylammonium
chloride; NH 4 CIO = 69.5 (5470-11-1)
Hydrogen Peroxide Solution, Dilute (7722-84-1)
White or almost white, crystalline powder, very soluble in
See Hydrogen peroxide solution (3 per cent) (0395).
water, soluble in ethanol (96 per cent).
Hydrogen Peroxide Solution, Strong (7722-84-1) Hydroxylamine Hydrochloride Solution R2
See Hydrogen peroxide solution (30 per cent) (0396).
Dissolve 2.5 g of hydroxylamine hydrochloride R in 4.5 mL of
Hydrogen Sulfide Hydrogen sulphide; H 2 S = 34.08 hot water R and add 40 mL of ethanol (96 per cent) R and
(7783-06-4) 0.4 mL of bromophenol blue solution R2. Add 0.5 M alcoholic
Gas, slightly soluble in water. potassium hydroxide until a greenish-yellow colour is obtained.
Hydrogen Sulfide Rt Hydrogen sulphide Rl; Dilute to 50.0 mL with ethanol (96 per cent) R.
H 2 S = 34.08 (7783-06-4) Hydroxylamine Solution, Alcoholic
Content: minimum 99.7 per cent V/V. Dissolve 3.5 g of hydroxylamine hydrochloride R in 95 mL of
Hydrogen Sulfide Solution Hydrogen sulphide solution ethanol (60 per cent Vlv'.) R, add 0.5 mL of a 2 g/L solution
A recently prepared solution of hydrogen sulfide R in water R. of methyl orange R in ethanol (60 per cent V/V) R and
The saturated solution contains about 0.4 per cent to sufficient 0. 5 M potassium hydroxide in alcohol
(60 per cent V/V) to give a pure yellow colour. Dilute to
0.5 per cent of H 2 S at 20 °C.
100 mL with ethanol (60 per cent Vlv'.) R.
Hydroquinone Quinol; C 6 H 6 O 2 = 110.1 (123-31-9)
Hydroxylamine Solution, Alkaline
Fine, colourless or white or almost white needles, darkening
on exposure to air and light, soluble in water and in ethanol Immediately before use, mix equal volumes of a 139 g/L
(96 per cent).
solution of hydroxylamine hydrochloride R and a 150 g/L
solution of sodium hydroxide R.
mp: about 173 ''C.
Hydroxylamine Solution Rt, Alkaline
Storage: protected from light and air.
Solution A. Dissolve 12.5 g of hydroxylamine hydrochloride R in
Hydroquinone Solution methanol R and dilute to 100 mL with the same solvent.
Dissolve 0.5 g of hydroquinone R in water R, add 20 µL of Solution B. Dissolve 12.5 g of sodium hydroxide R in
sulfuric acid R and dilute to 50 mL with water R.
methanol R and dilute to 100 mL with the same solvent.
4 1-Hydroxyacetophenone 1-( 4-Hydroxyphenyl)ethan-1- Mix equal volumes of solution A and solution B immediately
one; C 8 H 8 O 2 = 136.2 (99-93-4) before use.
4-Hydroxybenzaldehyde C 7 H 6 O 2 = 122.2 (123-08-0) Hydroxymethylfurfural 5-Hydroxymethylfurfural;
mp: about 118°. C 6 H 6 O 3 = 126.1 (67-47-0)
General reagent grade of commerce. Acicular crystals, freely soluble in water, in acetone and in
Colourless needles. ethanol (96 per cent).
2-Hydroxybenzimidazole lH-benzirnidazol-2-ol; mp: about 32 °C.
C 7 H 6N 2 O = 134.1 (615-16-7)
V-A86 Appendix I A 2023

Hydroxynaphthol Blue, Sodium Salt Trisodium 2,2'- Chromatography. Thin-layer chromatography (2.2.27) as
dihydroxy-l, l '-azonaphthalene-3 ',4,6 '-trisulfonate; prescribed in the monograph Mercaptopurine (0096); the
C 20 H 11 N 2Na 3O 11 S3 = 620 (63451-35-4') chromatogram shows only one principal spot.
4-(4-Hydroxyphenyl)butan-2-one C 10H 12 O 2 = 164.2 Ibuprofen (15687-27-1)
(5471-51-2) See Ibuprofen (0721).
mp: about 82°. Imidazole Glyoxaline; C 3H 4N 2 = 68.1 (288-32-4')
General reagent grade of commerce. White or almost white, crystalline powder, soluble in water
2-Hydroxypropylbetadex for Chromatography and in ethanol (96 per cent).
Betacyclodextrin modified by the bonding of (R) or (RS) mp: about 90 °C.
propylene oxide groups on the hydroxyl groups; Imidazole, Recrystallised
Hydroxypropyl-~-cyclodextrin (94035-02-6)
Twice recrystallise 2 5 g of imidazole from 100 mL of toluene,
See Hydroxypropylbetadex (1804). cool in ice with stirring, wash with ether and dry at room
pH (2.2.3): 5.0 to 7.5 for a 20 g/L solution. temperature at a pressure of 2 kPa over anhydrous silica gel, or
8-Hydroxyquinoline Quinolin-8-ol; Hydroxyquinoline; use a purified grade of commerce.
C 9 H 7NO = 145.2 (148-24-3) Complies with the following test.
White or slightly yellowish, crystalline powder, slightly Light absorption Absorbance of an 8% w/v solution at
soluble in water, freely soluble in acetone, in ethanol 325 nm, not more than 0.10, Appendix II B.
(96 per cent) and in dilute mineral acids. lmidazole Solution
mp: about 75 °C. Dissolve 8.25 g of recrystallised imidazole in 60 mL of water,
Sul,fated ash (2.4.14'): maximum 0.05 per cent. adjust the pH to 6.75 to 6.85 with 5M hydrochloric acid and
12-Hydroxystearic Acid 12-Hydroxyoctadecanoic acid; add sufficient water to produce 100 mL.
C1sH36O3 = 300.5 (106-14-9) The hydrochloric acid used in preparing this reagent must be
White or almost white powder. free from stabilising mercury compounds.
mp: 71 °C to 74 °C. lmidazole-Mercury Reagent
5-Hydroxyuracil 2,4,5-trihydroxypyrimidine; Dissolve 8.25 g of recrystallised imidazole in 60 mL of water
C 4 H 4 N 2O 3 = 128.1 (496-76-4') and add 10 mL of 5M hydrochloric acid. Stir the solution
White or almost white, crystalline powder. magnetically and add, dropwise, 10 mL of a 0.27% w/v
mp: about 310 °C, with decomposition. solution of mercury(II) chloride. If a cloudy solution results,
discard and prepare a further solution adding the mercury
Chromatography. Thin-layer chromatography (2.2.27) as chloride solution more slowly. Adjust the pH to 6.75 to 6.85
prescribed in the monograph Fluorouracil (0611); the with 5M hydrochloric acid (about 4 mL is required) and add
chromatogram shows a principal spot with an Rp of about sufficient water to produce 100 mL.
0.3.
lminodiacetic Acid 2,2'-Iminodiacetic acid;
Storage: in an airtight container. C 4H 7 NO 4 = 133.1 (142-73-4')
Hyoscine Hydrobromide Scopolamine hydrobromide; Iminodibenzyl 10, 11-Dihydrodibenz[bJ] azepine;
(6533-68-2) C 14 H 13N = 195.3 (494-19-9)
See Hyoscine hydrobromide (0106). Pale yellow, crystalline powder, practically insoluble in water,
Hyoscyamine Sulfate Hyoscyamine sulphate; (620-61-1) freely soluble in acetone.
See Hyoscyamine sulfate (0501). mp: about 106 °C.
Hypericin 1,3,4,6,8, 13-Hexahydroxy-l 0, 11- lmipramine Hydrochloride (113-52-0)
dimethylphenanthro [1, 10,9,8-opqra]perylene-7, 14-dione; See Imipramine hydrochloride (0029).
C30H16Os = 504.4 (548-04-9)
lmperatorin 9-[(3-Methylbut-2-enyl)oxy]-7H-furo[3,2-g]
Content: minimum 85 per cent. [l]benzopyran-7-one; C 16H 14 O 4 = 270.3 (482-44-0)
Hyperoside 2-(3,4-Dihydroxyphenyl)-3-~-D- 2-Indanamine Hydrochloride 2-Aminoindane
galactopyranosyloxy-5, 7-dihydroxychromen-4-one; hydrochloride; C 9H 12 ClN = 169.7 (2338-18-3)
C21H20O12 = 464.4
2,3-Dihydro-lH-inden-2-amine hydrochloride.
Faint yellow needles, soluble in methanol.
Indian Sandalwood Oil Sandalwood Oil; East Indian
Absorbance (2.2.25). A solution in methanol R shows Sandalwood Oil; Santalum Album; C 30 H 48 O 2 = 440.70
2 absorption maxima at about 257 nm and at about 359 nm. (8006-87-9)
Hypophosphorous Reagent General reagent grade of commerce.
Dissolve with the aid of gentle heat, 10 g of sodium Indigo Indigotin; 1,1',3,3'-Tetrahydro-2-2'-
hypophosphite R in 20 mL of water R and dilute to 100 mL bi(indolylidene)-3,3'-dione; C 16 H 10N 2 O 2 = 262.3 (482-89-3)
with hydrochloric acid R. Allow to settle and decant or filter
through glass wool. Indigo Carmine Acid blue 74; C 16H 8 N2Na2O 8 S2 = 466.3
(860-22-0)
Hypoxanthine 6-Hydroxypurine; C 5 H 4 N 4 O = 136.1
(68-94-0)
Schultz No. 1309
White or almost white, crystalline powder, very slightly Colour Index No. 73015
soluble in water, sparingly soluble in boiling water, soluble in It usually contains sodium chloride.
dilute acids and in dilute alkali hydroxide solutions, Blue or violet-blue powder or blue granules with a coppery
decomposes without melting at about 150 °C. lustre, sparingly soluble in water, practically insoluble in
2023 Appendix I A V-A87

ethanol (96 per cent). It is precipitated from an aqueous Iodine Bromide IBr = 206.8 (7789-33-5)
solution by sodium chloride. Bluish-black or brownish-black crystals, freely soluble in
Indigo Cannine Solution water, in ethanol (96 per cent) and in glacial acetic acid.
To a mixture of 10 mL of hydrochloric acid Rand 990 mL of bp: about 116 °C.
200 glL nitrogen-free sulfuric acid R add 0.2 g of indigo mp: about 40 cc.
carmine R.
Storage: protected from light.
The soluti.on complies with the following test: add 10 mL to a
Iodine Bromide Solution
solution of 1.0 mg of potassium nitrate R in 10 mL of water R,
rapidly add 20 mL of nitrogen-free sulfuric acid R and heat to Dissolve 20 g of iodine bromide R in glacial acetic acid R and
boiling. The blue colour is discharged within 1 min. dilute to 1000 mL with the same solvent.
Indigo Cannine Solution Rl Storage: protected from light.

Dissolve 4 g of indigo carmine R in about 900 mL of water R Iodine Chloride IC!= 162.4 (7790-99-0)
added in several portions. Add 2 mL of sulfuric acid R and Black crystals, soluble in water, in acetic acid and in ethanol
dilute to 1000 mL with water R. (96 per cent).
Assay. Place in a 100 mL conical flask with a wide neck bp: about 97.4 °C.
10.0 mL of nitrate standard solution (100 ppm NO_,) R, 10 mL Iodine Chloride Solution
of water R, 0.05 mL of the indigo carmine solution Rl, and Dissolve 1.4 g of iodine chloride R in glacial acetic acid R and
then in a single addition, but with caution, 30 mL of sulfuric dilute to 100 mL with the same acid.
acid R. Titrate the solution immediately, using the indigo
Storage: protected from light.
carmine solution Rl, until a stable blue colour is obtained.
Iodine Monochloride Reagent, Strong
The number of millilitres used, n, is equivalent to 1 mg of
NO3. Dissolve 10 g of potassium iodide and 6.4 g of potassium iodate
in 75 mL of water, add 75 mL of hydrochloric acid and 5 mL
Indirubin C 16H 10N 2 O 2 = 262.3 (479-41-4) of chloroform, shake and, if necessary, add dropwise with
1, 1 1,2 1,3-Tetrahydro-2,3 1-bi(indolylidene)-2 1,3-dione. vigorous shaking 0.lM potassium iodide until a faint iodine
Indometacin (53-86-1) colour appears in the chloroform layer. Add in the same
See Indometacin (0092). manner 0.001M potassium iodate until the chloroform is just
Industrial Methylated Spirit (95%) colourless.
Of the British Pharmacopoeia. Before use, readjust with either 0.lM potassium iodide or
0.001M potassium iodate as required.
Inosine 9-P-n-Ribofuranosylhypoxanthine; 9-P-D-
Ribofuranosyl-l, 9-dihydro-6H-purin-6-one; Store in a cool place protected from light.
C10H12N4Os = 268.2 (58-63-9) Iodine Monochloride Solution
mp: 222 °C to 226 °C. Dissolve 8 g of iodine trichloride in about 200 mL of glacial
myo-Inositol acetic acid and separately dissolve 9 g of iodine in 300 mL of
dichloromethane. Mix the two solutions and dilute to 1000 mL
See myo-Inositol (1805).
with glacial acetic acid.
Intermedine [(lR, 7aR)-l-Hydroxy-2,3,5, 7a-tetrahydro- Store in a stoppered bottle, protected from light and at a
1H-pyrrolizin-7-yl] methyl (2S,3R)-2,3-dihydroxy-2-(propan-
temperature not exceeding 15°.
2-yl) butanoate; 3 1-epi-Lycopsamine; C 15H 25 NO 5 = 299.4
(10285-06-0)
Iodine Pentoxide, Recrystallised Iodine pentoxide;
12 0 5 = 333.8 (12029-98-0)
Light brown powder.
Content: minimum 99.5 per cent.
Intermedine N-Oxide (1R,7aR)-7-[[[(2S,3R)-2,3-
Dihydroxy-2-(propan-2-yl) butanoyl] oxy] methyl]- l-hydroxy- White or almost white, crystalline powder, or white or
2,3,5, 7a-tetrahydro- lH-pyrrolizine 4-oxide; greyish-white granules, hygroscopic, very soluble in water
C1sH2 5NO6 = 315.4 (95462-14-9) forming HI03.
White or almost white powder, soluble in water and in Stability on heating. Dissolve 2 g, previously heated for 1 h at
methanol. 200 °C, in 50 mL of water R. A colourless solution is
obtained.
Iodic Acid HIO 3 = 175.9 (7782-68-5)
Assay. Dissolve 0.100 g in 50 mL of water R, add 3 g of
Analytical reagent grade of commerce.
potassium iodide R and 10 mL of dilute hydrochloric acid R.
Iodine (7553-56-2) Titrate the liberated iodine with 0.1 M sodium thiosulfate,
See Iodine (0031). using 1 mL of starch solution R as indicator.
To prepare 0.05M iodine dissolve 20 g of potassium iodide in 1 mL of 0.1 M sodium thiosulfate is equivalent to 2. 782 mg of
the minimum amount of water, add 13 g of iodine, allow to 1205.
dissolve and add sufficient water to produce 1000 mL. Storage: in an airtight container, protected from light.
Weaker solutions should be prepared using proportionately
Iodine Solution, Alcoholic
lesser amounts of reagents or by appropriate dilution.
A 10 glL solution of iodine R in ethanol (96 per cent) R.
Iodine-123 and Ruthenium-106 Spiking Solution
Storage: protected from light.
Prepare immediately before use. Mix 3.5 mL of an
18.5 kBq/mL solution of ruthenium-106 in the form of Iodine Solution, Chloroformic
ruthenium trichloride in a mixture of equal volumes of glacial A 5 glL solution of iodine R in chloroform R.
acetic acid R and water R with 200 µL of a 7 5 kBq/mL Storage: protected from light.
solution of iodine-123 in the form of sodium iodide in
water R.
V-A88 Appendix I A 2023

Iodine Solution Rl Storage: in an airtight container, protected from light.

To 10.0 mL of 0.05 M iodine add 0.6 g of potassium iodide R 2-lodohippuric Acid 2-(2-Iodobenzamido)acetic acid;
and dilute to 100.0 mL with water R. Prepare immediately C 9 H 8 IN03 ,2H2 0 = 341.1 (147-58-0)
before use. White or almost white, crystalline powder, sparingly soluble
Iodine Solution R2 in water.
To 10.0 mL of 0.05 M iodine add 0.6 g of potassium iodide R mp: about 170 °C.
and dilute to 1000.0 mL with water R. Prepare immediately Water (2.5.12): 9 per cent to 13 per cent, determined on
before use. 1.000 g.
Iodine Solution R3 Chromatography. Thin-layer chromatography (2.2.27), using
Dilute 2.0 mL of iodine solution Rl to 100.0 mL with cellulose for chromatography F 254 R as the coating substance:
water R. Prepare immediately before use. apply to the plate 20 µL of a solution of the 2-iodohippuric
Iodine Solution R4 acid, prepared by dissolving 40 mg in 4 mL of 0.1 M sodium
Dissolve 14 g of iodine R in 100 mL of a 400 g/L solution of hydroxide and diluting to 10 mL with water R. Develop over a
potassium iodide R, add 1 mL of dilute hydrochlom acid R and path of about 12 cm using as the mobile phase the upper
dilute to 1000 mL with water R. layer obtained by shaking together 20 volumes of water R,
40 volumes of glacial acetic acid R and 40 volumes of
Storage: protected from light.
toluene R. Allow the plate to dry in air and examine in
Iodine Solution R5 ultraviolet light at 254 nm. The chromatogram shows only
Dissolve 12. 7 g of iodine R and 20 g of potassium iodide R in one principal spot.
water Rand dilute to 1000.0 mL with the same solvent Iodomethane Methyl iodide; CH 3 I = 141.9 (74-88-4)
(0.05 M solution). Content: minimum 99.0 per cent.
Iodine Trichloride IC13 = 233.3 (865-44-1) Iodoplatinate Reagent
Analytical reagent grade of commerce. To 3 mL of a 100 g/L solution of chloroplatinic acid R add
Reddish orange crystals. 97 mL of water R and 100 mL of a 60 g/L solution of
Iodoacetamide 2-Iodoacetamide; C 2 H 4 INO = 185.0 potassium iodide R.
(144-48-9) Storage: protected from light.
Slightly yellow, crystalline powder, soluble in water. Iodoplatinate Reagent Rl
mp: about 92 °C. Mix 2.5 mL of a 50 g/L solution of chloroplatinic acid R,
Iodoacetic Acid C 2H 3I0 2 = 185.9 (64-69-7) 22.5 mL of a 100 g/L solution of potassium iodide R and
Colourless or white or almost white crystals, soluble in water 50 mL of water R.
and in ethanol (96 per cent). Storage: protected from light, at a temperature of 2-8 °C.
mp: 82 °C to 83 °C. 2-Iodopropane Isopropyl iodide; C 3 H 7 I = 170.0 (75-30-9)
2-Iodobenzoic Acid C 7H 5I0 2 = 248.0 (88-67-5) Content: minimum 99 per cent.
White or slightly yellow, crystalline powder, slightly soluble in 5-Iodouracil 5-Iodo-1H,3H-pyrimidine-2,4-dione;
water, soluble in ethanol (96 per cent). C4H 3 IN2 O2 = 238.0 (696-07-1)
mp: about 160 °C. mp: about 276 °C, with decomposition.
Chromatography. Thin-layer chromatography (2.2.27), using Chromatography. Thin-layer chromatography (2.2.27) as
cellulose for chromatography f254 R as the coating substance: prescribed in the monograph ldoxuridine (0669): apply 5 µL
apply to the plate 20 µL of a solution of the 2-iodobenzoic of a 0.25 g/L solution; the chromatogram obtained shows
acid, prepared by dissolving 40 mg in 4 mL of 0.1 M sodium only one principal spot.
hydroxide and diluting to 10 mL with water R. Develop over a Ion-exchange Resin, Strongly Acidic
path of about 12 cm using as the mobile phase the upper Resin in protonated form with sulfonic acid groups attached
layer obtained by shaking together 20 volumes of water R, to a lattice consisting of polystyrene cross-linked with
40 volumes of glacial acetic acid R and 40 volumes of 8 per cent of divinylbenzene. It is available as spherical
toluene R. Allow the plate to dry in air and examine in
beads; unless otherwise prescribed, the particle size is
ultraviolet light at 254 nm. The chromatogram shows only 0.3 mm to 1.2 mm.
one principal spot.
Capacity. 4.5 mmol to 5 mmol per gram, with a water
3-Iodobenzylanunonium Chloride 1-(3-Iodophenyl) content of 50 per cent to 60 per cent.
methanamine hydrochloride; 1-(3-Iodophenyl)
methanaminium chloride; m-Iodobenzylamine hydrochloride; Preparation of a column. Unless otherwise prescribed, use a
C 7 H 9 ClIN = 269.5 (3718-88-5) tube with a fused-in sintered glass disc having a length of
400 mm, an internal diameter of 20 mm and a filling height
White or almost white crystals. of about 200 mm. Introduce the resin, mixing it with water R
mp: 188 °C to 190 °C. and pouring the slurry into the tube, ensuring that no air
Iodoethane C 2 H 5 I = 156.0 (75-03-6) bubbles are trapped between the particles. When in use, the
Content: minimum 99 per cent. liquid must not be allowed to fall below the surface of the
Colourless or slightly yellowish liquid, darkening on exposure resin. If the resin is in its protonated form, wash with water R
to air and light, miscible with ethanol (96 per cent) and most until 50 mL requires not more than 0.05 mL of 0.1 M
organic solvents. sodium hydroxide for neutralisation, using 0.1 mL of methyl
orange solution Ras indicator.
d~g: about 1.95.
If the resin is in its sodium form or if it requires regeneration,
nt0 : about 1.513.
pass about 100 mL of a mixture of equal volumes of
bp: about 72 °C.
 2023 Appendix I A V-A89

hydrochloric acid Rl and water R slowly through the column Iron(m) Sulfate Ferric sulphate; Iron(rn) sulphate;
and then wash with water R as described above. Iron(rn)trisulfate hydrated; Iron(m)trisulphate hydrated;
Ion-exclusion Resin for Chromatography Ferric sulfate; Fe 2 (SO 4 ) 3 ,xH 2 O (15244-10-7)
Resin with a sulfonate funtionalised latex cross-linked with Yellowish-white powder, very hygroscopic, decomposes in
divinylbenzene. air, slightly soluble in water and in ethanol (96 per cent).
Irisflorentin 9-Methoxy-7-(3,4,5-trimethoxyphenyl)-8H- Storage: in an airtight container, protected from light.
l ,3-dioxolo [4,5-g][l ]benzopyran-8-one; C 20H 18 O 8 = 386.4 lron(m) Sulfate Pentahydrate Ferric sulphate
( 41743-73-1) pentahydrate; Iron(rn) sulphate pentahydrate; Ferric sulfate
Iron Fe = 55.85 (7439-89-6) pentahydrate; Fe2(SO 4 ) 3 ,5H 2O = 489.9 (142906-29-4)
Grey powder or wire, soluble in dilute mineral acids. White or yellowish powder.
Iron(m) Chloride, Anhydrous Iron(m) chloride; Iron(m) Sulfate Solution Ferric sulfate solution
anhydrous ferric chloride; FeC1 3 = 162.2 (7705-08-0) Dissolve 50 g of ferric sulfate R in an excess of water R, add
General reagent grade of commerce. 200 mL of sulfuric acid R and dilute to 1000 mL with
Greenish black crystals or crystalline powder turning orange water R.
on exposure to moist air. Iron(n) Sulfate Solution R2 Ferrous sulfate solution R2
Iron(m) Chloride Hexahydrate Iron trichloride Dissolve 0.45 g of ferrous sulfate R in 50 mL of 0.1 M
hexahydrate; Ferric chloride; FeC13,6H2 O = 270.3 hydrochloric acid and dilute to 100 mL with carbon dioxide-free
(10025-77-1) water R. Prepare immediately before use.
Yellowish-orange or brownish crystalline masses, Iron(n) Sulfate-Citrate Solution
deliquescent, very soluble in water, soluble in ethanol Iron(II) sulphate-citrate solution
(96 per cent). On exposure to light, ferric chloride and its Dissolve l g of sodium metabisulfite in 200 mL of water and
solutions are partly reduced. add 0.5 mL of 2M hydrochloric acid, 1.5 g of iron(II) sulfate
Swrage: in an airtight container. and 10 g of sodium citrate.
Iron(m) Chloride Solution Prepare immediately before use.
Analytical reagent grade of commerce, diluted to contain lsatin Indoline-2,3-dione; C 8H 5NO 2 = 147.1 (91-56-5)
about 15% w/v ofFeC1 3 • Small, yellowish-red crystals, slightly soluble in water, soluble
Iron(m) Chloride Solution, Ethanolic in hot water and in ethanol (96 per cent), soluble in solutions
Carefully add 25 mL of sulfuric acid dropwise to 75 mL of of alkali hydroxides giving a violet colour becoming yellow on
well-cooled absolute ethanol, stirring constantly. Add 2 g of standing.
anhydrous iron(III) chloride, stir and filter. mp: about 200 cc, with partial sublimation.
Iron(m) Chloride Solution Rl Ferric chloride Sulfated ash (2.4.14): maximum 0.2 per cent.
solution Rl Isatin Reagent
A 105 g/L solution of ferric chloride R. Dissolve 6 mg of ferric sulfate R in 8 mL of water R and add
Iron(m) Chloride Solution R2 Ferric chloride cautiously 50 mL of sulfuric acid R. Add 6 mg of isatin R and
solution R2 stir until dissolved.
A 13 g/L solution of ferric chloride R. The reagent should be pale yellow, but not orange or red.
Iron(m) Chloride-Sulfamic Acid Reagent Isoandrosterone Epiandrosterone; 3~-Hydroxy-Sa-
Ferric chloride-sulfamic acid reagent androstan-17-one; C 19 H 30 O 2 = 290.4 (481-29-8)
A solution containing 10 g/L of ferric chloride Rand 16 g/L of White or almost white powder, practically insoluble in water,
sulfamic acid R. soluble in organic solvents.
Iron(m) Nitrate Ferric nitrate; Fe(NO 3 h,9H 2 O = 404 [rt]~: + 88, determined on 20 g/L solution in methanol R.
(7782-61-8) mp: 172 °C to 174 cc.
Content: minimum 99.0 per cent mlm of Fe(NO 3 h,9H 2 O. M (2.2.41): 14.24 x 103, determined at 304 nm on a
Light-purple crystals or crystalline mass, very soluble in 1.25 g/L solution.
water. lsobutyl Acetate C 6H 12 O 2 = 116.2 (110-19-0)
Free acid: not more than 0.3 per cent (as HNO 3 ). bp: about 118°.
Iron(m) Nitrate Solution General reagent grade of commerce.
A 0.1 % w/v solution of iron(m) nitrate in 0.1 % v/v nitric acid. A colourless liquid; weight per mL, about 0.87 g.
Iron Salicylate Solution N-Isobutyldodecatetraenamide (2E,4E,8Z, 10EZ)-N-
Dissolve 0 .1 g of ferric ammonium sulfate R in a mixture of 2-(Methylpropyl)dodeca-2,4,8, 10-tetraenamide;
2 mL of dilute suljuric acid R and 48 mL of water R and C16H2sNO = 247.4 (866602-52-0)
dilute to 100 mL with water R. Add 50 mL of a 11.5 g/L White or almost white or non-coloured crystals.
solution of sodium salicylate R, 10 mL of dilute acetic acid R,
mp: about 70 °C.
80 mL of a 136 g/L solution of sodium acetate R and dilute to
500 mL with water R. The solution should be recently N-Isobutyldodecatetraenamide Solution
prepared. A solution of N-isobutyldodecatetraenamide R, exactly weighed,
Storage: in an airtight container, protected from light. in methanol R at a concentration of about 10 mg/mL.
Iron(n) Sulfate Ferrous sulphate; Iron(rn) sulphate; Isodrin 1,2,3,4, 10, 10-Hexachloro-1,4,4a,5,8,8a-hexahydro-
Ferrous sulfate; (7782-63-0) endo,endo- l ,4: 5,8-dimethanonaphthalene; C 12H 8 Cl6 = 364. 9
( 465-73-6)
See Ferrous sulfate heptahydrate (0083).
V-A90 Appendix I A 2023

Practically insoluble in water, soluble in common organic Isoniazid Solution

solvents such as acetone. Dissolve 0.1 g of isoniazid in 150 mL of methanol, add
A suitable certified reference solution may be used. 0.12 mL of hydrochloric acid and dilute to 200 mL with
Isoeugenol 2-Methoxy-4-[ ( IE)-prop-1-en- l-yl]phenol; methanol.
C10H12O 2 = 164.2 (97-54-1) Isonicotinamide 4-Pyridinecarboxamide; Pyridine-4-
Isoeugenyl Acetate C 12 H 14O 3 = 206.24 (93-29-8) carboxamide; C 6H 6N 2O = 122.1 (1453-82-3)
Analytical reagent grade of commerce. White or almost white, crystalline powder, soluble in water.
Isoimperatorin 4-[ (3-Methylbut-2-en-l-yl)oxy]-7H-furo Isonicotinic Acid Pyridine-4-carboxylic acid;
[3,2-g][l]benzopyran-7-one; C 16 H 14O 4 = 270.3 (482-45-1) C6H5 NO 2 = 123.1 (55-22-1)
Isoleucine (73-32-5) Creamish-white powder, sparingly soluble in water.
See Isoleucine (0770). mp: about 311 °C.
Isomalt C 12H 24O 11 = 344.3 (64519-82-0) Isopentyl Benzoate 3-Methylbutyl benzoate; Isoamyl
benzoate; C 12H 160 2 = 192.3 (94-46-2)
Mixture of 6-O-ix-n-glucopyranosyl-n-glucitol and of 1-O-ix-
D-glucopyranosyl-D-mannitol. n~: about 1.494.
White or almost white powder or granules, freely soluble in bp: about 261 °C.
water. Colourless or pale yellow liquid.
Isomaltitol 6-O-ix-n-Glucopyranosyl-n-glucitol; Isopropylamine 2-Propylamine; C 3 H 9 N = 59.1 (75-31-0)
C12H24O 11 = 344.3 (534-73-6) Colourless, highly volatile, flammable liquid.
White or almost white powder, freely soluble in water. n~: about 1.374.
Isomenthol (+ )-Isomentlwl: (1S,2R,5R)-2-isopropyl-5- bp: 32 °C to 34 °C.
methylcyclohexanol; (±)-lsomenthol: a mixture of equal parts
Isopropyl Methanesulfonate 1-methylethyl
of (1S,2R,5R)- and (1R,2S,5S)-2-isopropyl-5-
methanesulfonate; C 4 H 10 O 3 S = 138.2 (926-06-7)
methylcyclohexanol; C 10H 20O = 156.3 (23283-97-8)
Clear, colourless liquid.
Colourless crystals, practically insoluble in water, very soluble
in ethanol (96 per cent). Content: minimum 99.0 per cent.
[o:]~: (+ )-Isomenthol: about + 24, determined on a 100 giL Density: about 1.129 gicm 3 (20 °C).
solution in ethanol (96 per cent) R. ni 0 : 1.418-1.421.

bp: (+)-Isomenthol: about 218 °C. (±)-lsomenthol: about bp: about 82 °C at 6 mm Hg.
218 °C. lsopropyl Myristate Isopropyl tetradecanoate; (110-27-0)
mp: (+ )-Jsomenthol: about 80 °C. ( ± )-Jsomenthol: about See Isopropyl myristate (0725).
53 °C. 4-Isopropylphenol C 9H 12 O = 136.2 (99-89-8)
( + )-Isomenthone ( lR)-cis-p-Menthan-3-one; (lR)-cis-2- Content: minimum 98 per cent.
Isopropyl-5-methylcyclohexanone; C 10H 18 O = 154.2
bp: about 212 °C.
Contains variable amounts of menthone. A colourless liquid,
mp: 59 °C to 61 °C.
very slightly soluble in water, soluble in ethanol
(96 per cent). Isopropyl Toluenesulfonate 1-Methylethyl
4-methylbenzenesulfonate; Propan-2-yl
d~g: about 0.904. 4-methylbenzenesulfonate; Isopropyl tosilate;
n~: about 1.453. C1aH 14 O 3 S = 214.3 (2307-69-9)
[o:]~: about+ 93.2. Content: minimum 97.0 per cent.
Isomenthone used in gas chromatography complies with the Clear liquid.
following additional test. mp: about 20 °C.
Assay. Gas chromatography (2.2.28) as prescribed in the
Isopulegol (-)-Isopulegol; ( 1R,2S,5R)-2-Isopropenyl-5-
monograph Peppermint ozl (0405).
methylcyclohexanol; C 10H 180 = 154.2 (89-79-2)
Test solution. The substance to be examined.
dJ 0 : about 0.911.
Content: minimum 80.0 per cent, calculated by the
normalisation procedure.
ni0 : about 1.472.

bp: about 210 °C.

Isomethyleugenol 1,2-Dimethoxy-4-prop-l-enylbenzene;
C11H 14O 2 = 178.2 (93-16-3) Jsopulegol used in gas chromatography complies with the following
additional test.
Isomethyleugenol used in gas chromatography complies with
the following additional test. Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Mint oil, partly dementholised (1838).
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Niaouli oil, cineole type (2468). Content: minimum 99 per cent, calculated by the
normalisation procedure.
Content: minimum 97.0 per cent, calculated by the
normalisation procedure. Isoquercitrin 2-(3,4-Dihydroxyphenyl)-3-(~-D-
glucopyranosyloxy)-5, 7-dihydroxy-4H-1-benzopyran-4-one;
Isoniazid Isonicotinohydrazide; C 6H 7N 3 O = 137.1
C21H20 O 12 = 464.4 (482-35-9)
(54-85-3)
Isoquercitroside Isoquercitrin; 3,3',4',5,7-
mp: about 171 °.
Pentahydroxyflavone-3-glucoside; C 21 H 20 O 12 = 464.4
General reagent grade of commerce. (21637-25-2)
Colourless crystals or a white, crystalline powder.
2023 Appendix I A V-A9 l

lsorhamnetin-3-0-neohesperidoside 3-[ 6-Deoxy-C'l-L- Kerosene, Deodorised

mannopyranosyl-( I----> 2)-~-D-glucopyranosyloxy]-5, 7- General grade of commerce.
dihydroxy-2-( 4-hydroxy-3-methoxyphenyl)-4H- l-benzopyran-
11-Keto-~-boswellic Acid 31'.'l-Hydroxy-11-oxours-12-en-
4-one; C 28H 32 01 6 = 625 (55033-90-4)
24-oic acid; (4~)-31'.'l-Hydroxy- ll-oxours-12-en-23-oic acid;
Isorhamnetin-3-0-rutinoside 3-O-Methylquercetin-3- C30H4 60 4 = 470.7 (17019-92-0)
rutinoside; Narcissoside; C2 8H 32 O 16 = 625 (604-80-8)
White or almost white powder, insoluble in water, soluble in
Isorhynchophylline Methyl ( l 6E)- l 7-methoxy-2-oxo- acetone, in anhydrous ethanol and in methanol.
l 6, 17-didehydro-201'.'l-corynoxan-16-carboxylate; Methyl
mp: 195 °C to 197 °C.l l-Keto-f3-boswellic acid used in liquid
( l 6E)-l 6-(methoxymethylidene)-2-oxo-20C'l-corynoxan-l 7-
chromatography complies with the following additwnal test.
oate; C 22 H 28 N2O4 = 384.5 (6859-01-4)
Assay. Liquid chromatography (2.2.29) as prescribed in the
Isosilibinin 3,5, 7-Trihydroxy-2-[2-(4-hydroxy-3-
monograph Indian frankincense (2310).
methoxyphenyl)-3-hydroxymethyl-2,3-dihydro-l ,4-
benzodioxin-6-yl] chroman-4-one; C 25 H 22 O 10 = 482.4 Content: minimum 90 per cent, calculated by the
(72581-71-6) normalisation procedure.
White to yellowish powder, practically insoluble in water, Kieselguhr
soluble in acetone and in methanol. Acid-purified grade of commerce.
Isovitexin 6-~-D-Glucopyranosyl-5, 7-dihydroxy-2-( 4- Kieselguhr for Chromatography
hydroxyphenyl)-4H-l-benzopyran-4-one; Apigenin 6-C-~- White or yellowish-white, light powder, practically insoluble
glucopyranoside; C 21 H 20 O 10 = 432.4 (38953-85-4) in water, in dilute acids and in organic solvents.
Jacobine (lR,2' S,3' S,6R,7R,17R)-7-Hydroxy-3',6, 7- Filtratwn rate. Use a chromatography column 0.25 m long
trimethylspiro[2, 9-dioxa-14-azatricyclo [9 .5. l .0 14' 17 ]heptadec- and 10 mm in internal diameter with a sintered-glass (100)
ll-ene-4,2 '-oxirane]-3,8-dione; C 18 H 25 NO 6 = 351.4 plate and two marks at 0.10 m and 0.20 m above the plate.
( 6870-67-3) Place sufficient of the substance to be examined in the
White or almost white powder, soluble in methanol. column to reach the first mark and fill to the second mark
Jacobine N-Oxide ( lR,2 1S,3 'S,6R, 7R, 17R)-7-Hydroxy- with water R. When the first drops begin to flow from the
3 ',6, 7-trimethyl-3,8-dioxospiro[2, 9-dioxa-14-azatricyclo column, fill to the second mark again with water R and
[9.5. l.0 14' 17]heptadec-11-ene-4,2' -oxirane] 14-oxide; measure the time required for the first 5 mL to flow from the
C1sH2 5NO1 = 367.4 column. The flow rate is not less than I mUmin.
White or almost white powder, soluble in water and in Appearance of the eluate. The eluate obtained in the test for
methanol. filtration rate is colourless (2. 2. 2, Method I).
Kaempferol 3,5, 7-Trihydroxy-2-( 4-hydroxyphenyl)-4H-1- Acidity or alkalinity. To 1.00 g add I 0 mL of water R, shake
benzopyran-4-one; C 15 H 10O 6 = 286.2 (520-18-3) vigorously and allow to stand for 5 min. Filter the suspension
on a filter previously washed with hot water R until the
Kaolin, Light (1332-58-7)
washings are neutral. To 2.0 mL of the filtrate add 0.05 mL
A purified native hydrated aluminium silicate. It contains a of methyl red solutwn R; the solution is yellow. To 2.0 mL of
suitable dispersing agent. the filtrate add 0.05 mL of phenolphthalein solutwn Rl; the
Light, white or almost white powder free from gritty solution is at most slightly pink.
particles, unctuous to the touch, practically insoluble in water Water-soluble substances. Place 10.0 gin a chromatography
and in mineral acids. column 0.25 m long and 10 mm in internal diameter and
Coarse particles: maximum 0.5 per cent. elute with water R. Collect the first 20 mL of eluate,
Place 5.0 gin a ground-glass-stoppered cylinder about evaporate to dryness and dry the residue at 100 °C to
160 mm long and 35 mm in diameter and add 60 mL of a 105 °C. The residue weighs not more than 10 mg.
l 0 g/L solution of sodium pyrophosphate R. Shake vigorously Iron (2.4.9): maximum 200 ppm.
and allow to stand for 5 min. Using a pipette, remove 50 mL To 0.50 g add 10 mL of a mixture of equal volumes of
of the liquid from a point about 5 cm below the surface. hydrochloric acid Rl and water R, shake vigorously, allow to
To the remaining liquid add 50 mL of water R, shake, allow stand for 5 min and filter. 1.0 mL of the filtrate complies
to stand for 5 min and remove 50 mL as before. Repeat the with the test for iron.
operations until a total of 400 mL has been removed.
Loss on ignition: maximum 0.5 per cent. During heating to
Transfer the remaining suspension to an evaporating dish.
red heat (600 ± 50 °C) the substance does not become
Evaporate to dryness on a water-bath and dry the residue to
brown or black.
constant mass at 100-105 °C. The residue weighs not more
than 25 mg. Kieselguhr G
Fine particles. Disperse 5.0 gin 250 mL of water R by shaking Consists of kieselguhr treated with hydrochloric acid and
vigorously for 2 min. Immediately pour into a glass cylinder calcined, to which is added about 15 per cent of calcium
50 mm in diameter and, using a pipette, transfer 20 mL to a sulfate hemihydrate.
glass dish, evaporate to dryness on a water-bath and dry to A fine greyish-white powder; the grey colour becomes more
constant mass at 100-105 °C. Allow the remainder of the pronounced on triturating with water. The average particle
suspension to stand at 20 "C for 4 h and, using a pipette size is I 0-40 µm.
with its tip exactly 5 cm below the surface, withdraw a Calcium sulfate content. Determine by the method prescribed
further 20 mL without disturbing the sediment, place in a for silica gel G R.
glass dish, evaporate to dryness on a water-bath and dry to pH (2.2.3). Shake I g with 10 mL of carbon dioxide-free
constant mass at 100-105 °C. The mass of the second water R for 5 min. The pH of the suspension is 7 to 8.
residue is not less than 70 per cent of that of the first
Chromatographic separation. Thin-layer chromatography
residue.
(2. 2. 2 7). Prepare plates using a slurry of the kieselguhr G
V-A92 Appendix I A 2023

with a 2.7 g/L solution of sodium acetate R. Apply 5 µL of a Carrier gas: helium for chromatography R.
solution containing 0.1 g/L of lactose, sucrose, glucose and Temperature:
fructose in pyridine R. Develop over a path of 14 cm using a
mixture of 12 volumes of water R, 23 volumes of Time Temperature
2-propanol R and 65 volumes of ethyl acetate R. (min) CC)
The migration time of the solvent is about 40 min. Dry, Column 0 - 12.5 230 --+ 280
spray onto the plate about 10 mL of anisaldehyde solution R Injection pan 250
and heat for 5-10 min at 100-105 °C. The chromatogram Detector 280
shows four well-defined spots without tailing and well
separated from each other.
Detection: flame ionisation.
Lactic Acid (50-21-5)
Injection: an appropriate derivatised sample.
See Lactic acid (0458). Lactulose (4618-18-2)
Lactic Reagent Lactic acid reagent See Lactulose (1230).
Solution A. To 60 mL of lactic acid R add 45 mL of
Lanatoside C 3 ~-[(~-D-Giucopyranosyl-( 1---> 4)-3-O-acetyl-
previously filtered lactic acid R saturated without heating with 2,6-dideoxy-~-D-ribo-hexopyranosyl-(l-> 4)-2, 6-dideoxy-~-D-
Sudan red G R; as lactic acid saturates slowly without
ribo-hexopyranosyl-(1->4)-2,6-dideoxy-~-D-ribo-
heating, an excess of colorant is always necessary.
hexopyranosyl)oxy]-12~, 14-dihydroxy-5~-card-20(22)-
Solution B. Prepare 10 mL of a saturated solution of enolide; C 49H 76 0 20 = 985 (17575-22-3)
aniline R. Filter.
Long, flat prisms obtained after recrystallisation in ethanol
Solution C. Dissolve 75 mg of potassium iodide R in water and (96 per cent), freely soluble in pyridine and in dioxan.
dilute to 70 mL with the same solvent. Add 10 mL of ethanol
Lanthanum Chloride Heptahydrate
(96 per cent) Rand 0.1 g of iodine R. Shake.
LaCl3 ,7H2 0 = 371.4
Mix solutions A and B. Add solution C.
White or almost white powder or colourless crystals, freely
Lactobionic Acid C 12H 22 O 12 = 358.3 (96-82-2) soluble in water.
White or almost white, crystalline powder, freely soluble in Lanthanum Chloride Solution
water, practically insoluble in ethanol (96 per cent).
To 58.65 g of lanthanum trioxide R slowly add 100 mL of
mp: about 115 °C. hydrochloric acid R. Heat to boiling. Allow to cool and dilute
Lactose Lactose monohydrate; (5989-81-1) to 1000.0 mL with water R.
See Lactose monohydrate (OJ 87). Lanthanum Nitrate Lanthanum trinitrate hexahydrate;
~-Lactose ~-D-Lactose; C 12 H 22O 11 = 342.3 (5965-66-2) La(NO 3 h,6H 2 0 = 433.0 (10277-43-7)
White or slightly yellowish powder. Colourless crystals, deliquescent, freely soluble in water.
Content: minimum 99 per cent. Storage: in an airtight container.
rJ.-D-Lactose: not greater than 35 per cent. Lanthanum Nitrate Solution
Assay. Gas chromatography (2.2.28): use the normalisation A 50 g/L solution of lanthanum nitrate R.
procedure. Lanthanum Trioxide Lanthanum oxide; La2 O 3 = 325.8
Column: (1312-81-8)
=
- size: l 30 m, 0 = 0.25 mm; An almost white, amorphous powder, practically insoluble in
- stationary phase: cyanopropyl(3)phenyl(3)methyl(94) water R. It dissolves in dilute solutions of mineral acids and
polysiloxane R (film thickness 1 µm). absorbs atmospheric carbon dioxide.
Carrier gas: helium for chromatography R. Calcium: maximum 5 ppm.
Temperature: Lasiocarpine (1S,7aR)-7-[[[(2R,3R)-2-Hydroxy-2-(2-
hydroxypropan-2-yl)-3-methoxybutanoyl] oxy]methyl]-
Time Temperature 2,3,5, 7a-tetrahydro-lH-pyrrolizin-1-yl (2Z)-2-methylbut-2-
(min) CC) enoate; C 21 H 33NO1 = 411.5 (303-34-4)
Column 0 - 32.5 20--> 280 White or light brown powder, sparingly soluble in water,
Injection port 250 soluble in ethanol (96 per cent) and in methanol.
Detector 250
Lasiocarpine N-Oxide (1S,7aR)-7-[[[(2R,3R)-2-Hydroxy-
2-(2-hydroxypropan-2-yl)-3-methoxybutanoyl] oxy]methyl]-1-
Detection: flame ionisation. [[(2Z)-2-methylbut-2-enoyl] oxy]-2,3 ,5,7 a-tetrahydro-1H-
Injection: an appropriate derivatised sample. pyrrolizine 4-oxide; C 21 H 33NO 8 = 427.5 (127-30-0)
a-Lactose Monohydrate a.-D-Lactose monohydrate; White or almost white powder, soluble in water and in
C 12 H 22 0 11 ,H2 0 = 360.3 (5989-81-1) methanol, sparingly soluble in ethyl acetate.
White or almost white powder. Laurie Acid Dodecanoic acid; C 12H 240 2 = 200.3
Content: minimum 97 per cent. (143-07-7)
P-n-Lactose: less than 3 per cent. White or almost white, crystalline powder, practically
Assay. Gas chromatography (2.2.28): use the normalisation
insoluble in water, freely soluble in ethanol (96 per cent).
procedure. mp: about 44 °C.
Column: Laurie acid used in the assay of total fatty acids in Saw palmetto
=
- size: l 30 m, 0 =0.25 mm; fruit (1848) complies with the following additional test.
- stationary phase: methylpolysiloxane R (film thickness Assay. Gas chromatography (2.2.28) as prescribed in the
1 µm). monograph Saw palmetto fruit (1848).
 2023 Appendix I A V-A93

Content: minimum 98 per cent, calculated by the solution R. Centrifuge and use the clear colourless
normalisation procedure. supernatant solution.
Lavandulol 2-Isopropenyl-5-methylhex-4-en- l-ol; The solution remains clear when stored in a well-closed
C 10H 18 O = 154.2 (498-16-8) container.
Oily liquid with a characteristic odour. L-o:-Lecithin from egg yolk L-o:-Phosphatidylcholine;
Lavandulol used in gas chromatography complies with the (8002-43-5)
following additional test. General reagent grade of commerce.
Assay. Gas chromatography (2.2.28) as prescribed in the Leiocarposide 2-(~-o-Glucopyranosyloxy)benzyl 3-(~-o-
monograph Lavender oil (1338). glucopyranosyloxy)-6-hydroxy-2-methoxybenzoate;
Test solution. The substance to be examined. 2-[[[3-(~-o-Glucopyranosyloxy)-6-hydroxy-2-
Content: minimum 90.0 per cent, calculated by the methoxybenzoyl]oxy]methyl]phenyl-~-D-glucopyranoside;
normalisation procedure. C 27 H 34 O 16 = 614.5 (71953-77-0)
Lavandulyl Acetate 2-Isopropenyl-5-methylhex-4-en- l-yl White or almost white powder, soluble in water, freely
acetate; C 12H 20 O 2 = 196.3 (25905-14-0) soluble in methanol, slightly soluble in ethanol (96 per cent).
Colourless liquid with a characteristic odour. mp: 190 °C to 193 °C.
Lavandulyl acetate used in gas chromatography complies with the Lemon Oil See Lemon oil (0620).
following additional test. L-Leucine Leucine; (61-90-5)
Assay. Gas chromatography (2.2.28) as prescribed in the See Leucine (0771).
monograph Lavender ozl (1338). Levodopa (59-92-7)
Test solution. The substance to be examined. See Levod;Jpa (0038).
Content: minimum 93.0 per cent, calculated by the (Z)-Ligustilide (3Z)-3-Butylidene-1,3,4,5-
normalisation procedure. tetrahydroisobenzofuran-1-one; C 12H 140 2 = 190 .2
Lead(n) Acetate Lead di-acetate; Lead acetate; (81944-09-4)
C 4 H 6 O 4 Pb,3H2O = 379.3 (6080-56-4) Limonene o-Llmonene; (+)-p-Mentha-1,8-diene; (R)-4-
Colourless crystals, efflorescent, freely soluble in water, Isopropenyl-l-methylcyclohex-1-ene; C 10H 16 = 136.2
soluble in ethanol (96 per cent). (5989-27-5)
Lead Acetate Cotton Colourless liquid, practically insoluble in water, soluble in
Immerse absorbent cotton in a mixture of 1 volume of dilute ethanol (96 per cent).
acetic acid R and 10 volumes of lead acetate solution R. Drain dig: about 0.84.
off the excess of liquid, without squeezing the cotton, by nfi°: 1.471 to 1.474.
placing it on several layers of filter paper. Allow to dry in air. [ex]~: about+ 124.
Storage: in an airtight container. bp: 175 °C to 177 °c.
Lead Acetate Paper Limonene used in gas chromatography complies with the following
Immerse filter paper (about 80 g/m 2) in a mixture of additional test.
1 volume of dilute acetic acid R and 10 volumes of lead acetate Assay. Gas chromatography (2.2.28) as prescribed in the
solution R. Dry, then cut the paper into strips 15 mm by monograph Peppermint oil (0405).
40mm.
Test solution. The substance to be examined.
Lead Acetate Solution
Content: minimum 99.0 per cent, calculated by the
A 95 g/L solution of lead acetate R in carbon dioxide-free normalisation procedure.
water R.
Linalool (RS)-3, 7-Dimethylocta-1,6-dien-3-ol; Linalol;
Lead(n) Nitrate Lead dinitrate; Lead nitrate; C 10H1 8 O = 154.2 (78-70-6)
Pb(NO 3)z = 331.2 (10099-74-8)
Mixture of two stereoisomers (licareol and coriandrol).
White or almost white, crystalline powder or colourless
Liquid, practically insoluble in water.
crystals, freely soluble in water.
Lead Nitrate Solution
dig: about 0.860.
A 33 g/L solution of lead nitrate R.
nt0 : about 1.462.

bp: about 200 °C.

Lead(1v) Oxide Lead dioxide; PbO 2 = 239.2 (1309-60-0)
Linalol used in gas chromatography complies with the following
Dark brown powder, evolving oxygen when heated,
test.
practically insoluble in water, soluble in hydrochloric acid
with evolution of chlorine, soluble in dilute nitric acid in the Assay. Gas chromatography (2.2.28) as prescribed in the
presence of hydrogen peroxide, oxalic acid or other reducing monograph Anise oil (0804).
agents, soluble in hot, concentrated alkali hydroxide Test solution. The substance to be examined.
solutions. Content: minimum 98.0 per cent, calculated by the
Lead Subacetate Solution Basic lead acetate solution; normalisation procedure.
(1335-32-6) Linalyl Acetate (RS)-1,5-Dimethyl- l-vinylhex-4-enyl
Content: 16. 7 per cent m/m to 17 .4 per cent mlm of Pb acetate; C 12H 20 O 2 = 196.3 (115-95-7)
(Ar 207 .2) in a form corresponding approximately to the Colourless or slightly yellow liquid with a strong odour of
formula C 8 H14O10Pb3. bergamot and lavender.
Dissolve 40.0 g of lead acetate R in 90 mL of carbon dioxide- diJ: 0.895 to 0.912.
free water R. Adjust the pH to 7.5 with strong sodium hydroxide nt0 : 1.448 to 1.451.
V-A94 Appendix I A 2023

bp: about 215 °C. emulsifying agents in a suitable organic solvent or mixture of
Linalyl acetate used in gas chromatography complies with the organic solvents.
following additional test. Liquid Scintillation Cocktail Rl
Assay. Gas chromatography (2.2.28) as prescribed in the To 1000 mL of dioxan R, add 0.3 g of
monograph Bitter-orange-flower oil (1175). methylphenyloxazolylbenzene R, 7 g of diphenyloxazole R and
Test solution. The substance to be examined. 100 g of naphthalene R.
Content: minimum 95.0 per cent, calculated by the Lithium Li= 6.94 (7439-93-2)
normalisation procedure. A soft metal whose freshly cut surface is silvery-grey.
Lindane y-Hexachlorocyclohexane; C 6 H 6 Cl 6 = 290.8 It rapidly tarnishes in contact with air. It reacts violently with
(58-89-9) water, yielding hydrogen and giving a solution of lithium
For the monograph Wool fat (0134), a suitable certified hydroxide; soluble in methanol, yielding hydrogen and a
reference solution (10 ng/µL in cyclohexane) may be used. solution of lithium methoxide; practically insoluble in light
petroleum.
Linoleic Acid (9Z,12Z)-Octadeca-9,12-dienoic acid;
C 18H 32 O2 = 280.5 (60-33-3) Storage: under light petroleum or liquid paraffin.

Colourless, oily liquid. Lithium Carbonate Dilithium carbonate; Li2 CO3 = 73.9
(554-13-2)
d~ 0 : about 0.903.
White or almost white, light powder, sparingly soluble in
nt0 : about 1.470.
water, very slightly soluble in ethanol (96 per cent).
Linoleic acid used in the assay of total fatty acids in Saw palmetto A saturated solution at 20 °C contains about 13 g/L of
fruit (1848) complies with the following additional test. Li2 CO3 •
Assay. Gas chromatography (2.2.28) as prescribed in the Lithium Chloride LiCI = 42.39 (7447-41-8)
monograph Saw palmetto fruit (1848). Crystalline powder or granules or cubic crystals, deliquescent,
Content: minimum 98 per cent, calculated by the freely soluble in water, soluble in acetone and in ethanol
normalisation procedure. (96 per cent). Aqueous solutions are neutral or slightly
Linolenic Acid (9Z,12Z,15Z)-Octadeca-9,12,15-trienoic alkaline.
acid; ct-Linolenic acid; C 18H 30 O2 = 278.4 (463-40-1) Storage: in an airtight container.
Colourless liquid, practically insoluble in water, soluble in Lithium Hydroxide Lithium hydroxide monohydrate;
organic solvents. LiOH,H 2 O = 41.96 (1310-66-3)
dt 0 : about 0.915. White or almost white, granular powder, strongly alkaline, it
n]31: about 1.480. rapidly absorbs water and carbon dioxide, soluble in water,
Linolenic acid used in the assay of total fatty acids in Saw sparingly soluble in ethanol (96 per cent).
palmetto fruit (1848) complies with the following additional test. Storage: in an airtight container.
Assay. Gas chromatography (2.2.28) as prescribed in the Lithium Metahorate, Anhydrous LiBO 2 = 49.75
monograph Saw palmetto fruit (1848). (13453-69-5)
Content: minimum 98 per cent, calculated by the Lithium Sulfate Lithium sulphate; Li2 SO 4,H2 O = 128.0
normalisation procedure. (10102-25-7)
Linolenyl Alcohol C 18 H 32O = 264.4 (506-44-5) Colourless crystals, freely soluble in water, practically
(9Z, 12Z, l 5Z)-Octadeca-9, 12, 15-trien-l-ol. ct-Linolenyl insoluble in ethanol (96 per cent).
alcohol. Lithium Trifluoromethanesulfonate Lithium
Content: minimum 96 per cent. trifluoromethanesulphonate; CF3 LiO 3 S = 156.0 (33454-82-9)
Linoleyl Alcohol (9Z, l 2Z)-Octadeca-9,l 2-dien-1-ol; Litmus (1393-92-6)
C1 8H,4O = 266.5 (506-43-4) Schultz No. 1386
Relative density: 0.830. Indigo-blue fragments prepared from various species of
Content: minimum 85 per cent. Rocella, Lecanora or other lichens, soluble in water,
practically insoluble in ethanol (96 per cent).
Linsidornine Hydrochloride 3-(Morpholin-4-yl)
sydnonimine hydrochloride; 3-(Morpholin-4-yl)-1,2,3- Colour change: pH 5 (red) to pH 8 (blue).
oxadiazol-3-ium-5-aminide hydrochloride; Litmus Paper
C 6 H 11 ClN4 O 2 = 206.6 (16142-27-1) Use red litmus paper or blue litmus paper, as appropriate.
White or almost white powder. Litmus Paper, Blue
Lipase Solvent Maleate buffer solution pH 7.0 Boil 10 parts of coarsely powdered litmus R for 1 h with 100
Dissolve 10.0 g of sodium chloride R, 6.06 g of parts of ethanol (96 per cent) R. Decant the alcohol and add
tris(hydroxymethyl)aminomethane Rand 4.90 g of maleic to the residue a mixture of 45 parts of ethanol (96 per cent) R
anhydride R in 900 mL of water R. Adjust the pH using a and 55 parts of water R. After 2 days decant the clear liquid.
170 g/L solution of sodium hydroxide R. Dilute to 1000.0 mL Impregnate strips of filter paper with the solution and allow
with water R. to dry.
Storage: at 2 °C to 8 °C; use within 3 days. Test for sensitivity. Immerse a strip measuring 10 mm by
Liquid Scintillation Cocktail 60 mm in a mixture of 10 mL of 0.02 M hydrochloric acid
Commercially available solution for the determination of and 90 mL of water R. On shaking the paper turns red
radioactivity by liquid scintillation counting. It contains one within 45 s.
or more fluorescent agents and mostly one or more
2023 Appendix I A V-A95

Litmus Paper, Red Lysine Hydrochloride (2S)-2,6-Diaminohexanoic acid

To the blue litmus extract, add dilute hydrochloric acid R hydrochloride; C 6 H 15 ClN 2 O2 = 182.7 (657-27-2)
dropwise until the blue colour becomes red. Impregnate White or almost white, crystalline powder or colourless
strips of filter paper with the solution and allow to dry. crystals, freely soluble in water, slightly soluble in ethanol
Test for sensitivity. Immerse a strip measuring 10 mm by (96 per cent).
60 mm in a mixture of 10 mL of 0.02 M sodium hydroxide Lysyl Endopeptidase (78642-25-8)
and 90 mL of water R. On shaking the paper turns blue Achromobacter endoproteinase I. Lysyl bond specific
within 45 s. proteinase (EC 3.4.21.50).
Litmus Solution It belongs to the serine endopeptidase family. Initially
Boil 25 g of coarsely powdered litmus with 100 mL of ethanol isolated from Achromobacter lyticus. Enzymes with similar
(90%) under a reflux condenser for 1 hour, discard the clear specificity are produced by Lysobacter enzymogenes
liquid and repeat this operation using two 75-mL quantities (endoproteinase Lys-C) and Pseudomonas aeruginosa (Ps-1).
of ethanol (90%). Digest the extracted litmus with 250 mL of It cleaves peptide bonds at the carboxy-terminal of both
water and filter. lysine residues and S-aminoethylcysteine residues with a high
Loganin Methyl (lS,4aS,6S,7R,7aS)-l-(B-D- degree of specificity. 1 amidase unit (U) is the amount of
glucopyranosyloxy)-6-hydroxy-7-methyl-l ,4a,5,6, 7,7 a- enzyme that will produce 1 micromole of p-nitroaniline from
hexahydrocyclopenta [c] pyran-4-carboxylate; N-benzoyl-DL-lysine-p-nitroaniline per minute at 30 'C at
C17H26O10 = 390.4 (18524-94-2) pH 9.5.
mp: 220 °C to 221 °C. Lutetium Chloride Hexahydrate LuCl 3 ,6H 2 O = 389.4
Longifolene (lS,3aR,4S,8aS)-4,8,8-Trimethyl-9- (15230-79-2)
methylenedecahydro-l,4-methanoazulene; C 15H 24 = 204.4 White to yellow, crystalline powder, freely soluble in water.
(475-20-7) Macrogol 23 Laury! Ether See Macrogol lauryl
Oily, colourless liquid, practically insoluble in water, miscible ether (1124), the number of moles of ethylene oxide reacted
with ethanol (96 per cent). per mole of lauryl alcohol being 23 (nominal value).
d~ 8 : 0.9319. Macrogol 600 Polyethyleneglycol 600; (25322-68-3)
n1°: 1.5050. See Macrogols (1444).
[c,;Jgi: + 42.7. Macrogol 4000 Polyethyleneglycol 4000; (25322-68-3)
bp: 254 °C to 256 °C. See Macrogols (1444).
Longifolene used in gas chromatography complies with the Macrogol 6000 Polyethyleneglycol 6000; (25322-68-3)
foUowing additional test. White or almost white solid with a waxy or paraffin-like
Assay. Gas chromatography (2.2.28) as prescribed in the appearance, very soluble in water and in methylene chloride,
monograph Turpentine oil, Pinus pinaster type (1627). practically insoluble in ethanol (96 per cent), in fatry oils and
Content: minimum 98.0 per cent, calculated by the in mineral oils.
normalisation procedure. Macrogol 20,000 2-Nitroterephthalate Polyethylene
Lumiflavine 7,8,1 0-Trimethylbenzo[g]pteridine- Glycol 20,000 2-Nitroterephthalate
2,4(3H,10H)-dione; C 13 H 12N 4 O2 = 256.3 (1088-56-8) Polyethyleneglycol 20 000 with embedded
Yellow powder or orange crystals, very slightly soluble in 2-nitroterephthalate groups.
water, freely soluble in methylene chloride. Macrogol, Base-deactivated
Luteolin 2-(3,4-Dihydroxyphenyl)-5, 7-dihydroxy-4H- l- Base-deactivated polyethyleneglycol.
benzopyran-4-one; C 15 H 10O 6 = 286.2 (491-70-3) Macrogol Cetostearyl Ether See Macrogol cewsteaiyl ether
Luteolin-7-glucoside 2-(3,4-Dihydroxyphenyl)-7-(B-D- (1123).
glucopyranosyloxy)-5-hydroxy-4H-1-benzopyran-4-one; Macrogol, Polar-deactivated
C2 1H 20 O11 = 448.4 (5373-11-5) Polar-deactivated polyethyleneglycol.
Yel!ow powder. Magnesium Mg= 24.30 (7439-95-4)
Absorbance (2.2.25). A solution in methanol R shows Silver-white ribbon, turnings or wire, or a grey powder.
absorption maxima at 255 nm, 267 nm and 350 nm.
Magnesium Acetate Magnesium diacetate tetrahydrate;
mp: about 247 °C. C 4 H 6 MgO 4 ,4H2 O = 214.5 (16674-78-5)
Lycopsamine [(lR, 7aR)-l-Hydroxy-2,3,5,7a-tetrahydro- Colourless crystals, deliquescent, freely soluble in water and
lH-pyrrolizin-7-yl] methyl (2S,3S)-2,3-dihydroxy-2-(propan- in ethanol (96 per cent).
2-yl) butanoate; 3'-epi-Intermedine; C 15 H 25NO 5 = 299.4
Storage: in an airtight container.
(10285-07-1)
Magnesium Chloride (779 I-18-6)
Light brown powder.
See 1\1.agnesium chloride hexahydrate (0402).
Lycopsamine N-Oxide (lR,7aR)-7-[[[(2S,3S)-2,3-
Dihydroxy-2-(propan-2-yl) butanoyl] oxy] methyl]- l-hydroxy- Magnesium Nitrate Magnesium nitrate hexahydrate;
2,3,5, 7 a-tetrahydro- lH-pyrrolizine 4-oxide; Mg(NO 3 ) 2 ,6H 2 O = 256.4 (13446-18-9)
C 15 H 25 NO 6 = 315.4 (95462-15-0) Colourless, clear crystals, deliquescent, very soluble in water,
White or almost white powder, soluble in methanol. freely soluble in ethanol (96 per cent).
Storage: in an airtight container.
V-A96 Appendix I A 2023

Magnesium Nitrate Solution Malachite Green Solution

Dissolve 17.3 g of magnesium nitrate R in 5 mL of water R A 5 g/L solution of malachite green R in anhydrous acetu:
warming gently and add 80 mL of ethanol (96 per cent) R. acid R.
Cool and dilute to 100.0 mL with the same solvent. Malathion C 10H 19O 6 PS 2 = 330.3 (121-75-5)
Magnesium Oxide (1309-48-4) bp: about 156 °C.
See Light magnesium oxide (0040). A suitable certified reference solution (10 ng/µL in iso-
Magnesium Oxide, Heavy (1309-48-4) octane) may be used.
See Heavy magnesium oxide (0041). Maleic Acid (Z)-But-2-ene-1,4-dioic acid; (110-16-7)
Magnesium Oxide Rl See Maleic acid (0365).
Complies with the requirements prescribed for magnesium Maleic Anhydride Furan-2,5-dione; C 4 H 2 O 3 = 98.1
oxide R with the following modifications. (108-31-6)
Arsenu: (2.4.2, Method A): maximum 2 ppm. White or almost white crystals, soluble in water forming
Dissolve 0.5 gin a mixture of 5 mL of water Rand 5 mL of maleic acid, very soluble in acetone and in ethyl acetate,
hydrochloric acid Rl. freely soluble in toluene, soluble in ethanol (96 per cent)
Heavy metals (2.4.8): maximum 10 ppm. with ester formation, very slightly soluble in light petroleum.
mp: about 52 °C.
Dissolve 1.0 g in a mixture of 3 mL of water R and 7 mL of
hydrochloru: acid Rl. Add 0.05 mL of phenolphthalein Any residue insoluble in toluene does not exceed 5 per cent
solution R and concentrated ammonia R until a pink colour is (maleic acid).
obtained. Neutralise the excess of ammonia by the addition Maleic Anhydride Solution
of glacial acetic acid R. Add 0.5 mL in excess and dilute to Dissolve 5 g of maleic anhydride R in toluene R and dilute to
20 mL with water R. Filter, if necessary. 12 mL of the 100 mL with the same solvent. Use within one month. If the
solution complies with test A. Prepare the reference solution solution becomes turbid, filter.
using a mixture of 5 mL of lead standard solution
Malle Acid (6915-15-7)
(1 ppm Pb) Rand 5 mL of water R.
See Malic acid (2080).
Iron (2.4.9): maximum 50 ppm.
Maltitol (585-88-6)
Dissolve 0.2 gin 6 mL of dilute hydrochloru: acid Rand dilute
to 10 mL with water R. See Maltitol (1235).
Magnesium Silicate for Pesticide Residue Analysis Maltol 3-Hydroxy-2-methyl-4H-pyran-4-one;
(1343-88-0) C 6H 6 O 3 = 126.1 (118-71-8)
Magnesium silicate for chromatography (60-100 mesh). White or almost white crystalline powder, soluble in hot
water.
Magnesium Sulfate Magnesium sulphate; (10034-99-8)
mp: 161 °C to 162 °C.
See Magnesium sulfate heptahydrate (0044).
Maltose Monohydrate 4-O-a-o-glucopyranosyl-D-
Magneson Azo violet; 4-( 4-nitrophenylazo)resorcinol;
glucopyranose monohydrate; C 12H22O 11 ,H2O = 360.3
C12H9N3 O4 = 259.2 (74-39-5) (6363-53-7)
Indicator grade of commerce.
Maltotriose a-o-Glucopyranosyl-(1---->4)-ct-D-
When used for titration in non-aqueous media, it changes glucopyranosyl-(l -->4)-o-glucose; C 1 sH 32 O 16 = 504.4
from orange (acidic) through pink (neutral) to blue (basic). (1109-28-0)
MagnesonReagent White or almost white, crystalline powder, very soluble in
A 0.1 % w/v solution of magneson in a 1% w/v solution of water.
sodium hydroxide. mp: about 134 °C.
Magneson Solution Mandelic Acid 2-Hydroxy-2-phenylacetic acid;
A 0.2% w/v solution of magneson in toluene. CsHsO3 = 152.1 (90-64-2)
Magnolin C 23 H 28 O 7 = 416.5 (31008-18-1) White crystalline flakes, soluble in water.
(3S,3aR,6S,6aR)-3-(3,4-Dimethoxyphenyl)-6-(3,4,5- mp: 118 to 121 °C.
trimethoxyphenyl)-1,3,3a,4,6,6a-hexahydrofuro[3,4-c]furan. Manganese(1v) Oxide, Pre-washed Manganese(IV)
Magnolol 5,5 '-Di(prop-2-enyl)biphenyl-2,2 '-diol; 5,5 '- Oxide Activated; MnO 2 = 86.9 (1313-13-9)
Diallyl-2 ,2 '-dihydroxybiphenyl; 5,5 1-Di-2-propenyl-[ 1, 1 1- mp: about 535°.
biphenyl]-2,2'-diol; C 18H 18 O 2 = 266.3 (528-43-8)
General reagent grade of commerce.
Maize Oil See Maize oil, refined (1342). Manganese(n) Sulfate Manganese sulphate;
Makisterone A (22R)-2P,3P,14,20,22,25-Hexahydroxy-5p- Manganese(n) sulphate; Manganese sulfate; MnSO 4 ,
ergost-7-en-6-one; C 28 H 46 O 7 = 494.7 (20137-14-8) H 2 O = 169.0 (10034-96-5)
Malachite Green Basic green 4; C 23 H 25 CIN2 = 364.9 Pale-pink, crystalline powder or crystals, freely soluble in
(123333-61-9) water, practically insoluble in ethanol (96 per cent).
Schultz No. 754 Loss on ignition: 10.0 per cent to 12.0 per cent, determined
Colour Index No. 42000 on 1.000 g at 500 ± 50 °C.
Green crystals with a metallic lustre, very soluble in water o-Mannitol Mannitol; (69-65-8)
giving a bluish-green solution, soluble in ethanol See Mannitol (0559).
(96 per cent) and in methanol.
Absorbance (2.2.25). A 0.01 g/L solution in ethanol
(96 per cent) R shows an absorption maximum at 617 nm.
2023 Appendix I A V-A97

o-Mannose Mannose; C 6 H 12 O 6 = 180.2 (3458-28-4') ni0: about 1.450.

white or almost white, crystalline powder or small crystals, Menthone used in gas chromatography complies with the following
very soluble in water, slightly soluble in anhydrous ethanol. additional test.
[uJ;;: + 13.7 + 14.7, determined on a 200 g/L solution in Assay. Gas chromatography (2.2.28) as prescribed in the
water R containing about 0.05 per cent of NH 3 . monograph Peppermint oil (0405).
mp: about 132 °C, with decomposition. Test solution. The substance to be examined.
Marrubiin (2aS,5aS,6R, 7R,8aR,8bR)-6-[2-(Furan-3-yl) Content: minimum 90.0 per cent, calculated by the
ethyl)-6-hydroxy-2a,5a, 7-trimethyldecahydro-2H-naphtho normalisation procedure.
[l,8-bc)furan-2-one; C 20H 2 sO 4 = 332.4 (465-92-9) Menthyl Acetate (1R,2S,5R)-5-Methyl-2-(propan-2-yl)
Colourless, microcrystalline powder. cyclohexyl acetate; C 12H 22 0 2 = 198.3 (2623-23-6)
Marrubiin used in liquid chromatography complies with the Colourless liquid, slightly soluble in water, miscible with
following additional test. ethanol (96 per cent).
Assay. Liquid chromatography (2.2.29) as prescribed in the dig: about 0.92.
monograph White horehound (1835). ni0 : about 1.447.

Content: minimum 95.0 per cent, calculated by the bp: about 228 °C.
normalisation procedure.
Menthyl acetate used in gas chromatography complies with the
Mebendazole C 16H 13N 30 3 = 295.3 (31431-39-7) fallowing additional test.
General reagent grade of commerce. Assay. Gas chromatography (2.2.28) as prescribed in the
Meclozine Hydrochloride Meclozine dihydrochloride; monograph Peppermint oil (0405).
(1104-22-9) Test solution. The substance to be examined.
See Meclozine dihydrochloride (0622). Content: minimum 97.0 per cent, calculated by the
Medronic Acid (1984-15-2) normalisation procedure.
See Medronic acid for radiopharmaceutical preparations (2350). Mercaptoacetic Acid Thioglycollic acid; C 2H 4 O 2 S = 92.1
Melamine 1,3,5-Triazine-2,4,6-triamine; C 3 H 6 N 6 = 126.1 (68-11-1)
(108-78-1) Colourless liquid, miscible with water, soluble in ethanol
A white or almost white, amorphous powder, very slightly (96 per cent).
soluble in water and in ethanol (96 per cent). 2-Mercaptobenzimidazole lH-benzimidazole-2-thiol;
Menthofuran 3, 9-Epoxy-p-mentha-3,8-diene; C 7 H 6N 2 S = 150.2 (583-39-1)
3,6-Dimethyl-4,5,6, 7-tetrahydro-benzofuran; mp: about 302 °C.
C 10H 14O = 150.2 (17957-94-7) 2-Mercaptoethanol C 2H 6 OS = 78.1 (60-24-2)
Slightly bluish liquid, very slightly soluble in water, soluble in Liquid, miscible with water.
ethanol (96 per cent).
dig: about 1.116.
dfg: about 0.965. bp: about 157 °C.
ni0: about 1.480. 6-Mercaptopurine Purine-6-thiol; Mercaptopurine;
[a]~: about + 93. (6112-76-1)
bp: 196 °C. See Mercaptopurine monohydrate (0096).
Menthofuran used in gas chromatography complies with the Mercuric Oxide Yellow mercury(n) oxide; Mercury(n)
fallowing additional test. Oxide, Yellow; HgO = 216.6 (21908-53-2)
Assay. Gas chromatography (2.2.28) as prescribed in the A yellow to orange-yellow powder, practically insoluble in
monograph Peppermint oil (0405). water and in ethanol (96 per cent).
Test solution. The substance to be examined. Storage: protected from light.
Content: minimum 97.0 per cent, calculated by the Mercury Hg = 200.6 (7439-97-6)
normalisation procedure.
dig: about 13.5.
Menthol (-)-p-Menthan-3-ol (2216-51-5); (±)-p-menthan-
bp: about 357°.
3-ol; (2216-51-5)
Silver-white liquid, breaking into spherical globules which do
See Levomenthol (0619) and Racemic menthol (0623).
not leave a metallic trace when rubbed on paper.
Menthol used in gas chromatography complies with the following
Mercury(n) Acetate Mercury diacetate; Mercuric acetate;
additional test.
C4 H 6 HgO 4 = 318.7 (1600-27-7)
Assay. Gas chromatography (2.2.28) as prescribed in the
White or almost white crystals, freely soluble in water,
related substances test included in the monograph Racemic soluble in ethanol (96 per cent).
menthol (0623).
Mercury(n) Chloride Mercuric chloride; (7487-94-7)
Content: minimum 98.0 per cent, calculated by the
normalisation procedure. See Mercuric chloride (0120).
Menthone (2S,5R)-2-Isopropyl-5-methylcyclohexanone; Mercury(n) Chloride Solution Mercuric chloride
(-)-trans-p-Menthan-3-one; C 10 H 18 O = 154.2 (14073-97-3) solution
Contains variable amounts of isomenthone. A 54 g/L solution of mercuric chloride R.
Colourless liquid, very slightly soluble in water, very soluble Mercury(n) Iodide Mercury di-iodide; Mercuric iodide;
in ethanol (96 per cent). Hgl 2 = 454.4 (7774-29-0)
dig: about 0.897.
V-A98 Appendix I A 2023

Dense, scarlet, crystalline powder, slightly soluble in water, 5.0 mL of 0.02 M potassium bromate and heat on a water-
sparingly soluble in acetone and in ethanol (96 per cent), bath for 30 min. Allow to cool and add 0.5 g of potassium
soluble in an excess of potassium iodide solution R. iodide R. Titrate the liberated iodine with 0.1 M sodium
Storage: protected from light. thiosulfate, using 1 mL of starch solution R as indicator. Carry
Mercury(n) Nitrate Mercury dinitrate monohydrate; out a blank test.
Mercuric nitrate; Hg(NO 3 )z,H 2 O = 342.6 (7783-34-8) 1 mL of 0. 02 M potassium bromate is equivalent to 4.10 mg of
Colourless or slightly coloured crystals, hygroscopic, soluble H 3PO3.
in water in the presence of a small quantity of nitric acid. Storage: in an airtight container.
Storage: in an airtight container, protected from light. Methacrylic Acid 2-Methylprop-2-enoic acid;
Mercury, Nitric Acid Solution of C4H6O 2 = 86.1 (79-41-4)
Carefully dissolve 3 mL of mercury in 27 mL of fuming nitric Colourless liquid.
acid. Dilute the solution with an equal volume of water. n~: about 1.431.
Store protected from light, use within 2 months. bp: about 160 °C.
Mercury(n) Sulfate Solution Mercuric sulphate solution; mp: about 16 °C.
Mercury(n) sulphate solution; Mercuric sulfate solution; Methane CH4 = 16 (74-82-8)
(7783-35-9) Content: minimum 99.0 per cent V/V.
Dissolve 1 g of mercuric oxide R in a mixture of 20 mL of Methane Rl CH4 = 16 (74-82-8)
water R and 4 mL of sulfuric acid R.
Content: minimum 99.995 per cent V/V.
Mercury(n) Thiocyanate Mercury di(thiocyanate);
Methanesulfonic Acid Methanesulphonic acid;
Mercuric thiocyanate; Hg(SCN) 2 = 316.7 (592-85-8) CH4 O 3S = 96.1 (75-75-2)
White or almost white, crystalline powder, very slightly
Clear, colourless liquid, solidifying at about 20 °C, miscible
soluble in water, slightly soluble in ethanol (96 per cent),
with water, slightly soluble in toluene, practically insoluble in
soluble in solutions of sodium chloride.
hexane.
Mercury(n) Thiocyanate Solution Mercuric thiocyanate dfg: about 1.48.
solution
n~: about 1.430.
Dissolve 0.3 g of mercuric thiocyanate R in anhydrous ethanol R
and dilute to 100 mL with the same solvent. Methanesulfonic Acid, Methanolic
Storage: use within 1 week. Methanesulphonic acid, methanolic
Mesalazine (89-57-6) Solutions of the requisite molarity may be obtained by
dissolving the appropriate quantity of methanesulfonic acid in
See Mesalazine (1699).
methanol.
Mesityl Oxide 4-Methylpent-3-en-2-one; C 6H 10O = 98.1
Methanesulfonyl Chloride Methanesulphonyl chloride;
(141-79-7)
CH3 CIO2 S = 114.6 (124-63-0)
Colourless, oily liquid, soluble in 30 parts of water, miscible Clear, colourless or slightly yellow liquid.
with most organic solvents.
Content: minimum 99.0 per cent.
dfg: about 0.858. Density: 1.48 g/cm3 •
bp: 129 °C to 130 °C.
n~: about 1.452.
Metanil Yellow 4 1-anilinoazobenzene-3-sulfonic acid
sodium salt; C 18H 1 ~ 3 NaO 3 S = 375.4 (587-98-4) bp: about 161 °C.
Schultz No. 169 Methanol Methyl alcohol; CH4O = 32.04 (67-56-1)
Colour Index No. 13065 Clear, colourless, flammable liquid, miscible with water and
with ethanol (96 per cent).
A brownish-yellow powder, soluble in water and in ethanol
(96 per cent). dfg: 0.791 to 0.793.
Metanil Yellow Solution bp: 64 °C to 65 °C.When 'methanol' is followed by a
percentage figure, an instruction to use methanol diluted with
A 1 g/L solution of metanil yellow R in methanol R. water to produce the specified percentage v/v of methanol is
Test for sensitivity. To 50 mL of anhydrous acetic acid R add implied.
0.1 mL of the metanil yellow solution. Add 0.05 mL of Methanol, Acidified
0.1 M perchloric acid; the colour changes from pinkish-red to
violet. To 900 mL of methanol add 18 mL of glacial acetic acid and
dilute to 1000 mL with water.
Colour change: pH 1.2 (red) to pH 2.3 (orange-yellow).
Methanol, Aldehyde-free
Metaphosphoric Acid (HPO 3 )x (37267-86-0)
Dissolve 25 g of iodine R in 1 L of methanol R and pour the
Glassy lumps or sticks containing a proportion of sodium solution, with constant stirring, into 400 mL of 1 M sodium
metaphosphate, hygroscopic, very soluble in water.
hydroxide. Add 150 mL of water R and allow to stand for
Nitrates. Boil 1.0 g with 10 mL of water R, cool, add 1 mL of 16 h. Filter. Boil under a reflux condenser until the odour of
indigo carmine solution R, 10 mL of nitrogen-free sulfuric acid R iodoform disappears. Distil the solution by fractional
and heat to boiling. The blue colour is not entirely distillation.
discharged.
Aldehydes and ketones: maximum 0.001 per cent.
Reducing substances: maximmn 0.01 per cent, calculated as Methanol, Anhydrous (67-56-1)
H3PO3.
Treat 1000 mL of methanol R with 5 g of magnesium R.
Dissolve 35.0 g in 50 mL of water R. Add 5 mL of a 200 g/L If necessary initiate the reaction by adding 0 .1 mL of mercuric
solution of sulfuric acid R, 50 mg of potassium bromide R and
2023 Appendix I A V-A99

chloride solution R. When the evolution of gas has ceased, Assay. Gas chromatography (2.2.28) as prescribed in the
distil the liquid and collect the distillate in a dry container monograph Cassia oil (1496).
protected from moisture. Content: minimum 96.0 per cent, calculated by the
Water (2.5.12): maximum 0.3 g/L. normalisation procedure.
Methanol, Hydrochloric 2-Methoxyethanol Ethylene glycol monomethyl ether;
Dilute 1.0 mL of hydrochloric acid Rl to 100.0 mL with C 3 H 8 O 2 = 76.1 (109-86-4)
methanol R. Conrent: minimum 99.0 per cent.
Methanol Rl Clear, colourless liquid, miscible with water, with acetone
Complies with the requirements prescribed for methanol R and with ethanol (96 per cent).
\Vith the following additional requirement. dig: about 0.97.
Absorbance (2.2.25): maximum 0.70 at 210 nm, 0.30 at nt 0 : about 1.403.

220 nm, 0.13 at 230 nm, 0.02 at 250 nm, 0.01 at 260 nm bp: about 125 °C.
and higher wavelengths, determined using water R as
(lRS)-1-(6-Methoxynaphthalen-2-yl)ethanol
compensation liquid.
6-Methoxy-C(-methyl-2-naphthalenemethanol;
Methanol R2 C 13H 14 0 2 = 202.3 (77301-42-9)
Complies with the requirements prescribed for methanol R White or almost white powder.
and the following additional requirements. mp: about 113 °C.
Content: minimum 99.8 per cent.
1-(6-Methoxynaphthalen-2-yl)ethanone 6 '-Methoxy-2 ' -
Absorbance (2.2.25): maximum 0.17, determined at 225 nm acetonaphthone; C 13H 12O 2 = 200.2 (3900-45-6)
using water R as the compensation liquid. White or almost white powder.
Methanol R3 mp: about 108 °C.
Content: minimum 99.9 per cent.
6-Methoxy-2-naphthoic Acid 6-Methoxynaphthalene-2-
When used for mass spectrometry detection, a special grade may be carboxylic acid; C 12H 10 0 3 = 202.2 (2471-70-7)
needed.
White or almost white, crystalline powder.
Methimazole I-Methyl- I H-imidazole-2-thiol; Thiamazole; mp: 201 °C to 206 °C.
C 4 H 6N 2 S = 114.2 (60-56-0)
Methoxyphenylacetic Acid (RS)-2-Methoxy-2-
White or almost white, crystalline powder, freely soluble in
phenylacetic acid; C 9H 10O 3 = 166.2 (7021-09-2)
water, soluble in ethanol (96 per cent) and in methylene
chloride. White, crystalline powder or white or almost white crystals,
sparingly soluble in water, freely soluble in ethanol
mp: about 145 cc_
(96 per cent).
DL-Methionine (59-51-8)
mp: about 70 °C.
See DL-Methionine (0624).
Methoxyphenylacetic Reagent
L-Methionine (63-68-3)
Dissolve 2. 7 g of methoxyphenylacetic acid R in 6 mL of
See Methionine (1027). tetramethylammonium hydroxide solution R and add 20 mL of
L-Methionine Sulfoxide (2S)-2-Amino-4-[(RS)- anhydrous ethanol R.
methylsulfinyl]butanoic acid; C 5 H 11 NO 3 S = 165.2 Storage: in a polyethylene container.
(3226-65-1)
3-Methoxy-L-tyrosine C 10H 13NO4 H 2 O = 229.2
(RS)-Methotrexate (RS)-2-[4-[[(2,4-diaminopteridin-6-yl) (200630-46-2)
methyl]methylamino]benzoylaminoJpentanedioic acid; Off-white or yellow powder.
C 20H22N8 Os (60388-53-6)
Methyl Acetate C 3H 6 0 2 = 74.1 (79-20-9)
Content: minimum 96.0 per cent.
Clear, colourless liquid, soluble in water, miscible with
mp: about 195 °C.
ethanol (96 per cent).
Methoxyazobenzene C 1 ,H 12NO = 212.2 (2396-60-3)
dig: about 0.933.
mp: about 55°.
n~: about 1.361.
Use a grade of commerce suitable for chromatographic bp: 56 °C to 58 °C.
separations.
Methyl 4-acetylbenzoate C 10 H 100 3 = 178.2 (3609-53-8)
Methoxychlor 1, 1-(2,2,2-Trichloroethylidene)-bis(4-
methoxybenzene); C 16 H 15 Cl3 O 2 = 345.7 (72-43-5) mp: about 94 °C.
Practically insoluble in water, freely soluble in most organic Methyl 4-acetylbenzoate Reagent
solvents. Dissolve 0.25 g of methyl 4-acetylbenzoate R in a mixture of
bp: about 346 °C. 5 mL of sulfuric acid Rand 85 mL of cooled methanol R.
mp: 78 'C to 86 °C. Methyl Acrylate Methyl prop-2-enoate; C 4H 6 O 2 = 86.l
(96-33-3)
A suitable certified reference solution (10 ng/µL in iso-
octane) may be used. Clear, colourless liquid.
trans-2-Methoxycinnamaldehyde C 10H 10 0 2 = 162.2 bp: about 80 °C.
(60125-24-8) Methylal Dimethoxymethane; Dioxapentane;
mp: 44 °C to 46 °C. Formaldehyde dimethyl acetal; Methylene dimethyl ether;
C 3 H 8 0 2 = 76.1 (109-87-5)
trans-2-lvlethoxycinnamaldehyde used in gas chromatography
complies with the following additional test. Clear, colourless, volatile, flammable liquid, soluble in water
and miscible with ethanol (96 per cent).
V-AlOO Appendix I A 2023

dfg: about 0.860. 3-Methylbenzothiazolin-2-one Hydrazone

n~: about 1.354. Hydrochloride 3-Methylbenzothiazol-2 (3 H)-one
hydrazone hydrochloride monohydrate;
bp: about 41 °C.
Methylbenzothiazolone hydrazone hydrochloride;
Methylal used in gas chromatography complies with the fallowing C8 H 10 ClN3 S,H 2 0 = 233.7 (38894-11-0)
additional test.
Almost white or yellowish, crystalline powder.
Content: minimum 99.5 per cent, determined by gas
mp: about 270 °C.
chromatography.
Suitability for determination of aldehydes. To 2 mL of aldehyde-
Methylamine Hydrochloride Methanamine
free methanol R add 60 µL of a 1 g/L solution of
hydrochloride; CH 6 ClN = 67.5 (593-51-1)
propionaldehyde R in aldehyde-free methanol R and 5 mL of a
White or almost white powder. 4 g/L solution of methylbenzothiazolone hydrazone
Content: minimum 98.0 per cent. hydrochloride. Mix. Allow to stand for 30 min. Prepare a
Methyl 4-aminobenzoate C 8 H 9 N0 2 = 151.2 (619-45-4) blank omitting the propionaldehyde solution. Add 25.0 mL
mp: 110 °C to 113 °C. of a 2 g/L solution offerric chloride R to the test solution and
to the blank, dilute to 100.0 mL with acetone Rand mix.
4-Methylaminophenol Sulfate C 14H 20N 2 0 6 S = 344.4
The absorbance (2. 2. 25) of the test solution, measured at
(55-55-0)
660 nm using the blank as compensation liquid, is not less
Colourless crystals, very soluble in water, slightly soluble in than 0.62.
ethanol (96 per cent).
(R)-( +)-ix-Methylbenzyl Isocyanate (+)-(R)-ix-
mp: about 260 °C. Methylbenzyl isocyanate; (+)-[(lR)-1-lsocyanatoethyl]
Methylaminophenol-Sulfite Reagent benzene; (+)-(lR)-1-Phenylethyl isocyanate;
Methylaminophenol-sulfite reagent C 9 H 9NO = 147.2 (33375-06-3)
Dissolve 0.1 g of 4-methylaminophenol sulfate, 20 g of sodium Content: minimum 99.0 per cent.
metabisulfite and O.5 g of anhydrous sodium sulfite in sufficient Colourless liquid.
water to produce 100 mL. d;g: about 1.045.
3-(Methylamino)-1-phenylpropan-1-ol ng1: about 1.513.
C 10H 15NO = 165.2 (42142-52-9)
bp: 55 °C to 56 °C at 2.5 mm Hg.
White or almost white powder.
Enantiomeric purity: minimum 99.5.
mp: 59 °C to 64 °C.
Storage: at a temperature of 2 °C to 8 °C.
Methylamphetamine Hydrochloride Methamphetamine
(S)-(-)-ix-Methylbenzyl Isocyanate (-)-(S)-ix-
hydrochloride; C 10H 16 ClN = 185.7
Methylbenzyl isocyanate; H-[ (1S)-1-Isocyanatoethyl]
General reagent grade of commerce. benzene; (-)-(lS)-1-Phenylethyl isocyanate;
Methyl Anthranilate Methyl 2-aminobenzoate; C 9 H 9NO = 147.2 (14649-03-7)
C 8 H 9 N02 = 151.2 (134-20-3) Content: minimum 99.0 per cent.
Colourless crystals or a colourless or yellowish liquid, soluble Colourless liquid.
in water, freely soluble in ethanol (96 per cent).
mp: 24 °C to 25 °C.
d~g: about 1.045.
Methyl anthranilate used in gas chromatography complies with the
nt 0 : about 1.514.

bp: 55 °C to 56 °C at 2.5 mm Hg.

following additional test.
Enantiomeric purity: minimum 99.5 per cent.
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Bitter-orange-flower oil, (1175). Storage: at a temperature of 2 °C to 8 °C.
Test solution. The substance to be examined. NOTE: do not use the reagent if it is coloured.
Content: minimum 95.0 per cent, calculated by the 1-Methylbiguanide Hydrochloride C 3 H 9 N 5 = 151.6
normalisation procedure. (1674-62-0)
Methyl Arachidate Methyl eicosanoate; 2-Methylbutane Isopentane; C 5 H 12 = 72.2 (78-78-4)
C21H420 2 = 326.6 (1120-28-1) Content: minimum 99.5 per cent of C 5 H 12 .
Content: minimum 98.0 per cent, determined by gas Very flammable colourless liquid.
chromatography (2.4.22). dig: about 0.621.
White or yellow, crystalline mass, soluble in ethanol n~: about 1.354.
(96 per cent) and in light petroleum. bp: about 29 °C.
mp: about 46 °C. Water (2.5.12): maximum 0.02 per cent.
Methyl Benzenesulfonate Ethyl benzenesulfonate; Residue on evaporation: maximum 0.0003 per cent.
C 7Hs03S = 172.2 (80-18-2)
Absorbance (2.2.25): maximum 0.30 at 210 nm, 0.07 at
Content: minimum 98.0 per cent. 220 nm, 0.01 at 240 nm and higher wavelengths, determined
Clear, colourless liquid. using water R as compensation liquid.
bp: about 148 °C. 2-Methylbut-2-ene C 5H 10 = 70.1 (513-35-9)
Methyl Benzoate Benzoic acid, methyl ester; Very flammable liquid, practically insoluble in water, miscible
C 8Hs02 = 136.2 (93-58-3) with ethanol (96 per cent).
Colourless liquid. bp: 37.5 °C to 38.5 °C.
df0 : 1.088. Methyl 4-(butylamino)benzoate C 12H 17N0 2 = 207.3
bp: about 200 °C. (71839-12-8)
2023 Appendix I A V-AIOI

White or almost white solid. White or bluish-white crystals or leaflets, practically insoluble
Content: minimum 99.9 per cent. in water, slightly soluble in ethanol (96 per cent), soluble in
mineral acids.
Methyl Caprate Methyl Decanoate
mp: about 90 'C.
See Methyl decanoate R.
4,4' -Methylenebis-N ,N-dimethylaniline Reagent
Methyl Caproate Methyl hexanoate; C 7H14O2 = 130.2
Tetramethyldiaminodiphenylmethane reagent
(106-70-7)
Solution A. Dissolve 2.5 g of
d~g: about 0.885.
tetramethyldiaminodiphenylmethane R in 10 mL of glacial acetic
nL0 : about 1.405.
acid R and add 50 mL of water R.
bp: 150 'C to 151 "C. Solution B. Dissolve 5 g of potassium iodide R in 100 mL of
Methyl Caprylate Methyl octanoate; C 9 H 18 O 2 = 158.2 water R.
(111-11-5) Solution C. Dissolve 0.30 g of ninhydrin R in 10 mL of
diS: about 0.876. glacial acetic acid R and add 90 mL of water R.
ni)0 :
about 1.417. Mix solution A, solution B and 1.5 mL of solution C.
bp: 193°Cto 194°C. Methylene Blue 3, 7-Dimethylaminophenothiazin-5-ium
Methylcellulose 450 (9004-67-5) chloride; It occurs in different hydrated forms and may
See Methylcellulose (0345). contain up to 22 per cent of water; C 16 Hl8ClN3S,
xH 2 0 = 319.9 for the anhydrous substance (122965-43-9)
Nominal viscosity: 450 mPa-s.
Schultz No. 1038
Methyl Cinnamate C 10H 10O 2 = 162.2 (103-26-4')
Colour Index No. 52015
Colourless crystals practically insoluble in water, soluble in
ethanol (96 per cent). Dark-green or bronze, crystalline powder, freely soluble in
water, soluble in ethanol (96 per cent).
n6°:about 1.56.
Methylene Blue Solution
bp: about 260 °C.
Dissolve 3 mg of methylene blue R, 1.2 g of sulfuric acid R and
mp: 34 °C to 36 "C.
5.0 g of anhydrous sodium sulfate R in 100 mL of water R.
Methylcodeine C 19H 23 NO 3 = 313.4 g/mol (2859-16-7)
Methyl Erucate Methyl cis-13-docosenoate;
Analytical reagent grade of commerce. C 23 H 44 O 2 = 352.6 (1120-34-9)
Content: minimum 95.0 per cent d~g: about 0.871.
Methylcyclohexane C 7H 14 = 98.2 (108-87-2) nL 0 : about 1.456.
Methyl Decanoate Methyl Caprate; C 11 H 22 O 2 = 186.3 3-O-Methylestrone 3-Methoxy-1,3,5(1 0)-estratrien-17-
(110-42-9) one; C 19 H 24 O 2 = 284.4 (1624-62-0)
Content: minimum 99.0 per cent. White to yellowish-white powder.
Clear, colourless or yellow liquid, soluble in light petroleum.
[aJ~r about+ 157.
d~g: 0.871 to 0.876. mp: about 173 °C.
nL0 : 1.425 to 1.426.
Methyl Ethyl Ketone Ethyl methyl ketone; 2-Butanone;
Methyl Docosanoate Methyl behenate; C 1H 8 O = 72.1 (78-93-3)
C 23 H 46 O 2 = 354.6 (929-77-1) Clear, colourless, flammable liquid, very soluble in water,
mp: 54 °C to 55 cc. miscible with ethanol (96 per cent).
Methyldopa, Racemic C 10 H 13NO 4 ,1 ½H2O = 238.2 d~g: about 0.81.
Mixture of equal volumes of (2S)- and (2R)-2-amino-3-(3,4- bp: 79 °C to 80 'C.
dihydroxyphenyl)-2-methylpropanoic acids. Methyleugenol 1,2-Dimethoxy-4-prop-2-enylbenzene;
3-O-Methyldopamine Hydrochloride 4-(2-Aminoethyl)- C 11 H 14 0 2 = 178.2 (93-15-2)
2-methoxyphenol hydrochloride; C 9H 14 ClNO 2 = 203.7 Methyleugenol used in gas chromatography complies with
(1477-68-5) the following additional test.
mp: 213 ''C to 215 °C. Assay. Gas chromatography (2.2.28) as prescribed in the
4-O-Methyldopamine Hydrochloride 5-(2-Aminoethyl)- monograph Niaouli oil, cineole type (2468).
2-methoxyphenol hydrochloride; C 9H 14 CINO 2 = 203.7 Content: minimum 97.0 per cent, calculated by the
(645-33-0) normalisation procedure.
mp: 207 'C to 208 °C. N-Methylglucamine Meglumine; C1HnNO 5 = 195.2
Methyl Eicosenoate Methyl (1 IZ)-eicos-11-enoate; ( 6284-40-8)
C 21 H 40 O 2 = 324.5 (2390-09-2) mp: about 130°.
Methylenebisacrylamide N,N' - General reagent grade of commerce.
Methylenebispropenamide; C 7 H 10N 2 O 2 = 154.2 (110-26-9)
Methyl Green CI 42585; basic blue 20, 4-[[4-(Dimethyl-
Fine, white or almost white powder, slightly soluble in water, amino )phenyl] [4-( dimethyliminio )cyclohexa-2,5-
soluble in ethanol (96 per cent). dienylidene ]-methylphenyl] trimethylammonium dichloride;
mp: 300 'C, with decomposition. C26H 33 Cl2 N 3 = 458.5 (7114-03-6)
4,4 '-Methylenebis-N ,N-dimethylaniline Green powder, soluble in water, soluble in sulfuric acid
Tetramethyldiaminodiphenylmethane; C 17H 22N 2 = 254.4 giving a yellow solution turning green on dilution with water.
(101-61-1)
V-A102 Appendix I A 2023

Methyl Green-Iodomercurate Paper Methyl margarate used in the assay of total fatty acids in Saw
Immerse thin strips of suitable filter paper in a 4% w/v palmetto fruit (1848) complies with the following additional test.
solution of methyl green and allow to dry in air. Immerse the Assay. Gas chromatography (2.2.28) as prescribed in the
strips for 1 hour in a solution containing 14% w/v of monograph Saw palmetto fruit (1848).
potassium iodide and 20% w/v of mercuric iodide. Wash with Content: minimum 97 per cent, calculated by the
distilled water until the washings are practically colourless and normalisation procedure.
allow to dry in air. Methyl Methacrylate Methyl 2-methylprop-2-enoate;
Store protected from light; use within 48 hours. CsHsO 2 = 100.1 (80-62-6)
Methyl 4-Hydroxybenzoate (99-76-3) Colourless liquid.
See Methyl parahydroxybenzoate R. nt 0 : about 1.414.

1-Methylimidazole 1-Methyl-lH-imidazole; bp: about 100 °C.

C4H6N 2 = 82.1 (616-47-7) mp: about ~48 °C.
Colourless or slightly yellowish liquid. It contains a suitable stabilising reagent.
ng1: about 1.495. Methyl Methanesulfonate C 2H 6 O 3 S = 110.1 (66-27-3)
bp: 195 °C to 197 °C. Clear, colourless or slightly yellow liquid.
Storage: in an airtight container, protected from light.
Content: minimum 99.0 per cent.
1-Methylimidazole Rl Density: about 1.3 g/cm3 (25 °C).
Complies with the requirements prescribed for ng1: about 1.414.
1-methylimidazol,e R with the following additional
bp: about 202 °C.
requirement.
Methyl 2-Methoxybenzoate C 9 H 10 O 3 = 166.2 (606-45-1)
Content: minimum 95.0 per cent.
Colourless liquid.
2-Methylimidazole C 4H 6N 2 = 82.1 (693-98-1)
White or almost white, crystalline powder.
Methyl 4-Methoxybenzoate C 9 H 10O 3 = 166.2 (121-98-2)
White or almost white powder.
mp: about 145 °C.
Methyl N-methylanthranilate Methyl 2-(methylamino)
Methyl Isobutyl Ketone, Water-saturated
benzoate; C 9 H 11 NO 2 = 165.2 (85-91-6)
Shake methyl isobutyl ketone R with water R prior to use.
Pale yellow liquid.
Methyl Laurate Methyl dodecanoate; C 13H 26O 2 = 214.4
(111-82-0) dz 0 : about 1.128.

Content: minimum 98.0 per cent, determined by gas nt 0 : about 1.579.

chromatography (2.4.22). bp: 255 °C to 258 °C.

Colourless or yellow liquid, soluble in ethanol (96 per cent) Methyl N-methylanthranilate used in gas chromatography
and in light petroleum. complies with the following additional test.
d~g: about 0.87. Assay. Gas chromatography (2.2.28) as prescribed in the
nt0 : about 1.431. monograph Mandarin oil (2355).
Test solution. The substance to be examined.
mp: about 5 °C.
Content: minimum 97 per cent, calculated by the
Methyl Lignocerate Methyl tetracosanoate;
normalisation procedure.
C2sHsoO 2 = 382.7 (2442-49-1)
Methyl Myristate Methyl tetradecanoate;
Flakes.
C1sH30O 2 = 242.4 (124-10-7)
mp: about 58 °C.
Content: minimum 98.0 per cent, determined by gas
Methyl Linoleate Methyl (9Z,12Z)-octadeca-9,12- chromatography (2.4.22).
dienoate; C 19H 34O 2 = 294.5 (112-63-0)
Colourless or slightly yellow liquid, soluble in ethanol
d~g: about 0.888. (96 per cent) and in light petroleum.
ng1: about 1.466. d~g: about 0.87.
bp: 207 °C to 208 °C.
Methyl Linolenate Methyl (9Z,12Z,15Z)-octadeca-
nt0 : about 1.437.

mp: about 20 °C.

9,12,15-•trienoate; Methyl cx-linolenate; C 19 H 32O 2 = 292.5
(301-00-8)
2-Methyl-1,4-naphthoquinone Menadione; (58-27-5)
See Menadione (0507).
dig: about 0.901.
Methyl Nervonate (2733-88-2)
nfr about 1.471.
See Tetracos-15-enoic acid methyl ester R.
bp: about 207 °C.
Methyl y-linolenate Methyl (6Z,9Z,12Z)-octadeca-6,9,12- 2-Methyl-5-nitroimidazole C 4 H 5 N 3O 2 = 127.1
(88054-22-2)
trienoate; C 19 H 32 O 2 = 292.5 (16326-32-2)
mp: 252° to 254°.
Content: minimum 99.0 per cent, determined by gas
chromatography. White to light yellow powder.
Methyl Margarate Methyl heptadecanoate; Content, minimum 98.0%.
C1sH36O2 = 284.5 (1731-92-6) 2-Methyl-2-nitropropane-1,3-diol 2-Nitro-2-methyl-1,3-
White or almost white powder. propanediol; C~ 9NO4 = 135.1 (77-49-6)
mp: 32 'C to 34 °C. mp: about 148°.
2023 Appendix I A V-Al03

General reagent grade of commerce. Content: minimum 98 per cent, calculated by the
Methyl Oleate Methyl (9Z)-octadec-9-enoate; normalisation procedure.
C19H36O 2 = 296.4 (112-62-9) 2-Methylpentane C 6 H 14 = 86.2 (107-83-5)
Content: minimum 98.0 per cent, determined by gas Isohexane.
chromatography (2. 4. 22). Jig: about 0.653.
Colourless or slightly yellow liquid, soluble in ethanol bp: about 60.0 °C.
(96 per cent) and in light petroleum.
Colourless, flammable liquid, practically insoluble in water,
J?g: about 0.88. miscible with anhydrous ethanol.
ni 0 : about 1.452.
3-Methylpentan-2-one sec-Butyl methyl ketone;
Methylophiopogonanone A (3R)-3-[(l,3-Benzodioxol-5- C 6H 12 O = 100.2 (565-61-7)
yl)methyl]-2 ,3-dihydro-5, 7-dihydroxy-6,8-dimethyl-4H- l- Colourless, flammable liquid.
benzopyran-4-one; C 19H 18 O 6 = 342.3 (74805-92-8)
dig: about 0.815.
Methyl Orange Sodium 4'-(dimethylamino)azobenzene-4-
n~: about 1.400.
sulfonate; C 14H1 4 N 3NaO 3 S = 327.3 (547-58-0)
bp: about 118 °C
Schultz No. 176
4-Methylpentan-2-ol 4-Methyl-2-pentanol;
Colour Index No. 13025
C6H14O = 102.2 (108-11-2)
Orange-yellow, crystalline powder, slightly soluble in water,
Clear, colourless, volatile liquid.
practically insoluble in ethanol (96 per cent).
Methyl Orange Mixed Solution
J1°: about 0.802.
Dissolve 20 mg of methyl orange R and 0.1 g of bromocresol
ni0: about 1.411.
green R in 1 mL of 0. 2 M sodium hydroxide and dilute to bp: about 132 °C.
100 mL with water R. 4-Methylpentan-2-one Methyl isobutyl ketone;
Colour change: pH 3.0 (orange) to pH 4.4 (olive-green). C 6 H12O = 100.2 (108-10-1)
Methyl Orange Solution Clear, colourless liquid, slightly soluble in water, miscible
with most organic solvents.
Dissolve 0.1 g of methyl orange R in 80 mL of water Rand
dilute to 100 mL with ethanol (96 per cent) R. dig: about 0.80.
Test for sensitivity. A mixture of O.1 mL of the methyl orange bp: about 115 °C.
solution and 100 mL of carbon di-Oxide-free water R is yellow. Distillation range (2.2.11). Distil 100 mL. The range of
Not more than 0.1 mL of 1 M hydrochloric acid is required to temperature of distillation from 1 mL to 95 mL of distillate
change the colour to red. does not exceed 4.0 °C.
Colour change: pH 3.0 (red) to pH 4.4 (yellow). Residue on evaporation: maximum 0.01 per cent, determined
Methyl Orange-Xylene Cyanol FF Solution by evaporating on a water-bath and drying at 100-105 °C.
Dissolve 0.1 g of methyl orange and 0.26 g of xylene cyanol FF 4-Methylpentan-2-one Rl Methyl isobutyl ketone Rl
in 50 mL of ethanol (96%) and add sufficient water to Shake 50 mL of freshly distilled methyl isoburyl ketone R with
produce 100 mL. 0.5 mL of hydrochloric acid Rl for 1 min. Allow the phases to
Methyl Palmitate Methyl hexadecanoate; separate and discard the lower phase. Prepare immediately
C17H34O 2 = 270.5 (112-39-0) before use.
Content: minimum 98.0 per cent, determined by gas 4-Methylpentan-2-one R3 Methyl isobutyl ketone R3
chromatography (2.4.22). Complies with the requirements for methyl isobutyl ketone R
White or yellow, crystalline mass, soluble in ethanol and with the following limits.
(96 per cent) and in light petroleum. Gr: maximum 0.02 ppm.
mp: about 30 cc_ Cu: maximum 0.02 ppm.
Methyl Palmitoleate Methyl cis-9-hexadecenoate; Pb: maximum 0.1 ppm.
C17H12O 2 = 268.4 (1120-25-8) Ni: maximum 0.02 ppm.
Jig: about 0.876. Sn: maximum 0.1 ppm.
ni 0 : about 1.451. 4-Methylphenazone 1,5-Dimethyl-2-(4-methylphenyl)-
Methyl Parahydroxybenzoate (99-76-3) 1,2-dihydro-3H-pyrazol-3-one; C 12H 14N 2 O = 202.3
See Methyl parahydroxybenzoate (0409). (56430-08-1)

Methyl Pelargonate Methyl nonanoate; Methylphenyloxazolylbenzene 1,4-Bis (2-( 4-methyl-5-

C10H20O2 = 172.3 (1731-84-6) phenyl)oxazolyl]benzene; C 26 H 20N 2O 2 = 392.5 (3073-87-8)
Clear, colourless liquid. Fine, greenish-yellow powder with a blue fluorescence or
small crystals, soluble in ethanol (96 per cent), sparingly
Jl 0 : about 0.873. soluble in xylene.
ni 0 : about 1.422.
mp: about 233 °C.
bp: 91 'C to 92 °C.
Methylphenyloxazolylbenzene used for liquid scintillation is of a
Methyl pelargonate used in the assay of total fatty acids in Saw suitable analytical grade.
palmetto fruit (1848) complies with the following additional test.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP;
Assay. Gas chromatography (2.2.28) as prescribed in the C12H1sN = 173.3 (28289-54-5)
monograph Saw palmetto fruit (1848).
White or almost white, crystalline powder, slightly soluble in
water.
V-A104 Appendix I A 2023

mp: about 41 °C. d~g: about 1.028.

N-Methylpiperazine 1-Methylpiperazine; bp: about 202 °C.
Methylpiperazine; C 5H 12N 2 = 100.2 (109-01-3) mp: about -24 °C.
Colourless liquid, miscible with water and with ethanol Methyl Red 2-(4-Dimethylamino-phenylazo) benzoic acid;
(96 per cent). C1sH1sN3O2 = 269.3 (493-52-7)
d~g: about 0.90. Schultz No. 250
ng': about 1.466. Colour Index No. 13020
bp: about 138 °C. Dark-red powder or violet crystals, practically insoluble in
4-( 4-Methylpiperidin-1-yl)pyridine 4-( 4- water, soluble in ethanol (96 per cent).
Methylpiperidino)pyridine; C 11 H 16N 2 = 176.3 (80965-30-6) Methyl Red Mixed Solution
Clear liquid. Dissolve 0.1 g of methyl red R and 50 mg of methylene blue R
nt0: about 1.565. in 100 mL of ethanol (96 per cent) R.
Methylpolysiloxane Col.our change: pH 5.2 (red-violet) to pH 5.6 (green).
Polysiloxane substituted with 100 per cent of methyl groups. Methyl Red Solution
Methylprednisolone 11 ~' 17 ,21-Trihydroxy-fo- Dissolve 50 mg of methyl red R in a mixture of 1.86 mL of
methylpregna-1,4-diene-3,20-dione; C 22 H30O 5 = 374.5 0.1 M sodium hydroxide and 50 mL of ethanol (96 per cent) R
(83-43-2) and dilute to 100 mL with water R.
White or almost white, crystalline powder. Test for sensitivity. To 0.1 mL of the methyl red solution add
2-Methylpropan-1-ol Isobutyl alcohol; 2-Methylpropanol; 100 mL of carbon dioxide-free water Rand 0.05 mL of 0.02 M
C4H1 0O = 74.1 (78-83-1) hydrochloric acid. The solution is red. Not more than 0.1 mL
of 0. 02 M sodium hydroxide is required to change the colour
Clear colourless liquid, soluble in water, miscible with
ethanol (96 per cent). to yellow.
Colour change: pH 4.4 (red) to pH 6.0 (yellow).
d~g: about 0.80.
Methyl Salicylate (J 19-36-8)
n:J: 1.397 to 1.399.
See Methyl salicylate (0230)
bp: about 107 °C.
Di~tillation range (2.2.11). Not less than 96 per cent distils When used in the Assay of Methyl Salicylate preparations,
use a grade containing not less than 99.0% of C8 H 8 O3.
between 107 °C and 109 °C.
2-Methylpropan-2-ol tert-Butyl alcohol; 1,1-Dimethyl Methyl Stearate Methyl octadecanoate;
ethyl alcohol; 2-Methyl-2-propanol; C 4H 10O = 74.1 C19H3sO2 = 298.5 (112-61-8)
(75-65-0) Content: minimum 98.0 per cent, determined by gas
Clear, colourless liquid or crystalline mass, soluble in water, chromatography (2.4.22).
miscible with ethanol (96 per cent). White or yellow, crystalline mass, soluble in ethanol
Freezing point (2.2.18): about 25 °C. (96 per cent) and in light petroleum.
mp: about 38 °C.
Distillation range (2.2.11). Not less than 95 per cent distils
between 81 °C and 83 °C. Methyl Thymol Blue Tetrasodium 2,2',2",2"'-[3H-2,1-
benzoxathiol-3-ylidenebis [[ 6-hydroxy-2-methyl-5-( 1-
(lSR)-15-Methylprostaglandin F2 a (5Z)-7-
[(1R,2R,3R,5S)-3 ,5-Dihydroxy-2-[ ( 1E)-(3R)-3-hydroxy-3- methylethyl)-3, 1-phenylene] methylenenitrilo]]tetraacetate S,
methyloct-1-enyl] cyclopentyl] hept-5-enoic acid; S-dioxide; Methylthymol blue; C 37H4oN2Na4 O 13 S = 845
(1945-77-3)
C21H3 6 Os = 368.5 (35864-81-4)
Available as a 10 g/L solution in methyl acetate R. Produces a blue colour with calcium in alkaline solution.
Storage: at a temperature below -15 °C. Methylthymol Blue Mixture
2-Methylpyridine C 6 H 7 N = 93.1 (109-06-8) A mixture of 1 part of methylthymol blue R and 100 parts of
potassium nitrate R.
Colourless or light yellow liquid.
Methyl Toluenesulfonate Methyl
Content: minimum 97.5 per cent. 4-methylbenzenesulfonate; Methyl tosilate;
5-Methylpyridin-2-amine 6-Amino-3-picoline; CsH 10O 3 S = 186.2 (80-48-8)
C 6 HsN 2 = 108.1 (1603-41-4) Content: minimum 97.0 per cent.
White or yellow crystals or crystalline powder. Density: about 1.234 g/mL (25 °C).
mp: about 76 °C. bp: about 292 °C.
5-Methylpyridin-2(1H)-one C 6H 7 NO = 109.1 mp: 25 °C to 28 °C.
(1003-68-5)
N-Methyl-m-toluidine N,3-Dimethylaniline; N,3-
White or almost white powder, soluble in anhydrous ethanol Dimethylbenzenamine; Methyl-m-tolylamine;
and in methanol. C 8 H 11 N = 121.2 (696-44-6)
mp: about 181 °C. Content: minimum 97 per cent.
Storage: at a temperature of 2 °C to 8 °C. Methyl Tricosanoate Tricosanoic acid methyl ester;
N-Methylpyrrolidine C 5 H 11 N = 85.2 (120-94-5) C24H4sO2 = 368.6 (2433-97-8)
Content: minimum 97.0 per cent. Content: minimum 99.0 per cent.
bp: about 80 °C. White or almost white crystals, practically insoluble in water,
N-Methylpyrrolidone l-Methylpyrrolidin-2-one; soluble in hexane.
C 5H9NO = 99.1 (872-50-4)
2023 Appendix I A V-Al05

mp: 55 'C to 56 'C. Mordant Black 11 Mixed Triturate

Methyl Tridecanoate C 14H 28 O 2 = 228.4 (1731-88-0) A mixture of 1 g of mordant black 11, 0.4 g of methyl orange
Colourless or slightly yellow liquid, soluble in ethanol and 100 g of sodium chloride.
(96 per cent) and in light petroleum. Complies with the following test.
dfS: about 0.86. Sensitivity to magnesium Dissolve 50 mg in 100 mL of
nr: about 1.441. water, a brown colour is produced. Add 0.3 mL of
6M ammonia; the colour changes to pale green. Add 0.1 mL
mp: about 6 °C.
of a 1.0% w/v solution of magnesium sulfate; the colour
Methyl 3,4,5-Trimethoxybenzoate CllH 14 O 5 = 226.23 changes to red.
(1916-0 7-0)
Mordant Black 11 Solution
N-Methyltrimethylsilyl-trifluoroacetamide 2,2,2-
Trifiuoro-N-methyl-N-(trimethylsilyl)acetamide; A 0.1 % w/v solution of mordant black 11 in ethanol (96%).
C 6 H 12 F3 NOSi = 199.3 (24589-78-4) Mordant Black 11 Triturate
n~: about 1.380. Mix 1 g of mordant black 11 R with 99 g of sodium chloride R.
bp: 130 "C to 132 °C. Test for sensitivity. Dissolve 50 mg in 100 mL of water R.
Minocycline Hydrochloride The solution is brownish-violet. On addition of 0.3 mL of
dilute ammonia Rl the solution turns blue. On the subsequent
See Minocycline hydrochloride (1030). addition of O.1 mL of a 10 g/L solution of magnesium
Molecular Sieve (70955-01-0) sulfate R, it turns violet.
Molecular sieve composed of sodium aluminosilicate. It is Swrage: in an airtight container, protected from light.
available as beads or powder with a pore size of 0.4 nm. Mordant Black 11 Triturate Rl
When reused, it is recommended that the molecular sieve be Mix 1.0 g of mordant black 11 R, 0.4 g of methyl orange R and
regenerated according to the manufacturer's instructions. 100 g of sodium chloride R.
Molecular Sieve for Chromatography Mordant Blue 3 Chromoxane cyanine; (3564-18-9)
Molecular sieve composed of sodium aluminosilicate. Colour Index No. 43820
The pore size is indicated after the name of the reagent in
the tests where it is used. If necessary, the particle size is also General reagent grade of commerce.
indicated. A dark red or reddish brown powder.
Molybdenum(vr) Oxide Molybdenum trioxide; Produces an intense purple colour with aluminium in weakly
MoO 3 = 143.9 (1313-27-5) acidic solutions. When metal ions are absent, for example, in
Analytical reagent grade of commerce. the presence of an excess of disodium edetate, the solution is
pale pink.
Molybdovanadic Reagent
Morphine,Anhydrous
In a 150 mL beaker, mix 4 g of finely powdered ammonium
molybdate Rand 0.1 g of finely powdered ammonium
Add SM ammonia, in slight excess, to a solution of morphine
sulfate in water, wash the precipitated morphine with water
vanadate R. Add 70 mL of water Rand grind the particles
using a glass rod. A clear solution is obtained within a few until free from ammonium salts and dry at 110°.
minutes. Add 20 mL of nitric acid R and dilute to 100 mL Morphine Hydrochloride See Morphine
with water R. hydrochloride (0097).
Monocrotaline ( IR,4R,SR,6R, 16R)-5,6-Dihydroxy-4,5,6- Morpholine Tetrahydro-1,4-oxazine; C 4 H 9NO = 87.1
trimethyl-2,8-dioxa-13-azatricyclo[8.5. l .0 13' 16]hexadec- l 0- (110-91-8)
ene-3, 7-dione; C 16H 23 NO 6 = 325.4 (315-22-0) Colourless, hygroscopic liquid, flammable, soluble in water
White or almost white powder, soluble in methanol. and in ethanol (96 per cent).
Monocrotaline N-Oxide (IR,4R,SR,6R,16R)-S,6- dfg: about 1.01.
Dihydroxy-4,5 ,6-trimethyl-3, 7-dioxo-2,8-dioxa-13-azatricyclo Distillation range (2.2.11). Not less than 95 per cent distils
[8.5. l.0 13 ' 16]hexadec-10-ene 13-oxide; C 16 H23NO7 = 341.4 between 126 °C and 130 °C.
Brownish-white powder, soluble in methanol. Storage: in an airtight container.
Monodocosahexaenoin Monoglyceride of Morpholine for Chromatography
docosahexaenoic acid (C22:6); Glycerol Complies with the requirements prescribed for morpholine R
monodocosahexaenoate; (all-Z)-Docosa-4, 7, 10, 13, 16, 19- with the following additional requirement.
hexaenoic acid, monoester with propane-1,2,3-triol; Content: minimum 99.5 per cent.
C2 5 H3sO4 = 402.6 (124516-13-8)
2-[N-Morpholino]ethanesulfonic Acid 2-(Morpholin-4-
The reagent from Nu-Chek Prep, Inc. has been found yl)sulfonic acid; MES; C 6 H 13NO 4 S = 195.2 (4432-31-9)
suitable.
White or almost white, crystalline powder, soluble in water.
Mordant Black 11 Eriochrome black T; Solochrome
black; C 20 H 12N 3 NaO 7S = 461.4 (1787-61-7) mp: about 300 °C.
Schultz No. 241 Murexide 5,5 '-Nitrilobis(pyrimidine-2,4,6(1H,3H,SH)-
trione) monoammonium salt; C 8 H 8 N 6 O 6 ,H2 O = 302.2
Colour Index No. 14645
Brownish-red crystalline powder, sparingly soluble in cold
Bro½nish-black powder, soluble in water and in ethanol water, soluble in hot water, practically insoluble in ethanol
(96 per cent). (96 per cent), soluble in solutions of potassium hydroxide or
Storage: in an airtight container, protected from light. sodium hydroxide giving a blue colour.
Myosmine 3-(4,5-Dihydro-3H-pyrrol-2-yl)pyridine;
C 9 H 10N 2 = 146.2 (532-12-7)
V-A106 Appendix I A 2023

Colourless crystals. Naphthalene used for liquid scintillation is of a suitable analytical

mp: about 45 °C. grade.
~-Myrcene 7-Methyl-3-methylenocta-1,6-diene; Naphthalene-1,3-diol 1,3-Dihydroxynaphthalene;
C10H16 = 136.2 (123-35-3) Naphthoresorcinol; Dihydroxynaphthalene; (132-86-5)
Oily liquid with a pleasant odour, practically insoluble in See 1,3-dihydroxynaphthalene R.
water, miscible with ethanol (96 per cent), soluble in glacial Naphthalene-2, 7-diol 2, 7-Dihydroxynaphthalene;
acetic acid. It dissolves in solutions of alkali hydroxides. C10HsO2 = 160.2 (582-17-2)
df 0 : about 0.794. Needles, soluble in water and in ethanol (96 per cent).
n~r about 1.470. mp: about 190 °C.
fi-Myrcene used in gas chromatography complies with the 2,3-Naphthalenediamine Naphthalene-2,3-diamine;
fallowing additional test. 2,3-Diaminonaphthalene; C 10H 1oN 2 = 158.2 (771-97-1)
Assay. Gas chromatography (2.2.28) as prescribed in the Brownish-yellow crystalline powder, slightly soluble in
monograph Peppermint oil (0405). ethanol (96 per cent), practically insoluble in acetone.
Test solution. The substance to be examined. mp: 195 °C to 198 °C.
Content: minimum 90.0 per cent, calculated by the Naphthalenediol Reagent Solution
normalisation procedure. Dissolve 2.5 mg of naphthalene-2,7-diol in 90 mL of methanol
Myristic Acid Tetradecanoic acid; C 14H 28 O 2 = 228.4 and add 10 mg of potassium hexacyanoferrate (III) and 50 mg
(544-63-8) of potassium cyanide dissolved in I O mL of water. Allow to
Colourless or white or almost white flakes. stand for 30 minutes and add 100 mL of 0.05M sodium
hydroxide.
mp: about 58.5 °C.
Myristic acid used in the assay of total fatty acids inSaw palmetto
Naphthalenediol Solution
fruit (1848) complies with the following additional test.
2, 7-Dihydroxynaphthalene solution
Dissolve 10 mg of 2,7-dihydroxynaphthalene R in 100 mL of
Assay. Gas chromatography (2.2.28) as prescribed in the
sulfuric acid R and allow to stand until decolorised.
monograph Saw palmetto fruit (1848).
Storage: use within 2 days.
Content: minimum 97 per cent, calculated by the
normalisation procedure. Naphtharson Thorin; Disodium 4-[(2-arsonophenyl)azo]-
3-hydroxynaphthalene-2, 7-disulfonate;
Myristicine 5-Allyl-l-methoxy-2,3-
methylenedioxybenzene; 4-Methoxy-6-(prop-2-enyl)-1,3- C1 6H 11 AsN2Na2 O 10S2 = 576.3 (3688-92-4)
benzodioxole; C 11 H 12 O 3 = 192.2 (607-91-0) Red powder, soluble in water.
Oily colourless liquid, practically insoluble in water, slightly Naphtharson Solution
soluble in anhydrous ethanol, miscible with toluene and with A 0.58 g/L solution of naphtharson R.
xylene. Test for sensitivity. To 50 mL of ethanol (96 per cent) R, add
dig: about 1.144. 20 mL of water R, 1 mL of dilute sulfuric acid Rl and 1 mL
ni0 : about 1.540. of the naphtharson solution. Titrate with 0. 025 M barium
perchlorate; the colour changes from orange-yellow to orange-
bp: 276 °C to 277 °C.
pink.
mp: about 173 °C.
Storage: protected from light; use within 1 week.
Chromatography. Thin-layer chromatography (2.2.27) as
prescribed in the monograph Star anise (1153); the Naphtharson Solution Rl
chromatogram shows only one principal spot. A 1 g/L solution in dewnised distilled water R.
Myristicine used in gas chromatography complies with the Test for sensitivity. To 50 mL of ethanol (96 per cent) R, add
following additional test. 20 mL of water R, 1 mL of dilute sulfuric acid Rl and 1 mL
Assay. Gas chromatography (2.2.28) as prescribed in the
of naphtharson solution Rl. Titrate with 0.025 M barium
perchlorate; the colour changes from orange-yellow to orange-
monograph Nutmeg oil (1552).
pink.
Content: minimum 95.0 per cent, calculated by the
Storage: protected from light; use within 1 week.
normalisation procedure.
Storage: protected from light.
~-Naphthol Solution Rl
Dissolve 3.0 mg of fi-naphthol R in 50 mL of sulfuric acid R
Myristyl Alcohol Tetradecan-1-ol; C 14H 30 O = 214.4
(112-72-1)
and dilute to 100.0 mL with the same acid. Use the recently
prepared solution.
dfg: about 0.823. 1-Naphthol o:-Naphthol; C 10H 8 O = 144.2 (90-15-3)
mp: 38 °C to 40 °C.
White or almost white, crystalline powder or colourless or
Myrtillin Delphinidin 3-O-glucoside chloride; white or almost white crystals, darkening on exposure to
C21H21ClO12 = 500.8 (6906-38-3) light, slightly soluble in water, freely soluble in ethanol
Nalorphine Hydrochloride C 19H 2 1NO 3, HCl = 347.8 (96 per cent).
(57-29-4) mp: about 95 °C.
General reagent grade of commerce. Storage: protected from light.
Naphthalene C 10H 8 = 128.2 (91-20-3) 2-Naphthol ~-Naphthol; C 10 H 8 O = 144.2 (135-19-3)
White or almost white crystals, practically insoluble in water, White or slightly pink plates or crystals, very slightly soluble
soluble in ethanol (96 per cent). in water, very soluble in ethanol (96 per cent).
mp: about 80 °C. mp: about 122 °C.
2023 Appendix I A V-A107

Storage: protected from light. Naphthylethylenediamine Dihydrochloride Solution

1-Naphthol Solution a-Naphtha! solution Dissolve 0.1 g of naphthylethylenediamine dihydrochloride R in
Dissolve 0.10 g of IJ.-naphthol R in 3 mL of a 150 g/L water Rand dilute to 100 mL with the same solvent. Prepare
solution of sodium hydroxide R and dilute to 100 mL with immediately before use.
water R. Prepare immediately before use. Naringin 7-((2-0-(6-Deoxy-a-L-mannopyranosyl)-~-D-
1-Naphthol Solution, Strong glucopyranosyl] oxy]-5-hydroxy-2-( 4-hydroxyphenyl)-2,3-
Dissolve 1 g of I-naphtha[ in a solution of 6 g of sodium dihydro-4H-chromen-4-one; C 27 H 12 0 14 = 580.5
(10236-47-2)
hydroxide and 16 g of anhydrous sodium carbonate in 100 mL
of water. White or almost white crystalline powder, slightly soluble in
2-Naphthol Solution ~-Naphtha! solution water, soluble in methanol and in dimethylformamide.
Dissolve 5 g of freshly recrystallised /3-naphthol R in 40 mL of mp: about 171 °C.
dilute sodium hydroxide solution R and dilute to 100 mL with Absorbance (2.2.25). Naringin dissolved in a 5 g/L solution of
water R. Prepare immediately before use. dimethylformamide R in methanol R shows an absorption
1-Naphtholbenzein Phenylbis( 4-hydroxynaphthyl) maximum at 283 nm.
methanol; Naphtholbenzein; C 27 H 18 0 2 = 374.4 (145-50-6) Neohesperidin Hesperetin-7-neohesperidoside; (2S)-7-[[2-
Brownish-red powder or shiny brownish-black crystals, 0-( 6-Deoxy-a-L-mannopyranosyl)-~-D-glucopyranosyl] oxy]-
practically insoluble in water, soluble in ethanol (96 per cent) 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-2,3-dihydro-4H-
and in glacial acetic acid. l-benzopyran-4-one; C 28 H 34 0 15 = 610.6 (13241-33-3)
1-Naphtholbenzein Solution Naphtholbenzein solution trans-Nerolidol 3, 7, 11-Trimethyldodeca- l ,6, 1O-trien-3-ol;
C 15 H 26 0 = 222.4 (40716-66-3)
A 2 g/L solution of naphtholbenzein R in anhydrous acetic
acid R.
Slightly yellow liquid, slight odour of lily and lily of the
valley, practically insoluble in water and in glycerol, miscible
Test for sensitivity. To 50 mL of glacial acetic acid R add
with ethanol (96 per cent).
0.25 mL of the naphtholbenzein solution. The solution is
brownish-yellow. Not more than 0.05 mL of 0.1 M perchloric d~g: about 0.876.
acid is required to change the colour to green. nfi°; about 1.4 79.
Naphthol Yellow 2,4-Dinitro-l-naphthol, sodium salt; bp 12 : 145 'C to 146 'C.
C10H 5N2NaOs = 256.2 trans-Nerolidol used in gas chromatography complies with the
Orange-yellow powder or crystals, freely soluble in water, following additional test.
slightly soluble in ethanol (96 per cent). Assay. Gas chromatography (2. 2. 28) as prescribed in the
Naphthol Yellow S 8-Hydroxy-5,7-dinitro-2- monograph Bitter-orange-flower oil (1175).
naphthalenesulfonic acid disodium salt; Disodium Test solutwn. The substance to be examined.
5, 7-dinitro-8-oxidonaphthalene-2-sulfonate; Content: minimum 90.0 per cent, calculated by the
C10H4N2Na20sS = 358.2 (846-70-8) normalisation procedure.
Colour Index No. 10316 Neryl Acetate (Z)-3,7-Dimethylocta-2,6-dienyl acetate;
Yellow or orange-yellow powder, freely soluble in water. C12H2002 = 196.3 (141-12-8)
1-Naphthylacetic Acid (Naphthalen-1-yl)acetic acid; Colourless, oily liquid.
C12H1002 = 186.2 (86-87-3) d?g: about 0.907.
White or yellow crystalline powder, very slightly soluble in ni0 : about 1.460.
water, freely soluble in acetone.
bp2 5 : 134 °C.
mp: about 135 °C.
Neryl acetate used in gas chromatography complies with the
2-Naphthylacetic Acid 2-Naphthaleneacetic acid; following additional test.
C12H1 00 2 = 186.2 (581-96-4)
Assay. Gas chromatography (2.2.28) as prescribed in the
mp: about 142°. monograph Bitter-orange-flower oil (1175).
General reagent grade of commerce. Test solution. The substance to be examined.
Light yellow to light brown crystals. Content: minimum 93.0 per cent, calculated by the
1-Naphthylamine 1-Aminonaphthalene; Naphthylamine; normalisation procedure.
C1 0H 9 N = 143.2 (134-32-7) Neutral Red Cl 50040; basic red 5; C 15H 17 CIN4 = 288.8
White or almost white, crystalline powder, turning pink on (553-24-2)
exposure to light and air, slightly soluble in water, freely Produces a red colour with acids and an orange colour with
soluble in ethanol (96 per cent). alkalis.
mp: about 51 °C. Neutral Red Solution
Storage: protected from light. A 0.1 % w/v solution of neutral red in ethanol (50%) (pH
N-(1-Naphthyl)ethylenediamine Dihydrochloride range, 6.8 to 8.0).
N-(1-Naphthyl)ethane-diamine dihydrochloride; Nickel-Aluminium Alloy Raney nickel catalyst
Naphthylethylenediamine dihydrochloride;
Contains 48 per cent to 52 per cent of aluminium
C12H15Cl2N2 = 259.2 (1465-25-4) (Al; A, 26. 98) and 48 per cent to 52 per cent of nickel (Ni;
It may contain methanol of crystallisation. Ar 58.70).
White or yellowish-white powder, soluble in water, slightly Before use, reduce to a fine powder (180) (2.9.12).
soluble in ethanol (96 per cent).
It is practically insoluble in water and soluble in mineral
acids.
V-Al08 Appendix I A 2023

Nickel-Aluminium Alloy (Halogen-free) Test for sensitivity. To 50 mL of anhydrous acetic acid R add
Contains 48 per cent to 52 per cent of aluminium 0.25 mL of the Nile blue A solution. The solution is blue.
(Al; Ar 26. 98) and 48 per cent to 52 per cent of nickel On the addition of O.1 mL of 0.1 M perchloric acid, the colour
(Ni; Ar 58.71). changes to blue-green.
Fine, grey powder, practically insoluble in water, soluble in Colour change: pH 9.0 (blue) to pH 13.0 (red).
mineral acids with formation of salts. Aqueous Nile Blue A Solution
Chlorides: maximum 10 ppm. Dissolve 40 mg of Nz1e Blue A in 200 mL of water, shake
Dissolve 0.400 g in 40 mL of a mixture of 67 volumes of with 100 mL of n-heptane in a 500 mL separating funnel and
sulfuric acid R and 33 volumes of dilute nitric acid R. discard the heptane layer. Repeat the extraction with four
Evaporate the solution nearly to dryness, dissolve the residue 100 mL quantities of n-heptane and mix 20 mL of the
in water Rand dilute to 20.0 mL with the same solvent. aqueous solution with 180 mL of ethanol.
To one half-aliquot of the solution, add 1.0 mL of 0.1 M Ninhydrin Indane-1,2,3-trione; C 9 H 4 O 3 ,H 2O = 178.1
silver nitrate. Filter after 15 min and add 0.2 mL of sodium (485-47-2)
chloride solution (containing 10 µg of chlorides per millilitre) White or very pale yellow, crystalline powder, soluble in
to the filtrate. After 5 min the solution is more opalescent water and in ethanol (96 per cent).
than a mixture of the second half-aliquot of the solution with Storage: protected from light.
1.0 mL of 0.1 M silver nitrate.
Ninhydrin and Stannous Chloride Reagent
Nickel(n) Chloride Anhydrous nickel chloride; Nickel
chloride; NiC1 2 = 129.6 (7718-54-9) Dissolve 0.2 g of ninhydrin R in 4 mL of hot water R, add
5 mL of a 1.6 g/L solution of stannous chloride R, allow to
Yellow, crystalline powder, very soluble in water, soluble in stand for 30 min, then filter and store at a temperature of
ethanol (96 per cent). It sublimes in the absence of air and 2 °C to 8 °C. Immediately before use dilute 2.5 mL of the
readily absorbs ammonia. The aqueous solution is acid. solution with 5 mL of water R and 45 mL of 2-propanol R.
Nickel(n) Chloride Hexahydrate NiClz,6H2O = 237.7 Ninhydrin and Stannous Chloride Reagent Rl
(7791-20-0)
Dissolve 4 g of ninhydrin in I 00 mL of ethylene glycol
Analytical reagent grade of commerce. monomethyl ether. Shake gently with 1 g of cation exchange
Nickel Nitrate Hexahydrate Ni(NO 3 )2,6H2 O = 290.8 resin (300 µm to 840 µm) and filter (solution A). Dissolve
(13478-00-7) 0.16 g of stannous chloride in 100 mL of buffer solution pH 5.5
Nickel(n) Sulfate Nickel(n) sulphate; Nickel sulfate (solution B). Immediately before use, mix equal volumes of
heptahydrate; Nickel sulfate; NiSO 4 ,7H2O = 280.9 each solution.
(10101-98-1) Ninhydrin Reagent I Transfer 10 litres of
Green, crystalline powder or crystals, freely soluble in water, 2-methoxyethanol to a clean, dry 20-litre bottle and purge with
slightly soluble in ethanol (96 per cent). oxygen-free nitrogen for 2 to 3 minutes. Continue the nitrogen
Nicotinamide-adenine Dinucleotide NAD+; flow and add 10 g of hydrindantin and 100 g of ninhydrinand
C21H21N1O 14P2 = 663 (53-84-9) allow to dissolve
White or almost white powder, very hygroscopic, freely Transfer 10 litres of 2-methoxyethanol to a clean, dry 20-litre
soluble in water. bottle and purge with oxygen-free nitrogen for 2 to 3 minutes.
Continue the nitrogen flow and add 10 g of hydrindantin and
Nicotinamide-adenine Dinucleotide Solution
100 g of ninhydrin and allow to dissolve; the solution will be
Dissolve 40 mg of nicotinamide-adenine dinucleotide R in straw coloured when dissolution is complete. Add 2 litres of
water R and dilute to 10 mL with the same solvent. Prepare a solution prepared by dissolving 5.444 kg of sodium acetate
immediately before use. in 5 litres of water, adding 1 litre of glacial acetic acid,
Nicotinic Acid (59-67-6) adjusting the pH to 5.5 with glacial acetic acid and adding
See Nicotinic acid (0459). sufficient water to produce 10 litres. Add 6.5 litres of water
Nicotinoyl Hydrazide Pyridine-3-carbohydrazide; and 20 mL of a 20% w/v solution of polyoxyethylene 23 lauryl
C 6H 7N 3 0 = 137.1 (553-53-7) ether.
White or almost white powder or crystalline powder, soluble The deep red reagent is stable for at least 8 weeks when
in water. stored under nitrogen and protected from light; avoid
exposure to ammonia.
mp: about 160 °C.
Ninhydrin Solution
Nile Blue A 5-Amino-9-(diethylamino)benzo[a]
phenoxazinylium hydrogen sulfate; C 20H 21 N 3 O 5S = 415.5 A 2 g/L solution of Ninhydrin R in a mixture of 5 volumes of
(3625-57-8) dilute acetic acid R and 95 volumes of butanol R.
Schultz No. 1029 Ninhydrin Solution Rl
Colour Index No. 51180 Dissolve 1.0 g of ninhydrin R in 50 mL of ethanol
(96 per cent) R and add 10 mL of glacial acetic acid R.
Green, crystalline powder with a bronze lustre, sparingly
soluble in ethanol (96 per cent), in glacial acetic acid and in Ninhydrin Solution R2
pyridine. Dissolve 3 g of ninhydrin R in 100 mL of a 45.5 g/L solution
Absorbance (2.2.25). A 0.005 g/L solution in ethanol of sodium metabisulfite R.
(50 per cent V/V) R shows an absorption maximum at Ninhydrin Solution R3
640 nm. A 4 g/L solution in a mixture of 5 volumes of anhydrous acetic
Nile Blue A Solution acid Rand 95 volumes of butanol R.
A 10 g/L solution of Nile blue A R in anhydrous acetic acid R.
2023 Appendix I A V-A109

Ninhydrin Solution R4 Cadmium: maximum 0.1 ppm.

A 3 g/L solution of ninhydrin R in a mixture of 5 volumes of Atomic absorption spectrometry (2.2.23, Method II).
glacial acetic acid Rand 95 volumes of 2-propanol R. Source: cadmium hollow-cathode lamp.
Nitrazepam (146-22-5) Wavelength: 228.8 nm.
See Nitrazepam (0415). Atomisation device: air-acetylene or air-propane flame.
Nitrazepam Impurity A 3-Amino-6-nitro-4- Lead: maximum 0.1 ppm.
phenylquinol-2( ll-J)-one; J-J)-Quinolinone,-3-amino-6-nitro- Atomic absorption spectrometry (2.2.23, Method II).
4-phenyl; C 15 H 11 N 3 O3 = 281.3 (36020-93-6)
Source: lead hollow-cathode lamp.
General reagent grade of commenrce.
Wavelength: 283.3 nm or 217.0 nm.
Nitric Acid HNO 3 = 63.0 (7697-37-2)
Atomisation device: air-acetylene flame.
Content: 63.0 per cent m/m to 70.0 per cent mlm.
Nitric Acid, Dilute
Clear, colourless or almost colourless liquid, miscible with
water. Contains about 125 g/L of HNO 3 (Mr 63.0).
d?g: 1.384 to 1.416. Dilute 20 g of nitric acid R to 100 mL with water R.
A 10 g/L solution is strongly acid and gives the reaction of Nitric Acid, Dilute Rl
nitrates (2.3.1). Dilute 40 g of nitric acid R to 100 mL with water R.
Appearance. Nitric acid is clear (2.2.1) and not more intensely Nitric Acid, Dilute R2
coloured than reference solution Y6 (2.2.2, Method II). Dilute 30 g of nitric acid R to 100 mL with water R.
Chlorides (2.4.4): maximum 0.5 ppm. Nitric Acid, Fuming (7697-37-2)
To 5 g add 10 mL of water R and 0.3 mL of silver nitrate Clear, slightly yellowish liquid, fuming on contact with air.
solution R2 and allow to stand for 2 min protected from light. dfg: about 1.5.
Any opalescence is not more intense than that of a standard
Nitric Acid, Heavy Metal-free
prepared in the same manner using 13 mL of water R,
0.5 mL of nitric acid R, 0.5 mL of chloride standard solution Complies with the requirements prescribed for nim·c acid R
(5 ppm Cl) Rand 0.3 mL of silver nitrate solution R2. with the following maximum contents of heavy metals.
Sulfates (2. 4.13): maximum 2 ppm. As: 0.005 ppm.
Evaporate 10 g to dryness with 0.2 g of sodium carbonate R. Cd: 0.005 ppm.
Dissolve the residue in 15 mL of distilled water R. Prepare the Cu: 0.001 ppm.
standard using a mixture of 2 mL of sulfate standard solution Fe: 0.02 ppm.
(JO ppm SO,J Rand 13 mL of distilled water R. Hg: 0.002 ppm.
Arsenic (2.4.2, Method A): maximum 0.02 ppm. Ni: 0.005 ppm.
Gently heat 50 g with 0.5 mL of sulfuric acid R until white Pb: 0.001 ppm.
fumes begin to evolve. To the residue add 1 mL of a 100 g/L
Zn: 0.01 ppm.
solution of hydroxylamine hydrochloride R and dilute to 2 mL
with water R. Prepare the standard using 1.0 mL of arsenic Nitric Acid, Dilute, Heavy Metal-free
standard solution (1 ppm As) R. Complies with the requirements prescribed for dilute nitric
Iron (2.4. 9): maximum 1 ppm. acid R with the following maximum contents of heavy metals.
Dissolve the residue from the determination of sulfated ash in As: 0.005 ppm.
1 mL of dilute hydroch/,oric acid R and dilute to 50 mL with Cd: 0.005 ppm.
water R. Dilute 5 mL of this solution to 10 mL with water R. Cu: 0.001 ppm.
Heavy metals (2.4.8): maximum 2 ppm. Fe: 0.02 ppm.
Dilute 10 mL of the solution prepared for the limit test for Hg: 0.002 ppm.
iron to 20 mL with water R. 12 mL of the solution complies Ni: 0.005 ppm.
with test A. Prepare the reference solution using lead standard
Pb: 0.001 ppm.
solution (2 ppm Pb) R.
Zn: 0.01 ppm.
Sulfated ash: maximum 0.001 per cent.
Nitric Acid, Lead-free, Dilute
Carefully evaporate 100 g to dryness. Moisten the residue
with a few drops of suljuric acid R and heat to dull red. Dilute 5 g of lead-free nitric acid Rl to 100 mL with deionised
distilled water R.
Assay. To 1.50 g add about 50 mL of water R and titrate
with 1 M sodium hydroxide, using 0.1 mL of methyl red Nitric Acid, Lead-free
solution R as indicator. Complies with the requirements prescribed for Nitric acid R
1 mL of 1 M sodium hydroxide is equivalent to 63.0 mg of with the following additional test.
HNO3. Lead: maximum 0.1 ppm.
Storage: protected from light. Atomic absorption spectrometry (2.2.23, Method II).
Nitric Acid, Cadmium- and Lead-free Test solution. To 100 g add 0.1 g of anhydrous sodium
Complies with the requirements prescribed for nitric acid R carbonate R and evaporate to dryness. Dissolve the residue in
and with the following additional test. water R, heating slightly, and dilute to 50.0 mL with the
same solvent.
Test solution. To 100 g add 0.1 g of anhydrous sodium
carbonate R and evaporate to dryness. Dissolve the residue in Source: lead hollow-cathode lamp.
water R heating slightly, and dilute to 50.0 mL with the same Wavelength: 283.3 nm or 217.0 nm.
solvent.
V-AllO Appendix I A 2023

Atomisation device: air-acetylene flame. Yellow crystals or a crystalline mass, decomposing in moist
Nitric Acid, Lead-free Rl air, completely soluble in sodium hydroxide solution giving a
yellowish-orange colour.
Nitric acid R containing not more than I µg/kg of lead.
mp: about 72 °C.
Nitric Acid, Nickel-free
Complies with the requirements prescribed for nitric acid R
4-Nitrobenzyl Chloride Nitrobenzyl chloride;
C 7 H 6 CINO2 = 171.6 (100-14-1)
with the following additional requirement.
Nickel: maximum 0.005 ppm. Pale-yellow crystals, lachrymatory, practically insoluble in
water, very soluble in ethanol (96 per cent).
Nitrilotriacetic Acid C 6 H 9NO 6 = 191.1 (139-13-9)
4-(4-Nitrobenzyl)pyridine C 12HwN2 O 2 = 214.2
White or almost white crystalline powder, practically (1083-48-3)
insoluble in water and in most organic solvents.
Yellow powder.
mp: about 240 °C, with decomposition.
mp: about 70 °C.
4-Nitroaniline Nitroaniline; C 6 H 6N 2 O 2 = 138.1
(100-01-6) Nitrochromic Reagent
Bright yellow, crystalline powder, very slightly soluble in Dissolve 0. 7 g of potassium dichromate in nitric acid and dilute
water, sparingly soluble in boiling water, soluble in ethanol to I 00 mL with the same acid.
(96 per cent), forms water-soluble salts with strong mineral Nitroethane C 2 H 5NO 2 = 75.1 (79-24-3)
acids. Clear, oily, colourless liquid.
mp: about 147 °C. bp: about 114 °C.
Nitroaniline Solution, Diazotised 2-Nitroethanol C 2 H 5NO 3 = 91.1 (625-48-9)
Dissolve 0.4 g of 4-nitroaniline in 60 mL of IM hydrochloric bp: about 205°.
acid with the aid of heat, cool to I 5° and add a 10% w/v General reagent of commerce.
solution of sodium nitrite until one drop of the mixture turns
Nitrofurantoin (67-20-9)
starch iodide paper blue.
See Nitrofurantoin (0101).
Prepare immediately before use.
(5-Nitro-2-furyl)methylene Diacetate Nitrofurfural
2-Nitrobenzaldehyde Nitrobenzaldehyde; diacetate; 5-nitrofurfurylidene diacetate; C 9 H 9 NO 7 = 243.2
C1HsNO 3 = 151.1 (552-89-6) (92-55-7)
Yellow needles, slightly soluble in water, freely soluble in mp: about 90°.
ethanol (96 per cent), volatile in steam.
Yellow crystals.
mp: about 42 °C.
Nitrogen N 2 = 28.01 (7727-37-9)
4-Nitrobenzaldehyde C 7 H 5NO 3 = 151.1 (555-16-8)
Nitrogen, washed and dried.
Nitrobenzaldehyde Paper
Nitrogen Dioxide NO 2 = 46.01 (10102-44-0)
Dissolve 0.2 g of nitrobenzaldehyde R in 10 mL of a 200 g/L
Content: minimum 98.0 per cent V/V.
solution of sodium hydroxide R. Use the solution within 1 h.
Immerse the lower half of a slow filter paper strip 10 cm long Nitrogen for Chromatography N 2 = 28.01 (7727-37-9)
and 0.8-1 cm wide. Absorb the excess reagent between two Content: minimum 99.95 per cent VIV.
sheets of filter paper. Use within a few minutes of Nitrogen Gas Mixture Nitric oxide
preparation.
Nitrogen R containing 1 per cent V/V of each of the following
Nitrobenzaldehyde Solution gases: carbon dioxide R2, carbon monoxide Rl and oxygen Rl.
Add 0.12 g of powdered nitrobenzaldehyde R to 10 mL of Nitrogen Monoxide NO = 30.01
dilute sodium hydroxide solution R; allow to stand for 10 min
Content: minimum 98.0 per cent V/V.
shaking frequently and filter. Prepare immediately before use.
Nitrogen, Oxygen-free
Nitrobenzene C 6H 5 NO 2 = 123.1 (98-95-3)
Nitrogen R which has been freed from oxygen by passing it
Colourless or very slightly yellow liquid, practically insoluble
through alkaline pyrogallol solution R.
in water, miscible with ethanol (96 per cent).
Nitrogen Rl N 2 = 28.01 (7727-37-9)
bp: about 211 °C.
Content: minimum 99.999 per cent V/V.
Dinitrobenzene. To 0.1 mL add 5 mL of acetone R, 5 mL of
Carbon monoxide: less than 5 ppm.
water R and 5 mL of strong sodium hydroxide solution R. Shake
and allow to stand. The upper layer is almost colourless. Oxygen: less than 5 ppm.
4-Nitrobenzoic Acid C 7 H 5 NO4 = 167.1 (62-23-7) Nitromethane CH3 NO2 = 61.0 (75-52-5)
Yell ow crystals. Clear, colourless, oily liquid, slightly soluble in water,
mp: about 240 °C. miscible with ethanol (96 per cent).
4-Nitrobenzyl Bromide C 7 H 6 BrNO 2 = 216.0 (100-11-8) dfg: 1.132 to 1.134.
mp: about 99°. nt0: 1.381 to 1.383.
General reagent grade of commerce. Distillation range (2.2.11). Not less than 95 per cent distils
between I 00 °C and I 03 °C.
Pale yellow crystals with a lachrymatory vapour.
4-Nitrophenol p-Nitrophenol; C 6 H 5NO 3 = 139.1
Nitrobenzoyl Chloride 4-Nitrobenzoyl chloride; (100-02-7)
C 7 H4CINO 3 = 185.6 (122-04-3)
Content: minimum 95 per cent.
Colourless or slightly yellow powder, sparingly soluble in
water and in methanol.
2023 Appendix I A V-Al 11

mp: about 114 °C. Assay. Liquid chromatography (2.2.29) as prescribed in the
3-Nitrosalicylic Acid 2-Hydroxy-3-nitrobenzoic acid; monograph Capsicum (1859).
C 7 H 5NO 5 = 183.1 (85-38-1) Content: minimum 98.0 per cent, calculated by the
Yellowish crystals, slightly soluble in water, freely soluble in normalisation procedure.
ethanol (96 per cent). N onylamine N onan-1-amine; 1-Aminononane;
mp: 142 °C to 147 °c. C 9 H 21 N = 143.3 (112-20-9)
5-Nitrosalicylic Acid 2-Hydroxy-5-nitrobenzoic acid; Corrosive, colourless, clear liquid.
C 7 H 5NO 5 = 183 (96-97-9) dl 0 : about 0.788.
General reagent grade of commerce. n~: about 1.433.
N-Nitrosodiethanolamine 2,2 '-(Nitrosoimino )diethanol; Noradrenaline Acid Tartrate Noradrenaline bitartrate;
C 4 H1 0 N 2 03 = 134.l (1116-54-7) CsH11NO3,C4H6O 6 = 319.3 (69815-49-2)
Yellow liquid, miscible with anhydrous ethanol. mp: about 102°.
n~: about 1.485. General reagent grade of commerce.
bp: about 125 °C. A white, crystalline powder.
N-Nitrosodiisopropanolamine 1,1 '-(Nitrosoimino) Nordazepam 7-Chloro-2,3-dihydro-5-phenyl- lH-1,4-
bispropan-2-ol; C 6 H 14N 2 0 3 = 162.2 (53609-64-6) benzodiazepin-2-one; C 15 H 11 CIN 2 O = 270.7 (1088-11-5)
bp: 122-124 °C. White or pale yellow, crystalline powder, practically insoluble
N-Nitroso-ethylmethylamine N-Ethyl-N-methylnitrous in water, slightly soluble in ethanol (96 per cent).
amide; NEMA; C 3 H 8 N 2 0 = 88.1 (10595-95-6) mp: about 216 °C.
Yellow liquid. DL-Norleucine (RS)-2-Aminohexanoic acid;
bp: about 170 °C. C 6H 13NO 2 = 131.2 (616-06-8)
Nitrosodipropylamine Dipropylnitrosamine; Shiny crystals, sparingly soluble in water and in ethanol
C 6 H14N2 0 = 130.2 (621-64-7) (96 per cent), soluble in acids.
Liquid, soluble in anhydrous ethanol and in strong acids. Noroxymorphone C 16H 17NO 4 = 287.3 (33522-95-1)
dfg: about 0.915. General reagent grade of commerce.
bp: about 78 °C. Norpseudoephedrine Hydrochloride C 9 H 13NO,
Appropriate grade for chemiluminescence determination. HCI = 187.7 (53643-20-2)
Nitrosodipropylamine Solution mp: 180° to 181 °.
Inject 78.62 g of anhydrous ethanol R through the septum of a General reagent grade of commerce.
vial containing nitrosodipropylamine R. Dilute 1/100 in A crystalline powder.
anhydrous ethanol Rand place 0.5 mL aliquots in crimp- Noscapine Hydrochloride (912-60-7)
sealed vials. See Noscapine hydrochloride (0515).
Storage: in the dark at 5 °C. Nystose C 24 H 42 O 21 = 666.6 (13133-07-8)
Nitrotetrazolium Blue 3,3 '-(3,3'-Dimethoxy-4,4 1- ~-o-Fructofuranosyl-(2 ➔ 1)-~-D-fructofuranosyl-(2 ➔ 1)-~-D-
diphenylene )di [2-( 4-nitrophenyl)-5-phenyl-2H-tetrazolium) fructofuranosyl cx-D-glucopyranoside.
dichloride; p-Nitro-tetrazolium blue; C40H 30 Cl2 N 10O 6 = 818
Ochratoxin A Solution
(298-83-9)
50 µg/mL solution of (2S)-2-([[(3R)-5-chloro-8-hydroxy-3-
Crystals, soluble in methanol, giving a clear, yellow solution. methyl-l-oxo-3,4-dihydro- lH-2-benzopyran-7-yl) carbonyl]
mp: about 189 "C, with decomposition. amino)-3-phenylpropanoic acid (ochratoxin A) in a mixture
Nitrous Oxide N 2O = 44.01 of 1 volume of acetic acid R and 99 volumes of benzene R.
Content: minimum 99.99 per cent V/V. Octadecan-1-ol Stearyl alcohol; C 18H 38 O = 270.5
Nitrogen monoxide: less than 1 ppm. (I 12-92-5)
Carbon monoxide: less than 1 ppm. mp: about 60 °C.
Nitro-vanado-molybdic Reagent Nitro- Content: minimum 95 per cent.
molybdovanadic reagent Octadecyl [3-[3,5-bis (1, l-dimethylethyl)-4-
Solution A. Dissolve 10 g of ammonium molybdate R in hydroxyphenyl]-propionate] Octadecyl 3-(3,5-di-tert-
water R, add 1 mL of ammonia R and dilute to 100 mL with butyl-4-hydroxyphenyl)propionate; C 35H 62 O3 = 530.9
water R. (2082-79-3)
Solution B. Dissolve 2.5 g of ammonium vanadate R in hot White or slightly yellowish, crystalline powder, practically
water R, add 14 mL of nitric acid Rand dilute to 500 mL insoluble in water, very soluble in acetone and in hexane,
with water R. slightly soluble in methanol.
To 96 mL of nitric acid R add 100 mL of solution A and mp: 49 °C to 55 °C.
I 00 mL of solution B and dilute to 500 mL with water R. Octanal Octyl aldehyde; C 8 H 16 O = 128.2 (124-13-0)
Nonivamide N-[ (4-Hydroxy-3-methoxyphenyl)methyl) Oily, colourless liquid. Practically insoluble in water.
nonanamide; C 17H 27N03 = 293.4 (2444-46-4) Octanal used in gas chromatography complies with the following
White or almost white, crystalline powder, practically additional test.
insoluble in cold water, freely soluble in anhydrous ethanol. Assay. Gas chromatography (2.2.28) as prescribed in the
Nonivamide used in the test for nonivamide in the monograph monograph Sweet orange oil (1811).
Capsicum (1859) complies with the following additional test.
V-Al 12 Appendix I A 2023

Content: minimum 99 per cent, calculated by the bp: 175 °C to 179 °C.
normalisation procedure. Oleamide (9Z)-Octadec-9-enoamide; C 18H 35 NO = 281.5
N-Octane Octane; C 8 H 18 = 114.2 (111-65-9) Yellowish or white powder or granules, practically insoluble
Content: minimum 99 per cent. in water, very soluble in methylene chloride, soluble in
Octanoic Acid Caprylic acid; C 8 H 16O 2 = 144.2 anhydrous ethanol.
(124-07-2) mp: about 80 °C.
Slightly yellow, oily liquid. Oleanolic Acid 3~-Hydroxyolean-12-en-28-oic acid;
dz 0: about 0.910. Astrantiagenin C; C 30H 48 O 3 = 456.7 (508-02-1)
nfi°; about 1.428. Oleic Acid (9Z)-Octadec-9-enoic acid; C 18H 34 O 2 = 282.5
(112-80-1)
bp: about 239.7 °C.
mp: about 16.7 cc. Clear, colourless liquid, practically insoluble in water.
Caprylic acid used in the assay of total fatty acids in Saw
dz0 : about 0.891.

palmetto frnit (1848) complies with the following additional test. nb0 : about 1.459.

Assay. Gas chromatography (2.2.28) as prescribed in the mp: 13 °C to 14 °C.

monograph Saw palmetto frnit (1848). Oleic acid used in the assay of total fatty acids in the monograph
Content: minimum 98 per cent, calculated by the Saw palmetto frnit (1848) complies with the following additional
normalisation procedure. test.
Octan-1-ol Caprylic alcohol; 1-Octanol; Octanol; Assay. Gas chromatography (2.2.28) as prescribed in the
CsH1 8 O = 130.2 (111-87-5) monograph Saw palmetto frnit (1848).
Colourless liquid, practically insoluble in water, miscible with Content: minimum 98 per cent, calculated by the
ethanol (96 per cent). normalisation procedure.
dig: about 0.828. Oleuropein 2-(3,4-Dihydroxyphenyl)ethyl [(2S,3E,4S)-3-
bp: about 195 °C. ethylidene-2-(~-D-glucopyranosyloxy)-5-(methoxycarbonyl)-
3,4-dihydro-2H-pyran-4-yl]acetate; C 25 H 320 13 = 540.5
Octan-2-ol sec-Octyl alcohol; C 8H 18 O = 130.2 (32619-42-4)
(6169-06-8)
Powder, soluble in methanol.
bp: about 178°.
Oleuropein used in Olive leaf (1878) complies with the following
General reagent grade of commerce. test.
An oily liquid; weight per mL, about 0.82 g. Assay. Liquid chromatography (2.2.29) as prescribed in the
3-Octanone Octan-3-one; Ethylpentylketone; monograph Olive leaf (1878).
CsH 16O = 128.2 (106-68-3) Content: minimum 80 per cent, calculated by the
Colourless liquid with a characteristic odour. normalisation procedure.
d~g: about 0.822. Oleyl Alcohol (9Z)-Octadec-9-en-1-ol; C 18 H 36 O = 268.5
nff: about 1.415. (143-28-2)
bp: about 167 °C. bp: about 207 °C.
3-Octanone used in gas chromatography complies with the nft 1.460.
following additional test. Content: minimum 85 per cent.
Assay. Gas chromatography (2.2.28) as prescribed in the Olive Oil (8001-25-0)
monograph Lavender oil (1338).
See Olive oil, virgin (0518).
Test solution. The substance to be examined. Olive Oil Substrate Emulsion
Content: minimum 98.0 per cent, calculated by the
Homogenise 40 mL of olive oil, 330 mL of acacia solution and
normalisation procedure. 30 mL of water in an 800-mL beaker placed in a vessel
Octoxinol 10 Cl-[ 4-( 1, 1,3,3-Tetramethylbutyl)phenyl]-w- containing a mixture of ice and ethanol as cooling mixture.
hydroxypoly-(oxyethylene); C 34H 62 O 11 (average) = 647 Emulsify using a mixer at an average speed of 1000 to
(9002-93-1) 2000 revolutions per minute. Cool to 5° to 10°. Increase the
Clear, pale-yellow, viscous liquid, miscible with water, with mixing speed to 8000 revolutions per minute. Mix for
acetone and with ethanol (96 per cent), soluble in toluene. 30 minutes keeping the temperature below 25° by the
Storage: in an airtight container. continuous addition of crushed ice into the cooling mixture
(a mixture of calcium chloride and crushed ice is also
Octreotide Acetate (Acetate-free peptide: Mr 1019;
[83150-76-9]); D-Phenylalanyl-L-cysteinyl-L-phenylalanyl-D- suitable). Store this preparation (the stock emulsion) in a
refrigerator and use within 14 days. The emulsion must not
tryptophyl-L-lysyl-L-threonyl-N-[(1R,2R)-2-hydroxy-
separate into two distinct layers. Check the diameter of the
1-(hydroxymethyl)propyl]-L-cysteinamide cyclic (2-" 7)-
globules of the emulsion under a microscope. At least 90%
disulfide acetate; It contains a variable quantity of acetic
have a diameter below 3 µm and none has a diameter greater
acid; White or almost white powder, freely soluble in water
than I O µm. Shake the emulsion thoroughly before preparing
and acetic acid; C4 9H66N 10O 10S2,xC2H4O2 (79517-01-4)
the substrate emulsion.
Content: minimum 96.0 per cent.
For 10 determinations mix the following solutions in the
Octylamine I-Amino-octane; N-Octylamine; order indicated: 100 mL of the stock emulsion, 80 mL of
C 8H19N = 129.2 (111-86-4) tris-chloride buffer solution, 20 mL of a freshly prepared
Colourless liquid. 8% w/v solution of sodium taurocholate EPBRP and 95 mL of
d~g: about 0.782. water.
2023 Appendix I A V-Al 13

Use on the day of preparation. Organosilica Polymer for Mass Spectrometry,

Oracet Blue B Amorphous, Octadecylsilyl, End-capped Organosilica
Polymer for Chromatography, Amorphous, Octadecylsilyl,
Solvent blue 19
End-capped
A mixture of l-methylamino-4-anilinoanthraquinone
Synthetic, spherical hybrid particles containing both inorganic
(C 21 H 16N 20 2) and 1-amino-4-anilinoanthraquinone
(C 20 H 14N 2 O 2 ). When used for titration in non-aqueous (silica) and organic (organosiloxanes) components,
chemically modified at the surface by the bonding of
media, it changes from blue (basic) through purple (neutral)
octadecylsilyl groups. To minimise any interaction with basic
to pink (acidic).
compounds, they are carefully end-capped to cover most of
Oracet Blue B Solution the remaining silanol groups.
A 0.5% w/v solution of oracet blue Bin anhydrous acetic acid. Organosilica Polymer, Multi-layered, Octadecylsilyl,
Oracet Blue 2R l-Amino-4-(phenylamino)anthracene- End-capped
9,10-dione; Cl 61110; C 20 H14N 2O2 = 314.3 (4395-65-7) Synthetic, spherical hybrid particles, multi-layered, containing
mp: about 194°. both inorganic (silica) and organic (organosiloxanes)
Oracet Blue 2R Solution components, chemically modified at the surface by the
A 0.5% w/v solution of oracet blue 2R in anhydrous acetic acid. bonding of octadecylsilyl groups. To minimise any interaction
with basic compounds, they are carefully end-capped to
Orcinol 5-Methylbenzene-1,3-diol monohydrate; C 7 H 8 O 2,
cover most of the remaining silanol groups.
H 2 O = 142.2 (6153-39-5)
Orientin 2-(3,4-Dihydroxyphenyl)-8-~-D-g]ucopyranosyl-
Crystalline powder, sensitive to light.
5, 7-dihydroxy-4H-l-benzopyran-4-one; 8-~-D-
bp: about 290 °C. Glucopyranosyl-3 1,4 1,5, 7-tetrahydroxyflavone; Luteolin-8-C-
mp: 58 °C to 61 °C. ~-o-g]ucopyranoside; Luteolin-8-g]ucoside;
Organosilica Polymer, Amorphous, Octadecylsilyl C 21 H 20O 11 = 448.4 (28608-75-5)
Synthetic, spherical hybrid particles, containing both Orthophosphoric Acid Phosphoric acid; (7664-38-2)
inorganic (silica) and organic (organosiloxanes) components, See Concentrated phosphoric acid (0004).
chemically modified at the surface by trifunctionally bonded Orthophosphorous Acid Phosphorous acid;
octadecylsilyl groups. H 3 PO 3 = 82.0 (13598-36-2)
Organosilica Polymer, Amorphous, Octadecylsilyl, White or almost white, very hygroscopic and deliquescent
End-capped crystalline mass; slowly oxidised by oxygen (air) to H 3P0 4 .
Synthetic, spherical hybrid particles, containing both Unstable, orthorhombic crystals, soluble in water, in ethanol
inorganic (silica) and organic (organosiloxanes) components, (96 per cent) and in a mixture of 3 volumes of ether and
chemically modified at the surface by trifunctionally bonded 1 volume of ethanol (96 per cent).
octadecylsilyl groups. To minimise any interaction with basic
compounds, it is carefully end-capped to cover most of the df 1: 1.651.
remaining silanol groups. The particle size is indicated after mp: about 73 'C.
the name of the reagent in the tests where it is used. Osthole 7-Methoxy-8-(3-methylbut-2-enyl)-2H- l-
Organosilica Polymer, Amorphous, Polar-embedded benzopyran-2-one; 7-Methoxy-8-isopentenylcoumarin;
Octadecylsilyl, End-capped C1sH15O3 = 244.3 (484-12-8)
Synthetic, spherical hybrid particles containing both inorganic Oxalic Acid Ethanedioic acid dihydrate;
(silica) and organic (organosiloxanes) components, C 2 H 2 O4,2H 2 O = 126.1 (6153-56-6)
chemically modified at the surface by the bonding of polar- White or almost white crystals, soluble in water, freely
embedded octadecylsilyl groups. To minimise any interaction soluble in ethanol (96 per cent).
with basic compounds, they are carefully end-capped to Oxalic Acid and Sulfuric Acid Solution Oxalic acid and
cover most of the remaining silanol groups. sulphuric acid solution
Organosilica Polymer, Amorphous, Propyl-2- A 50 g/L solution of oxalic acid R in a cooled mixture of
phenylsilyl, End-capped equal volumes of sulfuric acid R and water R.
Synthetic, spherical hybrid particles containing both inorganic Oxazepam (604-75-1)
(silica) and organic (organosiloxanes) components, See Oxazepam (0778).
chemically modified at the surface by the bonding of propyl-
2-phenylsilyl groups. To minimise any interaction with basic Ox Brain, Acetone-dried
compounds, they are carefully end-capped to cover most of Cut into small pieces a fresh ox brain previously freed from
the remaining silanol groups. vascular and connective tissue. Place in acetone R for
Organosilica Polymer Compatible with 100% Aqueous preliminary dehydration. Complete the dehydration by
Mobile Phases, Octadecylsilyl, Solid Core, End- pounding in a mortar 30 g of this material with successive
quantities, each of 75 mL, of acetone R until a dry powder is
capped
obtained after filtration. Dry at 37 'C for 2 h or until the
Silica gel with spherical silica particles containing a solid non- odour of acetone is no longer present.
porous silica core surrounded by a thin outer organosilica
polymer coating with octadecylsilyl groups, suitable for use 2 ,2 1-Oxybis (N ,N-dimethylethylamine) Bis(2-
dimethylaminoethyl) ether; C 8 H 20N 2 O = 160.3 (3033-62-3)
with highly aqueous mobile phases including 100 per cent
aqueous phases. To minimise any interaction with basic Colourless, corrosive liquid.
compounds, it is carefully end-capped to cover most of the d~~: about 0.85.
remaining silanol groups. nf,0 : about 1.430.
V-Al 14 Appendix I A 2023

4,4 ' -[Oxybis[ (4,1-phenylene) sulfonyl]]dianiline Palmitoleic acid used in the assay of total fatty acids in Saw
C24H20N 2O 5 S2 = 480.6 (54616-64-7) palmetto fruit (1848) complies with the following additional test.
Light brown powder. Assay. Gas chromatography (2.2.28) as prescribed in the
mp: about 220 °C. monograph Saw palmetto fruit (1848).
0:r,,.-ygen 0 2 = 32.00 Content: minimum 98 per cent, calculated by the
Content: minimum 99.99 per cent V/V. normalisation procedure.
Nitrogen and argon: less than 100 ppm. Palmityl Alcohol Hexadecan-1-ol; Cetyl alcohol;
C16H34O = 242.4 (36653-82-4)
Carbon dioxide: less than 10 ppm.
mp: about 48 °C.
Carbon monoxide: less than 5 ppm.
Content: minimum 96 per cent.
Oxygen Rl 0 2 = 32.00
Pancreas Powder See Pancreas powder (0350).
Content: minimum 99 per cent V/V.
Papain (9001-73-4)
Oxytetracycline Hydrochloride
A proteolytic enzyme obtained from the latex of the green
See Oxytetracycline hydrochloride (0198). fruit and leaves of Garica papaya L.
Paeoniflorin [(IR,2S,3R,5R,6R,8S)-3-(~-D- Papaverine Hydrochloride (61-25-6)
Glucopyranosyloxy) 6-hydroxy-8-methyl-9, 10-dioxatetracyclo
[4.3.1.0 25 .03 ·8 ]decan-2-yl]methyl benzoate; See Papaverine hydrochloride (0102).
C23H23O11 = 480.5 (23180-57-6) Paper Chromatography Performance Test Solutions
Paeonol 1-( 2-Hydroxy-4-methoxyphenyl)ethan-1-one; 2 ' - Test solution (A). Sodium pertechnetate (9 9"'Tc) injection
Hydroxy-4 1-methoxyacetophenone; C 9 H 10 O 3 = 166.2 (fission) (0124) or Sodium pertechnetate (99mTc) injection (non-
(552-41-0) fission) (0283).
Palladium Pd = 106.4 (7440-05-3) Test solution (B). In a closed vial mix 100 µL of a 5 g/L
Grey white metal, soluble in hydrochloric acid. solution of stannous chloride R in 0. 05 M hydrochloric acui and
100 MBq to 200 MBq of Sodium pertechnetate (99mTc)
Palladium(n) Chloride Palladium chloride; injection (fission) (0124) or Sodium pertechnetate (99mTc)
PdC1 2 = 177.3 (7647-10-1) injection (non-fission) (0283) in a volume not exceeding 2 mL.
Red crystals. Paper for Chromatography
mp: 678 °C to 680 °C. Pure cellulose grade thin paper with a smooth surface and a
Palladium Chloride Solution thickness of about 0.2 mm.
Dissolve 1 g of palladium chloride R in 10 mL of warm Chromatographic separation. To 2 strips of paper for
hydrochloric acid R. Dilute the solution to 250 mL with a chromatography R apply separately 2-5 µL of test solution (a)
mixture of equal volumes of dilute hydrochloric acui R and and test solution (b) of paper chromatography performance test
water R. Dilute this solution immediately before use with solutions R. Develop over a pathlength of 3/4 of the paper
2 volumes of water R. height, using a mixture of equal volumes of methanol R and
Palmatine 2,3,9,10-Tetramethoxy-5,6-dihydro-7A,5 - water R. Allow to dry and determine the distribution of
isoquinolino [3,2-a] isoquinolin-7-ylium; 7 ,8, 13, 13a- radioactivity using a suitable detector. The paper is not
Tetradehydro-2,3 ,9, 10-tetramethoxyberbinium; satisfactory, unless the chromatogram obtained with test
C21H 22NO 4 + = 352.4 (3486-67-7) solution (a) shows a single radioactivity spot with an RF value
Palmatine Chloride Berbericine Chloride; C 21 H 22NO4, in the range 0.8-1.0 and the chromatogram obtained with
Cl = 387 .86 (J 0605-02-4) test solution (b) shows a single radioactivity spot at the
application point (RF value in the range 0.0-0.1).
General reagent grade of commerce.
Paracetamol (103-90-2)
Palmitic Acid Hexadecanoic acid; C 16H 32 O 2 = 256.4
(57-10-3) See Paracetamol (0049).
White or almost white, crystalline scales, practically insoluble Paracetamol, 4-Aminophenol-free
in water, freely soluble in hot ethanol (96 per cent). Recrystallise paracetamol R from water R and dry in vacuo at
mp: about 63 'C. 70 °C; repeat the procedure until the product complies with
the following test: dissolve 5 g of the dried substance in a
Chromatography. Thin-layer chromatography (2.2.27) as
prescribed in the monograph Chloramphenicol mixture of equal volumes of methanol R and water R and
dilute to 100 mL with the same mixture of solvents.
palmitate (0473); the chromatogram shows only one principal
spot. Add 1 mL of a freshly prepared solution containing 10 g/L of
sodium nitroprusside R and 10 g/L of anhydrous sodium
Palmitic acid used in the assay of total fatty acids in the carbonate R, mix and allow to stand for 30 min protected
monograph Saw palmetto fruit (1848) complies with the following from light. No blue or green colour is produced.
additional test.
Paraffin, Liquid (8042-47-5)
Assay. Gas chromatography (2.2.28) as prescribed in the
See Liquui paraffin (0239).
monograph Saw palmetto fruit (1848).
Content: minimum 98 per cent, calculated by the Paraffin, White Soft
normalisation procedure. A semi-liquid mixture of hydrocarbons obtained from
Palmitoleic Acid (9Z)-Hexadec-9-enoic acid; petroleum and bleached, practically insoluble in water and in
ethanol (96 per cent), soluble in light petroleum Rl, the
C16H 30O2 = 254.4 (373-49-9)
solution sometimes showing a slight opalescence.
Clear, colourless liquid.
Paraldehyde (123-63-7)
bp: about 162 °C.
See Paraldchydc (0351).
2023 Appendix I A V-A115

Pararosaniline Hydrochloride Pararosaniline chloride; penicillinase solution contains not less than 0.4 microkatals
Basic red 9; C 19H 18 ClN3 = 323.8 (569-61-9) (corresponding to the hydrolysis of not less than 500 mg of
Schultz No. 779 benzylpenicillin to benzylpenicilloic acid per hour) at 30 °C
Colour Index No. 42500 and pH 7, provided that the concentration of benzylpenicillin
does not fall below the level necessary for enzyme saturation.
Bluish-red, crystalline powder, slightly soluble in water,
soluble in anhydrous ethanol. Solutions in water and The Michaelis constant for benzylpenicillin of the
anhydrous ethanol are deep-red; solutions in sulfuric acid penicillinase in penicillinase solution is approximately
and in hydrochloric acid are yellow. 12 µg/mL.
mp: about 270 °C, with decomposition. Sterility (2.6.1). It complies with the test for sterility.

Pararosaniline Solution, Decolorised Storage: at a temperature between 0 °C and 2 °C for 2 to

3 days. When freeze-dried and kept in sealed ampoules, it
To 0.1 g of pararosaniline hydrochloride Rina ground-glass- may be stored for several months.
stoppered flask add 60 mL of water R and a solution of 1.0 g
of anhydrous sodium sulfite R or 2.0 g of sodium sulfite Pentaerythrityl Tetrakis[3-(3,5-di-tert-butyl-4-
heptahydrate R or 0.75 g of sodium metabisulfite R in 10 mL of
hydroxyphenyl)propionate] Pentaerythrityl tetrakis
water R. Slowly and with stirring add 6 mL of dilute (3-(3,5-di( 1, 1-dimethylethyl)-4-hydroxyphenyl)propionate];
hydrochloric acid R, stopper the flask and continue stirring C73H10sO12 = 1178 (6683-19-8)
until dissolution is complete. Dilute to 100 mL with water R. White or slightly yellow, crystalline powder, practically
Allow to stand for 12 h before use. insoluble in water, very soluble in acetone, soluble in
Storage: protected from light. methanol, slightly soluble in hexane.
Parthenolide (4E)-( 1aR, 7aS, 10aS, 10bS)-1 a,5-Dimethyl-8- mp: 110 °C to 125 °C.
methylene-2,3,6, 7, 7a,8, 10a, 10b-octahydro-oxireno (9, 1OJ a-form: 120 °C to 125 °C.
cyclodeca [1,2-b] furan-9( 1aH)-one; (E)-(5S, 6S)-4,5- ~-form: 110 °C to 115 °C.
Epoxygermacra-1(10), 11 (13)-dieno-12(6)-lactone; Pentafluoropropanoic Acid C 3HF 5O 2 = 164.0
C1sH 20O 3 = 248.3 (20554-84-1) (422-64-0)
White or almost white, crystalline powder, very slightly Clear, colourless liquid.
soluble in water, very soluble in methylene chloride, soluble
d~g: about 1.561.
in methanol.
[I)(]~: -71.4, determined on a 2.2 g/L solution in methylene
nt0 : about 1.284.

chloride R. bp: about 97 °C.

mp: 115 °C to 116 °C. Pentafluoropropionic Anhydride Pentafluoropropanoic
anhydride; C 6F 10 O3 = 310.0 (356-42-3)
Absorbance (2.2.25). A 0.01 g/L solution in ethanol
(96 per cent) R shows an absorption maximum at 214 nm. N-Pentane Pentane; C 5H 12 = 72.2 (109-66-0)
Assay. Liquid chromatography (2.2.29) as prescribed in the Clear, colourless, flammable liquid, very slightly soluble in
monograph Feveifew (1516), at the concentration of the water, miscible with acetone and with anhydrous ethanol.
reference solution. d~g: about 0.63.
Content: minimum 90 per cent, calculated by the nt0 : about 1.359.

normalisation procedure. bp: about 36 °C.

Peimine C 27 H4 5NO 3 = 431.7 (23496-41-5) Pentane used in spectrophotometry complies with the following
5a-Cevane-3 ~' 6a,20-triol. additional test.
Content: minimum 95.0 per cent. Absorbance (2.2.25): maximum 0.70 at 200 nm, 0.30 at
Peiminine C 27 H 43 NO 3 = 429.6 (18059-10-4) 210 nm, 0.07 at 220 nm, 0.03 at 230 nm, 0.01 at 240 nm,
determined using water R as compensation liquid.
3~,20-Dihydroxy-5a-cevan-6-one.
1,2-Pentanediol (2RS)-Pentane-1,2-diol;
Content: minimum 95.0 per cent.
CsH12O2 = 104.2 (5343-92-0)
L-Penicillamine Coated Silica Gel For Chiral
Separations
df 0 : about 0.971.
A very finely divided silica gel for chromatography coated
nt0 : about 1.439.

with L-penicillamine. bp: about 201 °C.

Penicillinase Solution 3-Pentanone Pentan-3-one; Diethyl ketone;
CsH1oO = 86.13 (96-22-0)
Dissolve 10 g of casein hydrolysate, 2. 72 g of potassium
dihydrogen phosphate R and 5.88 g of sodium citrate R in Pentan-1-ol N-Pentyl alcohol; Pentanol; C 5 H 12 O = 88.1
200 mL of water R, adjust to pH 7 .2 with a 200 g/L solution (71-41-0)
of sodium hydroxide R and dilute to 1000 mL with water R. Colourless liquid, sparingly soluble in water, miscible with
Dissolve 0.41 g of magnesium sulfate R in 5 mL of water R ethanol (96 per cent).
and add 1 mL of a 1.6 g/L solution of ferrous ammonium nt0 : about 1.410.

sulfate Rand sufficient water R to produce 10 mL. Sterilise bp: about 137 °C.
both solutions by heating in an autoclave, cool, mix,
Pentetic Acid [[(Carboxymethyl)imino]
distribute in shallow layers in conical flasks and inoculate
bis(ethylenenitrilo)]tetraacetic acid; White or almost white
with Bacillus cereus (NCTC 9946). Allow the flasks to stand
powder, slightly soluble in water; C 14 H 23 N 3O 10 = 393.3
at 18 °C to 37 °C until growth is apparent and then maintain
(67-43-6)
at 35 °C to 37 °C for 16 h, shaking constantly to ensure
maximum aeration. Centrifuge and sterilise the supernatant mp: 219 °C to 220 ''C, with decomposition.
by filtration through a membrane filter. 1.0 mL of
V-Al 16 Appendix I A 2023

tert-Pentyl Alcohol tert-Amyl alcohol; 2-Methyl-2- Perylene Dibenz[de,k~anthracene; C 20 H 12 = 252.3

butanol; C 5 H 12 O = 88.1 (75-85-4) (198-55-0)
Volatile, flammable liquid, freely soluble in water, miscible Orange powder.
with ethanol (96 per cent) and with glycerol. mp: about 279 °C.
dig: about 0.81. Petroleum, Light Petroleum ether 50-70 °C; (8032-32-4)
Distillation range (2.2.11). Not less than 95 per cent distils Clear, colourless, flammable liquid without fluorescence,
between 100 °C and 104 °C. practically insoluble in water, miscible with ethanol
Storage: protected from light. (96 per cent).
Pepsin d?g: 0.661 to 0.664.
A substance containing a proteolytic enzyme of the gastric Distillation range (2.2.11): 50 °C to 70 °C.
secretion of animals, diluted, if necessary, by admixture with Petroleum Rl, Light Petroleum ether 40-60 °C
Lactose or Sucrose. Use a grade of commerce capable of Complies with the requirements prescribed for light
digesting 2500 times its own weight of coagulated egg petroleum R, with the following modifications.
albumen.
d?g: 0.630 to 0.656.
Pepsin Powder (9001-75-6)
Distillation range (2.2.11): 40 °C to 60 °C. It does not
See Pepsin powder (0682). become cloudy at O °C.
Peptide N-glycosidase F (83534-39-8) Petroleum R2, Light Petroleum ether 30-40 °C
Peptide-N4-(N-acetyl-~-glucosaminyl)asparagine amidase Complies with the requirements prescribed for light
(EC 3.5.1.52). PNGase F. petroleum R, with the following modifications.
Perchloric Acid HC1O 4 = 100.5 (7601-90-3) dig: 0.620 to 0.630.
Content: 70.0 per cent m/m to 73.0 per cent mlm. Distillation range (2.2.11): 30 °C to 40 °C. It does not
Clear, colourless liquid, miscible with water. become cloudy at O °C.
d~g: about 1. 7. Petroleum R3, Light Petroleum ether 100-120 °C
Assay. To 2.50 g add 50 mL of water Rand titrate with 1 M Complies with the requirements prescribed for light
sodium hydroxide, using O.1 mL of methyl red solution R as petroleum R, with the following modifications.
indicator. dig: about 0.720.
1 mL of 1 M sodium hydroxide is equivalent to 100.5 mg of Distillation range (2.2.11): 100 °C to 120 °C.
HC1O 4 •
Water (2.5.12): maximum 0.03 per cent.
Perchloric Acid Solution
Petroleum R4, Light Petroleum ether 80-100 °C
Dilute 8.5 mL of perchloric acid R to 100 mL with water R.
Complies with the requirements prescribed for light
Perfluoroheptanoic Acid Tridecafluoroheptanoic acid; petroleum R, with the following modifications.
C 7 HF 13 O 2 = 364.1 (375-85-9)
d?g: about 0.70.
Periodic Acetic Acid Solution
Distillation range (2.2.11): 80 °C to 100 °C.
Dissolve 0.446 g of sodium periodate R in 2.5 mL of a
25 per cent V/V solution of sulfuric acid R. Dilute to Petroleum Spirit Petroleum ether
100.0 mL with glacial acetic acid R. Petroleum ether; light petroleum
Periodic Acid H 5106 = 227.9 (10450-60-9) Analytical reagent grades of commerce.
Crystals, freely soluble in water and soluble in ethanol Colourless, volatile, highly flammable liquids obtained from
(96 per cent). petroleum, consisting of a mixture of the lower members of
mp: about 122 °C. the paraffin series of hydrocarbons supplied in the following
fractions:
Periodic Acid Reagent
boiling range, 30° to 40°; weight per mL, about 0.63 g,
Dissolve 0.5 g of sodium periodate in 5 mL of water, add
1 mL of 2M sulfuric acid and dilute to 10 mL with water. boiling range, 40° to 60°; weight per mL, about 0.64 g,
Prepare immediately before use. boiling range, 50° to 70°; weight per mL, about 0.66 g,
Periodic Acid Solution boiling range, 60° to 80°; weight per mL, about 0.67 g,
General reagent grade of commerce containing about boiling range, 80° to 100°; weight per mL, about 0.70 g,
50% w/v of HI0 4 ,2H2 O. boiling range, 100° to 120°; weight per mL, about 0.72 g,
Permethrin C 21 H 20 Cl2 O 3 = 391.3 (52645-53-1) boiling range, 120° to 160°; weight per mL, about 0.75 g.
mp: 34 °C to 35 °C. Petroleum Spirit (boiling range, 40° to 60°), Aromatic-
A suitable certified reference solution (10 ng/µL in free
cyclohexane) may be used. Petroleum spirit (boiling range, 40° to 60°) complying with the
Peroxide Test Strips following additional test.
Use commercial test strips with a suitable scale in the range Light absorption Absorbance against air at 235 nm, not
from O ppm to 25 ppm peroxide. more than 0.7, Appendix II B.
Peroxyacetic Acid Solution pH Indicator Strip
Dilute 1 mL of hydrogen peroxide (100 volumes) to 100 mL Paper strip, or plastic strip containing multiple segments of
with glacial acetic acid. Mix and allow to stand for 12 hours different dye-impregnated papers, allowing visual
before use. determination of pH in the prescribed range, by comparison
with the corresponding master chart.
Discard 24 hours after preparation.
2023 Appendix I A V-Al 17

a-Phellandrene (R)-5-Isopropyl-2-methyl-cyclohexa-1,3- Add 25 mL of solution A to solution B. If necessary, adjust

diene; (-)-p-Mentha-1,5-diene; C 10H 16 = 136.2 (4221-98-1) the pH of the mixture to 4. 7.
nfi°: about 1.471. Phenol Red Solution R3
bp: 171 °C to 174 °C. Solution A. Dissolve 33 mg of phenol red R in 1.5 mL of dilute
c1.-Phellandrene used in gas chromatography complies with the sodium hydroxide solution R and dilute to 50 mL with water R.
following additional test. Solution B. Dissolve 50 mg of ammonium sulfate R in 235 mL
Assay. Gas chromatography (2. 2. 28) as prescribed in the of water R; add 105 mL of dilute sodium hydroxide solution R
monograph Eucalyptus oil (0390). and 135 mL of dilute acetic acid R.
Test solution. The substance to be examined. Add 25 mL of solution A to solution B; if necessary, adjust
Content: 95.0 per cent, calculated by the normalisation the pH of the mixture to 4.7.
procedure. Phenoldisulfonic Acid Solution
Phenacetin p-Ethoxyacetanilide; C 10H 13NO 2 = 179.2 Phenoldisulphonic acid solution
(62-44-2) A clear liquid which may develop a pale brown colour on
mp: about 135°. storage, prepared either by heating 3 g of phenol with 20 mL
General reagent grade of commerce. of sulfuric acid on a water bath for 6 hours and transferring
the resulting liquid to a stoppered vessel, or by diluting a
Phenanthrene C 14H 10 = 178.2 (85-01-8)
25% w/v solution of commerce with sulfuric acid to contain
White or almost white crystals, practically insoluble in water, 15% w/v of phenol. The solution complies with the following
sparingly soluble in ethanol (96 per cent). test.
mp: about 100 °C. Sensitivity to nitrate Evaporate a solution containing
Phenanthroline Hydrochloride 1, 10-Phenanthroline 0.1 mg of potassium nitrate to dryness in a porcelain dish on a
hydrochloride monohydrate; C 12H 9 CIN 2 ,H 2 O = 234.7 water bath. To the cooled residue add 1 mL of the reagent
(18851-33-7) and allow to stand for 10 minutes. Add 10 mL of water,
White or almost white, crystalline powder, freely soluble in cool, add 10 mL of SM ammonia and dilute to 25 mL with
water, soluble in ethanol (96 per cent). water. A distinct yellow colour is produced when compared
mp: about 215 °C, with decomposition. with a solution prepared in the same manner but omitting
the potassium nitrate.
Phenazone ( 60-80-0)
Phenolphthalein 3,3-Bis (4-hydroxyphenyl)-3H-
See Phenazone (0421). isobenzofuran-1-one; C 20 H 14 O 4 = 318.3 (77-09-8)
Phenol (J 08-95-2) White or yellowish-white powder, practically insoluble in
See Phenol (0631). water, soluble in ethanol (96 per cent).
Phenol, Liquefied Phenolphthalein Paper
General reagent grade of commerce. Immerse strips of filter paper for a few minutes in
A solution of phenol in water containing about 80% w/w of phenolphthalein solution R. Allow to dry.
C6H6O. Phenolphthalein Solution
Phenol Red 4,4'-(3H-2,l-Benzoxathiol-3-ylidene) Dissolve 0 .1 g of phenolphthalein R in 80 mL of ethanol
diphenol S,S-dioxide; Phenolsulfonphthalein; (143-74-8) (96 per cent) R and dilute to 100 mL with water R.
Bright red or dark red, crystalline powder, very slightly Test for sensitivity. To 0.1 mL of the phenolphthalein solution
soluble in water, slightly soluble in ethanol (96 per cent). add 100 mL of carbon dioxide-free water R. The solution is
Phenol Red Solution colourless. Not more than 0.2 mL of 0.02 M sodium
Dissolve 0.1 g of phenol red R in a mixture of 2.82 mL of hydroxide is required to change the colour to pink.
0.1 M sodium hydroxide and 20 mL of ethanol (96 per cent) R Colour change: pH 8.2 (colourless) to pH 10.0 (red).
and dilute to 100 mL with water R. Phenolphthalein Solution Rl
Test for sensitivity. Add 0.1 mL of the phenol red solution to A 10 g/L solution of phenolphthalein R in ethanol
100 mL of carbon dioxide-free water R. The solution is yellow. (96 per cent) R.
Not more than 0.1 mL of 0.02 M sodium hydroxide is Phenolphthalein-Thymol Blue Solution
required to change the colour to reddish-violet.
Dissolve 0.1 g of thymol blue in a mixture of 2.2 mL of
Colour change: pH 6.8 (yellow) to pH 8.4 (reddish-violet). 0.lM sodium hydroxide and 50 mL of ethanol (96%) and
Phenol Red Solution Rl dilute to 100 mL with water. Mix 3 volumes of this solution
Buffered phenol red solution with 2 volumes of phenolphthalein solution.
Dissolve 33 mg of phenol red in 1.5 mL of 2M sodium Phenoxyacetic Acid 2-Phenoxyethanoic acid;
hydroxide and dilute to 100 mL with water (solution A). C 8 H 8 O 3 = 152.1 (122-59-8)
To 250 mL of 2M sodium hydroxide add 325 mL of 2M acetic Almost white crystals, sparingly soluble in water, freely
acid and 575 mL of water (solution B). Mix 25 mL of soluble in ethanol (96 per cent), and in glacial acetic acid.
solution A with 4 75 mL of solution B. mp: about 98 °C.
Phenol Red Solution R2 Chromatography. Thin-layer chromatography (2.2.27) as
Solution A. Dissolve 33 mg of phenol red R in 1.5 mL of dilute prescribed in the monograph Phenoxymethylpenicillin (0148);
sodium hydroxide solution R and dilute to 100 mL with the chromatogram shows only one principal spot.
water R. 2-Phenoxyaniline 2-Phenoxybenzenamine;
Solution B. Dissolve 25 mg of ammonium sulfate R in 235 mL 2-Aminophenyl phenyl ether; C 12 H 11 NO = 185.2
of water R; add 105 mL of dilute sodium hydroxide solution R (2688-84-8)Phenoxybenzamine Hydrochloride N-(2-
and 135 mL of dilute acetic acid R.
V-A118 Appendix I A 2023

Chloroethyl)-N-( 1-methyl-2-phenoxyethyl)-benzylamine (2-Phenylbutanoyl)urea N-Carbamoyl-2-

hydrochloride; C 18H 23 Cl 2 NO = 340.3 phenylbutanamide; 2-Phenylbutyrylurea; Pheneturide;
Content: 97.0 per cent to 103.0 per cent (dried substance). C11H 14N 2O 2 = 206.2 (90-49-3)
White or almost white, crystalline powder, sparingly soluble General reagent grade of commerce containing> 95.0% of
in water, freely soluble in ethanol (96 per cent). C11H14N2O2.
mp: about 138 °C. (E)-4-Phenylbut-3-en-2-one Benzalacetone;
C10H 10O = 146.2 (122-57-6)
Loss on drying (2.2.32): maximum 0.5 per cent, determined
by drying over diphosphorns pentoxide R at a pressure not White or pale yellow mass.
exceeding 670 Pa for 24 h. Content: minimum 98.0 per cent.
Assay. Dissolve 0.500 g in 50.0 mL of ethanol-free bp: about 261 °C.
chloroform R and extract with three quantities, each of 20 mL, mp: about 39 °C.
of 0.01 M hydrochloric acid. Discard the acid extracts, filter N-Phenylcarbazole C 18 H 13N = 243.3 (1150-62-5)
the chloroform layer through cotton and dilute 5.0 mL of the
mp: about 96°.
filtrate to 500.0 mL with ethanol-free chloroform R. Measure
the absorbance of the resulting solution in a closed cell at the General reagent grade of commerce.
maximum at 272 nm. Calculate the content of A white to pale tan, crystalline powder.
C1 8 H 23 Cl2 NO, taking the specific absorbance to be 56.3. p-Phenylenediamine Dihydrochloride
Storage: protected from light. 1,4-Diaminobenzene dihydrochloride; C 6 H 10 Cl2 N 2 = 181.1
2-Phenoxyethanol Phenoxyethanol; C 8 H 10O 2 = 138.2 (624-18-0)
(122-99-6) Crystalline powder or white or slightly coloured crystals,
Clear, colourless, oily liquid, slightly soluble in water, freely turning reddish on exposure to air, freely soluble in water,
soluble in ethanol (96 per cent). slightly soluble in ethanol (96 per cent).
dfg: about 1.11. o-Phenylglycine (2R)-2-Amino-2-phenylacetic acid;
CsH9 NO2 = 151.2 (875-74-1)
nl31: about 1.537.
CMtent: minimum 99 per cent.
Freezing point (2.2.18): minimum 12 °C.
White or almost white, crystalline powder.
Phenyl Benzoate C 13H 10 O 2 = 198.2 (93-99-2)
mp: about 70°.
DL-Phenylglycine 2-Amino-2-phenylacetic acid;
CL-Phenylglycine; C 8 H 9 NO 2 = 151.2 (2835-06-5)
General reagent grade of commerce.
Phenylhydrazine C 6H 8 N 2 = 108.1 (100-63-0)
Phenyl Isothiocyanate C 7 H 5NS = 135.2 (103-72-0)
White or almost white, crystalline powder, becoming yellow
Liquid, insoluble in water, soluble in ethanol (96 per cent).
or dark red on exposure to air, melting at room temperature
Jig: about 1.13. giving an oily liquid, miscible with anhydrous ethanol,
n~}1; about 1.65. sparingly soluble in water.
bp: about 221 °C. bp: about 244 °C, with decomposition.
mp: about -21 °C. mp: about 20 °C.
Use a grade suitable for protein sequencing. Phenylhydrazine Hydrochloride Phenylhydrazinium
Phenyl(5)methyl(95)polysiloxane chloride; C 6 H 9 ClN 2 = 144.6 (59-88-1)
Polysiloxane substituted with 5 per cent of phenyl groups White or almost white, crystalline powder, becoming brown
and 95 per cent of methyl groups. on exposure to air, soluble in water and in ethanol
(96 per cent).
Phenyl(5)methyl(95)polysiloxane, Base-deactivated
mp: about 245 °C, with decomposition.
Base-deactivated polysiloxane substituted with 5 per cent of
phenyl groups and 95 per cent of methyl groups. Storage: protected from ligl1t.
Phenyl(50)methyl(50)polysiloxane Phenylhydrazine Hydrochloride Solution Strong
phenylhydrazine hydrochloride solution
Polysiloxane substituted with 50 per cent of phenyl groups
and 50 per cent of methyl groups. Dissolve 0.9 g of phenylhydrazine hydrochloride R in 50 mL of
water R. Decolorise with activated charcoal R and filter.
Phenylacetic Acid C 8 H 8 0 2 = 136.2 (103-82-2)
To the filtrate add 30 mL of hydrochloric acid Rand dilute to
White or almost white powder, soluble in water. 250 mL with water R.
bp: about 265 °C. Phenylhydrazine-Sulfuric Acid Solution
mp: about 75 °C. Phenylhydrazine-sulphuric acid solution
L-Phenylalanine Phenylalanine; (63-91-2) Dissolve 65 mg of phenylhydrazine hydrochloride R, previously
See Phenylalanine (0782). recrystallised from ethanol (85 per cent V/V) R, in a mixture
o-Phenylbenzoic Acid 2-Biphenylcarboxylic acid; of 80 volumes of water Rand 170 volumes of sulfuric acid R
C13H10O 2 = 198 (947-84-2) and dilute to 100 mL with the same mixture of solvents.
Prepare immediately before use.
mp: about 113°.
1-Phenylpiperazine C 10H 14N 2 = 162.2 (92-54-6)
General reagent grade of commerce.
Slightly viscous, yellow liquid, not miscible with water.
2-Phenylbutanoic acid (2RS)-2-Phenylbutanoic acid
(primidone impurity E); C 10H 12 O 2 = 164.2 (90-27-7) dJ 0: about 1.07.
General reagent grade of commerce containing > 95.0% of ngi: about 1.588.
C10H12O2 3-Phenylpropanoic Acid Diliydrocinnamic acid;
C 6 H 5CH2 CH2 CO 2H = 150.17 (501-52-0)
2023 Appendix I A V-All 9

mp: 47° - 50°. Phosphomolybdotungstic Reagent Lithium and sodium

White to off-white powder or crystals. molybdotungstophosphate solution; Folin Ciocalteau phenol
Analytical reagent grade of commerce. reagent of commerce
1-Phenylpropan-2-ol (2RS)- l-Phenylpropan-2-ol; Dissolve 100 g of sodium tungstate Rand 25 g of sodium
C 9 H 12 O = 136.2 (698-87-3) molybdate R in 700 mL of water R. Add 100 mL of
hydrochloric acid R and 50 mL of phosphoric acid R. Heat the
mp: 65 °C to 67 °C.
mixture under a reflux condenser in a glass apparatus for
1-Phenyl-1,2,3,4-tetrahydroisoquinoline 10 h. Add 150 g of lithium sulfate R, 50 mL of water Rand a
C1 5H1sN = 209.3 (22990-19-8) few drops of bromine R. Boil to remove the excess of bromine
Phloroglucide 2,3 1,4,5 1,6-Biphenylpentol; (15 min), allow to cool, dilute to 1000 mL with water Rand
C12H1 0 O 5 = 234.2 (491-45-2) filter. The reagent should be yellow in colour. If it acquires a
White or almost white powder, hygroscopic, light sensitive. greenish tint, it is unsatisfactory for use but may be
Slowly discolours on exposure to light. regenerated by boiling with a few drops of bromine R. Care
Phloroglucinol Benzene-1,3,5-triol; must be taken to remove the excess of bromine by boiling.
C 6 H 6 O 3 ,2H2 O = 162.1 (6099-90-7) Storage: at 2 °C to 8 °C.
White or yellowish crystals, slightly soluble in water, soluble Phosphomolybdotungstic Reagent, Dilute
in ethanol (96 per cent). To I volume of phosphomolybdotungstic reagent R add
mp: about 223 °C (instantaneous method). 2 volumes of water R.
Phloroglucinol Solution Phosphoric Acid, Dilute See Dilute phosphoric
To 1 mL of a 100 g/L solution of phloroglucinol R in ethanol acid (0005).
(96 per cent) R, add 9 mL of hydrochloric acid R. Phosphoric Acid, Dilute Rl
Storage: protected from light. Dilute 93 mL of dilute phosphoric acid R to 1000 mL with
Phosalone C 12H 15 CINO 4 PS 2 = 367.8 (2310-17-0) water R.
mp: 45 °C to 48 °C Phosphorus Pentoxide Diphosphorus pentoxide;
P2Os = 141.9 (1314-56-3)
A suitable certified reference solution ( 10 ng/µL in iso-
octane) may be used. White or almost white powder, amorphous, deliquescent.
It is hydrated by water with the evolution of heat.
Phospholipid
Storage: in an airtight container.
Wash a quantity of human or bovine brain freed from
meninges and blood vessels and macerate in a suitable Phosphotungstic Acid Solution
blender. Weigh 1000 to 1300 g of the macerate and measure Heat under a reflux condenser for 3 h, 10 g of sodium
its volume (V mL). Extract with three quantities, each of tungstate R with 8 mL of phosphoric acid R and 7 5 mL of
4 V mL, of acetone, filter by suction and dry the precipitate at water R. Allow to cool and dilute to I 00 mL with water R.
37° for 18 hours. Extract the dried precipitate with two Phthalaldehyde Benzene-1,2-dicarboxaldehyde;
quantities, each of 2 V mL, of a mixture of 2 volumes of C 8 H 6 O2 = 134.1 (643-79-8)
petroleum spirit (boiling range, 30° to 4(!') and 3 volumes of Yellow, crystalline powder.
petroleum spirit (boiling range, 40° to 60°), filtering each extract mp: about 55 °C.
through a filter paper previously washed with the petroleum
spirit mixture. Combine the extracts and evaporate to dryness Storage: protected from light and air.
at 45° at a pressure not exceeding 0.7 kPa. Dissolve the Phthalaldehyde Reagent
residue in O.2 V mL of ether and allow to stand at 4 ° until a Dissolve 2.47 g of boric acid R in 75 mL of water R, adjust to
deposit is produced. Centrifuge and evaporate the clear pH 10.4 using a 450 g/L solution of potassium hydroxide R
supernatant liquid under reduced pressure until the volume is and dilute to I 00 mL with water R. Dissolve 1.0 g of
about 100 mL per kg of the original macerate. Allow to phthalaldehyde R in 5 mL of methanol R, add 95 mL of the
stand at 4° until a precipitate is produced (12 to 24 hours) boric acid solution and 2 mL of thioglycollic acid R and adjust
and centrifuge. To the clear supernatant liquid add to pH 10.4 with a 450 g/L solution of potassium hydroxide R.
5 volumes of acetone, centrifuge, discard the supernatant Storage: protected from light; use within 3 days.
liquid, dry the precipitate and store protected from light in a
Phthalazine C 8 H 6N 2 = 130.1 (253-52-1)
vacuum desiccator.
Pale yellow crystals, freely soluble in water, soluble in
Phosphomolybdic Acid Dodecamolybdophosphoric acid;
anhydrous ethanol, in ethyl acetate and in methanol.
12MoO 3,H3 PO 4 ,xH 2 O (51429-74-4)
mp: 89 °C to 92 °C.
Orange-yellow, fine crystals, freely soluble in water, soluble in
ethanol (96 per cent). Phthalein Purple Metalphthalein; 2,2',2 11 ,2 111 -[o-
Cresolphthalein-3 ',3 11 -bis(methylenenitrilo) ]tetra-acetic acid;
Phosphomolybdic Acid Solution
( 1,3-Dihydro-3-oxo-isobenzofuran- l-ylidene) bis [(6-hydroxy-
Dissolve 4 g of phosphomolybdic acid R in water R and dilute 5-methyl-3, 1-phenylene)bis(methyleneimino)diacetic acid];
to 40 mL with the same solvent. Add cautiously and with C32H32N2O12,xH2O = 637, anhydrous substance
cooling 60 mL of sulfuric acid R. Prepare immediately before (2411-89-4)
use. Yellowish-white or brownish powder, practically insoluble in
Phosphomolybdic Acid Solution, Ethanolic water, soluble in ethanol (96 per cent). The product may be
A 20% w/v solution of phosphomolybdic acid in ethanol (96%). found in commerce in the form of the sodium salt: a
Prepare immediately before use. yellowish-white to pink powder, soluble in water, practically
insoluble in ethanol (96 per cent).
V-A120 Appendix I A 2023

Test for sensitivity. Dissolve 10 mg in 1 mL of concentrated Picrotoxinin (1R,3R,5S,8S,9R,12S,13R,14R)-1-hydroxy-

ammonia Rand dilute to 100 mL with water R. To 5 mL of 13-methyl-14-(prop-1-en-2-yl)-4, 7, 10-trioxapentacyclo
the solution add 95 mL of water R, 4 mL of concentrated [6.4. l. l 9' 12 .03 ' 5 .0 5 ' 13]tetradecane-6,11-dione;
ammonia R, 50 mL of ethanol (96 per cent) Rand 0.1 mL of C1 5H16O6 = 292.2 (17617-45-7)
0.1 M barium chloride. The solution is blue-violet. White or colourless crystalline powder or crystals, soluble in
Add 0.15 mL of 0.1 M sodium edetate. The solution becomes methylene chloride, in ethanol (96 per cent) and in alkaline
colourless. solutions.
Phthalic Acid Benzene-1,2-dicarboxylic acid; mp: 207 to 210 °C.
CsH6O4 = 166.1 (88-99-3) a-Pinene (1R,5R)-2,6,6-Trimethylbicyclo[3. l .1 ]hept-2-
White or almost white, crystalline powder, soluble in hot ene; C1oH1 6 = 136.2 (7785-70-8)
water and in ethanol (96 per cent). Liquid not miscible with water.
Phthalic Anhydride Isobenzofuran-1,3-dione; dig: about 0.859.
CsH 4 O 3 = 148.1 (85-44-9)
Content: minimum 99.0 per cent.
nt0 : about 1.466.
bp: 154 °C to 156 °C.
White or almost white flakes.
ct.-Pinene used in gas chromatography complies with the following
mp: 130 °C to 132 °C. additional test.
Assay. Dissolve 2.000 gin 100 mL of water Rand boil under Assay. Gas chromatography (2.2.28) as prescribed in the
a reflux condenser for 30 min. Cool and titrate with 1 M monograph Bitter-orange-flower oil (1175).
sodium hydroxide, using phenolphthalein solutwn R as indicator.
Test solution. The substance to be examined.
1 mL of 1 M sodium hydroxide is equivalent to 74.05 mg of
Content: minimum 99.0 per cent, calculated by the
CsH4O3.
normalisation procedure.
Phthalic Anhydride Solution
~-Pinene 6,6-Dimethyl-2-methylenebicyclo [3.1.1 ]heptane;
Dissolve 42 g of phthalic anhydride R in 300 mL of anhydrous
C10H16 = 136.2 (127-91-3)
pyridine R. Allow to stand for 16 h.
Colourless, oily liquid, odour reminiscent of turpentine,
Storage: protected from light; use within 1 week.
practically insoluble in water, miscible with ethanol
Picein 1-[4-(~-o-Glucopyranosyloxy)phenyl] ethanone; (96 per cent).
p-(Acetylphenyl)-~-D-glucopyranoside; C 14 H 18 O 7 = 298.3 /3-Pinene used in gas chromatography complies with the following
(530-14-3)
additional test.
mp: 194 °C to 195 °C. Assay. Gas chromatography (2.2.28) as prescribed in the
Picric Acid 2,4,6-Trinitrophenol; C 6 H 3N 3 O 7 = 229.1 monograph Bitter-orange-flower oil (1175).
(88-89-1)
Test solution. The substance to be examined.
Yellow prisms or plates, soluble in water and in ethanol Content: minimum 95.0 per cent.
(96 per cent).
1,4-Piperazinediethanesulfonic Acid Piperazine-1,4-
Storage: moistened with water R. bis (2-ethanesulfonic acid); 2,2 1-(Piperazine-1,4-diyl)
Picric Acid Solution bis(ethanesulfonic acid); Piperazine-N,N'-bis(2-
A 10 g/L solution of picric acid R. ethanesulfonic acid); PIPES; C 8 H 18N 2O 6 S 2 = 302.4
Picric Acid Solution Rl (5625-37-6)
Prepare 100 mL of a saturated solution of picric acid R and Content: minimum 99 per cent.
add 0.25 mL of strong sodium hydroxide solutwn R. White, crystalline powder.
Picrolonic Acid 3-Methyl-4-nitro-1-( 4-nitrophenyl)-5- Piperazine Dipicrate Solution
pyrazolone; C 10H 8 N 4 O 5 = 264.2 (550-74-3) Dissolve 0.2 g of piperazine hydrate in 3.5 mL of 0.5M sulfuric
mp: about 116°. acid and 10 mL of water. Add 100 mL of picric acid
General reagent grade of commerce. solution Rl, heat on a water bath for 15 minutes, cool and
Complies with the following test. filter. Wash the precipitate with water until the washings are
free from sulfate. Shake the precipitate with water to produce
Sensitivity Dissolve 25 mg in 10 mL of warm water a saturated solution and filter.
containing 0 .1 mL of glacial acetic acid; to 1 mL of this
solution add 1 mL of a 0.05% w/v solution of calcium chloride Piperazine Hydrate (142-63-2)
previously heated to 60°. A bulky precipitate is produced See Piperazine hydrate (0425).
within 5 minutes. Piperidine Hexahydropyridine; C 5 H 11 N = 85.2 (110-89-4)
A yellow or brownish yellow, crystalline powder. Colourless to slightly yellow, alkaline liquid, miscible with
Picrotin ( 1R,3R, 5S,8S, 9R, 12S, 13R, 14S)-1-hydroxy-14-(2- water, with ethanol (96 per cent) and with light petroleum.
hydroxypropan-2-yl)-13-methyl-4, 7, 10-trioxapentacyclo bp: about 106 °C.
[6.4.1.1. 9 ' 12 .0 3 ' 5 .0 5 ' 13]tetradecane-6,11-dione; Piperine (2E,4E)-l-(Piperidin-1-yl)-5-( 1,3-benzodioxol-5-
C1 5H 18 O 7 = 310.3 (21416-53-5) yl)penta-2,4-dien-1-one; 1-Piperoyl-piperidine; 1-[ (2E,4E)-
White or colourless crystalline powder or crystals, soluble in 5-(3,4-Methylenedioxyphenyl)-1-oxo-2 ,4-pentadienyl]
boiling water and in ethanol (96 per cent), practically piperidine; C 17 H 19 NO 3 = 285.3 (94-62-2)
insoluble in methylene chloride. Piperitone 6-lsopropyl-3-methyl-cyclohex-2-en-1-one;
mp: 248 °C to 250 °C. C10H15O = 152.2 (89-81-6)
Pirimiphos-ethyl C 13 H 24N 3 O 3 PS = 333.4 (23505-41-1)
mp: 15 °C to 18 °C.
 2023 Appendix I A V-A121

A suitable certified reference solution (10 ng/µL in the flasks with the exception of any that show obvious
cyclohexane) may be used. haemolysis or clots and keep the pooled blood at 10-15 °C.
Plasma, Citrated Rabbit As soon as possible and within 4 h of collection, centrifuge
Collect blood by intracardiac puncture from a rabbit that has the pooled blood at 1000-2000 g at 10-15 °C for 30 min.
been fasted for 12 hours prior to the collection, using a Separate the supernatant and centrifuge it at 5000 g for
plastic syringe with a No. 1 needle containing a suitable 30 min. (Faster centrifugation, for example 20 000 g for
volume of a 3.8% w/v solution of sodium citrate so that the 30 min, may be used if necessary to clarify the plasma, but
final volume ratio of citrate solution to blood is 1:9. Separate filtration procedures should not be used.) Separate the
the plasma by centrifugation at 1500 to 1800 g at 15° to 20° supernatant and, without delay, mix thoroughly and
for 30 minutes. distribute the plasma substrate into small stoppered
Store at 0° to 6° and use within 4 hours of collection. containers in portions sufficient for a complete heparin assay
(for example 10 mL to 30 mL). Without delay, rapidly cool
Plasma, Platelet-poor to a temperature below -70 °C (for example by immersing
Withdraw 45 mL of human blood into a 50 mL plastic the containers into liquid nitrogen) and store at a
syringe containing 5 mL of a sterile 38 g/L solution of sodium temperature below -30 °C.
citrate R. Without delay, centrifuge at 1500 g at 4 °C for The plasma is suitable for use as plasma substrate in the
30 min. Remove the upper two-thirds of the supernatant assay for heparin if, under the conditions of the assay, it gives
plasma using a plastic syringe and without delay centrifuge at a clotting time appropriate to the method of detection used
3500 g at 4 °C for 30 min. Remove the upper two-thirds of and if it provides reproducible, steep log dose-response
the liquid and freeze it rapidly in suitable amounts in plastic
curves.
tubes at or below -40 °C. Use plastic or silicone-treated
equipment. When required for use, thaw a portion of the plasma
substrate in a water-bath at 37 °C, gently swirling until
Plasma Substrate Substrate plasma thawing is complete; once thawed it should be kept at
Separate the plasma from human or bovine blood collected 10-20 °C and used without delay. The thawed plasma
into one-ninth its volume of a 38 g/L solution of sodium substrate may be lightly centrifuged if necessary; filtration
citrate R, or into two-sevenths its volume of a solution procedures should not be used.
containing 20 g/L of disodium hydrogen citrate Rand 25 g/L of Plasma Substrate R2
glucose R. With the former, prepare the substrate on the day
of collection of the blood. With the latter, prepare within Prepare from human blood containing less than 1 per cent of
two days of collection of the blood. the normal amount of factor IX. Collect the blood into one-
ninth its volume of a 38 g/L solution of sodium citrate R.
Storage: at -20 °C.
Storage: in small amounts in plastic tubes at a temperature of
Plasma Substrate Deficient in Factor V -30 °C or lower.
Use preferably a plasma which is congenitally deficient, or Plasma Substrate R3
prepare it as follows: separate the plasma from human blood
collected into one tenth of its volume of a 13.4 g/L solution Prepare from human blood containing less than 1 per cent of
of sodium oxalate R. Incubate at 37 °C for 24 h to 36 h. the normal amount of factor XI. Collect the blood into one-
The coagulation time determined by the method prescribed ninth its volume of a 38 g/L solution of sodium citrate R.
for coagulation factor V solution R should be 70 s to I 00 s. Storage: in small amounts in plastic tubes at a temperature of
If the coagulation time is less than 70 s, incubate again for -30 °C or lower.
12 h to 24 h. Plasminogen, Human (9001-91-6')
Storage: in small quantities at a temperature of -20 °C or A substance present in blood that may be activated to
lower. plasmin, an enzyme that lyses fibrin in blood clots.
Factor VII-deficient Plasma Platelet Substitute
Plasma that is deficient in factor VII. To 0.5 to 1 g of phospholipid add 20 mL of acetone and allow
Plasma Substrate Rl Substrate plasma Rl to stand for 2 hours with frequent shaking. Centrifuge for
Use water-repellent equipment (made from materials such as 2 minutes and discard the supernatant liquid. Dry the
suitable plastics or suitably silicone-treated glass) for taking and residue using a water pump, mix with 20 mL of chloroform
handling Mood. and shake for 2 hours. Filter under vacuum and suspend the
residue obtained in 5 to 10 mL of saline solution.
Collect a suitable volume of blood from each of at least five
sheep; a 285 mL volume of blood collected into 15 mL of Prepare a dilution in saline solution so that it will give clotting
anticoagulant solution is suitable but smaller volumes may be time differences between consecutive dilutions of the
collected, taking the blood, either from a live animal or at the reference preparation used in the Assay of factor IX fraction
time of slaughter, using a needle attached to a suitable of about 10 seconds.
cannula which is long enough to reach the bottom of the Store the dilute suspensions at -30° and use within 6 weeks.
collecting vessel. Discarding the first few millilitres and Plutonium-242 Spiking Solution
collecting only free-flowing blood, collect the blood in a Contains 50 Bq/L 242 Pu and a 134 mg/L solution of
sufficient quantity of an anticoagulant solution containing lanthanum chloride heptahydrate R in a 284 g/L solution of
8.7 g of sodium citrate Rand 4 mg of aprotinin R per 100 mL nitric acid R.
of water R to give a final ratio of blood to anticoagulant
Poloxamer 124
solution of 19 to 1. During and immediately after collection,
swirl the flask gently to ensure mixing but do not allow General reagent grade of commerce.
frothing to occur. When collection is complete, close the flask
and cool to 10-15 'C. When cold, pool the contents of all
V-A122 Appendix I A 2023

Poloxamer 188 See Poloxamers (1464). Polyoxyethylene 10 Lauryl Ether Decaethylene glycol
Polyamine Grafted Poly(vinyl alcohol) Copolymer monododecyl ether; C 3 2H 66 O 11 = 626.9 (9002-92-0)
Copolymer beads of poly(vinyl alcohol) to which polyamine bp: about 100°
is covalently bonded; The size range of the beads is specified mp: about 24°
after the name of the reagent in the tests where it is used; General reagent grade of commerce.
Polydatin 3-Hydroxy-5-[2-( 4-hydroxyphenyl)eth-1-en-1-yl]
phenyl ~-D-g]ucopyranoside; Resveratrol-3-~-mono-D- Polyoxyethylene 23 Lauryl Ether Brij 35;
glucoside; C 20 H 22 O 8 = 390.4 (65914-17-2) CssH120O24 = 1199.6 (9002-92-0)
Polyether Hydroxylated Gel for Chromatography bp: about 100°.
Gel with a small particle size having a hydrophilic surface mp: about 43°.
with hydroxyl groups. It has an exclusion limit for dextran of General reagent grade of commerce.
relative molecular mass 2 x 105 to 2.5 x 106 . Polysorbate 20 (9005-64-5)
Polyethylene Glycol 200 Macrogol 200; (25322-68-3) See Polysorbate 20 (0426).
Clear, colourless or almost colourless viscous liquid, very Polysorbate 65 (9005-71-4)
soluble in acetone and in anhydrous ethanol, practically Polysorbate 80 (9005-65-6)
insoluble in fatty oils. See Polysorbate 80 (0428).
dig: about 1.127. Polystyrene 900-1000 (9003-53-6)
n~: about 1.450.
Organic standard used for calibration in gas chromatography.
Polyethylene Glycol 200 Rl Macrogol 200 Rl
Mw: about 950.
Introduce 500 mL of macrogol 200 R into a 1000 mL round
MJM,,: 1.10.
bottom flask. Using a rotation evaporator remove any volatile
components applying for 6 h a temperature of 60 °C and a Potassium Acetate (127-08-2)
vacuum with a pressure of 1.5-2.5 kPa. See Potassium acetate (1139).
Polyethylene Glycol 300 Macrogol 300; (25322-68-3) Potassium Antimonate(v) Potassium pyroantimonate;
See Macrogols (1444). KSb(OH) 6 = 262.9 (12208-13-8)
Polyethylene Glycol 400 Macrogol 400; (25322-68-3) White or almost white, crystals or crystalline powder,
sparingly soluble in water.
See Macrogols (1444).
Potassium Antimonate(v) Solution Potassium
Polyethylene Glycol 1000 Macrogol 1000; (25322-68-3)
pyroantimonate solution
See Macrogols (1444).
Dissolve 2 g of potassium pyroantimonate R in 95 mL of hot
Polyethylene Glycol 1500 Macrogol 1500; (25322-68-3) water R. Cool quickly and add a solution containing 2.5 g of
See Macrogols (1444). potassium hydroxide R in 50 mL of water R and 1 mL of duute
Polyethylene Glycol 20,000 Macrogol 20 000 sodium hydroxide solution R. Allow to stand for 24 h, filter and
See Macrogols (1444). dilute to 150 mL with water R.
Polyethylene Glycol Ad.ipate Macrogol adipate; Potassium Bicarbonate (298-14-6)
Polyethyleneglycol adipate; (CsH12O4)n = Mr (l 72.2)n See Potassium hydrogen carbonate R.
White or almost white, wax-like mass, practically insoluble in Potassium Bicarbonate Solution, Saturated
water. Methanolic See potassium hydrogen carbonate solution,
mp: about 43 °C. saturated methanolic R.

Polyethylene Glycol Succinate Macrogol succinate; Potassium Borohydride Potassium tetrahydroborate;

Polyethyleneg]ycol succinate; (C6HsO4)n = Mr (144.l)n KBH 4 = 53.94 (13762-51-1)
White or almost white, crystalline powder, practically General reagent grade of commerce.
insoluble in water. Potassium Bromate KBrO 3 = 167.0 (7758-01-2)
mp: about 102 °C. White or almost white granular powder or crystals, soluble in
Polymethacrylate Gel water, slightly soluble in ethanol (96 per cent).
A methacrylate-based size-exclusion stationary phase for Potassium Bromide (7758-02-3)
water-soluble samples. See Potassium bromide (0184).
Butylated Polymethacrylate Gel Potassium bromide used for infrared absorption spectrophotometry
Gel based on butylated methacrylic acid polymer. (2. 2. 24) also complies with the following additional test.
Polymethacrylate Gel, Hydroxylated A disc 2 mm thick prepared from the substance previously
dried at 250 °C for 1 h, has a substantially flat baseline over
Stationary phase for size-exclusion chromatography. the range 4000 cm -I to 620 cm-l. It exhibits no maxima
Gel based on hydroxylated methacrylic acid polymer. with absorbance greater than 0.02 above the baseline, except
Polyorganosiloxane for Oxygen-Containing maxima for water at 3440 cm- 1 and 1630 cm- 1.
Compounds Potassium Carbonate Anhydrous potassium carbonate;
Combination of suitable polyorganosiloxanes with high K 2 CO 3 = 138.2 (584-08-7)
affinity for oxygen-containing compounds. White or almost white, granular powder, hygroscopic, very
Polyoxyethylated Castor Oil soluble in water, practically insoluble in anhydrous ethanol.
Light yellow liquid. It becomes clear above 26 °C. Storage: in an airtight container.
Potassium Carbonate Sesquihydrate
K 2 CO 3 ,l½H 2 O = 165.2 (6381-79-9)
2023 Appendix I A V-A123

General reagent grade of commerce. Potassium Dichromate Solution

Potassium Chlorate KC1O 3 = 122.6 (3811-04-9) A 106 g/L solution of potassium dichromate R.
A white or almost white powder, granules or crystals, soluble Potassium Dichromate Solution, Dilute
in water. A 7 .0% w/v solution of potassium dichromate.
Potassium Chloride (7447-40-7) Potassium Dichromate Solution Rl
See Potassium chloride (0185). A 5 g/L solution of potassium dichromate R.
Potassium chloride used for infra red absorption spectrophotometry Potassium Dihydrogen Citrate C 6 H 7 KO 7 = 230.2
(2. 2. 24) also complies with the fo/1,owing additional test.
General reagent grade of commerce.
A disc 2 mm thick, prepared from the substance previously Potassium Dihydrogen Orthophosphate Potassium
dried at 250 °C for 1 h, has a substantially flat baseline over dihydrogen phosphate; (7778-77-0)
the range 4000 cm- 1 to 620 cm-I. It exhibits no maxima
with absorbance greater than 0.02 above the baseline, except See Potassium dihydrogen phosphate (09 20).
maxima for water at 3440 cm- 1 and 1630 cm- 1• Potassium Dihydrogen Phosphate, 0.2M
Potassium Chloride, O.lM A solution of potassium dihydrogen phosphate R containing the
A solution of potassium chloride R containing the equivalent of equivalent of 27.22 g of KH2 PO 4 in 1000.0 mL.
7.45 g ofKCl in 1000.0 mL. Potassium Ferriperiodate Solution
Potassium Chromate Dipotassium chromate; Dissolve 1 g of potassium periodate R in 5 mL of a freshly
K 2 CrO 4 = 194.2 (7789-00-6) prepared 120 g/L solution of potassium hydroxide R.
Yellow crystals, freely soluble in water. Add 20 mL of water Rand 1.5 mL of ferric chloride
solution Rl. Dilute to 50 mL with a freshly prepared 120 g/L
Potassium Chromate Solution solution of potassium hydroxide R.
A 50 g/L solution of potassium chromate R. Potassium Fluoride KF = 58.1 (7789-23-3)
Potassium Citrate (6100-05-6) Colourless crystals or white or almost white crystalline
See Potassium citrate (0400). powder, deliquescent, soluble in water, practically insoluble
Potassium Cyanide KCN = 65.1 (151-50-8) in ethanol (96 per cent).
White or almost white, crystalline powder or white or almost Potassium Hexacyanoferrate(n) Potassium ferrocyanide;
white mass or granules, freely soluble in water, slightly ~[Fe(CN) 6],3H2 O = 422.4 (14459-95-1)
soluble in ethanol (96 per cent). Transparent yellow crystals, freely soluble in water,
Potassium Cyanide Solution practically insoluble in ethanol (96 per cent).
A 100 g/L solution of potassium cyanide R. Potassium Hexacyanoferrate(m) Potassium ferricyanide;
Potassium Cyanide Solution, Lead-free K 3 [Fe(CN) 6 ] = 329.3 (13746-66-2)
Dissolve 10 g of potassium cyanide R in 90 mL of water R, Red crystals, freely soluble in water.
add 2 mL of strong hydrogen peroxide solution R diluted 1 to 5. Potassium Hexacyanoferrate(n) Solution Potassium
Allow to stand for 24 h, dilute to 100 mL with water R and ferrocyanide solution
filter. A 53 g/L solution of potassium ferrocyanide R.
The solution complies with the following test: take 10 mL of Potassium Hexacyanoferrate(m) Solution Potassium
the solution, add 10 mL of water Rand 10 mL of hydrogen ferricyanide solution
sulfide solution R. No colour is evolved even after addition of Wash 5 g of potassium ferricyanide R with a little water R,
5 mL of dilute hydrochloric acid R.
dissolve and dilute to 100 mL with water R. Prepare
Potassium Cyanide Solution PbT immediately before use.
Dissolve 10 g of potassium cyanide in 90 mL of water, add Potassium Hexacyanoferrate(m) Solution, Dilute
2 mL of hydrogen peroxide solution (20 voO, allow to stand for
Wash about 1 g of potassium hexacyanoferrate(m) crystals with
24 hours, dilute to 100 mL with water and filter. a little water and dissolve the washed crystals in 100 mL of
Potassium Dichromate Dipotassium dichromate; water.
K 2 Cr2 O 7 = 294.2 (7778-50-9)
Produces a blue colour with solutions of iron(n) salts.
Potassium dichromate used for the calibration of Prepare immediately before use.
spectrophotometers (2.2.25) contains not less than
99. 9 per cent of K2 Cr2 O 7 , calculated with reference to the Potassium Hyaluronate (31799-91-4)
substance dried at 130 °C. General reagent grade of commerce obtained from human
Orange-red crystals, soluble in water, practically insoluble in umbilical cords and freeze dried.
ethanol (96 per cent). Protein, not more than 2%; chondroitin sulfate, not more
Assay. Dissolve 1.000 g in water R and dilute to 250.0 mL than 3%.
with the same solvent. To 50.0 mL of this solution add a Potassium Hyaluronate Stock Solution
freshly prepared solution of 4 g of potassium iodide R, 2 g of A 0.05% w/v solution of potassium hyaluronate.
sodium hydrogen carbonate R and 6 mL of hydrochloric acid R Store below 0° and use within 30 days.
in 100 mL of water R in a 500 mL flask. Stopper the flask
Potassium Hydrogen Carbonate Potassium bicarbonate;
and allow to stand protected from light for 5 min. Titrate KHC0 3 = 100.1 (298-14-6)
with 0.1 M sodium thiosulfate, using 1 mL of iodide-free starch
solution R as indicator. Transparent, colourless crystals, freely soluble in water,
practically insoluble in ethanol (96 per cent).
1 mL of 0.1 M sodium thiosulfate is equivalent to 4.903 mg of
K2Cr2O1,
V-A124 Appendix I A 2023

Potassium Hydrogen Carbonate Solution, Saturated Potassium Iodide Solution

Methanolic A 166 g/L solution of potassium iodide R.
Dissolve 0.1 g of potassium hydrogen carbonate R in 0.4 mL of Potassium Iodide Solution, Dilute
water R, heating on water-bath. Add 25 mL of methanol R A 10% w/v solution of potassium iodide.
and swirl, keeping the solution on the water-bath until
dissolution is complete. Use a freshly prepared solution.
Potassium Iodide Solution, lodinated
Dissolve 2 g of iodine R and 4 g of potassium iodide R in
Potassium Hydrogen Phthalate Potassium hydrogen
10 mL of water R. When solution is complete dilute to
benzene-1,2-dicarboxylate; C 8 H 5K0 4 = 204.2 (877-24-7)
100 mL with water R.
White or almost white crystals, soluble in water, slightly
soluble in ethanol (96 per cent).
Potassium Iodide Solution, lodinated Rl
Dissolve 500 mg of iodine R and 1.5 g of potassium iodide R
Potassium Hydrogen Phthalate, 0.2M
in water Rand dilute to 25 mL with the same solvent.
A solution of potassium hydrogen phthalate R containing the
equivalent of 40.84 g of C 8 H 5K0 4 in 1000.0 mL. Potassium Iodide Solution, Saturated
A saturated solution of potassium iodide R in carbon dioxide-free
Potassium Hydrogen Sulfate Potassium bisulfate;
water R. Make sure the solution remains saturated as
Potassium bisulphate; Potassium hydrogen sulphate;
indicated by the presence of undissolved crystals.
KHS0 4 = 136.2 (7646-93-7)
Test by adding to 0.5 mL of the saturated potassium iodide
Colourless, transparent, hygroscopic crystals, freely soluble in
solution 30 mL of a mixture of 2 volumes of chloroform R
water giving a strongly acid solution.
and 3 volumes of glacial acetic acid R, as well as 0.1 mL of
Storage: in an airtight container. starch solution R. Any blue colour formed should be
Potassium Hydrogen (+)-Tartrate Potassium hydrogen discharged by the addition of 0.05 mL of 0.1 M sodium
tartrate; C 4 H 5K0 6 = 188.2 (868-14-4) thiosulfate.
White or almost white, crystalline powder or colourless, Storage: protected from light.
slightly opaque crystals, slightly soluble in water, soluble in
Potassium lodobismuthate Solution
boiling water, practically insoluble in ethanol (96 per cent).
To 0.85 g of bismuth subnitrate R add 40 mL of water R,
Potassium Hydroxide (1310-58-3) 10 mL of glacial acetic acid R and 20 mL of a 400 g/L
See Potassium hydroxide (0840). solution of potassium iodide R.
Potassium Hydroxide, 2M Alcoholic Potassium Iodobismuthate Solution, Acid
Dissolve 12 g of potassium hydroxide R in 10 mL of water R Dissolve 1. 7 g of bismuth oxynitrate in a mixture of 80 mL of
and dilute to 100 mL with ethanol (96 per cent) R. water and 20 mL of glacial acetic acid, warming if necessary,
Potassium Hydroxide, Ethanolic cool, add 100 mL of a 50% w/v solution of potassium iodide
Solutions of the requisite molarity may be obtained by and mix. Dilute 10 mL to 100 mL with water, add 10 mL of
dissolving the appropriate amount of potassium hydroxide in glacial acetic acid, mix, add 0.12 g of iodine and shake until
sufficient ethanol (96%) to produce 1000 mL. the iodine has completely dissolved.
Potassium Hydroxide in Alcohol (10% v/v), 0.5M Store at a temperature of 2° to 8° and use within 2 weeks.
Dissolve 28 g of potassium hydroxide R in 100 mL of ethanol Potassium Iodobismuthate Solution, Dilute
(96 per cent) R and dilute to 1000 mL with water R. Dissolve 100 g of tartaric acid R in 500 mL of water R and
Potassium Hydroxide, Methanolic add 50 mL of potassium iodobismuthate solution Rl.
Solutions of the requisite molarity may be obtained by Storage: protected from light.
dissolving the appropriate amount of potassium hydroxide in Potassium Iodobismuthate Solution Rl
sufficient methanol to produce 1000 mL. Dissolve 100 g of tartaric acid R in 400 mL of water R and
Potassium Hydroxide Solution, Alcoholic add 8.5 g of bismuth subnitrate R. Shake for 1 h, add 200 mL
Dissolve 3 g of potassium hydroxide R in 5 mL of water R and of a 400 g/L solution of potassium iodide R and shake well.
dilute to 100 mL with aldehyde-free alcohol R. Decant the Allow to stand for 24 h and filter.
clear solution. The solution should be almost colourless. Storage: protected from light.
Potassium Hydroxide Solution Rl, Alcoholic Potassium Iodobismuthate Solution R2
Dissolve 6.6 g of potassium hydroxide R in 50 mL of water R Stock solution. Suspend 1. 7 g of bismuth subnitrate R and 20 g
and dilute to 1000 mL with anhydrous ethanol R. of tartaric acid R in 40 mL of water R. To the suspension add
Potassium Iodate KI0 3 = 214.0 (7758-05-6) 40 mL of a 400 g/L solution of potassium iodide R and stir for
1 h. Filter. The solution may be kept for several days in
White or almost white, crystalline powder, soluble in water.
brown bottles.
Potassium Iodide (7681-11-0)
Spray solution. Mix immediately before use 5 mL of the stock
See Potassium iodide (0186). solution with 15 mL of water R.
Potassium Iodide and Starch Solution Potassium lodobismuthate Solution R3
Dissolve 0. 7 5 g of potassium iodide R in 100 mL of water R. Dissolve 0.17 g of bismuth subnitrate R in a mixture of 2 mL
Heat to boiling and add whilst stirring a solution of 0.5 g of of glacial acetic acid R and 18 mL of water R. Add 4 g of
soluble starch R in 35 mL of water R. Boil for 2 min and allow potassium iodide R, 1 g of iodine R and dilute to 100 mL with
to cool. di7ute sulfuric acid R.
Test for sensitivity. A mixture of 15 mL of the potassium Potassium Iodobismuthate Solution R4
iodide and starch solution, 0.05 mL of glacial acetic acid R
Dissolve 1.7 g of bismuth subnitrate R in 20 mL of glacial
and 0.3 mL of iodine solution R2 is blue.
acetic acid R. Add 80 mL of distilled water R, 100 mL of a
400 g/L solution of potassium iodide R, 200 mL of glacial
2023 Appendix I A V-A125

acetic acid R and dilute to 1000 mL with disu7led water R. Potassium Sodium (+)-Tartrate Sodium potassium(+)-
Mix 2 volumes of this solution with 1 volume of a 200 g/L tartrate; Sodium potassium tartrate;
solution of barium chloride R. C 4 H 4 KNaO 6 ,4H2O = 282.2 (6381-59-5)
Potassium Iodobismuthate Solution R5 Colourless, prismatic crystals, very soluble in water.
To 0.85 g of bismuth subnitrate R add 10 mL of glacial acetic Potassium Sorbate Sorbic acid potassium salt;
acid R and gently heat until completely dissolved. C 6 H 7 KO 2 = 150.2 (110-44-1)
Add 40 mL of water Rand allow to cool. To 5 mL of this General reagent grade of commerce.
solution, add 5 mL of a 400 g!L solution of potassium
Potassium 4-sulfobenzoate 4-Sulfobenzoic acid
iodide R, 20 mL of glacial acetic acid R and 70 mL of water R.
potassium salt; Potassium 4-carboxybenzenesulfonate;
Potassium Iodoplatinate Solution C1HsKO 5 S = 240.3 (5399-63-3)
Add 5 mL of a 5% w/v solution of chloroplatinic(IV) acid to White, crystalline powder.
45 mL of dilute potassium iodide solution and dilute to 100 mL
Potassium Tetraiodomercurate Solution
with water.
Dissolve 1.35 g of mercuric chloride R in 50 mL of water R.
Store in an amber glass container.
Add 5 g of potassium iodide R and dilute to 100 mL with
Potassium Mercuri-iodide Solution, Alkaline water R.
To 3.5 g of potassium iodide add 1.25 g of mercury(II) chloride Potassium Tetraiodomercurate Solution, Alkaline
dissolved in 80 mL of water and a cold, saturated solution of
Dissolve 11 g of potassium iodide R and 15 g of mercuric
mercury(II) chloride in water, stirring continuously, until a
iodide R in water R and dilute to 100 mL with the same
slight red precipitate remains. Dissolve 12 g of sodium
solvent. Immediately before use, mix 1 volume of this
hydroxide in the resulting solution and add a little more of the
solution with an equal volume of a 250 g!L solution of
saturated solution of mercury(n) chloride and sufficient water
sodium hydroxide R.
to produce 100 mL. Allow to stand and decant the clear,
supernatant liquid. Potassium Tetroxalate Potassium trihydrogen dioxalate;
C 4 H¥O 8 ,2H2O = 254.2 (6100-20-5)
Potassium Nitrate KNO 3 = 101.1 (7757-79-1)
White or almost white, crystalline powder, sparingly soluble
Colourless crystals, very soluble in water.
in water, soluble in boiling water, slightly soluble in ethanol
Potassium Periodate Potassium metaperiodate; (96 per cent).
KIO 4 = 230.0 (7790-21-8)
Potassium Thiocyanate KSCN = 97.2 (333-20-0)
White or almost white, crystalline powder or colourless
Colourless crystals, deliquescent, very soluble in water and in
crystals, soluble in water.
ethanol (96 per cent).
Potassium Permanganate (7722-64-7)
Storage: in an airtight container.
See Potassium permanganate (0121).
Potassium Thiocyanate Solution
Potassium Permanganate and Phosphoric Acid
A 97 g/L solution of potassium thiocyanate R.
Solution
Povidone (9003-39-8)
Dissolve 3 g of potassium permanganate R in a mixture of
15 mL of phosphoric acid R and 70 mL of water R. Dilute to See Povidone (0685).
100 mL with water R. Prednisolone C 21 H 28 O 5 = 360.5 (50-24-8)
Potassium Permanganate Solution [1X]t0: about +97 (1% w/v in 1,4-dioxan).
A 30 g/L solution of potassium permanganate R. mp: about 230°, with decomposition.
Potassium Permanganate Solution, Dilute General reagent grade of commerce.
A 1.0% w/v solution of potassium permanganate. Hygroscopic crystalline powder.
Potassium Perrhenate KReO 4 = 289.3 (10466-65-6) Prednisolone 21-Acetate C 23 H 30 O 6 = 402.5 (52-21-1)
White or almost white, crystalline powder, soluble in water, General reagent grade of commerce.
slightly soluble in ethanol (96 per cent), in methanol and in Primeverin Methyl 4-methoxy-2-[ ( 6-O-P-D-xylopyranosyl-
propylene glycol. P-D-glucopyranosyl)oxy]benzoate; C20H28 O 13 = 4 76.4
Potassium Persulfate Dipotassium peroxodisulphate; (154-60-9)
Potassium persulphate; K 2 S2 0 8 = 270.3 (7727-21-1) Procaine Hydrochloride See Procaine
Colourless crystals or white or almost white, crystalline hydrochloride (0050).
powder, sparingly soluble in water, practically insoluble in Proline (147-85-3)
ethanol (96 per cent). Aqueous solutions decompose at room See Praline (0785).
temperature and more rapidly on warming.
n-Prolyl-L-phenylalanyl-L-arginine 4-Nitroanilide
Potassium Plumbite Solution Hydrochloride D-Prolyl-L-phenylalanyl-L-arginine
Dissolve 1. 7 g of lead acetate R, 3.4 g of potassium citrate R 4-nitroanilide dihydrochloride; C 26 H 36 Cl2 N 8 O 5 = 612
and 50 g of potassium hydroxide R in water R and dilute to Propane C 3H 8 = 44.10 (74-98-6)
100 mL with the same solvent.
Content: minimum 99.0 per cent V/V.
Potassium Pyroantimonate Solution Rl
Propane-1,2-diol Propylene glycol; (57-55-6)
Dissolve 2.0 g of potassium pyroantimonate R in 100 mL of
See Propylene glycol (0430).
hot water R. Boil for about 5 min, cool quickly and add
10 mL of a 150 g/L solution of potassium hydroxide R. Allow Propane-1,3-diol 1,3-Dihydroxypropane; C 3 H 8 O 2 = 76.1
to stand for 24 h and filter. (504-63-2)
Colourless, viscous liquid.
bp: about 214 'C.
V-A126 Appendix I A 2023

mp: about -27 °C. nt0: about 1.365.

Bis-tris Propane 2,2 1-(Propane-1,3-diyldiimino) bis bp: about 49 °C.
[2-(hydroxymethyl)-1,3-propanediol; C 11 H 26N 2 O 6 = 282.3 mp: about -81 °C.
(64431-96-5)
Propionic Acid C 3 H 6 O 2 = 74.1 (79-09-4)
Content: minimum 99.0 per cent.
Oily liquid, soluble in ethanol (96 per cent), miscible with
Propanol Rl (71-23-8) water.
See Propanol (2036). d~g: about 0.993.
2-Propanol Rl n~: about 1.387.
Complies with the requirements prescribed for 2-propanol R bp: about 141 °C.
with the following additional requirements.
mp: about -21 °C.
nf~: about 1.378. Propionic Anhydride C 6 H 10 O 3 = 130.1 (123-62-6)
Water (2.5.12): maximum 0.05 per cent, determined on 10 g.
Clear, colourless liquid, soluble in ethanol (96 per cent).
Absorbance (2.2.25): maximum 0.60 at 210 nm, 0.26 at
220 nm, 0.13 at 230 nm, 0.02 at 250 nm, 0.01 at 260 nm,
dfg: about 1.01.
determined using water R as compensation liquid. bp: about 167 °C.
2-Propanol R2 (67-63-0) Propionic Anhydride Reagent
See lsopropyl alcohol (0970). Dissolve 1 g of toluenesulfonic acid R in 30 mL of glacial acetic
acid R, add 5 mL of propionic anhydride R and allow to stand
Propan-1-ol N-Propyl alcohol; Propanol; C 3 H 8 O = 60.1
for at least 15 min before use.
(71-23-8)
Storage: use within 24 h.
Clear colourless liquid, miscible with water and with ethanol
(96 per cent). Propranolol Hydrochloride C 16H 21 NO 2 ,HC1 = 295.8
(318-98-9)
dig: about 0.802 to 0.806. mp: about 164°.
bp: about 97.2 'C.
General reagent grade of commerce.
Distillation range (2.2.11). Not less than 95 per cent distils
between 96 °C and 99 °C. Propyl Acetate C 5 H 10O 2 = 102.1 (109-60-4)
Propan-2-ol Isopropyl alcohol; 2-Propanol; C 3H 8 O = 60.1 dfg: about 0.888.
(67-63-0) bp: about 102 °C.
Clear, colourless, flammable liquid, miscible with water and mp: about -95 °C.
with ethanol (96 per cent). Propyl 4-Hydroxybenzoate Propyl parahydroxybenzoate;
dfg: about 0.785. (94-13-3)
bp: 81 °C to 83 °C. See Propyl parahydroxybenzoate (0431).
Propan-2-ol MB C 3 H 8 0 = 60.10 (67-63-0) Propylene Oxide C 3 H 6 O = 58.1 (75-56-9)
Analytical grade for molecular biology. Colourless liquid, miscible with ethanol (96 per cent).
Propan-2-ol Rl Protamine Sulfate (53597-25-4 (salmine) 9007-31-2
nt 0 , about 1.378. (clupeine))
Complies with the requirements prescribed for 2-propanol See Protamine sulfate (0569).
with the following additional requirements. Protopine Hydrochloride C 20 H 20 ClNO 5 = 389.8
(6164-47-2)
Water (2.5.12) Maximum 0.05%, determined on 10 g.
Absorbance (2.2.25) Maximum 0.60 at 210 nm, 0.26 at 5-Methyl-4,6, 7, 14-tetrahydrobis[ 1,3]benzodioxolo [4,5-
220 nm, 0.13 at 230 nm, 0.02 at 250 nm, 0.01 at 260 nm, c:51,61-g] azecin-13(5H)-one hydrochloride.
determined using water as compensation liquid. PSMA-11 (3S, 7S)-22-[3-[[[2-[[[5-(2-Carboxyethyl)-2-
Propanolamine 3-Amino-1-propanol; 3-Aminopropan-1- hydroxyphenyl]methyl] (carboxymethyl)amino]
ol; 3-Aminopropanol; C 3 H 9 NO = 75.1 (156-87-6) ethyl] (carboxymethyl) amino] methyl]-4-hydroxyphenyl]-
5, 13,20-trioxo-4,6, 12, 19-tetraazadocosane-l,3,7-tricarboxylic
Clear, colourless, viscous liquid.
acid supplied as trifluoroacetate salt; C 44 H 62N 6 O 17 = 947
d~g: about 0.99. (1366302-52-4)
ntl: about 1.461. White or almost white powder, freely soluble in water.
mp: about 11 °C. Content: minimum 96.0 per cent (anhydrous and
Propetamphos C 10 H 20NO 4 PS = 281.3 (31218-83-4) trifluoroacetic acid-free substance).
A suitable certified reference solution (10 ngiµL in PSMA-1007 . (3S, 1OS, 14S)-l-[4-[[(2S)-4-Carboxy-2-[(2S)-
cyclohexane) may be used. 4-carboxy-2-( 6-fluoropyridin-3-amido) butanamido]
Propidium Iodide 3,8-Diamino-5- butanamido] methyl] phenyl]-3-[ (naphthalen-2-yl)methyl]-
[3 (diethylmethylammonio )propyl]-6-phenylphenanthridinium 1,4, 12-trioxo-2,5,11, 13-tetraazahexadecane-10, 14,16-
diiodide; C21H 3 4l2N4 = 668.4 (25535-16-4) tricarboxylic acid; C 49 H 55 FN8 O 16 = 1031
Dark red solid. White or almost white powder.
Propionaldehyde Propanal; C 3H 6 O = 58.1 (123-38-6) Pteroic Acid 4-[ [(2-Amino-4-oxo-1,4-dihydropteridin-6-
Liquid freely soluble in water, miscible with ethanol yl)methyl] amino ]benzoic acid; C 14H 12N 6 O 3 = 312.3
(119-24-4)
(96 per cent).
Crystals, soluble in solutions of alkali hydroxides.
diS: about 0.81.
2023 Appendix I A V-A127

Puerarin 7,4 1-Dihydroxy-8-C-glucosyliso-haloprone; 8-~- Add 0.5 mL of pullulanase diluent and mix thoroughly. After
o-Glucopyranosyl-7-hydroxy-3-( 4-hydroxyphenyl)-4H-1- 30 s, transfer 1.0 mL of this solution to a test tube labelled
benzopyran-4-one; C 21 H 20 0 9 = 416.4 (3681-99-0) "pullulan test solution l ", add 2.0 mL of Somogyi reagent,
Pulegone (R)-2-Isopropylidene-5-methylcyclohexanone; and mix. After 30.5 min, transfer 1.0 mL of the mixture of
(+)-p-Menth-4-en-3-one; C 10H 16 0 = 152.2 (89-82-7) substrate and pullulanase diluent to a second test tube
Oily, colourless liquid, practically insoluble in water, miscible labelled "pullulan test solution 2", add 2.0 mL of Somogyi
with ethanol (96 per cent). reagent, and mix. In a third test tube labelled "standard
blank", mix 2.0 mL of Somogyi reagent and 1.0 mL of
d?~: about 0.936.
water R. In a fourth test tube labelled "glucose standard
nt 0 : 1.485 to 1.489.
solution", mix 2.0 mL of Somogyi reagent and 1.0 mL of
bp: 222 "C to 224 ·c. glucose standard solution, and add 1.0 mL of water R.
Pulegone used in gas chromawgraphy complies with the following Incubate the fourth test tube in a water-bath for exactly
additional test. 10 min. Remove the tube and allow it to cool under running
Assay. Gas chromatography (2.2.28) as prescribed in the water. Add 2.0 mL of Nelson reagent, mix well, and allow
monograph Peppermint oil (0405). the solution to stand for at least 15 min. Add 5.0 mL of
water R to each of the 4 test tubes and mix thoroughly.
Test solution. The substance to be examined.
Determine the absorbance at 520 nm of the standard blank
Content: minimum 98.0 per cent, calculated by the (AbianJ, the glucose standard solution (As,a), pullulan test
normalisation procedure. solution 1 (A 0 ) and pullulan test solution 2 (A 30), using
Pullulanase Pullulan-6-glucanohydrolase obtained from water R as the blank. One unit is defined as the enzymatic
Kl,ebsiella pneumoniae; (9075-68-7) activity required to produce 1 µmol of maltotriose (measured
Content: minimum 30 units/mg of protein. as glucose) from pullulan per minute. Calculate the
One unit represents the enzymatic activity required to pullulanase activity, P, in units/mL, using the following
produce 1.0 µmol of maltotriose from pullulan per minute at expression:
pH 5.0 at 30 °C.
[(A30 -Ao)/(As1d -AbJank)] x 0.185 x D
DETERNIINATION OF PULLULANASE ACTIVITY
Substrate. Dissolve 0.250 g of pullulan in 20.0 mL of water R, MEASUREMENT OF PROTEIN CONTENT (MEASURED AS
adding pullulan to the water. ALBUMINOID CONTENT) FOR THE CALCULATION OF SPECIFIC
Buffer solution A. 21 g/L solution of citric acid monohydrate R ACTNITY
adjusted to pH 5.0 with a 27 g/L solution of disodium Reagent A. Prepare a solution having known concentrations
hydrogen phosphate dodecahydrate R. of about 4 g/L of sodium hydroxide R and about 21 g/L of
Buffer solution B. Prepare a 136 g/L solution of sodium anhydrous sodium carbonate R.
acetate R adjusted to pH 6.0 with dilute acetic acid R. Dilute Reagent B. Transfer 0.5 g of copper sulfate pentahydrate R and
1 mL of this solution to 100 mL with water R. 1.0 g of sodium citrate R to a volumetric flask, dissolve in and
Somogyi reagent. To 28 g of anhydrous disodium hydrogen dilute with water R to 100.0 mL, and mix.
phosphate R and 40 g of sodium potassium tartrate R add about Lowry solution. Mix 50 volumes of reagent A and 1 volume of
700 mL of water R. Add 100 mL of a 42 g/L solution of reagent B.
sodium hydroxide R and mix. Add 80 mL of a 100 g/L
Diluted Folin-Ciocalteu's phenol reagent (for albuminoid
solution of copper sulfate pentahydrate R. Heat until complete
quantification). Prepare a two-fold dilution of the
dissolution. Add 180 g of anhydrous sodium sulfate Rand
commercially available 2 N Folin-Ciocalteu's phenol reagent
adjust the volume to 1 L with water R. Allow to stand at
or prepare a solution by making an appropriate dilution of
room temperature for 1 or 2 days to let insoluble matter
phosplwmolybdotungstic reagent R.
precipitate. Filter the solution and keep the filtrate in a
brown-glass bottle with a ground-glass stopper. Bovine albumin standard stock solution. Transfer 50.0 mg of
bovine albumin R to a volumetric flask, dissolve in and dilute
Nelson reagent. Dissolve 50 g of ammonium molybdate R in
with water R to 500.0 mL, and mix. It contains 100 µg/mL
900 mL of water R. Add 42 g of sulfuric acid R and mix.
of bovine albumin.
Dissolve 6 g of disodium arsenate R in 50 mL of water R.
Add the latter solution to the 1st solution, and allow to stand Standard solutions. Using appropriate dilutions of bovine
in a bro¼n-glass bottle with a ground-glass stopper at 37 °C albumin standard stock solution in water R, prepare 5
for 1 or 2 days. standard solutions having concentrations equally spaced
between 5 µg/mL and 100 µg/mL of bovine albumin.
Glucose standard solution. Dry glucose R at a pressure less than
6 kPa at 60 °C for 5 h, and calculate the water content. Test solution. Dilute pullulanase R with buffer solution B in
Transfer 10.00 g of dried glucose to a volumetric flask, order to obtain a solution having a concentration of
dissolve with water R, dilute to 1.0 L with the same solvent, 60-70 µg/mL of albuminoid. Water may be used as diluent.
and mix. Transfer 10.0 mL of this solution to a volumetric Record the dilution factor, Df,
flask and dilute to 1.0 L with water R. Each millilitre Procedure. Introduce into separate tubes 0.3 mL of each of
contains 100 µg of glucose. the standard solutions, the test solution and water R.
Pullulanase diluent. Dilute pullulanase R with buffer solution B Add 3.0 mL of Lowry solution to each tube and mix.
to prepare a solution with an enzyme activity of about Incubate at room temperature for 10 min. Add 0.3 mL of
0.2 units/mL. The measurement range is between 0.1 and diluted Folin-Ciocalteu's phenol reagent to each tube, mix
0.4 units/mL. Record the dilution factor (D).This diluent is immediately, and allow to stand at room temperature for
used as a diluted enzyme solution. 60 min. Determine the absorbances of the standard solutions
and the test solution at the wavelength of maximum
Procedure. Transfer 4.0 mL of substrate to a test tube and
absorbance, about 750 nm, using water Ras the blank.
add 0.5 mL of buffer solution A, mix, and incubate at 30 °C.
V-A128 Appendix I A 2023

Calculation. The relationship of absorbance to protein mp: about 58 °C.

concentration is non-linear; however, if the standard curve Pyridylazonaphthol PAN; C 15 H 11 N 3 O = 249.3 (85-85-8)
concentration range is sufficiently small, it will approach
Brick-red powder, practically insoluble in water, soluble in
linearity. Using linear regression method, plot the
ethanol (96 per cent), in methanol and in hot dilute alkali
absorbances of the standard solutions versus the protein
solutions.
(bovine albumin) concentrations, in µg/mL. Using the plot,
determine the concentration of protein (albuminoid content), mp: about 138 °C.
Catbuminoid, in µg/mL, in the test solution. Calculate the Pyridylazonaphthol Solution
albuminoid concentration, in mg/mL, in pullulanase R using A 1 g/L solution of pyridylazonaphthol R in anhydrous
the following expression: ethanol R.
Test for sensitivity. To 50 mL of water R add 10 mL of acetate
Cprotein = (Catbuminoid X Dr)/ 1000 buffer solution pH 4.4 R, 0.10 mL of 0.02 M sodium edetate
Calculate the specific activity, in units/mg, of pullulanase and 0.25 mL of the pyridylazonaphthol solution. After
using the formula: addition of O.15 mL of a 5 g/L solution of copper sulfate
pentahydrate R, the colour changes from light yellow to violet.
P/Cprotein 4-(2-Pyridylazo)resorcinol Monosodium Salt
P = pullulanase activity in units/mL. C11H8 N 3NaO2, H2O = 255.2 (16593-81-0)
Orange crystalline powder.
Pumice Powder
Pumice of commerce, powdered and sifted, which passes
Pyrogallol Benzene-1,2,3-triol; C 6 H 6 O 3 = 126.1 (87-66-1)
through a 710 µm sieve, but is retained on a 250 µm sieve. White or almost white crystals, becoming brownish on
exposure to air and light, very soluble in water and in ethanol
Putrescine 1,4-Butanediamine; Tetramethylenediamine;
(96 per cent), slightly soluble in carbon disulfide.
C4H12N2 = 88.15 (110-60-1)
On exposure to air, aqueous solutions, and more rapidly
Colourless oily liquid, very soluble in water. Strong alkaline solutions, become brown owing to the absorption of
piperidine-like odour. oxygen.
bp: about 159 °C. mp: about 131 °C.
mp: about 23 °C. Storage: protected from light.
Pyrazine-2-carbonitrile 2-Cyanopyrazine; Clear, pale PyrogallolSolution,Alkaline
yellow liquid; C 5 H 3N 3 = 105.1 (19847-12-2)
Dissolve O.5 g of pyrogallol R in 2 mL of carbon di.oxide-free
Content: minimum 99 per cent. water R. Dissolve 12 g of potassium hydroxide R in 8 mL of
Pyridine C 5H 5N = 79.1 (110-86-1) carbon dioxide-free water R. Mix the two solutions immediately
Clear, colourless liquid, hygroscopic, miscible with water and before use.
with ethanol (96 per cent). Pyrrolidine C 4H 9N = 71.1 (123-75-1)
bp: about 115 °C. Content: minimum 99 per cent.
Storage: in an airtight container. bp: 87 °C to 88 °C.
Pyridine, Anhydrous 2-Pyrrolidone Pyrrolidin-2-one; C 4 H 7NO = 85.1
Dry pyridine R over anhydrous sodium carbonate R. Filter and (616-45-5)
distil. Liquid above 25 °C, miscible with water, with anhydrous
Water (2.5.12): maximum 0.01 per cent mlm. ethanol and with ethyl acetate.
Pyridine Bromide Solution dl 5 :1.116.
Dissolve 8 g of pyridine and 5.4 mL of suljuric acid in 20 mL Water (2.5.12): maximum 0.2 per cent determined on 2.00 g.
of glacial acetic acid, keeping the mixture cool. Add 2.6 mL of Assay. Gas chromatography (2.2.28): use the normalisation
bromine dissolved in 20 mL of glacial acetic acid and dilute to procedure.
1000 mL with glacial acetic acid. Test solution. Dissolve 1.0 g in methanol R and dilute to
Prepare immediately before use. 10.0 mL with the same solvent.
Pyridine-4-carbonitrile 4-Cyanopyridine; Column:
C6H4 N 2 = 104.1 (100-48-1) - material: glass;
White or almost white crystalline powder. - size: l =30 m; 0 =0.53 mm;
bp: 194 °C to 196 °C. - stationary phase: macrogol 20 000 R (1.0 µm).
mp: 76 °C to 79 °C. Carrier gas: helium for chromatography R.
Pyridine-3-carboxaldehyde Nicotinaldehyde; Flow rate: adjusted so that the retention time of 2-pyrrolidone
C6H 5 NO = 107.1 (500-22-1) is about 10 min.
General reagent grade of commerce. Split ratio: 1:20.
Weight per mL, 1.14 g. Temperature:
Pyridinium Hydrobromide Perbromide Pyridinium
Time Temperature
tribromide(l-); C 5H 6 Br3N = 319.8 (39416-48-3)
(min) (C)
Red crystals. Column 0- I 80
2-Pyridylamine Pyrid-2-ylamine; 2-Aminopyridine; I - 12 80 ➔ 190
Pyridin-2-amine; C 5H 6N 2 = 94.1 (504-29-0) 12 - 32 190
Large crystals soluble in water and in ethanol (96 per cent). Injection port 200
bp: about 210 °C.
2023 Appendix I A V-A129

Detection: flame ionisation. Quinidine (S)-( 6-Methoxyquinol-4-yl) [(2R,4S,5R)-5-

Injection: 1 µL of the test solution. vinylquinuclidin-2-yl]methanol; C 20H 2 ,iN2 O 2 = 324.4
(56-54-2)
Content: minimum 98.0 per cent.
Pyruvic Acid 2-Oxopropanoic acid; C 3H 4 O 3 = 88.1 White or almost white crystals, very slightly soluble in water,
(127-17-3)
sparingly soluble in ethanol (96 per cent), slightly soluble in
methanol.
Yellowish liquid, miscible with water and with anhydrous
ethanol. [o:]~: about+ 260, determined on a 10 g/L solution in
anhydrous ethanol R.
dig: about 1.267.
mp: about 172 °c.
ng1: about 1.43.
Storage: protected from light.
bp: about 165 °C.
Quinidine Sulfate Quinidine sulphate; (6591-63-5)
Quercetin Dihydrate 2-(3,4-Dihydroxyphenyl)-3,5, 7-
trihydroxy-4H-l-benzopyran-4-one; See Quinidine sulfate (0017).
C1sH1oO1,2H2O = 338.2 Quinine (R)-( 6-Methoxyquinol-4-yl) [(2S,4S,5R)-5-
Yellow crystals or yellowish powder, practically insoluble in vinylquinuclidin-2-yl]methanol; C 20H 24N 2 0 2 = 324.4
(130-95-0)
water, soluble in acetone and in methanol.
Water (2.5.12): maximum 12.0 per cent, determined on
White or almost white, microcrystalline powder, very slightly
0.100 g. soluble in water, slightly soluble in boiling water, very soluble
in anhydrous ethanol.
Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Ginkgo leaf (1828). [o:Jt0 : about -167, determined on a 10 g/L solution in
anhydrous ethanol R.
Content:minimum 90 per cent (anhydrous substance)
calculated by the normalisation procedure. mp: about 175 °C.
Storage: protected from light.
Storage: protected from light.
Quercitrin Quercetin 3-L-rhamnopyranoside; 3-((6-Deoxy- Quinine Hydrochloride (6119-47-7)
Cl-L-mannopyranosyl) oxy]-2-(3,4-dihydroxyphenyl)-5, 7- See Quinine hydrochloride (0018).
dihydroxy-4H- l-benzopyran-4-one; Quercitroside; Quinine Sulfate Quinine sulphate; (6119-70-6)
C21H20O11 = 448.4 (522-12-3) See Quinine sulfate (0019).
Yellow crystals, practically insoluble in cold water, soluble in Quinoline C 9 H 7N = 129.2 (91-22-5)
ethanol (96 per cent).
ng1: 1.624 to 1.627.
mp: 176 °C to 179 °C. A pale yellow liquid.
Chromatography. Thin-layer chromatography (2.2.27) as
General reagent grade of commerce, suitable for phosphate
prescribed in the monograph Goldenrod (1892): apply 20 µL assay.
of the solution; after spraying, the chromatogram shows a
Complies with the foUowing requirement.
yellowish-brown fluorescent zone with an RF of about 0.6.
Storage: at a temperature of 2 °C to 8 °c. Tarry impurities Dissolve 1.0 g in a mixture of 2.5 mL
of water and 2.5 mL of hydrochloric acid. Add 3 mL of water
Quillaia Saponins, Purified and 1 mL of potassium chromate solution and heat to boiling.
A mixture of related saponins obtained from the bark of The solution shows no darkening and no black deposit.
QuillaJa saponaria Molina s.l.
Store protected from light.
Chromatography. Thin-layer chromatography (2.2.27) as
Quinoline Solution
prescribed in the monograph Quillaia bark (1843): apply
5 µL of the solution; after treating with a 10 per cent V/V Dissolve 50 mL of quinoline in a mixture of 60 mL of
hydrochloric acid and 300 mL of water previously heated to
solution of sulfun·c acid R in methanol R, heat at 120 °C for
5 min and examine in daylight; the chromatogram shows 70°, cool and filter.
3 principal zones in the upper part of the middle third. 3-Quinuclidinol (3R)-1-Azabicyclo [2.2.2] octan-3-ol;
Quinaldine Red 2-( 4-dimethylaminostyryl)quinoline C1H 13 NO = 127.2 (1619-34-7)
ethiodide; C 21 H 23 IN2 = 430.3 (117-92-0) Content: minimum 99 per cent.
Dark bluish-black powder, sparingly soluble in water, freely Light yellow powder.
soluble in ethanol (96 per cent). Rabbit Erythrocyte Suspension
Quinaldine Red Solution Prepare a 1.6 per cent V/V suspension of rabbit erythrocytes
Dissolve 0.1 g of quinaldine red R in methanol Rand dilute to as follows: defibrinate 15 mL of freshly drawn rabbit blood
100 mL with the same solvent. by shaking with glass beads, centrifuge at 2000 g for 10 min
Colour change: pH 1.4 (colourless) to pH 3.2 (red).
and wash the erythrocytes with three quantities, each of
30 mL, of a 9 g/L solution of sodium chloride R. Dilute
Quinhydrone Equimolecular compound of 1. 6 mL of the suspension of erythrocytes to 100 mL with a
1,4-benzoquinone and hydroquinone; C 12H 10 O4 = 218.2 mixture of 1 volume of phosphate buffer solution pH 7.2 Rand
(106-34-3) 9 volumes of a 9 g/L solution of sodium chloride R.
Dark green, lustrous crystals or a crystalline powder, slightly Rabies Antiserum, Fluorescein-conjugated
soluble in water, sparingly soluble in hot water, soluble in
ethanol (96 per cent) and in concentrated ammonia. lmmunoglobulin fraction with a high rabies antibody titre,
prepared from the sera of suitable animals that have been
mp: about 170 °C. immunised with inactivated rabies virus; the immunoglobulin
is conjugated with fluorescein isothiocyanate.
V-A130 Appendix I A 2023

Raclopride Tartrate Raclopride L-tartrate; White or almost white powder, sparingly soluble in water,
C1 9H 26 Cl 2N2O9 = 497.3 (98185-20-7) soluble in methanol.
White or almost white solid, sensitive to light, soluble in L-Rhamnose (2R,3R,4R,5R,6S)-6-Methyltetrahydro-2H-
water. pyran-2,3,4,5-tetrol monohydrate; 6-Deoxy-a.-L-
[0!]~: + 0.3, determined on a 3 g/L solution. mannopyranose monohydrate; a.-L-Rhamnopyranose
mp: about 141 "C. monohydrate; L-( + )-Rhamnose monohydrate; Rhamnose;
C 6 H 12 O 5,H 2O = 182.2 (6155-35-7)
Raffinose P-n-Fructofuranosyl a.-D-
galactopyranosyl-(1 ➔ 6)-a.-D-glucopyranoside; White or almost white, crystalline powder, freely soluble in
water.
C1sH32O16 = 504.4 (512-69-6)
Raffinose Pentahydrate P-D-Fructofuranosyl a.-D- [O!]~: + 7.8 to+ 8.3, determined on a 50 g/L solution in
water R containing about 0.05 per cent ofNH3.
galactopyranosyl-(1 ➔ 6)-et-D-glucopyranoside pentahydrate;
C1sH32O16,5H2O = 594.5 (17629-30-0) Rhaponticin Rhapontin; 4 1-methoxystilbene-3,3 ',5-triol
Content: minimum 98.0 per cent.
3-glucoside; C 21 H 24 O 9 = 420.4 (155-58-8)
Crystalline powder. Yellowish-grey, crystalline powder, soluble in ethanol
(96 per cent) and in methanol.
mp: about 80 °C.
Chromatography. Thin-layer chromatography (2.2.27) as
Raltegravir Potassium C 20 H 20FKN6 0 5 (871038-72-1) prescribed in the monograph Rhubarb (0291); the
See Raltegravir potassium (2887). chromatogram shows only one principal spot.
Rapeseed Oil See Rapeseed oil, refined (1369). Rhein 4,5-Dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-
Reducing Mixture Hydrazine reducing mixture 2-carboxylic acid; 1,8-Dihydroxy-3-carboxyanthraquinone;
Grind the substances added in the following order to obtain C1sH 8 O 6 = 284.2 (478-43-3)
a homogeneous mixture: 20 mg of potassium bromide R, 0.5 g Rhodamine 6 G 9-[2-(Ethoxycarbonyl)phenyl]-3,6-
of hydrazine sulfate R and 5 g of sodium chloride R. bis (ethyl amino )-2, 7-dimethylxanthenylium chloride;
Reichstein's Substance S C 21 H 30 O4 = 346.5 (152-58-9) C28H 31CIN2 O 3 = 479.0 (989-38-8)
Content: minimum 95.0 per cent. Colour Index No. 45160
mp: about 208 °C. Brownish-red powder.
Reserpine Methyl l l,l 7a.-dimethoxy-18P-[(3,4,5- Rhodamine B Basic violet 10; C 28H 31 CIN2O 3 = 479.0
trimethoxybenzoyl)oxy]-3 P,20a.-yohimban- l 6P-carboxylate; (81-88-9)
C33H40N2O9 = 609 (50-55-5) Schultz No. 864
General reagent grade of commerce. Colour Index No. 45170
Resin for Hydrophobic Interaction Chromatography Green crystals or reddish-violet powder, very soluble in water
Non-porous resin consisting of spherical polymethacrylate and in ethanol (96 per cent).
particles bonded with butyl groups. Rhynchophylline Methyl (16E)-l 7-methoxy-2-oxo-16,17-
pH limits of use: 2 to 12. didehydro-7P,20a.-corynoxan-l 6-carboxylate; Methyl ( l 6E)-
Resin for Reversed-phase Ion Chromatography l 6-(methoxymethylidene)-2-oxo-7 P,20a.-corynoxan-l 7-oate;
C22H2sN2O4 = 384.5 (76-66-4)
A neutral, macroporous, high specific surface area with a
non-polar character resin consisting of polymer lattice of Ribose D-Ribose; C 5H 10O 5 = 150.1 (50-69-1)
polystyrene cross-linked with divinylbenzene. Soluble in water, slightly soluble in ethanol (96 per cent).
Resin, Weak Cationic See weak cationic resin R. mp: 88 °C to 92 °C.
Resorcinol Benzene-1,3-diol; (108-46-3) Ricinoleic Acid (9Z,12R)-l 2-Hydroxyoctadec-9-enoic
acid; 12-Hydroxyoleic acid; C 18H 34 O 3 = 298.5 (141-22-0)
See Resorcinol (0290).
Resorcinol Reagent Yellow or yellowish-brown viscous liquid, consisting of a
mixture of fatty acids obtained by the hydrolysis of castor oil,
To 80 mL of hydrochloric acid Rl add 10 mL of a 20 g/L practically insoluble in water, very soluble in anhydrous
solution of resorc£nol R and 0.25 mL of a 25 g/L solution of ethanol.
copper sulfate pentahydrate Rand dilute to 100.0 mL with
water R. Prepare the solution at least 4 h before use.
dig: about 0.942.
Storage: at 2 cc to 8 °C for 1 week. nt 0 : about 1.472.

Resveratrol 3,4' ,5-Stilbenetriol; 5-[(E)-2-(4- mp: about 285 °C, with decomposition.
Hydroxyphenyl)ethenyl]benzene-1,3-diol; C 14H 12 O3 = 228.2 Rosmarinic Acid C 18H 16O 8 = 360.3 (20283-92-5)
(501-36-0) mp: 170 °C to 174 °C.
Retrorsine ( 1R,4Z,6R, 7S, 17R)-4-Ethylidene-7-hydroxy- Rosuvastatin Ethyl Ester Ethyl (3R,5S,6E)-7-[4-(4-
7-(hydroxymethyl)-6-methyl-2, 9-dioxa-14-azatricyclo fluorophenyl)-2-(N-methylmethanesulfonamido )-6-(propan-2-
[9.5. l.0 14' 17]heptadec-l l-ene-3,8-dione; yl)pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoate;
C1sH2sNO 6 = 351.4 (480-54-6) C24H32FN3O6S = 509.6 (851443-04-4)
Colourless or white or almost white crystalline powder, Content: minimum 98 per cent.
soluble in methanol. White or pale yellow powder.
Retrorsine N-Oxide ( IR,4Z,6R, 7S, 17R)-4-Ethylidene-7- Ruscogenins
hydroxy-7-(hydroxymethyl)-6-methyl-3,8-dioxo-2, 9-dioxa-14- Mixture of neoruscogenin (C 27 H 40 O 4 = 428.6) and
azatricyclo [9 .5. l. 014'17] heptadec-11-ene 14-oxide; ruscogenin (C 27 H 42 O 4 = 430.6)
C1sH 25NO 7 = 367.4 (15503-86-3)
General reagent grade of commerce.
2023 Appendix I A V-A131

Ruscogenins used in Ii.quid chromatography complies with the mp: 199 'C to 201 °C.
fol0wing test. Assay. Liquid chromatography (2.2.29) as prescribed in the
Assay Cany out the method described in the monograph monograph Wil0w bark (1583) at the concentration of the
for Butcher's Broom. The content is not less than 90% of reference solution.
ruscogenins of which at least 60% consists of neoruscogenin, Content: minimum 99.0 per cent, calculated by the
calculated by the normalisation procedure. normalisation procedure.
The reagent from Vinyals laboratory has been found suitable. Salicylaldehyde 2-Hydroxybenzaldehyde;
Rutecarpine 8,13-Dihydroindolo[2'_,3':3,4]pyrido[2,l-b] C 7 H 6 O 2 = 122.1 (90-02-8)
quinazolin-5(7 H)-one; C 18H 13 N 3O = 287.3 (84-26-4) Clear, colourless, oily liquid.
Ruthenium Red Ammoniated ruthenium oxychloride; d~g: about 1.167.
[(NH3) 5RuORu(NH3)4ORu(NH3) 5]Cl6,4H 2 O = 858
(11103-72-3)
nt 0:
about 1.574.
bp: about 196 °C.
Brownish-red powder, soluble in water.
mp: about -7 °C.
Ruthenium Red Solution
Salicylaldehyde Azine 2,2 '-Azinodimethyldiphenol;
A 0.8 g/L solution of rnthenium red R in lead acetate
C14H12N2O2 = 240.3 (959-36-4)
solutwn R.
Dissolve 0.30 g of hydrazine sulfate R in 5 mL of water R,
Rutin (250249-75-3) add 1 mL of glacial acetic acid R and 2 mL of a freshly
See Rutoside trihydrate R. prepared 20 per cent V/V solution of salicylaldehyde R in
Rutoside Trihydrate (250249-75-3) 2-propanol R. Mix, allow to stand until a yellow precipate is
See Rutoside trihydrate (1795). formed. Shake with two quantities, each of 15 mL, of
Sabinene Thuj-4(10)-ene; 4-Methylene-1-isopropylbicyclo methylene chloride R. Combine the organic layers and dry over
[3. l.0]hexane; C 10H 16 = 136.2 (3387-41-5) anhydrous sodium sulfate R. Decant or filter the solution and
evaporate to dryness. Recrystallise from a mixture of
A colourless, oily liquid.
40 volumes of methanol R and 60 volumes of toluene R with
Sabinene used in gas chromatography complies with the fol0wing cooling. Dry the crystals in vacua.
additional test.
mp: about 213 °C.
Assay. Gas chromatography (2.2.28) as prescribed in the Chromatography. Thin-layer chromatography (2.2.27) as
monograph Bitter-orange-flower oil (1175).
prescribed in the test for hydrazine in the monograph
Test solution. The substance to be examined. Povidone (0685); the chromatogram shows only one principal
Content: minimum 95.0 per cent, calculated by the spot.
normalisation procedure. Salicylic Acid (69-72-7)
Saccharin C 7 H 5NO 3S = 183.2 (81-07-2) See Salicylic acid (0366).
mp: about 230°. Saline Solution
General reagent grade of commerce. A 0.9% w/v solution of sodium chloride in freshly prepared
Saccharin Sodium (128-44-9) water, sterilised by heating in an autoclave.
See Saccharin sodium (0787). Salvianolic Acid B (2R)-2-[[(2E)-3-[(2S,3S)-3-[[(1R)-l-
Safrole 5-(Prop-2-enyl)-1,3-benzodioxole; 4-Allyl- Carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl]-2-(3,4-
1,2-(methylenedioxy)benzene; C 10H 10 0 2 = 162.2 (94-59-7) dihydroxyphenyl)-7-hydroxy-2,3-dihydrobenzofuran-4-yl]
prop-2-enoyl]oxy]-3-(3,4-dihydroxyphenyl)propanoic acid;
Colourless or slightly yellow, oily liquid, with the odour of
sassafras, insoluble in water, very soluble in ethanol C35H30O16 = 719 (121521-90-2)
(96 per cent), miscible with hexane. Sand
dfg: 1.095 to 1.096. White or slightly greyish grains of silica with a particle size
nt0: 1.537 to 1.538.
between 150 µm and 300 µm.
Sanguinarine Chloride Pseudochelerythrine chloride;
bp: 232 °C to 234 °C.
13-Methyl [ 1,3] benzodioxolo [5,6-c] [1,3] dioxolo [4,5-i]
Freezing point: about 11 °C. phenanthridin-13-ium chloride; C 20 H 14C!NO 4 = 367.8
Safrole used in gas chromatography complies with the fol0wing (5578-73-4)
additional test. mp: about 283 °C.
Assay. Gas chromatography (2.2.28) as prescribed in the Orange crystalline powder, soluble in methanol.
monograph Cinnamon bark oil, Ceylon (1501).
Storage: protected from light and moisture.
Content: minimum 96.0 per cent, calculated by the
normalisation procedure.
Santonin C1 5H 15 O 3 = 246.3 (481-06-1)
Saikosaponin A 13,28-Epoxy-l 6P,23-dihydroxy-4cx-olean- [o:Jh8 :
173 in ethanol.
l l-en-3 P-yl 6-deoxy-3-O-P-o-glucopyranosyl-P-o- mp: 174° to 176°.
galactopyranoside; C 42 H 68 O 13 = 781 (20736-09-8) General reagent grade of commerce.
Saikosaponin D 13,28-Epoxy-l fo,23-dihydroxy-4cx-olean- Chromatography Carry out Identification test C in the
l l-en-3P-yl 6-deoxy-3-O-P-o-glucopyranosyl-P-o- monograph for Amica Flower using a 0.025% w/v solution of
galactopyranoside; C 42 H 68 O 13 = 781 (20874-52-6) the reagent being examined in methanol. The chromatogram
Salicin 2-(Hydroxymethyl)phenyl-P-o-glucopyranoside; obtained with 10 µL of the solution shows a quenching zone
Salicoside; C 13 H 18 O 7 = 286.3 (138-52-3) with an Rf value of about 0.5. Spray the plate with
anisalaehyde solution and examine while heating at 105' for
[o:Jt0 : -62.5 ± 2.
V-A132 Appendix I A 2023

5 to I O minutes. In daylight the quenching zone is at first a pH 6.8 with hydrochloric acid R, and dilute to 250.0 mL with
yellow zone that quickly changes to a violet-red zone. water R.
Sarafloxacin Hydrochloride 6-Fluoro-1-(4- Alternatively, dithiothreitol may be used as reducing agent
fluorophenyl)-4-oxo-7-piperazin-1-yl-1,4-dihydroquinoline-3- instead of 2-mercaptoethanol. In this case prepare the sample
carboxylic acid hydrochloride; C 20 H 18 CIF 2N 3 O 3 = 421.8 buffer as follows: dissolve 3.78 g of tris(hydroxymethyl)
(91296-87-6) aminomethane R, 10.0 g of sodium dodecyl sulfate Rand
Schisandrin Schisandrol A; Wuweizichun A; 100 mg of bromophenol blue R in water R. Add 50.0 mL of
(6S, 7S,12aRa)-5,6,7,8-Tetrahydro-1,2,3,10,11, 12- glycerol R and dilute to 200 mL with water R. Adjust to
hexamethoxy-6, 7-dimethyldibenzo [a,c] cyclooctan-6-ol; pH 6.8 with hydrochloric acid R, and dilute to 250.0 mL with
C24H12O 7 = 432.5 (7432-28-2) water R. Immediately before use, add dithiothreitol R to a final
White or almost white, crystalline powder. concentration of 100 mM.
Schisandrin used in the assay in the monograph Schisandra Selenious Acid Selenous acid; Selenic(N) acid;
fruit (2428) complies with the following additional test.
H 2 SeO3 = 129.0 (7783-00-8)
Assay. Liquid chromatography (2.2.29) as prescribed in the Deliquescent crystals, freely soluble in water.
monograph Schisandra fruit (2428). Storage: in an airtight container.
Content: minimum 95 per cent, calculated by the Selenium Se = 79.0 (7782-49-2)
normalisation procedure. Brown-red or black powder or granules, practically insoluble
Storage: in an airtight container, at -20 °C or below. in water and in ethanol (96 per cent), soluble in nitric acid.
y-Schisandrin Schisandrin B; Wuweizisu B; mp: about 220 °C.
rac-(6R, 7S, 13aRa)-1,2,3,13-Tetramethoxy-6,7-dimethyl- Selenium Dioxide SeO 2 = 111.0 (7446-08-4)
5,6, 7,8-tetrahydrobenzo [3,4]cycloocta[l ,2-j] [1,3] General reagent grade of commerce.
benzodioxole; C 23 H 28O 6 = 400.5 (61281-37-6) Semicarbazide Acetate Solution Triturate 2.5 g of
White or almost white, crystalline powder. semicarbazide hydrochloride with 3.3 g of sodium acetate, add
Storage: in an airtight container, at -20 °C or below. 10 mL of methanol, mix, transfer to a flask with the aid of
Sclareol (1R,2R,4aS,8aS)-1-[(3R)-3-Hydroxy-3- 20 mL of methanoland allow to stand at a temperature of
methylpent-4-enyl]-2,5,5,8a- about 4 ° for 30 minutes
tetramethyldecahydronaphthalen-2-ol; C 20 H 36 O 2 = 308.5 Triturate 2.5 g of semicarbazide hydrochloride with 3.3 g of
(515-03-7) sodium acetate, add 10 mL of methanol, mix, transfer to a
Odourless crystals. flask with the aid of 20 mL of methanol and allow to stand at
[:x]~: 6.7, determined with a solution in anhydrous ethanol. a temperature of about 4° for 30 minutes; filter and add
sufficient methanol to produce I 00 mL.
bp19 mm: 218 °C to 220 "C.
Semicarbazide Hydrochloride CH5 N 3 O,HC1 = 111.5
mp: 96 °C to 98 °C. (563-41-7)
Sclareol used in the chromatographic profile test in the manograph mp: about 175°, with decomposition.
Clary sage oil (1850) complies with the following additional test.
Analytical reagent grade of commerce.
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Clary sage oil (1850). A white, crystalline powder.
Content: minimum 97 per cent, calculated by the
Senecionine ( I R,4Z, 6R, 7R, I 7R)-4-Ethylidene-7-hydroxy-
normalisation procedure. 6, 7-dimethyl-2, 9-dioxa-14-azatricyclo [9 .5.1.0 14' 17] heptadec-
11-ene-3,8-dione; C 18 H 25NO 5 = 335.4 (130-01-8)
Scopoletin 7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one;
7-Hydroxy-6-methoxycoumarin; C 10H 8 O 4 = 192.2 (92-61-5) White or almost white powder.
Faintly beige, fine crystals. Senecionine N-Oxide ( IR,4Z,6R, 7R, 17R)-4-Ethylidene-
7-hydroxy-6, 7-dimethyl-3,8-dioxo-2,9-dioxa-14-azatricyclo
mp: 202 °C to 208 °C. [9.5.l.0 14' 17 ]heptadec-11-ene 14-oxide; C1 8H 25 NO 6 = 351.4
SDS-PAGE Running Buffer (13268-67-2)
Dissolve 151.4 g of tris(hydroxymethyl)aminomethane R, White or almost white powder, soluble in methanol, sparingly
721.0 g of glycine Rand 50.0 g of sodium laurilsulfate R in soluble in ethyl acetate.
water R and dilute to 5000 mL with the same solvent. Seneciphylline (1R,4Z, 7R,17 R)-4-Ethylidene-7-hydroxy-7-
Immediately before use, dilute to 10 times its volume with dimethyl-6-methylidene-2, 9-dioxa-14-azatricyclo [9 .5.1.0 14•17]
water R and mix. Measure the pH (2. 2.3) of the diluted
heptadec-11-ene-3,8-dione; C 18H 23NO 5 = 333.4 (480-81-9)
solution. The pH is between 8.1 and 8.8.
White or almost white powder.
SDS-PAGE Sample Buffer (Concentrated)
Seneciphylline N-Oxide ( IR,4Z, 7R, 17R)-4-Ethylidene-7-
Dissolve 1.89 g of tris(hydroxymethyl)aminomethane R, 5.0 g hydroxy-7-dimethyl-6-methylidene-3,8-dioxo-2,9-dioxa-14-
of sodium laurilsulfate R and 50 mg of bromophenol blue R in azatricyclo [9 .5. l .014'17]heptadec-l l-ene 14-oxide;
water R. Add 25.0 mL of glycerol R and dilute to 100 mL
C1sH 23NO 6 = 349.4 (38710-26-8)
with water R. Adjust the pH to 6.8 with hydrochloric acid R,
and dilute to 125 mL with water R. White or almost white powder, soluble in methanol, sparingly
soluble in ethyl acetate.
SDS-PAGE Sample Buffer for Reducing Conditions
(Concentrated) Senecivernine (IR,5R,6R, 7R,l 7R)-7-Hydroxy-5,6,7-
trimethyl-4-methylidene-2,9-dioxa-14-azatricyclo [9. 5 .1. O14·17]
Dissolve 3.78 g of tris(hydroxymethyl)aminomethane R, 10.0 g heptadec-11-ene-3,8-dione; C 18H 25 NO 5 = 335.4
of sodium dodecyl sulfate R and 100 mg of bromophenol blue R (72755-25-0)
in water R. Add 50.0 mL of glycerol R and dilute to 200 mL
with water R. Add 25.0 mL of 2-mercaptoethanol R. Adjust to White or almost white powder, soluble in methanol.
2023 Appendix I A V-A133

Senecivernine N-Oxide (1R,5R,6R, 7R,17R)-7-Hydroxy- detection of the humidity status, for which the colour change
5,6, 7-trimethyl-4-methylidene-3,8-dioxo-2,9-dioxa-14- from the hydrated to anhydrous form is given on the label.
azatricyclo [9 .5. l .014' 17]heptadec- l 1-ene 14-oxide; Silica Gel BC for Chiral Chromatography
C 18H 25N0 6 = 351.4 (101687-28-9) A very finely divided silica gel for chromatography (5 µm)
White or almost white powder, soluble in water and in coated with ~-cyclodextrin. Higher selectivity may be
methanol. obtained when cyclodextrin has been derivatized with
Senkirkine ( 1R,4Z,6R, 7R)-4-Ethylidene-7-hydroxy-6, 7, 14- propylene oxide.
trimethyl-2, 9-dioxa-14-azabicyclo [9 .5 .1 ]heptadec-11-ene- Silica Gel for Chiral Chromatography, Urea Type
3,8, 17-trione; C 19 H 27N0 6 = 365.4 (2318-18-5)
A very finely divided silica gel (5 µm) coated with the
White or almost white powder, soluble in methanol. following derivative:
Sennoside A (9R, 9 'R)-5,5 '-Bis(~-o-glucopyranosyloxy)-
4,4 '-dihydroxy-l 0, 10 '-dioxo-9,9 ', 10,10 1-tetrahydro[9,9'-
bianthracene]-2,2 '-dicarboxylic acid; C 42 H 380 20 = 863
(81-27-6)
Sennoside B (9R,9' S)-5,5 '-Bis(~-o-glucopyranosyloxy)-
4,4'-dihydroxy-l 0, 10 '-dioxo-9,9 1, 10, 10 1-tetrahydro-9,9 1-
bianthracene-2,2 1-dicarboxylic acid; C 42 H 38 0 20 = 863
(128-57-4)
Pale yellow crystals, practically insoluble in water, very Silica Gel for Chiral Separation, Amylose Derivative
slightly soluble in ethanol (96 per cent), soluble in dilute of
solutions of alkali hydroxides. Substituted amylase coated on a very finely divided silica gel
mp: 180 °C to 186 'C. for chromatography.
L-Serine Serine; (56-45-1) Silica Gel for Chiral Separation, Cellulose Derivative
See Serine (0788). of Silica Gel OD for Chiral Separations
Sesame Oil Substituted cellulose coated on a very finely divided silica gel
for chromatography.
General reagent grade of commerce.
Silica Gel for Chiral Separation, Human Albumin
Sialic Acid (131-48-6)
Coated
See N-acetylneuraminic acid R.
A very finely divided silica gel, chemically modified at the
Silibinin Silybin; (2R,3R)-3,5, 7-Trihydroxy-2-[(2R,3R)- surface by the bonding of human albumin.
3-( 4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-
Silica Gel for Chiral Separation, Protein Derivative
dihydro-1,4-benzodioxin-6-yl]-2,3-dihydro-4H-1-benzopyran-
of
4-one; C 25 H 22 0io = 482.4 (22888-70-6)
A very finely divided silica gel for chromatography consisting
White or yellowish powder, practically insoluble in water,
of spherical particles coated with a protein derivative.
soluble in acetone and in methanol.
Silica Gel for Chiral Separation, Vancomycin-
Silibinin used in the assay of Milk thistle fruit (1860) complies
bonded
with the following additional test.
High-purity silica gel chemically modified by the bonding of
Assay. Liquid chromatography (2.2.29) as prescribed in the
vancomycin through multiple covalent linkages.
monograph Milk thistle fruit (1860).
Silica Gel for Chromatography
Test solution. Dissolve 5.0 mg of silibinin, dried in vacuo, in
methanol Rand dilute to 50.0 mL with the same solvent. A very finely divided silica gel.
Silibinin A and silibinin B content: minimum 95.0 per cent, Silica Gel for Chromatography, Alkyl-bonded for use
calculated by the normalisation procedure. with Highly Aqueous Mobile Phases
Silica for Chromatography, Porous A very finely divided silica gel with bonded alkyl groups
suitable for use with highly aqueous mobile phases.
Porous silica with porous layer open tubular (PLOT) design.
Silica Gel for Chromatography, Alkyl-bonded for use
Silica Gel with Highly Aqueous Mobile Phases, End-capped
A fine, white, homogeneous powder of an average particle
A very finely divided silica gel with bonded alkyl groups
size of about 15 µm containing a suitable binding agent.
suitable for use with highly aqueous mobile phases.
Silica Gel rr-Acceptor/n-Donor for Chiral Separations To minimise any interaction with basic compounds it is
A very finely divided silica gel for chromatography consisting carefully end-capped to cover most of the remaining silanol
of spherical particles to which 1-(3,5-dinitrobenzamido)- groups.
1,2,3,4-tetrahydrophenantrene has been covalently bound, Silica Gel for Chromatography, Alkylsilyl, Solid Core,
showing both 11:-electron acceptor and rr-electron donor End-capped
characteristics.
Silica gel with spherical silica particles containing a non-
Silica Gel AGP for Chiral Chromatography See al- porous solid silica core surrounded by a thin outer porous
Acid-glycoprotein silica gel for chiral separation R. silica coating with alkylsilyl groups. To minimise any
Silica Gel, Anhydrous (J 12926-00-8) interaction with basic compounds, it is carefully end-capped
Partly dehydrated polymerised, amorphous silicic acid, to cover most of the remaining silanol groups.
absorbing at 20 °C about 30 per cent of its mass of water. Silica Gel for Chromatography, Amido-alkylsilyl Silica
Practically insoluble in water, partly soluble in solutions of gel for chromatography, amidoalkylsilyl
sodium hydroxide. It contains a suitable indicator for A very finely divided silica gel, chemically modified at the
surface by the bonding of amidoalkylsilyl groups.
V-A134 Appendix I A 2023

Silica Gel for Chromatography, Amidohexadecylsilyl interaction with basic compounds it is carefully end-capped
A very finely divided silica gel with a fine particle size, to cover most of the remaining silanol groups.
chemically modified at the surface by the bonding of Silica Gel for Chromatography, Cyanosilyl, End-
amidohexadecylsilyl groups. capped, Base-deactivated
Silica Gel for Chromatography, Amidohexadecylsilyl, A very finely divided silica gel pre-treated before the bonding
End-capped of cyanosilyl groups by washing and hydrolysing most of the
A very finely divided silica gel, chemically modified at the superficial siloxane bridges, chemically modified at the
surface by the bonding of amidohexadecylsilyl groups. surface by bonding of cyano groups. To further minimise any
To minimise any interaction with basic compounds, it is interaction with basic compounds it is carefully end-capped
carefully end-capped to cover most of the remaining silanol to cover most of the remaining silanol groups.
groups. Silica Gel for Chromatography,
Silica Gel for Chromatography, Di-isobutyloctadecylsilyl
Aminopropylmethylsilyl A very finely divided silica gel chemically modified at the
Silica gel with a fine particle size, chemically modified by surface by the bonding of di-isobutyloctadecylsilyl groups.
bonding aminopropylmethylsilyl groups on the surface. Silica Gel for Chromatography,
Silica Gel for Chromatography, Aminopropylsilyl Diisopropylcyanosilyl Silica Gel for Chromatography,
Silica gel with a fine particle size, chemically modified by Diisopropylcyanopropylsilyl
bonding aminopropylsilyl groups on the surface. A very finely divided silica gel chemically modified at the
Silica Gel for Chromatography Rl, Aminopropylsilyl surface by the bonding of diisopropylcyanosilyl groups.
Silica gel with a particle size of about 55 µm, chemically Silica Gel for Chromatography,
modified by bonding aminopropylsilyl groups on the surface. 4-dimethylaminobenzylcarbamidesilyl
Silica Gel for Chromatography, Amylose-derivative A very finely divided silica gel, chemically modified at the
surface by the bonding of 4-dimethylaminobenzylcarbamide
of
groups.
A very finely divided (10 µm) silica gel, chemically modified
at the surface by the bonding of an amylase derivative.
Silica Gel for Chromatography,
Dimethyloctadecylsilyl
Silica Gel for Chromatography, Butylsilyl
A very finely divided silica gel, chemically modified at the
A very finely divided silica gel, chemically modified at the surface by the bonding of dimethyloctadecylsilyl groups.
surface by the bonding of butylsilyl groups.
Specific surface area: 300 m 2 /g.
Silica Gel for Chromatography, Butylsilyl, End-
capped Silica Gel for Chromatography, Diol
A very finely divided silica, chemically modified at the surface Spherical silica particles to which dihydroxypropyl groups are
by the bonding of butylsilyl groups. To minimise any bonded. Pore size 10 nm.
interaction with basic compounds, it is carefully end-capped Silica Gel for Chromatography, Dodecylsilyl, End-
to cover most of the remaining silanol groups. capped
Silica Gel for Chromatography, Carbamoylsilyl A very finely divided silica gel, chemically modified at the
A very finely divided silica gel, chemically modified at the surface by the introduction of dodecylsilyl groups.
surface by the bonding of carbamoylsilyl groups. To minimise any interaction with basic compounds, it is
carefully end-capped to cover most of the remaining silanol
Silica Gel for Chromatography Compatible with Highly groups.
Aqueous Mobile Phases, Octadecylsilyl Diol, End-
capped Silica Gel for Chromatography, Hexadecylamidylsilyl
A very finely divided silica gel, chemically modified at the A very finely divided (5 µm) silica gel, chemically modified at
the surface by the introduction of
surface by the bonding of octadecylsilyl groups and end-
capping. Free diol groups are also present. For use with hexadecylcarboxamidopropyldimethylsilyl groups.
highly aqueous mobile phases. Silica Gel for Chromatography, Hexadecylamidylsilyl,
Silica Gel for Chromatography, Crown-ether End-capped
A very finely divided (5 µm) silica gel, chemically modified at
Stationary phase for liquid chromatography.
the surface by the introduction of
Crown ether coated on silica gel. hexadecylcarboxamidopropyldimethylsilyl groups.
Silica Gel for Chromatography, Cyanopropylsilyl, End- To minimise any interaction with basic compounds it is
capped, Base-deactivated carefully end-capped to cover most of the remaining silanol
A very finely divided silica gel, pre-treated by various groups.
techniques before the bonding of cyanopropylsilyl groups. Silica Gel for Chromatography, Hexylsilyl
To minimise any interaction with basic compounds, it is A very finely divided silica gel, chemically modified at the
carefully end-capped to cover most of the remaining silanol surface by the bonding of hexylsilyl groups.
groups.
Silica Gel for Chromatography, Hexylsilyl, End-
Silica Gel for Chromatography, Cyanosilyl capped
A very finely divided silica gel chemically modified at the A very finely divided silica gel, chemically modified at the
surface by the bonding of cyanosilyl groups. surface by the bonding of hexylsilyl groups. To minimise any
Silica Gel for Chromatography, Cyanosilyl, End- interaction with basic compounds it is carefully end-capped
capped to cover most of the remaining silanol groups.
A very finely divided silica gel chemically modified at the
surface by the bonding of cyanosilyl groups. To minimise any
2023 Appendix I A V-A135

Silica Gel for Chromatography, Human Albumin Silica Gel for Chromatography, Nitrile
Coated A very finely divided silica gel, chemically modified at the
A very finely divided silica gel, chemically modified at the surface by the bonding of cyanopropylsilyl groups.
surface by the bonding of human albumin. Silica Gel for Chromatography,
Silica Gel for Chromatography (Hybrid Material), 4-Nitrophenylcarbamidesilyl
Octadecylsilyl, Ethylene-bridged, Charged Surface, A very finely divided silica gel, chemically modified at the
End-capped surface by bonding of 4-nitrophenylcarbamide groups.
Synthetic, spherical ethylene-bridged hybrid particles with a Silica Gel for Chromatography,
charged surface, containing both inorganic (silica) and Octadecanoylaminopropylsilyl
organic (organosiloxanes) components, chemically modified A very finely divided silica gel, chemically modified at the
at the surface by the bonding of octadecylsilyl groups. surface by the bonding of aminopropylsilyl groups which are
To minimise any interaction with basic compounds they are acylated with octadecanoyl groups.
carefully end-capped to cover most of the remaining silanol
groups. Silica Gel for Chromatography, Octadecylphenylsilyl,
End-capped
Silica Gel for Chromatography (hybrid material),
Octylsilyl, Ethylene-bridged, End-capped A very finely divided silica gel, chemically modified at the
surface by bonding of octadecylphenylsilyl groups.
Synthetic, spherical ethylene-bridged hybrid particles, To minimise any interaction with basic compounds it is
containing both inorganic (silica) and organic carefully end-capped to cover most of the remaining silanol
(organosiloxanes) components, chemically modified at the groups.
surface by the bonding of octylsilyl groups. To minimise any
interaction with basic compounds they are carefully end- Silica Gel for Chromatography, Octadecylsilyl
capped to cover most of the remaining silanol groups. A very finely divided silica gel, chemically modified at the
Silica Gel for Chromatography (Hybrid Material), surface by the bonding of octadecylsilyl groups.
Phenylhexylsilyl, Ethylene-bridged, Charged Surface, Silica Gel for Chromatography, Octadecylsilyl, Base-
End-capped deactivated
Synthetic, spherical ethylene-bridged hybrid particles with a A very finely divided silica gel, pretreated by various
charged surface, containing both inorganic (silica) and techniques before the bonding of octadecylsilyl groups to
organic (organosiloxanes) components, chemically modified minimise the interaction with basic components.
at the surface by the bonding of phenylhexylsilyl groups. Silica Gel for Chromatography, Octadecylsilyl, Cross-
To minimise any interaction with basic compounds they are linked, End-capped
carefully end-capped to cover most of the remaining silanol A very finely divided silica gel, chemically modified at the
groups. surface by the cross-linking and bonding of octadecylsilyl
Silica Gel for Chromatography (Hybrid Material), groups. To minimise any interaction with basic compounds it
Phenylsilyl, Ethylene-bridged, End-capped is carefully end-capped to cover most of the remaining silanol
Synthetic, spherical, ethylene-bridged hybrid particles, groups.
containing both inorganic (silica) and organic Silica Gel for Chromatography, Octadecylsilyl, End-
(organosiloxanes) components, chemically modified at the capped
surface by the bonding of phenylsilyl groups. To minimise A very finely divided silica gel, chemically modified at the
any interaction with basic compounds, they are carefully end- surface by the bonding of octadecylsilyl groups. To minimise
capped to cover most of the remaining silanol groups. any interaction with basic compounds it is carefully end-
Silica Gel for Chromatography (Hybrid Material), capped to cover most of the remaining silanol groups.
Polar-embedded, Octadecylsilyl, Ethylene-bridged, Silica Gel for Chromatography, Octadecylsilyl, End-
End-capped capped, Base-deactivated
Synthetic, spherical, ethylene-bridged hybrid particles A very finely divided silica gel, pretreated by various
containing both inorganic (silica) and organic techniques before the bonding of octadecylsilyl groups.
(organosiloxanes) components, chemically modified at the To further minimise any interaction with basic compounds, it
surface by the bonding of polar-embedded octadecylsilyl is carefully end-capped to cover most of the remaining silanol
groups. To minimise any interaction with basic compounds, groups.
they are carefully end-capped to cover most of the remaining
Silica Gel for Chromatography, Octadecylsilyl,
silanol groups. Ethylene-Bridged (Hybrid Material)
Silica Gel for Chromatography, Hydrophilic Synthetic, spherical, ethylene-bridged hybrid particles,
A very finely divided silica gel whose surface has been containing both inorganic (silica) and organic
modified to provide hydrophilic characteristics. (organosiloxanes) components, chemically modified at the
Silica Gel for Chromatography, Hydroxypropylsilyl surface by the bonding of octadecylsilyl groups. To minimise
A very finely divided silica gel, chemically modified at the any interaction with basic compounds, they are carefully end-
surface by the bonding of hydroxypropylsilyl groups. capped to cover most of the remaining silanol groups.
Silica Gel for Chromatography, Methylsilyl Silica Gel for Chromatography, Octadecylsilyl, Extra-
Liquid chromatographic reagent grade of commerce. dense Bonded, End-capped
A very finely divided silica gel (3 to l O µm) chemically A very finely divided silica gel, chemically modified at the
modified at the surface by the introduction of methylsilyl surface by the extra-dense bonding of octadecylsilyl groups.
groups. The particle size is specified after the name of the To minimise any interaction with basic compounds it is
reagent in tests where it is used. carefully end-capped to cover most of the remaining silanol
groups.
V-A136 Appendix I A 2023

Silica Gel for Chromatography, Octadecylsilyl, for Silica Gel for Chromatography, Octylsilyl and
Separation of Polycyclic Aromatic Hydrocarbons Octadecylsilyl Multi-Alkyl
A very finely divided ultrapure silica gel, chemically modified High purity base deactivated silica (10 µm particle sizes)
at the surface by the bonding of octadecylsilyl groups, chemically modified at the surface with binding of octylsilyl
optimised for the analysis of polycyclic aromatic and octadecylsilyl groups.
hydrocarbons. Silica Gel for Chromatography, Octylsilyl, Base-
Silica gel for Chromatography, Octadecylsilyl, deactivated
Monolithic A very finely divided silica gel, pretreated by various
Monolithic rods of highly porous (greater than 80 per cent) techniques before the bonding of octylsilyl groups to
metal-free silica with a bimodal pore structure, modified at minimise the interaction with basic components.
the surface by the bonding of octadecylsilyl groups. Silica Gel for Chromatography, Octylsilyl, End-
Silica Gel for Chromatography, Octadecylsilyl, capped
Monolithic, End-capped A very finely divided silica gel, chemically modified at the
Monolithic rods of highly porous (greater than 80 per cent) surface by the bonding of octylsilyl groups. To minimise any
metal-free silica with a bimodal pore structure, modified at interaction with basic compounds, it is carefully end-capped
the surface by the bonding of octadecylsilyl groups. to cover most of the remaining silanol groups.
To minimise any interaction with basic compounds, they are Silica Gel for Chromatography, Octylsilyl, End-
carefully end-capped to cover most of the remaining silanol capped, Base-deactivated
groups.
A very finely divided silica gel, pre-treated by various
Silica Gel for Chromatography, Octadecylsilyl, Polar- techniques before the bonding of octylsilyl groups. To further
embedded, Encapsulated minimise any interaction with basic compounds it is carefully
Silica gel chemically modified at the surface by the bonding end-capped to cover most of the remaining silanol groups.
of polar-embedded octadecylsilyl groups. To minimise any Silica Gel for Chromatography, Octylsilyl, Extra-dense
interaction with basic compounds, it is carefully encapsulated Bonded, End-capped
to cover most of the remaining silanol groups.
A very finely divided silica gel, chemically modified at the
Silica Gel for Chromatography, Octadecylsilyl, Polar surface by the extra-dense bonding of octylsilyl groups.
End-capped To minimise any interaction with basic compounds, it is
A very finely divided silica gel, chemically modified at the carefully end-capped to cover most of the remaining silanol
surface by the bonding of octadecylsilyl groups. To minimise groups.
any interaction with basic compounds it is carefully polar Silica Gel for Chromatography, Octylsilyl, Solid
end-capped to cover most of the remaining silanol groups. Core
Silica Gel for Chromatography, Octadecylsilyl, Solid Silica gel with spherical particles containing a non-porous
Core Silica gel with spherical silica particles containing a solid silica core surrounded by a thin outer porous silica
non-porous solid silica core surrounded by a thin outer coating with octylsilyl groups.
porous silica coating with octadecylsilyl groups; Silica Gel
Silica Gel for Chromatography, Octylsilyl, Solid Core,
for Chromatography, Octadecylsilyl, Solid Core, End- End-capped
capped
Silica gel with spherical silica particles containing a non-
Silica gel with spherical silica particles containing a non-
porous solid silica core surrounded by a thin outer porous
porous solid silica core surrounded by a thin outer porous
silica coating with octylsilyl groups. To minimise any
silica coating with octadecylsilyl groups. To minimise any
interaction with basic compounds it is carefully end-capped
interaction with basic compounds it is carefully end-capped
to cover most of the remaining silanol groups.
to cover most of the remaining silanol groups.
Silica Gel for Chromatography, Octylsilyl, with
Silica Gel for Chromatography, Octadecylsilyl, with
Embedded Polar Groups, End-capped
Embedded Polar Groups, End-capped
A very finely divided silica gel, chemically modified at the
A very finely divided silica gel, chemically modified at the
surface by the bonding of polar-embedded octylsilyl groups.
surface by the bonding of polar-embedded octadecylsilyl
To minimise any interaction with basic compounds, it is
groups. To minimise any interaction with basic compounds,
carefully end-capped to cover most of the remaining silanol
it is carefully end-capped to cover most of the remaining
groups.
silanol groups.
Silica Gel for Chromatography, Oxypropionitrilsilyl
Silica Gel for Chromatography, Octadecylsilyl, with
Extended pH Range, End-Capped A very finely divided silica gel chemically modified at the
surface by the bonding of oxypropionitrilsilyl groups.
A very finely divided silica gel, chemically modified at the
surface by the bonding of octadecylsilyl groups resistant to Silica Gel for Chromatography,
bases up to pH 11. To minimise any interaction with basic Palmitamidopropylsilyl, End-capped
compounds it is carefully end-capped to cover most of the A very finely divided silica gel, chemically modified at the
remaining silanol groups. surface by the bonding of palmitamidopropyl groups and
Silica Gel for Chromatography, Octylsilyl end-capped with acetamidopropyl groups.
A very finely divided silica gel, chemically modified at the Silica Gel for Chromatography,
surface by the bonding of octylsilyl groups. Pentafluorophenylpropylsilyl, Solid Core, End-
capped
Silica gel with spherical silica particles containing a non-
porous solid silica core surrounded by a thin outer porous
silica coating with pentafluorophenylpropylsilyl groups.
2023 Appendix I A V-A137

To minimise any interaction with basic compounds it is Silica Gel for Chromatography, Propylsilyl
carefully end-capped to cover most of the remaining silanol A very finely divided silica gel, chemically modified at the
groups. surface by the bonding of propylsilyl groups.
Silica Gel for Chromatography, Phenyl Silica Gel for Chromatography Compatible with 100%
Liquid chromatographic reagent grade of commerce. Aqueous Mobile Phases, Octadecylsilyl, End-capped
A very finely divided silica gel, chemically modified at the A very finely divided silica gel with bonded octadecylsilyl
surface by the bonding of phenyl groups. The particle size is groups suitable for use with highly aqueous mobile phases
indicated after the name of the reagent in the tests where it is including 100 per cent aqueous phases. To minimise any
used. interaction with basic compounds it is carefully end-capped
Silica Gel for Chromatography, Phenylethyl, End- to cover most of the remaining silanol groups.
capped Silica Gel for Chromatography compatible with
Liquid chromatographic reagent grade of commerce. 100 per cent Aqueous Mobile Phases, Octadecylsilyl
A very finely divided silica gel (2.5 to 10 µm) chemically A very finely divided silica gel with bonded octadecylsilyl
modified at the surface by the introduction of ether-linked groups suitable for use with highly aqueous mobile phases
phenyl groups. To minimise any interaction with basic including 100 per cent aqueous phases.
compounds it is carefully end-capped to cover most of the Silica Gel for Chromatography Rl, Nitrile
remaining silanol groups. The particle size is specified after A very finely divided silica gel consisting of porous, spherical
the name of the reagent in tests where it is used. particles with chemically bonded nitrite groups.
Silica Gel for Chromatography, Phenylhexylsilyl Silica Gel for Chromatography Rl, Octadecylsilyl
A very finely divided silica gel, chemically modified at the A very finely divided ultrapure silica gel, chemically modified
surface by the bonding of phenylhexyl groups. at the surface by the bonding of octadecylsilyl groups.
Silica Gel for Chromatography, Phenylhexylsilyl, End- Silica Gel for Chromatography R2, Octadecylsilyl
capped A very finely divided (15 nm pore size) ultrapure silica gel,
A very finely divided silica gel, chemically modified at the chemically modified at the surface by the bonding of
surface by the bonding of phenylhexylsilyl groups. octadecylsilyl groups (20 per cent carbon load), optimised for
To minimise any interaction with basic compounds it is the analysis of polycyclic aromatic hydrocarbons.
carefully end-capped to cover most of the remaining silanol Silica Gel for Chromatography Rl, Octadecylsilyl,
groups. End-capped
Silica Gel for Chromatography, Phenylhexylsilyl, Solid A very finely divided ultrapure silica gel, chemically modified
Core, End-capped at the surface by the bonding of octadecylsilyl groups.
Silica gel with spherical silica particles containing a non- To minimise any interaction with basic compounds it is
porous solid silica core surrounded by a thin outer porous carefully end-capped to cover most of the remaining silanol
silica coating with phenylhexylsilyl groups. To minimise any groups.
interaction with basic compounds it is carefully end-capped Silica Gel for Chromatography Rl, Octadecylsilyl,
to cover most of the remaining silanol groups. End-capped, Base-deactivated
Silica Gel for Chromatography, Phenylsilyl A very finely divided silica gel pre-treated before the bonding
A very finely divided silica gel, chemically modified at the of octadecylsilyl groups by washing and hydrolysing most of
surface by the bonding of phenyl groups. the superficial siloxane bridges. To further minimise any
Silica Gel for Chromatography, Phenylsilyl, End- interaction with basic compounds it is carefully end-capped
capped to cover most of the remaining silanol groups.
A very finely divided silica gel, chemically modified at the Silica Gel for Chromatography Rl, Octylsilyl
surface by the bonding of phenyl groups. To minimise any A very finely divided silica gel, chemically modified at the
interaction with basic compounds it is carefully end-capped surface by the bonding of octylsilyl and methyl groups
to cover most of the remaining silanol groups. (double bonded phase).
Silica Gel for Chromatography, Phenylsilyl, End- Silica Gel for Chromatography R2, Octylsilyl
Capped, Base-Deactivated Ultrapure very finely divided (10 nm pore size) silica gel,
A very finely divided silica gel, pre-treated by various chemically modified at the surface by the bonding of
techniques before the bonding of phenylsilyl groups. octylsilyl groups (19 per cent carbon load). Less than
To further minimise any interaction with basic compounds it 20 ppm of metals.
is carefully end-capped to cover most of the remaining silanol Silica Gel for Chromatography R3, Octylsilyl
groups.
A very finely divided ultrapure silica gel, chemically modified
Silica Gel for Chromatography, Phenylsilyl, Extra- at the surface by the bonding of octylsilyl groups and
dense Bonded, End-capped sterically protected with branched hydrocarbons at the
A very finely divided silica gel, chemically modified at the silanes.
surface by the extra-dense bonding of phenylsilyl groups. Silica Gel for Chromatography, Anion-exchange,
To minimise any interaction with basic compounds, it is Strong
carefully end-capped to cover most of the remaining silanol
A very finely divided silica gel, chemically modified at the
groups.
surface by the bonding of quaternary ammonium groups.
Silica Gel for Chromatography, Propoxybenzene, End-
pH limit of use: 2 to 8.
capped
A very finely divided silica gel, chemically modified at the
surface by the bonding of propoxybenzene groups.
V-A138 Appendix I A 2023

Silica Gel for Chromatography, Strong-anion- Calcium sulfate content. Place 0.25 g in a ground-glass
exchange stoppered flask, add 3 mL of dilute hydrochloric acid R and
A very finely divided silica gel, chemically modified at the 100 mL of water R and shake vigorously for 30 min. Filter
surface by the bonding of quaternary ammonium groups. through a sintered-glass filter (2.1.2) and wash the residue.
Silica Gel for Chromatography, Strong Cation- Carry out on the combined filtrate and washings the
complexometric assay of calcium (2. 5.11).
exchange
A very finely divided silica gel, chemically modified at the 1 mL of 0.1 M sodium edetate is equivalent to 14.51 mg of
surface by the bonding of sulfonic acid groups. CaSO4, 1/2H2O.
Silica Gel for Chromatography, Trimethylsilyl pH (2.2.3). Shake 1 g for 5 min with 10 mL of carbon
dioxide-free water R. The pH of the suspension is about 7.
A very finely divided silica gel, chemically modified at the
surface by the bonding of trimethylsilyl groups.
Silica Gel GF254 (112926-00-8)
Contains about 13 per cent of calcium sulfate hemihydrate
Crown-ether Silica Gel for Chiral Separation
and about 1.5 per cent of a fluorescent indicator having an
A very finely divided silica gel for chromatography coated
optimal intensity at 254 nm. The particle size is about
with the following chiral crown ether: 15 µm.
Calcium sulfate content. Determine by the method prescribed
for silica gel G R.
pH. Complies with the test prescribed for silica gel G R.
Fluorescence. Thin-layer chromatography (2.2.27) using silica
gel GF2s4 R as the coating substance. Apply separately to the
plate at ten points increasing volumes from 1 µL to 1O µL of
a 1 g/L solution of benzoic acid R in a mixture of 10 volumes
of anhydrous formic acid R and 90 volumes of 2-propanol R.
Develop over a path of 10 cm with the same mixture of
solvents. After evaporating the solvents examine the
chromatogram in ultraviolet light at 254 nm. The benzoic
acid appears as dark spots on a fluorescent background in the
upper third of the chromatogram for quantities of 2 µg and
(Ra)-6,23-Diphenyl-8,9,11, 12,14, 15,17, 18,20,21-
greater.
decahydrodinaphtho [2, 1-q: 1 ',2 '-s]
[1,4, 7, 1O,l 3,16]hexaoxacycloicosine. Silica Gel H (112926-00-8)
Silica Gel for HPTLC, Octadecylsilyl The particle size is of about 15 µm.
A finely divided silica gel, chemically modified at the surface pH (2.2.3). Complies with the test prescribed for silica
by the bonding of octadecylsilyl groups. gel GR.
Fine, white or almost white, homogeneous powder, Silica Gel H, Silanised
practically insoluble in water and in ethanol (96%). Preparation of a thin layer. See silanised silica gel HF254 R
Silica Gel for Size-exclusion Chromatography Chromatographic separation. Complies with the test prescribed
A very finely divided silica gel (10 µm) with a very for silanised silica gel HF254 R.
hydrophilic surface. The average diameter of the pores is Silica Gel HF254
about 30 nm. It is compatible with aqueous solutions Contains about 1.5 per cent of a fluorescent indicator having
between pH 2 and 8 and with organic solvents. It is suitable an optimal intensity at 254 nm. The particle size is about
for the separation of proteins with relative molecular masses 15 µm.
of 1 x 10 3 to 3 x 10 5 • pH. Complies with the test prescribed for silica gel G R.
Silica Gel F 254 Fluorescence. Complies with the test prescribed for silica
A fine, white, homogeneous powder of an average particle gel GF2 s4 R.
size of about 15 µm containing a suitable binding agent and Silica Gel HF254 , Silanised
about 1.5% of a florescent indicator having a maximum
Contains about 1.5 per cent of a fluorescent indicator having
intensity at 254 nm. It complies with the following
an optimal intensity at 254 nm.
requirement.
Preparation of a thin layer. Vigorously shake 30 g for 2 min
Fluorescence Carry out the method for thin-layer
with 60 mL of a mixture of 1 volume of methanol R and
chromatography using a mixture of 10 volumes of anhydrous
2 volumes of water R. Coat carefully cleaned plates with a
fomiic acid and 90 volumes of propan-2-ol as the mobile
layer 0.25 mm thick using a spreading device. Allow the
phase, but allowing the solvent front to ascend 10 cm above
coated plates to dry in air and then heat in an oven at
the line of application. Apply separately to the plate
100 °C to 105 °C for 30 min.
10 quantities from 1 to 10 uL of a 0.1 % w/v solution of
benzoic acid in the mobile phase. After removal of the plate, Chromatographic separation. Introduce 0.1 g each of methyl
dry it in a current of warm air and examine under ultraviolet laurate R, methyl myristate R, methyl palmitate R and methyl
light (254 nm). The benzoic acid appears as dark spots on a stearate R into a 250 mL conical flask. Add 40 mL of
fluorescent background in the upper third of the alcoholic potassium hydroxide solution R and heat under a reflux
chromatogram at levels of 2 µg and greater. condenser on a water-bath for 1 h. Allow to cool, transfer the
solution to a separating funnel by means of 100 mL of
Silica Gel G (112926-00-8)
water R, acidify (pH 2 to 3) with dilute hydrochloric acid Rand
Contains about 13 per cent of calcium sulfate hemihydrate. shake with three quantities, each of 10 mL of chloroform R.
The particle size is about 15 µm. Dry the combined chloroform extracts over anhydrous sodium
2023 Appendix I A V-A139

sulfate R, filter and evaporate to dryness on a water-bath. add 8 mL of a 200 g/L solution of silver nitrate R, dropwise,
Dissolve the residue in 50 mL of chloroform R. Examine by with stirring. Dilute to 200 mL with water R.
thin-layer chromatography (2.2.27), using silanised silica Silver Nitrate Solution
gel HF254 as the coating substance. Apply to the plate at
A freshly prepared 5.0% w/v solution of silver nitrate.
each of three separate points I 0 µL of the chlorofonnic
solution. Develop over a path of 14 cm with a mixture of Store protected from light.
10 volumes of glacial acetic acid R, 25 volumes of water R and Silver Nitrate Solution, Ammoniacal
65 volumes of dioxan R. Dry the plate at 120 °C for 30 min. Dissolve 2.5 g of silver nitrate R in 80 mL of water Rand add
Allow to cool, spray with a 35 g/L solution of phosphomolybdic dilute ammonia Rl dropwise until the precipitate has
acid R in 2-propanol R and heat at 150 °C until the spots dissolved. Dilute to 100 mL with water R. Prepare
become visible. Treat the plate with ammonia vapour until immediately before use.
the background is white. The chromatograms show four Silver Nitrate Solution in Pyridine
clearly separated, well-defined spots.
An 85 g/L solution of silver nitrate R in pyridine R.
Silica Gel OD for Chiral Separations Storage: protected from light.
See Cellulose derivative of silica gel for chiral separation R
Silver Nitrate Solution Rl
Silicotungstic Acid Dodecatungstosilicic acid; A 42.5 g/L solution of silver nitrate R.
H 4SiW 12O 40 ,xH2O (11130-20-4)
Storage: protected from light.
White or yellowish-white crystals, deliquescent, very soluble
in water and in ethanol (96 per cent). Silver Nitrate Solution R2
Storage: in an airtight container. A 17 g/L solution of silver nitrate R.
Silicristin (2R,3R)-3,5, 7-Trihydroxy-2-[ (2R,3S)-7- Storage: protected from light.
hydroxy-2-( 4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl- Silver Oxide Disilver oxide; Ag2O = 231.7 (20667-12-3)
2,3-dihydro- l -benzofuran-5-yl] chroman-4-one; Brownish-black powder, practically insoluble in water and in
C 25H 22 O 10 = 482.4 (33889-69-9) ethanol (96 per cent), freely soluble in dilute nitric acid and
White or yellowish powder, practically insoluble in water, in ammonia.
soluble in acetone and in methanol. Storage: protected from light.
Silidianin (3R,3aR,6R, 7aR,8R)-7 a-Hydroxy-8-(4-hydroxy- Silver Sulfate Ag2SO 4 = 311.8 (10294-26-5)
3-methoxyphenyl)-4-[(2R, 3R)-3,5, 7-trihydroxy-4- Content: minimum 99.0 per cent.
oxochroman-2-yl]-2,3,3a, 7a-tetrahydro-3,6-methano-l-
White or light grey powder, slightly soluble in water.
benzofuran-7 ( 6aH)-one; C 25 H 22 O 10 = 482.4 (29782-68-1)
mp: about 652 °C.
White or yellowish powder, practically insoluble in water,
soluble in acetone and in methanol. Storage: protected from light.
Silver Diethyldithiocarbamate Silver Sinensetin 3 ',4',5,6, 7-Pentamethoxyflavone;
diethylcarbamodithioate; C 5H 10AgNS 2 = 256.1 (1470-61-7) C 20 H2 0 O1 = 372.4 (2306-27-6)
Pale-yellow or greyish-yellow powder, practically insoluble in White or almost white, crystalline powder, practically
water, soluble in pyridine. insoluble in water, soluble in ethanol (96 per cent).
Storage below 8 °C is recommended. mp: about 177 °C.
It may be prepared as follows. Dissolve 1. 7 g of silver Absorbance (2.2.25). A solution in methanol R shows
nitrate R in 100 mL of water R. Separately dissolve 2.3 g of 3 absorption maxima, at 243 nm, 268 nm and 330 nm.
sodium diethyldithiocarbamate R in 100 mL of water R. Cool Assay. Liquid chromatography (2.2.29) as prescribed in the
both solutions to 10 °C, then mix, and while stirring, collect monograph Java tea (1229).
the yellow precipitate on a sintered-glass filter (16) (2. J.2) Content: minimum 95 per cent, calculated by the
and wash with 200 mL of cold water R. Dry the precipitate normalisation procedure.
in vacuo for 10 h (2.2.32). Sinomenine 7,8-Didehydro-4-hydroxy-3, 7-dimethoxy-l 7-
Silver Diethyldithiocarbamate Solution methyl-9o:, 13a, 14a-morphinan-6-one; Cucoline;
Prepare the solution immediately before use. Dissolve 0.100 g of C19H23NO4 = 329.4 (115-53-7)
silver diethyldithiocarbamate R in pyridine R and dilute to Sirolimus Rapamycin; C 51 H 79NO 13 = 914 (53123-88-9)
20.0 mL with the same solvent. mp: 183 °C to 185 °C.
Suitability test. The solution is clear (2.2. J). The absorbance Sitostanol Dihydro-P-sitosterol; C 29 H 52 O = 416.7
(2.2.25) of the solution is maximum 0.20 at 450 nm, (19466-47-8)
maximum 0.01 at 510 nm and maximum 0.010 at 538 nm.
Content: minimum 95.0 per cent.
Silver Manganese Paper
P-Sitosterol Stigrnast-5-en-3P-ol;
Immerse strips of slow filter paper into a solution containing 22,23-Dihydrostigrnasterol; C 29 H 50 O = 414.7 (83-46-5)
8.5 g/L of manganese sulfate Rand 8.5 g/L of silver nitrate R.
White or almost white powder, practically insoluble in water,
Maintain for a few minutes and allow to dry over an
sparingly soluble in tetrahydrofuran.
appropriate desiccant, protected from acid and alkaline
vapours. Content: minimum 75.0 per cent mlm (dried substance).
Silver Nitrate (7761-88-8) Assay. Gas chromatography (2.2.28), as prescribed in the
monograph Phytosterol (1911).
See Silver nitrate (0009).
Test solution. Dissolve 0.100 g of the substance to be
Silver Nitrate Reagent
examined in tetrahydrofuran R and dilute to 10.0 mL with the
Prepare immediately before use. To a mixture of 3 mL of same solvent. Introduce 100 µL of this solution into a
concentrated ammonia R and 40 mL of 1 M sodium hydroxide, suitable 3 mL flask and evaporate to dryness under
V-A140 Appendix I A 2023

nitrogen R. To the residue add 100 µL of a freshly prepared Sodium Carbonate (6132-02-1)
mixture of 50 µL of 1-methylimidazole Rand 1.0 mL of See Sodium carbonate decahydrate (0191).
heptafiuoro-N-methyl-N-(trimethylsilyObutanamide R. Close the
Sodium Carbonate, Anhydrous Disodium carbonate;
flask tightly and heat at 100 °C for 15 min. Allow to cool. Na2 CO3 = 106.0 (497-19-8)
Injection: l µL of the test solution. White or almost white powder, hygroscopic, freely soluble in
Sodium Na= 22.99 (7440-23-5) water.
A metal whose freshly cut surface is bright silver-grey. When heated to about 300 °C it loses not more than
It rapidly tarnishes in contact with air and is oxidised 1 per cent of its mass.
completely to sodium hydroxide and converted to sodium Storage: in an airtight container.
carbonate. It reacts violently with water, yielding hydrogen
and a solution of sodium hydroxide; soluble in anhydrous Sodium Carbonate Monohydrate (5968-11-6)
methanol, yielding hydrogen and a solution of sodium See Sodium carbonate monohydrate (0192).
methoxide; practically insoluble in light petroleum. Sodium Carbonate Solution
Storage: under light petroleum or liquid paraffin. A 106 glL solution of anhydrous sodium carbonate R.
Sodium Acetate (6131-90-4) Sodium Carbonate Solution, Dilute
See Sodium acetate trihydrate (0411). A 10% w/v solution of sodium carbonate.
Sodium Acetate, Anhydrous C 2 H3NaO2 = 82.0 Sodium Carbonate Solution Rl
(127-09-3) A 20 g/L solution of anhydrous sodium carbonate R in 0.1 M
Colourless crystals or granules, very soluble in water, sodium hydroxide.
sparingly soluble in ethanol (96 per cent). Sodium Carbonate Solution R2
Loss on drying (2.2.32). Not more than 2.0 per cent, A 40 g/L solution of anhydrous sodium carbonate R in 0.2 M
determined by drying in an oven at 105 °C. sodium hydroxide.
Sodium Arsenite Sodium metaarsenite; NaAsO 2 = 129.9 Sodium Cetostearyl Sultate See Sodium cetostearyl
(7784-46-5) sulfate (0847).
Sodium Arsenite Solution Sodium Chloride (7647-14-5)
Dissolve 5.0 g of sodium arsenite R in 30 mL of 1 M sodium See Sodium chloride (0193).
hydroxide. Cool to O °C and add, while stirring, 65 mL of
Sodium Chloride Solution
dilute hydrochloric acid R.
A 20 per cent m/m solution of sodium chloride R.
Sodium Ascorbate Solution (134-03-2)
Sodium Chloride Solution, Saturated
Dissolve 3.5 g of ascorbic acid R in 20 mL of 1 M sodium
hydroxide. Prepare immediately before use. Mix 1 part of sodium chloride R with 2 parts of water R, shake
from time to time and allow to stand. Before use, decant the
Sodium Azide NaN 3 = 65.0 (26628-22-8) solution from any undissolved substance and filter, if
White or almost white, crystalline powder or crystals, freely necessary.
soluble in water, slightly soluble in ethanol (96 per cent).
Sodium Cholate Cholic acid sodium salt hydrate;
Sodium Benzenesulfonate C 6 H 5 SO3Na = 180.16 C 24H 39NaO 5 = 430.5 (73163-53-8)
(515-42-4)
[et]~: +31.3 (0.5% w/v in water).
White crystalline powder, soluble in water.
General reagent grade of commerce.
Sodium Bicarbonate (144-55-8) Sodium Citrate Trisodium citrate; (6132-04-3)
See sodium hydrogen carbonate R.
See Sodium citrate (0412).
Sodium Bismuthate NaBiO 3 = 280.0 (12232-99-4) Sodium Cobaltinitrite Trisodium hexanitrocobaltate(III);
Content: minimum 85.0 per cent. Na 3[Co(NO 2 ) 6] = 403.9 (13600-98-1)
Yellow or yellowish-brown powder, slowly decomposing Orange-yellow powder, freely soluble in water, slightly
when moist or at a high temperature, practically insoluble in soluble in ethanol (96 per cent).
cold water. Sodium Cobaltinitrite Solution Sodium
Assay. Suspend 0.200 gin 10 mL of a 200 glL solution of hexanitritocobaltate(m)
potassium iodide R and add 20 mL of dilute suljuric acid R.
A 100 glL solution of sodium cobaltinitrite R. Prepare
Using 1 mL of starch solution Ras indicator, titrate with
immediately before use.
0.1 M sodium thiosulfate until an orange colour is obtained.
Sodium Decanesulfonate Sodium decanesulfonate;
1 mL of 0.1 M sodium thiosulfate is equivalent to 14. 00 mg of
C 10H 21 NaO 3S = 244.3 (13419-61-9)
NaBiO 3 •
Crystalline powder or flakes, white or almost white, freely
Sodium Bromide (7647-15-6)
soluble in water, soluble in methanol.
See Sodium bromide (0190).
Sodium Decyl Sulfate Sodium decyl sulfate;
Sodium Butanesulfonate 1-Butanesulfonic acid sodium C 10H 21 NaO 4 S = 260.3 (142-87-0)
salt; 1-Butanesulfonic acid sodium salt; Sodium
Content: minimum 95.0 per cent.
butanesulfonate; C 4 H 9 NaO 3 S = 160.2 (2386-54-1)
White or almost white powder, freely soluble in water.
White or almost white, crystalline powder, soluble in water.
Sodium Deoxycholate Deoxycholic acid, sodium salt;
mp: greater than 300 °C.
C 24 H 39NaO 4 = 414.6 (302-95-4)
Sodium Calcium Edetate (62-33-9)
Sodium Deoxyribonucleate About 85 per cent has a
See sodium calcium edetate (0231). relative molecular mass of 2 x 107 or greater (73049-39-5)
2023 Appendix I A V-Al41

White or almost white, fibrous preparation obtained from calf When used in the Assay of preparations containing Sodium
thymus. Fluoride, use a grade containing not less than 99.9% ofNaF.
Test for suitability. Dissolve 10 mg in imidazole buffer solution Sodium Formate Sodium methanoate; CHNaO 2 = 68.0
pH 6.5 Rand dilute to 10.0 mL with the same buffer (141-53-7)
solution (solution A). Dilute 2.0 mL of solution A to White or almost white, crystalline powder or deliquescent
50.0 mL with imidazole buffer solution pH 6.5 R. granules, soluble in water and in glycerol, slightly soluble in
The absorbance (2. 2. 25) of the solution, measured at ethanol (96 per cent).
260 nm, is 0.4 to 0.8. mp: about 253 °C.
To 0.5 mL of solution A add 0.5 mL of imidazole buffer Sodium Glucuronate D-Glucuronic acid, sodium salt;
solution pH 6. 5 R and 3 mL of perchloric acid (25 g/L
C 6H 9NaO 7,H2 O = 234.1
HC1O 4 ). A precipitate is formed. Centrifuge. The absorbance
of the supernatant, measured at 260 nm using a mixture of [(X]~: about+ 21.5, determined on a 20 g/L solution.
1 mL of imidazole buffer solution pH 6.5 Rand 3 mL of Sodium Glycocholate Sodium ((3,7,12-trihydroxy-5-
perchloric acid (25 g/L HC1O4 ) as compensation liquid, is cholan-24-oyl)amino] acetate dihydrate; N-[ (3,5, 7, 12)-3, 7, 12-
not greater than 0.3. Trihydroxy-24-oxocholan-24-yl]glycine monosodium salt
In each of two tubes, place 0.5 mL of solution A and 0.5 mL dihydrate; C 26 H 42 NNaO 6 ,2H 2 O = 523.6 (207300-80-9)
of a solution of a reference preparation of streptodornase Content: minimum 97 per cent of C 2 6H 42NNaO 6 ,2HzO.
containing 10 IU/mL in imidazole buffer solution pH 6.5 R. Sodium Heptanesulfonate 1-Heptanesulfonic acid
To one tube add immediately 3 mL of perchloric acid sodium salt; 1-Heptanesulphonic acid sodium salt; Sodium
(25 g/L HC1O 4 ). A precipitate is formed. Centrifuge and heptanesulphonate; C 7 H 15NaO 3 S = 202.3 (22767-50-6)
collect supernatant A. Heat the other tube at 37 °C for White or almost white, crystalline mass, freely soluble in
15 min and add 3 mL of perchloric acid (25 g/L HC1O 4). water, soluble in methanol.
Centrifuge and collect supernatant B. The absorbance of
Sodium Heptanesulfonate Monohydrate
supernatant B, measured at 260 nm with reference to
1-Heptanesulfonic acid sodium salt monohydrate;
supernatant A is not less than 0.15.
1-Heptanesulphonic acid sodium salt monohydrate; Sodium
Sodium Diethyldithiocarbamate heptanesulphonate monohydrate; C1H 15NaO 3S,H2O = 220.3
C 5 H 10NNaS 2,3H2O = 225.3 (20624-25-3)
Content: minimum 96 per cent (anhydrous substance).
White or almost white or colourless crystals, freely soluble in
White or almost white, crystalline powder, soluble in water,
water, soluble in ethanol (96 per cent). The aqueous solution
very slightly soluble in anhydrous ethanol.
is colourless.
Water (2.5.12): maximum 8 per cent, determined on 0.300 g.
Sodium Diethyldithiocarbamate Solution
Assay. Dissolve 0.150 gin SO mL of anhydrous acetic acid R.
A 0.1 % w/v solution of sodium diethyldithiocarbamate in water.
Titrate with 0.1 M perchloric acid, determining the end-point
Prepare immediately before use. potentiometrically (2.2.20).
Sodium Dihydrogen Orthophosphate Sodium 1 mL of 0.1 M perchloric acid is equivalent to 20.22 mg of
dihydrogen phosphate dihydrate; Sodium Dihydrogen C1H1sNaO3S.
Phosphate; (13472-35-0)
Sodium Hexanesulfonate Hexanesulfonic acid sodium
See Sodium dihydrogen phosphate dihydrate (0194). salt; Hexanesulphonic acid sodium salt; Sodium
Sodium Dihydrogen Orthophosphate, Anhydrous hexanesulphonate; C 6H 13 NaO 3S = 188.2 (2832-45-3)
Sodium dihydrogen phosphate, anhydrous; White or almost white powder, freely soluble in water.
NaH 2 PO 4 = 120.0 (7558-80-7)
Sodium Hexanesulfonate Monohydrate Hexanesulfonic
White or almost white powder, hygroscopic. acid sodium salt monohydrate; Sodium hexanesulfonate
Storage: in an airtight container. monohydrate; C 6H 13NaO 3 S,H2O = 206.2 (207300-91-2)
Sodium Dihydrogen Orthophosphate Monohydrate White or almost white powder, soluble in water.
Sodium dihydrogen phosphate monohydrate; NaH2PO4, Sodium Hexanesulfonate Monohydrate for Ion-pair
H 2 O = 138.0 (10049-21-5) Chromatography C 6 H 13NaO 3S,H2O = 206.2
White or almost white, slightly deliquescent crystals or (207300-91-2)
granules, freely soluble in water, practically insoluble in Content: minimum 99.0 per cent.
ethanol (96 per cent).
Sodium Hydrogen Carbonate Sodium bicarbonate;
Storage: in an airtight container. (144-55-8)
Sodium Dithionite Na 2 S 2 O 4 = 174.1 (7775-14-6) See Sodium hydrogen carbonate (0195).
White or greyish-white, crystalline powder, oxidises in air, Sodium Hydrogen Carbonate Solution
very soluble in water, slightly soluble in ethanol
A 42 g/L solution of sodium hydrogen carbonate R.
(96 per cent).
Sodium Hydrogen Sulfate Sodium bisulphate; Sodium
Storage: in an airtight container.
hydrogen sulphate; NaHSO 4 = 120.1 (7681-38-1)
Sodium Dodecyl Sulfate Sodium dodecyl sulphate; Freely soluble in water, very soluble in boiling water.
Sodium lauryl sulfate; Sodium lauryl sulphate; Sodium
It decomposes in ethanol (96 per cent) into sodium sulfate
laurilsulfate; (151-21-3)
and free sulfuric acid.
See Sodium laurilsulfate (0098).
mp: about 315 °C.
Content: minimum 99.0 per cent.
Sodium Hydrogensulfite Sodium hydrogensulphite;
Sodium Fluoride (7681-49-4) NaHO 3S = 104.1 (7631-90-5)
See Sodium fluoride (0514).
V-A142 Appendix I A 2023

White or almost white, crystalline powder, freely soluble in into the flask and titrate with 0.1 M sodium thiosulfate, using
water, sparingly soluble in ethanol (96 per cent). 1 mL of starch solution R as indicator.
On exposure to air, some sulfur dioxide is lost and the 1 mL of 0.1 M sodium thiosulfate is equivalent to 3.546 mg of
substance is gradually oxidated to sulfate. active chlorine.
Sodium Hydroxide (1310-73-2) Storage: protected from light.
See Sodium hydroxide (0677). see Strong Sodium Hypochlorite Solution
2M Sodium Hydroxide Sodium Hypochlorite Solution, Dilute
Dissolve 84 g of sodium hydroxide R in carbon dioxide-free Dilute 35 mL of sodium hypochlorite solution to 100 mL with
water Rand dilute to 1000.0 mL with the same solvent. water immediately before use.
4M Sodium Hydroxide The solution contains approximately 3.5% w/v of available
Dissolve 168 g of sodium hydroxide R in carbon dioxide-free chlorine.
water R and dilute to 1.0 L with the same solvent. Sodium Hypophosphite Sodium phosphinate
Sodium Hydroxide, Ethanolic monohydrate; NaH 2PO 2,H2O = 106.0 (J 0039-56-2)
Solutions of the requisite molarity may be obtained by White or almost white, crystalline powder or colourless
dissolving the appropriate amount of sodium hydroxide in crystals, hygroscopic, freely soluble in water, soluble in
sufficient ethanol (96%) to produce 1000 mL. ethanol (96 per cent).
Sodium Hydroxide, Methanolic Storage: in an airtight container.
Solutions of the requisite molarity may be obtained by Sodium Iodide (7681-82-5)
dissolving the appropriate amount of sodium hydroxide in See Sodium iodide (0196).
sufficient methanol to produce 1000 mL. Sodium Iodobismuthate Solution
Sodium Hydroxide Solution Boil for a few minutes a mixture of 2.6 g of bismuth
Dissolve 20.0 g of sodium hydroxide R in water Rand dilute to oxycarbonate, 7.0 g of sodium iodide and 25 mL of glacial acetic
100.0 mL with the same solvent. Verify the concentration by acid. Allow to stand for 12 hours and filter, if necessary,
titration with 1 M hydrochwric acid, using methyl orange through sintered glass. To 20 mL of the filtrate add 80 mL
solution R as indicator, and adjust if necessary to 200 g/L. of ethyl acetate (solution A). Immediately before use, mix
Sodium Hydroxide Solution, Carbonate-free 2 mL of solution A, 20 mL of glacial acetic acid and 40 mL
Dissolve sodium hydroxide R in carbon dioxide-free water R to of ethyl acetate.
give a concentration of 500 g/L and allow to stand. Decant For use in connection with thin-layer chromatography the
the clear supernatant, taking precautions to avoid the sensitivity may be increased by spraying first with this
introduction of carbon dioxide. solution and then with sulfuric acid (0.2%).
Sodium Hydroxide Solution, Dilute Solution A should be kept in a well-closed container.
Dissolve 8.5 g of sodium hydroxide R in water R and dilute to Sodium Laurilsulfate Sodium dodecyl sulphate;
100 mL with the same solvent. (151-21-3)
Sodium Hydroxide Solution, Methanolic See Sodium laurilsulfate (0098).
Dissolve 40 mg of sodium hydroxide R in 50 mL of water R. Sodium Laurilsulfate Rl (151-21-3)
Cool and add 50 mL of methanol R. Content: minimum 99.0 per cent.
Sodium Hydroxide Solution Rl, Methanolic Sodium Lauryl Sulfate (151-21-3)
Dissolve 200 mg of sodium hydroxide R in 50 mL of water R. See Sodium laurilsulfate R.
Cool and add 50 mL of methanol R. Sodium Laurylsulfonate for Chromatography Sodium
Sodium Hydroxide Solution, Strong laurylsulphonate for chromatography; C 12H 25NaO 3 S = 272.4
Dissolve 42 g of sodium hydroxide R in water R and dilute to (2386-53-0)
100 mL with the same solvent. White or almost white powder or crystals, freely soluble in
Sodium 2-Hydroxybutyrate Sodium (2RS)-2- water.
hydroxybutanoate; C 4 H 7 NaO 3 = 126.1 (19054-57-0) Absorbance Ai~m (2.2.25), determined in water R:
Sodium Hypobromite Solution about 0.05 at 210 nm; about 0.03 at 220 nm; about 0.02 at
In a bath of iced water mix 20 mL of strong sodium hydroxide 230 nm; about 0.02 at 500 nm.
solution R and 500 mL of water R, add 5 mL of bromine Sodium Metabisulfite Sodium metabisulphite; Sodium
solution R and stir gently until solution is complete. Prepare pyrosulfite; Sodium pyrosulphite; (7681-57-4)
immediately before use. See Sodium metabisulfite (0849).
Sodium Hypochlorite Solution Sodium Methanesulfonate Methanesulfonic acid, sodium
General reagent grade of commerce containing 10 to salt; Methanesulphonic acid, sodium salt; Sodium
14% w/v of available chlorine. methanesulphonate; CH3 SO 3 Na = 118.1 (2386-57-4)
Sodium Hypochlorite Solution (3% Cl) Sodium White or almost white, crystalline powder, hygroscopic.
hypochlorite solution, strong Storage: in an airtight container.
Content: 25 g/L to 30 g/L of active chlorine. Sodium 2-methyl-2-thiazoline-4-carboxylate
Yellowish liquid with an alkaline reaction. C 5H 6NNaO2S = 167.2 (15058-19-2)
Assay. Introduce into a flask, successively, 50 mL of water R, Sodium 2-methyl-4,5-dihydro- l ,3-thiazole-4-carboxylate.
1 g of potassium iodide Rand 12.5 mL of dilute acetic acid R. White solid.
Dilute 10.0 mL of the substance to be examined to Content: minimum 95 per cent.
100.0 mL with water R. Introduce 10.0 mL of this solution
2023 Appendix I A V-Al 43

Sodium Molybdate Disodium molybdate dihydrate; White or almost white powder or colourless crystals, very
Na 2MoO 4 ,2H2O = 242.0 (10102-40-6) soluble in water, very slightly soluble in ethanol
White or almost white, crystalline powder or colourless (96 per cent), practically insoluble in methylene chloride.
crystals, freely soluble in water. Sodium Pentanesulfonate 1-Pentanesulfonic acid sodium
Sodium 1,2-Naphthoquinone-4-sulfonate Sodium salt; 1-Pentanesulphonic acid sodium salt; Sodium
naphthoquinonesulfonate; C 10H 5 NaO 5 S = 260.2 (521-24-4) pentanesulphonate; C 5H 11 NaO 3 S = 174.2 (22767-49-3)
Yellow or orange-yellow, crystalline powder, freely soluble in White or almost white, crystalline solid, soluble in water.
water, practically insoluble in ethanol (96 per cent). Sodium Pentanesulfonate Monohydrate
Sodium Nitrate NaNO 3 = 85.0 (7631-99-4) 1-Pentanesulfonic acid sodium salt monohydrate;
White or almost white powder or granules or colourless, 1-Pentanesulphonic acid sodium salt monohydrate; Sodium
transparent crystals, deliquescent in moist air, freely soluble pentanesulphonate monohydrate; C 5 H 11 NaO 3 S,H2 O = 192.2
(207605-40-1)
in water, slightly soluble in ethanol (96 per cent).
Storage: in an airtight container.
White or almost white crystalline solid, soluble in water.
Sodium Nitrite NaNO 2 = 69.0 (7632-00-0) Sodium Pentanesulfonate Monohydrate Rl Sodium
pentanesulphonate monohydrate Rl; C 5H 11 NaO 3S,
Content: minimum 97.0 per cent.
H 2 O = 192.2 (207605-40-1)
White or almost white, granular powder or a slightly yellow, Content: minimum 99 per cent of C 5 H 11 NaO 3 S,H 2 O.
crystalline powder, freely soluble in water.
Sodium Perchlorate NaCIO 4 ,H2 O = 140.5 (7791-07-3)
Assay. Dissolve 0.100 gin 50 mL of water R. Add 50.0 mL
of 0. 02 M potassium permanganate and 15 mL of dilute sulfuric Content: minimum 99.0 per cent ofNaC1O 4 ,H 2 O.
acid R. Add 3 g of potassium iodide R. Titrate with 0.1 M White or almost white, deliquescent crystals, very soluble in
sodium thiosuifate, using 1.0 mL of starch solution R added water.
towards the end of the titration as indicator. Storage: in a well-closed container.
1 mL of 0.02 M potassium permanganate is equivalent to Sodium Periodate Sodium metaperiodate;
3.450 mg ofNaNOz. Nal0 4 = 213.9 (7790-28-5)
Sodium Nitrite Solution Content: minimum 99.0 per cent.
A 100 g/L solution of sodium nitrite R. Prepare immediately White or almost white, crystalline powder or crystals, soluble
before use. in water and in mineral acids.
Sodium Nitroprusside Sodium pentacyano- Sodium Periodate Solution
nitrosylferrate(III) dihydrate; Dissolve 1.07 g of sodium periodate R in water R, add 5 mL of
Na2 [Fe(CN) 5 (NO)],2H2 O = 298.0 (13755-38-9) dilute suifuric acid Rand dilute to 100.0 mL with water R.
Reddish-brown powder or crystals, freely soluble in water, Use a freshly prepared solution.
slightly soluble in ethanol (96 per cent). Sodium Phosphite Sodium phosphite pentahydrate;
Sodium Nitroprusside-Carbonate Solution Na2 HPO 3 ,5H 2O = 216.0 (13517-23-2)
Dissolve 1 g of sodium nitroprusside and 1 g of anhydrous White or almost white, crystalline powder, hygroscopic, freely
sodium carbonate in sufficient water to produce 100 mL. soluble in water.
Sodium Octanesulfonate Octanesulfonic acid sodium Storage: in an airtight container.
salt; Octanesulphonic acid sodium salt; Sodium Sodium Picrate Solution, A1kaline
octanesulphonate; C 8 H 17NaO 3 S = 216.3 (5324-84-5) Mix 20 mL of picric acid solution Rand 10 mL of a 50 g/L
Content: minimum 98.0 per cent. solution of sodium hydroxide R and dilute to 100 mL with
White or almost white, crystalline powder or flakes, freely water R.
soluble in water, soluble in methanol. Storage: use within 2 days.
Absorbance (2.2.25): maximum 0.10, determined at 200 nm Sodium 1-Propanesulfonate Sodium propane-1-sulfonate
and maximum 0.01, determined at 250 nm using a 54 g/L monohydrate; C 3 H 9 SO 4Na = 164.2 (304672-01-3)
solution. mp: about 250 °C.
Sodium Octanesulfonate Monohydrate Sodium Sodium Pyrophosphate Tetrasodium diphosphate
octanesulphonate monohydrate; C 8 H 17NaO 3 S,H2 O = 234.3 decahydrate; Na4 P 2 O 7 ,10H 2 O = 446.1 (13472-36-1)
(207596-29-0)
Colourless, slightly efflorescent crystals, freely soluble in
White or almost white powder.
water.
Sodium Octyl Sulfate Octyl sulfate sodium salt; 4-Octyl Sodium Pyruvate 2-Oxopropanoic acid sodium salt;
sulphate sodium salt; Sodium octyl sulphate; C 3 H 3NaO 3 = 110.0 (113-24-6)
C 8 H 17NaO 4 S = 232.3 (142-31-4)
White or faint yellow powder, soluble in water (100 mg/mL).
White or almost white, crystalline powder or flakes, freely
soluble in water, soluble in methanol. mp: greater than 300 °C.
Sodium Oxalate C 2 Na 2 O4 = 134.0 (62-76-0) Sodium Rhod.izonate Rhodizonic acid disodium salt;
C 6 Na 2 O 6 = 214.0 (523-21-7)
White or almost white, crystalline powder, soluble in water,
practically insoluble in ethanol (96 per cent). Violet crystals, soluble in water with an orange-yellow colour.
Sodium Oxidronate Sodium Solutions are unstable and must be prepared on the day of
hydroxymethylenediphosphonate; CH4 Na 2 O 7P 2 = 236.0 use.
(14255-61-9) Sodium Salicylate (54-21-7)
See Sodium salicylate (0413).
V-A144 Appendix I A 2023

Sodium Stearyl Fumarate C 22 H 39NaO 4 (4070-80-8) Sodium Tetraborate Borax; Disodium tetraborate;
See Sodium stearylfumarate (1567). (1303-96-4)
Sodium Sulfate Sodium sulfate decahydrate; See Borax (0013).
Na 2SO 4 ,10H 2 O = 322.2 (7727-73-3) Sodium Tetrahydroborate Sodium borohydride;
See Sodium sulfate decahydrate (0100). NaBH4 = 37.8 (16940-66-2)
Sodium Sulfate, Anhydrous Sodium sulphate, Colourless, hygroscopic crystals, freely soluble in water,
anhydrous; (7757-82-6) soluble in anhydrous ethanol, decomposing at higher
Ignite at 600 °C to 700 °C anhydrous sodium sulfate temperature or in the presence of acids or certain metal salts
complying with the requirements prescribed in the forming borax and hydrogen.
monograph on Anhydrous sodium sulfate (0099). Storage: in an airtight container.
Loss on drying (2.2.32): maximum 0.5 per cent, determined Sodium Tetrahydroborate Reducing Solution
by drying in an oven at 130 °C. Introduce about 100 mL of water R into a 5 00 mL
Sodium Sulfate, Anhydrous Rl volumetric flask containing a stirring bar. Add 5.0 g of sodium
Complies with the requirements prescribed for anhydrous hydroxide R in pellets and 2.5 g of sodium tetrahydroborate R.
sodium sulfate R with the following maximum contents. Stir until complete dissolution, dilute to 500.0 mL with
water R and mix. Prepare immediately before use.
Cl: 20 ppm.
Sodium Tetraphenylborate NaB(C 6H 5 ) 4 = 342.2
Pb: 10 ppm. (143-66-8)
As: 3 ppm. White or slightly yellowish, bulky powder, freely soluble in
Ca: 50 ppm. water and in acetone.
Fe: 10 ppm. Sodium Tetraphenylborate Solution
Mg: 10 ppm. Filter before use if necessary.
Sodium Sulfide Sodium sulphide; Na 2 S,9H 2 O = 240.2 A 10 g/L solution of sodium tetraphenylborate R.
(1313-84-4)
Storage: use within 1 week.
Colourless, rapidly yellowing crystals, deliquescent, very Sodium Thioglycollate Sodium mercaptoacetate;
soluble in water. C 2 H 3 NaO2 S = 114.1 (367-51-1)
Storage: in an airtight container.
White or almost white, granular powder or crystals,
Sodium Sulfide Solution Sodium sulphide solution hygroscopic, freely soluble in water and in methanol, slightly
Dissolve 12 g of sodium sulfide R with heating in 45 mL of a soluble in ethanol (96 per cent).
mixture of 10 volumes of water R and 29 volumes of glycerol Storage: in an airtight container.
(85 per cent) R, allow to cool and dilute to 100 mL with the
Sodium Thiosulfate Sodium thiosulphate; (10102-17-7)
same mixture of solvents.
See Sodium thiosulfate (0414).
The solution should be colourless.
Sodium Thiosulfate, Anhydrous Disodium thiosulfate;
Sodium Sulfide Solution Rl Sodium sulphide Na 2 S2 O 3 = 158.1 (7772-98-7)
solution Rl
Content: minimum 98.0 per cent.
Prepare by one of the following methods.
- Dissolve 5 g of sodium sulfide R in a mixture of 10 mL of Sodium Tungstate Disodium tungstate dihydrate;
water R and 30 mL of glycerol R.
Na2 WO 4 ,2H 2 O = 329.9 (10213-10-2)
- Dissolve 5 g of sodium hydroxide R in a mixture of 30 mL White or almost white, crystalline powder or colourless
of water R and 90 mL of glycerol R. Divide the solution crystals, freely soluble in water forming a clear solution,
into 2 equal portions. Saturate 1 portion with hydrogen practically insoluble in ethanol (96 per cent).
sulfide R, with cooling. Mix the 2 portions. Solochrome Dark Blue CI 15705; calcon; mordant black
Storage: in a well-filled container, protected from light; use 17; C 20H 13N 2 NaO 5 S = 416.4 (2538-85-4)
within 3 months. General reagent grade of commerce.
Sodium Sulfite Sodium sulphite; Sodium sulfite A brownish black powder with a violet sheen. Produces a
heptahydrate; (10102-15-5) purplish red colour with calcium ions in alkaline solutions.
See Sodium sulfite heptahydrate (0776). When metal ions are absent, for example, in the presence of
Sodium Sulfite, Anhydrous Sodium sulphite, anhydrous; an excess of disodium edetate, the solution is blue.
(7757-83-7) Solochrome Dark Blue Mixture
See Sodium sulfite (0775). A mixture of 1 part of solochrome dark blue with 99 parts of
Sodium (+)-Tartrate Disodium (2R,3R)-2,3- freshly ignited anhydrous sodium sulfate.
dihydroxybutanedioate dehydrate; Sodium tartrate; Complies with the following test.
C 4 H 4 Na 2 O6,2H 2 O = 230.1 (6106-24-7) Sensitivity to calcium Dissolve 0.2 gin 5 mL of water.
White or almost white crystals or granules, very soluble in To 1 mL of the solution add 50 mL of water, 10 mL of
water, practically insoluble in ethanol (96 per cent). lM sodium hydroxide and 1 mL of a 1% w/v solution of
Sodium Taurodeoxycholate Sodium 2-((3,12-dihydroxy- magnesium sulfate; a blue colour is produced. Add 0.1 mL of
5-cholan-24-oyl)amino] ethanesulfonate monohydrate; a 0.15% w/v solution of calcium chloride; a violet colour is
2-[[ (3,5, 12)-3, 12-Dihydroxy-24-oxocholan-24-yl] amino] produced. Add 0.1 mL of 0.01M disodium edetate VS; a pure
ethanesulfonic acid monosodium salt monohydrate; blue colour is produced.
C 26H4 4NNaO 6 S,H 2 O = 539.7 (110026-03-4) Sorbic Acid Hexa-2,4-dienoic acid; C 6 H 8 O2 = 112.1
(110-44-1)
Content: minimum 94 per cent of C 26H 44 NNaO 6 S,H2O.
2023 Appendix I A V-A145

mp: about 136°. Carry out the test for sensitivity each time the reagent is
General reagent grade of commerce. used.
o-Sorbitol Sorbitol; (50-70-4) Test for sensitivity. To a mixture of 1 mL of the starch
See Sorbitol (0435). solution and 20 mL of water R, add about 50 mg of
potassium iodide R and O.05 mL of iodine solution Rl.
Soya Bean Lecithin (8030-76-0) The solution is blue.
Soya-bean Oil, Refined See Soya-bean oil, refined (1473). Starch Solution, Iodide-free
Sphingomyelin from Egg Yolk (85187-10-6) Prepare the solution as prescribed for starch solution R
(2R,3S,4E)-2-(Acylamino )-3-hydroxyoctadec-4-en-1-yl omitting the mercuric iodide. Prepare immediately before
2-(trimethylazaniumyl)ethyl phosphate. use.
Squalane (63,10E,15E,19E)-2,6,10,15,19,23- Starch Solution Rl
Hexamethyltetracosane; Perhydrosqualene; C 30H 62 = 422.8 Mix 1 g of soluble starch R and a small amount of cold
(111-01-3)
water R. Add this mixture, while stirring, to 200 mL of
Colourless, oily liquid, freely soluble in fatty oils, slightly boiling water R. Add 0.25 g of salicylic acid R and boil for
soluble in acetone, in ethanol (96 per cent), in glacial acetic 3 min. Immediately remove from the heat and cool.
acid and in methanol. Storage: if long storage is required, the solution shall be
dig: 0.811 to 0.813. stored at 4 °C to 10 °C. A fresh starch solution shall be
nf)0 : 1.451 to 1.453. prepared when the end-point of the titration from blue to
Stanolone 17~-Hydroxy-5a-androstan-3-one; colourless fails to be sharp. If stored under refrigeration, the
C19H3 002 = 290.4 (521-18-6) starch solution is stable for about 2 to 3 weeks.
White or almost white powder. Test for sensitivity. A mixture of 2 mL of starch solution R 1,
mp: about 180 °C. 20 mL of water R, about 50 mg of potassium iodide R and
0.05 mL of iodine solution Rl is blue.
Staphylococcus Aureus Strain V8 Protease Type XVII-
B (66676-43-5) Starch Solution R2
Microbial extracellular proteolytic enzyme. A lyophilised Triturate 1.0 g of soluble starch R with 5 mL of water R and
powder containing 500 units to 1000 units per milligram of whilst stirring pour the mixture into 100 mL of boiling
water R. Use a freshly prepared solution.
solid.
Test for sensitim'ty. To a mixture of 1 mL of the starch
Starch
solution and 20 mL of water R, add about 50 mg of
Potato starch of commerce. potassium iodide Rand 0.05 mL of iodine solution Rl.
Starch, Hydrolysed The solution is blue.
Electrophoretic grade of commerce. Starch Substrate
Starch Iodate Paper Determine the water content of starch EPBRP by heating at
Immerse strips of filter paper in 100 mL of iodide-free starch 120° for 4 hours. Stir a quantity of starch EPBRP equivalent
solution R containing 0.1 g of potassium iodate R. Drain and to 2. 0 g of the dried substance with 10 mL of water and add,
allow to dry protected from light. stirring continuously, to 160 mL of boiling water. Rinse the
Starch Iodide Paper container with several 1O-mL quantities of water, add the
Immerse strips of filter paper in 100 mL of potassium iodide washings to the hot starch solution and heat to boiling,
and starch solution R. Drain and allow to dry protected from
stirring continuously. Cool to 20° and add sufficient water to
produce 200 mL.
light.
Test for sensitivity. Mix 0.05 mL of 0.1 M sodium nitrite with
Use on the day of preparation.
4 mL of hydrochloric acid R and dilute to 100 mL with Stavudine (3056-17-5)
water R. Apply one drop of the solution to starch iodide See Stavudine (2130).
paper; a blue spot appears. Stearic Acid Octadecanoic acid; C 18H 36 0 2 = 284.5
Starch Mucilage (57-11-4)
Triturate 0.5 g of starch or soluble starch with 5 mL of water White or almost white powder or flakes, greasy to the touch,
and add, stirring continuously, to sufficient water to produce practically insoluble in water, soluble in hot ethanol
about 100 mL. Boil for a few minutes, cool and filter. (96 per cent).
Produces a blue colour with free iodine in the presence of a mp: about 70 °C.
soluble iodide. Stearic acid used in the assay of total fatty acids in Saw palmetto
It must be recently prepared. fruit (1848) complies with the foUowing additional test.
Starch, Soluble (9005-84-9) Assay. Gas chromatography (2.2.28) as prescribed in the
White or almost white powder. monograph Saw palmetto fruit (1848).
Starch Solution Content: minimum 98 per cent, calculated by the
Triturate 1.0 g of soluble starch R with 5 mL of water R and normalisation procedure.
whilst stirring pour the mixture into 100 mL of boiling Stearic Anhydride C 36 H 700 3 = 551.0 (638-08-4)
water R containing 10 mg of mercuric iodide R. mp: about 70°.
NOTE: commercially available reagents may be used; General reagent grade of commerce.
including mercury-free solutions or those containing White, waxy flakes.
alternative preservatives.
V-Al 46 Appendix I A 2023

Stigmasterol (22E)-Stigmasta-5,22-dien-3~-ol; (22E)-24- Sudan Red G Sudan red I; C 17H 14N 2 O 2 = 278.3
Ethylcholesta-5,22-dien-3~-ol; C 29H 48 O = 412.7 (83-48-7) Schultz No. 149
White or almost white powder, insoluble in water. Colour Index No. 12150
mp: about 170 °C. Reddish-brown powder, practically insoluble in water.
[o:];;2: about - 51, determined with a 20 g/L solution in Chromatography. Thin-layer chromatography (2.2.27) using
chlorofonn R. silica gel G R as the coating substance: apply 10 µL of a
Streptomycin Sulfate Streptomycin sulphate; (3810-74-0) 0.1 g/L solution in methylene chloride R and develop over a
See Streptomycin sulfate (0053). path of 10 cm with the same solvent; the chromatogram
shows only one principal spot.
Strongly Acidic Ion-exchange Resin See ion-exchange
resin, strongly acidic R. Sudan Red Solution
Strontium Carbonate SrCO 3 = 147.6 (1633-05-2) A 0.5% w/v solution of sudan red in anhydrous acetic acid.
White or almost white, crystalline powder. Sudan Yellow 1-(phenylazo)naphthalen-2-ol; Sudan I;
Sudan orange; C 16H 12 N 2 O = 248.3 (842-07-9)
Content: minimum 99.5 per cent.
Colour Index No. 12055
Strontium Chloride Strontium chloride hexahydrate;
SrC12,6H2 O = 266.6 (10025-70-4) Orange-red powder, practically insoluble in water, soluble in
methylene chloride.
White or almost white crystals, very soluble in water.
mp: about 131 °C.
mp: about 115 °C (loss of water) and 872 °C.
Sudan Yellow Solution
Strontium Selective Extraction Resin
A 0.2% w/v solution of sudan yellow in a mixture of 1 volume
Commercially available resin prepared by loading a
of benzene and 4 volumes of petroleum spirit (boiling range, 60°
suspension of 4,4 '(5 ')-di-tert-butylcyclohexano-18-crown-6
to 80°).
(crown ether) in octanol onto an inert chromatographic
support. The bed density of this resin is approximately Sulfanilamide 4-Aminobenzenesulphonamide;
0.35 g/mL. Sulphanilamide; C 6H 8 N 2O 2 S = 172.2 (63-74-1)
Strontium-85 Spiking Solution White or almost white powder, slightly soluble in water,
freely soluble in boiling water, in acetone, in dilute acids and
Dilute strontium-85 standard solution R to a radioactivity
in solutions of the alkali hydroxides, sparingly soluble in
concentration of approximately 10 kBq/mL with a 0.27 g/L
ethanol (96 per cent) and in light petroleum.
solution of strontium chloride hexahydrate R in a 1.03 g/L
solution of hydrochloric acid R. mp: about 165 °C.
Strontium-85 Standard Solution Sulfathiazole 4-Amino-N-(thiazol-2-yl)
benzenesulfonamide; C9 H 9 N 3 0 2 S2 = 255.3 (72-14-0)
A solution of strontium-SS in the form of Sr2 + ions in a
51.5 g/L solution of hydrochloric acid R. White or yellowish-white powder or crystals, very slightly
soluble in water, soluble in acetone, slightly soluble in
Strychnine C21H 22N 2 O 2 = 334.4 (57-24-9)
ethanol (96 per cent). It dissolves in dilute mineral acids and
(4aR,4bR,5aS,8aR, 13aS, 15aS)-2,4a,4b,5,5a, 7,8, 13a, 15,15a- in solutions of alkali hydroxides and carbonates.
Decahydro-4,6-methano-6H-indolo [3 ,2, 1-h] oxepino [2,3,4-de]
mp: about 200 °C.
pyrrolo [2,3-h] quinolin-14-one. Strychnidin-10-one.
Sulfamic Acid H 3 NO 3 S = 97.1 (5329-14-6)
White or almost white, crystalline powder, sparingly soluble
in water. White or almost white crystalline powder or crystals, freely
soluble in water, sparingly soluble in acetone, in ethanol
mp: about 285 °C.
(96 per cent) and in methanol.
Styrene Ethenylbenzene; C 8 H 8 = 104.2 (100-42-5)
mp: about 205 °C, with decomposition.
bp: about 145 °C.
Sulfan Blue Sulphan blue; C 27 H 31 N 2NaO 6S2 = 566.6
Colourless, oily liquid, very slightly soluble in water. (129-17-9)
Styrene-Divinylbenzene Copolymer Schultz No. 769
Porous, rigid, cross-linked polymer beads. Several grades are Colour Index No. 42045
available with different sizes of beads. The size range of the
Violet powder, soluble in water. Dilute solutions are blue and
beads is specified after the name of the reagent in the tests
turn yellow on the addition of concentrated hydrochloric
where it is used.
acid.
Succinic Acid Butanedioic acid; C 4 H 6 O 4 = 118.1
Sulfanilic Acid Sulphanilic acid; C 6H 7 NO 3S = 173.2
(110-15-6)
(121-57-3)
White or almost white, crystalline powder or colourless
Colourless crystals, sparingly soluble in water, practically
crystals, soluble in water and in ethanol (96 per cent).
insoluble in ethanol (96 per cent).
mp: 184 °C to 187 °C.
Sulfanilic Acid Solution Sulphanilic acid solution
Sucrose (57-50-1)
Dissolve 0.33 g of sulfanilic acid R in 75 mL of water R
See Sucrose (0204). heating gently if necessary and dilute to 100 mL with glacial
Sudan Red CI 26100; sudan III; solvent red 23; 1-(4- acetic acid R.
phenylazophenylazo)-2-naphthol; C 22 H 16N 4 O = 352.4 Sulfanilic Acid Solution Rl Sulphanilic acid solution Rl
(85-86-9)
Dissolve 0.5 g of sulfanilic acid Rina mixture of 75 mL of
Technical reagent grade of commerce. dilute acetic acid Rand 75 mL of water R.
A reddish brown powder.
2023 Appendix I A V-A147

Sulfanilic Acid Solution, Diazotised Sulphanilic acid colour is less intense than that of a reference mixture
solution, diazotised prepared in the same manner and containing 12.5 mL of
Dissolve, with warming, 0.9 g of sulfanilic acid R in 9 mL of water R, 50 g of nitrogen-free sulfuric acid R, 2.5 mL of nitrate
hydrochloric acid R, and dilute to 100 mL with water R. Cool standard solution (10 ppm NO.,) Rand 0.2 mL of a 50 g/L
10 mL of this solution in iced water and add 10 mL of an solution of brucine R in glacial acetic acid R.
ice-cold 45 g/L solution of sodium nitrite R. Allow to stand at Ammonium: maximum 2 ppm.
0 "C for 15 min (if stored at this temperature, the solution is Pour 2.5 g, carefully and while cooling, into water Rand
stable for 3 days) and immediately before use add 20 mL of dilute to 20 mL with the same solvent. Cool, and add
a 100 g/L solution of sodium carbonate R. dropwise 10 mL of a 200 g/L solution of sodium hydroxide R,
Sulfomolybdic Reagent R2 Sulphomolybdic reagent R2 followed by 1 mL of alkaline potassium tetraiodomercurate
Dissolve about 50 mg of ammonium molybdate R in l 0 mL of solution R. The colour of the solution is less intense than that
sulfuric acid R. of a mixture of 5 mL of ammonium standard solution (1 ppm
NH,J R, 15 mL of water R, 10 mL of a 200 g/L solution of
Sulfomolybdic Reagent R3 Sulphomolybdic reagent R3
sodium hydroxide R and 1 mL of alkaline potassium
Dissolve with heating 2.5 g of ammonium molybdate R in tetraiodomercurate solution R.
20 mL of water R. Dilute 28 mL of sulfuric acid R in 50 mL
Arsenic (2.4.2, Method A): maximum 0.02 ppm.
of water R, then cool. Mix the two solutions and dilute to
100 mL with water R. To 50 g add 3 mL of nitric acid R and evaporate carefully
until the volume is reduced to about 10 mL. Cool, add to
Storage: in a polyethylene container.
the residue 20 mL of water R and concentrate to 5 mL.
Sulfosalicylic Acid 5-Sulfo-2-hydroxybenzoic acid; Prepare the standard using 1.0 mL of arsenic standard solution
5-Sulpho-2-hydroxybenzoic acid; 3-Sulpho-6-hydroxybenzoic (1 ppm As) R.
acid; 3-Sulpho-6-hydroxybenzoic acid; Sulfosalicylic acid;
Iron (2.4.9): maximum 1 ppm.
C 7H 6 O 6S,2H2O = 254.2 (5965-83-3)
White or almost white, crystalline powder or crystals, very Dissolve the residue on ignition with slight heating in 1 mL
soluble in water and in ethanol (96 per cent). of dilute hydrochloric acid Rand dilute to 50.0 mL with
water R. Dilute 5 mL of this solution to 10 mL with water R.
mp: about 109 °C.
Heavy metals (2.4.8): maximum 2 ppm.
Sulfur (7704-34-9)
Dilute 10 mL of the solution obtained in the test for iron to
See Sulfur (0953). 20 mL with water R. 12 mL of the solution complies with
Sulfur Dioxide Sulphur dioxide; SO 2 = 64.1 (7446-09-5) test A. Prepare the reference solution using lead standard
A colourless gas. When compressed it is a colourless liquid. solution (2 ppm Pb) R.
Sulfur Dioxide Rl Sulphur dioxide Rl; SO 2 = 64.1 Residue on ignition: maximum 0.001 per cent, determined on
(7446-09-5) 100 g by evaporating cautiously in a small crucible over a
Content: minimum 99.9 per cent V/V. naked flame and igniting the residue to redness.
Sulfur Dioxide Solution Sulphur dioxide solution, Assay. Weigh accurately a ground-glass-stoppered flask
sulfurous acid, sulphurous acid; H 2SO 3 = 82.08 containing 30 mL of water R, introduce 0.8 mL of the
sulfuric acid, cool and weigh again. Titrate with 1 M sodium
Use a solution of commerce containing about 5% w/v of
hydroxide, using 0.1 mL of methyl red solution Ras indicator.
SO 2; weight per mL, about 1.03 g.
1 mL of 1 M sodium hydroxide is equivalent to 49.04 mg of
Sulfuric Acid Sulphuric acid; H 2SO4 = 98.1 (7664-93-9)
H2SO4.
Content: 95.0 per cent mlm to 97.0 per cent mlm.
Storage: in a ground-glass-stoppered container made of glass
Colourless, caustic liquid with an oily consistency, highly or other inert material.
hygroscopic, miscible with water and with ethanol
(96 per cent) producing intense heat.
Sulfuric Acid, SM Sulphuric acid, SM
Dilute 28 mL of sulfuric acid R to 100 mL with water R.
d~g: 1.834 to 1.837.
A 10 g/L solution is strongly acid and gives the reactions of Sulfuric Acid, Dilute Sulphuric acid, dilute
sulfates (2.3.1). Contains 98 g/L of H2SO4.
Appearance. It is clear (2.2.1) and colourless (2.2.2, Add 5.5 mL of sulfuric acid R to 60 mL of water R, allow to
Method II). cool and dilute to 100 mL with the same solvent.
Oxidisable substances. Pour 20 g cautiously, with cooling, into Assay. Into a ground-glass-stoppered flask containing 30 mL
40 mL of water R. Add 0.5 mL of 0. 002 M potassium of water R, introduce 10.0 mL of the dilute sulfuric acid.
permanganate. The violet colour persists for at least 5 min. Titrate with 1 M sodium hydroxide, using 0.1 mL of methyl red
solution R as indicator.
Chlorides: maximum 0.5 ppm.
1 mL of 1 M sodium hydroxide is equivalent to 49.04 mg of
Pour 10 g, carefully and while cooling, into 10 mL of water R
and after cooling dilute to 20 mL with the same solvent. H2SO4.
Add 0.5 mL of silver nitrate solution R2. Allow to stand for Sulfuric Acid, Dilute Rl
2 min protected from bright light. The solution is not more Contains 4.9 g/L of H2SO 4.
opalescent than a standard prepared at the same time using a Prepared from sulfaric acid R.
mixture of 1 mL of chloride standard solution (5 ppm Cl) R, Sulfuric Acid, 2.5M Alcoholic Sulphuric acid,
19 mL of water Rand 0.5 mL of silver nitrate solution R2. 2.5M alcoholic
Nitrates: maximum 0.5 ppm. Carefully and with constant cooling, stir 14 mL of sulfuric
Pour 50 g or 27.2 mL, carefully and while cooling, into acid R into 60 mL of anhydrous ethanol R. Allow to cool and
15 mL of water R. Add 0.2 mL of a freshly prepared 50 g/L dilute to 100 mL with anhydrous ethanol R. Prepare
solution of brucine R in glacial acetic acid R. After 5 min any immediately before use.
V-A148 Appendix I A 2023

Sulfuric Acid, 0.25M Alcoholic Sulphuric acid, Sunflower Oil See Sunflower oil, refined (1371).
0.25M alcoholic Swertiamarin Swertiamaroside; (4R,5R,6S)-5-Ethenyl-
Dilute 10 mL of 2. 5 M alcoholic sulfuric acid R to 100 mL 6-(~-o-glucopyranosyloxy)-4a-hydroxy-4,4a,5,6-tetrahydro-
with anhydrous ethanol R. Prepare immediately before use. 1H,3H-pyrano[3,4-c]pyran-1-one; C 16H 22O 10 = 374.3
Sulfuric Acid, Alcoholic Solution of Sulphuric acid, (17388-39-5)
alcoholic solution of Tagatose D-(yxo-Hexulose; C 6H 12O 6 = 180.16 (87-81-0)
Carefully and with constant cooling, stir 20 mL of sulfuric White or almost white powder.
acid R into 60 mL of ethanol (96 per cent) R. Allow to cool [o,;]~: -2.3 determined on a 21.9 g/L solution.
and dilute to 100 mL with ethanol (96 per cent) R. Prepare mp: 134 °C to 135 °C.
immediately before use.
Talc (14807-96-6)
Sulfuric Acid, Ethanolic
See Talc (0438).
Sulphuric acid, ethanolic
Tannic Acid (1401-55-4)
Solutions of the requisite molarity may be obtained by
mixing sulfuric acid with ethanol (96%) as directed under Yellowish or light-brown, glistening scales or amorphous
sulfuric acid. powder, very soluble in water, freely soluble in ethanol
(96 per cent), soluble in acetone.
When 'ethanolic sulfuric acid' is followed by a percentage
figure, an instruction to use sulfuric acid diluted with ethanol Storage: protected from light.
(96%) to produce the specified percentage v/v proportion of Tannic Acid Reagent
sulfuric acid is implied. Dissolve 25 mg of tannic acid in 20 mL of glacial acetic acid
Sulfuric Acid-Formaldehyde Reagent Sulphuric acid- and add 80 mL of orthophosphoric acid.
formaldehyde reagent Prepare immediately before use.
Mix 2 mL of fonnaldehyde solution R with 100 mL of sulfuric Tanshinone HA C 19 H 18 O 3 = 294.3 (568-72-9)
acid R. 1,6,6-Trimethyl-6, 7,8, 9-tetrahydrophenanthro [1,2-b] furan-
Sulfuric Acid, Heavy Metal-free Sulphuric acid, heavy 10, ll -dione.
metal-free (+)-Tartaric Acid Tartaric acid; (87-69-4)
Complies with the requirements prescribed for suljuric acid R See Tartaric acid (0460).
with the following maximum contents of heavy metals.
Taurodeoxycholic Acid Sodium Salt
As: 0.005 ppm. C 26 H 4 ~aNO6S = 521.7 (1180-95-6)
Cd: 0.002 ppm. General reagent grade of commerce.
Cu: 0.001 ppm. Taxifolin (2R,3R)-2-(3,4-Dihydroxyphenyl)-3,5, 7-
Fe: 0.05 ppm. trihydroxy-2,3-dihydro-4H-1-benzopyran-4-one;
Hg: 0.005 ppm. C1sH12O1 = 304.3 (480-18-2)
Ni: 0.002 ppm. White or almost white powder, slightly soluble in anhydrous
Pb: 0.001 ppm. ethanol.
Zn: 0.005 ppm. Absorbance (2.2.25). A solution in anhydrous ethanol R shows
an absorption maximum at 290 nm.
Sulfuric Acid, Methanolic
Tecnazene C 6 HC1 4 NO 2 = 260.9 (117-18-0)
Sulphuric acid, methanolic
bp: about 304 °C.
Solutions of the requisite molarity may be obtained by
mixing sulfuric acid with methanol as directed under sulfuric mp: 99 °C to 100 °C.
acid. A suitable certified reference solution (10 ng/µL in
When 'methanolic sulfuric acid' is followed by a percentage cyclohexane) may be used.
figure, an instruction to use sulfuric acid diluted with methanol Terpinene l-lsopropyl-4-methylcyclohexa-1,3-diene;
to produce the specified percentage v/v proportion of sulfuric cx-Terpinene; C 10H 16 = 136.2 (99-86-5)
acid is implied. Clear, almost colourless liquid.
Sulfuric Acid, Nitrogen-free Sulphuric acid, nitrogen- dl 0 : about 0.837.
free n~: about 1.478.
Complies with the requirements prescribed for sulfuric acid R bp: about 17 4 °C.
with the following additional test. r:x-Terpinene used in gas chromatography complies with the
Nitrates. To 5 mL of water R add carefully 45 mL of the foUowing additional test.
sulfuric acid, allow to cool to 40 °C and add 8 mg of Assay. Gas chromatography (2.2.28) as prescribed in the
diphenylbenzidine R. The solution is colourless or very pale
monograph Tea tree oil (1837).
blue.
Content: minimum 90 per cent, calculated by the
Sulfuric Acid, Nitrogen-free Rl normalisation procedure.
Complies with the requirements prescribed for nitrogen-free y- Terpinene 1-Isopropyl-4-methylcyclohexa-1,4-diene;
sulfuric acid R. C 10H 16 = 136.2 (99-85-4)
Content: 95.0 per cent mlm to 95.5 per cent mlm. Oily liquid.
Sulfuric Acid Rl H 2 SO4 = 98.1 (7664-93-9)
y-Terpinene used in gas chromatography complies with the
Content: 75 per cent V/V. fallowing additional test.
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Peppennint oil (0405).
2023 Appendix I A V-Al49

Test solutwn. The substance to be examined. White or almost white crystals.

Content: minimum 93.0 per cent, calculated by the mp: 102 °C to 104 °C.
normalisation procedure. Tetrabutylammonium Dihydrogen Orthophosphate
Terpinen-4-ol 4-Methyl-1-( 1-methylethyl) cyclohex-3-en- l- Tetrabutylammonium dihydrogen phosphate;
ol; p-Menth-l-en-4-ol; C 10H 18 O = 154.2 (562-74-3) C15H3sNO4P = 339.5 (5574-97-0)
Oily, colourless liquid. White or almost white powder, hygroscopic.
Terpinen-4-ol used in gas chromatography complies with the pH (2.2.3): about 7.5 for a 170 g/L solution.
following additional test. Absorbance (2.2.25): about 0.10 determined at 210 nm using
Assay. Gas chromatography (2.2.28) as prescribed in the a 170 g/L solution.
monograph Lavender oil (1338). Storage: in an airtight container.
Test solutwn. The substance to be examined. Tetrabutylammonium Dihydrogen Phosphate
Content: minimum 90.0 per cent, calculated by the Solution
normalisation procedure. A 1.0 M solution of tetrabutylammonium dihydrogen
a-Terpineol (RS)-2-( 4-Methylcyclohex-3-enyl)-2-propanol; phosphate R. This solution is commercially available.
C 10H1 8O = 154.2 (98-55-5) Tetrabutylammonium Hydrogen Sulfate
Colourless crystals, practically insoluble in water, soluble in Tetrabutylammonium hydrogen sulphate;
ethanol (96 per cent). C15H31NO4S = 339.5 (32503-27-8)
Jig: about 0.935. Crystalline powder or colourless crystals, freely soluble in
ng1: about 1.483. water and in methanol.
[a]~: about 92.5. mp: 169 °C to 173 °C.
mp: about 35 °C. Absorbance (2.2.25): maximum 0.05, determined between
It may contain 1 to 3 per cent of P-terpineol. 240 nm and 300 nm using a 50 g/L solution.
a-Terpineol used in gas chromatography complies with the Tetrabutylammonium Hydrogen Sulfate Rl
following test. Tetrabutylammonium hydrogen sulphate Rl
Assay. Gas chromatography (2.2.28) as prescribed in the Complies with the requirements prescribed for
monograph Anise oil (0804). tetrabutylammonium hydrogen suljate R with the following
additional requirement.
Test solutwn. A 100 g/L solution in hexane R.
Absorbance (2.2.25): maximum 0.02, determined between
Content: minimum 97.0 per cent, calculated by the
215 nm and 300 nm using a 50 g/L solution.
normalisation procedure.
Tetrabutylammonium Hydroxide
Terpinolene p-Mentha-1,4(8)-diene; 4-Isopropylidene-1-
C 16 H31NO,30H2O = 800 (147741-30-8)
methylcyclohexene; C 10H 16 = 136.2 (586-62-9)
Content: minimum 98.0 per cent of C 16 H 37 NO,30H2 O.
Clear, almost colourless liquid.
White or almost white crystals, soluble in water.
Jl 0: about 0.863.
Assay. Dissolve 1.000 g in 100 mL of water R. Titrate
ni)0 : about 1.488. immediately with 0.1 M hydrochloric acid determining the
bp: about 184 °C. end-point potentiometrically (2.2.20). Carry out a blank
Terpinolene used in gas chromatography complies with the titration.
following additional test. 1 mL of 0.1 M hydrochloric acid is equivalent to 80.0 mg of
Assay. Gas chromatography (2.2.28) as prescribed in the C16H31NO,30H2O.
monograph Tea tree oil (1837). Tetrabutylammonium Hydroxide Solution
Content: minimum 90 per cent, calculated by the General reagent grade of commerce containing 40.0% w/v of
normalisation procedure. C15H31NO.
Testosterone (58-22-0) Tetrabutylammonium Hydroxide Solution (104 g/L)
See Testosterone (1373). A solution containing 104 g/L of C 16H 37NO (Mr 259.5),
Testosterone Propionate (57-85-2) prepared by dilution of a suitable reagent grade.
See Testosterone propwnate (0297). Tetrabutylammonium Hydroxide Solution (400 g/L)
1,2,3,4-Tetra-O-acetyl-P-o-glucopyranose A solution containing 400 g/L of C 16H 37NO (Mr 259 .5) of a
C14H20O10 = 348.3 (13100-46-4) suitable grade.
White or almost white powder, soluble in water with gentle Tetrabutylammonium Hydroxide, 0.4M
heating. Use a grade of commerce suitable for chromatography.
[:x]t0: + 11, determined on a 6 g/L solution in chloroform R. Tetrabutylammonium Iodide C 16H 36IN = 369.4
mp: 126 °C to 128 °C. (311-28-4)
1,3,4,6-Tetra-O-acetyl-P-o-mannopyranose Content: minimum 98.0 per cent.
C 14H20O1o = 348.3 (18968-05-3) White or slightly coloured, crystalline powder or crystals,
Colourless or white powder or crystals. soluble in ethanol (96 per cent).
mp: 160 °C to 161 "C. Su/fated ash (2.4.14): maximum 0.02 per cent.
[o:]g1: - 68, determined on a 7 g/L solution in methylene Assay. Dissolve 1.200 g in 30 mL of water R. Add 50.0 mL
chloride R. of 0.1 M silver nitrate and 5 mL of dz7ute nitric acid R. Titrate
Tetrabutylammonium Bromide C 16H 36 BrN = 322.4 the excess of silver nitrate with 0.1 M ammonium thiocyanate,
(1643-19-2) using 2 mL of ferric ammonium suljate solution R2 as indicator.
V-A150 Appendix I A 2023

1 mL of 0.1 M silver nitrate is equivalent to 36. 94 mg of Tetraheptylammonium Bromide C 28 H 60 BrN = 490.7

C15H36lN. (4368-51-8)
Tetrabutylammonium Perchlorate White or sligl1tly coloured, crystalline powder or crystals.
C16H 36 ClNO4 = 341.91 (1923-70-2) mp: 89 °C to 91 °C.
General reagent grade of commerce containing not less than Tetrahexylammonium Bromide N,N,N-Trihexylliexan-
98.0% of C1 6H36ClNO4. 1-aminium bromide; C 24 H 52BrN = 434.6 (4328-13-6)
Tetrachloroethane 1, 1,2,2-Tetrachloroethane; White or almost white, crystalline powder, hygroscopic.
C 2H2Cl4 = 167.9 (79-34-5) mp: about 100 °C.
Clear, colourless liquid, slightly soluble in water, miscible Tetrahexylammonium Hydrogen Sulfate
with ethanol (96 per cent). Tetrahexylammonium hydrogen sulphate;
d~g: about 1.59. C 24H 53NO4S = 451.8 (32503-34-7)
nfr about 1.495. White or almost white crystals.
Distillation range (2.2.11). Not less than 95 per cent distils mp: 100 °C to 102 °C.
between 145 °C and 147 °C. Tetrahydrofuran Tetramethylene oxide; C4H 8 O = 72.1
Tetrachlorvinphos C 10H 9 Cl 4 O4P = 366.0 (22248-79-9) (109-99-9)
mp: about 95 °C. Clear, colourless, flammable liquid, miscible with water, with
A suitable certified reference solution ( 10 ng/µL in iso- ethanol (96 per cent).
octane) may be used. dig: about 0.89.
Tetracos-15-enoic Acid Methyl Ester 15-Tetracosaenoic Do not distil if the tetrahydrofuran does not comply with the test
acid methyl ester; Methyl tetracos-15-enoate; Nervonic acid for peroxides.
methyl ester; C 25 H 48 O 2 = 380.7 (2733-88-2) Peroxides. Place 8 mL of potassium iodide and starch solution R
Content: minimum 99.0 per cent, determined by gas in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in
chromatography. diameter. Fill completely with the substance to be examined,
Liquid. shake vigorously and allow to stand protected from light for
Tetracycline C 22 H 24N 2O8 = 444.4 (60-54-8) 30 min. No colour is produced.
mp: about 176°. Tetrahydrofuran used in spectrophotometry complies with the
following additional test.
General reagent grade of commerce.
Absorbance (2.2.25): maximum 0.70 at 255 nm, 0.10 at
Store protected from light.
270 nm, 0.01 at 310 nm, determined using water Ras
Tetracycline Hydrochloride compensation liquid.
See Tetracycline hydrocliloride (0210). Tetrahydrofuran for Chromatography
N-Tetradecane Tetradecane; C 14H 30 = 198.4 (629-59-4) Complies with the requirements prescribed for
Content: minimum 99.5 per cent m!m. tetrahydrofuran R with the following additional requirements:
A colourless liquid. dl 0 = 0.8892.
dfg: about 0.76. bp: about 66 °C.
nf~: about 1.429. Content: minimum 99.8 per cent of C 4H 8 O.
bp: about 252 °C. Tetrahydrofuran, Stabiliser-free
mp: about -5 "C. Tetrahydrofuran that is free from stabilisers and inhibitors.
Tetradecylammonium Bromide Tetrakis (decyl) Reagent grade of commerce.
ammonium bromide; C 40 H 84BrN = 659 (14937-42-9) Tetrahydropalmatine (13aRS)-5,8,13,13a-Tetrahydro-
White or slightly coloured, crystalline powder or crystals. 2,3, 9, 10-tetramethoxy-6H-dibenzo [a,g] quinolizine;
mp: 88 °C to 89 °C. C21H2sNO4 = 355.4 (2934-97-6)
Tetraethylammonium Hydrogen Sulfate o-Tetrahydropalmatine Hydrochloride C 21 H 25NO4,
Tetraethylammonium hydrogen sulphate; HCl = 391.90 (6024-83-5)
C 8 H 21 NO 4 S = 227.3 (16873-13-5) General reagent grade of commerce.
Hygroscopic powder. cr-Tetralone 1-Oxotetraline; 3,4-Dihydronaphthalen-
mp: about 245 °C. 1(2H)-one; C 10H 10 O = 146.2 (529-34-0)
Tetraethylammonium Hydroxide Solution bp: about 115 °C.
C 8 H 21 NO = 147.3 (77-98-5) mp: about 5 °C.
A 200 g/L solution. Tetramethylammonium Bromide N,N,N-
Colourless liquid, strongly alkaline. Trimethylmethanaminium bromide; C 4H 12BrN = 154.1
dig: about 1.01. (64-20-0)
n5°: about 1.372. White or slightly yellow crystals, freely soluble in water.
HPLC grade. mp: about 285 °C, with decomposition.
Tetraethylene Pentamine 3,6,9-Triazaundecan-1,11- Tetramethylammonium Chloride C 4H 12ClN = 109.6
diamine; C 8 H 23N 5 = 189.3 (112-57-2) (75-57-0)
Colourless liquid, soluble in acetone. Colourless crystals, soluble in water and in ethanol
(96 per cent).
nt0 : aboutl.506.
mp: about 300 °C, with decomposition.
Storage: protected from humidity and heat.
2023 Appendix I A V-Al51

Tetramethylammonium Hydrogen Sulfate Tetrandrine C 38H 42N 2 O 6 = 623 (518-34-3)

Tetramethylammonium hydrogen sulphate; 1,2,3,4-Tetraphenylcyclopenta-1,3-diene
C 4H 13NO4S = 171.2 (80526-82-5) C 29 H 22 = 370.5 (15570-45-3)
Hygroscopic powder. mp: about 178°.
mp: about 295 °C. General reagent grade of commerce.
Tetramethylammonium Hydroxide Pentahydrate 1,2,3,4-Tetraphenylcyclopenta-1,3-dienone
Tetramethylammonium hydroxide; C 4 H 13NO,5H 2O = 181.2 Tetraphenylcyclopentadienone; C 29 H 20 O = 384.5 (479-33-4)
(10424-65-4)
mp: about 218°.
Suitable grade for HPLC. General reagent grade of commerce.
Tetramethylammonium Hydroxide Solution (75-59-2) Tetraphenylethylene C 26 H 20 = 332.5 (632-51-9)
Content: minimum 10.0 per cent mlm of C 4 H 13 NO.
mp: about 223°.
(Mr 91.2).
General reagent grade of commerce.
Clear, colourless or very pale yellow liquid, miscible with
water and with ethanol (96 per cent). Tetrapropylammonium Chloride C 12 H 28 CIN = 221.8
(5810-42-4)
Assay. To 1.000 g add 50 mL of water R and titrate with
0.05 M sulfuric acid, using 0.1 mL of methyl red solutian Ras
White or almost white, crystalline powder, sparingly soluble
indicator. in water.
1 mL of 0.05 M sulfuric acid is equivalent to 9.12 mg of mp: about 241 °C.
C4H13NO. Tetrapropylammonium Hydrogen Sulfate N,N,N-
Tetramethylammonium Hydroxide Solution, Dilute Tripropylpropan-1-arninium hydrogen sulfate;
C 12H 29NO4S = 283.4 (56211-70-2)
Dilute 10 mL of tetramethylammonium hydroxide solutian R to
100 mL with aldehyde-free alcohol R. Prepare immediately White or almost white, crystalline, hygroscopic powder.
before use. Tetrazolium Blue Blue tetrazolium salt;
Tetramethylbenzidine 3,3',5,5 '-Tetramethylbiphenyl- C 40 H 32 Cl2N8 0 2 = 728 (1871-22-3)
4,4 '-diamine; C1 6 H 20 N 2 = 240.3 (54827-17-7) Yellow crystals, slightly soluble in water, freely soluble in
Powder, practically insoluble in water, very soluble in ethanol (96 per cent) and in methanol, practically insoluble
methanol. in acetone.
mp: about 169 °C. mp: about 245 °C, with decomposition.
1,1,3,3-Tetramethylbutylamine 2-Amino-2,4,4- Tetrazolium Blue Solution, Alkaline
trimethylpentane; C 8 H 19N = 129.3 (107-45-9) Immediately before use mix 1 volume of a 0.2% w/v solution
Clear, colourless liquid. of tetrazolium blue in methanol with 3 volumes of a 12% w/v
solution of sodium hydroxide in methanol.
d~g: about 0.805. Tetrazolium Bromide 3-(4,5-Dimethylthiazol-2-yl)-2,5-
ni 0 : about 1.424.
diphenyltetrazolium bromide; MIT; C 18H 16BrN5 S = 414.3
bp: about 140 °C. (298-93-1)
T etramethylethylenediamine Tetramethylethane-1,2- Tetrazolium Salt 5-(3-Carboxymethoxyphenyl)-3-(4,5-
diarnine; C 6 H 16N2 = 116.2 (110-18-9) dimethylthiazol-2-yl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
Colourless liquid, miscible with water and with ethanol salt; MTS; C 20 H 17N 5 O 6S2 = 487.5 (138169-43-4)
(96 per cent). Thallium(I) Nitrate Thallous nitrate; TlNO 3 = 266.4
d~g: about 0.78. (10102-45-1)
nfr about 1.418. General reagent grade of commerce.
bp: about 121 °C. Thallium(I) Sulfate Thallium(!) sulphate; Thallous
N,N,N' ,N'-Tetramethyl-p-phenylenediamine sulphate; Thallous sulfate; Tl2 SO4 = 504.8 (7446-18-6)
Dihydrochloride C 10H1 6N 2 ,2HC1 = 237.2 (637-01-4) White or almost white, rhomboid prisms, slightly soluble in
General reagent grade of commerce. water, practically insoluble in ethanol (96 per cent).
Whitish grey crystals. Thebaine (SR, 9R, 13S)-4,5-Epoxy-3,6-dimethoxy-9a-
Tetramethylsilane TMS; C 4 H 12 Si = 88.2 (75-76-3) methylmorphina-6,8-diene; C 19H 21 NO 3 = 311.4 (115-37-7)
White or pale yellow, crystalline powder, very slightly soluble
Clear, colourless liquid, very slightly soluble in water, soluble
in acetone and in ethanol (96 per cent). in water, soluble in hot anhydrous ethanol and in toluene.
mp: about 193 °C.
dfg: about 0.64.
nt 0 : about 1.358. Chromatography (2.2.27). Thin-layer chromatography (2.2.27)
as prescribed in identification test B in the monograph Raw
bp: about 26 °C. opium (0777): apply to the plate as a band
Tetramethylsilane used in nuclear magnetic resonance spectrometry (20 mm x 3 mm) 20 µL of a 0.5 g/L solution; the
complies with the following additional test. chromatogram shows an orange-red or red principal band
In the nuclear magnetic resonance spectrum of an with an RF of about 0.5.
approximately 10 per cent V/V solution of the Theobromine C 7H 8N 4 O 2 = 180.2 (83-67-0)
tetramethylsilane in deuterated chloroform R, the intensity of A white or almost white powder, very slightly soluble in
any foreign signal, excluding those due to spinning side water and in anhydrous ethanol, slightly soluble in ammonia.
bands and to chloroform, is not greater than the intensity of It dissolves in dilute solutions of alkali hydroxides and in
the C-13 satellite signals located at a distance of 59 .1 Hz on mineral acids.
each side of the principal signal of tetramethylsilane.
V-A152 Appendix I A 2023

Content: minimum 99.0 per cent. Thrombin Solution, Human Rl

Theophylline (58-55-9) Reconstitute human thrombin R as directed by the
See Theophylline (0299). manufacturer and dilute to 2.5 IU/mL with phosphate buffer
solutwn pH 6. 5 R.
2-(2-Thienyl)acetic Acid 2-Thiopheneacetic acid;
C 6 H 6 O 2 S = 142.1 (1918-77-0) Thrombin Solution, Human R2
Brown powder. Reconstitute human thrombin R as directed by the
mp: about 65 °C. manufacturer and dilute to 5 IU/mL with tris(hydroxymethyl)
aminomethane-EDTA buffer solution pH 8.4 Rl.
Thioacetamide C 2 H 5NS = 75.1 (62-55-5)
Thromboplastin
Crystalline powder or colourless crystals, freely soluble in
water and in ethanol (96 per cent). A preparation containing the membrane glycoprotein tissue
factor and phospholipid, either purified from animal brain
mp: about 113 °C. (usually rabbit) or human placenta or manufactured using
Thioacetamide Reagent recombinant DNA technology with added phospholipid.
To 0.2 mL of thioacetamide solution R add 1 mL of a mixture The preparation is formulated for routine use in the
of 5 mL of water R, 15 mL of 1 M sodium hydroxide and prothrombin time test and may contain calcium.
20 mL of glycerol (85 per cent) R. Heat in a water-bath for Thromboplastin Reagent
20 s. Prepare immediately before use. Thrombokinase extract
Thioacetamide Solution Extract 1.5 g of acetone-dried ox brain with 60 mL of water
A 40 glL solution of thioacetamide R. for 10 to 15 minutes at 50°, centifuge for 2 minutes at
Thiobarbituric Acid 4,6-Dihydroxy-2-sulfanylpyrimidine; 1500 revolutions per minute and decant the supernatant
C4H4N 2 O 2 S = 144.2 (504-17-6) liquid. This extract will retain its activity for several days
Thiodiglycol 2,2 '-thiodiethanol; Thiodiethylene glycol; when stored in a refrigerator. It may contain 0.3% w/v of
C4H10O 2 S = 122.2 (111-48-8) o-cresol as an antimicrobial preservative.
Colourless or yellow, viscous liquid. Thujone 4-Methyl-1-(1-methylethyl)bicyclo [3. l .0]hexan-3-
Content: minimum 99.0 per cent.
one; C 10H 16 O = 152.2 (76231-76-0)
d;g: about 1.18. Colourless or almost colourless liquid, practically insoluble in
water, soluble in ethanol (96 per cent) and in many other
Thiomalic Acid (2RS)-2-Sulfanylbutanedioic acid; organic solvents.
C4H 6 O 4 S = 150.2 (70-49-5)
Thymidine l-(2-Deoxy-~-o-erythro-pentofuranosyl)-5-
mp: 150 °C to 152 °C. methylpyrimidine-2,4(1H,3H)-dione; C 10H 14N 2 O 5 = 242.2
Thiomersal Sodium mercurothiolate; Sodium Needles, soluble in water, in hot ethanol (96 per cent) and in
2-[(ethylmercurio)thio]benzoate; C 9 H 9 HgNaO 2 S = 404.8 glacial acetic acid.
(54-64-8)
Thymine 5-Methylpyrimidine-2,4(1H,3H)-dione;
Light, yellowish-white, crystalline powder, very soluble in CsH 6N 2 O 2 = 126.1 (65-71-4)
water, freely soluble in ethanol (96 per cent).
Short needles or plates, slightly soluble in cold water, soluble
Thiourea CH 4 N 2 S = 76.1 (62-56-6) in hot water. It dissolves in dilute solution of alkali
White or almost white, crystalline powder or crystals, soluble hydroxides.
in water and in ethanol (96 per cent). Thyminose 2-Deoxy-o-erythro-pentose; 2-Deoxy-o-ribose;
mp: about 178 °c. CsH 10 O 4 = 134.1 (533-67-5)
L- Threonine Threonine; ( 72-19-5) Thymol 2-Isopropyl-5-methylphenol; (89-83-8)
See Threonine (1049). Thymol used in gas chromatography complies with the following
Thrombin Thrombin, human; (9002-04-4) additional test.
Dried human thrombin. A preparation of the enzyme which Assay. Gas chromatography (2.2.28) as prescribed in the
converts human fibrinogen into fibrin. It is obtained from monograph Peppermint oil (0405).
liquid human plasma and may be prepared by precipitation Test solution. Dissolve 0.1 gin about 10 mL of acetone R.
with suitable salts and organic solvents under controlled Content: minimum 95.0 per cent, calculated by the
conditions of pH, ionic strength and temperature. normalisation procedure.
Yellowish-white powder, freely soluble in a 9 glL solution of Thymol Blue Thymolsulfonphthalein; 4,4'-(3H-2,1-
sodium chloride forming a cloudy, pale yellow solution. Benzoxathiol-3-ylidene)bis(2-isopropyl-5-methylphenol) S,S-
Storage: in a sealed, sterile container under nitrogen, dioxide; C 27H 30 O 5 S = 466.6 (76-61-9)
protected from light, at a temperature below 25 °C. Brownish-green or greenish-blue, crystalline powder, slightly
Thrombin, Bovine (9002-04-4) soluble in water, soluble in ethanol (96 per cent) and in
A preparation of the enzyme, obtained from bovine plasma, dilute solutions of alkali hydroxides.
that converts fibrinogen into fibrin. Thymol Blue Solution
A yellowish-white powder. Dissolve 0.1 g of thymol blue R in a mixture of 2.15 mL of
Storage: at a temperature below 0 °C. 0.1 M sodium hydroxide and 20 mL of ethanol (96 per cent) R
Thrombin Solution Thrombin solution, human and dilute to 100 mL with water R.
Reconstitute human thrombin R as directed by the Test for sensitivity. To 0.1 mL of the thymol blue solution add
manufacturer and dilute to 5 IU/mL with tris(hydroxymethyl) 100 mL of carbon dioxide-free water Rand 0.2 mL of 0.02 M
aminomethane sodium chloride buffer solution pH 7. 4 R. sodium hydroxide. The solution is blue. Not more than
2023 Appendix I A V-Al53

0.15 mL of 0. 02 M hydrochloric acid is required to change the dissolved, heating, if necessary, on a water-bath at 50 °C.
colour to yellow. Pass a current of nitrogen R for 15 min. Prepare immediately
Colour change: pH 1.2 (red) to pH 2.8 (yellow); pH 8.0 before use.
(olive-green) to pH 9.6 (blue). Tin Test Kit, Semi-quantitative
Thymolphthalein 3,3-Bis( 4-hydroxy-5-isopropyl-2- Commercially available set of reagents consisting of tin test
methylphenyl)phthalide; C 28 H 30 O 4 = 430.5 (125-20-2) strips and a reagent mixture for the determination of tin in
White or yellowish-white powder, practically insoluble in aqueous solutions, in a range of 10-200 µg/mL.
water, soluble in ethanol (96 per cent) and in dilute solutions Tiron 4,5-Dihydroxy-1,3-benzenedisulfonic acid disodium
of alkali hydroxides. salt monohydrate; C5H4Na 2 O8 S2,H2 O = 332 (270573-71-2)
Thymolphthalein Solution General reagent grade of commerce.
A 1 g/L solution of thymolphthalein R in ethanol Tiron Indicator Solution
(96 per cent) R. 2% w/v solution of tiron in water.
Test for sensitivity. To 0.2 mL of the thymolphthalein solution Titan Yellow Thiazol yellow; Disodium 2,2'-[(l-triazene-
add 100 mL of carbon dioxide-free water R. The solution is 1,3-diyl)di-4, 1-phenylene] bis-[6-methylbenzothiazole-7-
colourless. Not more than 0.05 mL of 0.1 M sodium sulfonate]; C 28 H 19NsNa2O 6 S4 = 696 (1829-00-1)
hydroxide is required to change the colour to blue. Schultz No. 280
Colour change: pH 9.3 (colourless) to pH 10.5 (blue). Colour Index No. 19540
Tin Granulated tin; Sn= 118.7 (7440-31-5) A yellowish-brown powder, freely soluble in water and in
Silvery-white granules, soluble in hydrochloric acid with ethanol (96 per cent).
release of hydrogen. Titan Yellow Paper
Arsenic (2.4.2, Method A): maximum 10 ppm, determined on Immerse strips of filter paper in titan yellow solution R and
0.1 g. leave for a few minutes. Allow to dry at room temperature.
Tin(n) Chloride Tin dichloride dehydrate; Stannous Titan Yellow Solution
chloride; SnC12,2H2 O = 225.6 (10025-69-1)
A 0.5 g/L solution of titan yellow R.
Content: minimum 97.0 per cent of SnCh,2H2O.
Test for sensitivity. To 0.1 mL of the titan yellow solution add
Colourless crystals, very soluble in water, freely soluble in 10 mL of water R, 0.2 mL of magnesium standard solution
ethanol (96 per cent), in glacial acetic acid and in dilute and (10 ppm Mg) Rand 1.0 mL of 1 M sodium hydroxide.
concentrated hydrochloric acid. A distinct pink colour is visible by comparison with a
Assay. Dissolve 0.500 gin 15 mL of hydrochloric acid Rina reference solution prepared in a similar manner omitting the
ground-glass-stoppered flask. Add 10 mL of water R and magnesium.
5 mL of chloroform R. Titrate rapidly with 0. 05 M potassium Titanium Ti= 47.88 (7440-32-6)
iodate until the chloroform layer is colourless.
Content: minimum 99 per cent.
1 mL of 0.05 M potassium iodate is equivalent to 22.56 mg of
SnC12,2H2 O. Metal powder, fine wire (diameter not more than 0.5 mm),
sponge.
Tin(n) Chloride Solution Stannous chloride solution
mp: about 1668 °C.
Heat 20 g of tin R with 85 mL of hydrochloric acid R until no
more hydrogen is released. Allow to cool. Density: about 4.507 g/cm3.
Storage: over an excess of tin R, protected from air. Titanium(m) Chloride Titanium trichloride;
TiC13 = 154.3 (1705-07-9)
Tin(n) Chloride Solution AsT
Reddish-violet crystals, deliquescent, soluble in water and in
Stannous chloride solution, low in arsenic, of commerce, or ethanol (96 per cent).
prepare from tin(II) chloride solution by adding an equal
volume of hydrochloric acid, reducing the original volume by mp: about 440 °C.
boiling and filtering through a fine-grain filter paper. Storage: in an airtight container.
Complies with the following test. Titanium(m) Chloride Solution Titanium trichloride
To 10 mL add 6 mL of water and 10 mL of hydrochloric acid, solution
distil and collect 16 mL. To the distillate add 50 mL of d~g: about 1.19.
water, 0.1 mL of the solution, 5 mL of0.IMpotassium iodide A 150 g/L solution of titanium trichloride R in hydrochloric
and 5 g of activated zinc. Using the apparatus and procedure acid (100 g/L HCl).
described for the limit test for arsenic, Appendix VII, the Titanium(m) Chloride-Sulfuric Acid Reagent
colour obtained in the test-tube with the test solution is not Titanium(m) chloride-sulphuric acid reagent; Titanium
more intense than that produced when the test is repeated trichloride--sulphuric acid reagent; Titanium trichloride-
with the addition of 1 mL of arsenic standard solution (1 ppm sulfuric acid reagent
As). Carefully mix 20 mL of titanium trichloride solution R with
Tin(u) Chloride Solution Rl Stannous chloride 13 mL of sulfuric acid R. Add sufficient strong hydrogen
solution Rl peroxide solution R to give a yellow colour. Heat until white
Immediately before use, dilute 1 volume of stannous chloride fumes are evolved. Allow to cool. Dilute with water R and
solution R with 10 volumes of dilute hydrochloric acid R. repeat the evaporation and addition of water R until a
Tin(n) Chloride Solution R2 Stannous chloride colourless solution is obtained. Dilute to 100 mL with
solution R2 water R.
To 8 g of stannous chloride R add 100 mL of a Titanium Dioxide Titanium(!V) oxide; (13463-67-7)
20 per cent V/V solution of hydrochloric acid R. Shake until See Titanium dioxide (0150).
V-A154 Appendix I A 2023

TLC Aluminium Oxide G Plate the middle of the chromatogram for quantities of 0.2 µg and
Support of metal, glass or plastic, coated with a layer of greater.
aluminium oxide (particle size 5-40 µm) containing about TLC Silica Gel F254 Silanised Plate
10 per cent of calcium sulfate hemihydrate as a binder. It complies with the requirements prescribed for
TLC Cellulose Plate TLC silanised silica gel plate R with the following modification.
Support of glass, metal or plastic, coated with a layer of It contains a fluorescent indicator having a maximum
cellulose. absorbance at 254 nm.
TLC Octadecylsilyl Silica Gel Plate TLC Silica Gel G Plate
Support of glass, metal or plastic coated with a layer of Complies with the requirements prescribed for TLC silica gel
octadecylsilyl silica gel. The plate may contain an organic plate R with the following modification.
binder. It contains calcium sulfate hemihydrate as binder.
TLC Octadecylsilyl Silica Gel F254 Plate TLC Silica Gel GF254 Plate
Support of glass, metal or plastic coated with a layer of Complies with the requirements prescribed for TLC silica gel
octadecylsilyl silica gel. plate R with the following modifications.
It contains a fluorescent indicator having a maximum It contains calcium sulfate hemihydrate as binder and a
absorbance in ultraviolet light at 254 nm. fluorescent indicator having a maximum absorbance at
TLC Performance Test Solution 254 nm.
Prepare a mixture of 1.0 mL of each of the following Fluorescence suppression. Complies with the test prescribed for
solutions and dilute to 10.0 mL with aceume R: a 0.5 g/L TLC silica gel F254 plate R.
solution of Sudan red GR in toluene R, a 0.5 g/L solution of TLC Silica Gel Plate for Aminopolyether Test
methyl orange R in ethanol R prepared immediately before use, Immerse a TLC silica gel plate R in iodoplatinate reagent Rl for
a 0.5 g/L solution of bromocresol green R in acetone Rand a 5-10 s. Dry at room temperature for 12 h, protected from
0.25 g/L solution of methyl red R in acetone R. light.
TLC Silica Gel Plate Silica gel Storage: protected from light, in an open container; use
Support of glass, metal or plastic, coated with a layer of silica within 30 days after preparation.
gel of a suitable thickness and particle size (usually 2 µm to TLC Silica Gel Plate for Chiral Separations,
10 µm for fine particle size [High Performance Thin-Layer Octadecylsilyl
Chromatography, HPTLC] plates and 5 µm to 40 µm for
normal TLC plates). If necessary, the particle size is Support of glass, metal or plastic, coated with a layer of
indicated after the name of the reagent in the tests where it is octadecylsilyl silica gel, impregnated with Cu2 + ions and
enantiomerically pure hydroxyproline. The plate may contain
used.
an organic binder.
The plate may contain an organic binder.
TLC Silica Gel Silanised Plate
Chromatographic separation. Apply to the plate an appropriate
volume ( 10 µL for a normal TLC plate and 1 µL to 2 µL for Support of glass, metal or plastic, coated with a layer of
a fine particle size plate) of TLC performance test solution R. silanised silica gel of a suitable thickness and particle size
Develop over a pathlength two-thirds of the plate height, (usually 2 µm to 10 µm for fine particle size [High
Performance Thin-Layer Chromatography, HPTLC] plates
using a mixture of 20 volumes of methanol R and 80 volumes
of toluene R. The plate is not satisfactory, unless the and 5 µm to 40 µm for normal TLC plates). If necessary, the
chromatogram shows four clearly separated spots, the spot of particle size is indicated after the name of the reagent in the
bromocresol green with an Rp value less than 0.15, the spot tests where it is used.
of methyl orange with an Rp value in the range of 0.1 to The plate may contain an organic binder.
0.25, the spot of methyl red with an Rp value in the range of Chromatographic separation. Introduce 0.1 g each of methyl
0.35 to 0.55 and the spot of Sudan red G with an Rp value laurate R, methyl myristate R, methyl palmitate R and methyl
in the range of 0.75 to 0.98. stearate R into a 250 mL conical flask. Add 40 mL of
TLC Silica Gel F254 Plate alcoholic potassium hydroxide solution R and heat under a reflux
Complies with the requirements prescribed for TLC silica gel condenser on a water-bath for 1 h. Allow to cool, transfer the
plate R with the following modification. solution to a separating funnel by means of 100 mL of
water R, acidify (pH 2 to 3) with dilute hydrochloric acid Rand
It contains a fluorescent indicator having a maximum shake with three quantitites each of 10 mL of methylene
absorbance at 254 nm. chloride R. Dry the combined methylene chloride extracts
Fluorescence suppression. Apply separately to the plate at five over anhydrous sodium sulfate R, filter and evaporate to
points increasing volumes ( 1 µL to 10 µL for normal dryness on a water-bath. Dissolve the residue in 50 mL of
TLC plates and 0.2 µL to 2 µL for fine particle size plates) methylene chloride R. Examine by thin-layer chromatography
of a 1 g/L solution of benzoic acid R in a mixture of (2.2.27), using TLC silanised silica gel plate R. Apply an
15 volumes of anhydrous ethanol Rand 85 volumes of appropriate quantity (about 10 µL for normal TLC plates
cyclohexane R. Develop over a pathlength half of the plate and about 1 µL to 2 µL for fine particle size plates) of the
height with the same mixture of solvents. After evaporating methylene chloride solution at each of three separate points.
the solvents examine the chromatogram in ultraviolet light at Develop over a pathlength two-thirds of the plate height with
254 nm. For normal TLC plates the benzoic acid appears as a mixture of 10 volumes of glacial acetic acid R, 25 volumes
dark spots on a fluorescent background approximately in the of water R and 65 volumes of dioxan R. Dry the plate at
middle of the chromatogram for quantities of 2 µg and 120 °C for 30 min. Allow to cool, spray with a 35 g/L
greater. For fine particle size plates the benzoic acid appears solution of phosphomolybdic acid R in 2-propanol R and heat at
as dark spots on a fluorescent background approximately in 150 °C until the spots become visible. Treat the plate with
ammonia vapour until the background is white.
2023 Appendix I A V-A155

The chromatograms show four clearly separated, well-defined o-Toluic Acid C 8 H 8 O2 = 136.2 (118-90-1)
spots. mp: about 104°.
a-Tocopherol (10191-41-0) General reagent grade of commerce.
See all-rac-a-Tocopherol (0692). A white, crystalline powder.
a-Tocopheryl Acetate (7695-91-2) o-Toluidine 2-Methylaniline; C 7 H 9 N = 107 .2 (95-53-4)
See all-rac-o.-Tocopheryl acetate (0439). Pale-yellow liquid becoming reddish-brown on exposure to
o-Tolidine 3,3'-Dimethylbenzidine; C 14H 1~ 2 = 212.3 air and light, slightly soluble in water, soluble in ethanol
(119-93-'T) (96 per cent) and in dilute acids.
Content: minimum 97.0 per cent. dig: about 1.01.
Light brownish, crystalline power. nt 0 : about 1.569.

mp: about 130 °C. bp: about 200 °C.

o-Tolidine Solution Storage: in an airtight container, protected from light.
Dissolve 0.16 g of o-tolidine R in 30.0 mL of glacial acetic p-Toluidine 4-Methylaniline; C 7 H 9N = 107.2 (106-49-0)
acid R, add 1.0 g of potassium iodide R and dilute to Lustrous plates or flakes, slightly soluble in water, freely
500.0 mL with water R. soluble in acetone and in ethanol (96 per cent).
Toluene Methylbenzene; C 7 H 8 = 92.1 (108-88-3) mp: about 44 °C.
Clear, colourless, flammable liquid, very slightly soluble in Toluidine Blue Toluidine Blue O; 3-Amino-7-
water, miscible with ethanol (96 per cent). dimethylamino-2-methylphenothiazin-5-ium chloride;
d~g: 0.865 to 0.870. C1sH16ClN3S = 305.8 (92-31-9)
bp: about 110 °C. Schultz No. 1041
Toluene, Sulfur-free Toluene, sulphur-free Colour Index No. 52040
Complies with the requirements prescribed for toluene R with Dark-green powder, soluble in water, slightly soluble in
the following additional requirements. ethanol (96 per cent).
Sulfur compounds. To 10 mL add 1 mL of anhydrous o-Toluidine Hydrochloride 2-Methylaniline
ethanol R and 3 mL of potassium plumbite solution R and boil hydrochloride; 2-Methylbenzenamine hydrochloride;
under a reflux condenser for 15 min. Allow to stand for C 7 H 10 ClN = 143.6 (636-21-5)
5 min. No darkening is produced in the aqueous layer. Content: minimum 98.0 per cent.
Thiophen-related substances. Shake 2 mL with 5 mL of isatin mp: 215 °C to 217 °C.
reagent R for 5 min and allow to stand for 15 min. No blue
Tosylarginine Methyl Ester Hydrochloride N-Tosyl-L-
colour is produced in the lower layer.
arginine methyl ester hydrochloride; Methyl (S)-5-guanidino-
p-Toluenesulfonamide (70-55-3) 2-( 4-methylbenzenesulfonamido)valerate hydrochloride;
See toluenesulfonamide R. C 14H 23 CIN4O4 S = 378.9 (1784-03-8)
Toluene-o-sulfonamide Toluene-a-sulphonamide; [a]t0 : -12 to -16, determined on a 40 g/L solution.
o-Toluenesulphonamide; 2-Methylbenzenesulfonamide; mp: about 145 °C.
2-Methylbenzenesulphonamide; o-Toluenesulfonamide;
Tosylarginine Methyl Ester Hydrochloride Solution
C1H 9N0 2 S = 171.2 (88-19-'T)
To 98.5 mg of tosylarginine methyl ester hydrochloride R add
White or almost white, crystalline powder, slightly soluble in
5 mL of tris(hydroxymethyl)aminomethane buffer solution
water, soluble in ethanol (96 per cent) and in solutions of
pH 8.1 R and shake to dissolve. Add 2.5 mL of methyl red
alkali hydroxides.
mixed solution Rand dilute to 25.0 mL with water R.
mp: about 156 °C.
Tosyl-lysyl-chloromethane Hydrochloride (3S)- 7-
Toluene-p-sulfonamide p-Toluenesulfonamide; Toluene Amino-1-chloro-3-tosylamido-2-heptanone hydrochloride;
sulfonamide; 4-Methylbenzenesulfonamide; C 14H 22 Cl 2N 2 O3 S = 369.3 (4238-41-9)
Toluenesulfonamide; C 7 H 9 NO 2 S = 171.2 (70-55-3)
[a]t0 : -7 to -9, determined on a 20 g/L solution.
Content: minimum 99.0 per cent.
mp: about 155 °C, with decomposition.
White or almost white, crystalline powder, slightly soluble in
Al~m: 310 to 340, determined at 230 nm in water R.
water, soluble in ethanol (96 per cent) and in solutions of
alkali hydroxides. Tosylphenylalanylchloromethane L-tosylaminophenethyl
chloromethyl ketone; C 17H 18 CINO 3 S = 351.9 (402-71-1)
mp: about 136 °C.
[a]~: -85 to -89, determined on a 10 g/L solution in ethanol
Toluene-p-sulfonic Acid Toluene-p-sulphonic acid;
(96 per cent) R.
Toluenesulphonic acid; 4-Methylbenzenesulfonic acid;
4-Methylbenzenesulphonic acid; Toluenesulfonic acid; mp: about 105 °C.
C7 H 8 0 3 S,H2 0 = 190.2 (6192-52-5) Al~m: 290 to 320, determined at 228.5 nm in ethanol
Content: minimum 87.0 per cent of C 7 HsO3S. (96 per cent) R.

White or almost white, crystalline powder or crystals, freely Toxaphene (8001-35-2)

soluble in water, soluble in ethanol (96 per cent). A mixture of polychloro derivatives.
T oluenesulfonylurea 4-Methylbenzenesulfonylurea; mp: 65 °C to 90 °C.
p-Toluenesulfonylurea; (4-Methylphenyl)sulfonylurea; A suitable certified reference solution (10 ng/µL in iso-
CsH 10N2O 3S = 214.2 (1694-06-0) octane) may be used.
White or almost white, crystalline powder. Tragacanth (9000-65-1)
mp: 196 to 198 °C. See Tragacanth (0532).
V-A156 Appendix I A 2023

Triacetin Propane-1,2,3-triyl triacetate; Glycerol triacetate; Distillatwn range (2.2.11). Not less than 98 per cent distils
C9H 14 O 6 = 218.2 (102-76-1) between 47 °C and 48 °C.
Almost clear, colourless to yellowish liquid, soluble in water, Trichodesmine (1R,4R,5R,6R, 16R)-5,6-Dihydroxy-5,6-
miscible with ethanol (96 per cent). dimethyl-4-(propan-2-yl)-2,8-dioxa- l 3-azatricyclo
d~g: about 1.16. [8.5. l.0 13' 16]hexadec-10-ene-3, 7-dione; C 18H 27NO 6 = 353.4
ni 0 : about 1.43. (548-90-3)

bp: about 260 °C. Yellowish-white powder, soluble in water and in methanol.
Triamcinolone 9-Fluoro-11 ~,lfo,17,21- Tricine N-[2-Hydroxy- l, 1-bis (hydroxymethyl)ethyl] glycine;
tetrahydroxypregna-l,4-diene-3,20-dione; C 6 H 13NO 5 = 179.2 (5704-04-1)
C21H 27 FO 6 = 394.4 (124-94-7) Use electrophoresis-grade reagent.
A crystalline powder. mp: about 183 °C.
mp: 262 °C to 263 °C. Tricosane C 23 H 48 = 324.6 (638-67-5)
Triamcinolone Acetonide (76-25-5) White or almost white crystals, practically insoluble in water,
See Triamcinolone acetonide (0533). soluble in hexane.
Tribromophenol 2,4,6-Tribromophenol; mp: about 48 °C.
C6H3 Br3 O = 330.8 (118-79-6) Tridecyl Alcohol Tridecanol; C 13H 28 O = 200.4
(112-70-9)
Tributyl Citrate Tributyl 2-hydroxypropane-1,2,3-
tricarboxylate; C 18H 32O 7 = 360.4 (77-94-1) Tridocosahexaenoin Triglyceride of docosahexaenoic acid
(C22:6); Glycerol tridocosahexaenoate; Propane-1,2,3-triyl
df 0 : about 1.043. tri-(all-Z)-docosa-4, 7, I 0, 13,16, 19-hexaenoate;
n;r about 1.445. C6 9 H 98 O6 = 1023.5 (124596-98-1)
Tributyl Orthophosphate Tributoxyphosphine oxide; The reagent from Nu-Chek Prep, Inc. has been found
Tributoxyphosphane oxide; Tributyl phosphate; suitable.
C12H 27 O4P = 266.3 (126-73-8)
Triethanolamine (102-71-6)
Colourless liquid, slightly soluble in water, soluble in the
usual organic solvents. See Trolamine (1577).
Triethyl Phosphonoformate Ethyl (diethoxyphosphoryl)
d1~: about 0.976.
formate; C1H1 5 O 5P = 210.2 (1474-78-8)
n&: about 1.422.
Colourless liquid.
bp: about 289 °C, with decomposition.
bp 12 mm: about 135 °C.
Tributylphosphine C 12H 27 P = 202.3 (998-40-3)
Triethylamine N,N-Diethylethanamine; C 6H 15N = 101.2
Clear, colourless liquid. (121-44-8)
bp: about 240 °C. Colourless liquid, slightly soluble in water at a temperature
mp: about -60 °C. below 18.7 °C, miscible with ethanol (96 per cent).
Trichlorethylene Trichloroethylene; C 2HC1 3 = 131.4 dig: about 0.727.
(79-01-6)
Colourless liquid, practically insoluble in water, miscible with
nt 0 : about 1.401.

bp: about 90 °C.

ethanol (96 per cent).
Triethylamine Rl N,N-Diethylethanamine;
d~g: about 1.46. C6H 15N = 101.2 (121-44-8)
n~;': about 1.4 77. Complies with the requirements prescribed for triethylamine R
Trichloroacetic Acid C 2HC1 3 O2 = 163.4 (76-03-9) with the following additional requirements.
Colourless crystals or a crystalline mass, very deliquescent, Content: minimum 99.5 per cent, determined by gas
very soluble in water and in ethanol (96 per cent). chromatography.
Storage: in an airtight container. Water: maximum 0.1 per cent.
Trichloroacetic Acid Solution Use freshly distilled or from a freshly opened container.
Dissolve 40.0 g of trichloroacetic acid R in water Rand dilute Triethylamine R2 N,N-Diethylethanamine;
to 1000.0 mL with the same solvent. Verify the C 6 H 15 N = 101.2 (121-44-8)
concentration by titration with 0.1 M sodium hydroxide and Complies with the requirements prescribed for triethylamine R
adjust if necessary to 40 ± 1 g/L. and with the following additional requirements.
1, 1, 1-Trichloroethane Methylchloroform; Content: minimum 99.5 per cent, determined by gas
C2H3 Cl 3 = 133.4 (71-55-6) chromatography.
Non-flammable liquid, practically insoluble in water, soluble Water: maximum 0.2 per cent.
in acetone and in methanol.
It is suitable for gradient elution in liquid chromatography.
dig: about 1.34. Use freshly distilled or from a freshly opened container.
ni 0 : about 1.438.
Triethylamine Hydrogen Carbonate Solution
bp: about 74 °C.
Pass a gentle current of carbon dioxide through a 5% w/v
Trichlorotrifluoroethane 1, l ,2-Trichloro-1,2,2- solution of triethylamine for 16 hours.
trifluoroethane; C 2ChF3 = 187.4 (76-13-1)
Triethylenediamine 1,4-Diazabicyclo[2.2.2]octane;
Colourless, volatile liquid, practically insoluble in water, C 6 H12N 2 = 112.2
miscible with acetone.
d~g: about 1.58.
2023 Appendix I A V-A157

Crystals, very hygroscopic, sublimes readily at room Absorbance (2.2.25): maximum 0.01 from 250 nm to 420 nm,
temperature, freely soluble in water, in acetone and in determined using water R as compensation liquid.
anhydrous ethanol. Trimethylpentane for Chromatography
bp: about 174 °C. Complies with the requirements prescribed for
mp: about 158 °C. trimethylpentane R with the following additional requirement.
Storage: in an airtight container. Residue on evaporation: maximum 2 mg/L.
Triflumuron 1-(2-Chlorobenzoyl)-3-(4- Trimethylpentane Rl
triflumoromethoxyphenyl)urea; C 15H 10 ClF 3N 2 O 3 = 358.7 Complies with the requirements prescribed for
( 6462 8-44-0) trimethylpentane R with the following modification.
White or almost white crystalline powder, practically Absorbance (2.2.25). Not more than 0.07 from 220 nm to
insoluble in water, sparingly soluble in acetone and in 360 nm, determined using water R as the compensation
methylene chloride. liquid.
Trifluoroacetic Acid C 2HF 3 O 2 = 114.0 (76-05-1) N-Trimethylsilylimidazole 1-Trimethylsilylimidazole;
Content: minimum 99 per cent. C 6H 12N 2Si = 140.3 (18156-74-6)
Liquid, miscible with acetone and with ethanol (96 per cent). Colourless, hygroscopic liquid.
dig: about 1.53. dig: about 0.96.
bp: about 72 °C. n;2;1: about 1.48.
Use a grade suitable for protein sequencing. Storage: in an airtight container.
Storage: in an airtight container. 3-Trimethylsilyl-1-propanesulfonic Acid, Sodium
Trifluoroacetic Anhydride C 4 F 6O 3 = 210.0 (407-25-0) Salt Sodium 3-(trimethylsilyl)-1-propanesulfonate; sodium
3-(trimethylsilyl)-1-propanesulphonate; 3-trimethylsilyl-1-
Colourless liquid.
propanesulphonic acid, sodium salt; C 6H 15NaO 3 SSi = 218.3
dig: about 1.5. (2039-96-5)
3-Trifluoromethylaniline 3-(Trifluoromethyl)aniline; rx,rx, mp: about 165°.
rx-Trifluoro-m-toluidine; 3-(Trifluoromethyl) benzenamide;
C 7 H 6 F 3N = 161.1 (98-16-8) Beige powder; content, minimum 97.0 %.
Trimethylsulfonium Hydroxide Trimethylsulfonium
Colourless liquid.
hydroxide; C 3 H 10OS = 94.2 (17287-03-5)
Density: 1.30 g/cm 3 (20 °C).
4-Trifluoromethylphenol C 7 H 5F 3 O = 162.1 (402-45-9)
df 0 : about 0.81.
Trimethyltin Chloride Chlorotrimethylstannane;
White or light yellow, crystalline solid or powder. C 3H 9C1Sn = 199 .3 (1066-4 5-1)
mp: about 46 °C. 2,4,6-Trinitrobenzenesulfonic Acid Picrylsulfonic acid;
Trifluoropropylmethylpolysiloxane Picrylsulphonic acid; 2,4,6-Trinitrobenzenesulphonic acid;
Polysiloxane substituted with trifluoropropyl groups and C 6H 3 N 3 O 9S,3H2O = 347.2 (2508-19-2)
methyl groups. White or almost white, crystalline powder, soluble in water.
T riglycine 2-[[2-[ (2-Aminoacetyl)amino] acetyl] amino] mp: 190 °C to 195 °C.
acetic acid; Glycylg]ycylglycine; C 6HuN 3 O 4 = 189.2 Triolein Propane-1,2,3-triyl tris [(9Z)-octadec-9-enoate];
(556-33-2) sn-Glyceryl trioleate; Glycerol trioleate; Oley! triglyceride;
Trigonelline Hydrochloride 1-Methylpyridinium-3- Cs1H104 O 6 = 885.4 (122-32-7)
carboxylate hydrochloride; C 7 H 8 ClNO2 = 173.6 (6138-41-6) Content: minimum 99.0 per cent.
Crystalline powder, very soluble in water, soluble in ethanol Triphenylamine C 18H 15N = 245.3 (603-34-9)
(96 per cent).
mp: about 126°.
mp: about 258 °C.
General reagent grade of commerce.
1,2,4-Trimethylbenzene Pseudocumene; C 9H 12 = 120.2
(95-63-6)
A white, crystalline solid.
Trimethylchlorosilane Chlorotrimethylsilane; Triphenylethylene C 20H 16 = 256.4 (58-72-0)
C 3H 9C1Si = 108.6 (75-77-4) mp: about 70°.
Clear, colourless liquid, fuming in air. General reagent grade of commerce.
dig: about 0.86. Triphenylmethanol Triphenylcarbinol; C 19H 16 O = 260.3
(76-84-6)
ni0 : about 1.388.
Colourless crystals, practically insoluble in water, freely
bp: about 57 °C.
soluble in ethanol (96 per cent).
2 ,2 ,4-Trimethylpentane Iso-octane; Trimethylpentane;
2,3,5-Triphenyltetrazolium Chloride Tetrazolium Salt;
CsHrn = 114.2 (540-84-1)
Triphenyltetrazolium Chloride; C 19H 15 ClN 4 = 334.8
Colourless, flammable liquid, practically insoluble in water, (298-96-4)
soluble in anhydrous ethanol.
Pale or dull-yellow powder, soluble in water, in acetone and
dig: 0.691 to 0.696. in ethanol (96 per cent).
ni0 : 1.391 to 1.393. mp: about 240 °C, with decomposition.
Distillation range (2.2.11). Not less than 95 per cent distils Storage: protected from light.
between 98 °C and 100 °C.
Analytical reagent grade of commerce containing not less
Trimethylpentane used in spectrophotometry complies with the
than 98.0 per cent of C19H1sC1N4
following additional test.
V-A158 Appendix I A 2023

Assay Dissolve 1 g in a mixture of 5 mL of dilute nitric acid Trisodium Orthophosphate Trisodium phosphate
and 45 mL of water, add 50 mL of 0.lM silver nitrate VS and dodecahydrate; Na3 PO 4 ,12H2 O = 380.1 (10101-89-0)
heat to boiling. Allow to cool, add 3 mL of dibutyl phthalate, Colourless or white or almost white crystals, freely soluble in
shake vigorously and titrate with 0. lM ammonium thiocyanate water.
VS using 2 mL of ammonium iron(III) sulfate solutwn R2 as Trometamol (77-86-1)
indicator. Each mL of O. lM silver nitrate VS is equivalent to
See Tris(hydroxymethyl)aminomethane R.
33.48 mg ofC19H 1 sC1N4.
Triphenyltetrazolium Chloride Solution Tropic Acid (2RS)-3-Hydroxy-2-phenylpropanoic acid;
C9H10O3 = 166.17 (529-64-6)
A 0.5% w/v solution of 2,3,5-triphenyltetrazolium chloride in
Tropine (1R,3r,5S)-Tropan-3-ol; C 8 H 15NO = 141.2
aldehyde-free ethanol (96%).
(120-29-6)
Store protected from light.
mp: about 54°.
Tripotassium Phosphate Trihydrate
K 3PO 4,3H2O = 266.3 (22763-03-7) General reagent grade of commerce.
White or almost white crystalline powder, freely soluble in Colourless crystals.
water. Troxerutin Trihydroxyethylrutin; 3',4',7-Tris[O-(2-
Triscyanoethoxypropane l ,2,3-Tris(2-cyanoethoxy) hydroxyethyl)] rutin; 2-[3,4-Bis(2-hydroxyethoxy)phenyl]-3-
propane; C12H 17N 3O3 = 251.3 [[6-0-( 6-deoxy-lX-L-mannopyranosyl)-/3-D-glucopyranosyl]
oxy]-5-hydroxy-7-(2-hydroxyethoxy)-4H- l-benzopyran-4-one;
Viscous, brown-yellow liquid, soluble in methanol. Used as a
C33H42O19 = 743 (7085-55-4)
stationary phase in gas chromatography.
mp: 168 °C to 176 °C.
dig: about 1.11. Trypsin (9002-07-7)
Viscosity (2.2. 9): about 172 mPa·s.
A proteolytic enzyme obtained by activation of trypsinogen
1,3 ,5- Tris(3, 5-di-(1,1-dimethylethyl)-4-hydroxybenzyl)- extracted from the pancreas of beef (Bos taurus L.).
1H,3H,5H-1,3,5-triazine-2,4,6-trione Tris(3,5-di-tert-
butyl-4-hydroxybenzyl isocyanurate; l,3,5-Tris[3,5-di(l,l- White or almost white, crystalline or amorphous powder,
dimethylethyl)-4-hydroxybenzyl]-l,3 ,5-triazine- sparingly soluble in water.
2,4,6 ( 1H,3H,5H)-trione; C48 H 69 O 6N 3 = 784.1 (27676-62-6) Trypsin for Peptide Mapping (9002-07-7)
White or almost white, crystalline powder. Trypsin of high purity treated to eliminate chymotryptic
mp: 218 °C to 222 °C. activity.
Tris(2,4-di-(l,l-dimethylethyl)phenyl) Phosphite Tryptophan C 11 H 12N 2O 2 = 204.2 (73-22-3)
C42H53O 3 P = 647 (31570-04-4) White or yellowish-white, crystalline powder or colourless
crystals, slightly soluble in water, very slightly soluble in
White or almost white powder.
ethanol (96 per cent).
mp: 182 °C to 186 °C.
Tris(hydroxymethyl)nitromethane C 4H 9NO 5 = 151
[e<Jfr about -30, determined on a 10 g/L solution.
(126-11-4)
Typhaneoside 3-[6-Deoxy-lX-L-mannopyranosyl-(l-+2)-[6-
deoxy-lX-L-mannopyranosyl-( 1-> 6) ]-/3-D-glucopyranosyloxy]-
General reagent of commerce. 5, 7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H- l-
Tris(hydroxymethyl)aminomethane Hydrochloride benzopyran-4-one; C 3 4H 42 O 20 = 771 (J 04472-68-6)
2-Amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride; Tyramine 4-(2-Aminoethyl)phenol; C 8 H 11 NO = 137 .2
NH2C(CH2OHh,HCI = 157.60 (1185-53-1) (51-67-2)
Analytical reagent grade of commerce. Crystals, sparingly soluble in water, soluble in boiling
Tris(hydroxymethyl)aminomethane MB anhydrous ethanol.
C4H 11 NO 3 = 121.14 (77-86-1) mp: 164 °C to 165 °C.
Molecular biology grade. L-Tyrosine 2-Amino-3-( 4-hydroxyphenyl)propionic acid;
Tris(hydroxymethyl)aminomethane Solution Rl Tyrosine; C 9H 11 NO 3 = 181.2 (60-18-4)
Dissolve 60.6 mg of tris(hydroxymethyl)aminomethane Rand White or almost white, crystalline powder, or colourless or
0.234 g of sodium chloride R in water Rand dilute to 100 mL white or almost white crystals, slightly soluble in water,
with the same solvent. practically insoluble in acetone and in anhydrous ethanol,
Storage: at 2 °C to 8 °C; use within 3 days. soluble in dilute hydrochloric acid and in solutions of alkali
Tris(hydroxymethyl)methylamine Tris(hydroxymethyl) hydroxides.
aminomethane; (77-86-1) Umbelliferone 7-Hydroxycoumarin; 7-Hydroxy-2H-l-
See Trometamol (1053). benzopyran-2-one; C 9H 6O 3 = 162.1 (93-35-6)
Tris(hydroxymethyl)methylamine Solution Needles from water.
Tris(hydroxymethyl)aminomethane solution mp: 225 °C to 228 °C.
A solution containing the equivalent of 24.22 g of Undecanoic Acid Hendecanoic acid; Undecylic acid;
C4H 11 NO 3 in 1000.0 mL. C11H22O2 = 186.29 (112-37-8)
Tris(hydroxymethyl)methylamine Solution, mp: about 30 °C.
Methanolic Content: minimum 97.0 per cent of C 11 H 22 O 2 •
Dissolve 6.07 g of tris(hydroxymethyl)methylamine in 900 mL Uracil C 4 H 4N 2 0 2 = 112.1 (66-22-8)
of methanol, add 50 mL of 0.5M hydrochloric acid and dilute Content: minimum 95.0 per cent.
to 1000 mL with water.
Urea (57-13-6)
See Urea (0743).
2023 Appendix I A V-A159

Urease-active Meal (9002-13-5) Vanillin Solution, Phosphoric

General reagent grade of commerce. Dissolve 1.0 g of vanillin R in 25 mL of ethanol
Activity Each mg of urease-active meal hydrolyses 3 mg of (96 per cent) R. Add 25 mL of water Rand 35 mL of
urea in 30 minutes at 37°. phosphoric acid R.
Uridine 1-f3-D-Ribofuranosyluracil; C 9 H 12N 2O 6 = 244.2 Veratric Acid 3,4-Dimethoxybenzoic acid;
(58-96-8) C 9 H 10 O4 = 182.2 (93-07-2)
White or almost white, crystalline powder, soluble in water. mp: about 180°.
mp: about 165 °C. General reagent grade of commerce.
Ursolic Acid 3f3-Hydroxyurs-12-en-28-oic acid; Veratrole 1,2-Dimethoxybenzene; C 8 H 10 O 2 = 138.2
C 3 0H4 8 O, = 456.7 (77-52-1) (91-16-7)
White or almost white powder, practically insoluble in water, df0 : 1.085.
sparingly soluble in methanol, slightly soluble in ethanol nt 0:1.534.
(96 per cent). bp: about 206 °C.
[o:]~: about 67.50, determined on a 10 g/L solution in a mp: about 22 °C.
56.1 g/L solution of potassium hydroxide R in ethanol Verbenone (1S,5S)-4,6,6-Trimethylbicyclo[3.1. l]hept-3-
(96 per cent) R.
en-2-one; C 10H 14O = 150.2 (1196-01-6)
mp: 285 °C to 288 °C. Oil with a characteristic odour, practically insoluble in water,
Urushiol I 3-Pentadecyl-1,2-benzenediol; miscible with organic solvents.
C 21H3 6O 2 = 320.51 (492-89-7) dig: about 0.978.
General reagent grade of commerce.
n~i3; about 1.49.
Urushiol n 3-(8-Pentadecenyl)-l,2-benzenediol;
[o:]b3: about+ 249.6.
C21H34O2 = 318.26 (2764-91-2)
bp: 227 °C to 228 °C.
General reagent grade of commerce.
mp: about 6.5 °C.
Valencene 4f3H,5cx-Eremophila-1(10),1 l-diene;
( lR, 7R,8aS)- l ,8a-Dimethyl-7-(1-methylethenyl)- Verbenone used in gas chromatography complies with the following
1,2,3,5,6, 7,8,8a-octahydronaphthalene; C 15H 24 = 204.4 additional test.
( 4630-07-3) Assay. Gas chromatography (2.2.28) as prescribed in the
Oily, colourless or pale yellow liquid, with a characteristic monograph Rosemary oil (1846).
odour, practically insoluble in water, soluble in ethanol Content: minimum 99 per cent, calculated by the
(96 per cent). normalisation procedure.
df 0 : about 0.918. Vinyl Acetate Ethenyl acetate; C 4 H 6 O 2 = 86,10
(108-05-4)
nt 0: about 1.508.
bp: about 123 °C. d?g: about 0.930.
Valencene used in gas chromatography complies with the following bp: about 72 °C.
additional test. Vinyl Chloride C 2 H 3Cl = 62.5 (75-01-4)
Assay. Gas chromatography (2.2.28) as prescribed in the Colourless gas, slightly soluble in organic solvents.
monograph Sweet orange oil (1811). Vinyl Polymer for Chromatography, Amino Alkyl
Content: minimum 80 per cent, calculated by the Spherical particles (5 µm) of a vinyl alcohol copolymer
normalisation procedure. chemically modified by bonding of amino alkyl groups.
Valerenic Acid (2E)-3-[(4S,7R,7aR)-3,7-Dimethyl- Vinyl Polymer for Chromatography, Octadecyl
2,4,5,6, 7, 7a-hexahydro-lH-inden-4-yl]-2-methylprop-2-enoic Spherical particles (5 µm) of a vinyl alcohol copolymer
acid; C 15H 22 O2 = 234.3 (3569-10-6) chemically modified by bonding of octadecyl groups on the
mp: 134 °C to 138 °C. hydroxyl groups.
Valerie Acid N-Valeric acid; C 5H 10O 2 = 102.1 (109-52-4) Vinyl Polymer for Chromatography, Octadecylsilyl
Colourless liquid, soluble in water, freely soluble in ethanol Spherical particles (5 µm) of a vinyl alcohol copolymer
(96 per cent). bonded to an octadecylsilane. Carbon content of 17 per cent.
dig: about 0.94. Vinyl(l)phenyl(5)methyl(94)polysiloxane
nfi°; about 1.409. Polysiloxane substituted with 1 per cent of vinyl groups,
bp: about 186 °C. 5 per cent of phenyl groups and 94 per cent of methyl
Valine (72-18-4) groups.
See Valine (0796). 2-Vinylpyridine C 7H 7 N = 105.1 (100-69-6)
Vanillin 4-Hydroxy-3-methoxybenzaldehyde; (121-33-5) Yellow liquid, miscible in water.
See Vanillin (0747). dig: about 0.97.
Vanillin Reagent nt0 : about 1.549.

Carefully add, dropwise, 2 mL of sulfuric acid R to 100 mL 4-Vinylpyridine 4-Ethenylpyridine; C 7H 7 N = 105.l

(100-43-6)
of a 10 g/L solution of vanz1lin R in ethanol (96 per cent) R.
Storage: use within 48 h. Clear, deep yellowish-brown liquid.
bp: 58-61 °C.
V-A160 Appendix I A 2023

1-Vinylpyrrolidin-2-one 1-Ethenylpyrrolidin-2-one; Water, Carbon Dioxide-free

C 6 H 9 NO = 111.1 (88-12-0) Water R which has been boiled for a few minutes and
Content: minimum 99.0 per cent. protected from the atmosphere during cooling and storage or
Clear colourless liquid. deionised water R with a resistivity of not less than
Water (2.5.12): maximum 0.1 per cent, determined on 2.5 g. 0.18 Mil·m, determined at 25 °C.
Use as the solvent, a mixture of 50 mL of anhydrous Water, Distilled
methanol R and 10 mL of butyrolactone R. Water R prepared by distillation.
Assay. Gas chromatography (2.2.28): use the normalisation Water, Distilled, Deionised
procedure. Deionised water R prepared by distillation with a resistivity of
Column: not less than 0.18 MQ-m, determined at 25 °C.
- material: fused-silica; Water for Chromatography
- size: l = 30 m, 0 = 0.5 mm; Deionised water with a resistivity of not less than
- stationary phase: macrogol 20 000 R. 0.18 MQ-m, determined at 25 °C, prepared by distillation,
Carrier gas: helium for chromatography R. ion exchange, reverse osmosis or any other suitable method,
Temperature: using water that complies with the regulations on water
intended for human consumption, as laid down by the
Time Temperature competent authority.
(min) CC) Its quality is such that no significant interfering peaks or loss
Column 0- 1 80 of sensitivity are observed when used in chromatography.
I - 12 80--, 190 Isocratic elution with UV detection at low wavelengths
12 - 27 190 (i.e. less than 230 nm), with evaporative detectors (e.g. light
Injection port 190 scattering detector, particle counter detector, charged aerosol
detector) or mass detectors, or gradient elution, may require
Detection: flame-ionisation. the use of water with a total organic carbon content of
Injection: 0.3 µL of the substance to be examined. maximum 5 ppb.
Adjust the flow rate of the carrier gas so that the retention Water for Injections See Water for injections (0169).
time of the peak corresponding to 1-vinylpyrrolidin-2-one is Water, Nitrate-free
about 17 min. To 100 mL of water R add a few milligrams of potassium
Vitexin Apigenin 8-glucoside; C 21 H 20 0 10 = 432.4 permanganate R and of barium hydroxide R. Distil using the
(3681-93-,f; apparatus described for the determination of Distillation range
Yellow powder. (2. 2.11). Reject the first 10 mL and collect the following
Storage: in an airtight container, protected from light. 50 mL.
Vitexin-2 11 -0-rhamnoside 8-[2-0-(6-Deoxy-o:-L- Water, Particle-free
mannopyranosyl)-~-o-glucopyranosyl]-5, 7-dihydroxy-2-( 4- Filter water R through a membrane with a pore size of
hydroxyphenyl)-4H-1-benzopyran-4-one; CnH30 0 14 • 0.22 µm.
(Mr 578.5) (64820-99-1) Water, Standard Solution for the Micro Determination
Water (7732-18-5) of
See Purified water (0008). Commercially available standard solution for the coulometric
titration of water, containing a certified content of water in a
Water MB
suitable solvent.
Deionised, filtered and autoclaved water.
Wedelolactone 1,8, 9-Trihydroxy-3-methoxy-6H-benzofuro
Water Rl (3,2-c] [l]benzopyran-6-one; C 16H 100 7 = 314.3 (524-12-9)
Prepared from distilled water R by multiple distillation. White Beeswax See White beeswax (0069).
Remove carbon dioxide by boiling for at least 15 min before
Xanthine 2,6-Dihydroxypurine; C 5 H 4 N 4 0 2 = 152.11
use in a boiling flask of fused silica or borosilicate glass and
(69-89-6)
cool. Any other suitable method may be used. The boiling
flask has been already used for the test or has been filled with Xanthydrol 9-Hydroxyxanthene; Xanthen-9-ol;
water Rand kept in an autoclave at 121 °C for at least 1 h C13H 10 0 2 = 198.2 (90-46-0)
prior to first use. When tested immediately before use, Content: minimum 90.0 per cent.
water Rl is neutral to methyl red solution R, i.e. it shall White or pale-yellow powder, very slightly soluble in water,
produce an orange-red (not a violet-red or yellow) colour soluble in ethanol (96 per cent) and in glacial acetic acid.
corresponding to pH 5.5 ± 0.1 when 0.05 mL of methyl red It is also available as a methanolic solution containing 90 g/L
solution R is added to 50 mL of the water to be examined. to 110 g/L of xanthydrol.
Conductivity: maximum 1 µS-cm -I, determined at 25 °C by mp: about 123 °C.
an in-line conductivity meter (see Purified water (0008)).
Assay. In a 250 mL flask dissolve 0.300 g in 3 mL of
Water, Anunonia-free Water, ammonium-free methanol R or use 3.0 mL of solution. Add 50 mL of glacial
To 100 mL of water R add 0.1 mL of sulfuric acid R. Distil acetic acid R and, dropwise with shaking, 25 mL of a 20 g/L
using the apparatus described for the determination of solution of urea R. Allow to stand for 12 h, collect the
Distillation range (2.2.11). Reject the first 10 mL and collect precipitate on a sintered-glass filter (16) (2.1.2), wash with
the following 50 mL. 20 mL of ethanol (96 per cent) R, dry in an oven at 100 °C to
105 °C and weigh.
1 g of precipitate is equivalent to 0. 9429 g of xanthydrol.
2023 Appendix I A V-A161

Storage: protected from light. If a methanolic solution is used, Xylitol C 5H 12O 5 = 152.1 (87-99-0)
store in small sealed ampoules and filter before use if White or almost white, crystalline powder or crystals.
necessary.
Content: minimum 96.0 per cent.
Xanthydrol Rl
o-Xylose Xylose; (58-86-6)
Complies with the requirements prescribed for xanthydrol R
See Xylose (1278).
with the following requirement.
Yohimbine Hydrochloride C 21 H 27 CIN2NO 3 = 390.9
Content: minimum 98.0 per cent of C 13H 10 O 2. (65-19-0)
Xanthydrol Reagent
Methyl 17 o:-hydroxyyohimban-16o:-carboxylate
Dissolve about 0.125 g of xanthydrol in 100 mL of anhydrous hydrochloride.
acetic acid. Add 1 mL of hydrochloric acid immediately before
Zinc Zn = 65.4 (7440-66-6)
use.
Content: minimum 99.5 per cent.
Xanthydrol Solution
Silver-white cylinders, granules, pellets or filings with a blue
To 0.1 mL of a 100 g/L solution of xanthydrol R in
sheen.
methanol R add 100 mL of anhydrous acetic acid R and 1 mL
of hydrochloric acid R. Allow to stand for 24 h before using. Arsenic (2.4.2, Method A): maximum 0.2 ppm.
Xylene A mixture of o-, m- and p- isomers; Dissolve 5.0 g in a mixture of the 15 mL of hydrochloric
CsH10 = 106.2 (1330-20-7) acid R and 25 mL of water R prescribed.
Mixture of isomers. Clear, colourless, flammable liquid, Zinc Acetate Zinc acetate dihydrate;
practically insoluble in water, miscible with ethanol (C2H3O2)2Zn,2H2O = 219.5 (5970-45-6)
(96 per cent). Bright white or almost white crystals, slightly efflorescent,
dig: about 0.867. freely soluble in water, soluble in ethanol (96 per cent).
It loses its crystallisation water at 100 °C.
ni0 : about 1.497.
dig: about 1.735.
bp: about 138 °C.
mp: about 237 °C.
m-Xylene 1,3-Dimethylbenzene; C 8 H 10 = 106.2
(108-38-3) Zinc Acetate Solution
Clear, colourless, flammable liquid, practically insoluble in Mix 600 mL of water R with 150 mL of glacial acetic acid R,
water, miscible with ethanol (96 per cent). 54. 9 g of zinc acetate R and stir to dissolve. Continue stirring
while adding 150 mL of concentrated ammonia R. Cool to
dig: about 0.884.
room temperature and adjust with ammonia R to pH 6.4.
ni0 : about 1.497.
Dilute the mixture to 1 L with water R.
bp: about 139 °C. Zinc, Activated
mp: about -47 °C. Place the zinc cylinders or pellets to be activated in a conical
o-Xylene 1,2-Dimethylbenzene; C 8 H 10 = 106.2 (95-47-6) flask and add a sufficient quantity of a 50 ppm solution of
Clear, colourless, flammable liquid, practically insoluble in chloroplatinic acid R to cover the metal. Allow the metal to
water, miscible with ethanol (96 per cent). remain in contact with the solution for 10 min, wash, drain
dig: about 0.881. and dry immediately.
n~: about 1.505.
Arsenic (2.4.2, Method A). To 5 g of the activated zinc add
15 mL of hydrochloric acid R, 25 mL of water R, 0.1 mL of
bp: about 144 °C.
stannous chloride solution R and 5 mL of potassium iodide
mp: about -25 °C. solution R. No colour is produced during the test.
Xylene Cyanol FF (2650-17-1) Activity. The requirements of the suitability test for arsenic
Colour Index No. 42135 (2.4.2, Method A) are met.
A blue, alcohol-soluble dye used as a screening agent in Zinc Chloride (7646-85-7)
methyl orange-xylene cyanol FF solution. See Zinc chloride (0110).
Xylenol Orange [3H-2,1-Benzoxathiol-3-ylidenebis(6- Zinc Chloride-Formic Acid Solution
hydroxy-5-methyl-m-phenylene )methylenenitrilo ]tetra-acetic
Dissolve 20 g of zinc chloride R in 80 g of an 850 g/L solution
acid S,S-dioxide tetrasodium salt; C 31 H 28N 2Na4 O 13 S = 761
of anhydrous formic acid R.
(3618-43-7)
Zinc Chloride Solution, Iodinated
Reddish-brown crystalline powder, soluble in water.
Dissolve 20 g of zinc chloride R and 6.5 g of potassium
Xylenol Orange Solution iodide R in 10.5 mL of water R. Add 0.5 g of iodine Rand
Dissolve 50.8 mg of xylenol orange R in water R and dilute to shake for 15 min. Filter if necessary.
100.0 mL with the same solvent.
Storage: protected from light.
Xylenol Orange Triturate Zinc Iodide Znl 2 = 319.2 (10139-47-6)
Triturate 1 part of xylenol orange R with 99 parts of potassium
General reagent grade of commerce.
nitrate R.
Zinc Iodide and Starch Solution
Test for sensitivity. To 50 mL of water R add 1 mL of dilute
acetic acid R, 50 mg of the xylenol orange triturate and To a solution of 2 g of zinc chloride R in 10 mL of water R
0.05 mL of lead nitrate solution R. add 0.4 g of soluble starch R and heat until the starch has
Add hexamethylenetetramine R until the colour changes from dissolved. After cooling to room temperature add 1.0 mL of
yellow to violet-red. After addition of 0.1 mL of 0.1 M a colourless solution containing 0.10 g zinc R as filings and
sodium edetate the colour changes to yellow. 0.2 g of iodine R in water R. Dilute the solution to 100 mL
with water Rand filter.
V-A162 Appendix I A 2023

Swrage: protected from light.

B. Volumetric Reagents and Solutions
Test for sensitivity. Dilute 0.05 mL of sodium nitrite solution R
to 50 mL with water R. To 5 mL of this solution add 0.1 mL Terminology
of dilute sulfuric acid R and 0.05 mL of the zinc iodide and Volumetric solutions are prepared according to the usual
starch solution and mix. The solution becomes blue. chemical analytical methods. The accuracy of the apparatus
Zinc Oxide (1314-13-2) used is verified to ensure that it is appropriate for the
See Zinc oxide (0252). intended use.
Zinc Powder Zn = 65.4 (7440-66-6) The concentration of volumetric solutions is indicated in
terms of molarity. Molarity expresses, as the number of
Content: minimum 90.0 per cent.
moles, the amount of substance dissolved in 1 L of solution.
Very fine, grey powder, soluble in dilute hydrochloric acid R. A solution which contains x moles of substance per litre is
Zinc Shot Zn = 65.38 said to be x M.
Analytical reagent grade of commerce. Volumetric solutions do not differ from the prescribed
Shot, 0.5 to 2.0 mm (about 8 to 30 mesh). strength by more than 10 per cent. The molarity of the
Zinc Sulfate Zinc sulphate; Zinc Sulfate Heptahydrate; volumetric solutions is determined by an appropriate number
(7446-20-0) of titrations. The repeatability does not exceed 0.2 per cent
(relative standard deviation).
See Zinc sulfate (0111).
Volumetric solutions are standardised by the methods
Zirconyl Chloride ZrOClz,8H 2 O = 322.3 (15461-27-5)
described below. When a volumetric solution is to be used in
Content, minimum 96.0% of ZrC1 2 O,8H2 O. an assay in which the end-point is determined by an
White or almost white, crystalline powder or crystals, freely electrochemical process (for example, amperometry or
soluble in water and in ethanol (96%). potentiometry) the solution is standardised by the same
Assay Dissolve 0.600 g in a mixture of 5 mL of nitric acid method. The composition of the medium in which a
and 50 mL of water. Add 50.0 mL of 0.1 M silver nitrate and volumetric solution is standardised should be the same as
3 mL of dibutyl phthalate and shake. Using 2 mL of ferric that in which it is to be used.
ammonium sulfate solution R2 as indicator, titrate with Solutions more dilute than those described below are either
0.1 M ammonium thiocyanate until a reddish-yellow colour is prepared by adapting the quantities stated or by dilution,
obtained. with carbon dioxide-free water R (unless otherwise prescribed),
1 mL of 0.1 M silver nitrate is equivalent to 16.11 mg of of a more concentrated solution that has been previously
ZrC12 O,8H 2 O. standardised. In the first case, the correction factor is
Zirconyl Nitrate A basic salt corresponding approximately determined on the volumetric solution to be used in the
to the formula ZrO(NO 3) 2 ,2H 2 O (14985-18-3) monograph. In the latter case, the correction factor of the
dilute solution is the same as that of the standardised
A white or almost white powder or crystals, hygroscopic, solution from which it was prepared.
soluble in water. The aqueous solution is a clear or at most
slightly opalescent liquid. Commercially available volumetric solutions traceable to a
primary standard may be used provided their titre is
Swrage: in an airtight container.
determined or verified prior to first use.
Zirconyl Nitrate Solution Titres of volumetric solutions are verified at appropriate
A 1 g/L solution in a mixture of 40 mL of water R and intervals that are defined in the quality system procedures.
60 mL of hydrochloric acid R.
British Pharmacopoeia monographs In assays and other
quantitative tests, solutions to be standardised before use;
include the letters VS after the name of the reagent.

Primary Standards
The following materials, after drying under the specified
conditions, are recommended for use as primary standards in
the standardisation of volumetric solutions. Analytical reagent
grade materials of commerce must be used.
For primary standards from commercial sources, a pre-
treatment step may be necessary. Follow the supplier's
instructions.
A secondary standard may be used provided its traceability to
a primary standard has been demonstrated.
For monographs of the European Pharmacopoeia, primary
standards are indicated by the suffix RV.
Arsenic Trioxide Arsenious trioxide; As 2 O 3 = 197 .8
(1327-53-3)
Sublime arsenious trioxide R in a suitable apparatus.
Swrage: over anhydrous silica gel R.
Benzoic Acid C 7 H 6 O 2 = 122.1 (65-85-0)
Sublime benzoic acid R in a suitable apparatus.
2023 Appendix I B V-A163

Ferrous Ethylenediammonium Sulfate solution must be determined in the same way.

Ethylenediammonium iron(II) disulfate tetrahydrate; The composition of the medium in which a volumetric
Ethylenediammonium tetraaquabis(sulfato)iron(II); solution is standardised should be the same as that in which
Fe(C 2 H 10N 2)(SO 4 ) 2,4H2O = 382.1 (113193-60-5) it is to be used.
Content: minimum 99.5 per cent. All volumetric solutions should, if practicable, be prepared,
Potassium Bromate KBrO 3 = 167.0 (7758-01-2) standardised and used at 20°; if a titration is carried out at a
Crystallise potassium bromate R from boiling water R. Collect markedly different temperature from that at which the
the crystals and dry to constant mass in an oven at standardisation took place, a suitable temperature correction
180 ± 10 °C (2.2.32). should be made.
Potassium Dichromate Acetic Acid VS C 2H 4 O 2 = 60.1
Dry to constant weight at 150°. Dilute 6.0 g of glacial acetic acid R to 1000.0 mL with
water R.
Potassium Hydrogen Phthalate Potassium
2-carboxybenzoate; C 8 H 5KO 4 = 204.2 (877-24-7) Standardisation. To 25.0 mL of acetic acid add 0.5 mL of
phenolphthalein solution Rand titrate with 0.lM sodium
Recrystallise potassium hydrogen phthalate R from boiling hydroxide.
water R, collect the crystals at a temperature above 35 °C
and dry to constant mass at 110 °C. O.lM Ammonium Cerium(IV) Nitrate VS 0. lM
ammonium and cerium nitrate
Potassium Iodate
Shake for 2 min a solution containing 56 mL of sulfuric
Dry to constant weight at 130°. acid R and 54.82 g of ammonium and cerium nitrate R, then
Sodium Carbonate, Anhydrous Sodium Carbonate; add 5 successive quantities, each of 100 mL, of water R,
Na2 CO3 = 106.0 (497-19-8) shaking after each addition. Dilute the clear solution to
Filter at room temperature a saturated solution of sodium 1000.0 mL with water R. Standardise the solution after
carbonate R. Introduce slowly into the filtrate a stream of 10 days.
carbon dioxide R with constant cooling and stirring. After Standardisation. Dissolve 0.300 g of ferrous
about 2 h, collect the precipitate on a sintered-glass filter ethylenediammonium sulfate RV in 50 mL of a diluted solution
(2.1. 2). Wash the filter with iced water R containing carbon of sulfuric acid R (49 g/L H 2 S0 4 ). Titrate with the
dioxide. After drying at 100 °C to 105 °C, heat to constant ammonium and cerium nitrate solution, determining the
mass at 270-300 °C, stirring from time to time. end-point potentiometrically (2.2.20) or using 0.1 mL of
Sodium Chloride NaCl= 58.44 (7647-14-5) ferroin R as indicator.
To 1 volume of the saturated sodium chloride solution R add 1 mL of 0.1 M ammonium and cerium nitrate is equivalent to
2 volumes of hydrochloric acid R. Collect the crystals formed 38.21 mg of Fe(C2H10N2)(SO4)2,4H2O.
and wash with hydrochloric acid Rl. Remove the hydrochloric Storage: protected from light.
acid by heating on a water-bath and dry the crystals to O.lM Ammonium Cerium(Iv) Sulfate VS 0.lM
constant mass at 300 °C. ammonium and cerium sulfate
Sulfanilic Acid 4-Aminobenzenesulfonic acid; Dissolve 65.0 g of ammonium and cerium sulfate Rina
C 6H 7NO 3 S = 173.2 (121-57-3) mixture of 500 mL of water R and 30 mL of sulfuric acid R.
Recrystallise sulfanilic acid R from boiling water R. Filter and Allow to cool and dilute to 1000.0 mL with water R.
dry to constant mass at 100-105 °C. Standardisation. Dissolve 0.300 g of ferrous
Trometamol 2-Amino-2-(hydroxymethyl)propane-1,3-diol; ethylenediammonium sulfate RV in 50 mL of a diluted solution
Tris(hydroxymethyl)aminomethane; C 4 HuNO 3 = 121.1 of sulfuric acid R (49 g/L H 2SO 4). Titrate with the
(77-86-1) ammonium and cerium sulfate solution, determining the
Content: minimum 99.5 per cent. end-point potentiometrically (2.2.20) or using 0.1 mL of
Zinc Zn = 65.4 (7440-66-6) ferroin R as indicator.
Content: minimum 99.9 per cent. 1 mL of 0.1 M ammonium and cerium sulfate is equivalent to
38.21 mg ofFe(C 2H10N2)(SO4)2,4H2O.
Preparation and Standardisation Dilution. Use a diluted solution of sulfuric acid R (59 g/L
For each solution the preparation and standardisation of the H 2SO 4 ) while cooling the solution.
most commonly used strengths are described. Solutions more 0.01M Ammonium Cerium(1v) Sulfate VS Ammonium
concentrated than those described are prepared and and cerium sulfate
standardised using proportionate amounts of the reagents. To 100 mL of 0.1M ammonium cerium(IV) sulfate VS add,
Aqueous solutions less concentrated than those described are while cooling, 30 mL of sulfuric acid and sufficient water to
prepared by making an exact dilution of a more concentrated produce 1000 mL.
solution with carbon dioxide-free water. The correction factors Ammonium Iron(n) Sulfate VS Ammonium Iron(n)
of these solutions are the same as those from which the Sulphate; Ferrous Ammonium Sulfate; (NH 4)zFe(SO 4) 2,
dilutions were prepared. Aqueous solutions of molarity below 6H2O = 392.l
0 .1 M are freshly prepared using carbon dioxide-free water. For a O.Ltt solution Dissolve 40 g of ammonium iron(II)
The water used in preparing volumetric solutions complies sulfate in 100 mL of 2M sulfuric acid and dilute with sufficient
with the requirements of the monograph for Purified Water. freshly boiled and cooled water to produce 1000 mL.
When used for the preparation of unstable solutions such as Ascertain its exact concentration in the follO\ving manner.
potassium permanganate and sodium thiosulfate, it should be To 25 mL add 10 mL of IM sulfuric acid and 1 mL of
freshly boiled and cooled. When a solution is to be used in orthophosphoric acid and titrate with 0.02M potassium
an assay in which the end point is determined by an permanganate VS. Each mL of 0.02M potassium permanganate
electrochemical process, the exact concentration of the VS is equivalent to 39.21 mg of (NH 4 ) 2Fe(SO 4 ) 2,6H2O.
V-A164 Appendix I B 2023

0.lM Ammonium Iron(rn) Sulfate VS 0.lM Ferric 0.01M Bismuth Nitrate VS

Ammonium Sulfate Dissolve 4.86 g of bismuth nitrate pentahydrate R in 60 mL of
Dissolve 50.0 g of ferric ammonium sulfate Rina mixture of dilute nitric acid Rand dilute to 1000.0 mL with water R.
6 mL of sulfuric acid R and 300 mL of water R and dilute to Standardisation. To 25.0 mL of the bismuth nitrate solution,
1000.0 mL with water R. add 50 mL of water Rand titrate with 0.01 M sodium edetate
Standardisation. To 10.0 mL of the ferric ammonium sulfate using 0.05 mL of a 1 g/L solution of xylenol orange R as
solution add 35 mL of water R, 3 mL of hydrochloric acid R indicator.
and 1 g of potassium iodide R. Allow to stand for 10 min. 0.05M Bromine VS 0.0167M bromide-bromate
Titrate with 0.1 M sodium thiosulfate, determining the Dissolve 2.7835 g of potassium bromate RVand 13 g of
end-point potentiometrically (2.2.20) or using 1 mL of starch potassium bromide R in water Rand dilute to 1000.0 mL with
solution R as indicator. the same solvent.
1 mL of 0.1 M sodium thiosulfate is equivalent to 48.22 mg of 0.lM Cerium(1v) Sulfate VS 0.lM Cerium Sulfate
FeNH4(SO 4)z, 12H2O.
Dissolve 40.4 g of cerium sulfate R in a mixture of 500 mL of
0.lM Ammonium Thiocyanate VS water R and 50 mL of sulfuric acid R. Allow to cool and
Dissolve 7.612 g of ammonium thiocyanate R in water Rand dilute to 1000.0 mL with water R.
dilute to 1000.0 mL with the same solvent. Standardisation. Dissolve 0 .300 g of ferrous
Standardisation. To 20.0 mL of 0.1 M silver nitrate add ethylenediammonium sulfate RV in 50 mL of a diluted solution
25 mL of water R, 2 mL of dilute nitric acid R and 2 mL of of sulfuric acid R (49 g/L H 2SO 4). Titrate with the cerium
ferric ammonium sulfate solution R2. Titrate with the sulfate solution, determining the end-point potentiometrically
ammonium thiocyanate solution until a reddish-yellow colour (2.2.20) or using 0.1 mL offerroin Ras indicator.
is obtained. 1 mL of 0.1 M cerium sulfate is equivalent to 38.21 mg of
0.lM Barium Chloride VS Fe(C2H10N2)(SO4)z,4H2O.
Dissolve 24.4 g of barium chloride R in water Rand dilute to Cetylpyridinium Chloride VS C 21 H 38 CIN,H 2O = 358.0
1000.0 mL with the same solvent. For a 0.005M solution Dissolve 1.8 g of cetylpyridinium
Standardisation. To 10. 0 mL of the barium chloride solution chloride in 10 mL of ethanol (96%) and dilute to 1000 mL
add 60 mL of water R, 3 mL of concentrated ammonia R and with water.
0.5-1 mg of phthalein purple R. Titrate with 0.1 M sodium Ascertain its exact concentration in the following manner.
edetate. When the solution begins to decolorise, add 50 mL Transfer 25 mL to a separating funnel, add 25 mL of
of ethanol (96 per cent) R and continue the titration until the chloroform, 10 mL of 0.01M sodium hydroxide and 10 mL of a
blue-violet colour disappears. freshly prepared 0.5% w/v solution of potassium iodide. Shake
0.05M Barium Perchlorate VS well, allow to separate and discard the chloroform layer.
Dissolve 15.8 g of barium hydroxide Rina mixture of 7.5 mL Shake the aqueous layer with three further 10-mL quantities
of perchloric acid R and 7 5 mL of water R, adjust the solution of chloroform and discard the chloroform solutions.
to pH 3 by adding perchloric acid R and filter if necessary. Add 40 mL of hydrochloric acid, cool and titrate with 0. 005M
Add 150 mL of ethanol (96 per cent) Rand dilute to 250 mL potassium iodate VS until the solution becomes pale brown in
with water R. Dilute to 1000.0 mL with buffer solution colour. Add 2 mL of chloroform and continue the titration,
pH 3.7 R. shaking vigorously and allowing the layers to separate after
Standardisation. To 5.0 mL of 0.05 M sulfuric acid add 5 mL each addition, until the chloroform becomes colourless.
of water R, 50 mL of buffer solution pH 3. 7 R and 0.5 mL of Titrate a mixture of 20 mL of water, 10 mL of the potassium
alizarin S solution R. Titrate with the barium perchlorate iodide solution and 40 mL of hydrochloric acid with 0. 005M
solution until an orange-red colour appears. Standardise potassium iodate VS in the same manner. The difference
immediately before use. between the titrations represents the amount of potassium
iodate required. Each mL of 0. 005M potassium iodate VS is
Dilution. Use buffer solution pH 3. 7 R.
equivalent to 3.580 mg of C 21 H 38 CIN,H2O.
0.025M Barium Perchlorate VS
0.02M Copper Sulfate VS 0.02M Copper Sulphate VS
Dilute 500 mL of 0. 05M banum perchlorate VS to 1000 mL
Dissolve 5.0 g of copper sulfate pentahydrate R in water Rand
with buffer solution pH 3. 7.
dilute to 1000.0 mL with the same solvent.
0.005M Barium Perchlorate VS
Standardisation. To 20.0 mL of the copper sulfate solution
Dilute 10.0 mL of 0.05 M barium perchlorate to 100.0 mL add 2 g of sodium acetate R and 0 .1 mL of pyridylazonaphthol
with a buffer solution prepared as follows: to 15.0 mL of solution R. Titrate with 0. 02 M sodium edetate until the colour
acetic acid R add 60.0 mL of 2-propanol R. Adjust to pH 3.7 changes from violet-blue to bright green. Titrate slowly
with ammonia Rand dilute to 100.0 mL with water R. towards the end of the titration.
0.004M Benzethonium Chloride VS Cupriethylenediamine Hydroxide Solution Use a lM
Dissolve in water R 1. 792 g of benzethonium chloride R, solution in which the molar ratio of ethylenediamine to
previously dried to constant mass at 100-105 °C, and dilute copper is 2.00 ± 0.04.
to 1000.0 mL with the same solvent.
Dioctyl Sodium Sulfosuccinate VS Dioctyl Sodium
Standardisation. Dissolve 0.350 g of the dried substance in Sulphosuccinate VS; C 20 H 37NaO 7 S = 444.6
35 mL of a mixture of 30 volumes of anhydrous acetic acid R For a 0.01M solution Dissolve 4.5 g of dioctyl sodium
and 70 volumes of acetic anhydride R. Titrate with 0.1 M sulfosuccinate in warm water, cool and dilute to 1000 mL with
perchloric acid, using 0.05 mL of crystal violet solution Ras water.
indicator. Carry out a blank titration.
Ascertain its exact concentration in the following manner.
1 mL of 0.1 M perchloric acid is equivalent to 44.81 mg of To 25 mL add 25 mL of a solution containing 20% w/v of
C21H42CINO2. anhydrous sodium sulfate and 2% w/v of sodium carbonate,
2023 Appendix I B V-A165

50 mL of chloroform and 1.5 mL of bromophenol blue solution 3M Hydrochloric Acid VS

and mix. Titrate with 0.01M tetrabutylammonium iodide VS Dilute 309.0 g of hydrochloric acid to 1000 mL with water.
until about 1 mL from the end point. Stopper the flask,
2M Hydrochloric Acid VS
shake vigorously for 2 minutes and continue the titration, in
0.05-mL increments, shaking vigorously and allowing the Dilute 206.0 g of hydrochloric acid to 1000 mL with water.
flask to stand for about 10 seconds after each addition. lM Hydrochloric Acid VS
Continue the titration until a blue colour just appears in the Dilute 103.0 g of hydrochloric acid R to 1000.0 mL with
chloroform layer. Each mL of 0.0 lM tetraburylammonium water R.
iodide VS is equivalent to 4.446 mg of C 20H 37NaO 7S. Standardisation. Dissolve 0. 950 g of trometamol RV in 50 mL
0.lM Disodium Edetate VS 0.lM Sodium Edetate VS of water R. Titrate with the hydrochloric acid solution,
Dissolve 3 7.5 g of sodium edetate R in 500 mL of water R, determining the end-point potentiometrically (2.2.20) or
add 100 mL of 1 M sodium hydroxide and dilute to using 0 .1 mL of methyl orange solutwn R as indicator until a
1000.0 mL with water R. yellowish-red colour is obtained.
Standardisation. Dissolve 0.120 g of zinc RV in 4 mL of 1 mL of 1 M hydrochloric acid is equivalent to 121.1 mg of
hydrochloric acid Rl. Add dilute sodium hydroxide solution R C4H11NO3.
until the solution is weakly acid and carry out the assay of 0.lM Hydrochloric Acid VS
zinc by complexometry (2. 5.11). Dilute 100.0 mL of 1 M hydrochloric acid to 1000.0 mL with
1 mL of 0.1 M sodium edetate is equivalent to 6.538 mg of carbon dioxide-free water R.
Zn. Standardisation. Carry out the titration described for 1 M
Storage: in a polyethylene container. hydrochloric acid using 95 mg of trometamol RV dissolved in
0.05M Disodium Edetate VS 0.05M Sodium Edetate 50 mL of water R.
Dissolve 18.6 g of disodium edetate in sufficient water to 1 mL of 0.1 M hydrochloric acid is equivalent to 12.11 mg of
produce 1000 mL. C4H11NO3.
Ascertain its exact concentration in the following manner. 0.5M Iodine VS
Dissolve 0.100 g of zinc, in granules, in 4 mL of Dissolve 127 g of iodine R and 200 g of potassium iodide R in
7M hydrochloric acid and add 0.1 mL of bromine water. Boil to water R and dilute to 1000.0 mL with the same solvent.
remove excess bromine, cool and dilute to 100 mL with Standardisation. To 2.0 mL of the iodine solution add 1 mL
water. Dilute 25 mL of this solution to 200 mL with water, of dilute acetic acid R and SO mL of water R. Titrate with
add about 50 mg of xylenol orange triturate and hexamine until 0.1 M sodium thiosulfate, using starch solution R as indicator.
the solution becomes violet-pink and add 2 g of hexamine in Storage: protected from light.
excess. Titrate with the disodium edetate solution until the
0.05M Iodine VS
violet-pink colour changes to yellow. Each mL of 0.0SM
disodium edetate VS is equivalent to 3.269 mg of Zn. Dissolve 12. 7 g of iodine R and 20 g of potassium iodide R in
water Rand dilute to 1000.0 mL with the same solvent.
0.02M Disodium Edetate VS 0.02M Sodium Edetate
Standardisation. To 10.0 mL of the iodine solution add 1 mL
Dissolve 7.444 g of disodium edetate in sufficient water to
of dilute acetic acid R and 40 mL of water R. Titrate with
produce 1000 mL.
0.1 M sodium thiosulfate, determining the end-point
Ascertain its exact concentration in the following manner. potentiometrically (2.2.20) or using starch solution Ras
Dissolve 0.100 g of zinc, in granules, in 4 mL of indicator.
7M hydrochloric acid and add 0.1 mL of bromine water. Boil to Storage: protected from light.
remove excess bromine, cool and dilute to 100 mL with
0.01M Iodine VS
water. Dilute 25 mL of this solution to 200 mL with water,
add about 50 mg of xylenol orange triturate and hexamine until Add 0.3 g of potassium iodide R to 20.0 mL of 0.05 M iodine
the solution becomes violet-pink and add 2 g of hexamine in and dilute to 100.0 mL with water R.
excess. Titrate with the disodium edetate solution until the 0.lM Iron(n) Sulfate VS 0.lM Ferrous sulfate; 0.lM
violet-pink colour changes to yellow. Each mL of 0.02M Iron(n) Sulphate VS
disodium edetate VS is equivalent to 1.308 mg of Zn. Dissolve 27.80 g of ferrous sulfate R in 500 mL of dilute
0.01M Disodium Edetate VS 0.01M Sodium Edetate sulfuric acid R and dilute to 1000.0 mL with water R.
Dissolve 3.722 g of disodium edetate in sufficient water to Standardisation. To 25.0 mL of the ferrous sulfate solution
produce 1000 mL. add 3 mL of phosphoric acid R and titrate immediately with
Ascertain its exact concentration in the following manner. 0. 02 M potassium permanganate. Standardise immediately
before use.
Dissolve 0.100 g of zinc, in granules, in 4 mL of 7M
hydrochloric acid and add 0.1 mL of bromine water. Boil to Karl Fischer Reagent VS
remove excess bromine, cool and dilute to 100 mL with Iodosulfurous reagent
water. Dilute 10 mL of this solution to 200 mL with water, The apparatus, which must be kept closed and dry during
add about SO mg of xylenol orange tn'turate and hexamine until the preparation, consists of a 3000- to 4000-mL round-
the solution becomes violet-pink and add 2 g of hexamine in bottomed flask with inlets for a thermometer and a stirrer
excess. Titrate with the disodium edetate solution until the and fitted with a drying tube. To 700 mL of anhydrous
violet-pink colour changes to yellow. Each mL of 0.01M pyridine and 700 mL of 2-methoxyethanol add, stirring
disodium edetate VS is equivalent to 0.6538 mg of Zn. constantly, 220 g of finely powdered iodine, previously dried
6M Hydrochloric Acid VS over phosphorus pentoxide. Continue stirring until the iodine
Dilute 618.0 g of hydrochloric acid to 1000 mL with water. has completely dissolved (about 30 minutes), cool to -10°
and add quickly, while stirring, 190 g of sulfur dioxide.
Do not allow the temperature to exceed 30°; cool.
V-A166 Appendix I B 2023

Determine the water-equivalent of the reagent immediately Magnesium Sulfate VS Magnesium Sulphate VS;
before use in the following manner. Transfer 20 mL of MgSO 4,7H2O = 246.5
anhydrous methanol to the titration vessel and titrate to the For a 0.05M solution Dissolve 12.5 g of magnesium sulfate
electrometric end point with the reagent, Appendix IX C. in sufficient water to produce 1000 mL.
Add in an appropriate form a suitable amount of water, Ascertain its exact concentration by carrying out the method
accurately weighed, and titrate to the end point. Calculate for the complexometric titration of magnesium,
the water-equivalent of the reagent in mg per mL. Appendix Vlll D, using 40 mL of the magnesium sulfate
The minimum water equivalent is 3.5 mg of water per mL of solution. Each mL of 0.lM disodium edetate VS is equivalent
reagent. Work protected from humidity.
to 24.65 mg of MgSO4 ,7HzO.
The composition of commercially available Karl Fischer reagents Mercury(n) Nitrate VS Mercuric nitrate
often differs from that above by the replacement of pyridine with
Hg(NO 3)z+ aq
other basic compounds. The use of these reagents must be validated
in order to verify in each individual case the stoichiometry and the For a 0.02M solution Dissolve 6.85 g of mercury(n) nitrate
absence of incompatibility between the substance under test and the in 20 mL of lM nitric acid and add sufficient water to
reagent. produce 1000 mL.
O.lM Lanthanum Nitrate VS Ascertain its exact concentration in the following manner.
Dissolve 15 mg of sodium chloride in 50 mL of water and
Dissolve 43.30 g of lanthanum nitrate R in water R and dilute
titrate with the mercury nitrate solution determining the end
to 1000.0 mL with the same solvent.
point potentiometrically, using a platinum or mercury
Standardisation. To 20.0 mL of the lanthanum nitrate indicator electrode and a mercury-mercury(!) sulfate
solution, add 15 mL of water Rand 25 mL of 0.1 M sodium
reference electrode. Each mL of 0.02M mercury(II) nitrate VS
edetate. Add about 50 mg of xylenol orange triturate R and
is equivalent to 2.338 mg of NaCl.
about 2 g of hexamethylenetetramine R. Titrate with 0.1 M
zinc sulfate until the colour changes from yellow to violet- lM Nitric Acid VS
pink. Dilute 96.6 g of nitric acid R to 1000.0 mL with water R.
1 mL of 0.1 M sodium edetate is equivalent to 43.30 mg of Standardisation. Dissolve 0. 950 g of trometamol RV in 50 mL
La(NO3)3,6H2O. of water R. Titrate with the nitric acid solution, determining
the end-point potentiometrically (2.2.20) or using 0.1 mL of
O.lM Lead Nitrate VS
methyl orange solution R as indicator until a reddish-yellow
Dissolve 33 g of lead nitrate R in water R and dilute to
colour is obtained.
1000.0 mL with the same solvent.
1 mL of 1 M nitric acid is equivalent to 121.1 mg of
Standardisation. Take 20.0 mL of the lead nitrate solution
C4H11NO3.
and carry out the determination of lead by complexometry
(2.5.JJ).
O.lM Perchloric Acid VS
Place 8.5 mL of perchloric acid R in a volumetric flask
0.05M Lead Nitrate VS
containing about 900 mL of glacial acetic acid R and mix.
Dissolve 16.5 g of lead(n) nitrate in sufficient water to Add 30 mL of acetic anhydride R, dilute to 1000.0 mL with
produce 1000 mL.
glacial acetic acid R, mix and allow to stand for 24 h.
Ascertain its exact concentration in the following manner. Determine the water content (2. 5.12) without addition of
To 50 mL of the solution add 300 mL of water and carry out methanol and, if necessary, adjust the water content to
the method for the complexometric titration of lead, 0.1-0.2 per cent by adding either acetic anhydride R or
Appendix VIII D. Each mL of 0.1M disodium edetate VS is water R. Allow to stand for 24 h.
equivalent to 33.12 mg of Pb(NO3)z. Standardisation. Dissolve 0.170 g of potassium hydrogen
O.lM Lithium Methoxide VS phthalate RV in 50 mL of anhydrous acetic acid R, warming
Dissolve 0.694 g of lithium R in 150 mL of anhydrous gently if necessary. Allow to cool protected from air, and
methanol Rand dilute to 1000.0 mL with toluene R. titrate with the perchloric acid solution, determining the
end-point potentiometrically (2.2.20) or using 0.05 mL of
Standardisation. To 10 mL of dimethylformamide R add
crystal violet solution Ras indicator. Note the temperature of
0.05 mL of a 3 g/L solution of thymol blue R in methanol R
the perchloric acid solution at the time of the titration. If the
and titrate with the lithium methoxide solution until a pure
temperature at which an assay is carried out is different from
blue colour is obtained. Immediately add O.100 g of benzoic
that at which the 0.1 M perchloric acid has been standardised,
acid RV. Stir to effect solution and titrate with the lithium
the volume used in the assay becomes:
methoxide solution until the pure blue colour is again
obtained. Protect the solution from atmospheric carbon
dioxide throughout the titration. From the volume of titrant
used in the second titration ascertain the exact strength of t1 temperature during standardisation,
the lithium methoxide solution. Standardise immediately r2 temperature during the assay,
before use. Ve corrected volume,
V observed volume.
1 mL of 0.1 M lithium methoxide is equivalent to 12.21 mg of
C1H6O2. 1 mL of 0.1 M perchloric acid is equivalent to 20.42 mg
O.lM Magnesium Chloride VS ofCsHsKO4.
Dissolve 20.33 g of magnesium chloride R in water R and Dilution. Use anhydrous acetic acid R.
dilute to 1000.0 mL with the same solvent. Other strengths of perchloric acid should be prepared by
Standardisation. Carry out the determination of magnesium diluting 0.1M perchloric acid VS appropriately with anhydrous
by complexometry (2.5.11). acetic acid.
2023 Appendix I B V-Al67

0.02M Perchloric Acid VS 0.1M Potassium Hydroxide VS

Dilute 20.0 mL of 0.lM perchloric acid to 100.0 mL with Dissolve 6 g of potassium hydroxide R in carbon dioxide-free
anhydrous acetic acid. water Rand dilute to 1000.0 mL with the same solvent.
Other strengths of perchloric acid should be prepared by Standardisation. Dissolve 0 .150 g of potassium hydrogen
diluting 0.lM perchloric acid VS appropriately with phthalate RV in 50 mL of water R. Titrate with the potassium
anhydrous acetic acid. hydroxide solution, determining the end-point
0.033M Potassium Bromate VS potentiometrically (2.2.20) or using 0.1 mL of phenolphthalein
Dissolve 5.5670 g of potassium bromate RV in water Rand solution R as indicator.
dilute to 1000.0 mL with the same solvent. 1 mL of 0.1 M potassium hydroxide is equivalent to 20.42 mg
0.02M Potassiwn Bromate VS of CsHsKO4.
Dissolve 3.340 g of potassium bromate in sufficient water to 0,5M Potassium Hydroxide, Ethanolic VS 0.5M
produce 1000 mL. Potassium hydroxide, alcoholic
0.0167M Potassium Bromate VS Dissolve 3 g of potassium hydroxide R in 5 mL of water R and
dilute to 100.0 mL with aldehyde-free alcohol R.
Dissolve 2. 783 g of potassium bromate in sufficient waterto
produce 1000 mL. Standardisation. Dissolve 0.500 g of benzoic acid RV in 10 mL
of water R and 40 mL of ethanol (96 per cent) R. Titrate with
0.0083M Potassium Bromate VS the potassium hydroxide solution, determining the end-point
Prepare by appropriate dilution of 0.033M Potassium Bromate potentiometrically (2.2.20) or using 0.1 mL of phenolphthalein
vs. solution R as indicator.
Potassium Dichromate VS K 2 Cr2 O 7 = 294.2 1 mL of 0. 5 M alcoholic potassium hydroxide is equivalent to
For a 0.0167M solution Dissolve 4.9 g of potassium 61.06 mg of C 7 H 6 Oz.
dichromate in sufficient water to produce 1000 mL. Dilution. Use aldehyde-free alcohol R.
Ascertain its exact concentration in the following manner. 0.lM Potassium Hydroxide, Ethanolic VS 0.lM
To 20 mL of the solution add 1 g of potassium iodide and Potassium hydroxide, alcoholic
7 mL of 2M hydrochloric acid. Add 250 mL of water and Dissolve 6 g of potassium hydroxide in 50 mL of water and
titrate with 0.lM sodium thiosulfate VS, using 3 mL of starch dilute to 1000 mL with aldehyde-free ethanol (96%).
solution as indicator, until the colour changes from blue to
light green. Each mL of 0.lM sodium thiosulfate VS is 0,01M Potassium Hydroxide, Ethanolic VS 0.01M
Potassium hydroxide, alcoholic
equivalent to 4.9 mg ofK2Cr2O7.
0.lM Potassiwn Hydrogen Phthalate VS Dilute 2.0 mL of 0.5M ethanolic potassium hydroxide to
100.0 mL with aldehyde-free ethanol (96%)).
In a conical flask containing about 800 mL of anhydrous
acetic acid R, dissolve 20.42 g of potassium hydrogen 0.05M Potassium Iodate VS
phthalate RV. Heat on a water-bath until completely Dissolve 10. 70 g of potassium iodate R in water R and dilute
dissolved, protected from humidity. Cool to 20 °C and dilute to 1000.0 mL with the same solvent.
to 1000.0 mL with anhydrous acetic acid R. Standardisation. To 3.0 mL of the potassium iodate solution
0.5M Potassiwn Hydroxide in Ethanol (60%) VS 0.5M add 40.0 mL of water R, I g of potassium iodide R and 5 mL
Potassium hydroxide in alcohol (60 per cent VN) of dilute sulfuric acid R. Titrate with 0.1 M sodium thiosulfate,
Dissolve 3 g of potassium hydroxide R in aldehyde-free alcohol R determining the end-point potentiometrically (2.2.20) or
(60 per cent VIV) and dilute to 100.0 mL with the same using 1 mL of starch solution R, added towards the end of the
titration, as indicator.
solvent.
Standardisation. Dissolve 0.500 g of benzoic acid RV in 10 mL 1 mL of 0.1 M sodium thiosulfate is equivalent to 3.567 mg of
of water R and 40 mL of ethanol (96 per cent) R. Titrate with KI03.
the potassium hydroxide solution, determining the end-point 0.001M Potassium Iodide VS
potentiometrically (2.2.20) or using 0.1 mL of phenolphthalein Dilute 10.0 mL of potassium iodide solution R to 100.0 mL
solution R as indicator. with water R. Dilute 5.0 mL of this solution to 500.0 mL
1 mL of 0. 5 M potassium hydroxide in alcohol with water R.
(60 per cent V/Tl) is equivalent to 61.06 mg of C 7 H 6 O2 . 0.02M Potassium Permanganate VS
Potassium Hydroxide in Ethanol (90%) VS Dissolve 3.2 g of potassium permanganate R in water Rand
For a lM solution Dissolve 60 g of potassium hydroxide in dilute to 1000.0 mL with the same solvent. Heat the solution
sufficient aldehyde-free ethanol (90%) to produce I 000 mL, for 1 h on a water-bath, allow to cool and filter through a
allow the solution to stand for 24 hours, decant the clear sintered-glass filter (2.1. 2).
solution and ascertain its exact concentration by the method Standardisation. Dissolve 0.300 g of ferrous
described under 0.5M ethanolic potassium hydroxide VS. ethylenediammonium suljate RV in 50 mL of a diluted solution
1M Potassium Hydroxide VS of sulfuric acid R (49 g/L H 2 SO4). Titrate with the potassium
permanganate solution, determining the end-point
Dissolve 60 g of potassium hydroxide in sufficient carbon
dioxide-free water to produce 1000 mL. Ascertain its exact potentiometrically (2.2.20) or by the colour of the solution
concentration in the following manner. Titrate 20 mL of the changing to pink. Standardise immediately before use.
solution with IM hydrochloric acid VS using 0.5 mL of 1 mL of 0. 02 M potassium permanganate is equivalent to
phenolphthalein solution as indicator. Each mL of 38.21 mg of Fe(C 2 H10N2)(SO4)2,4H2O.
lM hydrochloric acid VS is equivalent to 56.11 mg of KOH. Storage: protected from light.
V-A168 Appendix I B 2023

0.lM Silver Nitrate VS Standardisation. Carry out the titration described for 1 M
Dissolve 17.0 g of silver nitrate R in water Rand dilute to sodium hydroxide using 0 .150 g of potassium hydrogen
1000.0 mL with the same solvent. phthalate RV in 50 mL of water R.
Standardisation. Dissolve 50 mg of sodium chwride RV in 1 mL of 0.1 M sodium hydroxide is equivalent to 20.42 mg of
water R, add 5 mL of dilute nitric acid R and dilute to 50 mL CsHsKO4.
with water R. Titrate with the silver nitrate solution, Standardisation (for use in the assay of halide salts of organic
determining the end-point potentiometrically (2.2.20). bases). Dissolve 0 .100 g of benzoic acid RV in a mixture of
1 mL of 0.1 M silver nitrate is equivalent to 5.844 mg of 5 mL of 0.01 M hydrochloric acid and 50 mL of ethanol
NaCl. (96 per cent) R. Carry out the titration (2.2.20), using the
sodium hydroxide solution. Note the volume added between
Swrage: protected from light.
the 2 points of inflexion.
0.001M Silver Nitrate VS
1 mL of 0.1 M sodium hydroxide is equivalent to 12.21 mg of
Dilute 5 mL of O.lM silver nitrate VS to 500 mL with water. C 7H 6 0 2 .
O.lM Sodium Arsenite VS O.lM Sodium Hydroxide, Ethanolic VS
Dissolve arsenious trioxide RV equivalent to 4.946 g of As 2O 3 To 250 mL of anhydrous ethanol R add 3.3 g of strong sodium
in a mixture of 20 mL of strong sodium hydroxide solution R hydroxide solution R.
and 20 mL of water R, dilute to 400 mL with water R and
add dilute hydrochloric acid R until the solution is neutral to
Standardisation. Dissolve 0.100 g of benzoic acid RV in 10 mL
of water R and 40 mL of ethanol (96 per cent) R. Titrate with
blue litmus paper R. Dissolve 2 g of sodium hydrogen
the ethanolic sodium hydroxide solution, determining the
carbonate R in the solution and dilute to 500.0 mL with
end-point potentiometrically (2. 2. 20) or using 0 .2 mL of
water R.
thymolphthalein solution R as indicator. Standardise
Sodium Dodecyl Sulfate VS Sodium Dodecyl Sulphate immediately before use.
VS; C12H25 NaO4 S = 288.4
1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
For a 0.001M solution Dissolve 0.2884 g of sodium 12.21 mg of C1H 6O2.
dodecyl sulfate, calculated with reference to the substance
dried at 105° for 2 hours, in sufficient water to produce
O.lM Sodium Methoxide VS
1000 mL. Cool 175 mL of anhydrous methanol R in iced water Rand
add, in small portions, about 2.5 g of freshly cut sodium R.
Ascertain its exact concentration in the following manner.
When the metal has dissolved, dilute to 1000.0 mL with
To 50 mL add 15 mL of chloroform, 10 mL of lM sulfuric
acid and 1 mL of a solution containing 0.003% w/v of each toluene R.
of dimethyl yellow and oracet blue 2R in chloroform and titrate Standardisation. To 10 mL of dimethylformamide R add
with 0.004M benzethonium chloride VS, shaking vigorously and 0.05 mL of a 3 g/L solution of thymol blue R in methanol R,
allowing the layers to separate after each addition, until the and titrate with the sodium methoxide solution until a pure
chloroform layer acquires a permanent clear green colour. blue colour is obtained. Immediately add 0.100 g of benzoic
Each mL of 0.004M benzethonium chloride VS is equivalent to acid RV. Stir until dissolution and titrate with the sodium
1.154 mg ofC12H2 5NaO 4 S. methoxide solution until the pure blue colour is again
obtained. Protect the solution from atmospheric carbon
lM Sodium Hydroxide VS
dioxide throughout the titration. From the volume of titrant
Dissolve 42 g of sodium hydroxide R in carbon dioxide-free used in the second titration ascertain the exact strength of
water Rand dilute to 1000.0 mL with the same solvent. the sodium methoxide solution. Standardise immediately
Standardisation. Dissolve 1.50 g of potassium hydrogen before use.
phthalate RV in 50 mL of water R. Titrate with the sodium 1 mL of 0.1 M sodium methoxide is equivalent to 12.21 mg of
hydroxide solution, determining the end-point
C1H6O2.
potentiometrically (2.2.20) or using 0.1 mL of phenolphthalein
solution R as indicator. O.lM Sodium Nitrite VS
Dissolve 7.5 g of sodium nitrite R in water Rand dilute to
1 mL of J M sodium hydroxide is equivalent to 204.2 mg of
1000.0 mL with the same solvent.
CsHsKO4.
Standardisation. Dissolve 0.150 g of sulfanilic acid RV in
If sodium hydroxide free from carbonate is prescribed,
50 mL of dilute hydrochloric acid R and carry out the
prepare it as follows. Dissolve sodium hydroxide R in water R
determination of primary aromatic amino-nitrogen (2.5.8),
to give a concentration of 400-600 g/L and allow to stand.
using the sodium nitrite solution and determining the
Decant the clear supernatant, taking precautions to avoid the
end-point electrometrically. Standardise immediately before
introduction of carbon dioxide, and dilute with carbon
dioxide-free water R to the required molarity. The solution use.
complies with the following test. Titrate 20.0 mL of 1 mL of 0.1 M sodium nitrite is equivalent to 17.32 mg of
hydrochloric acid of the same molarity with the solution of C6H1NO 3 S.
sodium hydroxide, using 0 .1 mL of phenolphthalein solution R O.lM Sodium Periodate VS
as indicator. At the end-point add just sufficient of the acid Dissolve 21.4 g of sodium periodate R in about 500 mL of
to discharge the pink colour and concentrate the solution to water Rand dilute to 1000.0 mL with the same solvent.
20 mL by boiling. During boiling add just sufficient acid to Standardisation. In a stoppered flask, introduce 5.0 mL of the
discharge the pink colour, which should not reappear after
sodium periodate solution and add 100 mL of water R.
prolonged boiling. The volume of acid used does not exceed Add 10 mL of potassium iodide solution R and 5 mL of
0.1 mL. hydrochloric acid Rl, close, shake and allow to stand for
0.lM Sodium Hydroxide VS 2 min. Titrate with 0.1 M sodium thiosulfate until the yellow
Dilute 100.0 mL of JM sodium hydroxide to 1000.0 mL with colour almost disappears. Determine the end-point
carbon dioxide-free water R.
2023 Appendix I B V-A169

potentiometrically (2.2.20) or add 2 mL of starch solutwn R the reaction vessel and filter with three quantities, each of
and titrate slowly until the colour is completely discharged. 50 mL, of toluene R. Add the washings to the filtrate and
1 mL of 0.1 M sodium thiosulfate is equivalent to 2.67 4 mg of dilute to 1000.0 mL with toluene R. Pass dry carbon dioxide-
Nal0 4 or 0.125 mL of 0.1 M sodium perwdate. free nitrogen through the solution for 5 min.
Sodium Tetraphenylborate VS B(C 6H 5 ) 4Na = 342.2 Standardisation. To 10 mL of dimethylformamide R add
0.05 mL of a 3 giL solution of thymol blue R in methanol R
For a 0.01M solution Dissolve 3.5 g of sodium
and titrate with the tetrabutylammonium hydroxide solution
tetraphenylborate in 50 mL of water, shake for 20 minutes with
until a pure blue colour is obtained. Immediately add
0.5 g of aluminium hydroxide gel, add 250 mL of water and
0.100 g of benzoic acid RV. Stir to effect solution, and titrate
16.6 g of sodium chloride and allow to stand for 30 minutes.
with the tetrabutylammoniurn hydroxide solution until the
Filter, add 600 mL of water, adjust the pH to 8.0 to 9.0 with
pure blue colour is again obtained. Protect the solution from
0.lM sodium hydroxide and dilute to 1000 mL with water.
atmospheric carbon dioxide throughout the titration. From
Ascertain its exact concentration in the following manner. the volume of titrant used in the second titration ascertain
Dissolve 7 mg of potassium chloride, previously dried at 150° the exact strength of the tetrabutylammonium hydroxide
for 1 hour, in 5 mL of acetate buffer pH 3. 7 and 5 mL of solution. Standardise immediately before use.
water, add 15 mL of the sodium tetraphenylborate solution,
1 mL of 0.1 M tetrabutylammonium hydroxick is equivalent to
allow to stand for 5 minutes and filter through a dry,
sintered-glass filter. To 20 mL of the filtrate add 0.5 mL of 12.21 mg of C 7 H 6 02.
bromophenol blue solutwn and titrate the excess of sodium O.lM Tetrabutylammonium Hydroxide in 2-propanol
tetraphenylborate with 0.005M cetylpyridinium chloride VS to vs
the blue colour of the indicator. Repeat the procedure Prepare as described for 0.1 M tetrabutylammonium hydroxick
without the potassium chloride. The molarity of the solution using 2-propanol R instead of toluene R and standardise as
is given by the expression: described.
awi[l 5(a - b)0.07 455] Tetrabutylammonium Iodide VS C 16H 36IN = 369.4
where a is the volume of 0.005M cetylpyridinium chloride VS For a 0.01M solution Dissolve 4 g of tetrabutylammonium
required when the potassium chloride is omitted, b is the iodide in sufficient water to produce 1000 mL.
volume of 0.005M cetylpyridinium chloride VS required when Ascertain its exact concentration in the following manner.
the potassium chloride is present and w is the weight, in g, of To 25 mL add 50 mL of 0.01M silver nitrate VS and 0.5 mL
potassium chloride taken. of 2M nitric acid and titrate the excess of silver nitrate with
O.lM Sodium Thiosulfate VS 0.01M ammonium thiocyanate VS using ammonium iron(III)
Dissolve 25 g of sodium thiosulfate Rand 0.2 g of sodium sulfate solution Rl as indicator. Each mL of 0.0 lM silver nitrate
carbonate R in carbon dioxide-free water R and dilute to VS is equivalent to 3.694 mg of C 16H 36IN.
1000.0 mL with the same solvent. Titanium(m) Chloride VS TiC13 = 154.3
Standardisation. To 10.0 mL of 0.033 M potassium bromate, For a 0.1M solution Dilute 100 mL of titanium(III)
add 40 mL of water R, 10 mL of potassium iodide solutwn R chloride solutwn with 200 mL of hydrochloric acid and add
and 5 mL of hydrochloric acid Rl. Titrate with the sodium sufficient freshly boiled and cooled water to produce
thiosulfate solution, using 1 mL of starch solution R, added 1000 mL.
towards the end of the titration, as indicator. Ascertain its exact concentration immediately before use by
1 mL of 0.1 M sodium thiosulfate is equivalent to 2.783 mg of titrating with it, in an atmosphere of carbon dioxide, 25 mL
KBrO 3 or 0.5 mL of 0.033 M potassium bromate. of 0. lM ammonium iron(III) sulfate VS acidified with suljuric
0.5M Sulfuric Acid VS 0.5M Sulphuric Acid VS acid, using ammonium thiocyanate solutwn, added just before
the end point, as indicator. Each mL of 0. lM ammonium
Dissolve 28 mL of suljuric acid R in water R and dilute to
iron(III) sulfate VS is equivalent to 15.43 mg of TiCh.
1000.0 mL with the same solvent.
Standardisation. Dissolve 0.950 g of trometamol RV in 50 mL
0.05M Zinc Chloride VS
of water R. Titrate with the sulfuric acid solution, Dissolve 6.82 g of zinc chloride R, weighed with appropriate
determining the end-point potentiometrically (2.2.20) or precautions, in water R. If necessary, add dropwise dilute
using 0 .1 mL of methyl orange solutwn R as indicator until the hydrochloric acid R until the opalescence disappears. Dilute to
solution turns reddish-yellow. 1000.0 mL with water R.
1 mL of 0. 5 M sulfuric acid is equivalent to 121.1 mg of Standardisation. To 20.0 mL of the zinc chloride solution add
C4HuNO3. 5 mL of dilute acetic acid R and carry out the determination
of zinc by complexometry (2.5.11).
0.05M Sulfuric Acid VS 0.05M Sulphuric Acid VS
Dilute 100 mL of 0.5M Suljuric Acid VS to 1000 mL with
O.lM Zinc Sulfate VS 0.lM Zinc Sulphate VS
water. Dissolve 29 g of zinc sulfate R in water R and dilute to
1000.0 mL with the same solvent.
Use the exact concentration as ascertained for 0.5M Suljuric
Acid VS. Standardisation. To 20.0 mL of the zinc sulfate solution add
5 mL of dilute acetic acid R and carry out the determination
O.lM Tetrabutylammonium Hydroxide VS
of zinc by complexometry (2.5. 11).
Dissolve 40 g of tetraburylammonium iodide R in 90 mL of
anhydrous methanol R, add 20 g of finely powdered silver
oxide R and shake vigorously for 1 h. Centrifuge a few
millilitres of the mixture and test the supernatant for iodides.
If a positive reaction is obtained, add an additional 2 g of
silver oxide R and shake for a further 30 min. Repeat this
procedure until the liquid is free from iodides, filter the
mixture through a fine sintered-glass filter (2.1.2) and rinse
V-Al 70 Appendix IC 2023

Ammonium Standard Solution (1 ppm NH4)

C. Standard Solutions Immediately before use, dilute ammonium standard solution
(2.5 ppm NH,J R to 2.5 times its volume with water R.
The following solutions are used as reference standards in Antimony Standard Solution (100 ppm Sb)
limit tests and should, unless experience has shown it to be Dissolve antimony potassium tartrate R equivalent to 0.274 g
unnecessary, be prepared immediately before use.
of C 8 H 4 K 2 O 12Sb2 ,3H 2O in 500 mL of 1 M hydrochlonc acid
In monographs of the European Pharmacopoeia, the symbol and dilute the clear solution to 1000 mL with water R.
'%'maybe replaced by the words 'per cent'. In such cases
Antimony Standard Solution (1 ppm Sb)
the reagent described below is to be used.
Dissolve antimony potassium tartrate R equivalent to 0.274 g
Acetaldehyde Standard Solution (100 ppm C2H4O) of C 8 H 4 K 2O 12Sb2,3H 2 O in 20 mL of hydrochloric acid Rl
Dissolve 1.0 g of acetaldehyde R in 2-propanol R and dilute to and dilute the clear solution to 100.0 mL with water R.
100.0 mL with the same solvent. Dilute 5.0 mL of the To 10.0 mL of this solution add 200 mL of hydrochloric
solution to 500.0 mL with 2-propanol R. Prepare immediately acid RI and dilute to 1000.0 mL with water R. To 100.0 mL
before use. of this solution add 300 mL of hydrochloric acid RI and dilute
Acetaldehyde Standard Solution (100 ppm to 1000.0 mL with water R. Prepare the dilute solutions
C2H4O) Rl immediately before use.
Dissolve 1.0 g of acetaldehyde R in water R and dilute to Arsenic Standard Solution (10 ppm As)
100.0 mL with the same solvent. Dilute 5.0 mL of the Immediately before use, dilute with water R to 100 times its
solution to 500.0 mL with water R. Prepare immediately volume a solution prepared by dissolving arsenious trioxide R
before use. equivalent to 0.330 g of As 2 O 3 in 5 mL of dilute sodium
Aluminium Standard Solution (200 ppm Al) hydroxide solution Rand diluting to 250.0 mL with water R.
Dissolve in water R a quantity of aluminium potassium Arsenic Standard Solution (1 ppm As)
sulfate R equivalent to 0.352 g of AlK(SO4)z,12H2O. Immediately before use, dilute arsenic standard solution
Add 10 mL of dilute sulfuric acid Rand dilute to 100.0 mL (1 0 ppm As) R to 10 times its volume with water R.
with water R. Arsenic Standard Solution (0.1 ppm As)
Aluminium Standard Solution (100 ppm Al)
Immediately before use, dilute arsenic standard solution (1 ppm
Immediately before use, dilute with water R to 10 times its As) R to 10 times its volume with water R.
volume a solution containing 8.947 g of aluminium chloride R
Barium Standard Solution (0.1% Ba)
in 1000.0 mL of water R.
Dissolve barium chloride R equivalent to 0 .1 78 g of
Aluminium Standard Solution (25 ppm Al)
BaC12 ,2H 2 O in distilled water Rand dilute to 100.0 mL with
Immediately before use, dilute with water to 40 times its the same solvent.
volume a solution containing 8.947 g of aluminium chloride in
Barium Standard Solution (50 ppm Ba)
1000.0 mL of water.
Immediately before use, dilute with distilled water R to
Aluminium Standard Solution (10 ppm Al)
20 times its volume a solution in distilled water R containing
Immediately before use, dilute with water R to 100 times its barium chloride R equivalent to 0.178 g of BaC12 ,2H2O in
volume in a solution containing aluminium nitrate R 100.0 mL.
equivalent to 1.39 g of Al(NO 3h,9H2O in 100.0 mL.
Barium Standard Solution (2 ppm Ba)
Aluminium Standard Solution (5 ppm Al)
Immediately before use, dilute barium standard solution
Immediately before use, dilute with water R to 100 times its (50 ppm Ba) R to 25 times its volume with distilled water R.
volume in a solution containing aluminium nitrate R
Bismuth Standard Solution (100 ppm Bi)
equivalent to 0.695 g of Al(NO 3 h,9H2O in 100.0 mL.
Alternatively, use a commercially available standard solution Dissolve bismuth subnitrate R equivalent to 0.500 g of Bi in
containing a known amount of aluminium (5 ppm Al). 50 mL of nitric acid Rand dilute to 500.0 mL with water R.
Dilute the solution to 10 times its volume with dilute nitric
Aluminium Standard Solution (2 ppm Al)
acid R immediately before use.
Immediately before use, dilute with water R to 100 times its
Cadmium Standard Solution (0.1% Cd)
volume a solution containing aluminium potassium sulfate R
equivalent to 0.352 g of AIK(SO 4) 2,12H2 O and 10 mL of Dissolve cadmium R equivalent to 0 .100 g of Cd in the
dilute sulfuric acid R in 100.0 mL. smallest necessary amount of a mixture of equal volumes of
hydrochloric acid Rand water Rand dilute to 100.0 mL with a
Ammonium Standard Solution (100 ppm NH4)
1 per cent V/V solution of hydrochloric acid R.
Immediately before use, dilute to 25 mL with water R 10 mL
Cadmium Standard Solution (10 ppm Cd)
of a solution containing ammonium chloride R equivalent to
0.741 g ofNH4 Cl in 1000 mL. Immediately before use, dilute cadmium standard solution
(0.1 per cent Cd) R to 100 times its volume with a
Ammonium Standard Solution (3 ppm NH4)
1 per cent V/V solution of hydrochloric acid R.
Immediately before use, dilute with water R to 100 times its
Calcium Standard Solution (1000 ppm Ca)
volume a solution containing ammonium chloride R equivalent
to 0.889 g ofNH4 Cl in 1000.0 mL. Dissolve 2.5 g of calcium carbonate in 23 mL of
lM hydrochloric acid and add sufficient distilled water to
Ammonium Standard Solution (2.5 ppm NH4)
produce 100 mL. Dilute 1 volume of this solution to
Immediately before use, dilute with water R to 100 times its 10 volumes with distilled water immediately before use.
volume a solution containing ammonium chloride R equivalent Calcium Standard Solution (400 ppm Ca)
to 0.741 g ofNH4 Cl in 1000.0 mL.
Immediately before use, dilute with distilled water R to
10 times its volume a solution in distilled water R containing
2023 Appendix IC V-Al 71

calcium carbonate R equivalent to 1.000 g of CaCO 3 and Copper Standard Solution (10 ppm Cu)
23 mL of 1 M hydrochloric acid in 100.0 mL. Immediately before use, dilute copper standard solution
Calcium Standard Solution (100 ppm Ca) (0.1 per cent Cu) R to 100 times its volume with water R.
Immediately before use, dilute with distz7led water R to Copper Standard Solution (0.1 ppm Cu)
10 times its volume a solution in distilled water R containing Immediately before use, dilute copper standard solution
calcium carbonate R equivalent to 0.624 g of CaCO 3 and (10 ppm Cu) R to 100 times its volume with water R.
3 mL of acetic acid R in 250.0 mL.
Copper Standard Solution (0.1 per cent Cu) for ICP
Calcium Standard Solution (100 ppm Ca), Alcoholic A copper standard solution (1000 mg/L) suitable for
Immediately before use, dilute with ethanol (96 per cent) R to inductively coupled plasma (ICP) applications and traceable
10 times its volume a solution in distilled water R containing to national or international standards.
calcium carbonate R equivalent to 2.50 g of CaCO 3 and
Elementary Standard Solutions for Atomic
12 mL of acetic acid R in 1000.0 mL. Spectrometry, 1.000 g/L
Calcium Standard Solution (100 ppm Ca) Rl
This solution is prepared, generally in acid conditions, from
Immediately before use, dilute with water R to 10 times its the element or a salt of the element whose minimum content
volume a solution containing anhydrous calcium chloride R is not less than 99.0 per cent. The quantity per litre of
equivalent to 2.769 g of CaCl2 in 1000.0 mL of dilute solution is greater than 0.995 g throughout the guaranteed
hydrochloric acid R. period, as long as the vial has not been opened. The starting
Calcium Standard Solution (10 ppm Ca) material (element or salt) and the characteristics of the final
Immediately before use, dilute with distilled water R to solvent (nature and acidity, etc.) are mentioned on the label.
100 times its volume a solution in distilled water R containing Ferricyanide Standard Solution (50 ppm Fe(CN)6)
calcium carbonate R equivalent to 0.624 g of CaCO 3 and Immediately before use, dilute with water R to 100 times its
3 mL of acetic acid R in 250.0 mL. volume a solution containing potassium ferricyanide R
Chloride Standard Solution (50 ppm Cl) equivalent to 0.78 g ofK3 Fe(CN) 6 in 100.0 mL.
Immediately before use, dilute with water R to 10 times its Ferrocyanide Standard Solution (100 ppm Fe(CN) 6)
volume a solution containing sodium chloride R equivalent to Immediately before use, dilute with water R to 10 times its
0.824 g of NaCl in 1000.0 mL. volume a solution containing potassium ferrocyanide R
Chloride Standard Solution (8 ppm Cl) equivalent to 0.20 g of ~Fe(CN) 6,3H2 O in 100.0 mL.
Immediately before use, dilute with water R to 100 times its Fluoride Standard Solution (10 ppm F)
volume a solution containing sodium chloride R equivalent to Dissolve in water R sodium fluoride R previously dried at
1.32 g of NaCl in 1000.0 mL. 300 °C for 12 h, equivalent to 0.442 g ofNaF, and dilute to
Chloride Standard Solution (5 ppm Cl) 1000.0 mL with the same solvent (1 mL == 0.2 mg F). Store
Immediately before use, dilute with water R to 100 times its in a polyethylene container. Immediately before use, dilute
volume a solution containing sodium chloride R equivalent to the solution to 20 times its volume with water R.
0.824 g of NaCl in 1000.0 mL. Fluoride Standard Solution (1 ppm F)
Chromium Liposoluble Standard Solution (1000 ppm Immediately before use, dilute fluoride standard solution
Cr) (10 ppm F) R to 10 times its volume with water R.
A chromium (metal) organic compound in an oil. Formaldehyde Standard Solution (5 ppm CH2 0)
Chromium Standard Solution (0.1% Cr) Immediately before use, dilute with water R to 200 times its
Dissolve potassium dichromate R equivalent to 2.83 g of volume a solution containing 1.0 g of CH2 O per litre
K 2 Cr2 O 7 in water Rand dilute to 1000.0 mL with the same prepared from formaldehyde solution R.
solvent. Germanium Standard Solution (100 ppm Ge)
Chromium Standard Solution (100 ppm Cr) Dissolve ammonium hexafluorogermanate(IV) R equivalent to
Dissolve potassium dichromate R equivalent to 0.283 g of 0.307 g of (NH4 hGeF6 in a 0.01 per cent V/Vsolution of
K 2 Cr2 O 7 in water Rand dilute to 1000.0 mL with the same hydrofluoric acid R. Dilute the clear solution to 1000 mL with
solvent. water R.
Chromium Standard Solution (0.1 ppm Cr) Glucose Standard Solution
Immediately before use, dilute chromium standard solution Dissolve 0.10 g of glucose in a saturated solution of benzoic
(100 ppm Gr) R to 1000 times its volume with water R. acid in water, dilute to 100 mL with the saturated benzoic
Cobalt Standard Solution (100 ppm Co) acid solution and dilute 2 mL of this solution to 100 mL
with water.
Dissolve cobalt nitrate R equivalent to 0.494 g of
Co(NO3 ) 2 ,6H2 O in 500 mL of 1 M nitn·c acid and dilute the Glucose Standard Solution contains 20 µg of glucose
clear solution to 1000 mL with water R. per mL.
Copper Liposoluble Standard Solution (1000 ppm Glyoxal Standard Solution (20 ppm C2H 2 0 2)
Cu) In a 100 mL graduated flask weigh a quantity of glyoxal
A copper (metal) organic compound in an oil. solution R corresponding to 0.200 g of C 2 H 2 O 2 and make up
to volume with anhydrous ethanol R. Immediately before use
Copper Standard Solution (0.1% Cu)
dilute the solution to 100 times its volume with the same
Dissolve copper sulfate pentahydrate R equivalent to 0.393 g of solvent.
CuSO 4 ,5H 2 O in water R and dilute to 100.0 mL with the
same solvent.
V-Al 72 Appendix IC 2023

Glyoxal Standard Solution (2 ppm C2H 2 O 2 ) Lead Standard Solution (10 ppm Pb) Rl
Immediately before use, dilute glyoxal standard solution Immediately before use, dilute with water R to 10 times its
(20 ppm C 2H 2 0;J R to 10 times its volume with anhydrous volume a solution containing 0.160 g of lead nitrate R in
ethanol R. 100 mL of water R, to which is added 1 mL of lead-free nitric
Hydrogen Peroxide Standard Solution (2 ppm H 2 O 2 ) acid Rand dilute to 1000.0 mL.
Dilute 10.0 mL of dilute hydrogen peroxide solution R to Lead Standard Solution (2 ppm Pb)
300.0 mL with water R. Dilute 2.0 mL of this solution to Immediately before use, dilute lead standard solution
1000.0 mL with water R. Prepare immediately before use. (10 ppm Pb) R to 5 times its volume with water R.
Iodide Standard Solution (20 ppm I) Lead Standard Solution (1 ppm Pb)
Dilute 10.0 mL of a 0.026% w/v solution of potassium iodide Immediately before use, dilute lead standard solution
to 100.0 mL with water. (10 ppm Pb) R to 10 times its volume with water R.
Iodide Standard Solution (10 ppm I) Lead Standard Solution (0.25 ppm Pb)
Immediately before use, dilute with water R to 100 times its Immediately before use, dilute lead standard solution
volume a solution containing potassium iodide R equivalent to (1 ppm Pb) R to 4 times its volume with water R.
0.131 g of Kl in 100.0 mL. Lead Standard Solution (0.1 ppm Pb)
Iron Standard Solution (0.1% Fe) Immediately before use, dilute lead standard solution
Dissolve 0.100 g of Fe in the smallest amount necessary of a (1 ppm Pb) R to 10 times its volume with water R.
mixture of equal volumes of hydrochloric acid R and water R Lithium Standard Solution (100 ppm Li)
and dilute to 100.0 mL with water R.
Dissolve 0. 6109 g of lithium chloride in sufficient water to
Iron Standard Solution (250 ppm Fe) produce 1000 mL.
Immediately before use, dilute with water R to 40 times its Lutetium Standard Solution (20 ppm Lu)
volume a solution containing 4.840 g of ferric chloride Rina
Immediately before use, dissolve 0.445 g of lutetium chloride
150 g/L solution of hydrochloric acid R diluted to 100.0 mL. hexahydrate R in a mixture of equal volumes of heavy metal-
Iron Standard Solution (20 ppm Fe) free nitric acid R and water R, and dilute to 100.0 mL with
Immediately before use, dilute with water R to 10 times its the same mixture of solvents.
volume a solution containing ferric ammonium sulfate R Dilute 1.0 mL of this solution to 100.0 mL with water R.
equivalent to 0.863 g of FeNH4 (SO 4) 2 ,12H2 O and 25 mL of Magnesium Standard Solution (0.1% Mg)
dilute suljuric acid R in 500.0 mL.
Dissolve magnesium sulfate R equivalent to 1.010 g of
Iron Standard Solution (10 ppm Fe) MgSO 4 ,7H2 O in distilled water Rand dilute to 100.0 mL
Immediately before use, dilute with water R to 100 times its with the same solvent.
volume a solution containing ferrous ammonium sulfate R
Magnesium Standard Solution (1000 ppm Mg)
equivalent to 7 .022 g of Fe(NH4 )z(SO 4 )z,6H 2 O and 25 mL
of dilute sulfuric acid R in 1000.0 mL. Dissolve 5.275 g of magnesium nitrate R in 16 mL of dilute
nitric acid R and dilute to 500.0 mL with water R.
Iron Standard Solution (8 ppm Fe)
Standardisation: carry out the determination of magnesium by
Immediately before use, dilute with water R to 10 times its complexometry (2. 5.11).
volume a solution containing 80 mg of iron R and 50 mL of
hydrochloric acid R (220 g/L HCl) in 1000.0 mL. Magnesium Standard Solution (100 ppm Mg)
Iron Standard Solution (2 ppm Fe) . Immediately before use, dilute with water R to 10 times its
volume a solution containing magnesium sulfate R equivalent
Immediately before use, dilute iron standard solution to 1.010 g of MgSO 4 ,7H2 O in 100.0 mL.
(20 ppm Fe) R to 10 times its volume with water R.
Magnesium Standard Solution (10 ppm Mg)
Iron Standard Solution (1 ppm Fe)
Immediately before use, dilute magnesium standard solution
Immediately before use, dilute iron standard solution
(100 ppm Mg) R to 10 times its volume with water R.
(20 ppm Fe) R to 20 times its volume with water R.
Magnesium Standard Solution (10 ppm Mg) Rl
Lead Liposoluble Standard Solution (1000 ppm Pb)
Immediately before use, dilute with water R to 100 times its
A lead (metal) organic compound in an oil. volume a solution containing 8.365 g of magnesium chloride R
Lead Standard Solution (0.1% Pb) in 1000.0 mL of dilute hydrochloric acid R.
Dissolve lead nitrate R equivalent to 0.400 g of Pb(NO 3 ) 2 in Manganese Standard Solution (1000 ppm Mn)
water R and dilute to 250.0 mL with the same solvent.
Dissolve manganese sulfate R equivalent to 3.08 g of MnSO 4,
Lead Standard Solution (100 ppm Pb) H 2 O in 500 mL of 1 M nitric acid and dilute the solution to
Immediately before use, dilute lead standard solution 1000 mL with water R.
(0.1 per cent Pb) R to 10 times its volume with water R. Manganese Standard Solution (100 ppm Mn)
Lead Standard Solution (20 ppm Pb) Dissolve manganese sulfate R equivalent to 0.308 g of
Dissolve 0.80 g of lead(11) nitrate in water containing 2 mL of MnSO 4 ,H2 O in 500 mL of J M nitric acid and dilute the
nitric acid and add sufficient water to produce 250 mL. Dilute clear solution to 1000 mL with water R.
1 volume to 100 volumes with water immediately before use. Mercury Standard Solution (1000 ppm Hg)
Lead Standard Solution (10 ppm Pb) Dissolve mercuric chkiride R equivalent to 1.354 g of HgClz in
Immediately before use, dilute lead standard solution 50 mL of dilute nitric acid Rand dilute to 1000.0 mL with
(100 ppm Pb) R to 10 times its volume with water R. water R.
2023 Appendix IC V-Al 73

Mercury Standard Solution (100 ppm Hg) Phosphate Standard Solution (5 ppm P0 4)
Dissolve 1.080 g of yellow mercury(II) oxide in the minimum Immediately before use, dilute with water R to 100 times its
volume of 2M hydrochloric acid and add sufficient water to volume a solution containing potassium dihydrogen phosphate R
produce I 000 mL. equivalent to 0.716 g ofKH 2 PO 4 in 1000.0 mL.
Mercury Standard Solution (10 ppm Hg) Platinum Standard Solution (30 ppm Pt)
Immediately before use, dilute with water to 100 times its Immediately before use, dilute with 1 M hydrochloric acid to
volume a solution containing mercuric chloride R equivalent to 10 times its volume a solution containing 80 mg of
0.338 g of HgC1 2 in 250.0 mL. chloroplatinic acid R in 100.0 mL of 1 M hydrochloric acid.
Mercury Standard Solution (5 ppm Hg) Potassium Standard Solution (0.2 % K)
Dilute 1.0 mL of a 0.0675% w/v solution of mercury(!!) Dissolve dipotassium sulfate R equivalent to 0.446 g of K 2 S0 4
chloride to 100.0 mL with water. in distilled water Rand dilute to 100.0 mL with the same
Nickel Liposoluble Standard Solution (1000 ppm Ni) solvent.
A nickel (metal) organic compound in an oil. Potassium Standard Solution (600 ppm K)
Nickel Standard Solution (10 ppm Ni) Immediately before use, dilute with water R to 20 times its
Immediately before use, dilute with water R to 100 times its volume a solution containing dipotassium sulfate R equivalent
volume a solution containing nickel sulfate R equivalent to to 2.676 g ofK2 SO 4 in 100.0 mL.
4.78 g ofNiSO 4 ,7H 2 O in 1000.0 mL. Potassium Standard Solution (100 ppm K)
Nickel Standard Solution (5 ppm Ni) Immediately before use, dilute with water R to 20 times its
Immediately before use dilute nickel standard solution (10 ppm volume a solution containing dipotassium sulfate R equivalent
Ni) R to twice its volume with water for chromatography R. to 0.446 g of K 2 SO 4 in 100.0 mL.
Nickel Standard Solution (0.2 ppm Ni) Potassium Standard Solution (20 ppm K)
Immediately before use, dilute nickel standard solution (10 ppm Immediately before use, dilute potassium standard solution
Nz) R to 50 times its volume with water R. (100 ppm K) R to 5 times its volume with water R.
Nickel Standard Solution (0.1 ppm Ni) Scandium Standard Solution (0.1 per cent Sc) for
Immediately before use, dilute nickel standard solution (10 ppm ICP
Nz) R to 100 times its volume with water R. A scandium standard solution ( 1000 mg/L) suitable for
Nitrate Standard Solution (100 ppm N0 3) inductively coupled plasma (ICP) applications and traceable
Immediately before use, dilute with water R to 10 times its to national or international standards.
volume a solution containing potassium nitrate R equivalent to Selenium Standard Solution (100 ppm Se)
0.815 g of KNO 3 in 500.0 mL. Dissolve 0.100 g of selenium R in 2 mL of nitric acid R.
Nitrate Standard Solution (10 ppm N0 3) Evaporate to dryness. Take up the residue in 2 mL of
Immediately before use, dilute nitrate standard solution water R and evaporate to dryness; carry out three times.
(100 ppm NOJ) R to 10 times its volume with water R. Dissolve the residue in 50 mL of dilute hydrochloric acid R and
Nitrate Standard Solution (2 ppm N0 3 ) dilute to 1000.0 mL with the same acid.
Immediately before use, dilute nitrate standard solution Selenium Standard Solution (1 ppm Se)
(10 ppm NOJ) R to 5 times its volume with water R. Immediately before use, dilute with water R to 40 times its
Nitrite Standard Solution (20 ppm N0 2 ) volume a solution containing selenious acid R equivalent to
6.54 mg of H 2 SeO 3 in 100.0 mL.
Dissolve 0.6 g of sodium nitrite in sufficient water to produce
I 00 mL and dilute 1 mL of this solution to 200 mL with Silver Standard Solution (5 ppm Ag)
water. Immediately before use, dilute with water R to 100 times its
Palladium Standard Solution (500 ppm Pd) volume a solution containing silver nitrate R equivalent to
Dissolve 50.0 mg of palladium R in 9 mL of hydrochloric 0.790 g of AgNO 3 in 1000.0 mL.
acid Rand dilute to 100.0 mL with water R. Sodium Standard Solution (1000 ppm Na)
Palladium Standard Solution (20 ppm Pd) Dissolve a quantity of anhydrous sodium carbonate R
Dissolve 0.333 g of palladium chloride R in 2 mL of warm equivalent to 2.305 g of Na 2 CO 3 in a mixture of 25 mL of
hydrochloric acid R. Dilute the solution to 1000.0 mL with a water R and 25 mL of nitric acid R and dilute to 1000.0 mL
mixture of equal volumes of dilute hydrochloric acid R and with water R.
water R. Immediately before use dilute to 10 times its volume Sodium Standard Solution (200 ppm Na)
with water R. Immediately before use, dilute with water R to 10 times its
Palladium Standard Solution (0.5 ppm Pd) volume a solution containing sodium chloride R equivalent to
Dilute 1 mL of palladium standard solution (500 ppm Pd) R to 0.509 g of NaCl in 100.0 mL.
1000 mL with a mixture of 0.3 volumes of nitric acid Rand Sodium Standard Solution (50 ppm Na)
99.7 volumes of water R. Dilute the sodium standard solution (200 ppm Na) R to four
Phosphate Standard Solution (200 ppm P0 4 ) times its volume with water R.
Dissolve potassium dihydrogen phosphate R equivalent to Strontium Standard Solution (1.0 per cent Sr)
0.286 g of KH 2 PO 4 in water Rand dilute to 1000.0 mL with Cover with water R, strontium carbonate R equivalent to
the same solvent. 1.6849 g of SrCO 3 • Cautiously add hydrochloric acid R until
Phosphate Standard Solution (100 ppm P0 4) all the solid has dissolved and there is no sign of further
Dilute 10.0 mL of a 0.143% w/v solution of potassium effervescence. Dilute to 100.0 mL with water R.
dihydrogen orthophosphate to 100.0 mL with water immediately
before use.
V-Al 7 4 Appendix I C 2023

Sulfate Standard Solution (100 ppm S0 4) Zinc Standard Solution (10 ppm Zn)
Immediately before use, dilute with distilled water R to Immediately before use, dilute zinc standard solution (100 ppm
10 times its volume a solution in distilled water R containing Zn) R to 10 times its volume with water R.
dipotassium sulfate R equivalent to 0 .181 g of K 2 SO 4 in Zinc Standard Solution (5 ppm Zn)
100.0 mL. Immediately before use, dilute zinc standard solution (100 ppm
Sulfate Standard Solution (10 ppm S04) Zn) R to 20 times its volume with water R.
Immediately before use, dilute with distilled water R to Zirconium Standard Solution (1 g/L Zr)
100 times its volume a solution in distilled water R containing Dissolve zirconyl nitrate R equivalent to 0.293 g of
dipotassium sulfate R equivalent to 0.181 g ofK2 SO 4 in ZrO(NO 3)z,2H2O in a mixture of 2 volumes of hydrochloric
100.0 mL. acid Rand 8 volumes of water Rand dilute to 100.0 mL with
Sulfate Standard Solution (10 ppm S0 4) Rl the same mixture of solvents.
Immediately before use, dilute with ethanol
(30 per cent Vlv,J R to 100 times its volume a solution
containing dipotassium sulfate R equivalent to 0.181 g of
K2SO4 in 100.0 mL of ethanol (30 per cent Vlv,J R. D. Buffer Solutions
Sulfite Standard Solution (80 ppm S0 2)
Dissolve 3 .150 g of anhydrous sodium sulfite R in freshly
Buffer solutions should be prepared using carbon dioxide-free
prepared distilled water R and dilute to 100.0 mL with the
water.
same solvent. Dilute 0.5 mL to 100.0 mL with freshly
prepared distilled water R. Acetate Buffer pH 2.45
Sulfite Standard Solution (1.5 ppm S0 2) Mix 200 mL of lM hydrochloric acid with 200 mL of
1M sodium acetate and dilute to 1000 mL with water.
Dissolve sodium metabisulfite R equivalent to 0.152 g of
Immediately before use adjust the pH to 2.45 by the addition
Na2S2O 5 in water R and dilute to 100.0 mL with the same
of IM hydrochloric acid or IM sodium acetate, as required.
solvent. Dilute 5.0 mL of this solution to 100.0 mL with
water R. To 3.0 mL of the resulting solution, add 4.0 mL of Acetate Buffer pH 2.8
0.1 M sodium hydroxide and dilute to 100.0 mL with water R. Dissolve 4 g of anhydrous sodium acetate in about 840 mL of
Thallium Standard Solution (10 ppm TI) water, add sufficient glacial acetic acid to adjust the pH to 2.8
(about 155 mL) and dilute to 1000 mL with water.
Dissolve thallous sulfate R equivalent to 0.1235 g ofT12SO4 in
a 9 g/L solution of sodium chloride Rand dilute to 1000.0 mL Acetate Buffer pH 3.4
with the same solution. Dilute 10.0 mL of the solution to Mix 5 volumes of O. lM sodium acetate with 95 volumes of
100.0 mL with the 9 g/L solution of sodium chloride R. O. IM acetic acid.
Tin Liposoluble Standard Solution (1000 ppm Sn) Acetate Buffer pH 3.5
A tin (metal) organic compound in an oil. Buffer solution pH 3.5
Tin Standard Solution (5 ppm Sn) Dissolve 25 g of ammonium acetate in 25 mL of water and
Dissolve tin R equivalent to 0.500 g of Sn in a mixture of add 38 mL of 7M hydrochloric acid. Adjust the pH to 3.5 with
5 mL of water Rand 25 mL of hydrochloric acid Rand dilute either 2M hydrochloric acid or 6M ammonia and dilute to
to 1000.0 mL with water R. Dilute the solution to 100 times 100 mL with water.
its volume with a 2.5 per cent V/V solution of hydrochloric Acetate Buffer pH 3.7
acid R immediately before use. Dissolve 10 g of anhydrous sodium acetate in 300 mL of water,
Tin Standard Solution (0.1 ppm Sn) adjust to pH 3.7 with glacial acetic acid and dilute to
Immediately before use, dilute tin standard solution (5 ppm 1000 mL with water. If necessary, readjust to pH 3.7 with
Sn) R to 50 times its volume with water R. glacial acetic acid or anhydrous sodium acetate as required,
before use.
Titanium Standard Solution (100 ppm Ti)
Acetate Buffer pH 4,4 Acetate buffer solution pH 4.4
Dissolve 100.0 mg of titanium R in 100 mL of hydrochloric
acid R diluted to 150 mL with water R, heating if necessary. Dissolve 136 g of sodium acetate Rand 77 g of ammonium
Allow to cool and dilute to 1000 mL with water R. acetate R in water Rand dilute to 1000.0 mL with the same
solvent; add 250.0 mL of glacial acetic acid R and mix.
Vanadium Standard Solution (1 g/L V)
Acetate Buffer pH 4.6 Acetate buffer solution pH 4.6
Dissolve in water R ammonium vanadate R equivalent to
0.230 g ofNH4 VO 3 and dilute to 100.0 mL with the same Dissolve 5.4 g of sodium acetate R in 50 mL of water R, add
solvent. 2.4 g of glacial acetic acid R and dilute to 100.0 mL with
water R. Adjust the pH if necessary.
Zinc Standard Solution (5 mg/mL Zn)
Acetate Buffer pH 5.0
Dissolve 3.15 g of zinc oxide R in 15 mL of hydrochloric
acid R and dilute to 500.0 mL with water R. Dissolve 13.6 g of sodium acetate and 6 mL of glacial acetic
acid in sufficient water to produce 1000 mL.
Zinc Standard Solution (100 ppm Zn)
Acetate Buffer pH 6.0 Acetate buffer solution pH 6.0
Immediately before use, dilute with water R to 10 times its
volume a solution containing zinc sulfate R equivalent to Dissolve 100 g of ammonium acetate R in 300 mL of water R,
0.440 g of ZnSO 4, 7H2O and 1 mL of acetic acid R in add 4.1 mL of glacial acetic acid R, adjust the pH if necessary
100.0 mL. using ammonia R or acetic acid Rand dilute to 500.0 mL
with water R.
Zinc Standard Solution (25 ppm Zn)
Dilute 25.0 mL of zinc standard solution (100 ppm Zn) to
100. 0 mL with water immediately before use.
2023 Appendix ID V-Al 75

Acetate Buffer Solution pH 4.5 Barbitone Buffer pH 8.4 Barbital buffer solution pH 8.4
Dissolve 77 .1 g of ammonium acetate R in water R. Dissolve 8.25 g of barbital sodium R in water R and dilute to
Add 70 mL of glacial acetic acid Rand dilute to 1000.0 mL 1000.0 mL with the same solvent.
with water R. Barbitone Buffer pH 8.6 Rl Barbital buffer solution
Acetate Buffer Solution pH 4. 7 pH 8.6 Rl
Dissolve 136. l g of sodium acetate R in 500 mL of water R. Dissolve in water R 1.38 g of barbital R, 8.76 g of barbital
Mix 250 mL of this solution with 250 mL of dilute acetic sodium Rand 0.38 g of calcium lactate pentahydrate Rand
acid R. Shake twice with a freshly prepared, filtered, 0.1 g/L dilute to 1000.0 mL with the same solvent.
solution of dithizone R in chloroform R. Shake with carbon Borate Buffer pH 7.5 Borate buffer solution pH 7.5
tetrachloride R until the extract is colourless. Filter the
Dissolve 2.5 g of sodium chloride R, 2.85 g of disodium
aqueous layer to remove traces of carbon tetrachloride.
tetraborate Rand 10.5 g of boric acid R in water Rand dilute
Acetate Buffer Solution pH 4. 7 Rl to 1000.0 mL with the same solvent. Adjust the pH if
Dissolve 136.1 g of sodium acetate R in 500 mL of water R. necessary.
Mix 250 mL of this solution with 250 mL of dilute acetic Storage: at 2 °C to 8 °C.
acid R.
Borate Buffer pH 8.0
Acetate-edetate Buffer Solution pH 5.5
To 50 mL of a solution containing 0.6189 g of boric acid and
Dissolve 250 g of ammonium acetate R and 15 g sodium 0.7456 g of potassium chloride add 3.97 mL of 0.2M sodium
edetate R in 400 mL of water Rand add 125 mL of glacial hydroxide VS and dilute to 200 mL with water.
acetic acid R.
At 20°, the solution may be used as a solution of standard
Acetone Solution, Buffered pH.
Dissolve 8.15 g of sodium acetate R and 42 g of sodium Borate Buffer pH 8.4, 0.2M
chloride R in water R, add 68 mL of 0.1 M hydrochloric acid
Dissolve 2.0 g of sodium hydroxide and 12.1 g of boric acid in
and 150 mL of acetone R and dilute to 500 mL with water R. 250 mL of water, adjust to pH 8.4, if necessary, by adding a
Ammonia Buffer pH 10.0 Ammonium chloride buffer few granules of boric acid.
solution pH 10.0 Borate Buffer pH 9.0
Dissolve 5.4 g of ammonium chloride R in 20 mL of water R, Buffer solution pH 9.0
add 35.0 mL of ammonia Rand dilute to 100.0 mL with
water R.
Dissolve 6.18 g of boric acid R in 0.1 M potassium chloride R
and dilute to 1000.0 mL with the same solvent.
Ammonia Buffer pH 10.9 Buffer solution pH 10.9
Mix 1000.0 mL of this solution and 420.0 mL of
Dissolve 6.75 g of ammonium chloride R in ammonia Rand 0.1 M sodium hydroxide.
dilute to 100.0 mL with the same solvent. Borate Buffer pH 9.6
Ammonia Buffer pH 10.9, Dilute
To 50 mL of a solution containing 0.6189 g of boric acid and
Dilute 2 mL of ammonia buffer pH 10. 9 to 1000 mL with 0.7456 g of potassium chloride add 36.85 mL of 0.2M sodium
water. hydroxide VS and dilute with water to 200 mL.
Ammonium Acetate Buffer pH 4.5, 0.5M At 20°, the solution may be used as a solution of standard
Mix 14.3 mL of glacial acetic acid Rand 470 mL of water R pH.
and adjust to pH 4.5 with concentrated ammonia R. Dilute to Borate Buffer Solution pH 8.0, 0.0015M
500.0 mL with water R. Dissolve 0.572 g of disodium tetraborate R and 2.94 g of
Ammonium Carbonate Buffer Solution pH 10.3, 0.lM calcium chloride R in 800 mL of water R. Adjust the pH with
Dissolve 7. 91 g of ammonium carbonate R in 800 mL of 1 M hydrochloric acid. Dilute to 1000.0 mL with water R.
water R. Adjust the pH with dilute sodium hydroxide solution R. Borate Buffer Solution pH 10.0
Dilute to 1000.0 mL with water R.
Introduce 12.4 g of boric acid R into a 500.0 mL volumetric
Ammonium Chloride Buffer Solution pH 9.5 flask. Add 300 mL of water R to suspend the boric acid.
Dissolve 33.5 g of ammonium chloride R in 150 mL of Add 100 mL of a 56 g/L solution of potassium hydroxide R
water R, add 42.0 mL of concentrated ammonia Rand dilute and mix to dissolve the boric acid. Adjust to pH 10.0 by
to 250.0 mL with water R. slowly adding a 56 g/L solution of potassium hydroxide R
Storage: in a polyethylene container. (about 60 mL is usually needed). Mix. Dilute almost to
Ammonium Chloride Buffer Solution pH 10.4 volume with water R. If necessary, adjust the pH with boric
acid R or with a 56 g/L solution of potassium hydroxide R.
Dissolve 70 g of ammonium chloride R in 200 mL of water R,
Dilute to 500.0 mL with water R.
add 330 mL of concentrated ammonia R and dilute to
1000.0 mL with water R. If necessary, adjust to pH 10.4 with Borate Buffer Solution pH 10.4
ammonia R. Dissolve 24.64 g of boric acid R in 900 mL of distilled
Ammonium Chloride Buffer Solution pH 10.7 water R. Adjust the pH using a 400 g/L solution of sodium
hydroxide R. Dilute to 1000 mL with distilled water R.
Dissolve 67.5 g of ammonium chloride R in water R, add
570 mL of concentrated ammonia Rand dilute to 1000.0 mL Boric Buffer pH 9.0 Buffer solution pH 9.0 Rl
with water R. Dissolve 6.20 g of boric acid R in 500 mL of water R and
Barbitone Buffer pH 7.4 Barbital buffer solution pH 7 .4 adjust the pH with 1 M sodium hydroxide (about 41.5 mL).
Dilute to 1000.0 mL with water R.
Mix 50 mL of a solution in water R containing 19.44 g/L of
sodium acetate R and 29.46 g/L of barbital sodium R with Buffer (Acetate) Solution pH 5.0
50.5 mL of 0.1 M hydrochloric acid, add 20 mL of an 85 g/L To 120 mL of a 6 g/L solution of glacial acetic acid R add
of sodium chloride R and dilute to 250 mL with water R. 100 mL of 0.1 M potassium hydroxide and about 250 mL of
V-Al 76 Appendix ID 2023

water R. Mix. Adjust the pH to 5.0 with a 6 g/L solution of Buffer Solution pH 7.4
acetic acid R or with 0.1 M potassium hydroxide and dilute to Dissolve 0.6 g of potassium dihydrogen phosphate R, 6.4 g of
1000.0 mL with water R. disodium hydrogen phosphate dodecahydrate Rand 5.85 g of
Buffer (HEPES) Solution pH 7.5 sodium chloride R in water R, and dilute to 1000.0 mL with
Dissolve 2.38 g of HEPES R in about 90 mL of water R. the same solvent. Adjust the pH if necessary.
Adjust the pH to 7.5 with sodium hydroxide solution R. Dilute Buffer Solution pH 8.0
to 100 mL with water R. To 50.0 mL of 0.2 M potassium dihydrogen phosphate R add
Buffer Solution pH 2.2 46.8 mL of 0.2 M sodium hydroxide. Dilute to 200.0 mL with
Mix 6.7 mL of phosphoric acid R with 55.0 mL of a 40 g/L water R.
solution of sodium hydroxide Rand dilute to 1000.0 mL with Buffer Solution pH 8.0 Rl
water R. Dissolve 20 g of dipotassium hydrogen phosphate R in 900 mL
Buffer Solution pH 2.5 of water R. Adjust the pH with phosphoric acid R. Dilute to
Dissolve 100 g of potassium dihydrogen phosphate R in 800 mL 1000 mL with water R.
of water R; adjust to pH 2.5 with hydrochloric acid R and Buffer Solution pH 9.0
dilute to 1000.0 mL with water R. Dissolve 6.18 g of boric acid R in 0.1 M potassium chloride R
Buffer Solution pH 2.5 Rl and dilute to 1000.0 mL with the same solvent.
To 4.9 g of dilute phosphoric acid R add 250 mL of water R. Mix 1000.0 mL of this solution and 420.0 mL of 0.1 M
Adjust the pH with dilute sodium hydroxide solution R and sodium hydroxide.
dilute to 500.0 mL with water R. Buffer Solution pH 11
Buffer Solution pH 3.0 Dissolve 6.21 g of bone acid R, 4.00 g of sodium hydroxide R
Dissolve 21.0 g of citric acid monohydrate R in 200 mL of and 3.70 g of potassium chloride R in 500 mL of water Rand
1 M sodium hydroxide and dilute to 1000 mL with water R. dilute to 1000 mL with the same solvent.
Dilute 40.3 mL of this solution to 100.0 mL with 0.1 M Buffered Salt Solution pH 7.2
hydrochloric acid. Dissolve in water R 8.0 g of sodium chloride R, 0.2 g of
Buffer Solution pH 3.5 potassium chloride R, 0.1 g of anhydrous calcium chloride R,
Dissolve 25.0 g of ammonium acetate R in 25 mL of water R 0.1 g of magnesium chloride R, 3.18 g of disodium hydrogen
and add 38.0 mL of hydrochloric acid Rl. Adjust the pH if phosphate dodecahydrate Rand 0.2 g of potassium dihydrogen
necessary with dilute hydrochloric acid R or dilute ammonia Rl. phosphate Rand dilute to 1000.0 mL with water R.
Dilute to 100.0 mL with water R. Carbonate Buffer pH 9. 7
Buffer Solution pH 3. 7 Dissolve 8.4 g of sodium hydrogen carbonate and 10.6 g of
To 15.0 mL of acetic acid R add 60 mL of ethanol sodium carbonate in sufficient water to produce 500 mL.
(96 per cent) R and 20 mL of water R. Adjust to pH 3. 7 by Chloride Buffer pH 2.0, 0.lM Buffer solution pH 2.0
the addition of ammonia R. Dilute to 100.0 mL with water R. Dissolve 6.57 g of potassium chloride R in water R and add
Buffer Solution pH 5.2 119. 0 mL of 0.1 M hydrochloric acid. Dilute to 1000. 0 mL
Dissolve 1. 02 g of potassium hydrogen phthalate R in 30. 0 mL with water R.
of 0.1 M sodium hydroxide. Dilute to 100.0 mL with water R. Citrate Buffer Solution pH 5.0
Buffer Solution pH 5.5 Prepare a solution containing 20.1 g/L of citric acid
Dissolve 54.4 g of sodium acetate R in 50 mL of water R, monohydrate R and 8.0 g/L of sodium hydroxide R. Adjust the
heating to 35 "C if necessary. After cooling, slowly add pH with dilute hydrochloric acid R.
10 mL of anhydrous acetic acid R. Shake and dilute to Citrate Buffer Solution pH 3.0, 0.25M
100.0 mL with water R. Dissolve 5.3 g of citric acid monohydrate R in 80 mL of
Buffer Solution pH 6.5 water R. Adjust the pH with 1 M sodium hydroxide and dilute
Dissolve 60.5 g of disodium hydrogen phosphate to 100.0 mL with water R.
dodecahydrate R and 46 g of potassium dihydrogen phosphate R Citro-phosphate Buffer pH 4.5
in water R. Add 100 mL of 0. 02 M sodium edetate and 20 mg To 30 volumes of 0.2M disodium hydrogen orthophosphate add
of mercun·c chloride R and dilute to 1000. 0 mL with water R. sufficient 0.lM citric acid to give a pH of 4.5 (about
Buffer Solution pH 6.6 36 volumes).
To 250.0 mL of 0.2 M potassium dihydrogen phosphate R add Citro-phosphate Buffer pH 5.0
89.0 mL of 0.2 M sodium hydroxide. Dilute to 1000.0 mL Mix 48.5 mL of 0.lM citric acid with sufficient 0.2M disodium
with water R. hydrogen orthophosphate to produce 100 mL.
Buffer Solution pH 7 .0 Citro-phosphate Buffer pH 6.0 Phosphate buffer
To 1000 mL of a solution containing 18 g!L of disodium solution pH 6.0
hydrogen phosphate dodecahydrate R and 23 g/L of sodium Mix 63.2 mL of a 71.5 g/L solution of disodium hydrogen
chloride R add sufficient (about 280 mL) of a solution phosphate dodecahydrate Rand 36.8 mL of a 21 g/L solution
containing 7 .8 g!L of sodium dihydrogen phosphate R and of citn'c acid monohydrate R.
23 g/L of sodium chloride R to adjust the pH. Dissolve in the
Citro-phosphate Buffer pH 6.5
solution sufficient sodium azide R to give a 0.2 g/L solution.
Mix 29.0 mL of 0. lM citric acid with sufficient 0.2M disodium
Buffer Solution pH 7 .2 hydrogen orthophosphate to produce 100 mL.
To 250.0 mL of 0.2 M potassium dihydrogen phosphate R add
175.0 mL of 0.2 M sodium hydroxide. Dilute to 1000.0 mL
with water R. Adjust the pH if necessary.
2023 Appendix I D V-A 177

Citro-phosphate Buffer pH 6.8 Phosphate buffer Glycine Buffer Solution

solution pH 6.8 Mix 42 g of sodium hydrogen carbonate and 50 g of potassium
Mix 77 .3 mL of a 71.5 g/L solution of disodium hydrogen hydrogen carbonate with 180 mL of water and add a solution
phosphate dodecahydrate R with 22.7 mL of a 21 g/L solution containing 37.5 g of glycine and 15 mL of 13.5M ammonia in
of citric acid monohydrate R. 180 mL of water. Dilute to 500 mL with water and stir until
Citro-phosphate Buffer pH 7.0 Phosphate buffer solution is complete.
solution pH 7.0 Guanidine-tris(hydroxymethyl)aminomethane Buffer
Mix 82.4 mL of a 71.5 g/L solution of disodium hydrogen Solution pH 8.3
phosphate dodecahydrate R with 17. 6 mL of a 21 g/L solution Dissolve 1.21 g of tris(hydroxymethyl)aminomethane R in
of citric acid monohydrate R. 87.5 mL of a 764 g/L solution of guanidine hydrochloride R.
Citro-phosphate Buffer pH 7.2 Phosphate buffer Adjust to pH 8.3 with hydrochloric acid R and dilute to
solution pH 7.2 100 mL with water R.
Mix 87 .0 mL of a 71.5 g/L solution of disodium hydrogen Guanidine-tris(hydroxymethyl)aminomethane-EDTA
phosphate dodecahydrate R with 13.0 mL of a 21 g/L solution Buffer Solution pH 8.5
of citric acid monohydrate R. Dissolve 1.0 g of sodium edetate R, 12.1 g of
Citro-phosphate Buffer pH 7.6 tris(hydroxymethyl)aminomethane Rand 57.0 g of guanidine
Dissolve 67.1 g of disodium hydrogen orthophosphate and hydrochloride R in 35 mL of water R. Adjust to pH 8.5 with
1.33 g of citric acid in sufficient water to produce 1000 mL.
hydrochloric acid R and dilute to 100 mL with water R.
Copper Sulfate Solution pH 2.0 Guanidine-tris(hydroxymethyl)aminomethane-EDTA
Buffer Solution pH 8.6
Copper Sulphate Solution pH 2.0.
Dissolve 0.018 g of sodium edetate R, 2.2 g of
Mix together 53 mL of 0.2M hydrochloric acid, 250 mL of tris(hydroxymethyl)aminomethane Rand 28.7 g of guanidine
0.2M potassium chloride and 40 mL of a 0.393% w/v solution hydrochloride R in 20 mL of water R. Adjust to pH 8.6 with
of cof)per(II) sulfate and dilute with water to 1000 mL.
acetic acid R and dilute to 50 mL with water R.
Copper Sulfate Solution pH 4.0, Buffered lmidazole Buffer Solution pH 6.5
Dissolve 0.25 g of copper sulfate pentahydrate Rand 4.5 g of
Dissolve 6.81 g of imidazole R, 1.23 g of magnesium sulfate R
ammonium acetate R in dilute acetic acid R and dilute to and 0.73 g of calcium sulfate R in 752 mL of 0.1 M
100.0 mL with the same solvent. hydrochloric acid. Adjust the pH if necessary and dilute to
Copper Sulfate Solution pH 5.2, Buffered 1000.0 mL with water R.
Copper Sulphate Solution pH 5.2, Buffered Imidazole Buffer Solution pH 7.3
Dissolve 15.22 g of anhydrous disodium hydrogen Dissolve 3.4 g of imidazole R and 5.8 g of sodium chloride R in
orthophosphate in sufficient water to produce 536 mL and add water R, add 18.6 mL of 1 M hydrochloric acid and dilute to
a 2.1 % w/v solution of citric acid until the pH of the solution 1000.0 mL with water R. Adjust the pH if necessary.
is between 5.15 and 5.25 (about 464 mL). Mix 985 mL of
lM Morpholinoethanesulfonate Buffer Solution
the resulting solution with 15 mL of a 0.393% w/v solution pH6.0
of copper(II) sulfate.
Dissolve 48.8 g of 2-[N-morpholino}ethanesulfonic acid R in
Deuterated Sodium Phosphate Buffer Solution pH 5.0, 160 mL of water Rand add 25 mL of 2 M sodium
0,2M
hydroxide R. Adjust to pH 6.0 with 2 M sodium hydroxide R.
Dissolve 2.76 g of sodium dihydrogen phosphate monohydrate R Dilute to almost 250 mL with water R. Adjust the pH, if
in 90 mL of deuterium oxide R, adjust the pH with a necessary, with 2 M sodium hydroxide R and dilute to
deuterated solution of phosphoric acid R or a deuterated 1 M 250.0 mL with water R.
solution of sodium hydroxide R, dilute to 100 mL with
Octylamine Phosphate Buffer pH 3.0
deuterium oxide R and mix.
Dilute 3.32 mL of ocrylamine to 1000 mL, adjust the pH to
Diethanolamine Buffer Solution pH 10.0 3.0 using orthophosphoric acid and filter through a membrane
Dissolve 96.4 g of diethanolamine R in water Rand dilute to filter with a nominal pore size not greater than 0.5 µm.
400 mL with the same solvent. Add 0.5 mL of an 186 g/L Phosphate Buffers
solution of magnesium chloride R and adjust the pH with 1 M
Solutions from pH 5.8 to pH 8.0 may be prepared by mixing
hydrochloric acid. Dilute to 500.0 mL with water R.
50 mL of 0.2M potassium dihydrogen orthophosphate with the
Diethylammonium Phosphate Buffer Solution pH 6.0 quantities of 0.2M sodium hydroxide VS specified in the
Dilute 68 mL of phosphoric acid R to 500 mL with water R. following table and diluting to 200 mL with water.
To 25 mL of this solution add 450 mL of water R and 6 mL At 20c the solutions may be used as solutions of standard
of diethylamine R, adjust to pH 6 ± 0.05, if necessary, using pH.
diethylamine R or phosphoric acid Rand dilute to 500.0 mL
with water R.
pH 5.8 6.0 6.2 6.4 6.6 6.8
Glycine Buffer pH 2.9
ml of0.2M sodium 3.72 5.70 8.60 12.60 17.80 23.65
Dissolve 6.0 g of glycine and 4.68 g of sodium chloride in
hydroxide VS
10 litres of water. Adjust the pH with lM hydrochloric acid
(about 30 mL). pH 7.0 7.2 7.4 7.6 7.8 8.0
Glycine Buffer pH 11.3 ml of 0.2M sodium 29.63 35.00 39.50 42.80 45.20 46.80
hydroxide VS
Mix a solution containing 0.75% w/v of glycine and
0.58% w/v of sodium chloride with an equal volume of
0.lM sodium hydroxide and adjust the pH if necessary.
V-A178 Appendix ID 2023

Phosphate Buffer pH 3.0 Phosphate Buffer pH 7 .5, 0,2M

Dissolve 34 g of potassium dihydrogen orthophosphate in Dissolve 27 .22 g of potassium dihydrogen phosphate R in
250 mL of water and adjust the pH of the solution to 3.0 930 mL of water R, adjust to pH 7.5 with a 300 g/L solution
with orthophosphoric acid. of potassium hydroxide Rand dilute to 1000.0 mL with
Phosphate Buffer pH 3.5 Phosphate buffer solution water R.
pH 3.5 Phosphate Buffer pH 10, Mixed
Dissolve 68.0 g of potassium dihydrogen phosphate R in water R To 100 mL of 0.2M disodium hydrogen orthophosphate add
and dilute to 1000.0 mL with the same solvent. Adjust the 6.0 mL of 0.25M trisodium orthophosphate.
pH with phosphoric acid R. Phosphate Buffer, 0.025M Standard
Phosphate Buffer pH 4.0 Dissolve 3.40 g of potassium dihydrogen orthophosphate and
Dissolve 6.8 g of potassium dihydrogen orthophosphate in 3.55 g of anhydrous disodium hydrogen orthophosphate, both
700 mL of water, adjust the pH, if necessary, with a 10% v/v previously dried at 110° to 130° for 2 hours, in sufficient
solution of orthophosphoric acid and add sufficient water to water to produce 1000 mL.
produce 1000 mL. Phosphate Buffer Solution pH 2.0
Phosphate Buffer pH 4.0, Mixed Dissolve 8.95 g of disodium hydrogen phosphate
Dissolve 5.04 g of disodium hydrogen orthophosphate and dodecahydrate Rand 3.40 g of potassium dihydrogen
3.01 g of potassium dihydrogen orthophosphate in sufficient phosphate R in water Rand dilute to 1000.0 mL with the
water to produce 1000 mL and adjust the pH with glacial same solvent. If necessary adjust the pH with phosphoric
acetic acid. acid R.
Phosphate Buffer pH 4. 75 0.125M Phosphate Buffer Solution pH 2.0
Dilute 100 mL of0.5M potassium dihydrogen orthophosphate to Dissolve 17 .0 g of potassium dihydrogen phosphate R and
800 mL with water, adjust to pH 4.75 with 0.lM sodium 17.8 g of anhydrous disodium hydrogen phosphate R in water R
hydroxide and dilute to 1000 mL with water. and dilute to 1.0 L with the same solvent. If necessary adjust
Phosphate Buffer pH 4.9 the pH with phosphoric acid R.
Dissolve 40 g of sodium dihydrogen orthophosphate and 1.2 g of Phosphate Buffer Solution pH 2.5, 0.2M
sodium hydroxide in sufficient water to produce 100 mL. Dissolve 27.2 g of potassium dihydrogen phosphate R in about
If necessary, adjust the pH with lM sulfuric acid or lM sodium 900 mL of water R, adjust to pH 2.5 with phosphoric acid R
hydroxide as required. and dilute to 1.0 L with water R.
Phosphate Buffer pH 5.4, Mixed Phosphate Buffer Solution pH 2.8
Dissolve 1.76 g of disodium hydrogen orthophosphate and Dissolve 7 .8 g of sodium dihydrogen phosphate R in 900 mL of
13.61 g of potassium dihydrogen orthophosphate in sufficient water R, adjust to pH 2.8 with phosphoric acid R and dilute to
water to produce 1000 mL. Adjust the pH with 1000 mL with the same solvent.
0.05M orthophosphoric acid, if necessary. Phosphate Buffer Solution pH 3.0
Phosphate Buffer pH 6.8, Mixed Mix 0. 7 mL of phosphoric acid R with 100 mL of water R.
Dissolve 28.80 g of disodium hydrogen orthophosphate and Dilute to 900 mL with the same solvent. Adjust to pH 3.0
11.45 g of potassium dihydrogen orthophosphate in sufficient with strong sodium hydroxide solutwn R and dilute to 1000 mL
water to produce 1000 mL. with water R.
Phosphate Buffer pH 6.8, 0.2M Mixed Phosphate buffer Phosphate Buffer Solution pH 3.0, 0.lM
solution pH 6.8 Rl Dissolve 12.0 g of anhydrous sodium dihydrogen phosphate R in
To 51.0 mL of a 27 .2 g/L solution of potassium dihydrogen water R, adjust the pH with dilute phosphoric acid RJ and
phosphate R add 49.0 mL of a 71.6 g/L solution of disodium dilute to 1000 mL with water R.
hydrogen phosphate dodecahydrate R. Adjust the pH if Phosphate Buffer Solution pH 3.0 Rl
necessary.
Dissolve 3.40 g of potassium dihydrogen phosphate R in
Storage: at 2 °C to 8 °C. 900 mL of water R. Adjust to pH 3.0 with phosphoric acid R
Phosphate Buffer pH 7.0, Mixed and dilute to 1000.0 mL with water R.
Dissolve 0.50 g of anhydrous disodium hydrogen orthophosphate Phosphate Buffer Solution pH 3.2
and 0.301 g of potassium dihydrogen orthophosphate in To 900 mL of a 4 g/L solution of sodium dihydrogen
sufficient water to produce 1000 mL. phosphate R, add 100 mL of a 2.5 g/L solution of phosphoric
Phosphate Buffer pH 7.o, 0.067M Mixed 0.067M acid R. Adjust the pH if necessary.
Phosphate buffer solution pH 7.0 Phosphate Buffer Solution pH 3.2 Rl
Dissolve 0.908 g of potassium dihydrogen phosphate R in Adjust a 35.8 g/L solution of disodium hydrogen phosphate
water Rand dilute to 100.0 mL with the same solvent dodecahydrate R to pH 3.2 with dilute phosphoric acid R.
(solution A). Dissolve 2.38 g of disodium hydrogen phosphate Dilute 100.0 mL of the solution to 2000.0 mL with water R.
dodecahydrate R in water R and dilute to 100.0 mL with the Phosphate Buffer Solution pH 3.25
same solvent (solution B). Mix 38. 9 mL of solution A and
61.1 mL of solution B. Adjust the pH if necessary. Dissolve about 1.36 g of potassium dihydrogen phosphate R in
1000 mL of water Rand adjust to pH 3.25 ± 0.05 with
Phosphate Buffer pH 7.0, 0.lM Mixed 0.lM Phosphate dilute phosphoric acid R. Filter through a membrane filter
buffer solution pH 7.0
(nominal pore size 0.45 µm or finer).
Dissolve 1.361 g of potassium dihydrogen phosphate R in
water Rand dilute to 100.0 mL with the same solvent.
Adjust the pH using a 35 g/L solution of disodium hydrogen
phosphate dodecahydrate R.
2023 Appendix ID V-Al 79

Phosphate Buffer Solution pH 3.4 using a 400 g/L solution of sodium hydroxide R. Dilute to
Dissolve 68.0 g of potassium dihydrogen phosphate R in water R 1000 mL with distilled water R.
and dilute to 1000.0 mL with the same solvent. Adjust the Phosphate Buffer Solution pH 6.7, 0.lM
pH with phosphoric acid R. Dissolve 15.6 g of sodium dihydrogen phosphate R in water R
Phosphate Buffer Solution pH 4.5, 0.05M and dilute to 1.0 L with the same solvent. Dissolve 17.8 g of
Dissolve 6.80 g of potassium dihydrogen phosphate R in disodium hydrogen phosphate dihydrate R in water R and dilute
1000.0 mL of water R. The pH of the solution is 4.5. to 1.0 L with the same solvent. Mix the solutions, check the
pH and if necessary adjust to pH 6.7.
Phosphate Buffer Solution pH 5.0
Phosphate Buffer Solution pH 7.0 Rl
Dissolve 2.72 g of potassium dihydrogen phosphate R in
800 mL of water R. Adjust the pH with a 1 M potassium Mix 250.0 mL of 0.2 M potassium dihydrogen phosphate Rand
hydroxide solution prepared from potassium hydroxide R and 148.2 mL of a 8 g/L solution of sodium hydroxide R, adjust
dilute to 1000 mL with water R. the pH if necessary. Dilute to 1000.0 mL with water R.
Phosphate Buffer Solution pH 5.4, 0.067M Phosphate Buffer Solution pH 7 .0 R2
Mix appropriate volumes of a 23.99 g/L solution of disodium Mix 50.0 mL of a 136 g/L solution of potassium dihydrogen
hydrogen phosphate dodecahydrate R with a 9 .12 g/L solution of phosphate R with 29.5 mL of 1 M sodium hydroxide and dilute
sodium dihydrogen phosphate monohydrate R to obtain pH 5.4. to 100.0 mL with water R. Adjust the pH to 7.0 ± 0.1.
Phosphate Buffer Solution pH 5.5 Phosphate Buffer Solution pH 7 .0 R3
Dissolve 13.61 g of potassium dihydrogen phosphate R in Dissolve 5 g of potassium dihydrogen phosphate R and 11 g of
water Rand dilute to 1000.0 mL with the same solvent dipotassium hydrogen phosphate R in 900 mL of water R.
(solution A). Dissolve 35.81 g of disodium hydrogen phosphate Adjust to pH 7.0 with dilute phosphoric acid R or dilute sodium
dodecahydrate R in water R and dilute to 1000.0 mL with the hydroxide solution R. Dilute to 1000 mL with water R and
same solvent (solution B). Mix 96.4 mL of solution A and mix.
3.6 mL of solution B. Phosphate Buffer Solution pH 7.0 R4
Phosphate Buffer Solution pH 5.6 Dissolve 28.4 g of anhydrous disodium hydrogen phosphate R
Dissolve O. 908 g of potassium dihydrogen phosphate R in and 18.2 g of potassium dihydrogen phosphate R in water R and
water Rand dilute to 100.0 mL with the same solvent dilute to 500 mL with the same solvent.
(solution A). Dissolve 1.161 g of dipotassium hydrogen Phosphate Buffer Solution pH 7.0 R5
phosphate R in water Rand dilute to 100.0 mL with the same Dissolve 28.4 g of anhydrous disodium hydrogen phosphate R in
solvent (solution B). Mix 94.4 mL of solution A and 5.6 mL 800 mL of water R. Adjust the pH using a 30 per cent mlm
of solution B. If necessary, adjust to pH 5.6 using solution A solution of phosphoric acid R and dilute to 1000 mL with
or solution B. water R.
Phosphate Buffer Solution pH 5.8 Phosphate Buffer Solution pH 7.0 R6
Dissolve 1.1 9 g of disodium hydrogen phosphate dihydrate R Dissolve 3.56 g of disodium hydrogen phosphate dihydrate R in
and 8.25 g of potassium dihydrogen phosphate R in water R and 950 mL of water for chromatography R. Adjust the pH with
dilute to 1000.0 mL with the same solvent. phosphoric acid R and dilute to 1.0 L with water for
Phosphate Buffer Solution pH 6.0 Rl chromatography R.
Dissolve 6.8 g of sodium dihydrogen phosphate R in water R Phosphate Buffer Solution pH 7.0 R7
and dilute to 1000.0 mL with water R. Adjust the pH with Dissolve 35 g of dipotassium hydrogen phosphate R in 900 mL
strong sodium hydroxide solution R. of water R, adjust to pH 7. 0 with dilute phosphoric acid R and
Phosphate Buffer Solution pH 6.0 R2 dilute to 1.0 L with water R.
To 250.0 mL of 0.2 M potassium dihydrogen phosphate R add Phosphate Buffer Solution pH 7 .0, 0.025M
28.5 mL of 0.2 M sodium hydroxide and dilute to 1000.0 mL Mix 1 volume of 0. 063 M phosphate buffer solution pH 7. 0 R
with water R. with 1.5 volumes of water R.
Phosphate Buffer Solution pH 6.3, 0.lM Phosphate Buffer Solution pH 7.0, 0.03M
Dissolve 15.6 g of sodium dihydrogen orthophosphate in Dissolve 5.2 g of dipotassium hydrogen phosphate R in 900 mL
900 mL of water, adjust the pH to 6.3 with O.lM sodium of water for chromatography R. Adjust the solution to
hydroxide and add sufficient water to produce 1000 mL. pH 7 .0 ± 0.1 using phosphoric acid R and dilute to 1000 mL
Phosphate Buffer Solution pH 6.4 with water for chromatography R.
Dissolve 2.5 g of disodium hydrogen phosphate dodecahydrate R, Phosphate Buffer Solution pH 7 .0, 0.05M
2.5 g of sodium dihydrogen phosphate R and 8.2 g of sodium Mix 34 mL of water R and 100 mL of 0. 067 M phosphate
chloride R in 950 mL of water R. Adjust the pH of the buffer solution pH 7. 0 R.
solution to 6.4 with 1 M sodium hydroxide or 1 M hydrochloric Phosphate Buffer Solution pH 7.0, 0.063M
acid, if necessary. Dilute to 1000.0 mL with water R.
Dissolve 5.18 g of anhydrous disodium hydrogen phosphate R
Phosphate Buffer Solution pH 6.5 and 3.65 g of sodium dihydrogen phosphate monohydrate R in
Dissolve 2.75 g of sodium dihydrogen phosphate Rand 4.5 g of 950 mL of water R and adjust the pH with phosphoric acid R;
sodium chloride R in 500 mL of water R. Adjust the pH with dilute to 1000.0 mL with water R.
phosphate buffer solution pH 8.5 R. Phosphate Buffer Solution pH 7.4
Phosphate Buffer Solution pH 6.5, 0.lM Add 250.0 mL of 0.2 M potassium dihydrogen phosphate R to
Dissolve 13.80 g of sodium dihydrogen phosphate 393.4 mL of 0.1 M sodium hydroxide.
monohydrate R in 900 mL of distilled water R. Adjust the pH
V-AISO Appendix ID 2023

Phosphate Buffer Solution pH 7 .5, 0.05M Potassium Phosphate Buffer Solution pH 7.0
Dissolve 0.89 g of disodium hydrogen phosphate dihydrate R in Dissolve 10 mg of bovine albumin R and 68 mg of potassium
about 80 mL of water R. Adjust to pH 7.5 with an dihydrogen phosphate R in 30 mL of water R. If necessary,
8.5 per cent V/V solution of phosphoric acid Rand dilute to adjust to pH 7 .0 with potassium hydroxide R. Dilute to 50 mL
100.0 mL with water R. with water Rand filter.
Phosphate Buffer Solution pH 7 .5, 0.33M Saline pH 6.4, Phosphate-buffered
Dissolve 119. 31 g of disodium hydrogen phosphate Dissolve 1.79 g of disodium hydrogen orthophosphate, 1.36 g of
dodecahydrate R in water Rand dilute to 1000.0 mL with the potassium dihydrogen orthophosphate and 7 .02 g of sodium
same solvent (solution A). Dissolve 45.36 g of potassium chloruie in sufficient water to produce 1000 mL.
dihydrogen phosphate R in water Rand dilute to 1000.0 mL Saline pH 6.8, Phosphate-buffered
with the same solvent (solution B). Mix 85 mL of solution A
Dissolve 1.0 g of potassium dihydrogen phosphate R, 2.0 g of
and 15 mL of solution B. Adjust the pH if necessary.
dipotassium hydrogen phosphate R and 8.5 g of sodium
Phosphate Buffer Solution pH 8.0, 0.02M chloruie R in 900 mL of water R, adjust the pH if necessary
To 50.0 mL of 0.2 M potassium dihydrogen phosphate R add and dilute to 1000.0 mL with the same solvent.
46.8 mL of 0.2 M sodium hydroxuie. Dilute to 500.0 mL with Saline pH 7.2, Phosphate-albumin Buffered
water R. Dissolve 10. 75 g of disodium hydrogen phosphate
Phosphate Buffer Solution pH 8.0, 0.lM dodecahydrate R, 7.6 g of sodium chloruie Rand 10 g of bovine
Dissolve 0.523 g of potassium dihydrogen phosphate Rand albumin R in water Rand dilute to 1000.0 mL with the same
16. 73 g of dipotassium hydrogen phosphate R in water R and solvent. Immediately before use adjust the pH using dilute
dilute to 1000.0 mL with the same solvent. sodium hydroxuie solution R or dilute phosphoric acid R.
Phosphate Buffer Solution pH 8.0, 0.05M Saline pH 7.2 Rl, Phosphate-albumin Buffered
Dissolve 0.31 g of sodium dihydrogen phosphate R in 70 mL of Dissolve 10.75 g of disodium hydrogen phosphate
water R and adjust to pH 8.0 with 1 M sodium hydroxuie, dodecahydrate R, 7 .6 g of sodium chloruie R and 1 g of bovine
then dilute to 100 mL with water R. albumin R in water Rand dilute to 1000.0 mL with the same
Phosphate Buffer Solution pH 8.0, lM solvent. Immediately before use adjust the pH using dilute
Dissolve 136.1 g of potassium dihydrogen phosphate R in sodium hydroxuie solution R or dilute phosphoric acid R.
water R, adjust the pH with 1 M sodium hydroxide. Dilute to Saline pH 7.4, Phosphate-buffered
1000.0 mL with water R. Dissolve 2.38 g of disodium hydrogen phosphate
Phosphate Buffer Solution pH 8.5 dodecahydrate R, 0 .1 9 g of potassium dihydrogen phosphate R
Dissolve 3.5 g of dipotassium hydrogen phosphate Rand 4.5 g and 8.0 g of sodium chloride R in water. Dilute to 1000.0 mL
with the same solvent. Adjust the pH if necessary.
of sodium chloruie R in 500 mL of water R. Adjust the pH
with a mixture of equal volumes of dilute phosphoric acid R Sodium Acetate Buffer pH 7.0.
and water R. Dissolve 1.64 g of anhydrous sodium acetate in 1 L of water,
Phosphate Buffer Solution pH 9.0 adjust to pH 7 .0 using dilute acetic acid
Dissolve 1. 7 4 g of potassium dihydrogen phosphate R in 80 mL Sodium Acetate Buffer Solution pH 4.0, 0.lM
of water R, adjust the pH with a 1 M potassium hydroxide Dissolve 822 mg of sodium acetate R in 100 mL of water R
solution prepared from potassium hydroxide R and dilute to (solution A). Dilute 1.44 mL of glacial acetic acid R in
100.0 mL with water R. 250 mL of water R (solution B).Titrate 100 mL of
Phosphate Buffer Solution pH 11.3, 0.lM solution B using about 20 mL of solution A.
Dissolve 17. 4 g of dipotassium hydrogen phosphate R in about Sodium Acetate Buffer Solution pH 4.5
950 mL of water R, adjust to pH 11.3 using a 100 g/L Dissolve 63 g of anhydrous sodium acetate R in water R, add
solution of potassium hydroxide R and dilute to 1.0 L with 90 mL acetic acid Rand adjust to pH 4.5, and dilute to
water R. Filter through a membrane filter (nominal pore size 1000 mL with water R.
0.45 µm). Sodium Acetate Buffer Solution pH 5.0
Phosphate-Citrate Buffer Solution pH 5.5 Dissolve 50.0 g of sodium acetate R in 10.0 mL of glacial
Mix 56.85 mL of a 28.4 g/L solution of anhydrous disodium acetic acid Rand add water R. Adjust to pH 5.0 with a
hydrogen phosphate R and 43.15 mL of a 21 g/L solution of 4.2 g/L solution of sodium hydroxide R or with glacial acetic
citric acid monohydrate R. acid Rand dilute to 1000.0 mL with water R.
Phthalate Buffer pH 3.6 Buffer solution pH 3.6 Sodium Acetate Solution pH 6.0, Buffered
To 250.0 mL of 0.2 M potassium hydrogen phthalate R add Dissolve 4.1 g of anhydrous sodium acetate in 1000 mL of
11.94 mL of 0.2 M hydrochloric acui. Dilute to 1000.0 mL water and adjust the pH to 6.0 with glacial acetic acid.
with water R. Sodiumlcalcium Acetate Buffer Solution pH 7.0
Phthalate Buffer Solution pH 4.4 Dissolve 10 mg of bovine albumin R and 32 mg of calcium
Dissolve 2.042 g of potassium hydrogen phthalate R in 50 mL acetate R in 60 mL of water R. Add 580 µL of glacial acetic
of water R, add 7.5 mL of 0.2 M sodium hydroxuie and dilute acid R and adjust to pH 7 .0 with 2 M sodium hydroxuie R.
to 200.0 mL with water R. Dilute to 100 mL with water Rand filter.
Phthalate Buffer Solution pH 6.4, 0.5M Sodium Citrate Buffer Solution pH 7.8 (0.034M Sodium
Dissolve 100 g of potassium hydrogen phthalate R in water R Citrate, 0.101M Sodium Chloride)
and dilute to 1000.0 mL with the same solvent. Adjust the Dissolve 10.0 g of sodium citrate Rand 5.90 g of sodium
pH if necessary, using strong sodium hydroxide solution R. chloruie R in 900 mL of water R. Adjust the pH by addition
of hydrochloric acid R and dilute to 1000 mL with water R.
2023 Appendix ID V-A181

Sodium Phosphate Buffer Solution pH 7.5, 0.25M Tris-chloride Buffer pH 7.4 Tris(hydroxymethyl)
Dissolve 3.90 g of sodium dihydrogen phosphate R in 70 mL of aminomethane sodium chloride buffer solution pH 7.4
water R, adjust to pH 7.5 with a 300 g/L solution of sodium Dissolve 6.08 g of tris(hydroxymethyl)aminomethane R, 8.77 g
hydroxide R and dilute to 100 mL with water R. of sodium chloride R in 500 mL of distilled water R.
Sodium Phosphate Buffer Solution pH 8.0, 0.02M Add 10.0 g of bovine albumin R. Adjust the pH using
Dissolve 0.31 g of sodium dihydrogen phosphate R in 70 mL of hydrochloric acid R. Dilute to 1000.0 mL with distilled water R.
water R and adjust to pH 8.0 with J M sodium hydroxide, Tris-chloride Buffer pH 7.5 Tris(hydroxymethyl)
then dilute to 100 mL with water R. aminomethane buffer solution pH 7.5
Succinate Buffer Solution pH 4.6 Dissolve 7.27 g of tris(hydroxymethyl)aminomethane Rand
5.27 g of sodium chloride R in water R, and adjust the pH if
Disssolve 11.8 g of succinic acid Rina mixture of 600 mL of
necessary. Dilute to 1000.0 mL with water R.
water R and 82 mL of 1 M sodium hydroxide and dilute to
1000.0 mL with water R. Tris-chloride Buffer pH 7.5 Rl 0.05M Tris-
hydrochloride buffer solution pH 7.5
Sulfate Buffer Solution pH 2.00
Dissolve 6.057 g of tris(hydroxymethyl)aminomethane R in
Dissolve 132.1 g of ammonium sulfate R in water R and dilute
water R and adjust the pH with hydrochloric acid R. Dilute to
to 500.0 mL with the same solvent (Solution A). Carefully
1000.0 mL with water R.
and with constant cooling stir 14 mL of sulfuric acid R into
about 400 mL of water R; allow to cool and dilute to Tris-chloride Buffer pH 8.1 Tris(hydroxymethyl)
500.0 mL with water R (Solution B).Mix equal volumes of aminomethane buffer solution pH 8.1
solutions A and B. Adjust the pH if necessary. Dissolve 0.294 g of calcium chloride R in 40 mL of
Tetrabutylammonium Buffer Solution pH 7 .0 tris(hydroxymethyl)aminomethane solution Rand adjust the pH
with 1 M hydrochloric acid. Dilute to 100.0 mL with water R.
Dissolve 6.16 g of ammonium acetate Rina mixture of
15 mL of tetrabutylammonium hydroxide solution ( 400 g/L) R Tris-chloride Buffer pH 8.6
and 185 mL of water R. Adjust the pH with nitric acid R. Dissolve 2.0 g of tris(hydroxymethyl)methylamine and 2.4 g of
Thiobarbituric Acid-citrate Buffer sodium chloride in about 100 mL of water, adjust the pH to
8.6 with lM sodium hydroxide or lM hydrochloric acid and
Dissolve 5.0 g of thiobarbituric acid in 5 mL of 4M sodium
hydroxide and dilute to 500 mL with water. Dissolve dilute with water to 200 mL.
separately 37 g of sodium citrate in 32 mL of hydrochloric acid Tris-chloride Buffer Solution
and dilute to 250 mL with water. Mix the two solutions and Dissolve 0.606 g of tris(hydroxymethyl)methylamine and 2.34 g
adjust the pH of the resulting solution to 2.0. of sodium chloride in sufficient water to produce 1000 mL.
Total Ionic Strength Adjustment Buffer Store at 2° to 8° and use within 3 days of preparation.
Dissolve 58.5 g of sodium chloride R, 57.0 mL of glacial acetic Tris-EDTA Buffer Solution pH 8.0 MB
acid R, 61.5 g of sodium acetate Rand 5.0 g of Dissolve 60.57 g of tris(hydroxymethyl)aminomethane MB in
cyclohexylenedinitrilotetra-acetic acid R in water R and dilute to 500 mL of distilled water Rand adjust the pH (2.2.3) to 8.4
500.0 mL with the same solvent. Adjust to pH 5.0 to 5.5 using hydrochloric acid R (IM Tris buffer).
with a 335 g/L solution of sodium hydroxide R and dilute to Dissolve 18.60 g of ethylenediaminetetra-acetic acid R
1000.0 mL with distilled water R. inl00 mL of distilled water Rand adjust the pH (2.2.3) to 8.0
Total Ionic Strength Adjustment Buffer Rl using sodium hydroxide R (0.5M EDTA solution). (Use
Dissolve 210 g of citric acid monohydrate R in 400 mL of vigorous stirring and moderate heat if desired).
distilled water R. Adjust to pH 7.0 with concentrated Mix appropriate volumes of the two solutions to achieve the
ammonia R. Dilute to 1000.0 mL with distilled water R required concentration. (e.g. Mix 5 mL of lM Tris buffer
(solution A). Dissolve 132 g of ammonium phosphate R in with 1 mL of 0.SM EDTA solution and dilute with 496 mL
distilled water Rand dilute to 1000.0 mL with the same of distilled water R).
solvent (solution B). To a suspension of 292 g of Tris-EDTA Buffer pH 8.4 Tris(hydroxymethyl)
(ethylenedinitrilo)tetra-acetic acid R in about 500 mL of aminomethane-EDTA Buffer Solution pH 8.4
distilled water R, add about 200 mL of concentrated
Dissolve 5.12 g of sodium chloride R, 3.03 g of
ammonia R to dissolve. Adjust the pH to 6 to 7 with
tris(hydroxymethyl)aminomethane Rand 1.40 g of sodium
concentrated ammonia R. Dilute to 1000.0 mL with distilled
edetate R in 250 mL of distilled water R. Adjust the pH to 8.4
water R (solution C). Mix equal volumes of solution A, B,
using hydrochloric acid R. Dilute to 500.0 mL with distilled
and C and adjust to pH 7.5 with concentrated ammonia R.
water R.
Tris-acetate Buffer Solution pH 8.5 Tris-EDTA BSA Buffer Solution pH 8.4
Dissolve 0.294 g of calcium chloride Rand 12.11 g of
Dissolve 6.1 g of tris(hydroxymethyl)aminomethane R, 2.8 g of
tris(hydroxymethyl)aminomethane R in water R. Adjust the pH
sodium edetate R, 10.2 g of sodium chloride Rand 10 g of
with acetic acid R. Dilute to 1000.0 mL with water R. bovine albumin R in water R, adjust to pH 8.4 using 1 M
Tris-borate-EDTA Buffer Solution pH 8.4 MB hydrochloric acid and dilute to 1000.0 mL with water R.
Dissolve 10.80 g of tris(hydroxymethyl)aminomethane MB, Tris-glycine Buffer Solution pH 8.3
5.50 g boric acid Rand 0.584 g of ethylenediaminetetra-acetic
Dissolve 6.0 g of tris(hydroxymethyl)aminomethane Rand
acid R in 250 mL of distilled water R. Adjust the pH (2.2.3)
28.8 g of glycine R in water R and dilute to 1000.0 mL with
to 8.4 using hydrochloric acid R. Dilute to 1000.0 mL with
the same solvent. Dilute 1 volume to 10 volumes with
distilled water R.
water R immediately before use.
V-A182 Appendix I E 2023

Tris-hydrochloride Buffer Solution pH 6.8, lM Tris(hydroxymethyl)aminomethane Buffer Solution

Dissolve 60.6 g of tris(hydroxymethyl)aminomethane R in pH 9.0 Rl
400 mL of water R. Adjust the pH with hydrochwric acid R Dissolve 12.1 g of tris(hydroxymethyl)aminomethane R in
and dilute to 500.0 mL with water R. 950 mL of water R. Adjust to pH 9.0 with acetic acid Rand
Tris-hydrochloride Buffer Solution pH 7.5, 0.lM dilute to 1000. 0 mL with water R.
Dissolve 3.03 g of tris(hydroxymethyl)aminomethane R in Tris(hydroxymethyl)aminomethane-EDTA Buffer
200 mL of water R, adjust to pH 7.5 with hydrochloric acid R Solution pH 8.4 Rl
and dilute to 250 mL with water R. Dissolve 10.20 g of sodium chloride R, 6.10 g of
Tris-hydrochloride Buffer Solution pH 7.5, lM tris(hydroxymethyl)aminomethane R, 2.80 g of sodium edetate R
Dissolve 12.11 g of tris(hydroxymethyl)aminomethane R in and 1.00 g of macrogol 6000 R or 2.00 g of bovine albumin R
90 mL of water R, adjust to pH 7.5 with hydrochloric acid R or of human albumin R in 800 mL of water R. Adjust to
and dilute to 100.0 mL with water R. pH 8.4 with hydrochloric acid R and dilute to 1.0 L with
water R.
Tris-hydrochloride Buffer Solution pH 8.0
Tris(hydroxymethyl)aminomethane Sodium Chloride
Dissolve 1.21 g of tris(hydroxymethyl)aminomethane Rand Buffer Solution pH 7.4 Rl
29.4 mg of calcium chloride R in water R. Adjust the pH with
1 M hydrochloric acid and dilute to 100.0 mL with water R. Dissolve 0.1 g of bovine albumin Rina mixture containing
2 mL of tris(hydroxymethyl)aminomethane buffer solution
Tris-hydrochloride Buffer Solution pH 8.0, lM pH 7.4 Rand 50 mL of a 5.84 mgimL solution of sodium
Dissolve 121.1 g of tris(hydroxymethyl)aminomethane Rand chloride R. Dilute to 100.0 mL with water R.
1.4 7 g of calcium chloride R in 900 mL of water R. Adjust the Tris-sodium Acetate Buffer Solution pH 7.4
pH with hydrochloric acid Rand dilute to 1000.0 mL with
water R.
Dissolve 6.3 g of tris(hydroxymethyl)aminomethane Rand
4.9 g of anhydrous sodium acetate R in 900 mL of water R.
Tris-hydrochloride Buffer Solution pH 8.3 Adjust to pH 7.4 with suifuric acid R and dilute to 1000 mL
Dissolve 9.0 g of tris(hydroxymethyl)aminomethane R in 2.9 L with water R.
of water R. Adjust the pH with 1 M hydrochloric acid. Adjust Tris-sodium Acetate Buffer Solution pH 8.0
the volume to 3 L with water R.
Dissolve 6.3 g of tris(hydroxymethyl)aminomethane Rand
Tris-hydrochloride Buffer Solution pH 8.8, 1.5M 4.9 g of anhydrous sodium acetate R in 900 mL of water R.
Dissolve 90.8 g of tris(hydroxymethyl)aminomethane R in Adjust to pH 8.0 with sulfuric acid R and dilute to 1000 mL
400 mL of water R. Adjust the pH with hydrochwric acid R with water R.
and dilute to 500.0 mL with water R. Tris-sodium Acetate-sodium Chloride Buffer Solution
Tris-hydrochloride Buffer Solution pH 8.8, 3M pH7.4
Dissolve 363.3 g of tris(hydroxymethyl)aminomethane R in Dissolve 30.0 g of tris(hydroxymethyl)aminomethane R, 14.5 g
500 mL of water R. Adjust the pH with hydrochloric acid R of anhydrous sodium acetate Rand 14.6 g of sodium chloride R
and dilute to 1 L with water R. in 900 mL of water R. Add 0.50 g of bovine albumin R.
Tris-hydrochloride Buffer Solution pH 9.0, 0.05M Adjust to pH 7.4 with sulfuric acid R and dilute to 1000 mL
Dissolve 0.605 g of tris(hydroxymethyl)aminomethane R in with water R.
water R. Adjust the pH with 1 M hydrochloric acid and dilute Tris-sodium Acetate-Sodium Chloride Buffer Solution
to 100.0 mL with water R. pH8.0
Tris(hydroxymethyl)aminomethane Buffer Solution Dissolve 30.0 g of tris(hydroxymethyl)aminomethane R, 14.5 g
pH 7.4 of anhydrous sodium acetate Rand 14.6 g of sodium chloride R
Dissolve 30.3 g of tris(hydroxymethyl)aminomethane R in in 900 mL of water R. Add 0.50 g of bovine albumin R.
approximately 200 mL of water R. Add 183 mL of 1 M Adjust to pH 8.0 with suljuric acid Rand dilute to 1000 mL
hydrochloric acid. Dilute to 500.0 mL with water R. Note: the with water R.
pH is 7. 7-7.8 at room temperature and 7.4 at 37 °C. This
solution is stable for several months at 4 °C.
Tris(hydroxymethyl)aminomethane Buffer Solution
pH 7.5 Rl E. Reference Materials
Dissolve 1.21 g of tris(hydroxymethyl)aminomethane R in Where the letters BPCRS appear after the name of a
900 mL of water R and add 10 mL of 0.01 M calcium chloride substance in a test or assay, the British Pharmacopoeia
solution R. Adjust the pH if necessary with sodium hydroxide Chemical Reference Substance is to be used.
solution R or hydrochlon·c acid R, and dilute to 1000.0 mL A comprehensive and up-to-date list of British
with water R. Pharmacopoeia Chemical Reference Substances, together
Tris(hydroxymethyl)aminomethane Buffer Solution with terms of trade and supply, is available on our website at
pH 9.0 www.pharmacopoeia.com. The substances are obtainable
Dissolve 1.21 g of tris(hydroxymethyl)aminomethane R in from the BPCRS Sales Office, MHRA, 10th Floor, 10 South
950 mL of water for chromatography R. Adjust to pH 9.0 with Colonnade, Canary Wharf, London E14 4PU, United
acetic acid Rand dilute to 1000.0 mL with water for Kingdom (telephone +44 (0)20 3080 6561, e-mail:
chromatography R. bpcom@mhra.gov.uk, website www.pharmacopoeia.com).
In addition to BPCRS, monographs of the British
Pharmacopoeia may also refer to reference substances
available from other suppliers. These are denoted by the
letters CRS.
2023 Appendix IF V-A183

Where the letters CRS or EPCRS appear, the chemical occurrence of solvates, allotropy or occurrence of the
reference substance issued by the European Pharmacopoeia amorphous form.
Commission is to be used; where the letters ERP or EPBRP The identity of chemical composition implies that all
appear, the Biological Reference Preparation issued by the crystalline and amorphous forms of a given species have the
European Pharmacopoeia Commission is to be used; where same chemical behaviour in solution or as a melt; in contrast,
the letters HRS or EPHRS appear, the herbal reference their physico-chemical and physical characteristics (solubility,
substance issued by the European Pharmacopoeia hardness, compressibility, density, melting point, etc.), and
Commission is to be used. The substances, as well as therefore their reactivity and bioavailability may be different
European Pharmacopoeia infrared reference spectra, are at the solid state.
obtainable from the Council of Europe, European When a compound shows polymorphism, the form for which
Directorate for the Quality of Medicines & HealthCare, CRS
the free enthalpy is lowest at a given temperature and
Sales Team, 7 allee Kasmer, CS 30026, F-67081, Strasbourg
pressure is the most thermodynamically stable. The other
Cedex, France (facsimile +33 (0)3 88 41 27 71, e-mail:
forms are said to be in a metastable state. At normal
crs@pheur.org, website www.edqm.eu).
temperature and pressure, a metastable form may remain
Other sources of specific reference substances are shown unchanged or may change to a thermodynamically more
below. stable form.
Astragaloside I CRS, Astragaloside II CRS, If there are several crystalline forms, one form is
Astragaloside IV CRS, azadirachtin A CRS, bacopaside thermodynamically more stable at a given temperature and
I CRS, bacopaside II CRS, bacoside A CRS, pressure. A given crystalline form may constitute a phase that
Paeonol CRS, Rosmarinic acid CRS, salannin CRS, can reach equilibrium with other solid phases and with the
Salvianolic Acid B CRS, Tanshinone IIA CRS, liquid and gas phases.
Withaferin A CRS, Withanolide A CRS, Withanolide
If each crystalline form is the more stable within a given
B CRS, Z-Ligustilide CRS may be obtained from
temperature range, the change from one form to another is
Chromadex Inc. through LGC Standards, Queen's Road,
reversible and is said to be enantiotropic. The change from
Teddington, TWl 1 0LY, United Kingdom (telephone +44
one phase to another is a univariate equilibrium, so that at a
(0)20 8943 8480, facsimile +44 (0)20 8943 7554, e-mail: given pressure this state is characterised by a transition
uksales@lgcstandards. corn).
temperature. However, if only one of the forms is stable over
Opacity, Standard Preparation of The Standard the entire temperature range, the change is irreversible or
Preparation is the 5th International Reference Preparation, monotropic.
established in 197 5, and consists of a rod of plastic
Different crystalline forms or solvates may be produced by
simulating the optical properties of a bacterial suspension (10 varying the crystallisation conditions (temperature, pressure,
Units of opacity). It may be obtained from the National
solvent, concentration, rate of crystallisation, seeding of the
Institute for Biological Standards and Control (NIBSC),
crystallisation medium, presence and concentration of
Blanche Lane, South Mimms, Potters Bar, Hertfordshire, impurities, etc.).
EN6 3QG, United Kingdom (telephone +44 (0)
1707 641000, e-mail: enquiries@nibsc.org). The following techniques may be used to study
polymorphism:
Piperonyl Butoxide CRS may be obtained from LGC - X-ray diffraction of powders (2. 9.33),
Standards, Queen's Road, Teddington, TWl 1 0LY, United
- X-ray diffraction of single crystals,
Kingdom (telephone +44 (0)20 8943 8480, facsimile +44
- thermal analysis (2.2.34) (differential scanning
(0)20 8943 7554, e-mail: uksales@lgcstandards.com).
calorimetry, thermogravimetry, thermomicroscopy),
- microcalorimetry,
- moisture absorption analysis,
- optical and electronic microscopy,
F. Polymorphism - solid-state nuclear magnetic resonance,
(Ph. Bur. method 5.9) - infrared absorption spectrophotometry (2.2.24),
- Raman spectroscopy (2.2.48),
Polymorphism (or crystal polymorphism) is a phenomenon - measurement of solubility and intrinsic dissolution rate,
related to the solid state; it is the ability of a compound in - density measurement.
the solid state to exist in different crystalline forms having the
These techniques are often complementary and it is
same chemical composition. Substances that exist in a non-
indispensable to use several of them.
crystalline solid state are said to be amorphous.
Pressure/temperature and energy/temperature diagrams based
When this phenomenon is observed for a chemical element
on analytical data are valuable tools for fully understanding
(for example, sulfur), the term allotropy is used instead of
the energetic relationship (enantiotropism, monotropism) and
polymorphism.
the thermodynamic stability of the individual modifications of
The term pseudopolymorphism is used to describe solvates a polymorphic compound.
(including hydrates), where a solvent is present in the crystal
For solvates, differential scanning calorimetry and
matrix in stoichiometric proportions; the term may also be
thermogravimetry are preferable, combined with
extended to include compounds where the solvent is trapped
measurements of solubility, intrinsic dissolution rate and
in the matrix in variable proportions. However the term
X-ray diffraction.
pseudopolymorphism is ambiguous because of its use in
different circumstances. It is therefore preferable to use only For hydrates, water sorption/desorption isotherms are
the terms "solvates" and "hydrates". determined to demonstrate the zones of relative stability.
Where a monograph indicates that a substance shows In general, hydrates are less soluble in water than anhydrous
polymorphism, this may be true crystal polymorphism, forms, and likewise solvates are less soluble in their solvent
than unsolvated forms.
V-A184 Appendix II A 2023

Chemical analysis:
Appendix II - identification of active substances, excipients, dosage
forms, manufacturing intermediates, chemicals and
A. lnfrared Spectrophotometry packaging materials;
- quality assessment of active substances, excipients, dosage
(Absorption Spectrophotometry, Infrared, Ph. Bur. method forms, manufacturing intermediates and packaging
2.2.24) materials, including batch-to-batch spectral comparison
PRINCIPLE and supplier change assessment;
Infrared absorption spectrophotometry (also known as - quantification of active substances in a sample matrix,
infrared (IR) spectroscopy) is based on the interaction of determination of water and solvent content;
infrared radiation with matter. As a result of interaction - quantification of impurities, e.g. in gases, inorganic
between a molecule and IR radiation, absorption of materials;
frequencies specific to that molecule can occur, and some - reaction monitoring, e.g. chemical synthesis.
intermolecular and intramolecular vibrations can be excited Physical analysis:
to higher vibrational levels. This results in an infrared - determination of solid-state properties such as
absorption spectrum with characteristic bands that polymorphism.
correspond to the functional groups of the molecule.
LIMITATIONS
The infrared wavelength region can be further divided into 3 Notable limitations to the use ofIR spectroscopy include the
subregions, namely near-infrared, mid-infrared and far- following:
infrared. These subregions have wavelength ranges that are - it may be necessary to use additional techniques to
generally accepted by convention to be 0.8-2.5 µm, unambiguously identify a substance;
2.5-25 µm and 25-1000 µm respectively. However, in IR - pure enantiomers of a substance cannot be discriminated;
spectroscopy, wavenumber is more commonly used than - it may not be a suitable method for trace analysis;
wavelength, and can be calculated using the following - sample preparation conditions (e.g. pressure, solvent) may
equation: change the crystalline form of a substance that exhibits
polymorphism;
-
V = -1 · 104 - for heterogeneous samples, the limited sampling volume
/4 may be problematic.
where v is the wavenumber in reciprocal centimetres (cm- 1) MEASUREMENT MODES
and ), is the wavelength in micrometres. Thus IR measurements are based on passing radiation through or
12 500-4000 cm 1 is near-infrared, 4000-400 cm- 1 is mid- into a sample and measuring the attenuation of the emerging
infrared and 400-10 cm- 1 is far-infrared. beam at various wavelengths. This corresponds to 2 main
This chapter concerns only spectroscopy in the mid-infrared measurement modes, i.e. transmission and attenuated total
region, i.e. 4000-400 cm- 1 (2.5-25 µm), which hereafter is reflection (ATR). However, other modes also exist for
referred to as infrared for simplicity. This region is where the specific applications (e.g. diffuse and specular reflection).
fundamental molecular vibrations of functional groups appear TRANSMISSION MODE
in the spectrum as absorption bands. The region below This mode is based on determination of the transmittance
1500 cm- i is known as the 'fingerprint region', a very (1), namely the ability of the sample to transmit IR radiation
complex and informative part of the spectrum which at a given wavelength (wavenumber). It is defined by the
characterises the molecule being investigated. following ratio:
The mid-infrared region is flanked by the near-infrared
region, where overtones and combinations of fundamental I
T=-
vibrations, mainly C-H, N-H and 0-H functional groups, are Io
detected (2.2.40) and the far-infrared region, where
/0 intensity of incident radiation;
absorption bands associated with crystal lattice modes, J intensity of transmitted radiation.
hydrogen bonds, angle deformation vibrations of heavy atoms
and molecular rotations are observed. The resulting spectrum is presented in terms of transmittance
APPLICATIONS (1) on the y-axis versus wavelength or wavenumber on the
As the absorption bands in IR spectra are characteristic of x-axis. It can also be presented in terms of absorbance (A)
the constituent functional groups of a compound, IR on the y-axis, which is related to transmittance (1) by the
spectroscopy is widely used to identify substances and following equation:
provide information on their structure. It can also be used for
quantitative applications, which requires establishing a
mathematical relationship between the intensity of the
radiation absorbed by the sample and the concentration of
molar absorption coefficient of the sample, in square
the investigated component in the sample. a
centimetres per mole (cm2 -mor 1);
IR spectroscopy is widely used in the pharmaceutical field for sample thickness, in centimetres;
chemical and physical analysis in the laboratory, and has a sample concentration, in moles per cubic centimetre (mol-cm- 3).
wide variety of applications during the manufacturing process
as outlined below. IR spectroscopy thereby enables the ATTENUATED TOTAL REFLECTION MODE
application of Process Analytical Technology (PAT) as part ATR mode is based on the phenomenon of total internal
of an advanced control strategy. reflection. The sample, with a refractive index n 2 , is brought
into close contact with a crystal (diamond, germanium, zinc
selenide or any other suitable material), having a refractive
index n 1 which is greater than n 2 • A beam of IR light is then
2023 Appendix II A V-A185

passed through the crystal. When the angle a between the Table 2.2.24.-.1 - Band positions and associated acceptable
incident beam and the sample-crystal interface exceeds a tolerances of the pdystyrene film used to verify wavenumber
critical value x0 theoretically all of the radiation is reflected accuracy
(total internal reflection). However, an evanescent wave is
Band position (cm- 1)
produced which slightly penetrates the sample and part of the Tolerance (cm- 1)
energy is absorbed. The total reflection is attenuated, which Transmission ATR
makes it possible to generate an absorption spectrum.
906.6 906.l ± 1.0
In practice, multiple internal reflections are often used to
amplify the absorption intensity, although some accessories 1028.3 1027.7 ± 1.0
allow absorption measurements with a single reflection.
1601.2 1601.0 ± 1.0
The penetration depth dp is usually of the order of a few
micrometres and is given for a wavelength A by the following 3060.0 3059.7 ± 1.0
equation:
For measurement modes other than transmission or ATR,
reference materials must be defined by the user.
SPECTRAL RESOLUTION
where dp is the penetration depth, t, is the wavelength, rx is
1.4 1.4
the angle of incidence and n1, n2 are the refractive indices of
the reflection element and the sample, respectively.
Due to the relationship between these parameters, the L2 1.2

absorption intensity in ATR is greater at higher wavelengths

(i.e. smaller wavenumbers) and slight band shifts occur
1.0
compared to the corresponding transmission spectrum. It is 1.0

therefore not advisable to compare ATR spectra with

transmission spectra when identifying compounds.
0.8 0.8
EQUIPMENT
The most commonly used IR spectrometers are Fourier-
0.6 0.6
transform (FT-IR) spectrometers which typically consist of:
- a suitable polychromatic light source, e.g. a conducting
ceramic rod;
0.4 0.4
- an interferometer;
- a sample presentation accessory, e.g. a sample holder; D
- a detector; D.2
{I
0.2
- appropriate software for controlling the spectrometer, and i
for spectral evaluation and data processing. ,J t)
Other spectrometers based on alternative principles may also (M) --,.--,--,--..-~ 0.0 '-,--..--,.-..-~
3200 3000 2800 2600 1800 1700 1600 1500 1400
be used if the requirements described under Control of
Wavenumber (cm·') Wavenumber (cm- 1)
equipment performance are fulfilled.
IR spectrometers can also be used in association with a
microscope for the study of a small part of the sample or for Figure 2.2.24.-1. - Typical IR absorbance spectrum of
chemical imaging. polystyrene used to verify spectral resolution
IR spectroscopy can be coupled to other analytical
techniques such as thermal analysis or chromatography. Spectra recorded in transmission mode
The spectral resolution is typically verified using a
CONTROL OF EQUIPMENT PERFORMANCE polystyrene film approximately 35 µm thick.
Accuracy of wavenumber scale and spectral resolution are Acceptance criteria (see Figure 2.2.24.-1) The
critical parameters and must be verified. The tests described difference between the absorbance values at the absorption
below can be used for the control of instrument performance minimum at 2870 cm- 1 (A) and the absorption maximum at
and for qualification. They can also be used as system 2849.5 cm- 1 (B) is greater than 0.33; the difference between
suitability tests. the absorbance values at the absorption minimum at
These parameters are checked using suitable reference 1589 cm- 1 (C) and the absorption maximum at 1583 cm- 1
materials which are selected and presented depending on the (D) is greater than 0.08.
measurement mode (e.g. transmission or ATR).
Spectra recorded in ATR mode
For quantitative analysis, appropriate assessment criteria for Appropriate assessment criteria for the control of spectral
the control of absorption intensity must also be defined. resolution according to the specifications of each instrument
WAVENUMBER SCALE need to be defined.
The wavenumber scale is typically verified using a For measurement modes other than transmission or ATR,
polystyrene film that exhibits IR absorption bands at the reference materials have to be defined by the user.
wavenumbers shown in Table 2.2.24.-1.
PROCEDURE
SAMPLE PREPARATION AND PRESENTATION
Sample preparation and presentation vary according to the
physical state of the sample and the measurement mode.
V-A186 Appendix II A 2023

Transmission mode is applied to transparent samples, such uniform transparency or where the spectrum shows features
as neat liquids, solutions, gases or suitably prepared mulls such as:
and alkali halide discs. For liquids and gases, cells with fixed - low transmission at 4000 cm- 1;
or variable pathlength and IR transparent windows can be - a strongly sloping baseline between 4000 and about
used. For alkali-halide disks, specific sample holders are 2500 cm-';
used. Reflection mode, e.g. ATR, is appropriate for the - a ratio of relative intensities of some absorption bands
measurement of a wide range of samples in the solid and that is less than expected.
liquid state. Molten solids If prescribed in the monograph, make a film
Some preparation modes (e.g. for discs and mulls in of a molten mass and fix it on a suitable mount.
transmission mode or for solids in ATR mode) involve Evaporated solution If prescribed in the monograph,
grinding and/or the application of pressure, which may dissolve the substance to be examined in a suitable solvent.
induce unexpected crystal modifications. Prepare a film by evaporating the solvent on a suitable carrier
Transmission mode and fix it on a suitable mount.
Prepare the substance by one of the following methods Gases Use a suitable cell transparent to infrared radiation.
depending on the sample state (solid, liquid or gas). Sample Evacuate the air from the cell and fill to the desired pressure
bands in a spectrum have a minimum transmittance not through a stopcock or needle valve using a suitable gas
lower than 5 per cent, unless otherwise justified. transfer line between the cell and the container of the gas to
Liquids Examine liquids either in the form of a film be examined. If necessary, adjust the pressure in the cell to
between 2 plates transparent to infrared radiation or in a cell atmospheric pressure using a gas transparent to infrared
of suitable pathlength with windows that are transparent to radiation (e.g. nitrogen R or argon R), or purge with carbon
infrared radiation. dioxide-free air. An appropriate measurement protocol must
Liquids or solids in solution Prepare a solution of the be followed to compensate for water, carbon dioxide or other
substance to be examined in a suitable solvent. Choose a atmospheric gases.
concentration and a pathlength that give a satisfactory ATRmode
spectrum. Generally, good results are obtained with ATR is suitable for liquid and solid samples, and requires no
concentrations of 10-100 g/L for a pathlength of 0.5-0.1 mm. preparation apart from simple treatments such as the
The absorption due to the solvent is usually compensated by grinding of large crystals and coarse material. Proceed as
successively recording the spectra of the solvent and the follows depending on the sample state (liquid or solid).
sample solution and subtracting the solvent absorption bands Liquids Place the sample in contact with the crystal.
from the spectrum of the sample solution. Solids Ensure close and uniform contact between the
Solids dispersed in a solid (disc) Grind the substance to substance to be examined and the whole crystal surface,
be examined taking into consideration any possible changes either by applying pressure or by dissolving the substance in
(e.g. crystalline form) and mix with a suitable amount of an appropriate solvent, then covering the crystal with the
finely powdered and dried potassium bromide R or potassium resulting solution and evaporating to dryness.
chwride R, unless otherwise specified. A mixture of a few
METHODS
milligrams (e.g. 1-2 mg) of the substance to be examined in
Infrared spectroscopy is mostly used to identify substances,
a few hundred milligrams (e.g. 300-400 mg) of halide is
but it may also be carried out for quantitative applications.
normally sufficient to give a disc of 10-15 mm diameter and
Quantitative analysis (based on the Beer-Lambert law, which
a spectrum of suitable intensity. If the substance is a
relates the absorbance of a sample to its concentration) will
hydrochloride salt, it is recommended to use potassium
not be described in this chapter.
chloride R. Carefully grind the mixture, spread it uniformly in
a suitable die and apply a suitable pressure. A compacting The measurement is performed on an appropriately prepared
force of about 800 MPa is generally sufficient to prepare a sample. The data is then processed and evaluated, either to
disc. For substances that are unstable under normal identify substances or quantify them (e.g. based on
atmospheric conditions or are hygroscopic, the disc may be integration of IR-absorption bands).
pressed under vacuum. Several factors may cause the Spectral quality may be enhanced by mathematical
formation of faulty discs, such as insufficient or excessive pretreatments. In practice, these are limited to spectral
grinding, humidity or impurities in the dispersion medium. normalisation and subtraction of bands caused by carbon-
For example, any water in either the sample or the potassium dioxide and water vapour. The same pretreatments are
bromide will cause clouding of the disc and produce a low performed on both the sample and the reference spectra.
transmission spectrum. A disc is rejected if visual Identification
examination shows a lack of uniform transparency or when, Prepare the substance to be examined appropriately and
in the absence of a specific absorption band, the record the spectra between 4000 and 650 cm-1, unless
transmittance is less than 60 per cent or the absorbance is otherwise prescribed.
more than 0.22 at about 2000 cm- 1 (5 µm) and without Identification testing is performed by comparing the
compensation, unless otherwise prescribed. spectrum of the substance to be examined with the spectrum
Solids dispersed in a liquid (mull) Triturate a small obtained from a Ph. Eur. chemical reference substance
quantity of the substance to be examined with the minimum (CRS) or with a Ph. Eur. reference spectrum.
quantity of liquid paraffin R or other suitable liquid. The spectrum of the current batch of the Ph. Eur. CRS may
A mixture of a few milligrams (e.g. 5-10 mg) of the be recorded for immediate use or stored, for example, in a
substance to be examined in 1 drop of liquid paraffin R is spectral library for future consultation. A stored spectrum
generally sufficient to make an adequate mull. Compress the may be used, provided traceability to the current batch
mull between 2 plates transparent to infrared radiation. of CRS is ensured.
A mull is rejected if a visual examination shows lack of
In the case of substances that are not covered by individual
monographs, a suitable reference standard may be used.
2023 Appendix II A V-Al87

In all cases, spectra must be recorded using the same NIR spectroscopy has a wide variety of applications for
operating conditions and procedure, and especially the same chemical, physical and process analysis, for example:
measurement mode. Chemical analysis:
When comparison of the spectra recorded in the solid state - identification of active substances, excipients, dosage
show differences (see below), treat the substance to be forms, manufacturing intermediates, chemical materials
examined and the reference substance in the same manner so and packaging materials;
that they recrystallise or are produced in the same crystalline - qualification of active substances, excipients, dosage
form, or proceed as prescribed in the monograph, then forms, manufacturing intermediates and packaging
record the spectra again. However, this procedure must only materials, including batch-to-batch spectral comparison
be done for substances where the monograph does not cover and supplier change assessment;
a particular form of a substance that exhibits polymorphism. - quantification of active substances in a sample matrix,
Several comparison procedures may be used, and the analyst determination of chemical values such as hydroxyl value,
must document and justify the method used and the specific determination of absolute water content, determination of
acceptance criteria that allow a conclusion for identification. degree of hydroxylation and control of solvent content.
The spectra can be compared either by overlaying the spectra Physical analysis:
(in the whole spectral range or in the region of interest - crystalline form and crystallinity, polymorphism, solvates,
specified in the monograph) or by using mathematical particle size;
calculations from the sofrware. It is possible for example to - disintegration, hardness;
perform: - film properties.
- visual comparison based on band positions and relative Process analysis:
intensities unless otherwise specified - the transmission - monitoring of unit operations such as synthesis,
minima (or absorption maxima) in the spectrum obtained crystallisation, blending, drying, granulation and coating,
with the substance to be examined correspond in position for the purpose of process control;
and relative size to those of the reference; - control and endpoint detection.
- calculation of the correlation coefficient between the 2 Measurements in the NIR region are influenced by many
spectra - this value is calculated by the sofrware and the
chemical and physical factors as described below; the
identification threshold is defined by the user; reproducibility and relevance of results depend on control of
- evaluation by chemometric methods (e.g. Euclidean these factors and measurements are usually valid only for a
distance, Mahalanobis distance, classification methods);
defined calibration model.
these methods involve the set-up, assessment and
validation of the chemometric model by the analyst (see APPARATUS
5.21. Chemometric methods app/i,ed to analytical data). All NIR measurements are based on passing light through or
into a sample and measuring the attenuation of the emerging
Impurities in gases
(transmitted or reflected) beam. Spectrometers for
For the analysis of impurities, use a cell transparent to
measurement in the NIR region consist of a suitable light
infrared radiation and of suitable optical pathlength
source (such as a highly-stable quartz-tungsten lamp), a
(e.g. 1-20 m). Fill the cell as prescribed under Gases.
monochromator or interferometer, and a detector. Common
For detection and quantification of the impurities, proceed as
monochromators are acousto-optic tunable filters (AOTF),
prescribed in the monograph.
gratings or prisms. Traditionally, many NIR instruments have
Near-infrared Spectrophotometry a single-beam design, though some process instruments use
(Ph. Eur. method 2.2.40) internal referencing and can therefore be dual-beam (for
Near-infrared (NIR) spectroscopy is a technique with wide example in diode array instruments). Silicon, lead sulfide,
and varied applications in pharmaceutical analysis. The NIR and indium gallium arsenide are examples of detector
spectral range extends from 780 nm to 2500 nm (from materials. Conventional cuvette sample holders, fibre-optic
12 800 cm- 1 to 4000 cm- 1). NIR spectra are dominated by probes, transmission dip cells, neutral borosilicate vials and
C-H, N-H, 0-H and S-H overtones and combinations of spinning or traversing sample holders are a few examples of
fundamental mid-infrared (MIR) vibrations. They contain sampling devices. The selection is based on the intended
composite chemical and physical information and in most application, paying particular attention to the suitability of
cases this information can be extracted by suitable the sampling system for the type of sample to be analysed.
mathematical data treatment. NIR bands are much weaker Suitable data processing and evaluation units (e.g. sofrware
than the fundamental MIR vibrations from which they and computer) are usually part of the system.
originate. Because absorptivities in the NIR range are low, It is common to express the wavelength (t,) in nanometres
radiation can penetrate up to several millimetres into and the wavenumber (v) in reciprocal centimetres (cm- 1),
materials, including solids. Furthermore, many materials such depending on the measurement technique and apparatus.
as glass are relatively transparent in this region. Conversion between nm and cm- I is performed according to
Measurements can be made directly in situ, in addition to the following expression:
standard sampling and testing procedures.
NIR measurements can be performed off-line, and also Vcm-1 = 107 X -,I-
Am11
at-line or in-line, and on-line for process analytical
technology (PAT). Suitable chemometric methods may be MEASUREMENT METHODS
required for identification. However, when the specificity Transmission mode
criteria for a qualitative method are met, chemical Transmittance (7) is a measure of the decrease in radiation
identification or solid-state characterisation is possible by intensity at given wavelengths when radiation is passed
direct comparison of the untreated or pre-treated spectra through the sample. The sample is placed in the optical
obtained with the chemical substance being examined with a beam between the source and the detector. The arrangement
spectrum of a reference substance. is analogous to that in many conventional spectrometers.
V-Al88 Appendix II A 2023

The resulting spectrwn can be presented directly in terms of - find the best orientation of the sample (e.g. to minimise
transmittance (T) and/or absorbance (A) (y-axis) versus the the impact of debossing on tablets);
wavelength or wavenumber (x-axis). - find the best suitable accessory (e.g. transmission cell or
immersion probe);
I - optimise pathlength in transmission and transflection
T=-
Io modes;
- find a suitable spectroscopic background reference
lo intensity of incident radiation;
I intensity of transmitted radiation; material;
- show that the background reference material is reliable
over time and that the measurement of the background is
reproducible and stable over time;
- when measuring moving materials or samples (for
process-related measurements) it is important to obtain a
Diffuse reflection mode representative spectrum (e.g. by adjusting the measuring
The diffuse reflection mode gives a measure of time, the number of scans, co-adding individual spectra,
reflectance (R), the ratio of the intensity oflight reflected or increasing the beam size);
from the sample (/) to that reflected from a background or - ensure there is no fouling of the sensor, for example with
reference reflective surface Cir). Depending on the chemical build-up of material or contamination;
composition and physical characteristics of the sample, NIR - the measuring conditions (measuring time, beam size) in
radiation can penetrate a more or less substantial distance relation to the minimal sample size should be justified.
into the sample, where it can be absorbed by the overtones
In some process analysis situations it may be impossible to
and combinations of the fundamental vibrations of the
remove a probe for reference background data collection;
analyte species present in the sample. Non-absorbed
various options are therefore to be considered, including
radiation is partially reflected back from the sample to the
internal referencing, measurement of a background reference
detector. NIR reflectance spectra are typically obtained by
using a 2nd detector, etc. Only spectra measured against a
calculating and plotting log 10 (1/R) (y-axis) versus the
background possessing the same optical properties can be
wavelength or wavenumber (x-axis).
directly compared with one another.
Transmission mode
The measurement of transmittance (T) is dependent on a
background transmittance spectrum for its calculation.
I intensity of light diffusively reflected from the sample; Examples of background references include air, a polymeric
I, intensity of light reflected from the background or reference
reflective surface; disc, an empty cell, a solvent blank or in special cases a
reference sample. The method generally applies to liquids
(diluted or undiluted), dispersions, solutions and solids
(including tablets and capsules). For transmittance
measurements of solids, a suitable sample accessory is used.
Liquid samples are examined in a cell of suitable pathlength
Transflection mode (typically 0.5-4 mm), transparent to NIR radiation, or
This mode is a combination of transmittance and reflectance. alternatively by immersion of a fibre-optic probe of a suitable
In the measurement of transflectance ('P), a mirror or a configuration.
diffuse reflectance surface is used to reflect the transmitted
Diffuse reflection mode
radiation back through the sample, thus doubling the
This mode generally applies to solids. The sample is
pathlength. Non-absorbed radiation is reflected back from
examined directly, or in a suitable device (for example a
the sample to the detector. The resulting spectrum can be
sample holder), or in direct contact with a fibre-optic probe.
presented directly in terms of transflectance (T~ and/or
For process monitoring, material can be analysed through a
absorbance (A~ (y-axis) versus the wavelength or
polished window interface (e.g. sapphire), or using an in-line
wavenumber (x-axis).
fibre-optic probe. Care must be taken to ensure that the
measuring conditions are as reproducible as possible from
one sample to another. The reflected radiation of a
background reference is scanned to obtain the baseline, and
I intensity of cransflected radiation measured from the sample; then the reflectance of one or more analytical samples is
h intensity of transflected radiation of the reference material as measured. Common reflectance references include ceramic,
background;
thermoplastic resins and gold. Other suitable materials may
be used.
Transflection mode
A* = log10 (;.) = log10 (;)
This mode generally applies to liquids, suspensions and clear
plastic materials. A reflector is placed behind the sample so
SAMPLE PREPARATION/PRESENTATION as to double the pathlength. This configuration can be
Sample preparation and presentation may vary according to adopted to share the same instrument geometry with
the measurement mode. The following requirements are reflectance and fibre-optic probe systems where the source
necessary for all sampling techniques: and the detector are on the same side of the sample.
- optimise the measuring time and number of scans to The sample is examined through a cell with a mirror or a
optimise the signal-to-noise ratio; suitable diffusive reflector, made either of metal or of an inert
- find the best suitable measurement mode for the intended substance (for example, dried titanium dioxide) not
application (transmission, diffuse reflection or
transflection);
2023 Appendix II A V-A189

absorbing in the NIR region. Liquids can also be measured and noise reduction and the numerical calculation of the
using in-line transflectance probes. first- or second-order derivative of the spectrum. Higher-
FACTORS AFFECTING SPECTRAL RESPONSE order derivatives are not recommended because of increased
spectral noise.
Environment
The environment temperature and humidity must be taken CONTROL OF INSTRUMENT PERFORMANCE
into consideration before carrying out measurements. Use the apparatus according to the manufacturer's
Sample presentation area instructions and carry out the prescribed verification at
The sample presentation area or probe end must be clean of regular intervals, according to the use of the apparatus and
residue prior to measurement. Similarly, the in-line or on-line the application. For in-line and on-line applications, the use
interface to the sample should not have significant product or of alternative means of control of instrument performance
contamination build-up, which would interfere with the must be scientifically justified. For example, utilise the
desired measurement. standards built into the instrument or separate
channels/probes to demonstrate instrument performance
Sample temperature
(pending practicality).
This parameter is important for aqueous solutions and many
liquids, where a difference of a few degrees can result in System suitability tests may be required prior to sample
measurable spectral changes which may have a significant scanning, and the instrument attributes with potential impact
effect on the analysis. Temperature is also an important on suitability of the final measurement (typically photometric
parameter for solids and powders containing water. noise and wavelength accuracy) must be tested.
The frequency at which each performance test is carried out
Moisture and solvent residues must be risk-assessed depending on the instrument type and
Moisture and solvent residues present in the samples will its environment. For example, instruments placed in harsh
contribute significant absorption bands in the NIR region. environments with variations in temperature and humidity
Sample thickness may need frequent performance testing. Cases where the
Sample thickness is a known source of spectral variability and measurement system cannot be removed such as an in-line
must be assessed and/or controlled, particularly for tablet and probe or flow cell are also to be considered.
capsule analysis in transmittance mode. For the measurement Some accessories are custom designed and therefore require
of compressed powders, an infinite thickness is typically adequate performance testing.
reached after 5 mm of sample depth (e.g. in a vial).
Verification and calibration of the wavelength or
Sample optical properties wavenumber scale (except for filter apparatus) Verify
In solids, both surface and bulk scattering properties of the wavelength scale employed, generally in the region
samples must be taken into account. Spectra of physically, between 780 nm and 2500 nm ( 12 800 cm- 1 to 4000 cm- 1)
chemically or optically heterogeneous samples may require or in the intended spectral range using one or more suitable
increasing the beam size, or examining multiple samples or wavelength standards which have characteristic maxima or
spinning the sample to obtain a representative spectrum of minima within the wavelength range to be used.
the sample. Certain factors such as differing degree of For example, methylene chloride R, talc R, wavelength
compaction or particle size in powdered materials and surface reference lamps or a mixture of rare-earth oxides are suitable
finish can cause significant spectral differences. reference materials. Other suitable standards may also be
Solid-state forms used. Obtain a spectrum and measure the position of at least
Variations in solid-state forms (polymorphs, hydrates, 3 peaks distributed over the range used. For rare-earth
solvates and amorphous forms) influence vibrational spectra. oxides, the National Institute of Standards and Technology
Hence, different crystalline forms as well as the amorphous (NIST) provides suitable reference materials. Fourier
form of a solid may be distinguished from one another on the transform (FT) instruments have a linear frequency range,
basis of their NIR spectra. Where multiple crystalline forms therefore wavelength certification at a single frequency is
are present, care must be taken to ensure that the calibration sufficient.
samples have a distribution of forms relevant to the intended Verification and calibration of photometric linearity
application. The photometric linearity is demonstrated with a set of
Age of samples transmittance or reflectance standards with known percentage
Samples may exhibit changes in their chemical, physical or values of transmittance or reflectance. For reflectance
optical properties over time. Depending on the storage measurements, carbon-doped polymer standards are
conditions, solid samples may either absorb or desorb water, available. Ensure that the absorbance of the materials used is
and portions of amorphous material may crystallise. Materials relevant to the intended linear working range of the method.
used for NIR calibration should be representative of future Subsequent verifications of photometric linearity can use the
samples and their matrix variables. initial observed absorbance values as the reference values.
Non-linear calibration models and hence non-linear
PRE-TREATMENT OF NIR SPECTRAL DATA responses are acceptable with understanding demonstrated by
In many cases, and particularly for reflection mode spectra, the user.
some form of mathematical pre-treatment of the spectrum
Spectra obtained from reflectance and transmittance
may be useful prior to the development of a classification or
standards are subject to variability due to the differences
calibration model. The aim can be, for example, to reduce
between the experimental conditions under which they were
baseline variations, to reduce the impact of known variations
factory-calibrated and those under which they are
that are interfering in the subsequent mathematical models,
subsequently put to use. Hence, the percentage reflectance
or to simplify data before use. In some cases spectra may also
values supplied with a set of calibration standards may not be
be normalised or corrected for scatter, for example using
useful in the attempt to establish an 'absolute' calibration for
standard normal variate (SNV) transformation. Spectral pre-
a given instrument. As long as the standards do not change
treatment techniques may include, for example, windowing
chemically or physically and the same reference background
V-Al 90 Appendix II A 2023

is used as was used to obtain the certified values, subsequent Validation of the model
measurements of the same standards under identical Identification methods using direct spectral comparison must
conditions, including precise sample positioning, give be validated in accordance with identification method
information on long-term stability of the photometric validation procedures.
response. A tolerance of ± 2 per cent of the absorbance The validation parameters for qualitative methods are
value is acceptable for long-term stability; this verification is robustness and specificity.
only necessary if the spectra are used without pre-treatment.
LIMIT ANALYSIS
Recommendations for the conditions used to control
Relative comparison of spectra
instrument performance for the various measurement modes
A calibration is not required when comparing a set of spectra
are summarised in Table 2.2.40.-1.
for limit analysis purposes, such as the maximum or
QUALITATIVE ANALYSIS (IDENTIFICATION AND minimum absorbance at which an analyte absorbs. Also,
CHARACTERISATION) dryer end point control may use a qualitative approach
Establishment of a spectral reference library around a specific absorbing wavelength. Appropriate spectral
Record the spectra of a suitable number of representative ranges and pre-treatments (if used) must be shown to be fit
samples of the substance which have known, traceable for purpose.
identities, and that exhibit the variation typical for the Specificity
substance to be analysed (for example, solid-state form, The relative discriminatory power for a limit test must be
particle size, etc.). Libraries are built using representative demonstrated. The extent of specificity testing is dependent
samples under appropriate environmental conditions. The set on the application and the risks being controlled. Variations
of spectra obtained represents the information which can be in matrix concentrations within the operating range of the
used for identification of the sample to be analysed. method must not affect the measurement.
The collection of spectra in the library may be represented in
TREND ANALYSIS
different ways defined by the mathematical technique used
Relative comparison of spectra
for identification. These may be:
A calibration is not necessarily required when comparing a
- all individual spectra representing the substance;
- a mean spectrum of the measured batches for each set of spectra for trend analysis purposes, such as the moving
block approach to estimate statistical parameters such as
chemical substance;
- if necessary, a description of the variability within the mean, median and standard deviation. For example, blend
uniformity monitoring using NIR spectroscopy has adopted
substance spectra.
such data analysis approaches. Appropriate spectral ranges
The number of substances in the library depends on the and algorithms must be used for trend analyses.
specific application. All spectra in the library used have the
same: Specificity
- spectral range and number of data points; The relative discriminatory power for trend analysis must be
- technique of measurement; demonstrated. The extent of specificity testing is dependent
- data pre-treatment. on the application and the risks being controlled. Variations
in matrix concentrations within the operating range of the
If sub-groups (sub-libraries) are created, the above criteria
method must not affect the trend analysis.
are applied independently for each group. Sub-libraries are
individually validated. Original spectral data for the QUANTITATIVE ANALYSIS
preparation of the spectral library must be archived. Caution Establishment of a spectral reference library for a
must be exercised when performing any mathematical calibration model
transformation, as artefacts can be introduced or essential Calibration is the process of constructing a mathematical
information (important with qualification methods) can be model to relate the response from a sample scanned using an
lost. The suitability of the algorithm used should be analytical instrument to the properties of the samples.
demonstrated by successful method validation and in all Any calibration model that can be clearly defined in a
cases the rationale for the use of transform must be mathematical expression and gives suitable results can be
documented. used. Record the spectra of a suitable number of
Direct comparison of substance and reference spectra representative samples with known or future-established
Direct comparison of representative spectra of the substance values of the attribute of interest throughout the range to be
to be examined and of a reference substance for qualitative measured (for example, content of water). The number of
chemical or physical identification purposes may not require samples for calibration will depend on the complexity of the
use of a reference spectral library where specificity permits. sample matrix and interferences (e.g. temperature, particle
size, etc.). All samples must give quantitative results within a
Data evaluation calibration interval as defined by the intended purpose of the
Direct comparison of the representative spectrum of the method. Multiple linear regression (MLR), principal
substance to be examined is made with the individual or component regression (PCR) and partial least squares
mean reference spectra of all substances in the database on regression (PLS) are commonly used algorithms. For PLS or
the basis of their mathematical correlation or other suitable PCR calibrations, the regression coefficients and/or the
algorithms. A set of known reference mean spectra and the loadings should be plotted and the regions of large
variability around this mean can be used with an algorithm coefficients or loadings compared with the spectrum of the
for classification; alternatively, this can be achieved visually analyte. Predicted residual error sum of squares (PRESS)
by overlaying spectral data if specificity is inherent. There are plots (or similar) are useful to facilitate the optimising of the
different techniques available, such as principal component number of PCR or PLS factors.
analysis (PCA), cluster analysis, and soft independent
modelling by class analogy (SIMCA). The reliability of the Pre-treatment of data
technique chosen for a particular application has to be Wavelength selection or excluding certain wavelength ranges
validated according to the following: may enhance the accuracy and robustness of calibration
2023 Appendix II A V-Al91

Table 2.2.40.-1 - Control of instrument peiformance

Measurement mode Reflection Transflection Transmission

Verification of wavelength Typical tolerances for agreement with standard values are:
scale ( except for filter ± 1.0 nm at 780 nm(± 16 cm 1 at 12 800 cm- 1);
apparatus) ± 1.0 nm at 1200 nm(± 8 cm- 1 at 8300 cm 1);
± 1.0 nm at 1600 nm(± 6 cm I at 6250 cm- 1);
± 1.5 nm at 2000 nm(± 4 cm- 1 at 5000 cm- 1);
± 1.5 nm at 2500 nm(± 2 cm- 1 at 4000 cm 1).
For the reference material used, apply the tolerance for the nearest wavelength or wavenumber for each peak used. For diode array
instruments, most often the pixel resolution (wavelength between pixels) can be as large as 10 nm. The pixel resolution must be
adapted to match the spectral resolution. The peak-finding algorithms are critical to wavelength accuracy. Practically, ± 2 nm is
appropriate for peak wavelength accuracy using such instrumentation. Alternativelyi refer to manufacturer's specifications for
acceptance.

Bench/mobile instrument Measure talc R via a suitable medium or A suspension of l .2 g of dry titanium Methylene eh/on& R may be used and has
by fibre-optic probe. Talc R has dioxide R in about 4 mL of methylene characteristic sharp bands at 115 5 nm,
characteristic peaks at 948 nm, 139 I nm chloride R is used directly with a cell or a 1366 nm, 1417 nm, 1690 nm, 1838 nm,
and 2312 nm suitable for calibration. probe. Titanium dioxide has no 1894 nm, 2068 nm and 2245 nm. Choose 3
Alternatively, other suitable standards may absorption in the NIR range. Spectra arc peaks across the wavelength range for
also be used that ensure wavelength recorded with a maximum nominal calibration. Other suitable standards may
accuracy in the region of working instrument bandwidth of I O nm at also be used.
methodology. For example, measure an 2500 nm (16 cm- 1 at 4000 cm 1).
internal polystytene standard if built-in, or Methylene chloride has characteristic
measure a NIST standard or other sharp bands at 1155 nm, 1366 nm,
traceable material, and assess 3 peaks 1417 nm, 1690 nm, 1838 nm,
across the wavelength range for 1894 nm, 2068 nm and 2245 nm.
calibration. Choose 3 peaks across the wavelength
range for calibration. Other suitable
standards may also be used, such as a
liquid transflection standard mixed with
titanium dioxide or some other reflective
medium.

Process instrument If it is not practically possible to measure a traceable reference material at the point of sample measurement, use internal material
such as polystyrene, fibreglass or solvent and/or water vapour. Alternatively, adopt a second external channel/probe.
For FT instruments, the calibration of the wavenumber scale may be performed using a narrow, isolated water-vapour line, for
example, the line at 7306.74 cm- 1 , or 7299.45 cm- 1, or 7299.81 cm 1 or a narrow line from a certified reference material.

Verification of wavekng1h The standard deviation of the wavelength is consistent with the specifications of the instrument manufacturer, or otherwise
repeatability (except for scientifically justified.
filter apparatus)

Bench/mobile instrument Verify the wavelength repeatability using a suitable external or internal standard.

Process instrument Verify the wavelength repeatability using a suitable external or internal standard.

Verification of photomenic Measure 4 photometric standards across the working method absorbance range.
linearity and response
stabilit/ 1)

Bench/mobile instrument Analyse 4 reflectance standards, for Transflection measurements can use Analyse 4 transmittance standards to cover
example in the range of 10-99 per cent, appropriate reflectance or transmittance the absorbance values over the working
including I O per cent, 20 per cent, standards and criteria. absorbance range of the modelled data.
40 per cent and 80 per cent. In some Evaluate the observed absorbance values
circumstances 2 per cent may be against the reference absorbance values, for
appropriate. Evaluate the observed example perform a linear regression.
absorbance values against the reference Acceptable tolerances are 1.00 ± 0.05 for
absorbance values, for example perform a the slope and 0.00 ± 0.05 for the intercept
linear regression. Acceptable tolerances are for the 1st verification of photometric
1.00 ± 0.05 for the slope and linearity of an instrument. Subsequent
0.00 ± 0.05 for the intercept for the 1st verifications of photometric linearity can use
verification of photometric linearity of an the initial observed absorbance values as the
instrument. Subsequent verifications of reference values.
photometric linearity can use the initial
observed absorbance values as the
reference values.

Process instrument If photometric reflectance and transmittance standards cannot be measured at the point of sample measurement, use the photometric
standards built into the instrument.
Process instruments can use internal photometric standards for photometric linearity. Follow the manufacturer's verified tolerances in
such cases.

(l) Verification of photometric linean"t:>: and Venfication uf photometr£, noise are not required for instruments using methods to perform simple identifications which do

not use the photometric absorbances as part of model strategy (for example, simple correlation with absorbing wavelengths).
V-A192 Appendix II B 2023

Measurement mode Reflection

I T ransflection Transmission

Verification of photometric Determine the photometric noise at a relevant photometric region of the spectrum using a suitable reflectance standard, for example,
nois/ 1) white reflective ceramic tiles or carbon-doped polymer standards. Follow the manufacturer's methodology and specifications.

Bench/mobile instrument Scan the reflectance low flux standard (e.g. 5 or JO per cent, carbon-doped polymer Scan the transmittance high flux standard
standard) over a suitable wavelength range in accordance with the manufacturer's (e.g. 90 or 99 per cent, carbon-doped
recommendation and calculate the photometric noise as peak-to-peak noise. polymer standard) over a suitable
wavelength/wavenumber range in
accordance with the manufacturer's
recommendation and calculate the
photometric noise as peak-to-peak noise.

Process instrument As above, or if not practically possible, use the standard built into the instrument for As above, or if not practically possible, use
noise testing and manufacturer specifications. the standard built into the instrument for
noise testing and manufacturer
specifications.

Cll Verification of photometric linearity and Verification of photometric noise are not required for instruments using methods to perform simple identifications which do
not use the photometric absorbances as part of model strategy (for example, simple correlation with absorbing wavelengths).

models. Wavelength compression (wavelength averaging) either at a single wavelength or over a specified wavelength
techniques may be applied to the data. range.
Model validation parameters APPLICATIONS
Analytical performance characteristics to be considered for UV-Vis spectroscopy is traditionally used for the quantitative
demonstrating the validation of NIR methods are similar to and qualitative analysis of liquid samples, but is also suitable
those required for any analytical procedure. Specific for solid and gaseous analytes and has other applications
acceptance criteria for each validation parameter must be such as the determination of physical or chemical properties.
consistent with the intended use of the method. Validation UV-Vis spectroscopy as described in this chapter can be
parameters for quantitative methods are accuracy, linearity, applied in various ways:
precision (repeatability and intermediate precision), - when a monograph or general chapter refers to this
robustness and specificity. chapter, the requirements described in the relevant
ONGOING MODEL EVALUATION paragraphs of this chapter are mandatory;
NIR models validated for use are subjected to ongoing - when used as the detection method in chromatographic
performance evaluation and monitoring of validation systems as described in general chapter 2.2.46, the
parameters. requirements listed in the relevant paragraphs of this
chapter are mandatory;
TRANSFER OF DATABASES
- when used as a process analytical technology (PAT) tool
When databases are transferred to another instrument, for PAT applications similar to the applications described
spectral range, number of data points, spectral resolution and in this chapter, the provisions herein apply; for other PAT
other parameters have to be taken into consideration. Further applications, the principles are the same, however the
procedures and criteria must be applied to demonstrate that criteria are established bearing in mind the intended
the model remains valid with the new database or new purpose of the analysis, using a risk-based approach.
instrument.
EQUIPMENT
Spectrophotometers used for carrying out measurements in
the UV-Vis region typically consist of:
B. Ultraviolet and Visible Absorption a suitable light source (such as a deuterium lamp for the
UV region, a tungsten-halogen lamp for the visible region
Spectrophotometry or a xenon lamp to cover the entire UV-Vis range);
(Ph. Bur. method 2.2.25) UV-Vis spectrophotometers often have 2 sources;
a monochromator such as a grating system;
PRINCIPLE - other optical components, such as lenses or mirrors, that
Ultraviolet and visible (UV-Vis) spectroscopy (or relay light through the instrument and that may also be
spectrophotometry) is based on the ability of atoms, used to generate more than one beam of light,
molecules and ions to absorb light at specific wavelengths in i.e. in double-beam spectrophotometers, as opposed to
the ultraviolet (approximately 180-400 nm) and visible single-beam spectrophotometers;
(approximately 400-800 nm) range. This absorption is - a sample container, holder or sampling device; examples
associated with changes in electronic energy in the form of include conventional cuvettes, fibre-optic probes and
temporary transitions of electrons to an excited state at a immersed transmission cells (e.g. high-purity quartz or
higher energy orbital. As each energy level of a molecule or sapphire transparent to UV-Vis radiation); the choice
molecular ion also has associated vibrational and rotational depends on the intended application, paying particular
sub-levels, this results in many permitted transitions, which attention to its suitability for the type of sample to be
are generally impossible to separate, thereby producing analysed;
absorption bands rather than sharp lines. These bands are - a single-channel (e.g. photomultiplier, photodiode) or
characteristic of the functional groups and bonds in a multi-channel detector (e.g. photodiode array (PDA) or
molecule. charge-coupled device (CCD));
UV-Vis spectroscopy measurements involve exposing a suitable computerised data processing and evaluation
sample to light and measuring the attenuation and/or systems.
scattering of the emerging (transmitted or reflected) light
2023 Appendix II B V-Al 93

Control of cuvettes The specific absorbance (A\ f~~ cent) of the substance is
For benchtop instruments, cuvettes or cells with a defined generally used in monographs and is related to absorbance
path length are used. These can be made of different (A) as follows:
materials such as quartz or glass. The tolerance for the path
length of quartz and glass cuvettes is ± 0.5 per cent
(e.g. ± 0.005 cm for a 1 cm cuvette). Plastic cuvettes may Cm mass concentration of the substance in solution, in grams per
also be used but the tolerance interval is wider; therefore, I 00 millilitres.
their use must be thoroughly justified and based on a risk
assessment. A; ~: cent represents the specific absorbance of a dissolved

The following method may be applied to check the substance and refers to the absorbance of a 1 g/ 100 mL (or
cleanliness of optical cuvette windows and any significant 1 per cent m/V) solution in a 1 cm cuvette or cell and is
differences in their thickness or parallelism: fill the cuvette measured at a defined wavelength. The relationship between
with water R and measure its apparent absorbance against air A;~: cent and 8 is:

at 240 nm for quartz cuvettes and 650 nm for glass cuvettes;

A' per cent _ 108
rotate the cuvette 180° in its holder and measure the I cm - lvfr
apparent absorbance again at the same wavelength.
When using scanning instruments, it is recommended to scan Mr relative molecular mass.

over the optical region of interest.

Transmittance or absorbance measurements are generally
When using double-beam spectrophotometers, measures
used for liquids (dispersions and solutions), but can also be
should be taken (e.g. matching the cuvettes) to ensure that
used for solids (including tablets and capsules).
any difference between the absorbance of the cuvettes will
For measurements of solids, a suitable sample accessory is
not have a significant impact on the analysis to be performed.
used. Liquid samples are examined using a cell or cuvette
Acceptance criteria: with a suitable path length (typically 0.01-1 cm) and made of
- the apparent absorbance is not greater than 0.093 for a material that is transparent to UV-Vis radiation, or by
1 cm quartz cuvettes (UV region) and 0.035 for 1 cm using a fibre-optic probe of a suitable configuration immersed
glass cuvettes (visible region); in the liquid.
- the absorbance measured after rotation ( 180°) does not
Diffuse reflection mode
differ by more than 0.005 from the value previously
Diffuse reflection mode provides a measure of reflectance
obtained.
(R), which is given by the following equation:
MEASUREMENT
Transmission mode
Transmission mode provides a measure of the transmittance
(7), at a given UV-Vis wavelength, of a sample placed I intensity of light reflected and/or scattered from the sample;
between the light source and the detector. Transmittance is lo intensity of light reflected and/or scattered from a blank or
the ratio of the intensity of the transmitted light to the reference reflective surface.
intensity of the incident light and is given by the following
equation: Depending on the chemical composition and physical
characteristics of the sample, the UV-Vis radiation may be
absorbed as it passes through the sample. In diffuse reflection
mode, it is the non-absorbed radiation which is partially
reflected and/or scattered back from the sample that is
I intensity of transmitted radiation;
intensity of incident radiation.
measured by the detector. UV-Vis reflectance spectra are
lo
typically obtained by calculating and plotting log 10 (1/R) as a
A spectrum may be obtained by plotting the variation in function of the wavelength.
transmittance (7) or absorbance (A) as a function of This measurement mode is generally selected for solids.
wavelength. The sample is examined either in a suitable device
The absorbance is defined as the logarithm to base 10 of the (e.g. a sample holder) or in direct contact with a probe.
reciprocal of the transmittance for monochromatic radiation. For process monitoring, the material can be analysed through
It is a dimensionless quantity expressed in absorbance units a polished window interface (e.g. quartz or sapphire), or
(AU), given by the following equation: in-line using a probe. Care must be taken to ensure that the
measurement conditions are as reproducible as possible from
one sample to another.
Operation of the equipment
The factors below affect the spectral response and must
According to the Beer-Lambert law, which applies to clear always be taken into account.
diluted solutions, in the absence of interfering physico-
Choose a measurement mode that is appropriate for the
chemical factors, the absorbance (A) is proportional to the
intended application and the sample type.
path length ([) of the radiation through the sample, and to
the concentration (c) of the substance in solution in Define the measuring conditions taking into account the
accordance with the following equation: sample size and sample probe in such a way as to obtain a
satisfactory signal-to-noise ratio (e.g. beam size, measurement
time and number of measurements). For scanning
spectrophotometers, also select the scan range, scan rate and
molar absorption coefficient, in litres per mole per centimetre;
molar concentration of the substance in solution, in moles per
slit-width that provide the necessary optical resolution for the
litre; intended application without losing the required signal-to-
absorption path length, in centimetres. noise ratio or the linearity of the analytical method. When
V-Al 94 Appendix II B 2023

Table 2.2.25.-1. - Minimum tests to be canied out for the control of equipment performance
Wavelength Absorbance Photometric Resolution/spectral
Purpose Method Stray light
accuracy accuracy linearity bandwidth

Based on
measurement of the
absorbance at one or
Quantitative or
more identified X X X X If required in the monograph
limit test
wavelengths
(e.g. assay or
impurities test)

Based on wavelength
of absorption maxima X - - X -
and minima

Based on absorption
measurement and
Identification test
wavelength of
X X - X -
absorption maxima

Based on comparison
of spectrum with that X X - - -
of reference substance

using spectrophotometers with array sensors, there is no need For reflectance measurements, common reflectance blank
to adjust the beam size, scan range, scan rate or slit-width samples include ceramics, fluoropolymers such as
since the optical resolution is typically fixed and the full polytetrafluoroethylene (PTFE) and powders such as barium
spectrum is always recorded. sulfate (BaSO 4) and magnesium oxide (MgO), but other
Before an absorbance measurement is carried out, the zero suitable materials may also be used.
position of the absorption (baseline correction) should be set MATHEMATICAL TREATMENT OF SPECTRAL
or determined for the wavelengths of interest or over the DATA
appropriate range of wavelengths. In the case of single wavelength analysis used to determine
For PAT applications, when measuring moving materials or the concentration of an unknown sample (e.g. as prescribed
samples, ensure that there is no fouling of the sensor in monographs), mathematical treatment consists in
(e.g. no contamination or build-up of material). determining the regression of the photometric reading
Unless otherwise prescribed in the monograph, measure the (absorbance) on the concentration of the standard samples.
absorbance using a path length of 1 cm at the prescribed In the case of full range spectra, data for both diffuse
wavelength. If a single value for the position of an absorption reflection and transmission modes may have to be treated
maximum or minimum is given in a monograph, the user before a classification or calibration model can be developed.
must determine the wavelength position. The value obtained The aim can be, for example, to reduce baseline drift or to
may differ by not more than ± 2 nm, unless otherwise correct for scatter caused by particle size changes in solid
prescribed. samples. For example, first-, second- or higher-order
Quantitative measurements relying on absorption values derivative spectra can typically be used to improve resolution
above 2.0 should be avoided. or sensitivity. This pretreatment may be a useful means of
simplifying the data and thereby reducing the variations that
Background correction
Select a suitable spectroscopic blank (e.g. air, blank solvent, may cause interference in subsequently applied mathematical
models.
solid material). Unless otherwise prescribed, all
measurements are carried out with reference to the same A wide range of treatment methods, such as scaling,
solvent or the same mixture of solvents (blank). smoothing, normalisation and derivatisation, can be applied
either singly or in combination. More information is available
Measure the blank and the sample within a short time-frame
either in parallel in double-beam spectrophotometers or in general chapter 5.21. Chemometric methods applied to
analytical data.
sequentially in single-beam spectrophotometers.
The absorbance values of both blank and sample must be in CONTROL OF EQUIPMENT PERFORMANCE
the working range of the equipment as specified by the Spectrophotometer performance is controlled (automatically
manufacturer. or manually) at regular intervals as defined in the quality
For benchtop instruments, the absorbance of the solvent management system and dictated by the use of the
measured against air and at the prescribed wavelength must equipment and the application. For example, equipment
not exceed 0.4 and is preferably less than 0.2. exposed to variations in temperature and humidity may need
For chromatographic systems, the transmittance of the more frequent performance testing.
mobile phase may be used as the blank. Requirements for control of equipment performance for the
In some PAT applications, it may be impossible to remove various measurement modes are summarised in
the probe for background data collection. Various options are Table 2.2.25.-1. Further such tests may be performed if
therefore to be considered, including the use of internal appropriate.
references, measurement of a blank using a second detector, Wavelength accuracy, absorbance accuracy and linearity are
etc. Only spectra measured against a blank possessing the controlled using either certified reference materials such as
same optical properties can be directly compared with one solid filters or liquid filters in appropriate sealed cells, or
another. solutions prepared in the laboratory as described below.
2023 Appendix II B V-A195

Control of wavelength accuracy filter or the absorbance value that is calculated from a
Control the wavelength accuracy of an appropriate number certified specific absorbance. Nicotinic acid for equipment
of bands in the intended spectral range using one or more qualification CRS may be used.
reference materials; for example, use solid or liquid filters It is recommended to test absorbance accuracy at selected
(e.g. holmium perchwrate solution R) to verify the position of wavelengths using one or more solid or liquid filters with
absorption bands, or measure the emission from a light different absorbance levels; as a minimum, values at
source to check emission-line position. Table 2.2.25.-2 shows approximately the 2 limits of the expected absorbance range
examples of wavelengths used to check wavelength accuracy. should be verified.
When certified reference materials are used, the reference
For chromatographic systems and PAT applications, the
wavelength is that stated on the corresponding certificate.
testing of absolute absorbance accuracy may not be
Some instruments may have an automatic or inbuilt necessary, providing that a standard curve is measured as
wavelength accuracy control feature. required.
Table 2.2.25.-2. - Examples of wavelengths used for the control For measurements using nicotinic acid for equipment
of wavelength accuracy'Note: the wavelength varies with the qualification CRS, the certified specific absorbance is given in
resolution of the instrument the corresponding leaflet.
The solution of nicotinic acid can be prepared as follows:
Material Wavelengths (nm)'
dissolve 57.0-63.0 mg of nicotinic acid for equipment
Solutions Cerium in sulfuric acid 201.1; 211.4; 222.6; 240.4; qualification CRS in a 0.1 M hydrochloric acid solution
253.7 prepared from hydrochloric acid Rand dilute to 200.0 mL
Didymium in perchloric with the same acid solution; dilute 2.0 mL of the solution to
acid 511.8; 731.6; 794.2
50.0 mL with the same acid solution to obtain a final
Holmium in perchloric 241.1; 287.2; 361.3; 451.4;
concentration of 12 mg/L. These volumes can be adjusted to
acid 485.2; 536.6; 640.5 obtain nicotinic acid solutions with other concentrations (up
to about 40 mg/L), for the purposes of testing different
Solid filters Didymium glass 513.5 absorbance levels. The absorbance is measured at 213 nm
Holmium glass 279.3; 360.9; 453.4; 637.5 and 261 nm.
Acceptance criteria
Lamps Deuterium 486.0; 656.1
The difference between the measured absorbance and the
184.9; 253.7; 312.5; 365.0; absorbance of the certified material is ± 0.010 or
Mercury (low pressure) 404.7; 435.8; 546.1; 577.0;
579.1
± 1 per cent, whichever is greater, for each combination of
wavelength and absorbance assessed (applies to absorbance
Neon 717.4 values not greater than 2). Tolerances for higher absorbance
Xenon 541.9; 688.2; 764.2 values should be defined on the basis of a risk assessment.
Control of photometric linearity
For chromatographic systems, it is also possible to control Control the photometric linearity in the intended spectral
wavelength accuracy by measuring the absorbance of a range. In the ultraviolet range, the filters used to control
0.05 mg/mL solution of caffeine R in methanol R; the absorbance accuracy may be used, as can solutions of
absorption maximum is obtained at 272 nm and the nicotinic acid or caffeine. In the visible range, neutral glass
minimum at 244 nm. filters may be used. Prior to performing the test, ensure that
the absorbance of the standards is compatible with the
Acceptance criteria
intended linear range.
It is recommended to test at least 2 wavelengths that bracket
the intended spectral range. Solutions with increasing concentrations (e.g. 5-40 mg/L) of
nicotinic acid for equipment qualification CRS in a O.1 M
For benchtop instruments, the tolerance for wavelength
hydrochloric acid solution prepared from hydrochwric acid R
accuracy of UV-Vis spectroscopy in cuvettes is± 1 nm at may be used. The absorbance is measured at 213 nm and
wavelengths below 400 nm and ± 3 nm at wavelengths of
261 nm.
400 nm and above.
For chromatographic systems, it is also possible to check
For chromatographic systems, the tolerance for wavelength
photometric linearity using 0.5-50 mg/L solutions of
accuracy is ± 2 nm for the whole UV-Vis range. caffeine R in water for chromatography R. The absorbance is
For PAT applications, a tolerance of± 2 nm for the UV-Vis measured at 273 nm.
range is recommended. However, wider tolerance intervals Acceptance criterion
may be needed for some PAT applications, in which case the
requisite wavelength accuracy must be defined by the user The coefficient of determination (R) is not less than 0.999.
depending on the intended purpose, and using a risk-based Limit of stray light
approach. Stray light is determined at an appropriate wavelength using
The instrument parameters (especially the entrance optics suitable solid or liquid filters or solutions prepared in-house.
such as slit-width or optical fibre diameter) influence the The instrument parameters used for the test, such as slit-
resolution and must be the same as those intended for the width and type of light source (e.g. deuterium or tungsten
actual measurements. lamp), must be the same as those intended for the actual
measurements.
Control of absorbance accuracy
Control the absorbance accuracy at an appropriate number
Acceptance criterion
of wavelengths in the intended spectral range, using suitable The acceptance criterion depends on the filters or solutions
solid or liquid filters to check that the absorbance measured used, for example:
at the test wavelength matches the certified absorbance of the
V-A196 Appendix II C 2023

- the absorbance is not less than 3.0 when using a 10 g/L frequency (ro 1) of the B 1 field may be varied to achieve
solution of sodium iodide R at 220 nm, a 10 g/L solution a spectrum (continuous wave (CW) method). Nowadays the
of potassium iodide R at 250 nm or a 50 g/L solution of B 1 irradiation is achieved by the use of a radiofrequency (RF)
sodium nitrite Rat 340 nm and 370 nm; pulse (Fourier transform (FT) method). The coherent
- the absorbance is not less than 2.0 when using a 12 g/L radiation emitted during the return to the initial state is
solution of potassium chloride R at 198 nm. observed in the form of a decay curve, called the free
These values apply when using a 1 cm cell and water R as induction decay (FID). Subsequent Fourier transformation
the compensation liquid. gives the spectrum in the frequency domain, providing
information about the molecular structure. Additional
Control of resolution
radiofrequency fields may be applied during acquisition of
Where prescribed in a monograph, measure the resolution of
the FID signal to suppress scalar (through-bond) interactions
the equipment either using suitable certified reference
between nuclei (called 'decoupling'). One- and multi-
materials, or by recording the spectrum of a
dimensional techniques can be applied for qualitative and
0.02 per cent V/V solution of toluene R in hexane R or
quantitative purposes, on samples in either the liquid or the
heptane R, with respectively hexane R or heptane R as the
solid state.
compensation liquid.
Important structural information is derived from the
Acceptance criterion
following spectroscopic features:
For measurements taken with a solution prepared as
described above, the minimum ratio of the absorbance at the
resonance frequency kind of nuclei observed
maximum (269 nm) to that at the minimum (266 nm) is
stated in the monograph. number of resonance signals number of chemically distinct groups
(singlets, multiplets) of nuclei
SYSTEM SUITABILITY
System suitability tests may be required prior to sample chemical shift o (ppm) chemical nature and environment of
the structural group observed
measurement to verify critical parameters that may have an
impact on the result. intensity of resonance signals relative number of resonant nuclei
These tests may cover wavelength accuracy, absorbance per chemically distinct group
accuracy, stray light and photometric linearity. System multiplicity of coupling pattern number of nuclei that are scalar
functionality tests, for example those performed as part of coupled to the observed nucleus
equipment autotesting, may be considered part of the system
suitability tests. coupling constant "J (Hz) number of bonds in the coupling
pathway, and its geometry
In the case of UV-Vis detection for chromatographic systems,
additional system suitability tests are applicable if prescribed
in the monograph and/or in general chapter Correlations of different spectral parameters (e.g. chemical
2.2.46. Chromatographic separation techniques. shift and coupling constant, or chemical shifts of different
nuclei within one molecular system) can be performed by
homo- and hetero-nuclear two- and higher-dimensional
methods. Information about the relaxation times T 1 and T2 ,
nuclear Overhauser effects (NOEs) and the kinetics of time-
C. Nuclear Magnetic Resonance dependent processes are also accessible from appropriate
Spectrometry experiments.
(Ph. Bur. method 2.2.33) APPARATUS
A high-resolution NMR spectrometer consists of at least the
INTRODUCTION
following parts:
Nuclear magnetic resonance (NMR) spectrometry is an
a magnet to deliver the constant magnetic field B0 ;
analytical method in particular suitable for the elucidation of
a temperature-controlled probe to contain the sample, to
the chemical structure of organic molecules by means of
deliver the radiofrequency pulse and to detect radiation
interpretation of their NMR spectra, arising from, for
emitted by the sample;
example, 1 H or the X-nuclei 13 C, 19F, 15 N, 31 P. The spectra
an electronic console to generate high-power
can be used for qualitative and quantitative purposes.
radiofrequency pulses and to collect and digitise the FID
Under suitable experimental conditions, the integrated NMR signal; this unit also maintains the stability of the
intensities of the signals are directly proportional to the instrument electronics;
number of nuclear spins of the molecular group responsible a data acquisition and processing unit (computer);
for the signal. These integrals can be used for quantitative
and may also include:
analysis.
- a continuous flow cell for coupled liquid
GENERAL PRINCIPLES chromatographic-NMR or flow injection analysis;
Placing an ensemble of nuclei with angular momentum and a - a system for pulsed field gradient NMR.
magnetic moment in a static magnetic field (B0 ) causes the The high magnetic field is generated by a superconducting
nuclei to arrange themselves in different, quantum- coil in a Dewar flask filled with liquid helium. The probe
mechanically controlled orientations in relation to the axis of typically contains the sample in a 5 mm-outer-diameter
the magnetic field. These orientations are different in energy. sample tube or in a flow cell, and is connected to the
An oscillating high-frequency magnetic field (B 1 ), electronics cabinet by RF cables carrying lock, 1H-, and
perpendicular to B0 , will cause transitions between these X-nucleus frequencies. Additional devices for tuning and
orientations with net energy absorption. According to the matching the electronic circuits are essential, and sample
resonance condition ro 0 = yB0 (y =gyromagnetic ratio, temperature control is often used.
ro 0 = Larmor frequency), either the Bo magnetic field or the
2023 Appendix II C V-Al 97

The NMR spectrometer should be demonstrated to be Maximal signal intensity and signal-to-noise ratio will be
operating correctly. Appropriate tests to demonstrate this are, achieved if tac ·::::: 1.21(1tv112), where Vv 2 is the full width at
typically, measurement of linewidths at half height for half-height (fwhh), but it should be set to greater than
defined peaks under defined acquisition conditions, signal-to- 5/(Tivu2) to minimise signal distortion.
noise ratios (SIN) for standard mixtures, pulse power Repetition time (tr)
(measured as a 90° pulse width), and pulse reproducibility. The spin-lattice relaxation (T1 ) governs the time required for
All instrument manufacturers publish specifications and the spin system to return to equilibrium after a pulse.
measurement protocols for these parameters for specific Relaxation can be reduced by the use of special reagents.
instrument/probe combinations, and compliance with these For quantitative purposes, the repetition time used should be
specifications should be demonstrated. set relative to T1 and 0 to avoid saturation effects.
FOURIER TRANSFORM NMR (FT-NMR) Receiver gain
Contemporary spectrometers generally operate according to The analogue signal detected by the probe is amplified prior
the Fourier transform (FT) principle: after exciting the to digitisation and storage. The amplification, or receiver
sample with a radiofrequency pulse of appropriate frequency gain, should be set, either automatically or manually, so that
(v), amplitude (B 1) and duration (,p) and a succeeding short the signal does not overload the ADC, which causes signal
dead time (td) (to enable the electronics to recover), the distortion, but allows random noise generated in the probe to
amplified analogue FID signal is sampled during the be digitised (i.e. is non-zero).
acquisition time (tac) and digitised with an analogue-to-digital
OPTIMISATION OF ACQUISITION AND
converter (ADC), and the results are stored in the
PROCESSING PARAMETERS FOR QUANTITATIVE
spectrometer memory. The receiver output is amplified prior
PURPOSES
to digitisation to maximise sensitivity without saturating the
Besides the acquisition parameters, signal intensity is also
ADC. In case of observation of X-nuclei, the standard
influenced by several processing parameters. After collecting
experiment includes, if necessary, broadband 1 H decoupling,
a sufficient number of scans, the resulting FID is Fourier
i.e. irradiation of all the protons during the experiment.
transformed. For reliable quantitative purposes the following
To increase the SIN, multiple FID signals may be
parameters have to be optimised.
accumulated coherently and summed. Fourier transformation
of this time-domain data gives the frequency-domain Digital resolution
spectrum. The digital resolution is the frequency separation between
data points. The processed signal should have at least 5
PARAMETERS
digital points above half-height of the signals to be integrated.
The following acquisition parameters influence the result of
To improve the digital resolution additional points of zero
an FT experiment, and should be adjusted and controlled.
intensity may be added to the end of the experimental FID
Pulse width ( tp) before transformation ('zero filling').
The excitation pulse is directed along the x-axis of the
Signal-to-noise ratio (SIN)
so-called rotating frame, its duration (or 'width', "tp)
This is the ratio between the intensities (as peak height) of a
determines the flip angle (0) and thus the intensity (I) of the
specified signal in the NMR spectrum and the random
resonance signal:
fluctuations in that signal, which is usually measured in a
region of the spectrum that contains no signals from the
(1)
analyte. A poor signal-to-noise ratio (SIN) limits the accuracy
of peak integrations and quantitative analyses. An SIN equal
to or greater than 150: 1 allows peak integrations with a
My =M, xsin A (2) standard deviation of less than 1 per cent. Contemporary
spectrometers have software algorithms to report the SIN of
appropriate peaks. A sufficiently high SIN can be difficult to
The observed magnetisation My is maximum at 0 = 90°. obtain when analysing dilute solutions, and especially when
The pulse duration should be short to guarantee that all detecting nuclei other than 1H. Methods to enhance the SIN
signals in the spectral width (SW') are excited to a similar include:
degree. The magnetisation decays due to relaxation - increasing the number of accumulated scans (n), as SIN
processes. increases with Jn;
Dead time (td) - use of exponentional multiplication on the FID signal
The dead time is the time between the end of the pulse and before Fourier transformation; the exponentional
start of the acquisition, it is necessary for technical reasons multiplication factor should be in the order of the peak
and care should be taken as it may influence signal intensities full width at half-height (j-tJJhh);
and peak phase. Rapidly decaying signals (giving rise to - use of spectrometers with a higher magnetic field B0 ,
broad spectral lines) are reduced in intensity by more than since SIN is proportional to Ba312 ;
slowly decaying signals (which give rise to narrow spectral - use of digital filtering to reduce noise;
lines). - use of probes that maximise the filling factor;
Acquisition time (tac) - use of cryoprobes that reduce thermal noise.
The acquisition time (tac) is related to the spectral width Integration region
(i.e. the whole observed region) and the number of digital The intensity of the NMR signals is obtained by a quasi-
data points (DP) collected during signal acquisition. analogue signal integration either by a stepped-line plot or,
more accurately, by separate line integration and digital data
DP
la,·= 2SU7 (3) presentation. In liquid state, NMR signals have Lorentzian
line shape. Unless otherwise specified in the monograph or
when peak overlap occurs, the same integration range,
V-Al 98 Appendix II C 2023

expressed as a multiple of the peak fwhh, should be used for Other

Nucleus Water• Bx.reference Sx,reference
the monitored peak and the reference peak. solvents

Dynamic range 'H DSSb 1.00000000 TMS 1.00000000

The dynamic range of the analogue-to-digital converter
(ADC) detem1ines the minimum intensity line that can be "c DSSb 0.25144953 TMS 0.25145020

observed or quantified when integrating 2 signals with the "N NH, 0.10132912 CH3N0 2 0.10136767
same linewidth in a spectrum. A 16-bit ADC allows
19F CF3 COOH not reported CCl3 F 0.94094011
identification of a signal of 0.003 per cent intensity relative to
a strong signal completely filling the dynamic range of the 31p H 3 P0 4 0.40480742 (CH3 0) 3 PO 0.40480864
ADC. (85 per cent)

NMR OF SAMPLES IN SOLUTION achemical shift depends on pH

Most NMR experiments are performed on dilute solutions
(about 1 per cent) of the analyte in an appropriate solvent, bDSS = sodium 2,2-dimethyl-2-silapentane-5-sulfonate

which can be spiked with a suitable standard for chemical

shift calibration. In practice, X chemical shifts are referenced directly using an
Solvents appropriate standard. In 1H and 13 C NMR, internal
The solvent should be able to dissolve the analyte without referencing is mainly used, where the reference is added
further interaction if not otherwise intended. To minimise directly to the system under study. In 15N, 19F and 31 P
the intense solvent signals, fully deuterated solvents NMR, external referencing is often suitable, involving sample
(deuterium oxide R, deuterated chloroform R, deuterated dimethyl and reference contained separately in coaxial cylindrical
sulfoxide R, deuterated acetone R, deuterated methanol R, etc.) sample tubes.
should be used. The solvent atoms give signals that are easily Lock
identified by their chemical shift and can be used to calibrate In order to prevent drifting of the spectrum over time, a
the chemical shift axis (secondary reference). stabilising procedure, called field-frequency locking, is
Referencing performed. The 2 H (deuterium) signal arising from
The spectral feature most dependent on the chemical deuterated solvents is used to achieve this, unless otherwise
environment of the atom in the molecule is the chemical specified in the monograph.
shift, designated as Ii and reported in parts per million. QUAUTATIVE ANALYSIS
The chemical shift between the resonance for an NMR active The principal use for qualitative NMR spec.tra is as an
nucleus X (lix,sampie) is measured in parts per million as the identity test, in which the 1H or 13 C spectrum of a test
difference between the resonance frequency of that nucleus sample is compared to the spectrum of a reference sample or,
(vx,sampie) and that of an internal shift reference standard (vx, less commonly, with a published reference spectrum. Spectra
reference), both in hertz, divided by the basic spectrometer of reference and test samples should be acquired using the
operating frequency (vx,refcrence), in megahertz, at a given B0 : same procedure and operational conditions. The peaks in the
2 spectra, or characteristic regions of the spectra, should
( Vx,~amplc - vx,rcfcrence) (4) correspond in position, intensity and multiplicity.
VX.reference In appropriate cases, mathematical comparison, such as
calculation of a correlation coefficient, may be appropriate.
By convention, the standard for exact chemical shift In the absence of a reference standard, an in-house reference
referencing is the 1H resonance of tetramethylsilane R (TMS), may be used, whose identity has been demonstrated by
setting ◊TMs = 0 ppm. Formally, once the 1H shift scale has alternative methods, or the demonstration that the NMR
been referenced relative to TMS, the exact frequency of any spectrum is fully consistent with the reported structure for
other X resonance can be calculated and its chemical shift that material.
scale calibrated. The frequency of a (secondary) reference QUANTITATIVE ANALYSIS
standard at Ox = 0 ppm (vx,refcrcnce) is calculated from the Signal intensity in the basic NMR experiment is the
1H frequency of TMS (vH,TMs) and a tabulated value of the
integrated area under the signal curve measured. Only when
ratio (Bx.reference) of the isotope-specific frequency in relation 2 signals have equal fwhh and the same multiplicity may
to that of' 1 H in TMS: signal height serve as a measure of intensity. Under
conditions of essentially complete relaxation between scans,
l'X,rcfcrcnu: = VJ-1.TMS X - 1OOOOfilx.rcfc:n:m.:c (5)
the signal intensity (JA) is a true measure of the number (NA)
of nuclei responsible for the respective signal:
Reference standards at lix = 0 ppm and corresponding Bx,
(6)
reference values are shown below:

The constant Ks includes fundamental constants, properties

of the sample and receiver parameters, and can be omitted in
cases where signal intensities are compared, giving the direct
relation between the numbers of nuclei in the 2 compared
structure groups A and B:

(7)
2023 Appendix II D V-Al 99

The numbers (N,) of nuclei belonging to different structure introduced into the NMR magnet, the experimental
groups of 1 molecule are small integers. The values measured parameters are loaded and the experiment is executed.
are rounded to the closest integer numbers. However, the Key experimental parameters are indicated in the
proper operation of acquisition and processing of the monograph.
spectrometer is easily checked by comparing exact intensities The measurement procedure
within a spectrum of any suitable organic compound of Equilibrate the sample in the probe, and optimise the
known structure. instrument to achieve best resonance conditions and to
In addition to the fact that the intensities of signals arising maximise the SIN by tuning and matching the probe, and
from each component in a mixture are related to each other make adjustments to maximise magnetic field homogeneity
by small integer numbers, the relative molar amounts of over the sample volume (called 'shimming'). Record, or save
these components can be measured by comparison of the to computer, the parameter settings. An experiment may be
normalised intensities of resonances from different composed of multiple pulse-acquisition-delay sequences, and
components. The molar ratio of 2 components of a mixture the individual FIDs are summed in the computer memory,
is calculated according to the following equation: with random noise being averaged out. When an appropriate
SIN has been achieved, the FID is stored and the frequency-
nA IA Ns
-=-X- (8) domain spectrum is generated by Fourier transformation of
nB ls 1VA
the summed FIDs.
NMR IN THE SOLID STATE
The determination is only valid in cases where the structure
Samples in the solid state can be analysed using NMR
of the molecules for which JA and / 8 are determined are
spectrometers specially equipped for that purpose. Certain
known (or at least the values of Nfor the monitored groups).
technical procedures make observable individual lines for
Determinations are made using either an internal standard
individual atomic sites with a valuable extension of the
method or a peak-normalisation procedure.
applicability of NMR to inorganic materials as well.
Internal standard method
One technique is the rapid rotation (4-30 kHz) of the
The mass (mA) of an analyte (A) can be determined if a
powdered sample in a rotor (about 4 mm outer diameter)
known mass (mn) of a substance (B) with a known
inclined at an angle of 54. 7° (the 'magic angle') to the
percentage content (Pn) is added to the solution as an
direction of the B 0 magnetic field axis. This technique is
intensity standard. Equation (8) can be converted to equation
named magic angle spinning (MAS). Another effective tool is
(9):
high-power decoupling and a 3 rd method is the transfer of
polarisation from easily excitable nuclei towards less-
(9)
polarisable nuclei, i.e. cross polarisation (CP).
The combination of these techniques makes available high-
resolution spectra containing much information about
Here, M; are the molecular masses.
chemical and structural details in solid glassy, amorphous,
The intensity standard has to be carefully chosen; it should and crystalline materials of ceramic, polymeric or
be completely soluble in the solvent used for the analyte, mineralogical origin.
should produce only a small number of signals, and the
If NMR is applied to a solid, full details of the procedure are
'monitor group' should have a signal in an empty spectral
provided in the monograph.
region. A compound of high purity and with a relatively high
molecular mass is recommended for this purpose.
Normalisation procedure
The relative proportions of components in a mixture, the
degree of substitution in a structurally modified polymer, or
D. Atomic Spectrophotometry: Emission
the amount of a contaminant can be determined by and Absorption
comparison of the relative intensities of resonances present.
The experimental method should be validated to ensure that Atomic Emission Spectrometry
there is no overlap of the relevant signals. When the (Ph. Bur. method 2.2.22)
contaminant is of poorly defined structure or molecular mass GENERAL PRINCIPLE
(e.g. an emulsifier), addition of known amounts of that Atomic emission is a process that occurs when
material to the NMR tube will allow a calibration curve to be electromagnetic radiation is emitted by excited atoms or ions.
constructed. In atomic emission spectrometry the sample is subjected to
METHOD temperatures high enough to cause not only dissociation into
Sample handling atoms, but also to cause significant amounts of collisional
Dissolve the sample in the solvent to which the appropriate excitation and ionisation of the sample atoms to take place.
reference material may have been added to calibrate chemical Once the atoms and ions are in the excited states, they can
shift, as prescribed in the monograph. For quantitative decay to lower states through thermal or radiative (emission)
analysis, the solutions must be free from solid particles. Some energy transitions and electromagnetic radiation is emitted.
quantitative analyses may require an internal standard to be An emission spectrum of an element contains several more
included, so that integrations of resonances from the test lines than the corresponding absorption spectrum.
sample and the reference material can be compared. Atomic emission spectrometry is a technique for determining
Appropriate references and concentrations are indicated in the concentration of an element in a sample by measuring
the specific monographs. In other quantitative analyses, the the intensity of one of the emission lines of the atomic
result is obtained by comparing the relative intensities of 2 or vapour of the element generated from the sample.
all of the resonances that arise from the test sample. After The determination is carried out at the wavelength
loading the sample into a tube and capping, the sample is corresponding to this emission line.
V-A200 Appendix II D 2023

In this chapter only atomisation in flame is dealt with. Prepare the solution of the substance to be examined (test
The method of inductively coupled plasma-atomic emission solution) as prescribed in the monograph. Prepare not fewer
spectrometry (ICP-AES) is described in a different general than 3 reference solutions of the element to be determined,
chapter. the concentrations of which span the expected value in the
APPARATUS test solution. For assay purposes, optimal calibration levels
are between O. 7 and 1.3 times the expected content of the
This consists essentially of:
element to be determined or the limit prescribed in the
- a sample introduction and nebulisation system;
monograph. For purity determination, calibration levels are
- a flame to generate the atoms to be determined;
between the limit of detection and 1.2 times the limit
- a monochromator;
specified for the element to be determined. Any reagents
- a detector;
used in the preparation of the test solution are added to the
- a data-acquisition unit.
reference solutions and to the blank solution at the same
Oxygen, air and a combustible gas such as hydrogen, concentration.
acetylene, propane or butane may be used in flames.
Introduce each of the solutions into the instrument using the
The atomisation source is critical, since it must provide
same number of replicates for each solution, to obtain a
sufficient energy to excite and atomise the atoms. The atomic
steady reading.
spectra emitted from flames have the advantage of being
simpler than those emitted from other sources, the main Calculation
limitation being that the flames are not powerful enough to Prepare a calibration curve from the mean of the readings
cause emission for many elements allowing their obtained with the reference solutions by plotting the means
determination. Acidified water is the solvent of choice for as a function of concentration. Determine the concentration
preparing test and reference solutions, although organic of the element in the test solution from the curve obtained.
solvents may also be used if precautions are taken to ensure METHOD II - STANDARD ADDITIONS
that the solvent does not interfere with the stability of the Add to at least 3 similar volumetric flasks equal volumes of
flame. the solution of the substance to be examined (test solution)
INfERFERENCES prepared as prescribed. Add to all but 1 of the flasks
Spectral interference is reduced or eliminated by choosing an progressively larger volumes of a reference solution
appropriate emission line for measurement or by adjusting containing a known concentration of the element to be
the slit for spectral band-width. Physical interference is determined to produce a series of solutions containing
corrected by diluting the sample solution, by matching the steadily increasing concentrations of that element known to
matrix or by using the method of standard additions. give responses in the linear part of the curve, if at all
Chemical interference is reduced by using chemical modifiers possible. Dilute the contents of each flask to volume with
or ionisation buffers. solvent.
Introduce each of the solutions into the instrument using the
MEMORY EFFECT
same number of replicates for each solution, to obtain a
The memory effect caused by deposit of analyte in the steady reading.
apparatus may be limited by thoroughly rinsing between
runs, diluting the solutions to be measured if possible and Calculation
thus reducing their salt content, and by aspirating the Calculate the linear equation of the graph using a least-
solutions through as swiftly as possible. squares fit, and derive from it the concentration of the
element to be determined in the test solution.
METHOD
Use of plastic labware is recommended wherever possible. VALIDATION OF THE METHOD
Satisfactory performance of methods prescribed in
Operate an atomic emission spectrometer in accordance with
monographs is verified at suitable time intervals.
the manufacturer's instructions at the prescribed wavelength.
Optimise the experimental conditions (flame temperature, LINEARIIY
burner adjustment, use of an ionic buffer, concentration of Prepare and analyse not fewer than 4 reference solutions over
solutions) for the specific element to be analysed and in the calibration range and a blank solution. Perform not fewer
respect of the sample matrix. Introduce a blank solution into than 5 replicates.
the atomic generator and adjust the instrument reading to The calibration curve is calculated by least-square regression
zero or to its blank value. Introduce the most concentrated from all measured data. The regression curve, the means, the
reference solution and adjust the sensitivity to obtain a measured data and the confidence interval of the calibration
suitable reading. curve are plotted. The operating method is valid when:
It is preferable to use concentrations which fall within the - the correlation coefficient is at least 0. 99,
linear part of the calibration curve. If this is not possible, the - the residuals of each calibration level are randomly
calibration plots may also be curved and are then to be distributed around the calibration curve.
applied with appropriate calibration software. Calculate the mean and relative standard deviation for the
Determinations are made by comparison with reference lowest and highest calibration level.
solutions with known concentrations of the element to be When the ratio of the estimated standard deviation of the
determined either by the method of direct calibration lowest and the highest calibration level is less than 0.5 or
(Method I) or the method of standard additions (Method II). greater than 2.0, a more precise estimation of the calibration
METHOD I - DIRECT CALIBRATION curve may be obtained using weighted linear regression. Both
For routine measurements 3 reference solutions of the linear and quadratic weighting functions are applied to the
element to be determined and a blank are prepared and data to find the most appropriate weighting function to be
examined. employed. If the means compared to the calibration curve
show a deviation from linearity, two-dimensional linear
regression is used.
2023 Appendix II D V-A201

ACCURACY consisting of support-gas atoms, electrons and support-gas

Verify the accuracy preferably by using a certified reference ions. The plasma is then sustained within the torch and load
material (CRM). Where this is not possible, perform a test coil as radio-frequency energy is continually transferred to it
for recovery. through the inductive coupling process.
Recovery The ICP appears as an intense, very bright, plume-shaped
For assay determinations a recovery of 90 per cent to plasma. At the base the plasma is toroidal, and this is
110 per cent is to be obtained. For other determinations, for referred to as the induction region (IR), i.e. the region in
example for trace element determination, the test is not valid which the inductive energy transfer from the load coil to the
if recovery is outside of the range 80 per cent to 120 per cent plasma takes place. The sample is introduced through the
at the theoretical value. Recovery may be determined on a induction region into the centre of the plasma.
suitable reference solution (matrix solution) which is spiked APPARATUS
with a known quantity of analyte (middle concentration of
The apparatus consists essentially of the following elements:
the calibration range). - sample-introduction system consisting of a peristaltic
REPEATABIU"IY pump delivering the solution at constant flow rate into a
The repeatability is not greater than 3 per cent for an assay nebuliser;
and not greater than 5 per cent for an impurity test. - radio-frequency (RF) generator;
LIMIT OF QUANTIFICATION - plasma torch;
Verify that the limit of quantification (for example, - transfer optics focussing the image of the plasma at the
determined using the 10 cr approach) is below the value to entrance slit of the spectrometer; radial viewing is better
be measured. for difficult matrices (alkalis, organics), whereas axial
viewing gives more intensity and better detection limits in
Inductively Coupled Plasma-atomic Emission simple matrices;
Spectrometry - wavelength dispersive devices consisting of diffraction
(Ph. Bur. method 2.2.57) gratings, prisms, filters or interferometers;
GENERAL PRINCIPLE - detectors converting radiant energy into electrical energy;
Inductively coupled plasma-atomic emission spectrometry - data-acquisition unit.
(ICP-i\ES) is an atomic emission spectrometry method that INTERFERENCE
uses an inductively coupled plasma (ICP) as the excitation Interference is anything that causes the signal from an analyte
source. in a sample to be different from the signal for the same
An ICP is a highly ionised inert gas (usually argon) with concentration of that analyte in a calibration solution.
equal numbers of electrons and ions sustained by a radio- The well-known chemical interference that is encountered in
frequency (RF) field. The high temperature reached in the flame atomic absorption spectrometry is usually weak in ICP-
plasma successively desolvates, vaporises, excites - atomic AES. In rare cases where interference occurs, it may be
emission spectrometry (AES) detection - and ionises - mass necessary to increase the RF power or to reduce the inner
spectrometry (MS) detection - atoms from the sample. support-gas flow to eliminate it. The interference in ICP-
Detection limits are, generally, in the lower nanogram (ICP- AES can be of spectral origin or even the result of high
MS) to microgram (ICP-AES) per litre range. concentrations of certain elements or matrix compounds.
The plasma is formed by a tangential stream of support gas Physical interference (due to differences in viscosity and
through a 'torch', i.e. a system consisting of 3 concentric surface tension of the sample and calibration standards) can
quartz tubes. A metal coil (the load coil) surrounds the top be minimised by dilution of the sample, matrix matching, use
end of the torch and is connected to a radio-frequency (RF) of internal standards or through application of the method of
generator. Power (usually 700-1500 W) is applied through standard additions.
the coil and an oscillating magnetic field corresponding to the Another type of interference occasionally encountered in
frequency of the generator (in most cases 27 MHz, 40 MHz) ICP-AES is the so-called 'easily ionised elements (EIEs)
is formed. The plasma forms when the support gas is made effect'. The EIEs are those elements that are ionised much
conductive by exposing it to an electric discharge, which more easily, for example alkaline metals and alkaline earths.
produces seed electrons and ions. Inside the induced In samples that contain high concentrations of EIEs (more
magnetic field, the charged particles (electrons and ions) are than 0.1 per cent), suppression or enhancement of emission
forced to flow in a closed annular path. As they meet signals is likely to occur.
resistance to their flow, heating takes place producing Spectral interference
additional ionisation. The process occurs almost This may be due to other lines or shifts in background
instantaneously, and the plasma expands to its full strength intensity. These lines may correspond to argon (observed
and dimensions. The radio-frequency oscillation of the power above 300 nm), OH bands due to the decomposition of
applied through the coil causes radio-frequency electric and water (at about 300 nm), NO bands due to the interaction of
magnetic fields to be set up in the area at the top of the the plasma with the ambient air (between 200 nm and
torch. When a spark (produced by a Tesla tube or some 300 nm), and other elements in the sample, especially those
other seeding device) is applied to the support gas flowing present at high concentrations. The interference falls into 4
through the torch, some electrons are stripped from the different categories: simple background shift, sloping
support gas atoms. These electrons are then caught up in the background shift, direct spectral overlap, and complex
magnetic field and accelerated. Adding energy to the background shift.
electrons by the use of a coil is known as inductive coupling.
Absorption interference
These high-energy electrons in tum collide with other
This arises when part of the emission from an analyte is
support-gas atoms, stripping off still more electrons.
absorbed before it reaches the detector. This effect is
The collisional ionisation of the support gas continues in a
observed particularly when the concentration of a strongly
chain reaction, breaking down the gas into a physical plasma
V-A202 Appendix II D 2023

emitting element is so high that the atoms or ions of that CONTROL OF INSTRUMENT PERFORMANCE
element that are in the lower energy state of transition absorb System suitability
significant amounts of the radiation emitted by the relevant The following tests may be carried out with a multi-element
excited species. This effect, known as self-absorption, control solution to ensure the adequate performance of the
determines the upper end of the linear working range for a ICP-AES system:
given emission line. - energy transfer (generator, torch, plasma); measurement
Multicomponent spectral fitting of the ratio Mg II (280.270 nm)/Mg I (285.213 nm) may
Multiple emission-line determinations are commonly used to be used;
overcome problems with spectral interferences. A better, - sample transfer, by checking nebuliser efficiency and
more accurate method for performing spectral interference stability;
corrections is to use the information obtained with advanced - resolution (optical system), by measuring peak widths at
detector systems through multicomponent spectral fitting. half height, for example As (189.042 nm), Mn
This quantifies not only the interference, but also the (257.610 nm), Cu (324.754 nm) or Ba (455.403 nm);
background contribution from the matrix, thereby creating a - analytical performance, by calculating detection limits of
correction formula. Multicomponent spectral fitting utilises a selected elements over the wavelength range.
multiple linear-squares model based on the analysis of pure VALIDATION OF THE METHOD
analyte, the matrix and the blank, creating an interference- Satisfactory performance of methods prescribed in
corrected mathematical model. This permits the monographs is verified at suitable time intervals.
determination of the analyte emission in a complex matrix
LINEARITY
with improved detection limits and accuracy.
Prepare and analyse not fewer than 4 reference solutions over
PROCEDURE the calibration range plus a blank. Perform not fewer than
SAMPLE PREPARATION AND SAMPLE 5 replicates.
INTRODUCTION The calibration curve is calculated by least-square regression
The basic goal for the sample preparation is to ensure that from all measured data of the calibration test. The regression
the analyte concentration falls within the working range of curve, the means, the measured data and the confidence
the instrument through dilution or preconcentration, and that interval of the calibration curve are plotted. The operating
the sample-containing solution can be nebulised in a method is valid when:
reproducible manner. - the correlation coefficient is at least 0. 99;
Several sample-introduction systems tolerate high acid - the residuals of each calibration level are randomly
concentrations, but the use of sulfuric and phosphoric acids distributed around the calibration curve.
can contribute to background emission observed in the ICP Calculate the mean and relative standard deviation for the
spectra. Therefore, nitric and hydrochloric acids are lowest and for the highest calibration level.
preferable. The availability of hydrofluoric acid-resistant (for
When the ratio of the estimated standard deviations of the
example perfluoroalkoxy polymer) sample-introduction
lowest and the highest calibration level is less than 0.5 or
systems and torches also allows the use of hydrofluoric acid.
greater than 2.0, a more precise estimation of the calibration
In selecting a sample-introduction method, the requirements
curve may be obtained using weighted linear regression. Both
for sensitivity, stability, speed, sample size, corrosion
linear and quadratic weighting functions are applied to the
resistance and resistance to clogging have to be considered.
data to find the most appropriate weighting function to be
The use of a cross-flow nebuliser combined with a spray
employed.
chamber and torch is suitable for most requirements.
The peristaltic pumps used for ICP-AES usually deliver the If the means compared to the calibration curve show a
standard and sample solutions at a rate of 1 mUmin or less. deviation from linearity, two-dimensional linear regression is
used.
In the case of organic solvents being used, the introduction
of oxygen must be considered to avoid organic layers. ACCURACY
Verify the accuracy preferably by using a certified reference
CHOICE OF OPERATING CONDITIONS
material (CRM). Where this is not possible, perform a test
The standard operating conditions prescribed by the
for recovery.
manufacturer are to be followed. Usually, different sets of
operating conditions are used for aqueous solutions and for Recovery
organic solvents. Suitable operating parameters are to be For assay determinations a recovery of 90 per cent to
properly chosen: 110 per cent is to be obtained. The test is not valid if
- wavelength selection; recovery, for example for trace-element determination, is
- support-gas flow rates (outer, intermediate and inner outside of the range 80 per cent to 120 per cent of the
tubes of the torch); theoretical value. Recovery may be determined on a suitable
- RF power; reference solution (matrix solution) spiked with a known
- viewing position (radial or axial); quantity of analyte (concentration range that is relevant to
- pump speed; the samples to be determined).
- conditions for the detector (gain/voltage for REPEATABILITY
photomultiplier tube detectors, others for array detectors); The repeatability is not greater than 3 per cent for an assay
- integration time (time set to measure the emission and not greater than 5 per cent for an impurity test.
intensity at each wavelength). LIMIT OF QUANTIFICATION
Verify that the limit of quantification (for example,
determined using the 10 cr approach) is below the value to
be measured.
2023 Appendix II D V-A203

Atomic Absorption Spectrometry The flow of an inert gas during the pyrolysis step in the
(Ph. Bur. method 2.2.23) graphite tube furnace allows a better performance of the
subsequent atomisation process.
GENERAL PRINCIPLE
- Cold vapour and hydride technique
Atomic absorption is a process that occurs when a ground
state-atom absorbs electromagnetic radiation of a specific The atomic vapour may also be generated outside the
wavelength and is elevated to an excited state. The atoms in spectrometer. This is notably the case for the cold-vapour
the ground state absorb energy at their resonant frequency method for mercury or for certain hydride-forming elements
and the electromagnetic radiation is attenuated due to such as arsenic, antimony, bismuth, selenium and tin.
resonance absorption. The energy absorption is virtually a For mercury, atoms are generated by chemical reduction
direct function of the number of atoms present. with stannous chloride or sodium borohydride and the
atomic vapour is swept by a stream of an inert gas into a cold
This chapter provides general information and defines the
quartz cell mounted in the optical path of the instrument.
procedures used in element determinations by atomic
Hydrides thus generated are swept by an inert gas into a
absorption spectrometry, either atomisation by flame, by
heated cell in which they are dissociated into atoms.
electrothermal vaporisation in a graphite furnace, by hydride
generation or by cold vapour technique for mercury. INTERFERENCES
Atomic absorption spectrometry is a technique for Chemical, physical, ionisation and spectral interferences are
determining the concentration of an element in a sample by encountered in atomic absorption measurements. Chemical
measuring the absorption of electromagnetic radiation by the interference is compensated by addition of matrix modifiers,
atomic vapour of the element generated from the sample. of releasing agents or by using high temperature produced by
The determination is carried out at the wavelength of one of a nitrous oxide-acetylene flame; the use of specific ionisation
the absorption (resonance) lines of the element concerned. buffers (for example, lanthanum and caesium) compensates
The amount of radiation absorbed is, according to the for ionisation interference; by dilution of the sample, through
Lambert-Beer law, proportional to the element the method of standard additions or by matrix matching,
concentration. physical interference due to high salt content or viscosity is
eliminated. Spectral interference results from the overlapping
APPARATUS of resonance lines and can be avoided by using a different
This consists essentially of: resonance line. The use of Zeeman background correction
- a source of radiation; also compensates for spectral interference and interferences
- a sample introduction device; from molecular absorption, especially when using the
- a sample atomiser; electrothermal atomisation technique. The use of multi-
- a monochromator or polychromator; element hollow-cathode lamps may also cause spectral
- a detector; interference. Specific or non-specific absorption is measured
- a data-acquisition unit. in a spectral range defined by the band-width selected by the
The apparatus is usually equipped with a background monochromator (0.2-2 nm).
correction system. Hollow-cathode lamps and electrodeless
BACKGROUND CORRECTION
discharge lamps (EDL) are used as radiation source.
Scatter and background in the flame or the electrothermal
The emission of such lamps consists of a spectrum showing
atomisation technique increase the measured absorbance
very narrow lines with half-width of about 0.002 nm of the
values. Background absorption covers a large range of
element being determined.
wavelengths, whereas atomic absorption takes place in a very
There are 3 types of sample atomisers: narrow wavelength range of about 0.005-0.02 nm.
- Flame technique Background absorption can in principle be corrected by using
A flame atomiser is composed of a nebulisation system with a a blank solution of exactly the same composition as the
pneumatic aerosol production accessory, a gas-flow regulation sample, but without the specific element to be determined,
and a burner. Fuel-oxidant mixtures are commonly used to although this method is frequently impracticable. With the
produce a range of temperatures from about 2000 K to 3000 electrothermal atomisation technique the pyrolysis
K. Fuel gases include propane, hydrogen and acetylene; air temperature is to be optimised to eliminate the matrix
and nitrous oxide are used as oxidants. The configuration of decomposition products causing background absorption.
the burner is adapted to the gases used and the gas flow is Background correction can also be made by using 2 different
adjustable. Samples are nebulised, acidified water being the light sources, the hollow-cathode lamp that measures the
solvent of choice for preparing test and reference solutions. total absorption (element + background) and a deuterium
Organic solvents may also be used if precautions are taken to lamp with a continuum emission from which the background
ensure that the solvent does not interfere with the stability of absorption is measured. Background is corrected by
the flame. subtracting the deuterium lamp signal from the hollow-
- Electrothermal atomisation technique cathode lamp signal. This method is limited in the spectral
An electrothermal atomiser is generally composed of a range on account of the spectra emitted by a deuterium lamp
graphite tube furnace and an electric power source. from 190-400 nm. Background can also be measured by
Electrothermal atomisation in a graphite tube furnace taking readings at a non-absorbing line near the resonance
atomises the entire sample and retains the atomic vapour in line and then subtracting the results from the measurement
the light path for an extended period. This improves the at the resonance line. Another method for the correction of
detection limit. Samples, liquid as well as solid, are background absorption is the Zeeman effect (based on the
introduced directly into the graphite tube furnace, which is Zeeman splitting of the absorption line in a magnetic field).
heated in a programmed series of steps to dry the sample and This is particularly useful when the background absorption
remove major matrix components by pyrolysis and to then shows fine structure. It permits an efficient background
atomise all of the analyte. The furnace is cleaned using a correction in the range of 185-900 nm.
final temperature higher than the atomisation temperature.
V-A204 Appendix II D 2023

CHOICE OF THE OPERATING CONDITIONS Introduce each of the solutions into the instrument using the
After selecting the suitable wavelength and slit width for the same number of replicates for each of the solutions to obtain
specific element, the need for the following has to be a steady reading.
ascertained: Calculation
- correction for non-specific background absorption, Prepare a calibration curve from the mean of the readings
- chemical modifiers or ionisation buffers to be added to obtained with the reference solutions by plotting the means
the sample as well as to blank and reference solutions, as a function of concentration. Determine the concentration
- dilution of the sample to minimise, for example, physical of the element in the test solution from the curve obtained.
interferences,
METHOD II - STANDARD ADDITIONS
- details of the temperature programme, preheating, drying,
Add to at least 3 similar volumetric flasks equal volumes of
pyrolysis, atomisation, post-atomisation with ramp and
the solution of the substance to be examined (test solution)
hold times,
prepared as prescribed. Add to all but 1 of the flasks
- inert gas flow,
progressively larger volumes of a reference solution
- matrix modifiers for electrothermal atomisation (furnace),
containing a known concentration of the element to be
- chemical reducing reagents for measurements of mercury
determined to produce a series of solutions containing
or other hydride-forming elements along with cold vapour
steadily increasing concentrations of that element known to
cell or heating cell temperature,
give responses in the linear part of the curve, if possible.
- specification of furnace design (tank, L'vov platform, etc).
Dilute the contents of each flask to volume with solvent.
METHOD Introduce each of the solutions into the instrument, using the
Use of plastic labware is recommended wherever possible. same number of replicates for each of the solutions, to obtain
The preparation of the sample may require a dissolution, a a steady reading.
digestion (mostly microwave-assisted), an ignition step or a
Calculation
combination thereof in order to clear up the sample matrix
Calculate the linear equation of the graph using a least-
and/or to remove carbon-containing material. If operating in
squares fit and derive from it the concentration of the
an open system, the ignition temperature should not exceed
element to be determined in the test solution.
600 °C, due to the volatility of some metals, unless otherwise
stated in the monograph. VALIDATION OF THE METHOD
Operate an atomic absorption spectrometer in accordance Satisfactory performance of methods prescribed in
with the manufacturer's instructions at the prescribed monographs is verified at suitable time intervals.
wavelength. Introduce a blank solution into the atomic LINEAR/IT
generator and adjust the instrument reading so that it Prepare and analyse not fewer than 4 reference solutions over
indicates maximum transmission. The blank value may be the calibration range and a blank solution. Perform not fewer
determined by using solvent to zero the apparatus. Introduce than 5 replicates.
the most concentrated reference solution and adjust the The calibration curve is calculated by least-square regression
sensitivity to obtain a maximum absorbance reading. Rinse in from all measured data. The regression curve, the means, the
order to avoid contamination and memory effects. After measured data and the confidence interval of the calibration
completing the analysis, rinse with water R or acidified water. curve are plotted. The operating method is valid when:
If a solid sampling technique is applied, full details of the - the correlation coefficient is at least 0. 99,
procedure are provided in the monograph. - the residuals of each calibration level are randomly
Ensure that the concentrations to be determined fall distributed around the calibration curve.
preferably within the linear part of the calibration curve. Calculate the mean and relative standard deviation for the
If this is not possible, the calibration plots may also be lowest and highest calibration level.
curved and are then to be applied with appropriate When the ratio of the estimated standard deviation of the
calibration software. lowest and the highest calibration level is less than 0.5 or
Determinations are made by comparison with reference greater than 2.0, a more precise estimation of the calibration
solutions with known concentrations of the element to be curve may be obtained using weighted linear regression. Both
determined either by the method of direct calibration linear and quadratic weighting functions are applied to the
(Method I) or the method of standard additions (Method II). data to find the most appropriate weighting function to be
METHOD I - DIRECT CALIBRATION employed. If the means compared to the calibration curve
For routine measurements 3 reference solutions and a blank show a deviation from linearity, two-dimensional linear
solution are prepared and examined. regression is used.
Prepare the solution of the substance to be examined (test ACCURACY
solution) as prescribed in the monograph. Prepare not fewer Verify the accuracy preferably by using a certified reference
than 3 reference solutions of the element to be determined, material (CRM). Where this is not possible, perform a test
the concentrations of which span the expected value in the for recovery.
test solution. For assay purposes, optimal calibration levels Recovery
are between 0.7 and 1.3 times the expected content of the For assay determinations a recovery of 90 per cent to
element to be determined or the limit prescribed in the 110 per cent is to be obtained. For other determinations, for
monograph. For purity determination, calibration levels are example, for trace element determination the test is not valid
tl1e limit of detection and 1.2 times the limit specified for the if recovery is outside of the range 80 per cent to 120 per cent
element to be determined. Any reagents used in the at the theoretical value. Recovery may be determined on a
preparation of the test solution are added to the reference suitable reference solution (matrix solution) which is spiked
and blank solutions at the same concentration. with a known quantity of analyte (middle concentration of
the calibration range).
2023 Appendix II F V-A205

REPEATABILI1Y
The repeatability is not greater than 3 per cent for an assay
F. X-Ray Fluorescence Spectrometry
and not greater than 5 per cent for an impurity test. (Ph. Bur. method Spectrometry, X.ray fluorescence (2.2.37))
LIMIT OF QUANTIFICATION PRINCIPLE OF THE TECHNIQUE
Verify that the limit of quantification (for example, X-ray fluorescence (XRF) analysis is based on measurements
determined using the 10 cr approach) is below the value to of the X-rays emitted by the constituent atoms of a sample
be measured. when it is excited by an external source of radiation.
If sufficiently energetic radiation impinges on an atom of the
sample material, it may eject 1 of the inner-shell electrons of
that atom. The vacancy created is filled by 1 of the electrons
E. Fluorescence Spectrophotometry from an outer, higher-energy shell. The energy difference
between the 2 electron shells involved in the process is
[Fluorimetry] released in the form of fluorescent X-rays. These X-rays are
(Ph. Bur. method 2.2.21) characteristic since their energy is specific to each element
(atom). By measuring their energy and intensity, qualitative
Fluorimetry is a procedure which uses the measurement of
and quantitative data about the elemental composition of the
the intensity of the fluorescent light emitted by the substance
test material is obtained.
to be examined in relation to that emitted by a given
standard. XRF spectrometry is suitable for liquid, solid and powdered
materials and is widely used as a means of screening
Method Dissolve the substance to be examined in the
pharmaceutical ingredients and products for toxic elements
solvent or mixture of solvents prescribed in the monograph,
or elemental impurities, for quality control and in-process
transfer the solution to the cell or the tube of the fluorimeter
testing. It is also used to identify inorganic foreign elements
and illuminate it with an excitant light beam of the
within falsified medicinal products. As XRF can be non-
wavelength prescribed in the monograph and as near as
invasive, it lends itself to process analytical technology (PAT)
possible monochromatic.
applications, such as the analysis of an unwanted trace
Measure the intensity of the emitted light at an angle of 90° catalyst in an active pharmaceutical ingredient.
to the excitant beam, after passing it through a filter which
transmits predominantly light of the wavelength of the EQUIPMENT
fluorescence. Other types of apparatus may be used provided An XRF spectrometer (or analyser) consists of 3 essential
that the results obtained are identical. components:
For quantitative determinations, first introduce into the - a source of exciting radiation (e.g. an X-ray tube, an
apparatus the solvent or mixture of solvents used to dissolve electron beam if a scanning electron microscope is used
or, more rarely, a radioisotope);
the substance to be examined and set the instrument to zero.
Introduce the standard solution and adjust the sensitivity of - a means for reproducible sample presentation;
the instrument so that the reading is greater than 50. If the - a detector.
second adjustment is made by altering the width of the slits, Depending on the X-ray detection method employed, either
a new zero setting must be made and the intensity of the a wavelength-dispersive (WD) or an energy-dispersive (ED)
standard must be measured again. Finally introduce the XRF spectrometer is used.
solution of unknown concentration and read the result on the In a WD-XRF spectrometer the X-rays from the sample are
instrument. Calculate the concentration cx of the substance in directed at a crystal, which diffracts them at precise angles
the solution to be examined, using the formula: depending on their energy. The intensity of the diffracted
X-rays is measured sequentially by a detector counter.
In an ED-XRF spectrometer the X-rays from the sample are
directed at a solid-state detector, which generates an electric
pulse of an amplitude proportional to the energy of each
ex concentration of the solution to be examined,
c5 concentration of the standard solution, X-ray photon detected. During measurement, the
Ix intensity of the light emitted by the solution to be examined, spectrometer acquires an X-ray spectrum of the sample that
/, intensity of the light emitted by the standard solution. contains complete information about its composition.
An ED-XRF spectrometer can also be combined with a
If the intensity of the fluorescence is not strictly proportional scanning electron microscope (SEM). Substantial advances in
to the concentration, the measurement may be effected using miniaturisation and automation have led to the development
a calibration curve. of hand-held ED-XRF spectrometers for field measurements.
In some cases, measurement can be made with reference to a Some instruments are supplied with an initial factory-set
fixed standard (for example a fluorescent glass or a solution calibration that allows semi-quantitative analyses to be
of another fluorescent substance). In such cases, the carried out.
concentration of the substance to be examined must be
determined using a previously drawn calibration curve under MATRIX EFFECTS AND INTERFERENCE
the same conditions. The intensity of the characteristic X-rays of the analysed
elements is not necessarily linear with concentration, owing
to matrix effects. The intensity of the fluorescence measured
for a given element depends not only on the concentration of
that element in the sample but also on the absorption of the
incident and fluorescent radiations by the matrix.
The presence and concentration of other elements (analytes)
in the sample, the composition of the sample matrix, and the
particle size of the sample material are known to contribute
V-A206 Appendix II F 2023

to matrix effects. The presence of matrix effects must be reference materials (CRMs). Standards with a high carbon
taken into account in any calibration method utilised for load may be more representative for pharmaceutical
quantitative determination. applications.
At low concentrations the linearity is usually well preserved, Calibration
which greatly facilitates calibration of the spectrometer. The calibration model selected must be fit for purpose.
The intensity of the fluorescent radiation emitted by an Various calibration methods are available, including the
element in a given matrix and at a given wavelength is then 'fundamental parameters' approach, empirical calibration,
proportional to the concentration of that element and Compton/Rayleigh normalisation and multiple linear
inversely proportional to the mass absorption coefficient of regression (MLR).
the matrix at that wavelength. System suitability test
SAMPLE PREPARATION This test must be carried out before the analysis to ensure
It is essential that the sample is sufficiently thick that the that the performance of the measurement system is
intensities of characteristic X-rays measured are not affected satisfactory. It may also be performed to verify the calibration
by variations in sample thickness. of the system.
Liquid samples Samples are analysed 'as is', provided that Acceptance criteria The measurement system is suitable
the solution consists of a clear, single phase and has if the concentration obtained for a check material containing
sufficiently low volatility. A special liquid-sample holder and the element(s) of interest within the used concentration range
a commercially available support window composed of a does not differ from the actual concentration by more than
suitable polymer film (transparent to X-rays and solvent 5 per cent for an assay, and 20 per cent for impurity tests;
resistant) are required. Alternatively, liquid samples can be when using concentrations determined from reference
transferred onto the surface of a disc and dried before methods such as atomic absorption spectrometry, the
analysis. accuracy of the XRF calibration should be aligned to that of
Powdered samples Samples may be analysed 'as is' in the reference method; in this case an acceptance criterion of
special X-ray sample cups with bottoms made of a thin 10 per cent for the assay can be more realistic.
polymer film, transparent to X-rays. After transferring the Analysis
powder to a sample cup, the cup is tapped gently until no The samples are measured with the same parameters as used
further settling of the powder is observed. If necessary, more during calibration of the instrument.
powder can be added to the cup after tapping. Another
CONTROL OF EQUIPMENT PERFORMANCE
alternative for preparation of a powdered sample, which is
These parameters are also applicable for equipment qualification.
better suited to WD-XRF analysis, is to press the sample
powder into a pellet, with a binder (for example cellulose Specific procedures, acceptance criteria and time intervals for
powder, wax or ethylcellulose) or without a binder. The mass characterising XRF performance depend on the instrument
of reference material and sample material must be about the and its intended application. Demonstrating stable
same and the resulting pellets must be approximatively 5 mm instrument performance over extended periods of time
thick or more. provides some assurance that reliable measurements can be
obtained.
Solid samples Samples are analysed by placing them
directly on the spectrometer measuring window, making sure The following tests may be carried out at appropriate
they cover it completely. Solid samples for WD-XRF may intervals defined according to the user's quality system
need to be cut into a uniform shape with a flat surface for procedures to ensure the adequate performance of the XRF
reproducible analysis, whereas samples can be measured 'as instrument.
is' when using ED-XRF, provided there is adequate sample x- and y-axes
depth. It is recommended that the x-axis (energy or peak angle) and
Fusion The fusion bead method can be used to prepare y-axis (intensity) are verified at least on installation and
solid and powdered samples (e.g. minerals or oxides) if the thereafter at appropriate intervals that are defined according
element of interest is not volatile. The sample is to the user's quality system procedures. Consideration is to
homogeneously mixed with a flux reagent (e.g. dilithium be given to peak position in ED-XRF and to peak angle in
tetraborate) and transferred to a platinum vessel; a releasing WD-XRF when verifying the x-axis, and to the count rate
agent and/or oxidising agent may be added if necessary, for when verifying the y-axis.
example to prevent damage of the vessel. The mixture is Detector resolution
heated at an appropriate temperature while swirling the vessel Calculate the resolution (total width at half-height) at the
until a homogeneous melt is obtained. If necessary, the melt energy used during calibration of the instrument.
is then transferred to a flat-bottomed mould, kept in a Acceptance criteria The resolution value does not deviate
horizontal position and cooled under conditions maintaining by more than 20 per cent for an assay, and 25 per cent for
its property as a glass. identity and elemental impurities tests, from the value
PROCEDURE determined during calibration of the instrument.
Measuring conditions VALIDATION REQUIREMENTS
The instrument is set up and used in accordance with the The objective of an XRF method validation is to
manufacturer's instructions. The measurements may be demonstrate that the measurement procedure is fit for
carried out under vacuum, nitrogen or helium to improve the purpose (assay, content uniformity, limit tests and
sensitivity of the method for the quantitation of light identification tests). Where sample preparation is necessary, it
elements. is essential that test materials are spiked before any
Reference standards preparatory steps. For example, if a test material is to be
Standards required for the calibration, system suitability or digested, the material must be spiked at the beginning of the
control of equipment performance are prepared from certified digestion procedure.
2023 Appendix II G V-A207

For the determination of impurities, the validation neutral form, reach the ion source of the apparatus; this
requirements are given in general chapter 2.4. 20. For other technique is used for compounds of relatively low polarity
purposes, validation is performed according to the relevant with molecular masses of less than 1000 Da,
ICH guidelines. - moving-belt inteiface: the mobile phase, which may flow at
a rate of up to 1 mIJmin, is applied to the surface of a
moving belt; after the solvent evaporates, the components
to be analysed are successively carried to the ion source of
G. Mass Spectrometry the apparatus where they are ionised; this technique is
rather poorly suited to very polar or heat-labile
(Ph. Bur. method 2.2.43) compounds.
Mass spectrometry is based on the direct measurement of the Other types of coupling (electrospray, thermospray,
ratio of the mass to the number of positive or negative atmospheric-pressure chemical ionisation) are considered to
elementa1y charges of ions (ml z) in the gas phase obtained be ionisation techniques in their own right and are described
from the substance to be analysed. This ratio is expressed in in the section on modes of ionisation.
atomic mass units (1 a.m.u. = one twelfth the mass of 12C)
Supercritical fluid chromatography/mass spectrometry
or in daltons ( 1 Da = the mass of the hydrogen atom).
The mobile phase, usually consisting of supercritical carbon
The ions, produced in the ion source of the apparatus, are dioxide enters the gas state after passing a heated restrictor
accelerated and then separated by the analyser before between the column and the ion source.
reaching the detector. All of these operations take place in a
Capillary electrophoresis/mass spectrometry
chamber where a pumping system maintains a vacuum of
The eluent is introduced into the ion source, in some cases
10 3 to 10-6 Pa.
after adding another solvent so that flow rates of the order of
The resulting spectrum shows the relative abundance of the a few microlitres per minute can be attained. This technique
various ionic species present as a function of mlz. The signal is limited by the small quantities of sample introduced and
corresponding to an ion will be represented by several peaks the need to use volatile buffers.
corresponding to the statistical distribution of the various
isotopes of that ion. This pattern is called the isotopic profile MODES OF IONISATION
and (at least for small molecules) the peak representing the Electron impact
most abundant isotopes for each atom is called the The sample, in the gas state, is ionised by a beam of
monoisotopic peak. electrons whose energy (usually 70 eV) is greater than the
ionisation energy of the sample. In addition to the molecular
Information obtained in mass spectrometry is essentially
ion M+, fragments characteristic of the molecular structure
qualitative (determination of the molecular mass, information
are observed. This technique is limited mainly by the need to
on the structure from the fragments observed) or quantitative
vaporise the sample. This makes it unsuited to polar, heat-
(using internal or external standards) with limits of detection
labile or high molecular mass compounds. Electron impact is
ranging from the picomole to the femtomole.
compatible with the coupling of gas chromatography to mass
INTRODUCTION OF THE SAMPLE spectrometry and sometimes with the use of liquid
The very first step of an analysis is the introduction of the chromatography.
sample into the apparatus without overly disturbing the Chemical ionisation
vacuum. In a common method, called direct liquid This type of ionisation involves a reagent gas such as
introduction, the sample is placed on the end of a cylindrical methane, ammonia, nitrogen oxide, nitrogen dioxide or
rod (in a quartz crucible, on a filament or on a metal oxygen. The spectrum is characterised by ions of the (M
surface). This rod is introduced into the spectrometer after + H)+ or (M -Hf types, or adduct ions formed from the
passing through a vacuum lock where a primary intermediate analyte and the gas used. Fewer fragments are produced than
vacuum is maintained between atmospheric pressure and the with electron impact. A variant of this technique is used
secondary vacuum of the apparatus. when the substance is heat-labile: the sample, applied to a
Other introduction systems allow the components of a filament, is very rapidly vaporised by the Joule-Thomson
mixture to be analysed as they are separated by an effect (desorption chemical ionisation).
appropriate apparatus connected to the mass spectrometer.
Fast-atom bombardment (FAB) or fast-ion
Gas chromatography/mass spectrometry bombardment ionisation (liquid secondary-ion mass
The use of suitable columns (capillary or semi-capillary) spectrometry LSIMS)
allows the end of the column to be introduced directly into The sample, dissolved in a viscous matrix such as glycerol, is
the source of the apparatus without using a separator. applied to a metal surface and ionised by a beam of neutral
Liquid chromatography/mass spectrometry atoms such as argon or xenon or high-kinetic-energy caesium
This combination is particularly useful for the analysis of ions. Ions of the (M + H)+ or (M -Hf types or adduct ions
polar compounds, which are insufficiently volatile or too formed from the matrix or the sample are produced. This
heat-labile to be analysed by gas chromatography coupled type of ionisation, well suited to polar and heat-labile
with mass spectrometry. This method is complicated by the compounds, allows molecular masses of up to 10 000 Da to
difficulty of obtaining ions in the gas phase from a liquid be obtained. The technique can be combined with liquid
phase, which requires very special interfaces such as: chromatography by adding 1 per cent to 2 per cent of
- direct liquid introduction: the mobile phase is nebulised, and glycerol to the mobile phase; however, the flow rates must be
the solvent is evaporated in front of the ion source of the very low (a few microlitres per minute). These ionisation
apparatus, techniques also allow thin-layer chromatography plates to be
- particle-beam inteiface: the mobile phase, which may flow analysed by applying a thin layer of matrix to the surface of
at a rate of up to 0.6 mIJmin, is nebulised in a these plates.
desolvation chamber such that only the analytes, in
V-A208 Appendix II G 2023

Field desorption and field ionisation defined as the ratio between the molecular mass and peak
The sample is vaporised near a tungsten filament covered width at half height (50 per cent valley definition).
with microneedles (field ionisation) or applied to this filament Magnetic and electrostatic analysers
(field desorptwn). A voltage of about 10 kV, applied between The ions produced in the ion source are accelerated by a
this filament and a counter-electrode, ionises the sample. voltage V, and focused towards a magnetic analyser
These two techniques mainly produce molecular ions M+, (magnetic field B) or an electrostatic analyser (electrostatic
and (M + H)+ ions and are used for low polarity and/or field E), depending on the configuration of the instrument.
heat-labile compounds. They follow a trajectory of radius r according to Laplace's
Matrix-assisted laser desorption ionisation (MAI.DI) law:
The sample, in a suitable matrix and deposited on a metal
support, is ionised by a pulsed laser beam whose wavelength
may range from UV to IR (impulses lasting from a
picosecond to a few nanoseconds). This mode of ionisation
plays an essential role in the analysis of very high molecular Two types of scans can be used to collect and measure the
mass compounds (more than 100 000 Da) but is limited to various ions produced by the ion source: a scan of B holding
time-of flight analysers (see below). V fixed or a scan of V with constant B. The magnetic
analyser is usually followed by an electric sector that acts as a
Electrospray
kinetic energy filter and allows the resolving power of the
This mode of ionisation is carried out at atmospheric
instrument to be increased appreciably. The maximum
pressure. The samples, in solution, are introduced into the
resolving power of such an instrument (double sector) ranges
source through a capillary tube, the end of which has a
from 10 000 to 150 000 and in most cases allows the value
potential of the order of 5 kV. A gas can be used to facilitate
nebulisation. Desolvation of the resulting microdroplets of mlz ratios to be calculated accurately enough to determine
the elemental composition of the corresponding ions.
produces singly or multiply charged ions in the gas phase.
The flow rates vary from a few microlitres per minute to For monocharged ions, the mass range is from 2000 Da to
15 000 Da. Some ions may decompose spontaneously
1 mUmin. This technique is suited to polar compounds and
(metastable transitions) or by colliding with a gas (collision-
to the investigation of biomolecules with molecular masses of
activated dissociation (CAD)) in field-free regions between
up to 100 000 Da. It can be coupled to liquid
the ion source and the detector. Examination of these
chromatography or capillary electrophoresis.
decompositions is very useful for the determination of the
Atmospheric-pressure chemical ionisation (APCI) structure as well as the characterisation of a specific
Ionisation is carried out at atmospheric pressure by the compound in a mixture and involves tandem mass
action of an electrode maintained at a potential of several spectrometry. There are many such techniques depending on
kilovolts and placed in the path of the mobile phase, which is the region where these decompositions occur:
nebulised both by thermal effects and by the use of a stream - daughter-wn mode (determination of the decomposition
of nitrogen. The resulting ions carry a single charge and are ions of a given parent ion): BIB= constant, MIKES
of the (M + H) + type in the positive mode and of the (M (Mass-analysed Ion Kinetic Energy Spectroscopy),
-Hf type in the negative mode. The high flow rates that can - parent-ion mode (determination of all ions which by
be used with this mode of ionisation (up to 2 mUmin) make decomposition give an ion with a specific mlz ratio):
this an ideal technique for coupling to liquid B 2IE = constant,
chromatography. - neutral-loss mode (detection of all the ions that lose the
Thermospray same fragment):
The sample, in the mobile phase consisting of water and BIE(l -EIE0 ) 112 = constant, where E0 is the basic voltage of
organic modifiers and containing a volatile electrolyte the electric sector.
(generally ammonium acetate) is introduced in nebulised
Quadrupoles
form after having passed through a metal capillary tube at
The analyser consists of four parallel metal rods, which are
controlled temperature. Acceptable flow rates are of the order
cylindrical or hyperbolic in cross-section. They are arranged
of 1 mUmin to 2 mUmin. The ions of the electrolyte ionise
symmetrically with respect to the trajectory of the ions; the
the compounds to be analysed. This ionisation process may
pairs diagonally opposed about the axis of symmetry of rods
be replaced or enhanced by an electrical discharge of about
are connected electrically. The potentials to the two pairs of
800 volts, notably when the solvents are entirely organic.
rods are opposed. They are the resultant of a constant
This technique is compatible with the use of liquid
component and an alternating component. The ions
chromatography coupled with mass spectrometry.
produced at the ion source are transmitted and separated by
ANALYSERS varying the voltages applied to the rods so that the ratio of
Differences in the performance of analysers depend mainly continuous voltage to alternating voltage remains constant.
on two parameters: The quadrupoles usually have a mass range of I a.m.u.
- the range over which mlz ratios can be measured, ie, the to 2000 a.m.u., but some may range up to 4000 a.m.u.
mass range, Although they have a lower resolving power than magnetic
- their resolving power characterised by the ability to separate sector analysers, they nevertheless allow the monoisotopic
two ions of equal intensity with mlz ratios differing by profile of single charged ions to be obtained for the entire
fu'W, and whose overlap is expressed as a given percentage mass range. It is possible to obtain spectra using three
of valley definition; for example, a resolving power quadrupoles arranged in series, Q 1, Q2 , Q 3 (Q2 serves as a
(Ml fu'W) of 1000 with 10 per cent valley definition allows collision cell and is not really an analyser; the most
the separation of mlz ratios of 1000 and 1001 with the commonly used collision gas is argon).
intensity returning to 10 per cent above baseline. The most common types of scans are the following:
However, the resolving power may in some cases (time- - daughter-wn mode: Q1 selects an mlz ion whose fragments
of-flight analysers, quadrupoles, ion-trap analysers) be obtained by collision in Q2 are analysed by Q3,
2023 Appendix II G I V-A209

- parent-ion mode: Q3 filters only a specific mlz ratio, while deuterated standards). This type of analysis can be
Qi scans a given mass range. Only the ions decomposing performed only on apparatus fitted with three quadrupoles in
to give the ion selected by Q3 are detected, series, ion-trap analysers or cyclotron-resonance analysers.
- neutral loss mode: Qi and Q3 scan a certain mass range but
CALIBRATION
at an offset corresponding to the loss of a fragment
Calibration allows the corresponding mlz value to be
characteristic of a product or family of compounds.
attributed to the detected signal. As a general rule, this is
It is also possible to obtain spectra by combining quadrupole done using a reference substance. This calibration may be
analysers with magnetic or electrostatic sector instruments; external (acquisition file separate from the analysis) or
such instruments are called hybrid mass spectrometers. internal (the reference substance(s) are mixed with the
Ion-trap analyser substance to be examined and appear on the same
The principle is the same as for a quadrupole, this time with acquisition file). The number of ions or points required for
the electric fields in three dimensions. This type of analyser reliable calibration depends on the type of analyser and on
allows product-ion spectra over several generations (MS") to the desired accuracy of the measurement, for example, in the
be obtained. case of a magnetic analyser where the mlz ratio varies
Ion-cyclotron resonance analysers exponentially with the value of the magnetic field, there
Ions produced in a cell and subjected to a uniform, intense should be as many points as possible.
magnetic field move in circular orbits at frequencies which SIGNAL DETECTION AND DATA PROCESSING
can be directly correlated to their mlz ratio by applying a Ions separated by an analyser are converted into electric
Fourier transform algorithm. This phenomenon is called ion- signals by a detection system such as a photomultiplier or an
cyclotron resonance. Analysers of this type consist of electron multiplier. These signals are amplified before being
superconducting magnets and are capable of very high re-converted into digital signals for data processing, allowing
resolving power (up to 1000 000 and more) as well as MS'' various functions such as calibration, reconstruction of
spectra. However, very low pressures are required (of the spectra, automatic quantification, archiving, creation or use
order of 10- 7 Pa). of libraries of mass spectra. The various physical parameters
Time-of-flight analysers required for the functioning of the apparatus as a whole are
The ions produced at the ion source are accelerated at a controlled by computer.
voltage V of 10 kV to 20 kV. They pass through the analyser,
consisting of a field-free tube, 25 cm to 1.5 m long, generally
called a flight tube. The time (t) for an ion to travel to the
detector is proportional to the square root of the m/z ratio. G1. Inductively Coupled Plasma-Mass
Theoretically the mass range of such an analyser is infinite.
In practice, it is limited by the ionisation or desorption Spectrometry
method. Time-of-flight analysers are mainly used for high (Ph. Bur. method 2.2.58)
molecular mass compounds (up to several hundred thousand
Inductively coupled plasma-mass spectrometry (ICP-MS) is a
daltons). This technique is very sensitive (a few picomoles of
mass spectrometry method that uses an inductively coupled
product are sufficient). The accuracy of the measurements
plasma (ICP) as the ionisation source. The basic principles of
and the resolving power of such instruments may be
ICP formation are described in chapter 2.2.57 on inductively
improved considerably by using an electrostatic mirror
coupled plasma-atomic emission spectrometry (ICP-AES).
(reflectron).
ICP-MS utilises the ability of the ICP to generate charged
SIGNAL ACQUISITION ions from the element species within a sample. These ions
There are essentially three possible modes. are then directed into a mass spectrometer, which separates
Complete spectrum mode them according to their mass-to-charge ratio (mlz). Most
The entire signal obtained over a chosen mass range is mass spectrometers have a quadrupole system or a magnetic
recorded. The spectrum represents the relative intensity of sector. Ions are transported from the plasma through 2 cones
the different ionic species present as a function of m/z. (sampler and skimmer cones, forming the interface region) to
The results are essentially qualitative. The use of spectral the ion optics. The ion optics consist of an electrostatic lens,
reference libraries for more rapid identification is possible. which takes ions from an area at atmospheric pressure to the
Fragmentometric mode (Selected-ion monitoring) mass filter at a vacuum of 1o- 8 Pa or less, maintained with a
The acquired signal is limited to one (single-ion monitoring turbomolecular pump. After their filtration, ions of the
(SIM)) or several (multiple-ion monitoring (MIM)) ions selected mass/charge ratio are directed to a detector (channel
characteristic of the substance to be analysed. The limit of electromultiplier, Faraday cup, dynodes), where ion currents
detection can be considerably reduced in this mode. are converted into electrical signals. The element is
Quantitative or semiquantitative tests can be carried out quantified according to the number of ions arriving and
using external or internal standards (for example, deuterated generating electrical pulses per unit time.
standards). Such tests cannot be carried out with time-of- The sample-introduction system and data-handling
flight analysers. techniques of an ICP-AES system are also used in ICP-MS.
Fragmentometric double mass spectrometry mode APPARATUS
(multiple reaction monitoring (MRM)) The apparatus consists essentially of the following elements:
The unimolecular or bimolecular decomposition of a chosen - sample-introduction system, consisting of a peristaltic
precursor ion characteristic of the substance to be analysed is pump delivering the solution at constant flow rate into a
followed specifically. The selectivity and the highly specific nebuliser;
nature of this mode of acquisition provide excellent sensitivity - radio-frequency (RF) generator;
levels and make it the most appropriate for quantitative - plasma torch;
studies using suitable internal standards (for example,
V-A210 Appendix II Gl 2023

- interface region including cones to transport ions to the ISOTOPE SELECTION

ion optics; Isotope selection is made using several criteria. The most
- mass spectrometer; abundant isotope for a given element is selected to obtain
- detector; maximum sensitivity. Furthermore, an isotope with the least
- data-acquisition unit. interference from other species in the sample matrix and
INTERFERENCE from the support gas should be selected. Information about
isobaric interferences and interferences from polyatomic ions
Mass interference is the major problem, for example by
of various types, for example hydrides, oxides, chlorides, etc.,
isobaric species that significantly overlap the mass signal of
is usually available in the software of ICP-MS instrument
the ions of interest, especially in the central part of the mass
manufacturers.
range (for example 40-80 a.m.u.). The combination of
atomic ions leads to polyatomic or molecular interferences CONTROL OF INSTRUMENT PERFORMANCE
(i.e. 40Ar 16O with 56Fe or 40 Ar40Ar with 80Se). Matrix System suitability
interference may also occur with some analytes. Some - Tuning of the instrument allows to monitor and adjust
samples have an impact on droplet formation or on the the measurement before running samples. ICP-MS mass
ionisation temperature in the plasma. These phenomena may accuracy is checked with a tuning solution containing
lead to the suppression of analyte signals. Physical several isotopes covering the whole range of masses, for
interference is to be circumvented by using the method of example 9 Be, 59 Co, 89 Y, 1151n, 14°Ce and 209Bi.
internal standardisation or by standard addition. The element - Sensitivity and short- and long-term stability are recorded.
used as internal standard depends on the element to be The instrument parameters (plasma condition, ion lenses
measured: 59 Co and 1151n, for example, can be used as and quadrupole parameter) are to be optimised to obtain
internal standards. the highest possible number of counts.
The prime characteristic of an ICP-MS instrument is its - Tuning for resolution and mass axis is to be done with a
resolution, i.e. the efficiency of separation of 2 close masses. solution of Ll, Y and TI to ensure an acceptable response
Quadrupole instruments are, from this point of view, inferior over a wide range of masses.
to magnetic-sector spectrometers. - Evaluation of the efficiency of the plasma to decompose
oxides has to be performed in order to minimise these
PROCEDURE interferences. The ratio Ce/CeO and/or Ba/BaO is a good
SAMPLE PREPARATIONS AND SAMPLE indicator, and a level less than about 3 per cent is
INTRODUCTION required.
The sample preparation usually involves a step of digestion of - Reduction of the formation of double-charged ions is
the matrix by a suitable method, for example in a microwave made with Ba and Ce. The ratio of the signal for double-
oven. Furthermore, it is important to ensure that the analyte charged ions to the assigned element should be less than
concentration falls within the working range of the 2 per cent.
instrument through dilution or preconcentration, and that the - Long-term stability is checked by running a standard first
sample-containing solution can be nebulised in a and at the end of the sample sequence, controlling
reproducible manner. whether salt deposits on the cones have reduced the
Several sample-introduction systems tolerate high acid signal throughout the run.
concentrations, but the use of sulfuric and phosphoric acids
can contribute to background emission. Therefore, nitric and
VALIDATION OF THE METHOD
hydrochloric acids are preferable. The availability of Satisfactory performance of methods prescribed in
hydrofluoric acid-resistant (for example perfluoroalkoxy monographs is verified at suitable time intervals.
polymer) sample-introduction systems and torches also allows LINEAR/IT
the use of hydrofluoric acid. In selecting a sample- Prepare and analyse not fewer than 4 reference solutions over
introduction method, the requirements for sensitivity, the calibration range plus a blank. Perform not fewer than
stability, speed, sample size, corrosion resistance and 5 replicates.
resistance to clogging have to be considered. The use of a The calibration curve is calculated by least-square regression
cross-flow nebuliser combined with a spray chamber and from all measured data of the calibration test. The regression
torch is suitable for most requirements. The peristaltic curve, the means, the measured data and the confidence
pumps usually deliver the standard and sample solutions at a interval of the calibration curve are plotted. The operating
rate of 20-1000 µUmin. method is valid when:
In the case of organic solvents being used, the introduction - the correlation coefficient is at least O. 99;
of oxygen must be considered to avoid organic layers. - the residuals of each calibration level are randomly
CHOICE OF OPERATING CONDITIONS distributed around the calibration curve.
The standard operating conditions prescribed by the Calculate the mean and relative standard deviation for the
manufacturer are to be followed. Usually, different sets of lowest and for the highest calibration level.
operating conditions are used for aqueous solutions and for When the ratio of the estimated standard deviations of the
organic solvents. Suitable operating parameters are to be lowest and the highest calibration level is less than 0.5 or
properly chosen: greater than 2.0, a more precise estimation of the calibration
- selection of cones (material of sampler and skimmer); curve may be obtained using weighted linear regression. Both
- support-gas flow rates (outer, intermediate and inner linear and quadratic weighting functions are applied to the
tubes of the torch); data to find the most appropriate weighting function to be
- RF power; employed.
- pump speed; If the means compared to the calibration curve show a
- selection of one or more isotopes of the element to be deviation from linearity, two-dimensional linear regression is
measured (mass). used.
2023 Appendix II H V-A211

ACCUR4CY wavelength, for example in the near-infrared region but at the

Verify the accuracy preferably by using a certified reference cost of lower Raman scattering efficiency and thus longer
material (CRM.). Where this is not possible, perform a test analysis times. In addition, special hardware or software can
for recovery. contribute to limit the impact of fluorescence.
Recovery Apart from different collection geometries (90° scattering or
For assay determinations a recovery of 90 per cent to 180° backscattering), Raman spectroscopy encompasses
110 per cent is to be obtained. The test is not valid if many methodologies, including transmission Raman
recovery, for example for trace-element determination, is spectroscopy (TRS), resonance Raman (RR) spectroscopy,
outside the range 80 per cent to 120 per cent of the surface-enhanced Raman spectroscopy (SERS), tip-enhanced
theoretical value. Recovery may be determined on a suitable Raman spectroscopy (TERS), spatially offset Raman
reference solution (matrix solution) spiked with a known spectroscopy (SORS), Raman optical activity (ROA),
quantity of analyte (concentration range that is relevant to coherent anti-Stokes Raman spectroscopy (CARS),
the samples to be determined). stimulated Raman spectroscopy (SRS) and confocal (CF)
REPEATABILI1Y Raman spectroscopy. It also lends itself to chemical imaging
The repeatability is not greater than 3 per cent for an assay techniques offering the advantage of a high spatial resolution.
and not greater than 5 per cent for an impurity test. APPLICATIONS
LIMIT OF QUANTIFICATION Raman spectroscopy is commonly used for qualitative and
Verify that the limit of quantification (for example, quantitative applications and can be applied to solid, liquid
determined using the 10 cr approach) is below the value to and gaseous samples. Raman spectroscopy is a rapid and
be measured. non-invasive analytical method and can be performed off-
line, at-line, on-line or in-line, e.g. for process analytical
technology (PAT) (see general chapter 5.25. Process analytical
technology). Raman spectrometers can be situated far from
the point of measurement using long-distance optical fibres
H. Raman Spectroscopy to collect the Raman signal.
(Ph. Bur. method 2.2.48) Raman spectroscopy has a wide variety of applications, for
PRINCIPLE example:
Raman spectroscopy is a vibrational spectroscopic technique - identification of materials, e.g. active substances or
in which a sample is irradiated with an intense excipients;
monochromatic light source, usually a laser, although other - determination of solid-state properties, e.g. polymorphism
irradiation techniques are also possible. Most of the radiation and solvated state;
scattered from the sample has the same wavelength as that of - quality control, e.g. assay, uniformity of dosage units;
the incident light; this process is known as Rayleigh - process analysis, e.g. monitoring of biological and
scattering or elastic light scattering. Only a very small fraction chemical reactions, synthesis, crystallisation, granulation,
of the incident photons (about 10-6 - 10-8 ) is scattered from mixing, drying, lyophilisation, extrusion, encapsulation
the sample at wavelengths shifted from the excitation and coating;
wavelength; this scattered light is known as Raman scatter or - detection of falsified products;
inelastic light scatter. The differences between the excitation - mapping, imaging and depth profiling of pharmaceutical
wavelength and the Raman scattered wavelengths are known forms, e.g. distribution of chemical compounds, detection
as Raman shifts and are related to molecular vibrations of the of unknown substances.
sample. The light scattered with lower and higher energy is EQUIPMENT
called Stokes and anti-Stokes scatter, respectively. Two types of Raman spectrometers can be distinguished
For conventional Raman analysis, the Stokes scatter is more depending on the detection principle, dispersive and Fourier
commonly analysed. The Raman spectrum is the plot of the transform (FT) instruments. These may be benchtop
intensity of the Raman scattered radiation or the number of instruments (including microscope-coupled devices, portable
photons versus the Raman shift, usually expressed in instruments) or handheld instruments.
wavenumber (in cm- 1 ).
Raman spectrometers typically consist of the following
Raman and infrared (IR) spectroscopies are complementary components:
techniques. Both probe the fundamental molecular vibrations - a monochromatic light source, typically a laser, with a
of the investigated material. However, due to different wavelength in the ultraviolet, visible or near-infrared
excitation conditions, Raman and IR spectroscopies have region. The majority of instruments operated below
different sensitivities for functional groups within a material. 1064 nm are dispersive, while those operated at or above
Raman spectroscopy is particularly useful in examining 1064 nm are FT-based. In addition, laser wavelength and
polarisable bonds, functional groups and vibrations that are intensity should be selected based on the intended
highly symmetrical (e.g. C-C single or multiple bonds); but is application and the material to be examined, including
less sensitive to polar bonds (e.g. C=O) and vibrations that potentially fluorescent minor constituents;
are antisymmetric. For example, water, which has very - suitable optics (lens, mirrors or optical-fibre assembly)
intense absorption bands in the IR spectrum, exhibits that direct the monochromatic light at the material to be
relatively weak Raman scattering signals and interferes less examined and collect the Raman scattered light from it;
with Raman spectra. Raman spectroscopy can therefore be if applicable, a microscope focusing the laser beam on the
used for the investigation of aqueous solutions. material to be examined;
A major difficulty of Raman spectroscopy is that the material - an optical device (e.g. filters) that transmits the
(or its impurities) under examination may exhibit frequency-shifted Raman scattering but prevents the
fluorescence, which can mask the Raman signal. elastically scattered Rayleigh radiation from reaching the
Fluorescence may be avoided by choosing a longer excitation detector; and
V-A212 Appendix II H 2023

- a dispersing device (grating and/or prism polychromator) selected. For dispersive Raman spectrometers that use
combined with wavelength-selecting slits and a single or multiple gratings for different spectral resolutions, the
multi-channel detector (e.g. a charge-coupled device) or wavenumber scale should be verified at the same optical
- an interferometer with a detector that records the resolution as applied for sample measurement. All gratings
intensity of the scattered light over time, and a data- used for Raman measurements should be verified for
handling device (e.g. computer and appropriate software) accuracy of Raman shift.
that converts this data into the frequency or wavenumber RESPONSE-INTENSITY SCALE
domain by a Fourier-transform calculation; The absolute and relative intensities of Raman signals are
- for specific applications, a sample accessory (e.g. sample affected by variations of several factors including:
holder, moving tray) to position the sample in the path of - polarisation of the irradiating light;
the exciting radiation. - polarisation of the Raman scattered light;
Raman spectrometers are usually interfaced with dedicated - intensity of the irradiating light;
software for the acquisition of spectra. If required, the analyst - instrument/detector response;
should be able to inspect recorded spectra in order to - focus and geometry at sample;
confirm the validity of the measurement (e.g. integrity of the - packing density of particles in solid samples;
sample, fluorescence, saturated signals, signal-to-noise ratio) - refractive index nor change of n (~n) between analyte
and the validity of the analytical conclusion and the environment;
(e.g. identification using spectral comparison). - the particle size and particle-size distribution;
EQUIPMENT PERFORMANCE - the scattering cross-section;
Carry out the prescribed calibrations and/or system - the absorption cross-section.
performance tests according to the manufacturer's The verification of the response-intensity scale is principally
instructions at regular intervals, depending on the use of the performed for quantitative methods.
instrument. For example, emission spectra of a low-pressure Appropriate acceptance criteria will vary with the application.
lamp exhibiting characteristic maxima at wavenumbers over A maximum variation of ± 10 per cent in band intensities
the whole spectral range of the instrument are often used for compared to the previous instrument qualification is
calibration (e.g. neon lamps, but also mercury, argon, achievable in most cases. Response calibration may involve
krypton or xenon lamps). the use of white-light standards or luminescent glass
WAVENUMBER SCALE (e.g. NIST SRM 2241).
Verify the wavenumber scale for Raman shifts using a SPECTRAL RESOLUTION
suitable standard that has characteristic maxima at the Spectral resolution is the ability of a spectroscopic system to
wavenumbers under investigation, for example an organic separate adjacent bands, which makes it possible to
substance such as polystyrene, paracetamol or cyclohexane characterise complex samples (e.g. band analysis, crystallinity,
(see Table 2.2.48.-1). polymorphism).
Appropriate acceptance criteria for the control of spectral
Table 2.2.48.-1. - Raman shifts expressed in wavenumber (and
resolution according to the specifications of each instrument
tolerances) for polystyrene, paracetamol and cyclohexane
and the intended application need to be considered or are
Wavenumbe~ Tolerances defined in a monograph. For identity tests, unless otherwise
[cm- 1 ]
Benchtop Handheld prescribed in a monograph, the spectral resolution must be
[cm- 1] [cm- 1]
less than or equal to 15 cm-i (measured in the wavenumber
Polystyrene 8 620.9 ± 1.5 ± 2.5 range between 1000 cm-I and 1100 cm-I).
1001.4 ± 1.5 ± 2.0 The spectral resolution is verified using a suitable reference
1031.8 ± 1.5 ± 2.0 material. The instrument parameters used for the test, such
1602.3 ± 1.5 ± 3.0 as laser, slit-width and grating for dispersive instruments and
3054.3 ± 3.0 NAE
circular aperture (Jacquinot stop or J-stop) for FT-instruments,
Paracetamolc 797.2 ± 1.5 ± 2.5 must be the same as those applied for sample measurements.
857.9 ± 1.5 ± 2.0 For example, record the Raman spectrum of calcium
1168.5 ± 1.5 ± 2.0 carbonate for equipment qualification CRS, and determine the
1236.8 ± 1.5 ± 2.0 full width at half height (W1085) of the band located at
1323.9 ± 1.5 ± 2.5 1085 cm- 1 • The spectral resolution (R) using calcium
1648.4 ± 1.5 ± 3.0 carbonate is then given by the following relation:
2931.1 ± 2.0 NAE
R = (W10ss - 0.684)
Cyclohexane 0 801.3 ± 1.5 ± 2.5
1.0209
1028.3 ± 1.0 ± 2.0
1266.4 ± 1.0 ± 2.0 PROCEDURE
1444.4 ± 1.0 ± 2.5 PREPARATION OF THE SAMPLE
2852.9 ± 2.0 ± 3.0 Raman spectra can be obtained from solids, liquids and gases
A Standard guide for Raman shift standards for spectrometer calibration (American
generally without prior sample preparation or dilution.
Society for Testing and Materials ASTM E 1840). Measurements can be performed directly, or in suitable glass
8 Polystyrene film (e.g. 76 µm), pellets (e.g. NIST 706a) or rod.
or plastic containers, or through films, provided that
c Paracetamol for equipment qualification CRS (which represents monoclinic unwanted signal contributions are under control.
form I).
D Cyclohcxane R. When using Raman spectroscopy, the measured sample area
E NA: no data available. and volume may be small (in particular for microscope-
coupled devices) and care must be taken to ensure that the
A minimum of 3 wavenumbers covering the working range of measurement is representative. This can be achieved by, for
the instrument intended for measurements should be example, rotation of the sample, performing multiple
2023 Appendix II J V-A213

measurements on different preparations of the sample, using (in the whole spectral range or in the region of interest
orbital raster scanning (ORS), increasing the area of specified in the monograph) or by using mathematical
illumination by reducing the magnification (focus), by calculations of the software. It is possible for example to
demagnification (defocusing) of the laser beam or by perform:
changing the focal length between measurements to scan at - a visual comparison based on band positions and relative
different depths. TRS and SORS also sample a relatively intensities unless otherwise specified; the maxima in the
large volume owing to the separation of excitation and spectrum obtained with the material to be examined
collection regions. correspond in position and, if applicable, in relative
It is not always possible to consider Raman as a non- intensity to those in the spectrum obtained with the
destructive technique. The energy transmitted by the laser reference standard;
depends on the duration of exposure and the wavelength. - a statistical determination of the similarity between the
An excess of energy may change the physical state or degrade spectra of the material to be examined and the reference
the sample. standard - this value is calculated by the software and the
For chemical imaging analysis that scans the surface of the identification threshold is defined by the user;
sample, sample preparation must be adapted to the - evaluation by chemometric methods (e.g. similarity and
equipment. For example, spectrometers equipped with a distance measures, classification methods); these methods
microscope may require samples with a flat surface (see involve the set-up of a spectral reference library and the
general chapter 5.24. Chemical imaging). elaboration, assessment and validation of a chemometric
model (see general chapter 5.21. Chemometric methods
QUALITATIVE METHODS applied to analytical data).
Since frequency shift positions and relative intensities are
employed for identification, identical laser intensity for both QUANTITATIVE METHODS
the reference standard and the material to be examined may Quantitative determination requires that the reference
not be necessary. The material to be examined is measured standard and the material to be examined must be measured
in the same physical state (e.g. liquid, solid) as the reference at the same nominal laser excitation intensity and frequency.
or library material. Raman techniques offer the advantage of Ensure that the material to be examined is measured in the
non-invasive measurements of the material to be examined same physical state (e.g. liquid, solid) and concentration
without removal from the packaging. However, some range as the reference standard or library used for calibration.
packaging materials may lead to additional signals in the While the Beer-Lambert law is not valid for Raman
Raman spectrum. This is especially the case when the spectroscopy, Raman intensity is directly proportional to the
packaging material absorbs at the laser's excitation concentration of the Raman scattering analytes and
wavelength, superimposes the Raman signal, has a high calibration can be based on both univariate and multivariate
Raman scattering cross-section, or exhibits other chemometric methods (see general chapter 5.21. Chemometric
spectroscopic interference. methods applied to analytical data). However, for solid samples
and suspensions the Raman intensity may be affected by the
Identification can be performed using either a reference matrix (e.g. owing to fluorescence and self-absorption).
standard or a spectral reference library.
The Raman signal is influenced by the refractive index of the
Identification using a reference standard material, the particle size and the particle-size distribution
Prepare the material to be examined and the reference (where small particles give a relatively more intense Raman
standard by the same procedure and record the spectra with scattering than large particles), the packing density, the
the same (or equivalent) instrument type under the same scattering cross-section, the absorption cross-section, etc. (see
conditions and compare between 400 cm- 1 and 2000 cm- 1, also under Response-intensity scale).
unless otherwise prescribed. When the spectra recorded for
samples in the solid state show differences in the positions of
the signal maxima, treat the material to be examined and the
reference standard in the same manner so that they crystallise J. Flow Cytometry
or are produced in the same solid form, or proceed as
described in the monograph and then record the spectra. (Ph. Bur. method 2.7.24)
However, this procedure must not be performed for Flow cytometry consists of a multiparametric analysis of
substances where the monograph covers a particular optical properties of individual particles in a fluidic system.
polymorphic form. Cells or particles in suspension are individually distributed
Identification using a spectral reference library into a linear array (stream), which flows through a detection
Record the spectra of a suitable number of materials which device. Solid tissues have to be reduced to a single-cell
present typical variation (manufacturer, batch, particle size, suspension to be analysed.
impurity profile, etc.) and comply with the requirements of The spectrum of parameters measurable by flow cytometry
the monograph or established specifications. The number includes volume and morphological complexity of cells or
and selection of samples in the database depends on the cell-like structures, cell pigments, DNA content, RNA
specific application. Suitable mathematical pre-treatments of content, proteins, cell surface markers, intracellular markers,
the Raman spectrum may facilitate qualitative comparison of enzymatic activity, pH, membrane and fluidity.
spectra (e.g. baseline correction, normalisation, derivatives, It is possible to collect 2 morphological parameters plus 1 or
cosmic ray removal). more fluorescence signals per single cell. The multiparametric
Comparison procedures analysis allows the definition of cell populations by their
Several comparison procedures may be used, and the analyst phenotype.
must document and justify the method used and the specific APPARATUS
acceptance criteria that allow a conclusion for identification. Focusing, magnifying, and choice of light source are
The spectra can be compared either by overlaying the spectra optimised to allow the automatic detection and measurement
V-A214 Appendix II J 2023

of morphological differences and staining patterns. Flow nucleus, the amount and type of cytoplasmic granules or the
cytofluorimetric analysis meets the following criteria: membrane roughness, and correlates with the morphological
- choice of light source depending on the parameters to be complexity of the cell, so that the higher the SS intensity, the
analysed; higher the cell complexity. As a function of the
- adjustment of instrument settings depending on the cell morphological characteristics of cells, scatter signals will
type to be analysed (for example, cell cultures, leucocytes, always be generated during a flow analysis; they are defined
platelets, bacteria, spermatozoa, yeast) and the analysis to as intrinsic parameters.
be performed (for example, phenotyping, cell cycle, Fluorescence
apoptosis, cytokines, membrane fluidity, fluorescent Depending on the type and number of light sources, when a
protein). cell passes through the sensing area, it will emit fluorescent
Flow cytometry is characterised by the automated light. Fluorescence signals are generated from fluorescent
quantification of set parameters for a high number of single dyes naturally present in the cells (for example, co-enzymes,
cells during each analysis session. For example, chlorophyll, seaweed pigments) and/or from fluorescent
100 000 particles or more (practically unlimited) are analysed probes taken up by the cells when stained for the analysis of
one after the other, typically in about 1 min. The detection specific characteristics (for example, fluorescent antibodies,
limit is as low as 100 fluorescent molecules per cell. nucleic acid dyes, pH probes, calcium probes, fluorescent
A flow cytometer apparatus has 5 main components: proteins). Nowadays, there is a large number and a wide
- a fluidic system and a flow cell; range of different types of fluorescent probes available.
- a light source; The optical filters must be adapted to the fluorochromes
- a detection and Analogue to Digital Conversion (ADC) used and changed if necessary. Each fluorescent probe is
system; characterised by its excitation spectrum and its emission
- an amplification system; spectrum. They are chosen depending on the nature of the
- a computer provided with software for analysis of the excitation source and the detection system, and according to
signals. the specific purpose of the analysis.
FLU/DIC SYSTEM AND FLOW CELL SIGNALMANAGEMENTANDANALOGUETO
The single cell is exposed to the light source and detected in DIGITAL CONVERSION
the flow cell. The fluidic system carries the suspended cells Scatter and fluorescence signals emitted by cells when
individually from the sample tube to the laser intercept point. passing across the laser beam are sorted and addressed to
To achieve this, the sample stream is drawn out to a very their detectors using optical filters. The detectors are
thin fluid thread by a sheath fluid in the flow cell transducers (photomultiplier tubes (PMTs)) that convert
(hydrodynamic focusing). The light beam is focused in an light signals radiated from the cells into voltage pulses.
elliptical shape, by 2 confocal lenses, into the flow cell The process of counting each pulse in the appropriate
channel through which the cells pass. The flow rate must be channel is known as Analogue to Digital Conversion (ADC).
constant during routine cell surface marker analysis and must The process is finally shown as a frequency histogram.
ensure a suitable distance between the cells to allow Amplification
counting. Voltage pulses need to be amplified for optimal visualisation.
LIGHT SOURCES The amplification process accentuates the differences
Commonly used light sources are: between cell signals, and consequently increases the
- lamps (mercury, xenon); resolution among cell populations of different characteristics
- high power water-cooled lasers (argon, krypton, dye (for example, the differentiation of viable from non-viable
laser); cells, or non-specific fluorescence from antigen-specific
- low power air-cooled lasers (argon (488 nm), red helium- fluorescence after staining with a fluorescent monoclonal
neon (633 nm), green helium-neon, helium- antibody).
cadmium (UV)); There are 2 methods of amplification: linear or logarithmic;
- diode lasers (blue, green, red, violet). the choice between the 2 types is made for every single signal
SIGNAL DETECTION according to the morphological characteristics of the cells and
When a particle passes across the light beam, it scatters some the staining reagents used (for example, fluorescent
of the light in all directions. Fluorescent dyes, when added to monoclonal antibodies, nucleic acid dyes).
the particle, give off their own light (fluorescence), which is Linear amplification, which enhances the differences among
also radiated in all directions. 2 types of signals may thereby strong pulses, is used with those parameters that generate
be generated: high intensity signals, for example:
- scatter of light; - cell scatters;
- fluorescence emission. - fluorescence from nucleic acid dyes for cell cycle studies.
The instrument's light detectors collect some of this scattered Logarithmic amplification, in contrast, is for weak pulses and
and fluorescent light and produce electronic signals parameters or analysis conditions that may generate both
proportional to the amount of light collected. weak and strong pulses, for example:
Scatter - cell antigens;
2 parameters of light scattering are measured: - scatter from platelets, bacteria, yeast;
- the amount scattered mainly forward (forward scatter - fluorescence from nucleic acid dyes for apoptosis studies.
(FS)) Compensation of fluorescence signals
- the amount scattered at 90° from the direction of the light Each fluorescent dye has an absorption wavelength spectrum
beam (side scatter (SS)). and a higher emission wavelength spectrum. When using 2 or
Forward scatter correlates with the cell volume while side more fluorescent probes simultaneously for staining cells (for
scatter is influenced by parameters such as the shape of the example, 4-antigen immunophenotyping), the fluorochromes
2023 Appendix II K V-A215

emission spectra may overlap. As a consequence, each especially useful when studying rare cell populations or for
fluorescence detector will sense its own specific fluorescent sorting purposes.
light and a variable quantity of light emitted by the other CONTROLS
fluorescent probes. This results in signal over-evaluation and
poor separation of the cell populations. Internal control
The system's optical alignment must be validated before
The solution is in the use of an electronic matrix that allows analysis using adapted fluorospheres and the optimum fluidic
the selective subtraction of the interfering signals from each stability is checked. The data obtained are reported and allow
fluorescence signal after detector sensing (fluorescence the periodical review of control values against the mean
compensation). performance value. A positive control is highly desirable to
Fluorescence compensation requires the use of fluorescence prove that the test antibody is functional and to allow the
calibrators, preferably positive cell samples stained with the proper setting of the flow cytometer. The positive control
fluorochromes of interest, combined in a manner equivalent must include samples known to be positive for the marker of
to that for the antibody used for the analysis. interest.
SIGNAL PLOITING AND DISPLAY External control
After amplification and compensation, the signals are plotted To ensure reliability in the data obtained or to check inter-
in 2 or 3 dimensions. Histograms show the signal intensities laboratory reproducibility, participation in a proficiency
versus the cell counts for a given parameter. Cytograms, in testing study is recommended.
which each dot represents a cell, result from the combination
of 2 signal intensities (dual-parameter dot plots). The type
and number of plots and signal combinations are chosen on
the basis of the specimens and dyes used. When analysing
acquired data, the flow cytometry software can also generate
K. Peptide Identification by Nuclear
other kinds of graphs (such as overlays, surface plots, Magnetic Resonance Spectrometry
tomograms, contour plots, density plots, overlay plots). (Ph. Bur. method 2.2.64)
Statistical data such as mean fluorescent intensities (and their
shifts in time or their dependence on cell function) can also This general chapter is to be used in conjunction with
be used. general chapter 2.2.33. Nuclear magnetic resonance spectrometry
in the context of peptide identification. The approach to be
DATA ANALYSIS
followed is qualitative and consists of comparing the nuclear
Different kinds of cell populations may be present inside the
magnetic resonance (NMR) spectrum of a test sample with
cell suspensions to be analysed, some of which are unwanted
that of a reference sample acquired under identical
(such as dead cells, debris or macro-aggregates), or simply
conditions.
not relevant for the analysis (for example, granulocytes when
studying lymphocytes). This depends on the cell sample type This general chapter mainly applies to the use of proton
(whole blood, bone marrow, cell cultures, biological fluids, NMR (1H NMR) spectrometry, to confirm the identity of
cell suspensions from solid tissues) and on the handling small peptide products (up to approximately 15 amino
procedures (for example, staining methods, lysis, fixation, acids). It is also applicable when using 13 C NMR
cryopreservation, thawing, paraffin-embedded tissue spectrometry with some modifications. The scope is
preparation). restricted to the use of one-dimensional NMR spectrometry.
As a consequence, not all the signals generated during a flow GENERAL PRINCIPLES
cytometry analysis belong to the cells to be studied. Equipment
2 strategies are adopted to exclude unwanted and irrelevant Unless otherwise specified, an apparatus with a field strength
cell signals. giving an operating frequency for proton NMR of at least
The 1st is used during data acquisition. It is a noise 300 MHz.
threshold, applied to I (or more) significant parameter(s), set Spectral acquisition conditions and their optimisation
to acquire only the cells with signal intensities higher than the After introduction into the magnet, the sample is allowed to
pre-defined discrimination value for that parameter. Due to come to thermal equilibrium, especially if analysis is carried
its characteristics of a strong signal with a low grade of out at a temperature significantly different from room
interference, forward scatter is the parameter most often used temperature: monitoring the lock signal is often a valuable
as discriminator. visual guide to the progress of this process.
The 2nct, applied during data analysis, consists of the use of The spectral width must encompass the complete spectrum
gating regions to restrict the analysis only to signals from of the peptide, with an empty spectral region at each side.
those populations that satisfy given morphological and Typically, a spectral width of 12 ppm or 16 ppm is
expression profile characteristics. 2 types of logical gating are appropriate.
commonly used. The 1st is the morphological gate. The cell The following parameters may be optimised to improve
populations are identified using their morphological signals resolution of characteristic peaks: temperature and/or pH
(FS and SS). A region gate is drawn around the population primarily, buffer and peptide concentrations. Control of
of interest (for example, lymphocytes, viable cells) then the sample temperature is recommended but is not mandatory;
fluorescence plots are gated into the selected region. The 2nd if not used, the effect of small temperature changes on the
is the fluorescence-based gate. The cell population of interest appearance of the spectrum is validated.
is identified on the basis of the expression intensity of an
The number of data points collected is such as to define
antigen or a dye, then a gate region is drawn around it.
peaks adequately.
Afterwards the fluorescence plots are gated into the selected
region. Solvent suppression is not recommended but, if used, the
intensities of peaks close to the solvent resonance may be
The analysis software allows the creation of multiple gate
affected and this has to be validated when comparing spectra.
regions, using a sequential logic order. This feature is
V-A216 Appendix II L 2023

Chemical shift referencing

For samples in aqueous solution, sodium 2,2-dimethyl-2-
L. Chemical Imaging
silapentane-5-sulfonate (DSS), sodium 3-(trimethylsilyl) (Ph. Bur. general, text 5. 24)
propionate (TSP) or a deuterated analogue (TSP-d4 ) are 1 SCOPE
appropriate, and the chemical shift of the methyl signals is
Chemical imaging (Cl) combines spatially resolved sensing
often set to 0 ppm. Either the reference material is added at
technologies with data analysis techniques to characterise a
low amounts (10-100 ppm has been found to be appropriate)
sample in chemical and physical terms, using information
to the deuterated water used to dissolve the final sample, or
primarily obtained from its surface. Chemical imaging is
an easily recognised internal resonance that is consistently
particularly suited to the analysis of solid, semi-solid and
present (such as acetate anion) can be used as a secondary
liquid samples with regard to material properties, including
reference. In this case, a validation spectrum obtained under
component identity (active pharmaceutical ingredients and
the same spectral conditions is used to define the chemical
excipients), domain size and distribution, polymorphism, and
shift of the secondary standard.
particle morphology. Thus, imaging can be applied to assess
Sample size identity, quality and quantity of active ingredients,
Usually a few milligrams are used. If sample sizes are intermediates, and excipients in bulk or solid dosage forms,
variable, the effects of this variation on the appearance of the biological samples, packaging and devices. Imaging is used to
spectrum are validated. explore sample homogeneity, detect physical sample defects
Sample preparation (e.g. cracks in cores or coatings), and identify foreign
The test and reference samples must be comparable in terms particles or contaminants. It also facilitates process
of concentration, pH and buffer composition. Typically, understanding and root cause determination. Lastly, it is a
samples in solution are lyophilised, and the dried samples tool to evaluate falsified or counterfeit medicinal products.
dissolved in deuterated water or a buffer in deuterated water. This general chapter's primary focus is on chemical imaging
It may be worthwhile to lyophilise a solution in deuterated systems (CIS) based on surface analysis performed with
water one or more times ('deuterium exchange') as this vibrational spectroscopy, e.g. mid-infrared spectroscopy,
reduces the intensity of strong solvent signals; volatile process near-infrared spectroscopy and Raman spectroscopy.
impurities such as ethanol will also be lost. Use of buffer for However, it also applies to other techniques that supply
the final sample preparation can reduce aggregation and images.
improve spectral reproducibility by reducing batch-to-batch It offers specific recommendations to assess the performance
pH variation. Some probes are intolerant to high salt of chemical imaging systems for the qualitative and
concentrations, but ionic strengths up to 200 mM sodium quantitative exploitation of images. Where chemical imaging
chloride are normally tolerated. High salt concentrations tend systems are primarily intended for investigative purposes, the
to increase 90° pulse length. performance requirements for infrared absorption
VERIFICATION OF IDENTITY spectrophotometry (2.2.24), near-infrared spectroscopy
Determination of key spectral factors (2.2.40) and Raman spectroscopy (2.2.48) need not be
Use of a qualitative approach does not entail stringent applied. Instead, individual criteria have to be established
requirements on spectral parameters (for example, fast pulse using a risk-based approach.
repetition rates can be used, as full relaxation is not 2 ASPECTS OF CHEMICAL IMAGING
required). The use of short pulse widths (for example, a 30° 2-1 DEFINITION
pulse) and fast repetition rates will have no significantly Chemical imaging of pharmaceutical samples is a method
deleterious effect on spectra, and will allow faster acquisition that consists of a collection of responses to the illumination
of acceptable signal-to-noise ratios. Variation in the pulse at multiple wavelengths of positions spatially distributed
width and acquisition time within wide limits will not affect across the sample surface. For a given surface location
the ability to compare spectra. The number of scans (pixel), a set of responses will be associated with the
collected must give appropriate signal-to-noise ratios for low impinging wavelengths. When x and y positions are varied
intensity resonances and therefore a minimum signal-to-noise successively over the range of wavelengths and responses are
ratio of 50: 1 is recommended. collected at each location, an image of a wide surface of the
Identification of characteristic resonances sample is constructed.
It is possible to compare either the complete spectrum or a Imaging systems resolve spatial information primarily at the
portion of it. Comparison of spectra of relevant samples will sample surface. Chemical and morphological sample
highlight regions of the spectrum that are distinctive, and characteristics or features are collated into an image made up
comparison can be constrained to these regions. It is of contributions from multiple domains distributed on the
important to define resonances from impurities, such as surface of the sample. As a result each mapped sample point
residual solvents, which may be essentially irrelevant to (pixel) contains a wealth of information, and the recorded
product quality and which may vary in intensity between signal reflects chemical and physical properties such as
batches. ingredient identity, concentration, crystallinity, orientation,
Spectral comparison domain size and particle size.
See the provisions of general chapter 2.2.33. 2-2 IMAGING MODES
Chemical images may be acquired in broadband,
multispectral or hyperspectral modes. An example of the
broadband mode is the camera, where filters are used to
weight the relative importance of different wavelengths for
the 3 colour variables red, green, and blue (RGB).
Multispectral imaging involves sampling spectral bands that
may be spread unevenly, not unlike a combination of
different detectors in different frequency ranges.
2023 Appendix II L V-A21 7

•• •
Spectrum at pixel i,j • V· 'k

Optical image of the sample

22.S

2:0.C

175

15.0

12.S

l'J.0

Chemical image developed at vk_4

n x p x k datacube

Figure 5.24.-1. - Example of a n x p x k datacube obtained by recording spectra of k wavenumbers at n x p pixels of the sample;
a grey-scale image was developed at wavenumber vk_4

Hyperspectral imaging (HS imaging, HSI) is a two- Not unlike classical spectroscopy, which gathers chemical and
dimensional (2D) visualisation technique that records a full physical information about the single area under
range spectrum for each pixel. In practice, HSI expands examination, imaging captures spectral and spatial
spectroscopic single point analysis of samples into 2D characteristics of surface areas. Spectral and spatial analysis
projections of a slice of finite depth for each measured area of image pixels can be iterative, or combined in no particular
of the sample. Hyperspectral sensors take advantage of order.
numerous contiguous channels to uncover features that 2-4 HIGHER DIMENSIONAL CHEMICAL IMAGING
usually cannot be resolved using the average spectra obtained Spatially resolved three-dimensional imaging can be seen as
from a single measurement in classical spectroscopy. Images extending spatial resolution along a third direction (z) that
may be produced with a spectral quality similar to that of complements x-y imaging. It is the process of stacking a
conventionally obtained spectra. However, this comes at the collection of images covering a depth into the sample which
cost of longer measurement times, larger amounts of data, is larger compared to the analysis depth. This can be
and complex computation. The spectral quality is sometimes obtained indirectly (invasive, destructive process), by
lowered to increase the image throughput. Sample domains sequentially collecting images of appropriately prepared solid
may include complex spectral patterns embedded in the samples, e.g. by slicing the surface stepwise with a
individual pixel data that cannot be identified solely by visual microtome. Meanwhile there are techniques which enable
inspection, and appropriate image analysis is required to direct (non-invasive, non-destructive process) 3D imaging of
extract analytical information from such a multitude of solids using x-y-z spatially resolved spectroscopy. These
measurements. Computational and numerical methods are include tomography methods such as optical coherence
essential for processing, extracting and analysing content in tomography (OCT), far-infrared (FIR)/terahertz (THz) and
hyperspectral images. time-domain THz spectroscopy, confocal Raman, and others,
2-3 DATACUBE for example, X-ray tomography or nuclear magnetic
In hyperspectral imaging each pixel is associated with one resonance imaging (MRI). Ultimately 4D and higher
spectrum. Measurements are spread over both dimensions of dimensional imaging would mean simultaneous spatial (x, y,
the sample surface. At each measurement, a column vector of and z) and spectroscopically resolved mapping. Although
dimensions equal to the number of recorded spectral data higher dimensional imaging is not within the remit of the
points is associated. As a result, data is packaged into a current general chapter, similar recommendations may
three-dimensional array called a datacube or hypercube. initially apply.
The datacube is a signal intensity data set made up of the 2-5 APPLICATIONS
pixel x-y coordinates and the corresponding series of Visual display of the distribution of sample surface features
responses (e.g. absorbance or transmittance) to the scanned complements classical analytical methods by facilitating rapid
spectrum (see Figure 5.24.-1). and non-destructive comparison between samples. Cl can be
V-A218 Appendix II L 2023

I. acetylsalicylic acid 2. caffeine 3. paracetamol

Figure 5.24.-2. - Example of characterisation of ingredients with Raman imaging

used to analyse small to large surface areas of a sample. dispersions) can also be considered. The characterisation of
Imaging comes into its own as soon as constituents and nano- and micro-crystalline materials can be performed to
morphological characteristics differ from one position to the monitor structural changes under stress conditions and over
next. Thus, the technique is particularly suited to exploring time, and to evaluate defects in crystals, for example,
samples that are heterogeneous with regard to chemical resulting from milling and micronisation of the material.
content and physical morphology. Imaging captures the The chemical characterisation of samples based on chemical
distribution of selected components and features, images is mainly performed to identify and characterise the
i.e. attributes of various parts of a sample. For example, distribution and abundance of individual components in a
images can display the location of features that directly mixture based on characteristic spectral features (see Figure
impact product performance. They can show whether a 5.24.-2). This analysis can be performed to determine
component is evenly distributed on a relevant scale. molecular but also atomic elemental species present on the
Therefore, it is important to choose CI methods depending surface. The kinetics and mechanism of active substance
on the needs with regard to spatial resolution. dissolution and release can be modelled, and, as another
The main applications of chemical imaging focus on solid- example, the drug concentration gradient between the
state property analysis, determination of chemical or physical solid/solution interface and the bulk solution can be
features, contaminant identification, anti-counterfeit, and determined.
chemical identification. For example, the measurement of 2-6 CHEMICAL IMAGING SYSTEMS
thickness and uniformity of coating on tablets, and the The selection of a specific imaging instrument or technique
characterisation of surface properties, such as component depends on the particular analytical application intended.
mapping, determination of adhesion force and deformation A chemical imaging system is characterised by setup, spatial
depth, can be considered. Moreover, particle characterisation and spectral resolutions, magnification, type and size of
can be performed based on measurements of size, sample, sample preparation and presentation, moving or
agglomeration and morphology, determination of roughness resting sample, acquisition time, number of measurements,
of the surface, and detection of broken particles and foreign data analysis algorithms, software, etc.
particles. Analysis of particles can also be performed in Numerous techniques enable the production of hyperspectral
liquids. images. A brief description of techniques relying on
Spatial distribution of different polymorphic forms can be vibrational spectroscopy is given below, along with their
analysed with CI techniques and the investigation of potential uses and limitations.
polymorphic transitions and multiphasic materials (e.g. solid Mid-infrared (MIR)
2023 Appendix II L V-A2 l 9

MIR spectroscopy is based on the interaction of light with 2-8 RESOLUTION

the sample, for the study of intra-molecular vibrations of a Pixels contain a wealth of contributions from chemical and
material. The electromagnetic spectrum range usually spans physical features occurring at the surface and near sub-
the region of 4000-400 cm- 1 (2.5-25 µm). MIR imaging may surface of the sample. The performance of an imaging system
be used for the characterisation of chemical species in a is directly related to the spatial and spectral resolutions that
mixture of ingredients. Measurements are often carried out can be achieved. Linear line mapping systems are more
using attenuated total reflectance (ATR) microscopy where sensitive to spatial interference, whereas spectral resolution is
the sample is in contact with an IR-transparent crystal of more critical with focal plane acquisition.
higher refractive index than the sample. The measurement Depending on the imaging technique, spectral resolution may
may be difficult for samples with excessive moisture due to be affected for example by laser wavelength, grating, detector
interference with water bands. and spectrometer focal length. Spectral resolution has an
Near-infrared (NIR) impact on chemical feature extraction because it influences
NIR spectroscopy detects molecular vibrations originating the performance of qualitative and quantitative image
from C-H, N-H, O-H and S-H overtones, and combinations analysis, e.g. identity of ingredients.
of fundamental mid-infrared vibrations. The electromagnetic Spatial resolution is the smallest distance between two
spectral range usually extends from consecutive points which can be distinguished. It has an
12500-4000 cm- 1 (0.8-2.5 µm). The measurement is impact on image processing as information may appear as
contactless, usually in diffuse reflectance mode, and delivers unresolved. For example, the domain size of components of
physical and chemical information. interest should be consistent with the spatial resolution
Far-infrared (FIR) and terahertz (THz) achieved by the system. The spatial resolution obtainable is
In the FIR range, electromagnetic radiation typically spans limited by the instrument characteristics, for example spot
from 400 to approximately 10 cm- 1 (25-1000 µm). This size of the laser, diffraction limits, detector size,
allows spectra to cover inter-molecular and lattice vibration magnification, numerical aperture, etc. The spatial resolution
modes. The selectivity of the technique to the hydrogen obtained by a specific CI system is also affected by the
bonding networks makes possible the identification and diffusion of radiation across the sample surface. Scattering
characterisation of solid state forms, e.g. polymorphic forms occurring below the surface is likely to distort the image
and degree of crystallinity. obtained and limit the spatial resolution.
The radiation can pass through a wide variety of non- 2-9 REPRESENTATIVENESS OF SAMPLE SURFACE
conducting materials, but cannot pass through conducting Depending on the instrument setup, the topology of the
materials such as metal and water, and penetrates deep into sample surface may impact imaging performance.
the sample. The sample surface investigated has to be representative for
Raman the intended purpose of analysis. Depending on the latter,
the impact of sample homogeneity has to be evaluated.
Raman spectroscopy detects frequency shifts originating from
For example, with a design based on reflectance, due to the
the inelastic part of the light scattered by a sample previously
limitation of penetration depth and spatial resolution, a true
irradiated with an intense monochromatic light source
measure of morphology or distribution of particle shapes in
(usually a laser). Raman spectra contain numerous narrow
the sample is only estimated. Thus, results obtained from
bands which makes possible the identification of chemical
surface image analysis may not be representative of the whole
substances. As a result, Raman imaging gives information on
sample because it may not be homogeneous.
chemical species in a sample (including polymorphs).
One approach to overcome this limitation would be to
Water, air and glass are weak Raman scatterers and therefore
measure a number of cross-sections of the sample to improve
the analysis of aqueous samples may be performed in
estimations for the whole sample. Another approach would
atmospheric conditions or in sealed vials. However, the
be to use an alternative optical design where penetration
measured signal may be disturbed by fluorescence.
depth can be increased, e.g. confocal or spatially resolved
2-7 ACQUISITION MODES offset measurements to capture information near the surface,
Either spatial sequencing or wavelength sequencing tomography or transmission measurements to collect whole
techniques are required to produce an image. The 3 modes matrix information.
used to record datacubes are point mapping, line mapping
3 ELEMENTS OF A CHEMICAL IMAGING
and global imaging. Point mapping (point scanning) is the
simplest and typically follows a rectangular grid pattern. PROCESS
A spectrum is recorded at each point on the grid with the Proceeding with imaging encompasses numerous steps such
sample stage being moved to the next neighbouring location. as:
Scanning is computer-controlled along both spatial axes. - sample preparation;
In line mapping (linear scanning), the sample is illuminated - control of instrument components and system
along a line and the image signal is dispersed along one performance;
spatial axis onto the detector. The sample is moved along the - calibration of instrument and system suitability test;
other spatial axis to capture the next neighbouring line until - measurement, image processing, display and storage;
full mapping is eventually achieved. Here the speed and - image analysis and computation of numerical results.
flexibility of the experimental setup enable on-line application 3-1 SAMPLE
of continuous CI. Global imaging (focal plane scanning) is 3-1-1 Sample preparation
performed when all image points of a sample are imaged Sample preparation has to be in accordance with the imaging
simultaneously onto the detector array. This can be done on technique used. In situ measurements with probes can be
a per wavelength basis with filters or tuneable filters. performed without sample preparation. On the other hand,
setups for Raman scattering for example proceed by non-
contact focusing, meaning that the sample surface may need
V-A220 Appendix II L 2023

to be prepared to obtain a reasonably flat surface. If the Multispectral systems are verified for all signal sources
instrumentation is unable to compensate for topographical involved.
variation, sample surfaces may be mechanically modified, for 3-2-1-3 Mapping
example by flattening a concave tablet surface. Focusing When images from more than one instrument are going to be
adjustments by automatic refocusing during mapping aligned, it is important to have verified x-y scales.
compensates for slightly uneven surfaces. Sample surface
3-2-1-4 Magnification
preparation has also to be considered with ATR-IR imaging
for which contact between optics and sample is required. In the case where the CIS permits different magnifications,
the optical or electronic magnification level needs to be
3-1-2 Sample presentation
optimised. If magnification is inadequate for resolving
Appropriate sample presentation is dictated by instrument
relevant features, the value of imaging may be diminished;
setup. A particular setup will be more or less adapted to a
whereas if magnification is set higher than required, the field
specific sample type and analytical task. The sample is
of view is reduced.
positioned so that it is optimally imaged. The setup is
optimised to reduce specular reflection as much as possible. 3-2-2 Calibration
Angles and distances between probe (or beam), sample and The purpose of calibration is to transform the recorded signal
detector match the setup requirements. into data suitable for analysis, interpretation, or comparison
with reference data.
3-2 CONTROL OF INSTRUMENT PERFORMANCE
Evaluating both instrument performance and image analysis Basic instrument calibration of imaging systems consists of x-
methods is essential to avoid misinterpretation or artefacts. and y-axis calibration in both wavelengths and intensity.
Parameters include spectral as well as spatial components. 3-2-2-1 Spectralax~
The instrument is to be used according to the manufacturer's Optimal wavenumber accuracy is critical and should be
instructions. achieved at the same level as for a standard spectrometer.
Instrument performance verification consists of periodic Spectral axis calibration assigns wavelength and intensity
performance qualification as well as system suitability tests. values to every mapped pixel of the sample surface.
The intervals depend on the use of the instrument and its To determine these relationships, well-characterised light
application. The system suitability tests are carried out before sources or reference materials can be used. Calibration
measurements to check if the Cl system is operating properly should be carried out using referenced and
for the intended application. certified/normalised internal or external standards provided
The parameters to be assessed and the acceptance criteria either by the supplier or a third party. Technology and
applied during performance qualification and system environmental conditions are other critical points.
suitability tests have to be justified and depend on the Cl 3-2-2-2 Spatial axes
technique and the purpose of the analysis. Parameters which Spatial axis calibration has an impact on the actual field of
might be verified are described below. view of the CIS and the sampled surface area, i.e. the pixel
3-2-1 CIS component adjustment location. Calibration corrects deformations on the spatial axis
In a schematic setup, a typical CIS would be made up of such as those caused by the optics. For example, the pixel
components including source of radiation, optical devices, position may be accompanied by variation of the spectral
sample holder, detector, and software. resolution from the centre towards the detector boundaries.
The system and its individual components comply with all In addition, mixing of signals from adjacent pixels can occur.
expected requirements. Both errors change the appearance of the pixel spectra and
may reduce the accuracy of subsequent data analysis. If it is
3-2-1-1 Sources, optics and detectors
not possible to deduce the position of the imaged pixels on
Source intensity is monitored as part of the setup verification. the detector surface from the pattern on the target surface, a
In particular, the source intensity should be verified before special test target can be used or developed for the
starting calibration routines or a new series of sample application.
measurements. Optical path, confocality, wavelength
Spatial resolution in both x and y directions is also limited,
accuracy and energy throughput at any x-y position (pixel) for example, by image size and step size of the moving table
match the specifications. or the conveyor belt.
Alignment of optics, sample and detectors comply with
3-2-2-3 Method
measurement requirements in terms of distances, angles and
polarisation. This alignment may shift with the temperature. Appropriate data processing and modelling are needed to
In particular, illumination of the sample or regions of interest deliver information about the property that correlates with
has to be as homogeneous as possible and reproducible. the observed features in images. Calibration related to the
analytical objective requires, for example, identifying and
Parasitic or adverse effects, for example scattering, estimating a characteristic identity or morphological features
background, noise, bad pixels, cosmic rays and fluorescent of the sample.
lights in the laboratory, and side effects such as sample
fluorescence have to be controlled. Stray light, ghost lines, 3-3 IMAGE PROCESSING
and ghost images can be caused by reflections from imperfect Processing means that the image is treated for display and
surface elements. They constitute parasitic light that has to subsequent feature estimations. Imaging requires steps to
be dealt with. improve the image for analysis, e.g. to control brightness and
contrast, reduce noise, sharpen edges and borders, remove
3-2-1-2 Multi-wavelength and multispectral systems
unwanted features, etc. This improvement allows features to
Multi-wavelength systems should be tested at wavelengths be recognised i.e. separated and discriminated from the
spread over the wavelength scale using the peaks of the background.
selected reference standard with good signal-to-noise
properties.
2023 Appendix II L V-A221

Chemical images are generally processed by chemometric 3-4-2 Feature extraction

modelling for qualitative or quantitative purposes (see general The extraction of chemical or morphological features of
chapter 5.21. Chemometric methods applied to analytical data). samples involves supervised techniques to perform
3-3-1 Spectral selection dimensional reduction. It concentrates further image
Image contrast is built upon specific signatures of the processing on characteristics that are meaningful for the
components at the sample surface and derived from analysis. For example, a selected feature could be the
spectroscopic data such as peak intensity, peak position, quantity of a given substance or a solid-state form of interest.
baseline variations (scattering differences), or values If the sample is comprised of components whose spectral
calculated with multivariate analysis. features are well separated, it is possible to proceed directly
with a univariate method based on a single wavelength per
3-3-2 Image pre-processing
feature, thus utilising only a small fraction of available data.
Data analysis methods for imaging data sets usually begin
However, in many cases spectral overlap is significant and
with the same steps as for single point spectroscopy. Pre-
the univariate approach is insufficient. The extraction of
processing is utilised to separate chemical and physical
features can then be improved by including a greater
effects, unless an analysis is based on the physical effects
proportion of the spectral information and applying a
(e.g. scattering differences seen as baseline variations).
multivariate approach, such as clustering, classification, PLS
The collected data is typically affected by noise or
discriminant analysis (PLS-DA), linear discriminant analysis
instrumental distortions and pre-processing is often applied
(LDA), etc.
to improve image quality. There may also be a need to
remove irrelevant sources of variation such as cosmic ray 3-4-3 Quantification of ingredients
spikes or stripes due to detector configuration. Common pre- There are several ways to measure ingredient variation using
processing operations for spectra are: baseline correction, regression methods. A quantitative model can be computed
derivatives, smoothing, wavelet-based filtering, standard by using average spectra or binning of regions of interest at
normal variate (SNV), multiplicative scatter correction the surface of the sample. The known bulk concentration
(MSC), normalisation, truncation and Fourier-transform. may be used when the ingredient present varies in responses
It has to be verified that pre-processing does not cause for the analytical method used. This can be performed by
artefacts. peak integration at a single wavelength or alternative
chemometric methods.
3-4 IMAGE ANALYSIS
Pixel arrangements in an image of the sample represent a When individual pixel spectra become increasingly mixed,
map of the distribution of chemical or physical properties or chemometric tools, typically multivariate data analysis, can be
features. Depending on the objective of the analysis, data can deployed with similar data analytical strategies as those for
be explored for spectral features, spatial features, and a single-point spectroscopy.
combination of both. Examination of the datacube can be For example, it is possible to estimate relative component
performed in a direct way, as for conventional spectroscopy abundance by using pure component spectra as the starting
(e.g. by analysing height of peaks or area ratios), or by point. The reference spectra used to create the library are
techniques including image processing, multivariate data collected from separate pure component samples. These
analysis, etc. spectra can be measured with a non-imaging spectrometer, or
as separate images of pure components. The inherent
3-4-1 Visual exploration
variation in pure component spectra when acquired across a
A review by the user of an image displayed on screen may
large number of pixels provides additional statistical
deliver a first impression before beginning enhancement,
robustness. The reference spectra and the unknown sample
exploration and evaluation. Images can be analysed for
can also be measured simultaneously in the same field of
features at the surface of the sample, such as location,
view to create one single data set. If pure component
distribution, size and shape of domains, constituent
reference spectra can be derived directly from an isolated
identification, etc. Standard image processing tools, such as
area of the imaged sample, there is no need to acquire
morphological filtering and particle statistics, may be applied
additional reference spectra for calibration. A multivariate
directly for samples that are significantly heterogeneous.
model can be developed either based on external reference
For a chemical image, both spectral and spatial analyses can spectra or based on spectra from isolated areas of the
be equally important and there are different steps of chemical respective image.
image analysis. Spectral and spatial analyses are often
Both with simultaneously and separately acquired pure
iterative by nature. In practice, the methods used depend
component spectra, it is important to verify that they are
upon whether it is the spectral information or the spatial
representative for the present analysis by for example
information that is of more interest. The goal is to reduce
regression residuals.
data dimensionality and extract as many features as possible.
Unsupervised multivariate analysis such as principal 3-4-4 Spatial analysis of image features
component analysis (PCA) may be applied prior to feature Spatial analysis of the images can be performed with regard
extraction to describe the information contained in the to distributions of shapes, sizes and locations of domains, etc.
image. A preliminary segmentation of PCA images may be Analysis of shape and distribution relies on a variety of
used to facilitate identification of regions of interest in the numerical methods and algorithms.
image for further processing. If the ingredients of a mixture Images display the samples by setting a threshold value and
are known and available for reference measurements, the converting data into a set of colours or shades according to a
corresponding data is used to reduce the number of spectral monochrome or pseudo-colour intensity scale to underline
layers in the hypercube down to a few chemically or the presence or proportion of the analytes of interest at each
physically meaningful layers by using supervised regression pixel. Alternatively, the distribution of several analytes can be
methods, e.g. multivariate curve resolution-alternating least combined into colour images. Further analysis can be
squares (MCR-ALS), independent component analysis performed, such as counting pixels, estimating the size
(ICA), and partial least squares regression (PLS). distributions of particles of the same chemical origin, and
V-A222 Appendix II M 2023

Potential
segmentation which is a process to divide the image between
regions that correspond to surface areas of interest. Eoxd, toxd
3-4-5 Measuring dimensions
Images can be used for example to determine area,
dimensions, perimeter, aspect ratio and roundness.
3-4-6 Statistics
Pixel statistics can be performed to facilitate further image
analysis. Application of methods such as variance analysis Delay Integration
and box-plot for domain size, shape, distribution, and
distances between domains, enables estimation of the
variability within an image. These methods deliver a
condensed description of relations between domains in the Ered, Ired
images with simplification of the image analysis. Property Time
estimations can also be derived from these values.

Ea": detection potential applied during a period ta"

Eoxd: oxidation potential applied during a period toxd
E"d: reduction potential applied during a period teed
M. Direct Amperometric and Pulsed
Electrochemical Detection Figure 2.2.63.-1. - 3-Step potential-time waveform (pulse
sequence)
(Ph. Bur. method 2.2.63)
Direct amperometric detection and pulsed electrochemical EQUIPMENT
detection (PED) are used to detect electroactive compounds An electrochemical detection system comprises 3 electrodes:
by oxidation (or reduction) when coupled to a separation a measuring electrode (gold, platinum or glassy carbon);
technique such as liquid chromatography (2.2.29). a reference electrode (usually silver-silver chloride); and an
Electrochemical detection uses the redox potential of analytes auxiliary electrode (e.g. titanium cell body) (Figure
and is therefore only valid for electroactive substances. 2.2.63.-2).
PRINCIPLE The choice of measuring electrode depends on the substance
When a constant potential is applied between 2 electrodes, to be examined. The electrodes are coupled to a potentiostat,
the oxidation (or reduction) of an electroactive substance which controls the potentials applied to the measuring
generates a current proportional to the amount of analyte electrode without subjecting the reference electrode to a
passing the electrode surface. This analytical technique current, the auxiliary electrode functioning as a cathode
requires a supporting electrolyte, which can be added post- whenever the measuring electrode is operating as an anode
column if not already present in the mobile phase. Detection and vice versa.
usually involves oxidation of the compounds of interest.
The potential applied to the measuring electrode can serve as Mobile phase out
a selectivity parameter for the compounds of interest.
The upper limit of the electroactivity range is defined by the
oxidation curve of the mobile phase, the supporting t
electrolyte or the measuring electrode.
With direct amperometric detection, only a constant potential
A
is applied. However, this method may result in the surface of
the measuring electrode becoming fouled with adsorbed
carbon compounds, resulting in signal variability and a
decreased response. This passivation phenomenon is
particularly common with sugars, thiols and phenols. C
The problem of electrode fouling can be resolved by the B
application of a series of pulses, a process known by the
generic term of pulsed electrochemical detection (PED).
The most widely used PED mode is pulsed amperometric
Mobile phase in ~ . - - - - - - -
detection (PAD), which involves a 3-step series of pulses or
waveform (Figure 2.2.63.-1). After applying the detection
potential (Ede,), a high positive potential (Eoxd) (anodic
polarisation) is applied to clean the surface of the measuring
electrode, followed by a negative potential (Erect) (cathodic
A. reference B. auxiliary C. measuring
polarisation) to restore the surface of the measuring electrode electrode electrode
electrode. A new cycle of the waveform can then start.
Following the potential step from Bred to Edw background
signals will originate. Although these decay quickly, a brief
delay must be introduced before the analyte signal can be Figure 2.2.63.-2. - Electrochemical detection system
properly measured.
EQUIPMENT PERFORMANCE
Other waveforms comprising more than 3 voltage steps have
The potentials to be applied depend on the analyte and the
been developed.
type of electrodes used, and the settings must therefore be
adapted accordingly.
2023 Appendix II N V-A223

All electrodes should be in good condition and periodic facilitates process development in accordance with quality by
replacement is recommended. When the detector is used for design (QbD) principles, enables real-time release
quantitative analysis, it is advisable to check the linear range, testing (RTRT) and supports continuous manufacturing
sensitivity and repeatability of the detector. processes.
The quality of the reagents used is of utmost importance. Time delays between obtaining a sample for testing, analysis
It is advisable to employ reagents of the highest purity. of that sample and any consequent outcomes must be taken
ADDITIONAL INFORMATION into consideration when applying PAT. When the analytical
Maintenance of the electrochemual cell results are used on a continuous basis to monitor and control
a process, it is important to minimise such delays. This can
Although the application of an increased potential in PED
be achieved most effectively with sensor-based continuous
cleans the surface of the measuring electrode effectively, it
measurement systems directly interfacing with the process
can be necessary to polish the measuring electrode
stream during a specific unit operation. The sensors
mechanically from time to time when the background current
interfaced with the process continuously measure the process
increases and the sensitivity decreases. The cleaning of the
conditions and material characteristics within the process
measuring electrode must be carried out very carefully to
environment (in situ) and send the measured data
avoid creating pits or scratches. It is also advisable to wipe
(e.g. a spectrum) to an operating system where it is recorded
the auxiliary electrode at the same time to remove deposited
and analysed, and where any necessary adjustments to
substances. The cell requires some time to stabilise after
processing conditions can be determined on a continuous
polishing.
basis. These in situ measurements can generate very large
Sodium hydroxide solution volumes of data representative of the process.
Detection can be enhanced in alkaline media (at least
2 INTERFACING MODES
pH 12), as is the case with aminoglycoside antibiotics. When
the mobile phase is not sufficiently alkaline, this can be The interfacing of analytical techniques with the
achieved by post-column addition of a sodium hydroxide manufacturing process is central to the application of PAT.
solution to increase the pH of the column effluent. Results generated by analytical equipment interfaced with a
The solutions are mixed in a coil that is linked to the process can be used for monitoring purposes and to ensure
electrochemical cell. It is essential that the length of the that the process is stable, for example via automated
mixing coil is such as to produce a homogeneous solution feedback or feedforward control loops.
but with minimal peak broadening. It is important to avoid The terms 'off-line', 'at-line', 'on-line' and 'in-line' describe
carbonates in the sodium hydroxide solution. To avoid interfacing modes (see Figure 5.25.-1). Measurements using
baseline disturbances, the sodium hydroxide solution should on-line and in-line systems generally support rapid and
be degassed before use, it must be added pulse-free and its automated process adjustments since they move the analytical
flow should be constant between runs. The parts of the techniques into the process stream. In contrast, off-line and
chromatographic system coming into contact with the at-line measurements involve transferring the samples away
sodium hydroxide-containing solutions must be alkaline from the process stream to the analytical equipment.
resistant. Off-line measurements
These correspond to conventional analytical testing in which
the sample is removed from the manufacturing process
environment and tested in a laboratory, typically located
Process Analytical Technology away from the production environment. This transfer of
samples away from the process stream can result in a
(Ph. Bur. method 5.25) significant time lag and generally does not permit immediate
This general chapter focuses on the interfacing of analytical process adjustments. However, off-line measurements can
techniques with the manufacturing process as a means of nonetheless be useful for PAT purposes if relevant analytical
enhancing process control and improving process understanding. data can be obtained within a time frame that is compatible
It should be noted that it is not within the scope of the chapter to with process dynamics.
give specific instructions for all possible process analytual At-line measurements
technology sensors; instead, it offers a general approach to the With at-line measurements, the sample is also removed
integration of analytical technUJ.ues in the process environment (manually or automatically) from the process stream for
together with aspects to be taken into consideration for the testing, but the testing equipment is usually located within
application of process analytical technology. the production environment, i.e. in close physical proximity
1 INTRODUCTION to the process stream, and testing can therefore take place
Process analytical technology (PAT) can be defined as a with minimal delay. This results in a shorter time frame for
system for designing, analysing and controlling obtaining analytical data, and also differentiates at-line
manufacturing processes through timely measurements measurements from off-line measurements. It is therefore
(i.e. during processing) of critical quality attributes (CQA) of possible to make process adjustments based on the results of
raw and in-process materials and critical performance at-line measurements.
characteristics of processes in order to ensure the quality of On-line measurements
the final product. It is important to note that the term On-line measurements typically involve sensor-based
'analytical' in PAT is used in a broad sense to include measurements made under real-time conditions by diverting
chemical, physical and microbiological measurements a portion of the material from the process stream directly
conducted in an integrated manner and combined with data into a measuring device and the results are available after a
analysis. The goal of PAT is control of the manufacturing minimal time delay.
process and enhanced process understanding, guided by risk
management. Interfacing manufacturing processes with
analytical techniques is therefore essential in PAT, as it
V-A224 Appendix II N 2023

Depending on the nature of the test, e.g. whether or not it is In some cases, PAT measurements can be used qualitatively
detrimental to the product, the diverted portion may be to build models such as process trajectories or process
returned to the process stream, otherwise it is discarded. signatures which can be used to characterise process
In-line measurements variability and highlight unusual process behaviours. These
In-line measurements are taken within the process stream by models are not necessarily directly linked to a critical
placing measuring devices, typically sensors, directly in in-process quality attribute or performance characteristic. It is
contact with or into the process stream. No portions of the therefore recommended to demonstrate the causal
material are therefore removed from the process stream. relationship between the PAT measurements, the model and
the relevant quality attributes or performance characteristics
In-line measurements must also be non-detrimental to the
when using such models for process control purposes.
product.
General validation principles also apply to in-line or on-line
measurements although some validation procedures may be
PROCESS ENVIRONMENT LABORATORY
different to those for conventional quality control methods.
Analytical validation procedures for at-line and off-line
measurements assume homogenous and authentic samples,
which are crucial for the assessment of precision. However,
this requirement is rarely fulfilled for on-line or in-line
methods which are often used to measure processes in which
the material rapidly changes and/or moves during the
measurement, for example a fast-drying process or reaction
monitoring. Comparison of results from on-line/in-line
measurements with results from a reference test procedure
would normally be needed.
4 STATISTICAL PROCESS CONTROL
Statistical process control (SPC) comprises a set of data
analysis methods applied in order to monitor and control a
process based on analysis of process variability
(e.g. ofCQAs). For PAT purposes, the process can be
monitored using, for example, real-time data collected by
process analysers.
Based on this data, SPC can be used to make process
process stream adjustments, when necessary, to maintain or attain a desired
state and to ensure that the process remains under control.
This includes, for example, trend analysis of quality attributes
Figure 5.25.-1. - Interfacing modes
or performance characteristics. SPC can also be used to
measure process variability and process capability
3 COMPARISON OF INTERFACING MODES
(i.e. the ability of a process to produce a product that
Both off-line and at-line measurements are based on analysis
complies with requirements), with a view to enhancing
of discrete samples removed from a process stream or bulk
process understanding, thereby improving lifecycle
material. These samples are considered representative of the
management.
material in the process stream at the time they are removed.
Frequent at-line measurements also provide data supporting Rather than using univariate SPC methods to monitor several
the application of PAT, as they are carried out within a short individual variables, it is often preferable to use multivariate
time frame and in the immediate vicinity of the process statistical process control (MSPC) to analyse several variables
stream. simultaneously, taking into account potential correlations (see
general chapter 5. 28. Multivariate statistical process control).
Neither in-line nor on-line measurements involve sampling in
the conventional sense. The tested portion might not be 5 EUROPEAN PHARMACOPOEIA TEXTS
separated from the process stream, and is usually smaller SUPPORTING THE APPLICATION OF PAT
than a conventional sample. Typically, the analytical techniques described in the Ph. Eur.
Scale of scrutiny must be considered and will determine the include qualification criteria designed for off-line analytical
frequency and duration of measurements. Furthermore, systems. These criteria are not always relevant or practical in
measurements can be influenced by physical attributes a PAT setting, for instance when the equipment is
interfering with the acquisition properties of the measuring specifically designed for on-line or in-line measurements.
system. When measuring solids or suspensions, for example For this reason, certain general chapters (including 2. 2. 40.
using spectroscopic methods, consideration must be given to Near-infrared spectroscopy, 2.2.48. Raman spectroscopy and
the surface and bulk scattering properties of samples, and 2.9.47. Demonstration of uniformity of dosage units using large
movement of the material. Influencing factors such as particle sample sizes) have been revised or specifically elaborated to
size, surface roughness and solid density can cause significant support and promote the use of the techniques in
spectral differences, which must be taken into account when conjunction with PAT.
a method is designed and used in order to ensure that the General Notices
process and material are described adequately. Section 1.1, under Demonstration of compliance with the
In-line and on-line modes offer the advantage of fast data Pharmacopoeia, states that a substance can be demonstrated
acquisition, allowing high measurement frequency, and thus to be of pharmacopoeia! quality on the basis of product
enabling rapid continuous monitoring as well as immediate design, together with the control strategy applied and data
action and control of the process.
2023 Appendix II N V-A225

derived, for example, from validation studies of the Demonstration of uniformity of dosage units using
manufacturing process. large sample sizes
The section also states that an enhanced approach to quality General chapter 2. 9.47 allows the determination of
control could utilise PAT and/or real-time release testing dosage unit uniformity in a PAT environment where the
strategies (including parametric release) as alternatives to sample size is markedly greater than 30 units.
end-product testing alone. Compliance with the acceptance criteria of general chapter
Absorption spectrophotometry, infrared 2.9.47 is considered as evidence that the batch would also
General chapter 2.2.24 has a wide variety of applications in comply with general chapter 2. 9. 40. Unifonnity of dosage units
the manufacturing process that enable PAT, such as reaction if tested using a small sample size (e.g. 30-unit). Therefore,
monitoring in chemical synthesis. Wavenumber shifts and general chapter 2.9.47 does not constitute stand-alone
acceptable tolerances for the reference material polystyrene acceptance criteria.
are described for both transmission and ATR measurement Alternative methods for control of microbiological
modes. quality
Absorption spectrophotometry, ultraviolet and visible General chapter 5.1. 6 describes alternative methods that
General chapter 2.2.25 covers the use of PAT in the UV-Vis might be used for the application of PAT in order to
range using modern detectors such as photodiode contribute to real-time or near-real-time microbiological
arrays (PDA) or charge-coupled devices (CCD). Both quality control (e.g. test for microbiological examination of
transmission and diffuse reflection measurement modes are non-sterile products or for sterility based on laser-induced
possible with off-line, at-line, on-line and in-line fluorescence) of in-process samples, active substances,
measurements. The table with reference wavelengths for the medicinal products or excipients (particularly water).
control of wavelength accuracy includes wavelengths in the The chapter also provides guidance on how to validate these
UV range down to 180 nm. alternative methods.
X-ray fluorescence spectrometry Chemometric methods applied to analytical data
General chapter 2.2.37 includes references to modern Chemometrics (general chapter 5.21) has proven to be well
equipment and the current applications of the XRF suited for PAT. The investigation of large data tables and
technique. treatment of intricate signals, i.e. data collections with a
hidden structure, of the type accumulated during
Substantial advances in miniaturisation and automation have
measurements in PAT setups, requires alternative analytical
led to the development of hand-held energy dispersive
tools to those used in a one-variable-at-a-time approach.
XRF (ED-XRF) spectrometers for rapid field measurements.
XRF is potentially non-destructive, although it may cause Chemical imaging
sample instability. Hence, XRF lends itself to applications of Chemical imaging (Cl) (general chapter 5.24) can be used to
PAT such as the analysis of unwanted trace catalysts in support PAT applications. Cl measures spatial distribution
active pharmaceutical ingredients (API). and contributes to the understanding of the properties of
materials such as finished products, pharmaceutical
N ear-infrared spectroscopy
intermediates, excipients, APis and starting materials.
General chapter 2. 2. 40 takes into account the increasing use
of NIR spectroscopy for process monitoring and control, and The uses of Cl include, for example, detection of defects at
the development of modern NIR spectrometers. the sample surface such as cracks in tablet coatings and
NIR measurements can be performed off-line, but the identification of foreign particles. It is a versatile tool used in
technique also lends itself to at-line, on-line and in-line process development and improvement, root cause analysis
testing. (e.g. for out-of-specification results), and may also be used to
enhance process understanding.
The 'Sample preparation/presentation' section includes
moving materials or samples, and the use of fibre-optic probe Multivariate statistical process control
systems in different measurement modes (i.e. transmission, General chapter 5.28 gives insight into how MSPC can be
diffuse reflection and transflection). The use of internal used to control and improve manufacturing processes, based
referencing for process analysis purposes is permitted when it on the collection and processing of large amounts of data on
is impossible to remove a probe for reference background multiple variables. It describes the principle of MSPC and
data collection. A detailed table for the control of instrument the development and use of multivariate control charts, and
performance depending on measurement mode and provides an overview of the theoretical background of
instrument used (benchtop, mobile or process instrument) is multivariate statistical procedures.
also provided. The chapter includes a section on qualitative
analysis for identification and characterisation of a substance
in addition to sections on limit analysis (e.g. for dryer
end-point control) and trend analysis (for monitoring blend
uniformity).
Raman spectroscopy
General chapter 2.2.48 includes Raman technologies that
have potential uses for the application of PAT, including
hand-held instruments.
Wavenumber shifts and acceptable tolerances for the
reference materials cyclohexane, paracetamol and polystyrene
(values derived from an inter-laboratory study) are described
for both benchtop and hand-held Raman systems.
V-A226 Appendix III 2023

effluent concentration, versus time or volume. Idealised

Appendix III chromatograms are represented as a sequence of Gaussian
peaks on a baseline (Figure 2.2.46.-1).
Chromatographic Separation Techniques Peak
(Ph. Bur. method 2.2.46) The portion of a chromatogram recording the detector
response when a single component (or 2 or more unresolved
Chromatographic separation techniques are multi-stage components) is eluted from the column.
separation methods in which the components of a sample are The peak may be defined by the peak area, or the peak
distributed between 2 phases, one of which is stationary, height (h) and the peak width at half-height (wh), or the peak
while the other is mobile. The stationary phase may be a height (h) and the peak width between the points of
solid or a liquid supported on a solid or a gel. The stationary inflection (w;). In Gaussian peaks (Figure 2.2.46.-1) there is
phase may be packed in a column, spread as a layer, or the following relationship:
distributed as a film, etc. The mobile phase may be gaseous
or liquid or supercritical fluid. The separation may be based
on adsorption, mass distribution (partition), ion-exchange,
etc., or may be based on differences in the physico-chemical Retention time (tR)
properties of the molecules such as size, mass, volume, etc. Time required for elution of a component (Figure 2.2.46.-1,
This chapter contains definitions and calculations of common baseline scale being in minutes).
parameters and generally applicable requirements for system Retention volume (VR)
suitability. Principles of separation, apparatus and methods Volume of the mobile phase required for elution of a
are given in the following general methods: component. It may be calculated from the retention time and
- paper chromatography (2.2.26); the flow rate (F) in millilitres per minute using the following
- thin-layer chromatography (2.2.27); equation:
- gas chromatography (2.2.28);
- liquid chromatography (2.2.29);
- size-exclusion chromatography (2.2.30);
- supercritical fluid chromatography (2.2.45). Hold-up time (tM)
Time required for elution of an unretained component
DEFINITIONS
(Figure 2.2.46.-1, baseline scale being in minutes). In size-
The system suitability and acceptance criteria in monographs have exclusion chromatography, the symbol t0 (see below) is used.
been set using parameters as defined below. With some equipment,
certain parameters, such as the signal-to-noise ratio and resolution, Hold-up volume ( VM)
can be calculated using software provided by the manufacturer. Volume of the mobile phase required for elution of an
It is the responsibility of the user to ensure that the calculation unretained component. It may be calculated from the hold-
methods used in the software are equivalent to the requirements of up time and the flow rate (F) in millilitres per minute using
the European Pharmacopoeia and to make any necessary the following equation:
corrections if this is not the case.
Chromatogram
A graphical or other representation of detector response, In size-exclusion chromatography, the symbol Vo (see below)
effluent concentration or other quantity used as a measure of is used.

Peak 1 Peak2

illC
8.
rJl

&

.c::

Volume (V)
Time (t)
Figure 2.2.46.-1.
2023 Appendix III V-A227

tR2,vR2
Q)
(/)
C
0
c.
(/)
~
LC and GC ...
~
Q)
0

Size-exclusion
chromatography

10'

(/)
(/) 10 6
cc
E
ro 105
=i
(J
Q)
0
::i: 1Q4

103

Time (t)
Volume (V)

Figure 2.2.46.-2.

Retention factor (k)

The retention factor (also known as mass distribution ratio
(Dm) or capacity factor (k ')) is defined as:
Total mobile phase time (t,)
k = amount of component in stationary phase = K., ~
In size-exclusion chromatography, retention time of a
component whose molecules are smaller than the smallest gel
amount of component in mobile phase c VM
pores (Figure 2.2.46.-2).
Kc distribution constant (also known as equilibrium distribution Total mobile phase volume ( Vi)
coefficient); In size-exclusion chromatography, retention volume of a
Vs volume of tbe stationary phase;
VM volume of tbe mobile phase.
component whose molecules are smaller than the smallest gel
pores. It may be calculated from the total mobile phase time
The retention factor of a component may be determined and the flow rate (F) in millilitres per minute using the
from the chromatogram using the following equation: following equation:
V-A228 Appendix III 2023

V,=t,xF

Retention time of an unretained compound (t0)

In size-exclusion chromatography, retention time of a retention time of the peak corresponding to the component;
component whose molecules are larger than the largest gel width of the peak at half-height.
pores (Figure 2.2.46.-2).
Retention volume of an unretained compound ( V0) The plate number varies with the component as well as with
In size-exclusion chromatography, retention volume of a the column, the column temperature, the mobile phase and
component whose molecules are larger than the largest gel the retention time.
pores. It may be calculated from the retention time of an Dwell volume (D)
unretained compound and the flow rate (F) in millilitres per The dwell volume (also known as gradient delay volume) is
minute using the following equation: the volume between the point at which the eluents meet and
the top of the column. It can be determined using the
Vo=toxF following procedure.
Distribution constant (~) Column Replace the chromatographic column by an
In size-exclusion chromatography, the elution characteristics appropriate capillary tubing (e.g. 1 m x 0.12 mm).
of a component in a particular column may be given by the Mobile phase:
distribution constant (also referred to as distribution - mobile phase A: water R;
coefficient), which is calculated using the following equation: - mobile phase B: 0 .1 per cent V/V solution of acetone R;
tR - to
K,o=--- Time Mobile phase A Mobile phase B
t, - to (min) (per cent V/JJ) (per cent V/JJ)
0 - 20 100 ➔ 0 0 ➔ 100
Retardation factor (Rp)
20 - 30 0 100
The retardation factor (also known as retention factor (R1)),
used in planar chromatography, is the ratio of the distance
from the point of application to the centre of the spot and Flow rate Set to obtain sufficient back-pressure
the distance travelled by the solvent front from the point of (e.g. 2 mUmin).
application (Figure 2.2.46.-3). Detection Spectrophotometer at 265 nm.
Determine the time (t0 _5 ) in minutes when the absorbance
b
Rp=- has increased by 50 per cent (Figure 2.2.46.-4).
a
migration distance of the component; D= tvxF
a migration distance of the solvent front.
tn ro.s - O.StG (in minutes);
tc pre-defined gradient time (= 20 min);
F flow rate (in millilitres per minute).

-A 100 % ----------,'--:::;-;.;--,-----
/
/
9
~
-0-B C:
(tl
..Q
0
Jl
a <t:

-- _c /

Time

Figure 2.2.46.-4
A. mobile phase front B. spot C. line of application
Symmetry factor (As)
The symmetry factor of a peak (Figure 2.2.46.-5) is
Figure 2.2.46.-3.
calculated using the following equation:
Plate number (N)
A _ Wo.os
The column performance (apparent efficiency) may be ' - 2d
calculated from data obtained under either isothermal,
isocratic or isodense conditions, depending on the technique, Wo.os width of the peak at one-twentieth of the peak height;
as the plate number (also referred to as number of theoretical d distance between the perpendicular dropped from the peak
maximum and the leading edge of the peak at one-twentieth of
the peak height.

plates), using the following equation, the values of tR and wh

being expressed in the same units:
2023 Appendix III V-A229

An As value of 1.0 signifies symmetry. When As > 1.0, the

peak is tailing. When A, < 1.0, the peak is fronting.

IT\
/ I \
/ ! \
j \
I ,
I '
I \
--d--

Figure 2.2.46.-5

Resolution (R5 )
The resolution between peaks of 2 components
(Figure 2.2.46.-1) may be calculated using the following
equation:

l.18(tR2 - tR1)
R,=------
wh1 +wh2
Figure 2.2.46.-6

Relative retention (r)

retention times of the peaks;
Relative retention is calculated as an estimate using the
tRli tR2
U'hJ, Wh2 peak widths at half-height. following equation:
tR; - tM
In quantitative planar chromatography, using densitometry, r=----
tRsr - tM
the migration distances are used instead of retention times
and the resolution between peaks of 2 components may be tRi retention time of the peak of interest;
calculated using the following equation: t&, retention time of the reference peak (usually the peak
corresponding to the substance to be examined);
tM hold-up time.
R, = l.18a(Rn - RFI)
WhJ + Wh2 The unadjusted relative retention (r0 ) is calculated using the
retardation factors of the peaks;
following equation:
RF1,Rm
whl, Whz peak widths at half-height;
a migration distance of the solvent front.

Peak-to-valley ratio (p/v) Unless otherwise indicated, values for relative retention stated
The peak-to-valley ratio may be employed as a system in monographs correspond to unadjusted relative retention.
suitability criterion in a test for related substances when
In planar chromatography, the retardation factors RFsr and
baseline separation between 2 peaks is not achieved
Rp; are used instead of tRs, and tRi•
(Figure 2.2.46.-6).
Signal-to-noise ratio (SIN)
H The short-term noise influences the precision of
p/v = ___p_
Hv quantification. The signal-to-noise ratio is calculated using
the following equation:
height above the extrapolated baseline of the minor peak;
height above the extrapolated baseline at the lowest point of the
curve separating the minor and major peaks.
S/N=2H
h
H height of the peak (Figure 2 .2.46.-7) corresponding to the
component concerned, in the chromatogram obtained with the
prescribed reference solution, measured from the maximum of
the peak to the extrapolated baseline of the signal observed over
a distance equal to at least 5 times the width at half-height;
h range of the noise in a chromatogram obtained after injection or
application of a blank, observed over a distance equal to at least
5 times the width at half-height of the peak in the
chromatogram obtained with the prescribed reference solution
and, if possible, situated equally around the place where this
peak would be found.
V-A230 Appendix III 2023

K constant (0.349), obtained from the expression K =

~ x ~ in which ¾ represents the required percentage
H ;elativ~ 0standard de~iation after 6 injections for B = I .O;
B upper limit given in the definition of the individual
monograph minus l 00 per cent;
n number of replicate iujectious of the reference solution
(3 ::'. n ::'. 6);
Student's tat the 90 per cent probability level (double
sided) with n-1 degrees of freedom.

Unless otherwise prescribed, the maximum permiued relative

standard deviation does not exceed the appropriate value
given in Table 2.2.46.-1. This requirement does not apply to
tests for related substances.

Figure 2.2.46.-7. Table 2.2.46.-1. - Repeatability requirements

Number of individual injections
System repeatability
The repeatability of response is expressed as an estimated 3 4 5 6
percentage relative standard deviation (s,(%)) of a
B (per cent) Maximum pennitted relative standard deviation
consecutive series of measurements for not fewer than 3
injections or applications of a reference solution, and is 2.0 0.41 0.59 0.73 0.85
calculated using the following equation:
2.5 052 0.74 0.92 1.06

( 01
Sr /0 . -
) _
---v
100 /z_)Yi - y) 2
Y n- l
-
3.0 0.62 0.89 l.10

in a related substances test, the limit of quantification

1.27

Y; individual values expressed as peak area, peak height, or ratio of (corresponding to a signal-to-noise ratio of 10) is equal to
areas by the internal standardisation method; or less than the disregard limit.
y = mean of individual values;
Compliance with the system suitability criteria is required
n number of individual values. throughout the chromatographic procedure. Depending on
various factors, such as the frequency of use of the procedure
SYSTEM SUITABILITY and experience with the chromatographic system, the analyst
The various components of the equipment employed must be chooses an appropriate verification scheme to monitor this.
qualified and be capable of achieving the performance ADJUSTMENT OF CHROMATOGRAPHIC
required to conduct the test or assay. CONDITIONS
The system suitability tests represent an integral part of the The extent to which the various parameters of a
method and are used to ensure adequate performance of the chromatographic test may be adjusted to satisfy the system
chromatographic system. Apparent efficiency, retention factor suitability criteria without fundamentally modifying the
(mass distribution ratio), resolution and symmetry factor are methods are listed below. Adjustment of conditions with
the parameters that are usually employed in assessing the gradient elutions is more critical than with isocratic elutions,
performance of the column. Factors that may affect the since it may lead to shifts in peaks to a different step of the
chromatographic behaviour include: gradient, thus leading to the incorrect assignment of peaks,
- the composition, ionic strength, temperature and apparent and to the masking of peaks or a shift such that elution
pH of the mobile phase; occurs beyond the prescribed elution time. Changes other
- flow rate, column dimensions, column temperature and than those indicated require revalidation of the method.
pressure; The chromatographic conditions described have been
- stationary phase characteristics including type of validated during the elaboration of the monograph.
chromatographic support (particle-based or monolithic), The system suitability tests are included to verify that the
particle or macropore size, porosity, specific surface area; separation required for satisfactory performance of the test or
- reversed-phase and other surface-modification of the assay is achieved. Nonetheless, since the stationary phases are
stationary phases, the extent of chemical modification (as described in a general way and there is such a variety
expressed by end-capping, carbon loading etc.). available commercially, with differences in chromatographic
The following requirements and any supplementary behaviour, some adjustments of the chromatographic
requirements given in the individual monograph are to be conditions may be necessary to achieve the prescribed system
fulfilled unless otherwise prescribed: suitability requirements. With reversed-phase liquid
- in a related substances test or assay, for a peak in the chromatographic methods in particular, adjustment of the
chromatogram obtained with a reference solution used for various parameters will not always result in satisfactory
quantification, the symmetry factor is 0.8 to 1.5, unless chromatography. In that case, it may be necessary to replace
otherwise prescribed; the column with another of the same type (e.g. octadecylsilyl
- in an assay of an active substance where the value is silica gel), which exhibits the desired chromatographic
100 per cent for a pure substance, the maximum behaviour. The Knowledge database on the EDQM website
permiued relative standard deviation (sr(%),,,ax) for the usually contains information on the column(s) used during
defined limits is calculated for a series of injections of the monograph elaboration.
reference solution using the following equation:
2023 Appendix III V-A231

For critical parameters the adjustments are defined clearly in Injection volume May be decreased, provided detection
the monograph to ensure the system suitability. and repeatability of the peak(s) to be determined are
Thin-layer chromatography and paper satisfactory; no increase permitted.
chromatography Liquid chromatography: gradient elution
Composition of the mobile phase The amount of the Adjustment of chromatographic conditions for gradient
minor solvent component may be adjusted by ± 30 per cent systems requires greater caution than for isocratic systems.
relative or ± 2 per cent absolute, whichever is the larger; for Composition of the mobile phase/gradient elution
a minor component at 10 per cent of the mobile phase, a Minor adjustments of the composition of the mobile phase
30 per cent relative adjustment allows a range of and the gradient are acceptable provided that:
7-13 per cent whereas a 2 per cent absolute adjustment - the system suitability requirements are fulfilled;
allows a range of 8-12 per cent, the relative value therefore - the principal peak(s) elute(s) within± 15 per cent of the
being the larger; for a minor component at 5 per cent of the indicated retention time( s);
mobile phase, a 30 per cent relative adjustment allows a - the final composition of the mobile phase is not weaker in
range of 3.5-6.5 per cent whereas a 2 per cent absolute elution power than the prescribed composition.
adjustment allows a range of 3-7 per cent, the absolute value Where compliance with the system suitability requirements
being the larger in this case; no other component is altered
cannot be achieved, it is often preferable to consider the
by more than 10 per cent absolute.
dwell volume or to change the column.
pH of the aqueous component of the mobile phase Dwell volume The configuration of the equipment
± 0.2 pH, unless otherwise prescribed, or ± 1.0 pH when
employed may significantly alter the resolution, retention
non-ionisable substances are to be examined. time and relative retentions described. Should this occur, it
Concentration of salts in the buffer component of a may be due to excessive dwell volume. Monographs
mobile phase ± I O per cent. preferably include an isocratic step before the start of the
Application volume l 0-20 per cent of the prescribed gradient programme so that an adaptation can be made to
volume if using fine particle size plates (2-10 µm). the gradient time points to take account of differences in
Liquid chromatography: isocratic elution dwell volume between the system used for method
Composition of the mobile phase The amount of the development and that actually used. It is the user's
minor solvent component may be adjusted by ± 30 per cent responsibility to adapt the length of the isocratic step to the
relative or ± 2 per cent absolute, whichever is the larger (see analytical equipment used. If the dwell volume used during
example above); no other component is altered by more than the elaboration of the monograph is given in the monograph,
10 per cent absolute. the time points (t ruin) stated in the gradient table may be
replaced by adapted time points (tc ruin), calculated using the
pH of the aqueous component of the mobile phase
following equation:
± 0.2 pH, unless otherwise prescribed, or ± 1.0 pH when
non-ionisable substances are to be examined. (D-Do)
Concentration of salts in the buffer component of a tc = t- F
mobile phase ± l O per cent.
D dwell volume} in millilitres;
Flow rate ± 50 per cent; a larger adjustment is
Do dwell volume used for development of the method, in millilitres;
acceptable when changing the column dimensions (see the F flow rate, in millilitres per minute.
formula below).
Column parameters The isocratic step introduced for this purpose may be
Stationary phase: omitted if validation data for application of the method
- no change of the identity of the substituent of the without this step is available.
stationary phase permitted (e.g. no replacement of pH of the aqueous component of the mobile phase No
Cl8 by CS); adjustment permitted.
- particle size: maximum reduction of 50 per cent; Concentration of salts in the buffer component of a
no increase permitted. mobile phase No adjustment permitted.
Column dimensions: Flow rate Adjustment is acceptable when changing the
- lengt,h: ± 70 per cent; column dimensions (see the formula below).
- internal diameter. ± 25 per cent. Column parameters
When column dimensions are changed, the flow rate may be Stationary phase:
adjusted as necessary using the following equation: - no change of the identity of the substituent of the
stationary phase permitted (e.g. no replacement
of C18 by CS);
- particle size: no adjustment permitted.
flow rate indicated in the monograph, in millilitres per minute; Column dimensions:
adjusted flow ratei in millilitres per minute; - lengt,h: ± 70 per cent;
length of the column indicated in the monograph, in
- internal diameter. ± 25 per cent.
millimetres;
length of the column used, in millimetres; When column dimensions are changed, the flow rate may be
internal diameter of the column indicated in the monograph, in adjusted as necessary using the following equation:
millimetres;
d, internal diameter of the column used, in millimetres.

Temperature ± 10 'C, where the operating temperature

is specified, unless otherwise prescribed.
flow rate indicated in the monograph} in millilitres per minute;
Detector wavelength No adjustment permitted. adjusted flow rate 1 in millilitres per minute;
V-A232 Appendix III A 2023

length of the column indicated in the monograph, in to the response(s) (peak(s)) obtained with a reference
millimetres;
solution.
length of the column used, in millimetres;
internal diameter of the column indicated in the monograph, in - Intemal standard method. Equal amounts of a component
millimetres; that will be resolved from the substance to be examined
internal diameter of the column used, in millimetres. (the internal standard) are introduced into the test
solution and a reference solution. The internal standard is
Temperature ± 5 °C, where the operating temperature is chosen such that it does not react with the substance to
specified, unless otherwise prescribed. be examined, is stable and does not contain impurities
Detector wavelength No adjustment permitted. with the same retention time as that of the substance to
Injection volume May be decreased, provided detection be examined. The concentration of the substance to be
and repeatability of the peak(s) to be determined are examined is determined by comparing the ratio of the
satisfactory; no increase permitted. peak areas or peak heights due to the substance to be
examined and the internal standard in the test solution
Gas chromatography
with the ratio of the peak areas or peak heights due to the
Column parameters
substance to be examined and the internal standard in the
Stationary phase: reference solution.
- particle size: maximum reduction of SO per cent; - Normalisation procedure. The percentage content of a
no increase permitted (packed columns); component of the substance to be examined is calculated
- film thickness: ~so per cent to + 100 per cent by determining the area of the corresponding peak as a
(capillary columns). percentage of the total area of all the peaks, excluding
Column dimensions: those due to solvents or reagents or arising from the
- length: ± 70 per cent; mobile phase or the sample matrix, and those at or below
- intemal diameter. ± 50 per cent. the disregard limit.
Flow rate ± 50 per cent. - Calibration procedure. The relationship between the
Temperature ± 10 per cent. measured or evaluated signal (y) and the quantity
(concentration, mass, etc.) of substance (x) is determined
Injection volume and split volume May be adjusted,
and the calibration function is calculated. The analytical
provided detection and repeatability are satisfactory.
results are calculated from the measured signal or
Supercritical fluid chromatography evaluated signal of the analyte by means of the inverse
Composition of the mobile phase For packed columns, function.
the amount of the minor solvent component may be adjusted In tests for related substances for both the external standard
by ± 30 per cent relative or ± 2 per cent absolute, method, when a dilution of the test solution is used for
whichever is the larger; no adjustment is permitted for a comparison, and the normalisation procedure, any correction
capillary column system. factors indicated in the monograph are applied (i.e. when the
Detector wavelength No adjustment permitted. response factor is outside the range 0.8-1.2).
Column parameters When the related substances test prescribes the total of
Stationary phase: impurities or there is a quantitative determination of an
- particle size: maximum reduction of SO per cent; impurity, it is important to choose an appropriate threshold
no increase permitted (packed columns). setting and appropriate conditions for the integration of the
Column dimensions: peak areas. In such tests the disregard limir, i.e. the limit at or
- length: ± 70 per cent; below which a peak is disregarded, is generally 0.05 per cent.
- intemal diameter. Integration of the peak area of any impurity that is not
± 25 per cent (packed columns); completely separated from the principal peak is preferably
performed by valley-to-valley extrapolation (tangential skim).
± SO per cent (capillary columns).
Flow rate ± 50 per cent. Additional points for monographs of the British
Temperature ± 5 °C, where the operating temperature is Pharmacopoeia
specified. System suitability
Unless otherwise stated in the monograph, the maximum
Injection volume May be decreased, provided detection
permitted relative standard deviation for six replicate
and repeatability are satisfactory; no increase permitted.
injections of the prescribed reference solution does not
QUANTIFICATION exceed 2.0%. This requirement is applicable only to assays
Peaks due to solvents and reagents or arising from the mobile for formulated preparations.
phase or the sample matrix are disregarded during
quantification.
- Detector sensitivity. The detector sensitivity is the signal
output per unit concentration or unit mass of a substance A. Thin-layer Chromatography
in the mobile phase entering the detector. The relative
detector response factor, commonly referred to as response (Ph. Bur. method 2.2.27)
factor, expresses the sensitivity of a detector for a given Thin-layer chromatography is a separation technique in
substance relative to a standard substance. The correction which a stationary phase consisting of an appropriate material
factor is the reciprocal of the response factor. is spread in a uniform thin layer on a support (plate) of glass,
- Extemal standard method. The concentration of the metal or plastic. Solutions of analytes are deposited on the
component(s) to be analysed is determined by comparing plate prior to development. The separation is based on
the response(s) (peak(s)) obtained with the test solution adsorption, partition, ion-exchange or on combinations of
these mechanisms and is carried out by migration
2023 Appendix III A V-A233

(development) of solutes (solutions of analytes) in a solvent is performed in a saturated tank. Apply the prescribed
or a suitable mixture of solvents (mobile phase) through the volume of solutions as described above. When the solvent
thin-layer (stationary phase). has evaporated from the applied solutions, place the plate in
APPARATUS the chromatographic tank, ensuring that the plate is as
vertical as possible and that the spots or bands are above the
Plates
surface of the mobile phase. Close the chromatographic tank,
The chromatography is carried out using pre-coated plates as
maintain it at 20-25 °C and protect from sunlight. Remove
described under Reagents (4.1.1). The particle size of the
the plate when the mobile phase has moved over the
silica gel is indicated after the name of the reagent in the
prescribed distance, measured between the points of
tests where it is used.
application and the solvent front. Dry the plate and visualise
Pre-treatment of the plates It may be necessary to wash the chromatograms as prescribed.
the plates prior to separation. This can be done by migration
For two-dimensional chromatography, dry the plates after the
of an appropriate solvent. The plates may also be
first development and carry out a second development in a
impregnated by procedures such as development, immersion
direction perpendicular to that of the first development.
or spraying. At the time of use, the plates may be activated, if
necessary, by heating in an oven at 120 °C for 20 min. Horizontal development
Apply the prescribed volume of the solutions as described
Chromatographic tank
above. When the solvent has evaporated from the applied
With a flat bottom or twin trough, of inert, transparent
solutions, introduce a sufficient quantity of the mobile phase
material, of a size suitable for the plates used and provided
into the trough of the chamber using a syringe or pipette,
with a tightly fitting lid. For horizontal development the tank
place the plate in the chamber after verifying that the latter is
is provided with a trough for the mobile phase and it
horizontal and connect the mobile phase direction device
additionally contains a device for directing the mobile phase
according to the manufacturer's instructions. If prescribed,
to the stationary phase.
develop the plate starting simultaneously at both ends. Close
Micropipettes, microsyringes, calibrated disposable the chamber and maintain it at 20-25 °C. Remove the plate
capillaries when the mobile phase has moved over the distance
Or other application devices suitable for the proper prescribed in the monograph. Dry the plate and visualise the
application of the solutions. chromatograms as prescribed.
Fluorescence detection device For two-dimensional chromatography, dry the plates after the
To measure direct fluorescence or the inhibition of first development and carry out a second development in a
fluorescence. direction perpendicular to that of the first development.
Visualisation devices and reagents VISUAL EVALUATION
Suitable devices are used for derivatisation to transfer to the Identification
plate reagents by spraying, immersion or exposure to vapour The principal spot in the chromatogram obtained with the
and, where applicable, to facilitate heating for visualisation of test solution is visually compared to the corresponding spot
separated components. in the chromatogram obtained with the reference solution by
Documentation comparing the colour, the size and the retardation factor (Rp)
A device may be used to provide documentation of the of both spots.
visualised chromatogram, for example a photograph or a The retardation factor (Rp) is defined as the ratio of the
computer file. distance from the point of application to the centre of the
METHOD spot and the distance travelled by the solvent front from the
Sample application point of application.
Apply the prescribed volume of the solutions at a suitable Verification of the separating power for
distance from the lower edge and from the sides of the plate identification Normally the performance given by the
and on a line parallel to the lower edge; allow an interval of suitability test described in Reagents (4.1.1) is sufficient. Only
at least 10 mm (5 mm on high-performance plates) between in special cases an additional performance criterion is
the centres of circular spots and 5 mm (2 mm on high- prescribed in the monograph.
performance plates) between the edges of bands. Apply the Related substances test
solutions in sufficiently small portions to obtain circular spots The secondary spot(s) in the chromatogram obtained with
2-5 mm in diameter (1-2 mm on high-performance plates) or the test solution is (are) visually compared to either the
bands 10-20 mm (5-10 mm on high-performance plates) by corresponding spot(s) in the chromatogram obtained with the
1-2 mm. reference solution containing the impurity(ies) or the spot in
In a monograph, where both normal and high-performance the chromatogram obtained with the reference solution
plates may be used, the working conditions for high- prepared from a dilution of the test solution.
performance plates are given in the brackets [ ) after those Verification of the separating power The requirements
for normal plates. for the verification of the separating power are prescribed in
Vertical development the monographs concerned.
Line the walls of the chromatographic tank with filter paper. Verification of the detecting power The detecting power
Pour into the chromatographic tank a sufficient quantity of is satisfactory if a spot or band is clearly visible in the
the mobile phase for the size of the tank to give after chromatogram obtained with the most dilute reference
impregnation of the filter paper a layer of appropriate depth solution.
related to the dimension of the plate to be used.
For saturation of the chromatographic tank, replace the lid QUANTITATIVE MEASUREMENT
and allow to stand at 20-25 °C for 1 h. Unless othenvise The requirements for resolution and separation are
indicated in the monograph, the chromatographic separation prescribed in the monographs concerned.
V-A234 Appendix III A 2023

Substances separated by thin-layer chromatography and Additional points for monographs of the British
responding to UV-Vis irradiation can be determined directly Pharmacopoeia
on the plate, using appropriate instrumentation. While When the method prescribed in a monograph carries the
moving the plate or the measuring device, examine the plate instructions 'protected from light' or 'in subdued light' it is
by measuring the reflectance of the incident light. Similarly, intended that the entire procedure is carried out under these
fluorescence may be measured using an appropriate optical conditions.
system. Substances containing radionuclides can be Unless otherwise indicated in the monograph, the mobile
quantified in 3 ways: either directly by moving the plate phase should be allowed to ascend 15 cm above the line of
alongside a suitable counter or vice versa (see application.
Radiopharmaceutical preparations (0125)), by cutting the plates
The phrase ultraviolet light (254 nm) indicates that the plate
into strips and measuring the radioactivity on each individual
should be examined under an ultraviolet lamp having a
strip using a suitable counter or by scraping off the stationary
maximum output at 254 nm (see below); other wavelength
phase, dissolving it in a suitable scintillation cocktail and
maxima may be specified.
measuring the radioactivity using a liquid scintillation
counter. The term secondary spot means any spot other than the
principal spot. Similarly, a secondary band is any band other
Apparatus than the principal band.
The apparatus for direct measurement on the plate consists
Where a spraying technique is prescribed it is essential that
of:
- a device for exact positioning and reproducible dispensing the reagent is evenly applied as a fine spray. The following
method of visualisation is used when directed in the
of the amount of substances onto the plate;
monograph.
- a mechanical device to move the plate or the measuring
device along the x-axis or the y-axis; METHOD I
- a recorder and a suitable integrator or a computer; Spray the dried plate with ethanolic sulfuric acid (20%), heat
- for substances responding to UV-Vis irradiation: a at 105° for 30 minutes and immediately expose to nitrous
photometer with a source of light, an optical device able fumes in a closed glass tank for 15 minutes (the nitrous
to generate monochromatic light and a photo cell of fumes may be generated by adding 7M sulfuric acid dropwise
adequate sensitivity are used for the measurement of to a solution containing 10% w/v of sodium nitrite and
reflectance or transmittance; if fluorescence is measured, a 3% w/v of potassium iodide). Place the plate in a current of
suitable filter is required to prevent light used for warm air for 15 minutes and spray with a 0.5% w/v solution
excitation from reaching the detector while permitting of N-(1-naphthyl) ethylenediamine dihydrochloride in ethanol
emitted light or a specific portion thereof to pass; (96%). If necessary, allow to dry and repeat the spraying.
- for substances containing radionuclides: a suitable counter for
MATERIALS
radioactivity. The linearity range of the counting device is
The coating substances and precoated plates are described in
to be verified.
Appendix I A: General Reagents. Prepare suspensions of the
Method coating substances as recommended by the manufacturer
Prepare the solution of the substance to be examined (test unless otherwise prescribed. Commercial pre-coated plates
solution) as prescribed in the monograph and, if necessary, may be used for pharmacopoeia! tests where a coating
prepare the reference solutions of the substance to be substances is prescribed provided that they comply with the
determined using the same solvent as in the test solution. test for chromatographic separation described for the
Apply the same volume of each solution to the plate and corresponding coating substance and with any additional test
develop. for verification of separating power required in the
Substances responding to UV- Vis irradiation Prepare monograph test.
and apply not fewer than 3 reference solutions of the
substance to be examined, the concentrations of which span Ultraviolet Ray Lamps for Analytical Purposes
the expected value in the test solution (about 80 per cent, (Ph. Eur. method 2.1.3)
100 per cent and 120 per cent). Treat with the prescribed Mercury vapour in quartz lamps is used as the source of
reagent, if necessary, and record the reflectance, the ultraviolet light. A suitable filter may be fitted to eliminate
transmittance or fluorescence in the chromatograms obtained the visible part of the spectrum emitted by the lamp. When
with the test and reference solutions. Use the measured the Pharmacopoeia prescribes in a test the use of ultraviolet
results for the calculation of the amount of substance in the light of wavelength 254 nm or 365 nm, an instrument
test solution. consisting of a mercury vapour lamp and a filter which gives
Substances containing radionuclides Prepare and apply an emission band with maximum intensity at about 254 nm
a test solution containing about 100 per cent of the expected or 365 nm is used. The lamp used should be capable of
value. Determine the radioactivity as a function of the path revealing without doubt a standard spot of sodium salicylate
length and report the radioactivity in each resulting peak as a with a diameter of about 5 mm on a support of silica gel G R,
percentage of the total amount of radioactivity. the spot being examined while in a position normal to the
radiation.
Criteria for assessing the suitability of the system are
described in the chapter on Chromatographic separation For this purpose apply 5 µL of a 0.4 g/L solution of sodium
techniques (2.2.46). The extent to which adjustments of salicylate R in alcohol R 1 for lamps of maximum output at
parameters of the chromatographic system can be made to 254 nm and 5 µL of a 2 g/L solution in alcohol R(3J for lamps
satisfy the criteria of system suitability are also given in this of maximum output at 365 nm. The distance between the
chapter. lamp and the chromatographic plate under examination used
in a pharmacopoeia! test should never exceed the distance
used to carry out the above test.

1 The alcohol R used must be free from fluorescence.

2023 Appendix III B V-A235

Identification of Phenothiazines by Thin-Layer Identification of Steroids

Chromatography (No Ph. Bur. method)
(Ph. Bur. method 2.3.3) Carry out the method for thin-layer chromatography using
Examine by thin-layer chromatography (2.2.27) using kieselguhr Gas the coating substance. Impregnate the dry
kieselguhr G R as the coating substance. Impregnate the plate plate by placing it in a tank containing a shallow layer of the
by placing it in a closed tank containing the necessary specified impregnating solvent, allowing the solvent to ascend
quantity of the impregnation mixture composed of a solution to the top, removing the plate from the tank and allowing the
containing 10 per cent V/V of phenoxyethanol R and 50 g/L of solvent to evaporate; use within 2 hours, with the flow of the
macrogol 300 R in acetone R so that the plate dips about mobile phase in the direction in which impregnation was
5 mm beneath the surface of the liquid. When the carried out. Unless otherwise specified, apply separately to
impregnation mixture has risen at least 17 cm from the lower the plate 2 µL of each of the following three solutions in a
edge of the plate, remove the plate and use immediately for mixture of 9 volumes of chloroform and 1 volume of methanol.
chromatography. Carry out the chromatography in the same Solution (1) contains 0.25% w/v of the substance being
direction as the impregnation. examined. Solution (2) contains 0.25% w/v of the
Test solution Dissolve 20 mg of the substance to be corresponding British Pharmacopoeia Chemical Reference
examined in chloroform R and dilute to 10 mL with the same Substance or European Pharmacopoeia Chemical Reference
solvent. Substance. Solution (3) is a mixture of equal volumes of
solutions (1) and (2). Use the specified mobile phase. After
Reference solution Dissolve 20 mg of the corresponding
removal of the plate, allow the solvent to evaporate, heat at
chemical reference substance (CRS) in chloroform Rand
120° for 15 minutes and spray the hot plate with ethanolic
dilute to 10 mL with the same solvent.
sulfuric acid (20%). Heat at 120° for a further 10 minutes,
Apply separately to the plate 2 µL of each solution and allow to cool and examine in daylight and under ultraviolet
develop in the dark over a path of 15 cm using a mixture of light (365 nm). The principal spot in the chromatogram
50 mL of light petroleum R and 1 mL of diethylamine R obtained with solution ( 1) is similar in position, colour in
saturated with phenoxyethanol R (i.e. add about 3 mL to daylight, fluorescence in ultraviolet light (365 nm) and size to
4 mL of phenoxyethanol R to the above mixture of solvents to that in the chromatogram obtained with solution (2).
give a persistent cloudiness on shaking, decant, and use the The principal spot in the chromatogram obtained with
supernatant, even if it is cloudy). After development place the solution (3) appears as a single, compact spot.
plate under ultraviolet light at 365 nm and examine after a
few minutes. The spot in the chromatogram obtained with Impregnating solvents
the test solution is similar in position, fluorescence and size I. A mixture of 1 volume offormamide and 9 volumes of
to the spot in the chromatogram obtained with the reference acetone.
solution. Spray with a 10 per cent V/V solution of sulfuric II. A mixture of 1 volume of propane-1,2-diol and 9 volumes
acid R in alcohol R. The spot in the chromatogram obtained of acetone.
with the test solution is of the same colour as that in the III. A mixture of 1 volume of liquid paraffin and 9 volumes
chromatogram obtained with the reference solution and has of petroleum spirit (boiling range, 40° to 60° or 50° to 70°).
similar stability over a period of at least 20 min. Mobile phases
Related Substances in Phenothiazines A. Chloroform.
(No Ph. Bur. method) B. A mixture of 25 volumes of chloroform and 75 volumes of
Carry out the method for thin-layer chromatography protected toluene.
from light using silica gel GF254 as the coating substance and C. Toluene.
the mobile phase prescribed in the monograph, but allowing D. A mixture of 20 volumes of toluene and 80 volumes of
the solvent front to ascend 12 cm above the line of cyclohexane.
application. Unless otherwise specified, apply separately to E. A mixture of equal volumes of cyclohexane and
the plate 10 µL of each of two solutions of the substance petroleum spirit (boiling range, 40° to 60° or 50° to 70°).
being examined prepared immediately before use in a
F. A mixture of 40 volumes of glacial acetic acid and
mixture of 95 volumes of methanol and 5 volumes of
60 volumes of water.
dicthylamine containing (1) 2.0% w/v and (2) 0.010% w/v.
After removal of the plate, allow it to dry in air and examine G. A mixture of 20 volumes of 1,4-dioxan and 80 volumes
under ultraviolet light (254 nm). Disregard any spot remaining of hexane.
on the line of application. Unless otherwise specified any H. A mixture of 29 volumes of toluene, 56 volumes of
secondary spot in the chromatogram obtained with solution (1) chloroform and 115 volumes of cyclohexane.
is not more intense than the spot in the chromatogram
obtained with solution (2) (0.5%).
Mobile phases
A. A mixture of 10 volumes of acetone, 10 volumes of B. Gas Chromatography
diethylamine and 80 volumes of cyclohexane. (Ph. Bur. method 2.2.28)
B. A mixture of 5 volumes of diethylamine, 10 volumes of
PRINCIPLE
acetone and 85 volumes of hexane.
Gas chromatography (GC) is a chromatographic separation
C. A mixture of 18 volumes of lM ammonia and technique based on the difference in the distribution of
90 volumes of butan-1-ol. species between 2 non-miscible phases in which the mobile
phase is a carrier gas moving through or passing the
stationary phase contained in a column. It is applicable to
substances or their derivatives which are volatilised under the
temperatures employed.
V-A236 Appendix III B 2023

GC is mainly based on mechanisms of adsorption or mass Helium, nitrogen and hydrogen are commonly used carrier
distribution. gases.
EQUIPMENT DETECTORS
The equipment typically consists of: Flame-ionisation detectors are usually employed but other
- an injector; detectors such as electron-capture, nitrogen-phosphorus,
- a chromatographic column contained in an oven; mass spectrometric, thermal conductivity or infrared
- one or more detector(s); spectrophotometric detectors may also be used.
- a data acquisition system. PROCEDURE
The carrier gas flows through the column and then through Equilibrate the column, the injector and the detector at the
the detector at a controlled rate or pressure. temperatures and the gas flow rates/pressures specified in the
The chromatography is carried out either at a constant monograph until a stable baseline is achieved. Prepare the
temperature or according to a given temperature programme. test solution(s) and the reference solution(s) as prescribed.
The solutions injected must be free from solid particles.
INJECTORS
Injection may be carried out either into a vaporisation Criteria for assessing the suitability of the system are
chamber which may be equipped with a stream splitter, or described in general chapter 2.2.46 Chromatographic separatwn
directly at the head of the column using a syringe or an techniques. The extent to which adjustments of parameters of
injection valve. the chromatographic system can be made to satisfy the
Injections of vapour phase May be effected by static or criteria of system suitability are also given in this general
chapter.
dynamic head-space injection systems.
Dynamic head-space (purge and trap) injection systems
include a sparging device by which volatile substances in STATIC HEAD-SPACE GAS
solution are swept into an absorbent column maintained at a CHROMATOGRAPHY
low temperature. Retained substances are then desorbed into Static head-space gas chromatography is a technique
the mobile phase by rapid heating of the absorbent column. particularly suitable for separating and determining volatile
Static head-space Injection systems include a compounds present in solid or liquid samples. The method is
thermostatically controlled sample heating chamber in which based on the analysis of the vapour phase in equilibrium with
closed vials containing solid or liquid samples are placed for the solid or liquid phase.
a fixed period of time to allow equilibration of the volatile EQUIPMENT
components of the sample between the non-gaseous phase
The equipment consists of a gas chromatograph provided
and the vapour phase. After equilibration, a predetermined
with a sample-introduction device that may be connected to
amount of the head-space of the vial is flushed into the gas
a module that automatically controls the pressure and the
chromatograph.
temperature. If necessary, a device for eliminating solvents
STATIONARY PHASES can be added.
Stationary phases are contained in columns which may be: The sample to be analysed is introduced into a container
- a capillary column whose stationary phase may be a solid fitted with a suitable stopper and a valve-system which
coating the inner surface of the column permits the passage of the carrier gas. The container is
(e.g. macrogol 20 000), or a liquid deposited on the inner placed in a thermostatically controlled chamber at a
surface (e.g. dimethylpolysiloxane); in the latter case it temperature set according to the substance to be examined.
may be chemically bonded to the inner surface;
The sample is held at this temperature long enough to allow
- a column packed with the stationary phase which may be
equilibration between the solid or liquid phase and the
a solid phase (e.g. alumina, silica) or an inert solid
vapour phase.
support (usually a porous polymer) impregnated or
coated with a liquid. The carrier gas is introduced into the container and, after the
prescribed time, a suitable valve is opened so that the gas
Capillary columns, made of fused silica, are 0.1 mm to
expands towards the chromatographic column taking the
0.53 mm in internal diameter (0) and at least 5 m in length.
volatilised compounds with it.
The stationary phase is a film 0.1 µm to 5.0 µm thick.
Instead of using a chromatograph specifically equipped for
Packed columns, made of glass or metal, are usually 1 m to
the introduction of samples, it is also possible to use airtight
3 m in length with an internal diameter (0) of 2 mm to
syringes and a conventional chromatograph. Equilibration is
4mm.
then carried out in a separate chamber and the vapour phase
MOBILE PHASES is carried onto the column, while necessary precautions are
Retention time and peak efficiency depend on the carrier gas taken to avoid any changes in the equilibrium.
flow rate; retention time is directly proportional to column
length and resolution is proportional to the square root of the PROCEDURE
column length. Using the reference preparations, determine suitable
instrument settings to produce an adequate response.
The carrier gas flow rate is usually expressed in millilitres per
minute at atmospheric pressure and at the stated DIRECT CALIBRATION
temperature. Flow rate is measured at the detector outlet, Introduce into separate, identical containers the preparation
either with a calibrated mechanical device or with a bubble to be examined and each of the reference preparations, as
tube, while the column is at operating temperature. prescribed in the monograph, avoiding contact between the
The linear velocity of the carrier gas through a column is sampling device and the samples.
inversely proportional to the square of the internal diameter Close the containers hermetically and place in the
of the column for a given flow volume. thermostatically controlled chamber set to the temperature
and pressure prescribed in the monograph; after
2023 Appendix III C V-A237

equilibration, carry out the chromatography under the INTERNAL STANDARDS

prescribed conditions. Reagents used as internal standards should not contain any
STANDARD ADDITIONS impurity that would produce a peak likely to interfere in the
Add to a set of identical suitable containers equal volumes of determination described in the monograph.
the preparation to be examined. Add to all but one of the INJECTION VOLUME
containers, suitable quantities of a reference preparation Where no injection volume is specified in the monograph,
containing a known concentration of the substance to be the analyst should select an appropriate volume for their
determined so as to produce a series of preparations specific application. The volume chosen is dependent on the
containing steadily increasing concentrations of the response of the analyte, the detector used, the efficiency of
substance. the column and the overall performance of the
Close the containers hermetically and place in the chromatographic system. Where a volume is not indicated,
thermostatically controlled chamber set to the temperature 1 µL is usually appropriate; however this should be checked
and pressure prescribed in the monograph; after for suitability under the local operating conditions.
equilibration, carry out the chromatography under the
SECONDARY PEAKS
prescribed conditions.
Reference may be made to a secondary peak. A secondary
Calculate the linear equation of the graph using a least- peak is a peak in the chromatogram other than the principal
squares fit, and derive from it the concentration of the peak and any peaks due to internal standard, solvent and
substance to be determined in the preparation to be derivatising agents. Peaks identified as being due to the
examined. counter-ion and/or other excipients including preservatives in
Alternatively, plot on a graph the mean of readings against the material being examined may also be excluded.
the added quantity of the substance to be determined.
Extrapolate the line joining the points on the graph until it
meets the concentration axis. The distance between this
point and the intersection of the axes represents the
concentration of the substance to be determined in the
C. Size-exclusion Chromatography
preparation to be examined. (Ph. Bur. method 2.2.30)

Additional points for monographs of the British PRINCIPLE

Pharmacopoeia Size-exclusion chromatography is a liquid chromatography
APPARATUS (2.2.29) technique which separates molecules in solution
according to their size. With organic mobile phases, the
The design of a particular chromatograph may require
technique is known as gel-permeation chromatography and
modification of the conditions detailed in the monograph.
with aqueous mobile phases it is known as gel-filtration
In such a case, the analyst should be satisfied that the
chromatography. The sample is introduced into a column,
modified conditions produce comparable results. If necessary,
which is filled with a gel or a porous particle packing
adjust the flow rate of the carrier gas to improve the quality
material, and is carried by the mobile phase through the
of the chromatogram or to modify the retention times of the
column. The size separation takes place by repeated
peaks of interest.
exchange of the solute molecules between the solvent of the
METHOD mobile phase and the same solvent in the stagnant liquid
Unless otherwise stated in the monograph, use nitrogen as phase (stationary phase) within the pores of the packing
the carrier gas and a flame ionisation detector. Occasionally material. The pore-size range of the packing material
reference is made to on-column injection, in which case the determines the molecular-size range within which separation
sample is injected directly on to the packing material without can occur.
the use of an inlet heater. When non-volatile material is to be Molecules small enough to penetrate all the pore spaces elute
injected on to the column, a suitable interchangeable pre- at the total mobile phase volume V, (also known as total
column may be used. permeation volume). Molecules apparently larger than the
REAGENTS maximum pore size of the packing material migrate along the
Solvents and reagents used in the preparation of solutions for column only through the spaces between the particles of the
examination should be of a quality suitable for use in gas packing material without being retained and elute at the
chromatography. A wide range of chemical substances is retention volume of an unretained compound V0 (also known
used as stationary phases, including polyethylene glycols, as exclusion volume or void volume). Separation according to
high-molecular weight esters and amides, hydrocarbons, molecular size occurs between the retention volume of an
silicone gums and fluids (polysiloxanes often substituted with unretained compound and the total mobile phase volume,
methyl, phenyl, nitrilo, vinyl or fluoroalkyl groups or with useful separation usually occurring in the first two thirds
mixtures of these) and microporous cross-linked polyaromatic of this range.
beads. A suitable stationary phase, its concentration and the EQUIPMENT
nature and grade of a suitable solid support are stated in the The equipment consists essentially of a chromatographic
monograph. The column should be conditioned in column of varying length and internal diameter (0), if
accordance with the manufacturer's instructions. In most necessary temperature-controlled, packed with a separation
cases reference is made to a particular commercial brand that material that is capable of fractionation in the appropriate
has been found to be suitable for the purpose, but such range of molecular sizes. The packing material may be a soft
statements do not imply that a different but equivalent support such as a swollen gel or a rigid support composed of
commercial brand may not be used. a material such as glass, silica or a solvent-compatible, cross-
linked organic polymer. Rigid supports usually require
pressurised systems giving faster separations.
V-A238 Appendix III C 2023

One end of the column is usually fitted with a suitable device Molecular Mass Distribution in Dextrans
for applying the sample such as a flow adapter, a syringe (Ph. Eur. method 2.2.39)
through a septum or an injection valve and may also be Examine by size-exclusion chromatography (2.2.30).
connected to a suitable pump for controlling the flow of the
Test solution Dissolve 0.200 g of the substance to be
eluent. Alternatively, the sample may be applied directly to
examined in the mobile phase and dilute to 10 mL with the
the drained bed surface or, where the sample is denser than
mobile phase.
the eluent, it may be layered beneath the eluent.
Marker solution Dissolve S mg of glucose R and 2 mg of
The mobile phase is chosen according to sample type,
dextran V 0 CRS in 1 mL of the mobile phase.
separation medium and method of detection. The eluent is
passed through the column at a constant rate. Calibration solutions Dissolve separately in 1 mL of the
mobile phase 15 mg of dextran 4 for calibration CRS, 1S mg
The outlet of the column is usually connected to a suitable
of dextran 10 for calibration CRS, 20 mg of dextran 40 for
detector fitted with an automatic recorder which enables the
calibration CRS, 20 mg of dextran 70 for calibration CRS and
monitoring of the relative concentrations of separated
20 mg of dextran 250 for calibration CRS.
components of the sample. IBtraviolet/visible
spectrophotometers (2.2.25), differential refractometers (RI), System suitability solution Dissolve either 20 mg of
luminescent detectors, multi-angle light scattering (MALS) dextran 40 for peiformance test CRS (for dextran 40) or 20 mg
detectors and other detectors may be used. An automatic of dextran 60/70 for peiformance test CRS (for dextran 60 and
fraction collector may be attached, if necessary. dextran 70) in 1 mL of the mobile phase.
The chromatographic procedure may be carried out using:
PROCEDURE
- a column 0.3 m long and 10 mm in internal diameter,
Before carrying out the separation, the packing material is
packed with cross-linked agarose for chromatography R or a
treated, and the column is packed, as described in the
series of columns, 0.3 m long and 7.5 mm in internal
monograph, or according to the manufacturer's instructions.
diameter, packed with polyether hydroxylated gel for
Criteria for assessing the suitability of the system are chromatography R,
described in general chapter 2.2.46. Chromatographic as the mobile phase, at a flow rate of 0.5-1 mUmin, kept
separation techniques. The extent to which adjustments of constant to ± 1 per cent per hour, a solution containing
parameters of the chromatographic system can be made to 7 g of anhydrous sodium sulfate R and 1 g of
satisfy the criteria of system suitability are also given in this chlorobutanol R in 1000 mL of water for chromatography R,
general chapter. as detector a differential refractometer,
DETERMINATION OF REIATIVE COMPONENT - a 100 µL to 200 µL loop injector,
COMPOSITION OF MIXTURES maintaining the system at a constant temperature
Carry out the separation as stated in the monograph. (± 0.1 °C).
If possible, monitor the elution of the components
CALIBRATION OF THE CHROMATOGRAPHIC
continuously and measure the corresponding peak areas.
SYSTEM
If the sample is monitored by a physico-chemical property to
which all the components of interest exhibit equivalent Carry out replicate injections of the chosen volume of the
responses (for example if they have the same specific marker solution. The chromatogram shows 2 peaks, the first
absorbance), calculate the relative amount of each of which corresponds to dextran V 0 CRS and the second of
component by dividing the respective peak area by the sum which corresponds to glucose R. From the elution volume of
of the peak areas of all the components of interest. If the the peak corresponding to dextran V0 , calculate the void
responses to the property used for detection of the volume Vo and from the peak corresponding to dextrose,
components of interest are not equivalent, calculate the calculate the total volume V,.
content by means of calibration curves obtained with the Inject the chosen volume of each of the calibration solutions.
calibration standards prescribed in the monograph. Draw carefully the baseline of each of the chromatograms.
Divide each chromatogram into p (at least 60) equal vertical
DETERMINATION OF MOLECUIAR MASSES
sections (corresponding to equal elution volumes). In each
Size-exclusion chromatography may be used to determine
section i, corresponding to an elution volume V; measure the
molecular masses by comparison with appropriate calibration
height (y,) of the chromatogram line above the baseline and
standards specified in the monograph. The retention volumes
calculate the coefficient of distribution K, using the
of the calibration standards may be plotted against the
expression:
logarithm of their molecular masses. The plot usually
approximates a straight line within the exclusion and total (Vi - Vo)
(1)
permeation limits for the separation medium used. From the (Vi - Vo)
calibration curve, molecular masses may be estimated.
The molecular-mass calibration is valid only for the particular
macromolecular solute/solvent system used under the Vo void volume of the column, determined using the peak
specified experimental conditions. corresponding to dextran Vo CRS in the chromatogram obtained
DETERMINATION OF MOLECUIAR SIZE with the marker solution,
V, total volume of the column, determined using the peak
DISTRIBUTION OF POLYMERS corresponding to glucose in the chromatogram obtained with
Size-exclusion chromatography may be used to determine the the marker solution,
distribution of the molecular size of polymers. However, V, elution volume of section i in the chromatogram obtained with
sample comparison may be valid only for results obtained each of the calibration solutions.
under the same experimental conditions. The reference
standards used for the calibration and the methods for Carry out the calibration using either of the following
determination of the distribution of molecular sizes of methods.
polymers are specified in the monograph.
 2023 Appendix III C V-A239

Calibration by plotting of the curve M

For each of the dextrans for calibration calculate the
coefficient of distribution Kmax corresponding to the ...
maximum height of the chromatographic line, using
expression (1). Plot on semilogarithmic paper the values of 106
. ''
Kmax (on the x-axis) against the declared molecular mass at
the maximum height of the chromatographic line (Mmax) of '
each of the dextrans for calibration and glucose. Draw a first
--
'
'
calibration curve through the points obtained, extrapolating it ''
from the point Kmax obtained with dextran 250 for '' I
calibration CRS to the lowest K value obtained for this CRS , Dextra~ 250
(Figure 2.2.39.-1). Using this first calibration curve, 105
transform, for each chromatogram, all K; values into the ' +
• Dextran 70
corresponding molecular mass M;, thus obtaining the -", I
molecular mass distribution. Calculate for each dextran for '-
I
Dextran 40
calibration the average molecular mass Mw using equation \.
(3) below. If the calculated values for Mw do not differ by
more than 5 per cent from those declared for each of the
dextrans for calibration and the mean difference is within
104 \ Dextran 10 --:=

± 3 per cent, the calibration curve is approved. If not, move " -·

the calibration curve along the y-axis and repeat the '\
procedure above until the calculated and the declared values • Dextran4
for Mw do not differ by more than 5 per cent.
Calibration by calculation of the curve
Calculate from equations (2) and (3) below, using a suitable
\----
103
method 1, values for b1, b2 , b3 , b4 and b5 that give values of
Mw within 5 per cent of the declared values of each of the
dextrans for calibration and 180 ± 2 for glucose:

M;=bs+ (2) 250

7D 4(
1D 4
\ ► Glucose/dextrose

p 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 K
2_)y;M;)
Kfw=~ (3) Figure 2.2.39.-1. - Example of a calibration curve.
2>
i=I
The dotted line corresponds to the part of the curve that is
extrapolated. Horizontal lines at the bottom of the figure represent
the width and the position of the chromatographic line obtained
with each of the dextrans for calibration.
p number of sections dividing the chromatograms,
Yi height of the chromatographic line above the baseline in section
i, "
M; molecular mass in section i. I:;(y;M;)
Mw=~
SYSTEM SUITABILITY I:;Y,
i=l
(4)
Inject the chosen volume of the appropriate system suitability
solution.
in which n is defined by the expressions:
Average molecular mass of dextran for performance
test CRS
Calculate the average molecular mass Mw as indicated under (5)
Calibration of the chromatographic system, using either the
plotted calibration curve or the values obtained above for b1,
b2 , b3 , b4 and b5 • The test is not valid unless Mw is:
- 41 000 to 47 000 (dextran 40 for peiforrnance test CRS),
(6)
- 67 000 to 75 000 (dextran 60/70 for peiforrnance test CRS).
Average molecular mass of the 10 per cent high-
fraction dextran
Calculate Mw for the 10 per cent high-fraction dextran eluted p number of sections dividing the chromatograms,
Y, height of the chromatographic line above the baseline in section
through section n using the equation: i,
M; molecular mass in section i.

The test is not valid unless Mw of the 10 per cent high

fraction dextran is:
1 An iterative method such as the Gauss-Newton method modified by
- 110 000 to 130 000 (dextran 40 for peifonnance test CRS),
Hartley is suitable (see 0. Hartley, Tecnometrics, 3 (1961) and G. Nilsson - 190 000 to 230 000 (dextran 60/70 for peiformance
and K. N11sson, J. Chromat. 101, 137 (1974)). A curve-fitting programme test CRS).
for microcomputers, capable of non-linear regression, may be used.
V-A240 Appendix III D 2023

Average molecular mass of the 10 per cent low-fraction - 1 or more detectors;

dextran - a data acquisition system.
Calculate Mw for the 10 per cent low-fraction dextran eluted The mobile phase is supplied from 1 or more reservoirs and
in and after section m using the expression: is pumped to the injector, then through the column, usually
at a constant rate, and then through the detector(s).
p
L(y,M,) PUMPING SYSTEMS
M w =p~ LC pumping systems deliver the mobile phase at a controlled
flow rate. Pressure fluctuations are to be minimised, for
LYi (7)
example by passing the pressurised solvent through a pulse-
dampening device. Tubing and connections are capable of
in which m is defined by the expressions: withstanding the pressures developed by the pumping system.
LC pumps may be fitted with a facility for 'bleeding' the
system of entrapped air bubbles.
(8)
Microprocessor-controlled pumping systems are capable of
accurately delivering a mobile phase of either constant
(isocratic elution) or varying composition (gradient elution),
according to a defined programme. In the case of gradient
(9) elution, pumping systems which deliver solvent(s) from
several reservoirs are available and solvent mixing can be
achieved on either the low or high-pressure side of the
pump(s).
p number of sections dividing the chromatograms,
y, height of the chromatographic line above the baseline in section INJECTORS
i, The sample solution is introduced into the flowing mobile
Mi molecular mass in section i.
phase at or near the head of the column using an injection
system which can operate at high pressure. Fixed-loop and
The test is not valid unless Mw of the 10 per cent low-
variable volume devices operated manually or by an
fraction dextran is:
autosampler are used. Partial filling of loops during manual
- 6000 to 8500 (dextran 40 for performance test CRS),
injection may adversely affect injection volume precision.
- 7000 to 11 000 (dextran 60/70 for performance test CRS).
STATIONARY PHASES
MOLECULAR MASS DISTRIBUTION OF THE There are many types of stationary phases employed in LC,
DEXTRAN TO BE ANALYSED including:
Inject the chosen volume of the test solution and calculate - silica or alumina, commonly used in normal-phase LC
Mw of the total molecular mass distribution, Mw of the (polar stationary phase and non-polar mobile phase),
10 per cent high-fraction dextran and Mw of the 10 per cent where the separation is based on differences in adsorption
low-fraction dextran as indicated under System suitability. on the stationary phase and/or mass distribution between
the mobile phase and the stationary phase (partition
chromatography);
- a variety of chemically modified supports prepared from
D. Liquid Chromatography polymers, silica or porous graphite, used in normal-phase
(Ph. Eur. method 2.2.29) and reversed-phase LC (non-polar stationary phase and
polar mobile phase), where the separation is based
PRINCIPLE principally on partition of the molecules;
Liquid chromatography (LC) is a method of - resins or polymers with acidic or basic groups, used in
chromatographic separation based on the difference in the ion-exchange chromatography, where separation is based
distribution of species between 2 non-miscible phases, in on competition between the ions to be separated and
which the mobile phase is a liquid which percolates through those in the mobile phase;
a stationary phase contained in a column. - porous silica or polymers, used in size-exclusion
LC is mainly based on mechanisms of adsorption, mass chromatography (2.2.30), where separation is based on
distribution, ion exchange, size exclusion or stereochemical differences between the volumes of the molecules,
interaction. corresponding to steric exclusion;
Unless otherwise specified, all the information below is valid - specially modified stationary phases, e.g. cellulose or
for both standard LC and LC using reduced particle-size amylose derivatives, proteins or peptides, cyclodextrins
columns (e.g. sub-2.0 µm). etc., for the separation of enantiomers (chiral
chromatography).
The latter requires instrumentation that is able to withstand
higher pressures (typically up to 100 MPa, i.e. about 15 000 Most separations are based on reversed-phase LC utilising
psi), generates lower extra-column band broadening, provides chemically modified silica as the stationary phase.
improved gradient mixing and allows a higher sampling rate The surface of the support, i.e. the silanol groups of silica, is
in the detection system. reacted with various silane reagents to produce covalently
bound silyl derivatives covering a varying number of active
EQUIPMENT sites on the surface of the support. The nature of the bonded
The equipment typically consists of: phase is an important parameter for determining the
- a pumping system; separation properties of the chromatographic system.
- an injector;
Unless otherwise stated by the manufacturer, silica-based
- a chromatographic column (a column temperature
reversed-phase columns are considered to be stable in mobile
controller may be used);
phases having an apparent pH in the range 2.0 to 8.0.
2023 Appendix III D V-A241

Columns containing porous graphite or particles of polymeric Mobile phases may contain other components, for example a
materials such as styrene-divinylbenzene copolymer are stable counter-ion for ion-pair chromatography or a chiral selector
over a wider pH range. for chiral chromatography using an achiral stationary phase.
Analysis using normal-phase LC with unmodified silica or DETECTORS
polar chemically modified silica (e.g. cyanopropyl or diol) as Ultraviolet/visible (UVNis) spectrophotometers (including
the stationary phase, with a non-polar mobile phase is diode array detectors) (2.2.25), are the most commonly
applicable in certain cases. employed detectors. Fluorescence spectrophotometers,
For analytical separations, the particle size of the most differential refractometers (RI), electrochemical detectors
commonly used stationary phases varies between 2 and (ECD), light scattering detectors, charged aerosol detectors
10 µm. The particles may be spherical or irregular, and of (CAD), mass spectrometers (MS) (2.2.43), radioactivity
varying porosiry and specific surface area. These properties detectors, multi-angle light scattering (MALS) detectors or
contribute to the chromatographic behaviour of a particular other detectors may be used.
stationary phase. In the case of reversed phases, the nature of PROCEDURE
the stationary phase, the extent of bonding, e.g. expressed as Equilibrate the column with the prescribed mobile phase and
the carbon loading, and whether the stationary phase is end- flow rate, at 20-25 °C or at the temperature specified in the
capped (i.e. part of the residual silanol groups are silylated) monograph, until a stable baseline is achieved. Prepare the
are additional determining factors. Tailing of peaks, solution(s) of the substance to be examined and the reference
particularly of basic substances, can occur when residual solution(s) required. The solutions must be free from solid
silanol groups are present. particles.
In addition to porous particles, superficially porous or Criteria for assessing the suitability of the system are
monolithic materials may be used. described in general chapter 2.2.46. Chromatographic
Unless otherwise prescribed in the monograph, columns separation techniques. The extent to which adjustments of
made of stainless steel of varying length and internal diameter parameters of the chromatographic system can be made to
(0) are used for analytical chromatography. Columns with satisfy the criteria of system suitability are also given in this
internal diameters of less than 2 mm are often referred to as chapter.
microbore columns.
The temperature of the mobile phase and the column must Additional points for monographs of the British
be kept constant during the analysis. A column temperature Pharmacopoeia
may be specified in the monograph for optimal performance The composition and flow rate of the mobile phase are stated
but most separations are performed at 20-25 °C. in the monograph. It is advisable to use as the mobile phase
solvent mixtures that have been de-aerated using a vacuum
MOBILE PHASES pump or other suitable means of de-aeration that has no
For normal-phase LC, low-polarity organic solvents are effect on the composition of the mixture.
generally employed. The residual water content of the
In quantitative work, particularly where the use of an internal
solvents used in the mobile phase is to be strictly controlled
standard is not specified in the monograph, the use of a
to obtain reproducible results.
fixed-volume loop injector is recommended. In certain
In reversed-phase LC, aqueous mobile phases, usually with exceptional cases the use of peak heights alone is prescribed
organic solvents and/or modifiers, are employed. in the monograph; where this is the case peak heights should
The components of the mobile phase are usually filtered to be used irrespective of the symmetry factor.
remove particles greater than 0.45 µm in size (or greater than The column is usually made of stainless steel and its
0.2 µm when the stationary phase is made of sub-2.0 µm dimensions are stated in the monograph. The dimensions are
particles, and when special detectors, e.g. light scattering stated as (length x internal diameter). When the monograph
detectors, are used). Multicomponent mobile phases are prescribes the use of a stationary phase designated by a letter,
prepared by measuring the required volumes (unless masses the relevant stationary phase defined below is intended.
are specified) of the individual components, followed by The nominal diameter of the particles of the stationary phase
mixing. Alternatively, the solvents may be delivered by is stated in parentheses immediately following the designating
individual pumps controlled by proportioning valves, by letter. In most cases reference is made to a particular
which mixing is performed according to the desired commercial brand that has been found to be suitable for the
proportion. Solvents are normally degassed before pumping purpose, but such statements do not imply that a different
by sparging with helium, sonication and/or using in-line but equivalent commercial brand may not be used.
membrane/vacuum modules to avoid the creation of gas The separation should be carried out at a constant ambient
bubbles in the detector cell. temperature unless otherwise specified in the monograph.
Solvents for the preparation of the mobile phase are normally When using mobile phases of high pH with a silica-based
free of stabilisers and, if an ultraviolet detector is employed, column, it is advisable to use a pre-column before the
are transparent at the wavelength of detection. Solvents and analytical column.
other components employed are to be of appropriate quality. Unless otherwise specified in the monograph the detector
In particular, water for chromatography R is used for the consists of a photometric detector fitted with a low-volume
preparation of mobile phases when water, or an aqueous flow cell (about 10 µL is suitable); the wavelength setting is
solution, is 1 of the components. Any necessary adjustments specified in the monograph.
of the pH are made to the aqueous component of the mobile
The design of a particular chromatograph may require
phase and not the mixture. If buffer solutions or saline
modification of the conditions detailed in the monograph.
solutions are used, adequate rinsing of the system is carried
In such a case the analyst should be satisfied that the
out with a mixture of water and a small proportion of the
modified conditions produce comparable results.
organic part of the mobile phase (5 per cent V/V) to prevent
crystallisation of salts after completion of the analysis.
V-A242 Appendix III E 2023

INJECTION VOLUME than one chromatogram is to be run on the same strip of

Where no injection volume is specified in the monograph, paper, space the solutions along the pencil line at points not
the analyst should select an appropriate volume for their less than 3 cm apart. Insert the paper into the tank, close the
specific application. The volume chosen is dependent on the lid and allow to stand for 1 h 30 min. Lower the paper into
response of the analyte, the detector used, the efficiency of the mobile phase and allow elution to proceed for the
the column and the overall performance of the prescribed distance or time. Remove the paper from the tank
chromatographic system. Where a volume is not indicated, and allow to dry in air. Protect the paper from bright light
20 µL is usually appropriate; however this should be checked during the elution process.
for suitability under the local operating conditions. DESCENDING PAPER CHROMATOGRAPHY
RESOLUTION FACTOR Apparatus The apparatus consists of a glass tank of
Unless otherwise stated in the monograph, for monographs suitable size for the chromatographic paper used, ground at
with a stated resolution facwr read Resolution (Rs) as referred the top to take a closely fitting glass lid. The lid has a central
to in Appendix III Chromatographic Separation Techniques. hole about 1.5 cm in diameter closed by a heavy glass plate
or a stopper. In the upper part of the tank is suspended a
RUNTIME
solvent trough with a device for holding the chromatographic
Where no run time is specified in the monograph, the analyst
paper. On each side of the trough, parallel to and slightly
should select an appropriate run time for their specific
above its upper edges, are two glass guide rods to support the
application. The run time chosen is dependent on the type of
paper in such a manner that no part of it is in contact with
test. For example, where a run time is not indicated in a
the walls of the tank. The chromatographic paper consists of
Related substances test the analyst should ensure that the run
suitable filter paper, cut into strips of sufficient length, and of
time is greater than all known or likely secondary peaks;
any convenient width between 2.5 cm and the length of the
similarly in an Assay the run time should be chosen to allow
trough; the paper is cut so that the mobile phase runs in the
the baseline to stabilise following the elution of the peak of
direction of the grain of the paper.
interest.
Method Place in the bottom of the tank a layer 2.5 cm
SECONDARY PEAKS deep of the solvent prescribed in the monograph, close the
Reference may be made to secondary peaks. A secondary peak tank and allow to stand for 24 h at 20 °C to 25 °C. Maintain
is a peak in the chromatogram other than the principal peak the tank at this temperature throughout the subsequent
and any peak due to internal standard, solvent or derivatising procedure. Draw a fine pencil line horizontally across the
agents. Peaks identified as being due to the counter-ion paper at such a distance from one end that when this end is
and/or other excipients including preservatives in the material secured in the solvent trough and the remainder of the paper
being examined may also be excluded. is hanging freely over the guide rod, the line is a few
MATERIALS centimetres below the guide rod and parallel with it. Using a
Solvents and reagents used in the preparation of solutions for micro-pipette, apply on the pencil line the volume of the
examination should be of a quality suitable for use in liquid solution prescribed in the monograph. If the total volume to
chromatography. be applied would produce a spot more than 10 mm in
diameter, apply the solution in portions, allowing each to dry
before the next application. When more than one
chromatogram is to be run on the same strip of paper, space
the solutions along the pencil line at points not less than
E. Paper Chromatography 3 cm apart. Insert the paper in the tank, close the lid, and
(Ph. Bur. method 2.2.26) allow to stand for 1 h 30 min. Introduce into the solvent
ASCENDING PAPER CHROMATOGRAPHY trough, through the hole in the lid, a sufficient quantity of
the mobile phase, close the tank and allow elution to proceed
Apparatus The apparatus consists of a glass tank of
for the prescribed distance or time. Remove the paper from
suitable size for the chromatographic paper used, ground at
the tank and allow to dry in air. The paper should be
the top to take a closely fining lid. In the top of the tank is a
protected from bright light during the elution process.
device which suspends the chromatographic paper and is
capable of being lowered without opening the chamber.
In the bottom of the tank is a dish to contain the mobile
phase into which the paper may be lowered.
The chromatographic paper consists of suitable filter paper, F. Electrophoresis1
cut into strips of sufficient length and not less than 2.5 cm (Ph. Bur. method 2.2.31)
wide; the paper is cut so that the mobile phase runs in the
♦ 1 GENERAL PRINCIPLE
direction of the grain of the paper.
Under the influence of an electrical field, charged particles
Method Place in the dish a layer 2.5 cm deep of the
dissolved or dispersed in an electrolyte solution migrate in
mobile phase prescribed in the monograph. If prescribed in
the direction of the electrode bearing the opposite polarity.
the monograph, pour the stationary phase between the walls
In gel electrophoresis, the movements of the particles are
of the tank and the dish. Close the tank and allow to stand
retarded by interactions with the surrounding gel matrix,
for 24 h at 20 °C to 25 °C. Maintain the tank at this
which acts as a molecular sieve. The opposing interactions of
temperature throughout the subsequent procedure. Draw a
the electrical force and molecular sieving result in differential
fine pencil line horizontally across the paper 3 cm from one
migration rates according to sizes, shapes and charges of
end. Using a micro pipette, apply to a spot on the pencil line
particles. Because of their different physico-chemical
the volume of the solution prescribed in the monograph.
properties, different macromolecules of a mixture will migrate
If the total volume to be applied would produce a spot more
than 10 mm in diameter, apply the solution in portions
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter
allowing each to dry before the next application. When more
5. 8 Pharnzacopoeial harmonisation.
2023 Appendix III F V-A243

at different speeds during electrophoresis and will thus be and fixed on to a suitable carrier designed to prevent
separated into discrete fractions. Electrophoretic separations diffusion of the conducting electrolyte, such as a horizontal
can be conducted in systems without support phases frame, inverted-V stand or a uniform surface with contact
(e.g. free solution separation in capillary electrophoresis) and points at suitable intervals.
in stabilising media such as thin-layer plates, films or gels. Gel electrophoresis The device consists essentially of a
2 FREE OR MOVING BOUNDARY glass plate (for example, a microscope slide) over the whole
ELECTROPHORESIS surface of which is deposited a firmly adhering layer of gel of
This method is mainly used for the determination of uniform thickness. The connection between the gel and the
mobility, the experimental characteristics being directly conducting solution is effected in various ways according to
measurable and reproducible. It is chiefly employed with the type of apparatus used. Precautions must be taken to
substances of high relative molecular mass and low avoid condensation of moisture or drying of the solid layer.
diffusibility. The boundaries are initially located by a physical - measuring device or meam of detection.
process such as refractometry or conductimetry. After Method Introduce the electrolyte solution into the
applying a given electric field for an accurately measured electrode compartments. Place the support suitably
time, the new boundaries and their respective positions are impregnated with electrolyte solution in the chamber under
observed. The operating conditions must be such as to make the conditions prescribed for the type of apparatus used.
it possible to determine as many boundaries as there are Locate the starting line and apply the sample. Apply the
components. electric current for the prescribed time. After the current has
been switched off, remove the support from the chamber, dry
3 ZONE ELECTROPHORESIS USING A
and visualise.
SUPPORTING MEDIUM
This method requires the use of small samples only. 4 POLYACRYLAMIDE ROD GEL
The nature of the support, such as paper, agar gel, cellulose ELECTROPHORESIS
acetate, starch, agarose, methacrylamide, mixed gel, In polyacrylamide rod gel electrophoresis, the stationary
introduces a number of additional factors modifying the phase is a gel which is prepared from a mixture of acrylamide
mobility: and N,N' -methylenebisacrylamide. Rod gels are prepared in
tubes 7.5 cm long and 0.5 cm in internal diameter, one
a) owing to channelling in the supporting medium, the
solution being applied to each rod.
apparent distance covered is less than the real distance,
Apparatus This consists of 2 buffer solution reservoirs
b) some supporting media are not electrically neutral. As the
made of suitable material such as poly(methyl methacrylate)
medium is a stationary phase it may sometimes give rise to a
and mounted vertically one above the other. Each reservoir is
considerable electro-endosmotic flow,
fined with a platinum electrode. The electrodes are
c) any heating due to the joule effect may cause some connected to a power supply allowing operation either at
evaporation of the liquid from the supporting medium which, constant current or at constant voltage. The apparatus has in
by capillarity, causes the solution to move from the ends the base of the upper reservoir a number of holders
towards the centre. The ionic strength therefore tends to equidistant from the electrode.
increase gradually.
Method The solutions should usually be degassed before
The rate of migration then depends on four main factors: the polymerisation and the gels used iIIlIIlediately after
mobility of the charged particle, the electro-endosmotic flow, preparation. Prepare the gel mixture as prescribed and pour
the evaporation flow, and the field strength. Hence it is into suitable glass tubes, stoppered at the bonom, to an equal
necessary to operate under clearly defined experimental height in each tube and to about 1 cm from the top, taking
conditions and to use, wherever possible, reference care to ensure that no air bubbles are trapped in the tubes.
substances. Cover the gel mixture with a layer of water R to exclude air
An apparatus for electrophoresis consists of: and allow to set. Gel formation usually takes about 30 min
- a generator supplying direct current whose voltage can be and is complete when a sharp interface appears between the
controlled and, preferably, stabilised, gel and the water layer. Remove the water layer. Fill the
- an electrophoresis chamber. This is usually rectangular and lower reservoir with the prescribed buffer solution and
made of glass or rigid plastic, with 2 separate remove the stoppers from the tubes. Fit the tubes into the
compartments, the anodic and the cathodic, containing holders of the upper reservoir and adjust so that the bottom
the electrolyte solution. In each compartment is iIIlIIlersed of the tubes are immersed in the buffer solution in the lower
an electrode, for example of platinum or graphite. These reservoir. Carefully fill the tubes with the prescribed buffer
are connected by means of an appropriately isolated solution. Prepare the test and reference solutions containing
circuit to the corresponding terminal of the power supply the prescribed marker dye and make them dense by
to form the anode and the cathode. The level of the dissolving in them sucrose R, for example. Apply the solutions
liquid in the 2 compartments is kept equal to prevent to the surface of a gel using a different tube for each
siphoning. solution. Add the same buffer to the upper reservoir.
The electrophoresis chamber is fined with an airtight lid Connect the electrodes to the power supply and allow
which maintains a moisture-saturated atmosphere during electrophoresis to proceed at the prescribed temperature and
operation and reduces evaporation of the solvent. A safety using the prescribed constant voltage or current. Switch off
device may be used to cut off the power when the lid is the power supply when the marker dye has migrated almost
removed. If the electrical power measured across the strip into the lower reservoir. Immediately remove each tube from
exceeds 10 W, it is preferable to cool the support. the apparatus and extrude the gel. Locate the position of the
- a support-canying device: bands in the electropherogram as prescribed. ♦
Strip electrophoresis The supporting strip, previously
wetted with the same conducting solution and dipped at each
end into an electrode compartment is appropriately tightened
V-A244 Appendix III F 2023

5 SODIUM DODECYL SULFATE into their individual polypeptide subunits and that minimise
POLYACRYLAMIDE GEL ELECTROPHORESIS aggregation. Most commonly, the strongly anionic detergent
(SDS-PAGE) SDS is used in combination with heat to dissociate the
5-1 SDS-PAGE - UNIFORM PERCENTAGE GELS proteins before they are loaded on the gel. The denatured
Scope polypeptides bind to SDS, become negatively charged and
Polyacrylamide gel electrophoresis is used for the qualitative exhibit a consistent charge-to-mass ratio regardless of protein
characterisation of proteins in biological preparations, for type. Because the amount of SDS bound is almost always
control of purity and for quantitative determinations. proportional to the molecular mass of the polypeptide and is
independent of its sequence, SDS-polypeptide complexes
Purpose migrate through polyacrylamide gels with mobilities
Analytical gel electrophoresis is an appropriate method with dependent on the size of the polypeptide.
which to identify and to assess the homogeneity of proteins
The electrophoretic mobilities of the resultant detergent-
in pharmaceutical preparations. The method is routinely used
polypeptide complexes all assume the same functional
for the estimation of protein subunit molecular masses and
relationship to their molecular masses. SDS complexes will
for determination of the subunit compositions of purified
migrate toward the anode in a predictable manner, with low-
proteins.
molecular-mass complexes migrating faster than larger ones.
Ready-to-use gels and reagents are commercially available The molecular mass of a protein can therefore be estimated
and can be used instead of those described in this text, from its relative mobility in calibrated SDS-PAGE, and the
provided that they give equivalent results and that they meet intensity of a single band relative to other undesired bands in
the validity requirements given below under Validation of the such a gel can be a measure of purity.
test.
Modifications to the polypeptide backbone, such as N- or
5-1-1 Characteristics ofpolyacrylamide gels O-linked glycosylation, can change the apparent molecular
The sieving properties of polyacrylamide gels are established mass of a protein since SDS does not bind to a carbohydrate
by the three-dimensional network of fibres and pores which is moiety in a manner similar to a polypeptide; therefore, a
formed as the bifunctional bisacrylamide cross-links adjacent consistent charge-to-mass ratio is not maintained.
polyacrylamide chains. Polymerisation is usually catalysed by
Depending on the extent of glycosylation and other post-
a free radical-generating system composed of ammonium
translational modifications, the apparent molecular mass of
persulfate and N,N,N' ,N'-tetramethylethylenediamine
proteins may not be a true reflection of the mass of the
(TEMED).
polypeptide chain.
As the acrylamide concentration of a gel increases, its
Reducing conditions
effective pore size decreases. The effective pore size of a gel is
Polypeptide subunits and three-dimensional structure are
operationally defined by its sieving properties, i.e. by the
often maintained in proteins by the presence of disulfide
resistance it imparts to the migration of macromolecules.
bonds. A goal of SDS-PAGE analysis under reducing
There are limits on the acrylamide concentrations that can be
conditions is to disrupt this structure by reducing disulfide
used. At high acrylamide concentrations, gels break much
bonds. Complete denaturation and dissociation of proteins
more easily and are difficult to handle. As the pore size of a
by treatment with 2-mercaptoethanol (2-ME) or
gel decreases, the migration rate of a protein through the gel
dithiothreitol (DTT) will result in unfolding of the
decreases. By adjusting the pore size of a gel, through
polypeptide backbone and subsequent complexation with
manipulating the acrylamide concentration, the resolution of
SDS. Using these conditions, the molecular mass of the
the method can be optimised for a given protein product.
polypeptide subunits can reasonably be calculated by linear
Thus, a given gel is physically characterised by its respective
regression (or, more accurately, by non-linear regression)
composition of acrylamide and bisacrylamide.
with the aid of suitable molecular-mass standards.
In addition to the composition of the gel, the state of the
protein is an important component to the electrophoretic Non-reducing conditions
mobility. In the case of proteins, the electrophoretic mobility For some analyses, complete dissociation of the protein into
is dependent on the pK value of the charged groups and the subunit peptides is not desirable. In the absence of treatment
size of the molecule. It is influenced by the type, the with reducing agents such as 2-ME or DTT, disulfide
concentration and the pH of the buffer, by the temperature covalent bonds remain intact, preserving the oligomeric form
and the field strength, and by the nature of the support of the protein. Oligomeric SDS-protein complexes migrate
material. more slowly than their SDS-polypeptide subunits.
In addition, non-reduced proteins may not be completely
5-1-2 Denaturing polyacrylamide gel electrophoresis saturated with SDS and, hence, may not bind the detergent
The method cited as an example is limited to the analysis of in a constant mass ratio. Moreover, intra-chain disulfide
monomeric polypeptides with a mass range of 14 000 to bonds constrain the molecular shape, usually in such a way
100 000 daltons. It is possible to extend this mass range as to reduce the Stokes radius of the molecule, thereby
using various techniques (e.g. gradient gels, particular buffer reducing the apparent molecular mass Mr. This makes
system). For instance, tricine-SDS gels, with tricine as the molecular-mass determinations of these molecules by SDS-
trailing ion in the electrophoresis running-buffer (instead of PAGE Jess straightforward than analyses of fully denatured
glycine as in the method described here), can separate very polypeptides, since it is necessary that both standards and
small proteins and peptides under 10 000 to 15 000 daltons. unknown proteins be in similar configurations for valid
Denaturing polyacrylamide gel electrophoresis using glycine- comparisons.
SDS is the most common mode of electrophoresis used in
5-1-3 Characteristics of discontinuous buffer system gel
assessing the pharmaceutical quality of protein products and
electrophoresis
will be the focus of the example method. Typically, analytical
The most popular electrophoretic method for the
electrophoresis of proteins is carried out in polyacrylamide
characterisation of complex mixtures of proteins uses a
gels under conditions that ensure dissociation of the proteins
discontinuous buffer system involving 2 contiguous, but
2023 Appendix III F V-A245

distinct gels: a resolving or separating (lower) gel and a side of the gel mould thus forming the bottom of the gel
stacking (upper) gel. The 2 gels are cast with different mould. Verify that the tubing runs along the edge of the glass
porosities, pH, and ionic strengths. In addition, different plates and has not been extruded while placing the clamps.
mobile ions are used in the gel and electrode buffers. The gel mould is now ready for pouring the gel.
The buffer discontinuity acts to concentrate large volume Preparation of the gel
samples in the stacking gel, resulting in improved resolution. In a discontinuous buffer SOS polyacrylamide gel, it is
When power is applied, a voltage drop develops across the recommended to pour the resolving gel, let the gel set, and
sample solution which drives the proteins into the stacking then pour the stacking gel since the composition of the 2 gels
gel. Glycinate ions from the electrode buffer follow the in acrylamide-bisacrylamide, buffer and pH are different.
proteins into the stacking gel. A moving boundary region is
Preparation of the resolving gel In a conical flask,
rapidly formed with the highly mobile chloride ions in the
prepare the appropriate volume of solution containing the
front and the relatively slow glycinate ions in the rear.
desired concentration of acrylamide for the resolving gel,
A localised high-voltage gradient forms between the leading
using the values given in Table 2.2.31.-1. Mix the
and trailing ion fronts, causing the SOS-protein complexes to
components in the order shown. Where appropriate, before
form into a thin zone (stack) and migrate between the
adding the ammonium persulfate solution and the TEMEO,
chloride and glycinate phases. Within broad limits, regardless
filter the solution if necessary under vacuum through a
of the height of the applied sample, all SOS-proteins
cellulose acetate membrane (pore diameter 0.45 µm). Keep
condense into a very narrow region and enter the resolving
the solution under vacuum, while swirling the filtration unit,
gel as a well-defined, thin zone of high protein density.
until no more bubbles are formed in the solution.
The large-pore stacking gel does not retard the migration of
Add appropriate amounts of ammonium persulfate solution
most proteins and serves mainly as an anti-convective
and TEMEO as indicated in Table 2.2.31.-1, swirl and pour
medium. At the interface of the stacking and resolving gels,
immediately into the gap between the 2 glass plates of the
the proteins undergo a sharp increase in retardation due to
mould. Leave sufficient space for the stacking gel (the length
the restrictive pore size of the resolving gel and the buffer
of the teeth of the comb plus 1 cm). Using a tapered glass
discontinuity, which also contributes to the focusing of the
pipette, carefully overlay the solution with water-saturated
proteins. Once in the resolving gel, proteins continue to be
isobutanol. Leave the gel in a vertical position at room
slowed by the sieving of the matrix. The glycinate ions
temperature to allow polymerisation.
overtake the proteins, which then move in a space of uniform
pH formed by the tris(hydroxymethyl)aminomethane and Preparation of the stacking gel After polymerisation is
glycine. Molecular sieving causes the SOS-polypeptide complete (about 30 min), pour off the isobutanol and wash
complexes to separate on the basis of their molecular masses. the top of the gel several times with water to remove the
isobutanol overlay and any unpolymerised acrylamide. Drain
5-1-4 Preparing vertical discontinuous buffer SDS
as much fluid as possible from the top of the gel, and then
polyacrylamide gels
remove any remaining water with the edge of a paper towel.
This section describes the preparation of gels using particular
instrumentation. This does not apply to pre-cast gels. In a conical flask, prepare the appropriate volume of solution
For pre-cast gels or any other commercially available containing the desired concentration of acrylamide, using the
equipment, the manufacturer's instructions must be used for values given in Table 2.2.31.-2. Mix the components in the
guidance. order shown. Where appropriate, before adding the
ammonium persulfate solution and the TEMED, filter the
The use of commercial reagents that have been purified in
solution if necessary under vacuum through a cellulose
solution is recommended. When this is not the case and acetate membrane (pore diameter 0.45 µm). Keep the
where the purity of the reagents used is not sufficient, a pre-
solution under vacuum, while swirling the filtration unit,
treatment is applied. For instance, any solution sufficiently until no more bubbles are formed in the solution.
impure to require filtration must also be deionised with a
Add appropriate amounts of ammonium persulfate solution
mixed-bed (anion/cation exchange) resin to remove acrylic
and TEMED as indicated in Table 2.2.31.-2. Swirl and pour
acid and other charged degradation products. When stored immediately into the gap between the 2 glass plates of the
according to recommendations, acrylamide/bisacrylamide
mould directly onto the surface of the polymerised resolving
solutions and solid persulfate are stable for long periods. gel. Immediately insert a clean polytetrafluoroethylene comb
Assembling the gel moulding cassette into the stacking gel solution, being careful to avoid trapping
Clean the 2 glass plates (e.g. 10 cm by 8 cm in size), the air bubbles. Add more stacking gel solution to fill the spaces
polytetrafluoroethylene comb, the 2 spacers and the silicone of the comb completely. Leave the gel in a vertical position
rubber tubing (e.g. 0.6 mm in diameter by 35 cm) with mild and allow to polymerise at room temperature.
detergent and rinse extensively with water, followed by Preparation of the samples
anhydrous ethanol, and allow the plates to dry at room Unless otherwise stated in the specific monograph, the
temperature. Lubricate the spacers and the tubing with non-
samples can be prepared as follows:
silicone grease. Apply the spacers along each of the 2 short
sides of the glass plate 2 mm away from the edges and 2 mm Preparation of the samples (non-reducing
away from the long side corresponding to the bottom of the conditions) Mix equal volumes of a mixture comprising
gel. Begin to lay the tubing on the glass plate by using one water R plus the preparation to be examined or the reference
spacer as a guide. Carefully twist the tubing at the bottom of preparation, and concentrated SDS-PAGE sample buffer R.
the spacer and follow the long side of the glass plate. While Preparation of the samples (reducing conditions) Mix
holding the tubing with 1 finger along the long side, twist equal volumes of a mixture comprising water R plus the
again the tubing and lay it on the second short side of the preparation to be examined or the reference preparation, and
glass plate, using the spacer as a guide. Place the second concentrated SDS-PAGE sample buffer for reducing conditions R
glass plate in perfect alignment and hold the mould together containing 2-ME (or DTT) as the reducing agent.
by hand pressure. Apply 2 clamps on each of the 2 short The concentration prescribed in the monograph can vary
sides of the mould. Carefully apply four clamps on the longer depending on the protein and staining method.
V-A246 Appendix III F 2023

Table 2.2.31.-1. - Preparation of resolving gel

Component volumes (mL) per gel mould volume of
Solution components
5mL lOmL 15 mL 20mL 25 mL 30mL 40mL 50mL

6 per cent acrylamide

WaterR 2.6 5.3 7.9 10.6 13.2 15.9 21.2 26.5

Acrylamide solution (lJ 1.0 2.0 3.0 4.0 5.0 6.0 8.0 10.0

1.5 M Tris (pH 8.si< 2J 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5

100 g/L SDsC3l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

JOO glL APSC 4 l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

TEMEDC l 5
0.004 0.008 0.012 0.016 0.02 0.024 0.032 0.04

8 per cent acrylamide

WaterR 2.3 4.6 6.9 9.3 11.5 13.9 18.5 23.2

Acrylamide solution(!) 1.3 2.7 4.0 5.3 6.7 8.0 10.7 13.3

1.5 M Tris (pH 8.8]< 2l 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5

100 glL SDSC 3 l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

100 g/L APSC 4 l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

TEMEDC l 5
0.003 0.006 0.009 0.012 0.015 0.018 0.024 0.03

10 per cent acrylamide

Water R 1.9 4.0 5.9 7.9 9.9 11.9 15.9 19.8

Acrylamide solution(!) 1.7 3.3 5.0 6.7 8.3 10.0 13.3 16.7

1.5 M Tris (pH 8.8PJ 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5

100 glL SDSC 3 l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

100 glL APSC 4l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

TEMEDC5 l 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02

12 per cent acrylamide

WaterR 1.6 3.3 4.9 6.6 8.2 9.9 13.2 16.5

Acrylamide solutionl 1l 2.0 4.0 6.0 8.0 10.0 12.0 16.0 20.0

1. 5 M Tris (pH 8.sjl2 l 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5

100 giL SDsC3l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

100 g.lL APsC 4l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

TEMEDC 5l 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02

14 per cent acrylamide

WaterR 1.4 2.7 3.9 5.3 6.6 8.0 10.6 13.8

Acrylamide solutionC 1l 2.3 4.6 7.0 9.3 11.6 13.9 18.6 23.2

1.5 M Tris (pH s.sPJ 1.2 2.5 3.6 5.0 6.3 7.5 10.0 12.5

I 00 g/L SDSl3J 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

100 glL APSC4 l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

TEMED< 5 l 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02

15 per cent acrylamide

Water R 1.1 2.3 3.4 4.6 5.7 6.9 9.2 11.5

Acrylamide solutionl l 1
2.5 5.0 7.5 10.0 12.5 15.0 20.0 25.0

1.5 M Tris (pH s.8ic2 1 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5

100 glL SDSC 3 l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

100 g,IL APSC 4 l 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5

TEMED< 5 l 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02

(!) Acrylamidc solution: 30 per cent acrylamide/bisacrylamide (29: I) solution R.

(2) 1.5 M Tris (pH 8.8): 1.5 M tris-hydroch/oride buffer solutwn pH 8.8 R.
(3) 100 g/L SDS: a 100 g/L solution of sodium dodecyl sulfate R.
(4) 100 g/L APS: a 100 g/L solution of ammonium persulfate R. Ammonium persulfate provides the free radicals that drive polymerisation of acrylamide and
bisacrylamide. Since ammonium persulfate solution decomposes rapidly, fresh solutions must be prepared daily.
(5) TEMED: tetramethylethylenediamine R.
2023 Appendix III F V-A24 7

Table 2.2.31.-2. - Preparation of stacking gel

Component volumes (mL) per gel mould volume of
Solution components
1 mL 2mL 3mL 4mL 5mL 6mL 8mL lOmL

Water R 0.68 1.4 2.1 2.7 3.4 4.1 5.5 6.8

Acrylamide solutionC 1l 0.17 0.33 0.5 0.67 0.83 1.0 1.3 1.7

1.0 M Tris (pH 6.8)' 2 J 0.13 0.25 0.38 0.5 0.63 0.75 1.0 1.25

100 g/L SDsl 3 l 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1

100 g/L APsC 4l 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1

TEMEDC'i 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.01

(1) Acrylamide solution: 30 per cent acrylamide/bisacrylamide (29: !) solution R.

(2) 1.0 M Tris (pH 6.8): IM tris-hydrochlmide buffer solutwn pH 6.8 R.
(3) 100 g/L SDS: a 100 g/L solution of sodium dodecyl sulfate R.
(4) 100 g/L APS: a I 00 g/L solution of ammonium persulfate R. Ammonium persulfate provides the free radicals that drive polymerisation of acrylamide and
bisacrylamide. Since ammonium persulfate solution decomposes rapidly, fresh solutions must be prepared daily.
(5) TEMED: tetramethylethylenediamine R.

Sample treatment: keep for 5 min in a water-bath or in a Gradient gels offer some advantages, as some proteins which
block heater set at 100 °C, then cool. The temperature and co-migrate on fixed-concentration gels can be resolved within
time in the monograph may vary as protein cleavage may gradient gels. During electrophoresis, the proteins migrate
occur during the heat treatment. until the pore size prevents further progress and a stacking
Mounting the gel in the electrophoresis apparatus and effect therefore occurs, resulting in sharper bands. As shown
electrophoretic separation in Table 2.2.31.-3, gradient gels also allow separation of
After polymerisation is complete (about 30 min), remove the proteins with a wider range of molecular masses compared to
polytetrafluoroethylene comb carefully. Rinse the wells a single fixed concentration gel.
immediately with water or with the SDS-PAGE running The table gives suggested compositions of the linear gradient,
buffer R to remove any unpolymerised acrylamide. relating the range of acrylamide concentrations to the
If necessary, straighten the teeth of the stacking gel with a appropriate protein molecular mass ranges. Note that other
blunt hypodermic needle attached to a syringe. Remove the gradient shapes (e.g. concave) can be prepared for specific
clamps on one short side, carefully pull out the tubing and applications.
replace the clamps. Proceed similarly on the other short side.
Remove the tubing from the bottom part of the gel. Mount Table 2.2.31.-3
the gel in the electrophoresis apparatus. Add the Acrylamide Protein range
electrophoresis buffers to the top and bottom reservoirs. (per cent) (kDa)
Remove any bubbles that become trapped at the bottom of 5 - 15 20 - 250
the gel between the glass plates. This is best done with a 5 - 20 10 - 200
bent hypodermic needle attached to a syringe. Never pre-run 10 - 20 10 - 150
the gel before loading the samples, since this will destroy the 8 - 20 8 - 150
discontinuity of the buffer systems. Before loading the sample
carefully rinse each well with SDS-PAGE running buffer R. Gradient gels are also used for the determination of
Prepare the test and reference solutions in the recommended molecular mass and protein purity.
sample buffer and treat as specified in the individual
5-3 DETECTION OF PROTEINS IN GELS
monograph. Apply the appropriate volume of each solution
Coomassie and silver staining are the most common protein
to the stacking gel wells. staining methods and are described in more detail below.
Start the electrophoresis using the conditions recommended Several other commercial stains, detection methods and
by the manufacturer of the equipment. Manufacturers of commercial kits are available. For example, fluorescent stains
SDS-PAGE equipment may provide gels of different surface are visualised using a fluorescent imager and often provide a
area and thickness and electrophoresis running time and linear response over a wide range of protein concentrations,
current/voltage may vary in order to achieve optimal often several orders of magnitude depending on the protein.
separation. Check that the dye front is moving into the Coomassie staining has a protein detection level of
resolving gel. When the dye is near the bottom of the gel,
approximately 1 µg to 10 µg of protein per band. Silver
stop the electrophoresis. Remove the gel assembly from the
staining is the most sensitive method for staining proteins in
apparatus and carefully separate the glass plates. Remove the gels and a band containing 10 ng to 100 ng can be detected.
spacers, cut off and discard the stacking gel and immediately
These figures are considered robust in the context of these
proceed with staining.
gels. Improved sensitivity of 1 or 2 orders of magnitude has
5-2 SDS-PAGE - GRADIENT CONCENTRATION sometimes been reported in the literature.
GELS Coomassie staining responds in a more linear manner than
Gradient gels (resolving gels) are prepared with an increasing silver staining; however, the response and range depend on
concentration of acrylamide from the top to the bottom. the protein and development time. Both Coomassie and
Preparation of gradient gels requires a gradient-forming silver staining can be less reproducible if staining is stopped
apparatus. Ready-to-use gradient gels are commercially in a subjective manner, i.e. the point at which the staining is
available with specific recommended protocols. deemed satisfactory. The use of dynamic ranges of reference
V-A248 Appendix III F 2023

proteins is very important as they help assess the intra- 5-5 MOLECUIAR-MASS DETERMINATION
experimental sensitivity and linearity. All gel staining steps Molecular masses of proteins are determined by comparison
are carried out while wearing gloves, at room temperature, of their mobilities with those of several marker proteins of
with gentle shaking (e.g. on an orbital shaker platform) and known molecular weight. Mixtures of pre-stained and
using any convenient container. unstained proteins with precisely known molecular masses
Coomassie staining blended for uniform staining are available for calibrating gels.
Immerse the gel in a large excess of Coomassie staining They are available in various molecular mass ranges.
solution R and allow to stand for at least 1 h. Remove the Concentrated stock solutions of proteins of known molecular
staining solution. mass are diluted in the appropriate sample buffer and loaded
on the same gel as the protein sample to be examined.
Destain the gel with a large excess of destaining solution R.
Change the destaining solution several times, until the Immediately after the gel has been run, the position of the
stained protein bands are clearly distinguishable on a clear bromophenol blue tracking dye is marked to identify the
background. The more thoroughly the gel is destained, the leading edge of the electrophoretic ion front. This can be
smaller is the amount of protein that can be detected by the done by cutting notches in the edges of the gel or by
method. Destaining can be speeded up by including a few inserting a needle soaked in India ink into the gel at the dye
grams of anion-exchange resin or a small sponge in the front. After staining, measure the migration distances of each
destaining solution R. protein band (markers and unknowns) from the top of the
resolving gel. Divide the migration distance of each protein
NOTE: the acid-alcohol solutions used in this procedure do not
by the distance travelled by the tracking dye. The normalised
completely fix proteins in the gel. This can lead to losses of some
migration distances are referred to as the relative mobilities of
low-molecular-mass proteins during the staining and destaining of
the proteins (relative to the dye front), or Rp. Construct a
thin gels. Permanent fixation is obtainable by allowing the gel to
plot of the logarithm of the relative molecular masses (Mr) of
stand in a mixture of 1 volume of trichloroacetic acid R,
the protein standards as a function of the Rp values.
4 volumes of methanol R and 5 volumes of water R for 1 h before
Unknown molecular masses can be estimated by linear
it is immersed in the Coomassie staining solutwn R.
regression analysis (or more accurately by non-linear
Silver staining regression analysis) or interpolation from the curves of log
Immerse the gel in a large excess of fixing solution R and Mr against Rp if the values obtained for the unknown
allow to stand for 1 h. Remove the fixing solution, add fresh samples are positioned along the approximately linear part of
fixing solution and incubate either for at least 1 h or the graph.
overnight, if convenient. Discard the fixing solution and wash
5-6 VALIDATION OF THE TEST
the gel in a large excess of water R for 1 h. Soak the gel for
The test is not valid unless the target resolution range of the
15 min in a 1 per cent V/V solution of glutaraldehyde R.
gel has been demonstrated by the distribution of appropriate
Wash the gel twice for 15 rnin in a large excess of water R.
molecular mass markers, e.g. across 80 per cent of the length
Soak the gel in fresh silver nitrate reagent R for 15 min, in
of the gel. The separation obtained for the expected proteins
darkness. Wash the gel three times for 5 min in a large excess
must show a linear relationship between the logarithm of the
of water R. Immerse the gel for about 1 min in developer
molecular mass and the Rp. If the plot has a sigmoidal shape,
solution R until satisfactory staining has been obtained. Stop
then only data from the linear region of the curve can be
the development by incubation in the blocking solutwn R for
used in the calculations. Additional validation requirements
15 min. Rinse the gel with water R.
with respect to the test sample may be specified in individual
5-4 RECORDING OF THE RESULTS monographs.
Gels are photographed or scanned while they are still wet or Sensitivity must also be validated. A reference protein control
after an appropriate drying procedure. Currently, gel corresponding to the desired concentration limit that is run
scanning systems with data analysis software are in parallel with the test samples can serve to establish system
commercially available to immediately photograph and suitability.
analyse the wet gel.
5-7 QUANTIFICATION OF IMPURITIES
Depending on the staining method used, gels are treated in a
SDS-PAGE is often used as a limit test for impurities. When
slightly different way. For Coomassie staining, after the
impurities are quantified by normalisation to the main band
destaining step, allow the gel to stand in a 100 g/L solution
using an integrating densitometer or image analysis, the
of glycerol R for at least 2 h (overnight incubation is pos~ibl_e).
responses must be validated for linearity. Note that
For silver staining, add to the final rinsing a step of 5 mm m
depending on the detection method and protein as described
a 20 g/L solution of glycerol R.
in the introduction of section 5-3, the linear range can vary
Drying of stained SDS polyacrylarnide gels is one of the but can be assessed within each run by using one or more
methods used to obtain permanent documentation. This control samples containing an appropriate range of protein
method frequently results in the cracking of gel during drying concentration.
between cellulose films.
Where the impurity limit is specified in the individual
Immerse 2 sheets of porous cellulose film in water R and monograph, a reference solution corresponding to that level
incubate for 5 min to 10 min. Place one of the sheets on a of impurity should be prepared by diluting the test solution.
drying frame. Carefully lift the gel and place it on the For example, where the limit is 5 per cent, a reference
cellulose film. Remove any trapped air bubbles and pour a solution would be a 1:20 dilution of the test solution.
few millilitres of water R around the edges of the gel. Place No impurity (any band other than the main band) in the
the second sheet on top and remove any trapped air bubbles. electropherogram obtained with the test solution may be
Complete the assembly of the drying frame. Place in an oven more intense than the main band obtained with the reference
or leave at room temperature until dry. solution.
 2023 Appendix III G V-A249

Under validated conditions, impurities may be quantified by (capillary effective length) is given by the expression:
normalisation to the main band using an integrating
densitometer or by image analysis. l l XL
t=---=---~--
1',p + Vea (µ,p + µ, 0) X V

In general, uncoated fused-silica capillaries above pH 3 have

negative charge due to ionised silanol groups in the inner
G. Capillary Electrophoresis1 wall. Consequently, the electro-osmotic flow is from anode to
(Ph. Bur. method 2.2.47) cathode. The electro-osmotic flow must remain constant
from run to run if good reproducibility is to be obtained in
GENERAL PRINCIPLES
the migration velocity of the solutes. For some applications,
Capillary electrophoresis is a physical method of analysis
it may be necessary to reduce or suppress the electro-osmotic
based on the migration, inside a capillary, of charged analytes
flow by modifying the inner wall of the capillary or by
dissolved in an electrolyte solution, under the influence of a changing the concentration, composition and/or pH of the
direct-current electric field.
buffer solution.
The migration velocity of an analyte under an electric field of
After the introduction of the sample into the capillary, each
intensity E, is determined by the electrophoretic mobility of analyte ion of the sample migrates within the background
the analyte and the electro-osmotic mobility of the buffer
electrolyte as an independent zone, according to its
inside the capillary. The electrophoretic mobility of a solute electrophoretic mobility. Zone dispersion, that is the
({tep) depends on the characteristics of the solute (electric
spreading of each solute band, results from different
charge, molecular size and shape) and those of the buffer in
phenomena. Under ideal conditions the sole contribution to
which the migration takes place (type and ionic strength of
the solute-zone broadening is molecular diffusion of the
the electrolyte, pH, viscosity and additives).
solute along the capillary (longitudinal diffusion). In this ideal
The electrophoretic velocity (vep) of a solute, assuming a
case the efficiency of the zone, expressed as the number of
spherical shape, is given by the equation:
theoretical plates (N), is given by:

N=~~-~---
(µep + µ, 0 ) X V X l
2xDxL
effective charge of the solute, D molecular diffusion coefficient of the solute in the buffer.
viscosity of the electrolyte solution,
Stoke's radius of the solute,
V applied voltage,
In practice, other phenomena such as heat dissipation,
L total length of the capillary. sample adsorption onto the capillary wall, mismatched
conductivity between sample and buffer, length of the
When an electric field is applied through the capillary filled injection plug, detector cell size and unlevelled buffer
with buffer, a flow of solvent is generated inside the capillary, reservoirs can also significantly contribute to band dispersion.
called electro-osmotic flow. The velocity of the electro- Separation between 2 bands (expressed as the resolution, R,)
osmotic flow depends on the electro-osmotic mobility (µe 0) can be obtained by modifying the electrophoretic mobility of
which in tum depends on the charge density on the capillary the analytes, the electro-osmotic mobility induced in the
internal wall and the buffer characteristics. The electro- capillary and by increasing the efficiency for the band of each
osmotic velocity (v,0 ) is given by the equation: analyte, according to the equation:

R, = /N(µ,pb - µ,pa)
4(µ,p + µeo)
dielectric constant of the buffer, electrophoretic mobilities of the 2 analytes
zeta potential of the capillary surface. separated,

The velocity of the solute (v) is given by: mean electrophoretic mobility of the 2 analytes
li,p ='/, (µ,pb + µ,pa).
V = Vep + V, 0

The electrophoretic mobility of the analyte and the electro- APPARATUS

osmotic mobility may act in the same direction or in opposite An apparatus for capillary electrophoresis is composed of:
directions, depending on the charge of the solute. In normal - a high-voltage, controllable direct-current power supply;
capillary electrophoresis, anions will migrate in the opposite - 2 buffer reservoirs, held at the same level, containing the
direction to the electro-osmotic flow and their velocities will prescribed anodic and cathodic solutions;
be smaller than the electro-osmotic velocity. Cations will - 2 electrode assemblies (the cathode and the anode),
migrate in the same direction as the electro-osmotic flow and immersed in the buffer reservoirs and connected to the
their velocities will be greater than the electro-osmotic power supply;
velocity. Under conditions in which there is a fast electro- - a separation capillary (usually made of fused-silica) which,
osmotic velocity with respect to the electrophoretic velocity of when used with some specific types of detectors, has an
the solutes, both cations and anions can be separated in the optical viewing window aligned with the detector.
same run. The ends of the capillary are placed in the buffer
The time (t) taken by the solute to migrate the distance ([) reservoirs. The capillary is filled with the solution
from the injection end of the capillary to the detection point prescribed in the monograph;
- a suitable injection system;
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter - a detector able to monitor the amount of substances of
5. 8 Pharmacopoeia/ harmonisation. interest passing through a segment of the separation
V-A250 Appendix III G 2023

capillary at a given time; it is usually based on absorption only charged analytes with electrophoretic mobilities greater
spectrophotometry (UV and visible) or fluorimetry, but than the electro-osmotic flow will pass to the outlet.
conductimetric, amperometric or mass spectrometric Temperature The main effect of temperature is observed
detection can be useful for specific applications; indirect on buffer viscosity and electrical conductivity, and therefore
detection is an alternative method used to detect non- on migration velocity. In some cases, an increase in capillary
UV-absorbing and non-fluorescent compounds; temperature can cause a conformational change in proteins,
- a thermostatic system able to maintain a constant modifying their migration time and the efficiency of the
temperature inside the capillary is recommended to obtain separation.
a good separation reproducibility; Capillary The dimensions of the capillary (length and
- a recorder and a suitable integrator or a computer. internal diameter) contribute to analysis time, efficiency of
The definition of the injection process and its automation are separations and load capacity. Increasing both effective length
critical for precise quantitative analysis. Modes of injection and total length can decrease the electric fields (working at
include gravity, pressure or vacuum injection and constant voltage) which increases migration time. For a given
electrokinetic injection. The amount of each sample buffer and electric field, heat dissipation, and hence sample
component introduced electrokinetically depends on its band-broadening, depend on the internal diameter of the
electrophoretic mobility, leading to possible discrimination capillary. The latter also affects the detection limit,
using this injection mode. depending on the sample volume injected and the detection
Use the capillary, the buffer solutions, the preconditioning system employed.
method, the sample solution and the migration conditions Since the adsorption of the sample components on the
prescribed in the monograph of the considered substance. capillary wall limits efficiency, methods to avoid these
The employed electrolytic solution is filtered to remove interactions should be considered in the development of a
particles and degassed to avoid bubble formation that could separation method. In the specific case of proteins, several
interfere with the detection system or interrupt the electrical strategies have been devised to avoid adsorption on the
contact in the capillary during the separation run. A rigorous capillary wall. Some of these strategies (use of extreme pH
rinsing procedure should be developed for each analytical and adsorption of positively charged buffer additives) only
method to achieve reproducible migration times of the require modification of the buffer composition to prevent
solutes. protein adsorption. In other strategies, the internal wall of the
CAPILLARY ZONE ELECTROPHORESIS capillary is coated with a polymer, covalently bonded to the
PRINCIPLE silica, that prevents interaction between the proteins and the
In capillary zone electrophoresis, analytes are separated in a negatively charged silica surface. For this purpose, ready-to-
capillary containing only buffer without any anticonvective use capillaries with coatings consisting of neutral-hydrophilic,
medium. With this technique, separation takes place because cationic and anionic polymers are available.
the different components of the sample migrate as discrete Electrolytic solution parameters
bands with different velocities. The velocity of each band Buffer type and concentration Suitable buffers for
depends on the electrophoretic mobility of the solute and the capillary electrophoresis have an appropriate buffer capacity
electro-osmotic flow in the capillary (see General Principles). in the pH range of choice and low mobility to minimise
Coated capillaries can be used to increase the separation current generation.
capacity of those substances adsorbing on fused-silica Matching buffer-ion mobility to solute mobility, whenever
surfaces. possible, is important for minimising band distortion.
Using this mode of capillary electrophoresis, the analysis of The type of sample solvent used is also important to achieve
both small (Mr < 2000) and large molecules on-column sample focusing, which increases separation
(2000 <Mr< 100 000) can be accomplished. Due to the efficiency and improves detection.
high efficiency achieved in capillary zone electrophoresis, An increase in buffer concentration (for a given pH)
separation of molecules having only minute differences in decreases electro-osmotic flow and solute velocity.
their charge-to-mass ratio can be effected. This separation
Buffer pH The pH of the buffer can affect separation by
mode also allows the separation of chiral compounds by modifying the charge of the analyte or additives, and by
addition of chiral selectors to the separation buffer. changing the electro-osmotic flow. In protein and peptide
OPTIMISATION separation, changing the pH of the buffer from above to
Optimisation of the separation is a complex process where below the isoelectric point (pi) changes the net charge of the
several separation parameters can play a major role. solute from negative to positive. An increase in the buffer pH
The main factors to be considered in the development of generally increases the electro-osmotic flow.
separations are instrumental and electrolytic solution Organic solvents Organic modifiers (methanol,
parameters. acetonitrile, etc.) may be added to the aqueous buffer to
Instrumental parameters increase the solubility of the solute or other additives and/or
Voltage A Joule heating plot is useful in optimising the to affect the degree of ionisation of the sample components.
applied voltage and capillary temperature. Separation time is The addition of these organic modifiers to the buffer
inversely proportional to applied voltage. However, an generally causes a decrease in the electro-osmotic flow.
increase in the voltage used can cause excessive heat Additives for chiral separations For the separation of
production, giving rise to temperature and, as a result enantiomers, a chiral selector is added to the separation
thereof, viscosity gradients in the buffer inside the capillary. buffer. The most commonly used chiral selectors are
This effect causes band broadening and decreases resolution. cyclodextrins, but crown ethers, polysaccharides and proteins
Polarity Electrode polarity can be normal (anode at the may also be used. Since chiral recognition is governed by the
inlet and cathode at the outlet) and the electro-osmotic flow different interactions between the chiral selector and each of
will move toward the cathode. If the electrode polarity is the enantiomers, the resolution achieved for the chiral
reversed, the electro-osmotic flow is away from the outlet and compounds depends largely on the type of chiral selector
2023 Appendix III G V-A251

used. In this regard, for the development of a given CAPILLARY ISOELECTRIC FOCUSING
separation it may be useful to test cyclodextrins having a PRINCIPLE
different cavity size (a-, ~-, or y-cyclodextrin) or modified In isoelectric focusing, the molecules migrate under the
cyclodextrins with neutral (methyl, ethyl, hydroxyalkyl, etc.) influence of the electric field, so long as they are charged, in
or ionisable (aminomethyl, carboxymethyl, sulfobutyl ether, a pH gradient generated by ampholytes having pl values in a
etc.) groups. When using modified cyclodextrins, batch-to- wide range (poly-aminocarboxylic acids), dissolved in the
batch variations in the degree of substitution of the separation buffer.
cyclodextrins must be taken into account since it will The three basic steps of isoelectric focusing are loading,
influence the selectivity. Other factors controlling the focusing and mobilisation.
resolution in chiral separations are concentration of chiral
selector, composition and pH of the buffer and temperature. Loading step
The use of organic additives, such as methanol or urea can Two methods may be employed:
- loading in one step: the sample is mixed with ampholytes
also modify the resolution achieved.
and introduced into the capillary either by pressure or
CAPILLARY GEL ELECTROPHORESIS vacuum;
PRINCIPLE - sequential loading: a leading buffer, then the ampholytes,
In capillary gel electrophoresis, separation takes place inside a then the sample mixed with ampholytes, again
capillary filled with a gel that acts as a molecular sieve. ampholytes alone and finally the terminating buffer are
Molecules with similar charge-to-mass ratios are separated introduced into the capillary. The volume of the sample
according to molecular size since smaller molecules move must be small enough not to modify the pH gradient.
more freely through the network of the gel and therefore Focusing step
migrate faster than larger molecules. Different biological When the voltage is applied, ampholytes migrate toward the
macromolecules (for example, proteins and DNA fragments), cathode or the anode, according to their net charge, thus
which often have similar charge-to-mass ratios, can thus be creating a pH gradient from anode (lower pH) to cathode
separated according to their molecular mass by capillary gel (higher pH). During this step the components to be
electrophoresis. separated migrate until they reach a pH corresponding to
CHARACTERISTICS OF GELS their isoelectric point (pl) and the current drops to very low
2 types of gels are used in capillary electrophoresis: values.
permanently coated gels and dynamically coated gels. Mobilisation step
Permanently coated gels, such as cross-linked polyacrylamide, If mobilisation is required for detection, use one of the
are prepared inside the capillary by polymerisation of the following methods.
monomers. They are usually bonded to the fused-silica wall - in the first method, mobilisation is accomplished during
and cannot be removed without destroying the capillary. the focusing step under the effect of the electro-osmotic
If the gels are used for protein analysis under reducing
flow; the electro-osmotic flow must be small enough to
conditions, the separation buffer usually contains sodium allow the focusing of the components;
dodecyl sulfate and the samples are denatured by heating in a - in the second method, mobilisation is accomplished by
mixture of sodium dodecyl sulfate and 2-mercaptoethanol or applying positive pressure after the focusing step;
dithiothreitol before injection. When non-reducing conditions - in the third method, mobilisation is achieved after the
are used (for example, analysis of an intact antibody), focusing step by adding salts to the cathode reservoir or
2-mercaptoethanol and dithiothreitol are not used. the anode reservoir (depending on the direction chosen
Separation in cross-linked gels can be optimised by for mobilisation) in order to alter the pH in the capillary
modifying the separation buffer (as indicated in the capillary when the voltage is applied. As the pH is changed, the
zone electrophoresis section) and controlling the gel porosity proteins and ampholytes are mobilised in the direction of
during the gel preparation. For cross-linked polyacrylamide the reservoir which contains the added salts and pass the
gels, the porosity can be modified by changing the detector.
concentration of acrylamide and/or the proportion of cross-
linker. As a rule, a decrease in the porosity of the gel leads to The separation achieved, expressed as lip!, depends on the
a decrease in the mobility of the solutes. Due to the rigidity pH gradient (dpH/dx), the number of ampholytes having
of these gels, only electro kinetic injection can be used. different pl values, the molecular diffusion coefficient (D),
the intensity of the electric field (E) and the variation of the
Dynamically coated gels are hydrophilic polymers, such as electrophoretic mobility of the analyte with the pH
linear polyacrylamide, cellulose derivatives, dextran, etc.,
(-dµ/dpH):
which can be dissolved in aqueous separation buffers giving
rise to a separation medium that also acts as a molecular
sieve. These separation media are easier to prepare than D(dpH/dx)
lipl =3x
cross-linked polymers. They can be prepared in a vial and E(-dµ/dpH)
filled by pressure in a wall-coated capillary (with no electro-
osmotic flow). Replacing the gel before every injection OPTIMISATION
generally improves the separation reproducibility. The main parameters to be considered in the development of
The porosity of the gels can be increased by using polymers separations are:
of higher molecular mass (at a given polymer concentration) Voltage
or by decreasing the polymer concentration (for a given Capillary isoelectric focusing utilises very high electric fields,
polymer molecular mass). A reduction in the gel porosity 300 V/cm to 1000 V/cm in the focusing step.
leads to a decrease in the mobility of the solute for the same
buffer. Since the dissolution of these polymers in the buffer Capillary
gives low viscosity solutions, both hydrodynamic and The electro-osmotic flow must be reduced or suppressed
electrokinetic injection techniques can be used. depending on the mobilisation strategy (see above). Coated
capillaries tend to reduce the electro-osmotic flow.
V-A252 Appendix III G 2023

Solutions distribution ratio (Dm), which is the ratio of the number of

The anode buffer reservoir is filled with a solution with a pH moles of solute in the micelle to those in the mobile phase.
lower than the pI of the most acidic ampholyte and the For a neutral compound, k' is given by:
cathode reservoir is filled with a solution with a pH higher
, tR - to Vs
than the pI of the most basic ampholyte. Phosphoric acid for
the anode and sodium hydroxide for the cathode are k=
tQX
(1 --
tR)=KXVM
frequently used. 4nc
Addition of a polymer, such as methylcellulose, in the tR migration time of the solute,
ampholyte solution tends to suppress convective forces (if t0 analysis time of an unretained solute (determined by injecting
any) and electro-osmotic flow by increasing the viscosity. an electro-osmotic flow marker which does not enter the
micelle, for instance methanol),
Commercial ampholytes are available covering many pH
tmc micelle migration time (measured by injecting a micelle marker,
ranges and may be mixed if necessary to obtain an expanded such as Sudan III, which migrates while continuously associated
pH range. Broad pH ranges are used to estimate the in the micelle),
isoelectric point whereas narrower ranges are employed to K partition coefficient of the solute,
improve accuracy. Calibration can be done by correlating Vs volume of the micellar phase,
V:w volume of the mobile phase.
migration time with isoelectric point for a series of protein
markers.
Likewise, the resolution between 2 closely-migrating solutes
During the focusing step precipitation of proteins at their (R,) is given by:
isoelectric point can be prevented, if necessary, using buffer
additives such as glycerol, surfactants, urea or zwitterionic
buffers. However, depending on the concentration, urea
denatures proteins.
R, = ,/Fi X IX - l X __5z__ X l - (f.:)
4 IX k~+I l+k~x(~)
MICELLAR ELECTROKINETIC tmc
CHROMATOGRAPHY (MEKC)
N number of theoretical plates for one of the solutes,
PRINCIPLE a selectivity,
In micellar electrokinetic chromatography, separation takes k'aandk'b retention factors for both solutes, respectively
place in an electrolyte solution which contains a surfactant at (k'b > k'a),
a concentration above the critical micellar concentration
(cmc). The solute molecules are distributed between the Similar, but not identical, equations give k' and R, values for
aqueous buffer and the pseudo-stationary phase composed of electrically charged solutes.
micelles, according to the partition coefficient of the solute. OPTIMISATION
The technique can therefore be considered as a hybrid of The main parameters to be considered in the development of
electrophoresis and chromatography. It is a technique that separations by MEKC are instrumental and electrolytic
can be used for the separation of both neutral and charged solution parameters.
solutes, maintaining the efficiency, speed and instrumental Instrumental parameters
suitability of capillary electrophoresis. One of the most widely Voltage Separation time is inversely proportional to applied
used surfactants in MEKC is the anionic surfactant sodium voltage. However, an increase in voltage can cause excessive
dodecyl sulfate, although other surfactants, for example heat production that gives rise to temperature gradients and
cationic surfactants such as cetyltrimethylammonium salts, viscosity gradients of the buffer in the cross-section of the
are also used. capillary. This effect can be significant with high conductivity
The separation mechanism is as follows. At neutral and buffers such as those containing micelles. Poor heat
alkaline pH, a strong electro-osmotic flow is generated and dissipation causes band broadening and decreases resolution.
moves the separation buffer ions in the direction of the Temperature Variations in capillary temperature affect the
cathode. If sodium dodecyl sulfate is employed as the partition coefficient of the solute between the buffer and the
surfactant, the electrophoretic migration of the anionic micelles, the critical micellar concentration and the viscosity
micelle is in the opposite direction, towards the anode. As a of the buffer. These parameters contribute to the migration
result, the overall micelle migration velocity is slowed down time of the solutes. The use of a good cooling system
compared to the bulk flow of the electrolytic solution. In the improves the reproducibility of the migration time for the
case of neutral solutes, since the analyte can partition solutes.
between the micelle and the aqueous buffer, and has no
electrophoretic mobility, the analyte migration velocity will
Capillary As in capillary zone electrophoresis, the
dimensions of the capillary (length and internal diameter)
depend only on the partition coefficient between the micelle
contribute to analysis time and efficiency of separations.
and the aqueous buffer. In the electropherogram, the peaks
Increasing both effective length and total length can decrease
corresponding to each uncharged solute are always between
the electric fields (working at constant voltage), increase
that of the electro-osmotic flow marker and that of the
migration time and improve the separation efficiency.
micelle (the time elapsed between these two peaks is called
The internal diameter controls heat dissipation (for a given
the separation window). For electrically charged solutes, the
buffer and electric field) and consequently the sample band
migration velocity depends on both the partition coefficient
broadening.
of the solute between the micelle and the aqueous buffer,
and on the electrophoretic mobility of the solute in the Electrolytic solution parameters
absence of micelle. Surfactant type and concentration The type of
Since the mechanism in MEKC of neutral and weakly surfactant, in the same way as the stationary phase in
ionised solutes is essentially chromatographic, migration of chromatography, affects the resolution since it modifies
the solute and resolution can be rationalised in terms of the separation selectivity. Also, the log k' of a neutral compound
retention factor of the solute (k'), also referred to as mass increases linearly with the concentration of surfactant in the
mobile phase. Since resolution in MEKC reaches a
 2023 Appendix III G V-A253

maximum when k' approaches the value of jt,,,,/t0 , excluding those due to solvents or any added reagents
modifying the concentration of surfactant in the mobile phase (normalisation procedure). The use of an automatic
changes the resolution obtained. integration system (integrator or data acquisition and
Buffer pH Although pH does not modify the partition processing system) is recommended.
coefficient of non-ionised solutes, it can modify the electro- SYSTEM SUITABILITY
osmotic flow in uncoated capillaries. A decrease in the buffer In order to check the behaviour of the capillary
pH decreases the electro-osmotic flow and therefore increases electrophoresis system, system suitability parameters are
the resolution of the neutral solutes in MEKC, resulting in a used. The choice of these parameters depends on the mode
longer analysis time. of capillary electrophoresis used. They are: retention factor
Organic solvents To improve MEKC separation of (k') (only for micellar electrokinetic chromatography),
hydrophobic compounds, organic modifiers (methanol, apparent number of theoretical plates (N), symmetry factor
propanol, acetonitrile, etc.) can be added to the electrolytic (A,) and resolution (R,). In previous sections, the theoretical
solution. The addition of these modifiers usually decreases expressions for N and R, have been described, but more
migration time and the selectivity of the separation. Since the practical equations that allow these parameters to be
addition of organic modifiers affects the critical micellar calculated from the electropherograms are given below.
concentration, a given surfactant concentration can be used APPARENT NUMBER OF THEORETICAL PLATES
only within a certain percentage of organic modifier before The apparent number of theoretical plates (N) may be
the micellisation is inhibited or adversely affected, resulting in calculated using the expression:
the absence of micelles and, therefore, in the absence of
partition. The dissociation of micelles in the presence of a
high content of organic solvent does not always mean that
the separation will no longer be possible; in some cases the
hydrophobic interaction between the ionic surfactant tR migration time or distance along the baseline from the point of
monomer and the neutral solutes forms solvophobic injection to the perpendicular dropped from the maximum of
complexes that can be separated electrophoretically. the peak corresponding to the component,

Additives for chiral separations For the separation of

w,, width of the peak at half-height.

enantiomers using MEKC, a chiral selector is included in the

RESOLUTION
micellar system, either covalently bound to the surfactant or
The resolution (R,) between peaks of similar height of
added to the micellar separation electrolyte. Micelles that
2 components may be calculated using the expression:
have a moiety with chiral discrimination properties include
salts of N-dodecanoyl-L-amino acids, bile salts, etc. Chiral
resolution can also be achieved using chiral discriminators,
R, = 1.18 X (tR2 - tRJ)
such as cyclodextrins, added to the electrolytic solutions
WhJ + Whz
which contain micellised achiral surfactants.
Other additives Several strategies can be carried out to
modify selectivity, by adding chemicals to the buffer.
migration times or distances along the baseline
The addition of several types of cyclodextrins to the buffer from the point of injection to the perpendiculars
can also be used to reduce the interaction of hydrophobic dropped from the maxima of two adjacent peaks,
solutes with the micelle, thus increasing the selectivity for this w hl and wh2 peak widtbs at half-height.
type of compound.
The addition of substances able to modify solute-micelle When appropriate, the resolution may be calculated by
interactions by adsorption on the latter, is used to improve measuring the height of the valley (Hv) between 2 partly
the selectivity of the separations in MEKC. These additives resolved peaks in a standard preparation and the height of
may be a second surfactant (ionic or non-ionic) which gives the smaller peak (Hp) and calculating the peak-to-valley ratio:
rise to mixed micelles or metallic cations which dissolve in
the micelle and form co-ordination complexes with the
solutes.
QUANTIFICATION SYMMETRY FACTOR
Peak areas must be divided by the corresponding migration The symmetry factor (A,) of a peak may be calculated using
time to give the corrected area in order to: the expression:
- compensate for the shift in migration time from run to
run, thus reducing the variation of the response, A _ wo.os
' - 2d
- compensate for the different responses of sample
constituents with different migration times. w0 _05 width of tbe peak at one-twentietb of the peak height,
Where an internal standard is used, verify that no peak of the d distance between tbe perpendicular dropped from the peak
maximum and the leading edge of the peak at one-twentieth of
substance to be examined is masked by that of the internal the peak height.
standard.
CALCULATIONS Tests for area repeatability (standard deviation of areas or of
From the values obtained, calculate the content of the the area/migration-time ratio) and for migration time
component or components being examined. When repeatability (standard deviation of migration time) are
prescribed, the percentage content of one or more introduced as suitability parameters. Migration time
components of the sample to be examined is calculated by repeatability provides a test for the suitability of the capillary
determining the corrected area(s) of the peak(s) as a washing procedures. An alternative practice to avoid the lack
percentage of the total of the corrected areas of all peaks, of repeatability of the migration time is to use migration time
relative to an internal standard.
V-A254 Appendix III H 2023

A test for the verification of the signal-to-noise ratio for a columns). A capillary column has a maximum internal
standard preparation (or the determination of the limit of diameter (0) of 100 µm.
quantification) may also be useful for the determination of Mobile phases
related substances. Usually the mobile phase is carbon-dioxide which may
SIGNAL-TO-NOISE RATIO contain a polar modifier such as methanol, 2-propanol or
The detection limit and quantification limit correspond to acetonitrile. The composition, pressure (density),
signal-to-noise ratios of 3 and 10 respectively. The signal-to- temperature and flow rate of the prescribed mobile phase
noise ratio (S/1\~ is calculated using the expression: may either be constant throughout the whole
chromatographic procedure (isocratic, isodense, isothermic
S 2H elution) or may vary according to a defined programme
N h (gradient elution of the modifier, pressure (density),
temperature or flow rate).
H height of the peak corresponding to the component concerned,
in the electropherogram obtained with the prescribed reference Detectors
solution, measured from the maximum of the peak to the Ultraviolet/visible (UVNis) spectrophotometers and flame
extrapolated baseline of the signal observed over a distance ionisation detectors are tl1e most commonly employed
equal to twenty times the width at half-height,
h range of the background in an electropherogram obtained after
detectors. Light scattering detectors, infrared absorption
injection of a blank, observed over a distance equal to twenty spectrophotometers, thermal conductivity detectors or other
times the width at the half-height of the peak in the special detectors may be used.
electropherogram obtained with the prescribed reference
solution and, if possible, situated equally around the place METHOD
where this peak would be found. Prepare the test solution(s) and the reference solution(s) as
prescribed. The solutions must be free from solid particles.
Criteria for assessing the suitability of the system are
described in the chapter on Chromatographic separation
techni,ques (2.2.46). The extent to which adjustments of
H. Supercritical Fluid Chromatography parameters of the chromatographic system can be made to
(Ph. Bur. method 2.2.45) satisfy the criteria of system suitability are also given in this
chapter.
Supercritical fluid chromatography (SFC) is a method of
chromatographic separation in which the mobile phase is a
fluid in a supercritical or a subcritical state. The stationary
phase, contained in a column, consists of either finely divided
solid particles, such as a silica or porous graphite, a J. lsoelectric Focusing 1
chemically modified stationary phase, as used in liquid (Ph. Bur. method 2.2.54)
chromatography, or, for capillary columns, a cross-linked
liquid film evenly coated on the walls of the column. GENERAL PRINCIPLES
Isoelectric focusing (IEF) is a metl1od of electrophoresis that
SFC is based on mechanisms of adsorption or mass
separates proteins according to their isoelectric point.
distribution.
Separation is carried out in a slab of polyacrylamide or
APPARATUS agarose gel that contains a mixture of amphoteric electrolytes
The apparatus usually consists of a cooled pumping system, (ampholytes). When subjected to an electric field, tl1e
an injector, a chromatographic column, contained in an ampholytes migrate in the gel to create a pH gradient.
oven, a detector, a pressure regulator and a data acquisition In some cases gels containing an immobilised pH gradient,
device (or an integrator or a chart recorder). prepared by incorporating weak acids and bases to specific
Pumping system regions of the gel network during the preparation of the gel,
Pumping systems are required to deliver the mobile phase at are used. When the applied proteins reach the gel fraction
a constant flow rate. Pressure fluctuations are to be that has a pH that is the same as tl1eir isoelectric point (pi),
minimised, e.g. by passing the pressurised solvent through a their charge is neutralised and migration ceases. Gradients
pulse-damping device. Tubing and connections are capable can be made over various ranges of pH, according to tl1e
of withstanding the pressures developed by the pumping mixture of ampholytes chosen.
system. THEORETICAL ASPECTS
Microprocessor controlled systems are capable of accurately When a protein is at the position of its isoelectric point, it
delivering a mobile phase in either constant or varying has no net charge and cannot be moved in a gel matrix by
conditions, according to a defined programme. In the case of the electric field. It may, however, move from that position
gradient elution, pumping systems which deliver solvent(s) by diffusion. The pH gradient forces a protein to remain in
from several reservoirs are available and solvent mixing can its isoelectric point position, thus concentrating it; this
be achieved on either the low or high-pressure side of the concentrating effect is called "focusing". Increasing tl1e
pump(s). applied voltage or reducing the sample load result in
Injectors improved separation of bands. The applied voltage is limited
Injection may be carried out directly at the head of the by the heat generated, which must be dissipated. The use of
column using a valve. thin gels and an efficient cooling plate controlled by a
thermostatic circulator prevents the burning of the gel whilst
Stationary phases allowing sharp focusing. The separation is estimated by
Stationary phases are contained in columns which have been determining the minimum pl difference (Llpl), which is
described in the chapters on Li,quid chromatography (2.2.29)
(packed columns) and Gas chromatography (2.2.28) (capillary 1 This chapter has undergone pharmacopoeia! harmonisation. See chapter

5. 8 Pharmacopoeia! harmonisation.
2023 Appendix III J V-A255

necessary to separate 2 neighbouring bands: ISOELECTRIC FOCUSING IN POLYACRYLAMIDE

GELS: DETAILED PROCEDURE
D(dpH/dx) The fallowing method is a detailed description of an /BF procedure
~pI = 3 x in thick polyacrylamide slab gels, which is used unless otherwise
E(-dµ/dpH)
stated in the monograph.
D diffusion coefficient of the protein, PREPARATION OF THE GELS
dpH
pH gradient,
Mould
dx The mould (see Figure 2.2.54.-1) is composed of a glass
E intensity of the electric field, in volts per centimetre, plate (A) on which a polyester film (B) is placed to facilitate
d11 handling of the gel, one or more spacers (C), a second glass
variation of the solute mobility with the pH in the region
dpH plate (D) and clamps to hold the structure together.
close to the pl.

Since D and - ddµ for a given protein cannot be altered, the

pH
separation can be improved by using a narrower pH range
and by increasing the intensity of the electric field.
Resolution between protein bands on an IEF gel prepared
with carrier ampholytes can be quite good. Improvements in
resolution may be achieved by using immobilised pH
gradients where the buffering species, which are analogous to
carrier ampholytes, are copolymerised within the gel matrix.
Proteins exhibiting pis differing by as little as 0.02 pH units
may be resolved using a gel prepared with carrier ampholytes D
while immobilised pH gradients can resolve proteins differing
by approximately 0.001 pH units.
C
PRACTICAL ASPECTS
Special attention must be paid to sample characteristics
and/or preparation. Having salt in the sample can be
problematic and it is best to prepare the sample, if possible,
in deionised water or 2 per cent ampholytes, using dialysis or
gel filtration if necessary.
The time required for completion of focusing in thin-layer
polyacrylamide gels is determined by placing a coloured Figure 2.2.54.-1 - Mould
protein (e.g. haemoglobin) at different positions on the gel
surface and by applying the electric field: the steady state is 7.5 per cent polyacrylamide gel
reached when all applications give an identical band pattern. Dissolve 29.1 g of acrylamide Rand 0.9 g of
In some protocols the completion of the focusing is indicated methylenebisacrylamide R in 100 mL of water R.
by the time elapsed after the sample application. To 2.5 volumes of this solution, add the mixture of
The IEF gel can be used as an identity test when the ampholytes specified in the monograph and dilute to
migration pattern on the gel is compared to a suitable 10 volumes with water R. Mix carefully and degas the
standard preparation and IEF calibration proteins, the IEF solution.
gel can be used as a limit test when the density of a band on Preparation of the mould
IEF is compared subjectively with the density of bands Place the polyester film on the lower glass plate, apply the
appearing in a standard preparation, or it can be used as a spacer, place the second glass plate and fit the clamps. Before
quantitative test when the density is measured using a use, place the solution on a magnetic stirrer and add
densitometer or similar instrumentation to determine the 0.25 volumes of a 100 g/L solution of ammonium persulfate R
relative concentration of protein in the bands subject to and 0.25 volumes of tetramethylethylenediamine R.
validation. Immediately fill the space between the glass plates of the
APPARATUS mould with the solution.
An apparatus for IEF consists of: METHOD
- a controllable generator for constant potential, current Dismantle the mould and, making use of the polyester film,
and power; potentials of 2500 V have been used and are transfer the gel onto the cooled support, wetted with a few
considered optimal under a given set of operating millilitres of a suitable liquid, taking care to avoid forming air
conditions; a supply of up to 30 W of constant power is bubbles. Prepare the test solutions and reference solutions as
recommended; specified in the monograph. Place strips of paper for sample
- a rigid plastic IEF chamber that contains a cooled plate, application, about 10 mm x 5 mm in size, on the gel and
of suitable material, to support the gel; impregnate each with the prescribed amount of the test and
- a plastic cover with platinum electrodes that are reference solutions. Also apply the prescribed quantity of a
connected to the gel by means of paper wicks of suitable solution of proteins with known isoelectric points as pH
width, length and thickness, impregnated with solutions markers to calibrate the gel. In some protocols the gel has
of anodic and cathodic electrolytes. pre-cast slots where a solution of the sample is applied
instead of using impregnated paper strips. Cut 2 strips of
paper to the length of the gel and impregnate them with the
electrolyte solutions: acid for the anode and alkaline for the
V-A256 Appendix III K 2023

cathode. The compositions of the anode and cathode solutions should be used to prevent carbamylation of the
solutions are given in the monograph. Apply these paper protein;
wicks to each side of the gel several millimetres from the - the use of alternative staining methods;
edge. Fit the cover so that the electrodes are in contact with - the use of gel additives such as non-ionic detergents
the wicks (respecting the anodic and cathodic poles). Proceed (e.g. octylglucoside) or zwitterionic detergents (e.g.,
with the isoelectric focusing by applying the electrical CHAPS or CHAPSO), and the addition of ampholyte to
parameters described in the monograph. Switch off the the sample, to prevent proteins from aggregating or
current when the migration of the mixture of standard precipitating.
proteins has stabilised. Using forceps, remove the sample POINTS TO CONSIDER
application strips and the 2 electrode wicks. Immerse the gel
Samples can be applied to any area on the gel, but to protect
in fixing solution for isoelectric focusing in polyacrylamide gel R.
the proteins from extreme pH environments samples should
Incubate with gentle shaking at room temperature for
not be applied close to either electrode. During method
30 min. Drain off the solution and add 200 mL of destaining
development the analyst can try applying the protein in
solution R. Incubate with shaking for 1 h. Drain the gel, add
3 positions on the gel (i.e. middle and both ends); the
coomassie staining solutwn R. Incubate for 30 min. Destain the
pattern of a protein applied at opposite ends of the gel may
gel by passive diffusion with destaining solution R until the
not be identical.
bands are well visualised against a clear background. Locate
the position and intensity of the bands in the A phenomenon known as cathodic drift, where the pH
electropherogram as prescribed in the monograph. gradient decays over time, may occur if a gel is focused too
long. Although not well understood, electroendoosmosis and
VARIATIONS TO THE DETAILED PROCEDURE absorption of carbon dioxide may be factors that lead to
(SUBJECT TO VALIDATION) cathodic drift. Cathodic drift is observed as focused protein
Where reference to the general method on isoelectric migrating off the cathode end of the gel. Immobilised pH
focusing is made, variations in methodology or procedure gradients may be used to address this problem.
may be made subject to validation. These include: Efficient cooling (approximately 4 °C) of the bed that the gel
- the use of commercially available pre-cast gels and of lies on during focusing is important. High field strengths
commercial staining and destaining kits, used during isoelectric focusing can lead to overheating and
- the use of immobilised pH gradients, affect the quality of the focused gel.
- the use of rod gels,
- the use of gel cassettes of different dimensions, including
ultra-thin (0.2 mm) gels,
- variations in the sample application procedure, including
different sample volumes or the use of sample application K. Peptide Mapping 1
masks or wicks other than paper, (Ph. Bur. method 2.2.55)
- the use of alternate running conditions, including
Peptide mapping is an identity test for proteins, especially
variations in the electric field depending on gel
those obtained by rDNA technology. It involves the chemical
dimensions and equipment, and the use of fixed
or enzymatic treatment of a protein resulting in the formation
migration times rather than subjective interpretation of
of peptide fragments followed by separation and
band stability,
identification of these fragments in a reproducible manner.
- the inclusion of a pre-focusing step,
It is a powerful test that is capable of identifying almost any
- the use of automated instrumentation,
single amino acid changes resulting from events such as
- the use of agarose gels.
errors in the reading of complementary DNA (cDNA)
VALIDATION OF ISO-ELECTRIC FOCUSING sequences or point mutations. Peptide mapping is a
PROCEDURES comparative procedure because the information obtained,
Where alternative methods to the detailed procedure are compared to a reference substance similarly treated, confirms
employed they must be validated. The following criteria may the primary structure of the protein, is capable of detecting
be used to validate the separation: whether alterations in structure have occurred, and
- formation of a stable pH gradient of desired demonstrates process consistency and genetic stability. Each
characteristics, assessed for example using coloured pH protein presents unique characteristics which must be well
markers of known isoelectric points, understood so that the scientific and analytical approaches
- comparison with the electropherogram provided with the permit validated development of a peptide map that provides
chemical reference substance for the preparation to be sufficient specificity.
examined, This chapter provides detailed assistance in the application of
- any other validation criteria as prescribed in the peptide mapping and its validation to characterise the desired
monograph. protein, to evaluate the stability of the expression construct of
SPECIFIED VARIATIONS TO THE GENERAL cells used for recombinant DNA products and to evaluate
METHOD the consistency of the overall process, to assess product
Variations to the general method required for the analysis of stability as well as to ensure the identity of the protein, or to
specific substances may be specified in detail in monographs. detect the presence of protein variant.
These include: Peptide mapping is not a general method, but involves
- the addition of urea in the gel (3 M concentration is often developing specific maps for each unique protein. Although
satisfactory to keep protein in solution but up to 8 M can the technology is evolving rapidly, there are certain methods
be used): some proteins precipitate at their isoelectric
point; in this case, urea is included in the gel formulation
to keep the protein in solution; if urea is used, only fresh 1 This chapter has undergone phamiacopoeial hamionisation. See chapter
5. 8 Phamiacopoeial hamionisation.
2023 Appendix III K V-A257

Table 2.2.55.-1. - Examples of cleavage agents

Type Agent Specificity

Emymatic Trypsin (EC 3.4.21.4) C-terminal side of Arg and Lys

Chymotrypsin (EC 3.4.21.1) C-terminal side of hydrophobic residues (e.g. Leu, Met, Ala,
aromatics)
Pepsin (EC 3.4.23.1 and 2) Non-specific digest

Lysyl endopeptidase (Lys-C endopeptidase) (EC 3.4.21.50) C-terminal side of Lys

Glutamyl endopeptidase (from S. aureus strain VS) C-tenninal side of Glu and Asp
(EC 3.4.21.19)

Peptidyl-Asp metallo-endopeptidase (endoproteinase Asp- N-terminal side of Asp

N)

Clostripain (EC 3.4.22.8) C-tenninal side of Arg

Chemical Cyanogen bromide C-terminal side of Met

2-Nitro-5-thio-cyanobenzoic acid N-terminal side of Cys

O-Iodosobenzoic acid C-terminal side of Trp and Tyr

Dilute acid Asp and Pro

BNPS-skatole Trp

that are generally accepted. Variations of these methods will Pretreatment of the cleavage agent
be indicated, when appropriate, in specific monographs. Pretreatment of cleavage agents, especially enzymatic agents,
A peptide map may be viewed as a fingerprint of a protein might be necessary for purification purposes to ensure
and is the end product of several chemical processes that reproducibility of the map. For example, trypsin used as a
provide a comprehensive understanding of the protein being cleavage agent will have to be treated with tosyl-L-
analysed. 4 principal steps are necessary for the development phenylalanine chloromethyl ketone to inactivate
of the procedure: isolation and purification of the protein, if chymotrypsin. Other methods, such as purification of trypsin
the protein is part of a formulation; selective cleavage of the by high performance liquid chromatography (HPLC) or
peptide bonds; chromatographic separation of the peptides; immobilisation of enzyme on a gel support, have been
and analysis and identification of the peptides. A test sample successfully used when only a small amount of protein is
is digested and assayed in parallel with a reference substance. available.
Complete cleavage of peptide bonds is more likely to occur Pretreatment of the protein
when enzymes such as endoproteases (e.g., trypsin) are used, Under certain conditions, it might be necessary to
instead of chemical cleavage reagents. A map must contain concentrate the sample or to separate the protein from
enough peptides to be meaningful. On the other hand, if excipients and stabilisers used in formulation of the product,
there are too many fragments, the map might lose its if these interfere with the mapping procedure. Physical
specificity because many proteins will then have the same procedures used for pretreatment can include ultrafiltration,
profiles. column chromatography and lyophilization. Other
ISOLATION AND PURIFICATION pretreatments, such as the addition of chaotropic agents
Isolation and purification are necessary for analysis of bulk (e.g. urea) can be used to unfold the protein prior to
drugs or dosage forms containing interfering excipients and mapping. To allow the enzyme to have full access to cleavage
carrier proteins and, when required, will be specified in the sites and permit some unfolding of the protein, it is often
monograph. Quantitative recovery of protein from the dosage necessary to reduce and alkylate the disulfide bonds prior to
form must be validated. digestion.
Digestion with trypsin can introduce ambiguities in the
SELECTIVE CLEAVAGE OF PEPTIDE BONDS peptide map due to side reactions occurring during the
The selection of the approach used for the cleavage of digestion reaction, such as non-specific cleavage,
peptide bonds will depend on the protein under test. This deamidation, disulfide isomerisation, oxidation of methionine
selection process involves determination of the type of residues, or formation of pyroglutamic groups created from
cleavage to be employed, enzymatic or chemical, and the the deamidation of glutamine at the N-terminal side of a
type of cleavage agent within the chosen category. Several peptide. Furthermore, peaks may be produced by
cleavage agents and their specificity are shown in autohydrolysis of trypsin. Their intensities depend on the
Table 2.2.55.-1. This list is not all-inclusive and will be ratio of trypsin to protein. To avoid autohydrolysis, solutions
expanded as other cleavage agents are identified. of proteases may be prepared at a pH that is not optimal
Pretreatment of sample (e.g. at pH 5 for trypsin), which would mean that the
Depending on the size or the configuration of the protein, enzyme would not become active until diluted with the digest
different approaches in the pretreatment of samples can be buffer.
used. If trypsin is used as a cleavage agent for proteins with a Establishment of optimal digestion conditions
molecular mass greater than 100 000 Da, lysine residues Factors that affect the completeness and effectiveness of
must be protected by citraconylation or maleylation; digestion of proteins are those that could affect any chemical
otherwise, too many peptides will be generated. or enzymatic reactions.
V-A258 Appendix III K 2023

pH of the reaction milieu The pH of the digestion into the HPLC system, they can damage the sealing of pump
mixture is empirically determined to ensure the optimisation valves or clog the top of the chromatographic column. Both
of the performance of the given cleavage agent. For example, pre- and post-pump filtration is also recommended.
when using cyanogen bromide as a cleavage agent, a highly Chromatographic column
acidic environment (e.g. pH 2, formic acid) is necessary; The selection of a chromatographic column is empirically
however, when using trypsin as a cleavage agent, a slightly determined for each protein. Columns with I O nm or 30 nm
alkaline environment (pH 8) is optimal. As a general rule, the pore size with silica support can give optimal separation.
pH of the reaction milieu must not alter the chemical For smaller peptides, octylsilyl silica gel for chromatography R
integrity of the protein during the digestion and must not (3-10 µm) and octadecylsilyl silica gel for chromatography R
change during the course of the fragmentation reaction. (3-10 µm) column packings are more efficient than butylsilyl
Temperature A temperature between 25 °C and 37 °C is silica gel for chromatography R (5-10 µm).
adequate for most digestions. The temperature used is Solvent
intended to minimise chemical side reactions. The type of The most commonly used solvent is water with acetonitrile
protein under test will dictate the temperature of the reaction as the organic modifier to which not more than 0.1 per cent
milieu, because some proteins are more susceptible to trifluoroacetic acid is added. If necessary, add propyl alcohol
denaturation as the temperature of the reaction increases. or isopropyl alcohol to solubilise the digest components,
For example, digestion of recombinant bovine somatropin is provided that the addition does not unduly increase the
conducted at 4 °C, because at higher temperatures it will viscosity of the components.
precipitate during digestion.
Mobile phase
Time If sufficient sample is available, a time course study
Buffered mobile phases containing phosphate are used to
is considered in order to determine the optimum time to
provide some flexibility in the selection of pH conditions,
obtain a reproducible map and avoid incomplete digestion.
since shifts of pH in the 3.0-5.0 range enhance the separation
Time of digestion varies from 2 h to 30 h. The reaction is
of peptides containing acidic residues (e.g. glutamic and
stopped by the addition of an acid which does not interfere
aspartic acids). Sodium or potassium phosphates, ammonium
in the map or by freezing.
acetate, phosphoric acid at a pH between 2 and 7 (or higher
Amount of deavage agent used Although excessive for polymer-based supports) have also been used with
amounts of cleavage agent are used to accomplish a acetonitrile gradients. Acetonitrile containing trifluoroacetic
reasonably rapid digestion time (i.e. 6-20 hours), the amount acid is used quite often.
of cleavage agent is minimised to avoid its contribution to the
Gradient
chromatographic map pattern. A protein to protease ratio
Gradients can be linear, nonlinear, or include step functions.
between 20:1 and 200:1 is generally used. It is recommended
A shallow gradient is recommended in order to separate
that the cleavage agent is added in 2 or more stages to
complex mixtures. Gradients are optimised to provide clear
optimise cleavage. Nonetheless, the final reaction volume
resolution of 1 or 2 peaks that will become "marker" peaks
remains small enough to facilitate the next step in peptide
for the test.
mapping, the separation step. To sort out digestion artifacts
that might interfere with the subsequent analysis, a blank lsocratic elution
determination is performed, using a digestion control with all Isocratic HPLC systems using a single mobile phase are used
the reagents, except the test protein. on the basis of their convenience of use and improved
detector responses. Optimal composition of a mobile phase
CHROMATOGRAPHIC SEPARATION to obtain clear resolution of each peak is sometimes difficult
Many techniques are used to separate peptides for mapping. to establish. Mobile phases for which slight changes in
The selection of a technique depends on the protein being component ratios or in pH significantly affect retention times
mapped. Techniques that have been successfully used for of peaks in peptide maps must not be used in isocratic
separation of peptides are shown in Table 2.2.55-2. In this HPLC systems.
section, a most widely used reversed-phase HPLC method is
described as one of the procedures of chromatographic Other parameters
separation. Temperature control of the column is usually necessary to
achieve good reproducibility. The flow rates for the mobile
Table 2.2.55-2. - Techniques used for the separation of peptides phases range from 0.1-2.0 mUmin, and the detection of
Reversed-phase high performance liquid chromatography (HPLC)
peptides is performed with a UV detector at 200-230 nm.
Ion-exchange chromatography (!EC)
Other methods of detection have been used (e.g. post-
Hydrophobic interaction chromatography (HIC)
column derivatisation), but they are not as robust or versatile
Polyacrylamide gel electrophoresis (PAGE), non-denaturating
as UV detection.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Validation
Capillary electrophoresis (CE) This section provides an experimental means for measuring
Paper chromatography-high voltage (PCHV) the overall performance of the test method. The acceptance
High voltage-paper electrophoresis (HYPE) criteria for system suitability depend on the identification of
critical test parameters that affect data interpretation and
The purity of solvents and mobile phases is a critical factor in acceptance. These critical parameters are also criteria that
HPLC separation. HPLC-grade solvents and water that are monitor peptide digestion and peptide analysis. An indicator
commercially available, are recommended for reversed-phase that the desired digestion endpoint has been achieved is
HPLC. Dissolved gases present a problem in gradient shown by comparison with a reference standard, which is
systems where the solubility of the gas in a solvent may be treated in the same manner as the test protein. The use of a
less in a mixture than in a single solvent. Vacuum degassing reference substance in parallel with the test protein is critical
and agitation by sonication are often used as useful degassing in the development and establishment of system suitability
procedures. When solid particles in the solvents are drawn limits. In addition, a chromatogram is included with the
2023 Appendix III K V-A259

reference substance for additional comparison purposes. Such approaches are, for example, the automated
Other indicators may include visual inspection of protein or identification of compounds by IR spectroscopy and the
peptide solubility, the absence of intact protein, or application of diode-array UV spectral analysis for
measurement of responses of a digestion-dependent peptide. identification of peptides. These methods have limitations
The critical system suitability parameters for peptide analysis due to inadequate resolutions, co-elution of fragments, or
will depend on the particular mode of peptide separation and absolute peak response differences between reference
detection and on the data analysis requirements. substance and sample digest fragments.
When peptide mapping is used as an identification test, the The numerical comparison of the peak retention times and
system suitability requirements for the identified peptides peak areas or peak heights can be done for a selected group
cover selectivity and precision. In this case, as well as when of relevant peaks that have been correctly identified in the
identification of variant protein is done, the identification of peptide maps. Peak areas can be calculated using 1 peak
the primary structure of the peptide fragments in the peptide showing relatively small variation as an internal reference,
map provides both a verification of the known primary keeping in mind that peak area integration is sensitive to
structure and the identification of protein variants by baseline variation and likely to introduce error in the analysis.
comparison with the peptide map of the reference substance Alternatively, the percentage of each peptide peak height
for the specified protein. The use of a digested reference relative to the sum of all peak heights can be calculated for
substance for a given protein in the determination of peptide the sample under test. The percentage is then compared to
resolution is the method of choice. For an analysis of a that of the corresponding peak of the reference substance.
variant protein, a characterised mixture of a variant and a The possibility of auto-hydrolysis of trypsin is monitored by
reference substance can be used, especially if the variant producing a blank peptide map, that is, the peptide map
peptide is located in a less-resolved region of the map. obtained when a blank solution is treated with trypsin.
The index of pattern consistency can be simply the number The minimum requirement for the qualification of peptide
of major peptides detected. Peptide pattern consistency can mapping is an approved test procedure that includes system
be best defined by the resolution of peptide peaks. suitability as a test control. In general, early in the regulatory
Chromatographic parameters, such as peak-to-peak process, qualification of peptide mapping for a protein is
resolution, maximum peak width, peak area, peak tailing sufficient. As the regulatory approval process for the protein
factors, and column efficiency, may be used to define peptide progresses, additional qualifications of the test can include a
resolution. Depending on the protein under test and the partial validation of the analytical procedure to provide
method of separation used, single peptide or multiple peptide assurance that the method will perform as intended in the
resolution requirements may be necessary. development of a peptide map for the specified protein.
The replicate analysis of the digest of the reference substance
ANALYSIS AND IDENTIFICATION OF PEPTIDES
for the protein under test yields measures of precision and
This sectwn gives guidance on the use of peptide mapping during
quantitative recovery. Recovery of the identified peptides is
development in support of regulatory applications.
generally ascertained by the use of internal or external
peptide standards. The precision is expressed as the relative The use of a peptide map as a qualitative tool does not
standard deviation (RSD). Differences in the recovery and require the complete characterisation of the individual
precision of the identified peptides are to be expected; peptide peaks. However, validation of peptide mapping in
therefore, the system suitability limits will have to be support of regulatory applications requires rigorous
established for both the recovery and the precision of the characterisation of each of the individual peaks in the peptide
identified peptides. These limits are unique for a given map. Methods to characterise peaks range from N-terminal
protein and will be specified in the individual monograph. sequencing of each peak followed by amino acid analysis to
the use of mass spectroscopy (MS).
Visual comparison of the relative retentions, the peak
responses (the peak area or the peak height), the number of For characterisation purposes, when N-terminal sequencing
peaks, and the overall elution pattern is completed initially. and amino acids analysis are used, the analytical separation is
It is then complemented and supported by mathematical scaled up. Since scale-up might affect the resolution of
analysis of the peak response ratios and by the peptide peaks, it is necessary, using empirical data, to assure
chromatographic profile of a 1: 1 ( V/V) mixture of sample that there is no loss of resolution due to scale-up. Eluates
and reference substance digest. If all peaks in the sample corresponding to specific peptide peaks are collected,
digest and in the reference substance digest have the same vacuum-concentrated, and chromatographed again, if
relative retentions and peak response ratios, then the identity necessary. Amino acid analysis of fragments may be limited
of the sample under test is confirmed. by the peptide size. If the N-terminus is blocked, it may need
to be cleared before sequencing. C-terminal sequencing of
If peaks that initially eluted with significantly different relative
proteins in combination with carboxypeptidase and matrix-
retentions are then observed as single peaks in the 1: 1
assisted laser desorption ionisation coupled to time-of-flight
mixture, the initial difference would be an indication of
analyser (MALDI-TOF) can also be used for characterisation
system variability. However, if separate peaks are observed in
purposes.
the 1: 1 mixture, this would be evidence of the
nonequivalence of the peptides in each peak. If a peak in the The use of MS for characterisation of peptide fragments is by
1:1 mixture is significantly broader than the corresponding direct infusion of isolated peptides or by the use of on-line
peak in the sample and reference substance digest, it may LC-MS for structure analysis. In general, it includes
indicate the presence of different peptides. The use of electrospray and MALDI-TOF-MS, as well as fast-atom
computer-aided pattern recognition software for the analysis bombardment (FAB). Tandem MS has also been used to
of peptide mapping data has been proposed and applied, but sequence a modified protein and to determine the type of
issues related to the validation of the computer software amino acid modification that has occurred. The comparison
preclude its use in a compendia! test in the near future. of mass spectra of the digests before and after reduction
Other automated approaches have been used that employ provides a method to assign the disulfide bonds to the
mathematical formulas, models, and pattern recognition. various sulfydryl-containing peptides.
V-A260 Appendix III L 2023

If regions of the primary structure are not clearly use, keeping solvent reservoirs covered, and not placing
demonstrated by the peptide map, it might be necessary to amino acid analysis instrumentation in direct sunlight.
develop a secondary peptide map. The goal of a validated Laboratory practices can determine the quality of the amino
method of characterisation of a protein through peptide acid analysis. Place the instrumentation in a low traffic area
mapping is to reconcile and account for at least 95 per cent of the laboratory. Keep the laboratory clean. Clean and
of the theoretical composition of the protein structure. calibrate pipets according to a maintenance schedule. Keep
pipet tips in a covered box; the analysts may not handle pipet
tips with their hands. The analysts may wear powder-free
latex or equivalent gloves. Limit the number of times a test
L. Amino Acid Analysis 1 sample vial is opened and closed because dust can contribute
to elevated levels of glycine, serine, and alanine.
(Ph. Bur. method 2.2.56)
A well-maintained instrument is necessary for acceptable
Amino acid analysis refers to the methodology used to amino acid analysis results. If the instrument is used on a
determine the amino acid composition or content of proteins, routine basis, it is to be checked daily for leaks, detector and
peptides, and other pharmaceutical preparations. Proteins lamp stability, and the ability of the column to maintain
and peptides are macromolecules consisting of covalently resolution of the individual amino acids. Clean or replace all
bonded amino acid residues organised as a linear polymer. instrument filters and other maintenance items on a routine
The sequence of the amino acids in a protein or peptide schedule.
determines the properties of the molecule. Proteins are
considered large molecules that commonly exist as folded REFERENCE MATERIAL
structures with a specific conformation, while peptides are Acceptable amino acid standards are commercially available
smaller and may consist of only a few amino acids. Amino for amino acid analysis and typically consist of an aqueous
acid analysis can be used to quantify proteins and peptides, mixture of amino acids. When determining amino acid
to determine the identity of proteins or peptides based on composition, protein or peptide standards are analysed with
their amino acid composition, to support protein and peptide the test material as a control to demonstrate the integrity of
structure analysis, to evaluate fragmentation strategies for the entire procedure. Highly purified bovine serum albumin
peptide mapping, and to detect atypical amino acids that has been used as a protein standard for this purpose.
might be present in a protein or peptide. It is necessary to CALIBRATION OF INSTRUMENTATION
hydrolyse a protein/peptide to its individual amino acid Calibration of amino acid analysis instrumentation typically
constituents before amino acid analysis. Following involves analysing the amino acid standard, which consists of
protein/peptide hydrolysis, the amino acid analysis procedure a mixture of amino acids at a number of concentrations, to
can be the same as that practiced for free amino acids in determine the response factor and range of analysis for each
other pharmaceutical preparations. The amino acid amino acid. The concentration of each amino acid in the
constituents of the test sample are typically derivatised for standard is known. In the calibration procedure, the analyst
analysis. dilutes the amino acid standard to several different analyte
APPARATUS levels within the expected linear range of the amino acid
Methods used for amino acid analysis are usually based on a analysis technique. Then, replicates at each of the different
chromatographic separation of the amino acids present in the analyte levels can be analysed. Peak areas obtained for each
test sample. Current techniques take advantage of the amino acid are plotted versus the known concentration for
automated chromatographic instrumentation designed for each of the amino acids in the standard dilution. These
analytical methodologies. An amino acid analysis instrument results will allow the analyst to determine the range of amino
will typically be a low-pressure or high-pressure liquid acid concentrations where the peak area of a given amino
chromatograph capable of generating mobile phase gradients acid is an approximately linear function of the amino acid
that separate the amino acid analytes on a chromatographic concentration. It is important that the analyst prepare the
column. The instrument must have post-column samples for amino acid analysis so that they are within the
derivatisation capability, unless the sample is analysed using analytical limits (e.g., linear working range) of the technique
precolumn derivatisation. The detector is usually an employed in order to obtain accurate and repeatable results.
ultraviolet/visible or fluorescence detector depending on the 4 to 6 amino acid standard levels are analysed to determine a
derivatisation method used. A recording device (e.g., response factor for each amino acid. The response factor is
integrator) is used for transforming the analogue signal from calculated as the average peak area or peak height per
the detector and for quantitation. It is preferred that nanomole of amino acid present in the standard.
instrumentation be dedicated particularly for amino acid A calibration file consisting of the response factor for each
analysis. amino acid is prepared and used to calculate the
concentration of each amino acid present in the test sample.
GENERAL PRECAUTIONS
This calculation involves dividing the peak area
Background contamination is always a concern for the
corresponding to a given amino acid by the response factor
analyst in performing amino acid analysis. High purity
for that amino acid to give the nanomoles of the amino acid.
reagents are necessary (e.g., low purity hydrochloric acid can
For routine analysis, a single-point calibration may be
contribute to glycine contamination). Analytical reagents are
sufficient; however, the calibration file is updated frequently
changed routinely every few weeks using only high-pressure
and tested by the analysis of analytical controls to ensure its
liquid chromatography (HPLC) grade solvents. Potential
integrity.
microbial contamination and foreign material that might be
present in the solvents are reduced by filtering solvents before REPEATABILITY
Consistent high quality amino acid analysis results from an
analytical laboratory require attention to the repeatability of
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter the assay. During analysis of the chromatographic separation
5. 8 Pharmacopoeia/ harmonisation.
2023 Appendix III L V-A261

of the amino acids or their derivatives, numerous peaks can Ideally, an internal standard is an unnaturally occurring
be observed on the chromatogram that correspond to the primary amino acid that is commercially available and
amino acids. The large number of peaks makes it necessary inexpensive. It should also be stable during hydrolysis, its
to have an amino acid analysis system that can repeatedly response factor should be linear with concentration, and it
identify the peaks based on retention time and integrate the needs to elute with a unique retention time without
peak areas for quantitation. A typical repeatability evaluation overlapping other amino acids. Commonly used amino acid
involves preparing a standard amino acid solution and standards include norleucine, nitrotyrosine, and
analysing many replicates (e.g., 6 analyses or more) of the cx-aminobutyric acid.
same standard solution. The relative standard deviation
PROTEIN HYDROLYSIS
(RSD) is determined for the retention time and integrated
Hydrolysis of protein and peptide samples is necessary for
peak area of each amino acid. An evaluation of the
amino acid analysis of these molecules. The glassware used
repeatability is expanded to include multiple assays
for hydrolysis must be very clean to avoid erroneous results.
conducted over several days by different analysts. Multiple
Glove powders and fingerprints on hydrolysis tubes may
assays include the preparation of standard dilutions from
cause contamination. To clean glass hydrolysis tubes, boil
starting materials to determine the variation due to sample
tubes for 1 h in 1 M hydrochloric acid or soak tubes in
handling. The amino acid composition of a standard protein
concentrated nitric acid or in a mixture of equal volumes of
(e.g., bovine serum albumin) is often analysed as part of the
concentrated hydrochloric acid and nitric acid. Clean
repeatability evaluation. By evaluating the replicate variation
hydrolysis tubes are rinsed with high-purity water followed by
(i.e., RSD), the laboratory can establish analytical limits to
a rinse with HPLC grade methanol, dried overnight in an
ensure that the analyses from the laboratory are under
oven, and stored covered until use. Alternatively, pyrolysis of
control. It is desirable to establish the lowest practical
clean glassware at 500 °C for 4 h may also be used to
variation limits to ensure the best results. Areas to focus on
eliminate contamination from hydrolysis tubes. Adequate
to lower the variability of the amino acid analysis include
disposable laboratory material can also be used.
sample preparation, high background spectral interference
due to quality of reagents and/or laboratory practices, Acid hydrolysis is the most common method for hydrolysing
instrument performance and maintenance, data analysis and a protein sample before amino acid analysis. The acid
interpretation, and analyst performance and habits. hydrolysis technique can contribute to the variation of the
All parameters involved are fully investigated in the scope of analysis due to complete or partial destruction of several
the validation work. amino acids: tryptophan is destroyed; serine and threonine
are partially destroyed; methionine might undergo oxidation;
SAMPLE PREPARATION and cysteine is typically recovered as cystine (but cystine
Accurate results from amino acid analysis require purified recovery is usually poor because of partial destruction or
protein and peptide samples. Buffer components (e.g., salts, reduction to cysteine). Application of adequate vacuum (less
urea, detergents) can interfere with the amino acid analysis than 200 µm of mercury or 26. 7 Pa) or introduction of an
and are removed from the sample before analysis. Methods inert gas (argon) in the headspace of the reaction vessel can
that utilise post-column derivatisation of the amino acids are reduce the level of oxidative destruction. In peptide bonds
generally not affected by buffer components to the extent involving isoleucine and valine the amido bonds of Ile-Ile,
seen with pre-column derivatisation methods. It is desirable Val-Val, Ile-Val, and Val-Ile are partially cleaved; and
to limit the number of sample manipulations to reduce asparagine and glutamine are deamidated, resulting in
potential background contamination, to improve analyte aspartic acid and glutamic acid, respectively. The loss of
recovery, and to reduce labour. Common techniques used to tryptophan, asparagine, and glutamine during an acid
remove buffer components from protein samples include the hydrolysis limits quantitation to 17 amino acids. Some of the
following methods: (1) injecting the protein sample onto a hydrolysis techniques described are used to address these
reversed-phase HPLC system, removing the protein with a concerns. Some of the hydrolysis techniques described (i.e.,
volatile solvent containing a sufficient organic component, Methods 4-11) may cause modifications to other amino
and drying the sample in a vacuum centrifuge; (2) dialysis acids. Therefore, the benefits of using a given hydrolysis
against a volatile buffer or water; (3) centrifugal ultrafiltration technique are weighed against the concerns with the
for buffer replacement with a volatile buffer or water; technique and are tested adequately before employing a
(4) precipitating the protein from the buffer using an organic method other than acid hydrolysis.
solvent (e.g., acetone); (5) gel filtration.
A time-course study (i.e., amino acid analysis at acid
INTERNAL STANDARDS hydrolysis times of 24 h, 48 h and 72 h) is often employed to
It is recommended that an internal standard be used to analyse the starting concentration of amino acids that are
monitor physical and chemical losses and variations during partially destroyed or slow to cleave. By plotting the observed
amino acid analysis. An accurately known amount of internal concentration of labile amino acids (e.g., serine and
standard can be added to a protein solution prior to threonine) versus hydrolysis time, the line can be
hydrolysis. The recovery of the internal standard gives the extrapolated to the origin to determine the starting
general recovery of the amino acids of the protein solution. concentration of these amino acids. Time-course hydrolysis
Free amino acids, however, do not behave in the same way studies are also used with amino acids that are slow to cleave
as protein-bound amino acids during hydrolysis, whose rates (e.g., isoleucine and valine). During the hydrolysis time
of release or destruction are variable. Therefore, the use of an course, the analyst will observe a plateau in these residues.
internal standard to correct for losses during hydrolysis may The level of this plateau is taken as the residue
give unreliable results. It will be necessary to take this point concentration. If the hydrolysis time is too long, the residue
into consideration when interpreting the results. Internal concentration of the sample will begin to decrease, indicating
standards can also be added to the mixture of amino acids destruction by the hydrolysis conditions.
after hydrolysis to correct for differences in sample An acceptable alternative to the time-course study is to
application and changes in reagent stability and flow rates. subject an amino acid calibration standard to the same
V-A262 Appendix III L 2023

hydrolysis conditions as the test sample. The amino acid in 170-185 °C for about 12.5 min. After hydrolysis, dry the
free form may not completely represent the rate of hydrolysis tube in vacuo for 15 min to remove the residual
destruction of labile amino acids within a peptide or protein acid.
during the hydrolysis. This is especially true for peptide METHOD3
bonds that are slow to cleave (e.g., Ile-Val bonds). However, Tryptophan oxidation during hydrolysis is prevented by using
this technique will allow the analyst to account for some thioglycollic acid (TGA) as the reducing acid.
residue destruction. Microwave acid hydrolysis has been used
Hydrolysis solution
and is rapid but requires special equipment as well as special
7 M hydrochloric acid containing 1 per cent of phenol,
precautions. The optimal conditions for microwave hydrolysis
10 per cent of trifluoroacetic acid and 20 per cent of
must be investigated for each individual protein/peptide
sample. The microwave hydrolysis technique typically thioglycollic acid.
requires only a few minutes, but even a deviation of one Vapour phase hydrolysis
minute may give inadequate results (e.g., incomplete Dry about 10 µg to 50 µg of the protein/peptide under test in
hydrolysis or destruction of labile amino acids). Complete a sample tube. Place the sample tube in a larger tube with
proteolysis, using a mixture of proteases, has been used but about 200 µL of the hydrolysis solution. Seal the larger tube
can be complicated, requires the proper controls, and is in vacuo (about 50 µm of mercury or 6. 7 Pa) to vaporise the
typically more applicable to peptides than proteins. TGA. Heat the sample tube to 166 °C for about 15-30 min.
During initial analyses of an unknown protein, experiments After hydrolysis, dry the sample tube in vacuo for 5 min to
with various hydrolysis time and temperature conditions are remove the residual acid. Recovery of tryptophan by this
conducted to determine the optimal conditions. method may be dependent on the amount of sample present.
METHODl METHOD4
Acid hydrolysis using hydrochloric acid containing phenol is Cysteine/cystine and methionine oxidation is performed with
the most common procedure used for protein/peptide performic acid before the protein hydrolysis.
hydrolysis preceding amino acid analysis. The addition of Oxidation solution
phenol to the reaction prevents the halogenation of tyrosine. Use performic acid freshly prepared by mixing 1 volume of
Hydrolysis solution hydrogen peroxide solution (30 per cent) and 9 volumes of
6 M hydrochloric acid containing 0.1 per cent to anhydrous formic acid and incubating at room temperature
1.0 per cent of phenol. for 1 h.
Procedure Procedure
Liquid phase hydrolysis Place the protein or peptide Dissolve the protein/peptide sample in 20 µL of anhydrous
sample in a hydrolysis tube, and dry (the sample is dried so formic acid and heat at 50 °C for 5 min; then add 100 µL of
that water in the sample will not dilute the acid used for the the oxidation solution. Allow the oxidation to proceed for
hydrolysis). Add 200 µL of hydrolysis solution per 500 µg of 10-30 min. In this reaction, cysteine is converted to cysteic
lyophilised protein. Freeze the sample tube in a dry ice- acid and methionine is converted to methionine-sulfone.
acetone bath, and flame seal in vacuo. Samples are typically Remove the excess reagent from the sample in a vacuum
hydrolysed at 110 °C for 24 h in vacuo or in an inert centrifuge. The oxidised protein can then be acid hydrolysed
atmosphere to prevent oxidation. Longer hydrolysis times using Method 1 or Method 2. This technique may cause
(e.g., 48 h and 72 h) are investigated if there is a concern modifications to tyrosine residues in the presence of halides.
that the protein is not completely hydrolysed. METHODS
Vapour phase hydrolysis This is one of the most Cysteine/cystine oxidation is accomplished during the liquid
common acid hydrolysis procedures, and it is preferred for phase hydrolysis with sodium azide.
microanalysis when only small amounts of the sample are Hydrolysis solution
available. Contamination of the sample from the acid reagent To 6 M hydrochloric acid containing 0.2 per cent of phenol,
is also minimised by using vapour phase hydrolysis. Place add sodium azide to obtain a final concentration of 2 g/L.
vials containing the dried samples in a vessel that contains an The added phenol prevents halogenation of tyrosine.
appropriate amount of hydrolysis solution. The hydrolysis Liquid phase hydrolysis
solution does not come in contact with the test sample. Conduct the protein/peptide hydrolysis at about 110 °C for
Apply an inert atmosphere or vacuum (less than 200 µm of 24 h. During the hydrolysis, the cysteine/cystine present in
mercury or 26. 7 Pa) to the headspace of the vessel, and heat the sample is converted to cysteic acid by the sodium azide
to about 110 °C for a 24 h hydrolysis time. Acid vapour present in the hydrolysis solution. This technique allows
hydrolyses the dried sample. Any condensation of the acid in better tyrosine recovery than Method 4, but it is not
the sample vials is to be minimised. After hydrolysis, dry the quantitative for methionine. Methionine is converted to a
test sample in vacuo to remove any residual acid. mixture of the parent methionine and its 2 oxidative
METHOD2 products, methionine-sulfoxide and methionine-sulfone.
Tryptophan oxidation during hydrolysis is decreased by using METHOD6
mercaptoethanesulfonic acid as the reducing acid. Cysteine/cystine oxidation is accomplished with dimethyl
Hydrolysis solution sulfoxide (DMSO).
2.5 M mercaptoethanesulfonic acid solution. Hydrolysis solution
Vapour phase hydrolysis To 6 M hydrochloric acid containing 0.1 per cent to
Dry about 1 µg to I 00 µg of the protein/peptide under test in 1.0 per cent of phenol, add dimethyl sulfoxide to obtain a
a hydrolysis tube. Place the hydrolysis tube in a larger tube final concentration of 2 per cent VIV.
with about 200 µL of the hydrolysis solution. Seal the larger Vapour phase hydrolysis
tube in vacuo (about 50 µm of mercury or 6. 7 Pa) to Conduct the protein/peptide hydrolysis at about 110 °C for
vaporise the hydrolysis solution. Heat the hydrolysis tube to 24 h. During the hydrolysis, the cysteine/cystine present in
2023 Appendix III L V-A263

the sample is converted to cysteic acid by the DMSO present Buffer solution
in the hydrolysis solution. As an approach to limit variability Use the reducing solution, prepared as described for
and compensate for partial destruction, it is recommended to Method 8.
evaluate the cysteic acid recovery from oxidative hydrolysis of Procedure
standard proteins containing 1-8 mol of cysteine per mole of Dissolve the test sample in 50 µL of the buffer solution, and
protein. The response factors from protein/peptide add about 2.5 µL of stock solution C. Store under nitrogen
hydrolysates are typically about 30 per cent lower than those or argon for 2 h at room temperature in the dark. Add the
for non-hydrolysed cysteic acid standards. Because histidine, carboxymethylation solution in a ratio 1.5 fold per total
methionine, tyrosine, and tryptophan are also modified, a theoretical content of thiols, and incubate for an additional
complete compositional analysis is not obtained with this 30 min at room temperature in the dark. If the thiol content
technique. of the protein is unknown, then add 5 µL of 100 mM
METHOD7 iodoacetamide for every 20 nmol of protein present.
Cysteine/cystine reduction and alkylation is accomplished by The reaction is stopped by adding excess of
a vapour phase pyridylethylation reaction. 2-mercaptoethanol. Desalt the protein/peptide by collecting
Reducing solution the protein/peptide fraction from a reversed-phase HPLC
Transfer 83.3 µL of pyridine, 16. 7 µL of 4-vinylpyridine, separation. The collected sample can be dried in a vacuum
16. 7 µL of tributylphosphine, and 83.3 µL of water to a centrifuge before acid hydrolysis. The S-
suitable container and mix. carboxyamidomethyl-cysteine formed will be converted to
S-carboxymethyl-cysteine during acid hydrolysis.
Procedure
Add the protein/peptide (between 1 and 100 µg) to a METHOD JO
hydrolysis tube, and place in a larger tube. Transfer the Cysteine/cystine is reacted with dithiodiglycolic acid or
reducing solution to the large tube, seal in vacuo (about dithiodipropionic acid to produce a mixed disulfide.
50 µm of mercury or 6.7 Pa), and heat at about 100 °C for The choice of dithiodiglycolic acid or dithiodipropionic acid
5 min. Then remove the inner hydrolysis tube, and dry it in depends on the required resolution of the amino acid analysis
a vacuum desiccator for 15 min to remove residual reagents. method.
The pyridylethylated sample can then be acid hydrolysed Reducing solution
using previously described procedures. The pyridylethylation A 10 g/L solution of dithiodiglycolic acid (or
reaction is performed simultaneously with a protein standard dithiodipropionic acid) in 0.2 M sodium hydroxide.
sample containing 1-8 mol of cysteine per mole of protein to Procedure
evaluate the pyridylethyl-cysteine recovery. Longer incubation Transfer about 20 µg of the test sample to a hydrolysis tube,
times for the pyridylethylation reaction can cause and add 5 µL of the reducing solution. Add 10 µL of
modifications to the a-amino terminal group and the i:-amino isopropyl alcohol, and then remove all of the sample liquid
group of lysine in the protein. by vacuum centrifugation. The sample is then hydrolysed
METHODS using Method 1. This method has the advantage that other
Cysteine/cystine reduction and alkylation is accomplished by amino acid residues are not derivatised by side reactions, and
a liquid phase pyridylethylation reaction. that the sample does not need to be desalted prior to
Stock solutions hydrolysis.
Prepare and filter 3 solutions: 1 M Tris-hydrochloride METHODll
pH 8.5 containing 4 mM disodium edetate (stock Asparagine and glutamine are converted to aspartic acid and
solution A), 8 M guanidine hydrochloride (stock solution B), glutamic acid, respectively, during acid hydrolysis. Asparagine
and 10 per cent of 2-mercaptoethanol (stock solution C). and aspartic acid residues are added and represented by Asx,
Reducing solution while glutamine and glutamic acid residues are added and
Prepare a mixture of 1 volume of stock solution A and represented by Glx. Proteins/peptides can be reacted with bis
3 volumes of stock solution B to obtain a buffered solution of (1,1-tri:fluoroacetoxy)iodobenzene (BTI) to convert the
6 M guanidine hydrochloride in 0.25 M tris-hydrochloride. asparagine and glutamine residues to diaminopropionic acid
and diaminobutyric acid residues, respectively, upon acid
Procedure hydrolysis. These conversions allow the analyst to determine
Dissolve about I O µg of the test sample in 50 µL of the the asparagine and glutamine content of a protein/peptide in
reducing solution, and add about 2.5 µL of stock solution C. the presence of aspartic acid and glutamic acid residues.
Store under nitrogen or argon for 2 h at room temperature in
the dark. To achieve the pyridylethylation reaction, add Reducing solutions
about 2 µL of 4-vinylpyridine to the protein solution, and Prepare and filter 3 solutions: a solution of 10 mM
incubate for an additional 2 h at room temperature in the trifluoroacetic acid (Solution A), a solution of 5 M guanidine
dark. Desalt the protein/peptide by collecting the hydrochloride and 10 mM trifluoroacetic acid (Solution B),
protein/peptide fraction from a reversed-phase HPLC and a freshly prepared solution of dimethylformamide
separation. The collected sample can be dried in a vacuum containing 36 mg of BTI per millilitre (Solution C).
centrifuge before acid hydrolysis. Procedure
METHOD9 In a clean hydrolysis tube, transfer about 200 µg of the test
Cysteine/cystine reduction and alkylation is accomplished by sample, and add 2 mL of Solution A or Solution B and
a liquid phase carboxymethylation reaction. 2 mL of Solution C. Seal the hydrolysis tube in vacua. Heat
the sample at 60 °C for 4 h in the dark. The sample is then
Stock solutions dialysed with water to remove the excess reagents. Extract
Prepare as directed for Method 8. the dialysed sample 3 times with equal volumes of butyl
Carboxymethylation solution acetate, and then lyophilise. The protein can then be acid
Prepare a 100 g/L solution of iodoacetamide in alcohol. hydrolysed using previously described procedures. The o:,~-
V-A264 Appendix III L 2023

diaminopropionic and rx, y-diaminobutyric acid residues do When the amino acid reacts with ninhydrin, the reactant has
not typically resolve from the lysine residues upon ion- a characteristic purple or yellow colour. Amino acids, except
exchange chromatography based on amino acid analysis. imino acid, give a purple colour, and show an absorption
Therefore, when using ion-exchange as the mode of amino maximum at 570 nm. The imino acids such as proline give a
acid separation, the asparagine and glutamine contents are yellow colour, and show an absorption maximum at 440 nm.
the quantitative difference in the aspartic acid and glutamic The post-column reaction between ninhydrin and amino
acid content assayed with underivatised and BTI-derivatised acids eluted from the column is monitored at 440 nm and
acid hydrolysis. The threonine, methionine, cysteine, 570 nm, and the chromatogram obtained is used for the
tyrosine, and histidine assayed content can be altered by BTI determination of amino acid composition.
derivatisation; a hydrolysis without BTI will have to be The detection limit is considered to be 10 pmol for most of
performed if the analyst is interested in the composition of the amino acid derivatives, but 50 pmol for the proline
these other amino acid residues of the protein/peptide. derivative. Response linearity is obtained in the range of
METHODOLOGIES OF AMINO ACID ANALYSIS: 20-500 pmol with correlation coefficients exceeding 0.999.
GENERAL PRINCIPLES To obtain good composition data, samples larger than 1 µg
Many amino acid analysis techniques exist, and the choice of before hydrolysis are best suited for this amino acid analysis
any one technique often depends on the sensitivity required of protein/peptide.
from the assay. In general, about one-half of the amino acid METHOD 2 - POST-COLUMN OPA
analysis techniques employed rely on the separation of the DERIVATISATION
free amino acids by ion-exchange chromatography followed o-Phthalaldehyde (OPA) reacts with primary amines in the
by post-column derivatisation (e.g., with ninhydrin or presence of thiol compound, to form highly fluorescent
o-phthalaldehyde). Post-column derivatisation techniques can isoindole products. This reaction is used for the post-column
be used with samples that contain small amounts of buffer derivatisation in analysis of amino acids by ion-exchange
components, (such as salts and urea) and generally require chromatography. The rule of the separation is the same
between 5 µg and 10 µg of protein sample per analysis. as Method 1.
The remaining amino acid techniques typically involve pre- Although OPA does not react with secondary amines (imino
column derivatisation of the free amino acids (e.g., phenyl acids such as proline) to form fluorescent substances, the
isothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl oxidation with sodium hypochlorite or chloramine T allows
carbamate or o-phthalaldehyde; (dimethylamino) secondary amines to react with OPA. The procedure employs
azobenzenesulfonyl chloride; 9-fluorenylmethyl a strongly acidic cation-exchange column for separation of
chloroformate; and 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) free amino acids followed by post-column oxidation with
followed by reversed-phase HPLC. Pre-column derivatisation sodium hypochlorite or chloramine T and post-column
techniques are very sensitive and usually require between derivatisation using OPA and a thiol compound such as
0. 5 µg and 1. 0 µg of protein sample per analysis but may be N-acetyl-L-cysteine or 2-mercaptoethanol. The derivatisation
influenced by buffer salts in the samples. Pre-column of primacy amino acids is not noticeably affected by the
derivatisation techniques may also result in multiple continuous supply of sodium hypochlorite or chloramine T.
derivatives of a given amino acid, which complicates the Separation of the amino acids on an ion-exchange column is
result interpretation. Post-column derivatisation techniques accomplished through a combination of changes in pH and
are generally influenced less by performance variation of the cation strength. After post-column derivatisation of eluted
assay than pre-column derivatisation techniques. amino acids with OPA, the reactant passes through the
The following methods may be used for quantitative amino fluorometric detector. Fluorescence intensity of OPA-
acid analysis. Instruments and reagents for these procedures derivatised amino acids are monitored with an excitation
are available commercially. Furthermore, many modifications wavelength of 348 nm and an emission wavelength of
of these methodologies exist with different reagent 450 nm.
preparations, reaction procedures, chromatographic systems, The detection limit is considered to be a few tens of
etc. Specific parameters may vary according to the exact
picomole level for most of the OPA-derivatised amino acids.
equipment and procedure used. Many laboratories will use Response linearity is obtained in the range of a few picomole
more than one amino acid analysis technique to exploit the
level to a few tens of nanomole level. To obtain good
advantages offered by each. In each of these methods, the compositional data, samples larger than 500 ng of
analogue signal is visualised by means of a data acquisition protein/peptide before hydrolysis are recommended.
system, and the peak areas are integrated for quantification
purposes. METHOD 3 - PRE-COLUMN PITC
DERIVATISATION
METHOD 1 - POST-COLUMN NINHYDRIN Phenylisothiocyanate (PITC) reacts with amino acids to form
DERIVATISATION phenylthiocarbamyl (PTC) derivatives which can be detected
Ion-exchange chromatography with post-column ninhydrin with high sensitivity at 254 nm. Therefore, pre-column
derivatisation is one of the most common methods employed derivatisation of amino acids with PITC followed by a
for quantitative amino acid analysis. As a rule, a lithium- reversed-phase HPLC separation with UV detection is used
based cation-exchange system is employed for the analysis of to analyse the amino acid composition.
the more complex physiological samples, and the faster
sodium-based cation-exchange system is used for the more After the reagent is removed under vacuum, the derivatised
simplistic amino acid mixtures obtained with protein amino acids can be stored dry and frozen for several weeks
hydrolysates (typically containing 17 amino acid with no significant degradation. If the solution for injection is
components). Separation of the amino acids on an ion- kept cold, no noticeable loss in chromatographic response
exchange column is accomplished through a combination of occurs after 3 days.
changes in pH and cation strength. A temperature gradient is Separation of the PTC-amino acids on a reversed-phase
often employed to enhance separation. HPLC with an octadecylsilyl (ODS) column is accomplished
through a combination of changes in concentrations of
2023 Appendix III L V-A265

acetonitrile and buffer ionic strength. PTC-amino acids Pre-column derivatisation of amino acids with OPA is
eluted from the column are monitored at 254 nm. followed by a reversed-phase HPLC separation. Because of
The detection limit is considered to be 1 pmol for most of the instability of the OPA-amino acid derivative, HPLC
the PTC-amino acids. Response linearity is obtained in the separation and analysis are performed immediately following
range of 20-500 pmol with correlation coefficients derivatisation. The liquid chromatograph is equipped with a
exceeding 0.999. To obtain good compositional data, fluorometric detector for the detection of derivatised amino
samples larger than 500 ng of protein/peptide before acids. Fluorescence intensity of OPA-derivatised amino acids
hydrolysis are recommended. is monitored with an excitation wavelength of 348 nm and an
emission wavelength of 450 nm.
METHOD 4 - PRE-COLUMN AQC DERIVITISATION
Pre-column derivatisation of amino acids with Detection limits as low as 50 fmol via fluorescence have been
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reported, although the practical limit of analysis remains at
followed by reversed-phase HPLC separation with 1 pmol.
fluorometric detection is used. METHOD 6 - PRE-COLUMN DABS-CL
AQC reacts with amino acids to form stable, fluorescent DERIVATISATION
unsymmetric urea derivatives (AQC-amino acids) which are Pre-column derivatisation of amino acids with
readily amenable to analysis by reversed-phase HPLC. (dimethylamino )azobenzenesulfonyl chloride (DABS-Cl)
Therefore, pre-column derivatisation of amino acids with followed by reversed-phase HPLC separation with visible
AQC followed by reversed-phase HPLC separation with light detection is used.
fluorimetric detection is used to analyse the amino acid DABS-Cl is a chromophoric reagent employed for the
composition. labelling of amino acids. Amino acids labelled with DABS-Cl
Separation of the AQC-amino acids on a reversed-phase (DABS-amino acids) are highly stable and show an
HPLC with an ODS column is accomplished through a absorption maximum at 436 nm.
combination of changes in concentrations of acetonitrile and DABS-amino acids, all naturally occurring amino acid
buffer ionic strengh. Selective fluorescence detection of the derivatives, can be separated on an ODS column of a
derivatives with an excitation wavelength at 250 nm and an reversed-phase HPLC by employing gradient systems
emission wavelength at 395 nm allows for the direct injection consisting of acetonitrile and aqueous buffer mixture.
of the reaction mixture with no significant interference from Separated DABS-amino acids eluted from the column are
the only major fluorescent reagent by-product, detected at 436 nm in the visible region.
6-aminoquinoline. Excess reagent is rapidly hydrolysed This method can analyse the imino acids such as praline
(t 112 < 15 s) to yield 6-aminoquinoline, N-hydroxysuccinimide together with the amino acids at the same degree of
and carbon dioxide, and after 1 min no further derivatisation sensitivity, DABS-Cl derivatisation method permits the
can take place. simultaneous quantification of tryptophan residues by
Peak areas for AQC-amino acids are essentially unchanged previous hydrolysis of the protein/peptide with sulfonic acids
for at least 1 week at room temperature. Therefore AQC- such as mercaptoethanesulfonic acid, p-toluenesulfonic acid
amino acids have more than sufficient stability to allow for or methanesulfonic acid described in Method 2 under
overnight automated chromatographic analysis. Protein hydrolysis. The other acid-labile residues, asparagine
The detection limit is considered to range from about and glutamine, can also be analysed by previous conversion
40 fmol to 320 fmol for each amino acid, except for cystein. into diaminopropionic acid and diaminobutyric acid,
The detection limit for cystein is approximately 800 finol. respectively, by treatment of protein/peptide with BTI
Response linearity is obtained in the range of 2.5-200 µM described in Method 11 under Protein hydrolysis.
with correlation coefficients exceeding 0.999. Good The non-proteinogenic amino acid norleucine cannot be used
compositional data can be obtained from the analysis of as an internal standard in this method as this compound is
derivatised protein hydrolysates derived from as little as eluted in a chromatographic region crowded with peaks of
30 ng of protein/peptide. primary amino acids. Nitrotyrosine can be used as an internal
METHOD 5 - PRE-COLUMN OPA DERIVATISATION standard because it is eluted in a clean region.
Pre-column derivatisation of amino acids with The detection limit of DABS-amino acid is about 1 pmol.
o-phthalaldehyde (OPA) followed by reversed-phase HPLC As little as 2-5 pmol of an individual DABS-amino acid can
separation with fluorometric detection is used. This be quantitatively analysed with reliability, and only 10-30 ng
technique does not detect amino acids that exist as secondary of the dabsylated protein hydrolysate is required for each
amines (e.g., praline). analysis.
OPA in conjunction with a thiol reagent reacts with primary METHOD 7 - PRE-COLUMN FMOC-CL
amine groups to form highly fluorescent isoindole products. DERIVATISATION
2-Mercaptoethanol or 3-mercaptopropionic acid can be used Pre-column derivatisation of amino acids with
as the thiol. OPA itself does not fluoresce and consequently 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by
produces no interfering peaks. In addition, its solubility and reversed-phase HPLC separation with fluorometric detection
stability in aqueous solution, along with the rapid kinetics for is used.
the reaction, make it amenable to automated derivatisation FMOC-Cl reacts with both primary and secondary amino
and analysis using an autosampler to mix the sample with the acids to form highly fluorescent products. The reaction
reagent. However, lack of reactivity with secondary amino proceeds under mild conditions in aqueous solution and is
acids has been a predominant drawback. This method does completed in 30 s. The derivatives are stable, only the
not detect amino acids that exist as secondary amines (e.g., histidine derivative showing any breakdown. Although
praline). To compensate for this drawback, this technique FMOC-Cl is fluorescent itself, the reagent excess and
may be combined with another technique described in fluorescent side-products can be eliminated without loss of
Method 7 or Method 8. FMOC-amino acids.
V-A266 Appendix III L 2023

FMOC-amino acids are separated by a reversed-phase in which ru is the peak response, in nanomoles, of the amino
HPLC using an ODS column. The separation is carried out acid under test; and r is the sum of peak responses, in
by gradient elution varied linearly from a mixture of nanomoles, for all amino acids present in the test sample.
10 volumes of acetonitrile, 40 volumes of methanol and Comparison of the mole percent of the amino acids under
50 volumes of acetic acid buffer to a mixture of 50 volumes test to data from known proteins can help establish or
of acetonitrile and 50 volumes of acetic acid buffer and corroborate the identity of the sample protein.
20 amino acid derivatives are separated in 20 min. Each Unknown Protein Samples
derivative eluted from the column is monitored by a This data analysis technique can be used to estimate the
fiuorometric detector set at an excitation wavelength of protein concentration of an unknown protein sample using
260 nm and an emission wavelength of 313 nm. the amino acid analysis data. Calculate the mass, in
The detection limit is in the low femtomole range. A linearity micrograms, of each recovered amino acid using the formula:
range of0.1-50 µMis obtained for most of the amino acids.
mM,
METHOD 8 - PRE-COLUMN NBD-F
DERIVATISATION 1000
Pre-column derivatisation of amino acids with 7-fiuoro-4- in which m is the recovered quantity, in nanomoles, of the
nitrobenzo-2-oxa-1,3-diazole (NBD-F) followed by reversed- amino acid under test; and Mr is the average molecular mass
phase HPLC separation with fiuorometric detection is used. for that amino acid, corrected for the mass of the water
NBD-F reacts with both primary and secondary amino acids molecule that was eliminated during peptide bond formation.
to form highly fluorescent products. Amino acids are The sum of the masses of the recovered amino acids will give
derivatised with NBD-F by heating to 60 °C for 5 min. an estimate of the total mass of the protein analysed after
NBD-amino acid derivatives are separated on an ODS appropriate correction for partially and completely destroyed
column of a reversed-phase HPLC by employing a gradient amino acids. If the molecular mass of the unknown protein is
elution system consisting of acetonitrile and aqueous buffer available (i.e., by SDS-PAGE analysis or mass spectroscopy),
mixture, and 17 amino acid derivatives are separated in the amino acid composition of the unknown protein can be
35 min. i:-Aminocaproic acid can be used as an internal predicted. Calculate the number of residues of each amino
standard, because it is eluted in a clean chromatographic acid using the formula:
region. Each derivative eluted from the column is monitored m
by a fiuorometric detector set at an excitation wavelength of
480 nm and an emission wavelength of 530 nm.
The sensitivity of this method is almost the same as for the
pre-column OPA derivatisation method (Method 5), in which m is the recovered quantity, in nanomoles, of the
excluding proline to which OPA is not reactive, and might be amino acid under test; M is the total mass, in micrograms, of
advantageous for NBD-F against OPA. The detection limit the protein; and Mr, is the molecular mass of the unknown
for each amino acid is about 10 fmol. Profile analysis can be protein.
achieved with about 1.5 mg of protein hydrolysates in the
pre-column reaction mixture.
Known protein samples
This data analysis technique can be used to investigate the
DATA CALCULATION AND ANALYSIS amino acid composition and protein concentration of a
When determining the amino acid content of a protein sample of known molecular mass and amino acid
protein/peptide hydrolysate, it should be noted that the acid composition using the amino acid analysis data. When the
hydrolysis step destroys tryptophan and cysteine. Serine and composition of the protein being analysed is known, one can
threonine are partially destroyed by acid hydrolysis, while exploit the fact that some amino acids are recovered well,
isoleucine and valine residues may be only partially cleaved. while other amino acid recoveries may be compromised
Methionine can undergo oxidation during acid hydrolysis, because of complete or partial destruction (e.g., tryptophan,
and some amino acids (e.g., glycine and serine) are common cysteine, threonine, serine, methionine), incomplete bond
contaminants. Application of adequate vacuum (less than cleavage (i.e., for isoleucine and valine) and free amino acid
200 µm of mercury or 26. 7 Pa) or introduction of inert gas contamination (i.e., by glycine and serine).
(argon) in the headspace of the reaction vessel during vapour Because those amino acids that are recovered best represent
phase hydrolysis can reduce the level of oxidative destruction. the protein, these amino acids are chosen to quantify the
Therefore, the quantitative results obtained for cysteine, amount of protein. Well-recovered amino acids are, typically,
tryptophan, threonine, isoleucine, valine, methionine, glycine, aspartate-asparagine, g]utamate-glutamine, alanine, leucine,
and serine from a protein/peptide hydrolysate may be variable phenylalanine, lysine, and arginine. This list can be modified
and may warrant further investigation and consideration. based on experience with one's own analysis system. Divide
Amino Acid Mole Percent the quantity, in nanomoles, of each of the well-recovered
This is the number of specific amino acid residues per amino acids by the expected number of residues for that
100 residues in a protein. This result may be useful for amino acid to obtain the protein content based on each well-
evaluating amino acid analysis data when the molecular mass recovered amino acid. Average the protein content results
of the protein under investigation is unknown. This calculated. The protein content determined for each of the
information can be used to corroborate the identity of a well-recovered amino acids should be evenly distributed
protein/peptide and has other applications. Carefully identify about the mean. Discard protein content values for those
and integrate the peaks obtained as directed for each amino acids that have an unacceptable deviation from the
procedure. Calculate the mole percent for each amino acid mean. Typically greater than 5 per cent variation from the
present in the test sample using the formula: mean is considered unacceptable. Recalculate the mean
protein content from the remaining values to obtain the
IO0ru protein content of the sample. Divide the content of each
r amino acid by the calculated mean protein content to
2023 Appendix III M V-A267

determine the amino acid composition of the sample by - O-glycosylation, which involves the addition of
analysis. oligosaccharides to the hydroxyl groups of serine,
Calculate the relative compositional error, in percentage, threonine, and/or hydroxyproline;
using the formula: - C-glycosylation, which involves the addition of an
a-mannopyranose to the C2-carbon of the indole ring of
IOOm tryptophan.
ms Non-enzymatic additions, also known as glycation, can occur
when proteins are incubated with reducing sugars.
in which m is the experimentally determined quantity, in This chapter describes analytical methods for the N- and
nanomoles per amino acid residue, of the amino acid under O-linked glycosylations, which are the most commonly found
test; and ms is the known residue value for that amino acid. in glycoprotein medicinal products.
The average relative compositional error is the average of the
absolute values of the relative compositional errors of the 1-2 HETEROGENEITY OF THE PROTEIN
individual amino acids, typically excluding tryptophan and GLYCOSYL4TION
cysteine from this calculation. The average relative Different levels of glycan heterogeneity can appear during the
compositional error can provide important information on production of glycoproteins. This heterogeneity may result
the stability of analysis run over time. The agreement in the from variations:
amino acid composition between the protein sample and the - in the degree of occupancy (full, partial, unoccupied);
known composition can be used to corroborate the identity - in the type of glycosylation (N- or O-linked);
and purity of the protein in the sample. - in the oligosaccharide structures (extensions, branching
and linkage).
This heterogeneity in glycosylation results in a set of
glycoforms for one specific glycoprotein. These variations
M. Glycan Analysis of Glycoproteins arise because, unlike transcription and translation,
glycosylation is a non-template post-translational modification
(Ph. Bur. method 2.2.59) process. The glycosylation pattern at a given site depends on
1 INTRODUCTION many factors including the cell-specific and/or growth-
Glycan analysis is a test to analyse glycan moieties of dependent availability of glycosyltransferases and exo-
glycoproteins. It may involve: glycosidases found in the Golgi apparatus and endoplasmic
- whole glycoprotein analysis; reticulum. Protein glycosylation is also influenced by the
- separation and detection of protein glycoforms; protein structure, the production process, the host-vector
- analysis of glycopeptides obtained after enzymatic expression system and the cell culture conditions.
treatment of the glycoprotein; 2 GLYCAN ANALYSIS PROCEDURES
- analysis of released glycans obtained after chemical or Heterogeneity in glycosylation can be assessed by 4 distinct
enzymatic treatment of the glycoprotein. and complementaty approaches:
Monosaccharide analysis may complement information - analysis of the intact glycoprotein;
obtained by glycan analysis. - analysis of glycopeptides;
Glycosylation can play a predominant role in determining the - analysis of released glycans;
function, pharmacokinetics, pharmacodynamics, stability, and - monosaccharide analysis.
imrnunogenicity of biotherapeutics. Glycosylation, unlike The present section provides methods and general
transcription, is a non-template-driven enzymatic requirements used for glycan analysis of glycoproteins
modification process that results in glycan heterogeneity. containing N- and O-linked glycans.
The manufacturing procedure also has an influence on Glycan analysis is usually a multistep process. There are
glycan heterogeneity. Glycoprotein glycan analysis may numerous methodologies for glycan analysis. This variety is a
therefore be an important test to identify variations in the consequence of the diversity and complexity of glycan
glycosylation pattern of the glycoprotein and/or monitor the structures, of the available technologies and detection
consistency of the glycosylation pattern during production. systems, and of the wide range of approaches depending on
Glycan analysis can be a comparative procedure, because the the level of information required.
information obtained, compared to a similarly treated Figure 2.2.59.-1 provides an overview of glycan analysis
reference substance, confirms product consistency. analytical procedures that can be employed to apply the
This chapter provides approaches used for glycoprotein chosen approach(es). Many variations of the same techniques
glycan analysis and requirements for the application of and conditions are available depending on the glycan
methods and validation of methods. structures and origin.
Glycan analysis is not a single general method, but involves Isolation and purification
the application of specific procedures and the development of Isolation and purification may be necessary for analysis of
specific glycan maps for each unique glycoprotein. Specific bulk drug substances or dosage forms containing interfering
procedures are therefore indicated in relevant specific excipients, and, when required, will be described in the
monographs. specific monograph.
1-1 PROTEIN GLYCOSYLATION 2-1 ANALYSIS OF INTACT GLYCOPROTEIN
There are 3 main types of enzymatic glycosylation found in Analysis of the intact glycoprotein provides information on
proteins: the overall pattern of glycosylation of the glycoprotein.
- N-glycosylation, which involves the addition of This approach provides limited information when the
oligosaccharides to the nitrogen on the terminal amide molecule is large and contains multiple glycosylation sites.
group of asparagine;
V-A268 Appendix III M 2023

ANALYSIS OF INTACT GLYCOPROTEIN

MS
IEF
CE
___,. Ion-exchange chromatography
Size-exclusion chromatography
SDS-PAGE

ANALYSIS OF GLYCOPEPTIDES
Direct analysis
I GL YCOPROTEIN I -

!Separation prior to direct analysis! MS

Proteolysis
LCICE I
'
PRETREATMENT
(isolation & purification)
if necessary
___,. I •i
Deglycosylation
I of the digest

.,
ANALYSIS OF RELEASED GLYCANS Analysis of unlabelled glycans
-IHPAEC-PADI
~
• Desialylation~I PG~ MS 5
Release of glycans Analysis of labelled glycans

------
0- and/or N-linked glycans:
enzymes or chemicals 1 Exo-glycosidases
treatment

l
~

Labelling
T MS

( :er
of glycans
Separation
l LCICE
~
MS
LC

ANALYSIS OF MONOSACCHARIDES

HPAEC-PAD
___,. PGC-MS
Acid
hydrolysis ----I Fluorophore
labelling
LC
CE
~

--+I Colorimetric methods I

CE: Capillary electrophoresis LC: Liquid chromatography

HPAEC-PAD: High-pH anion-exchange chromatography with pulsed MS: Mass spectrometry
amperometric detection PGC: Porous graphite chromatography
IEF: Isoelectric focusing SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Figure 2.2.59.-1. - Overview of glycan analysis procedures

Methods such as capillary electrophoresis (CE) (2.2. 47) and reliable correlation between the degree of sialylation and the
mass spectrometry (MS) (2.2.43) can be used. Size-based bioactivity of the product.
techniques, such as size-exclusion chromatography (2.2.30) 2-2 ANALYSIS OF GLYCOPEPTIDES
and sodium dodecyl sulfate polyacrylamide gel Analysis of glycopeptides provides information on site-specific
electrophoresis (SDS-PAGE) (2.2.31), may provide glycosylation properties, on the degree of occupancy, and on
information on the glycosylation status of a protein. If the the oligosaccharide structures. It involves proteolytic
degree of sialylation significantly contributes to the biological digestion of the glycoprotein. Approaches to site-specific
activity of the glycoprotein, ion-exchange chromatography cleavage of the protein backbone are given in general chapter
(2.2.46), isoelectric focusing (IEF) (2.2.54) or CE (2.2.47) 2.2.55. Peptide mapping.
may be performed to monitor sialylation. The technique
must be chosen according to its suitability to provide a
2023 Appendix III M V-A269

After proteolysis of the glycoprotein, the following Table 2.2.59.-1. - Examples of enzymatic cleavage agents
approaches can be chosen.
Agents Specificity
Direct analysis by MS (2.2.43) Care should be taken that
the glycopeptide signal is not suppressed due to the presence N-linked glycans release
of other peptides, where glycopeptides represent a minor Hydrolysis of an N"-(acetyl-P-o-
portion of the total peptide mixture and where signal glucosaminyl)asparagine residue
intensities are lower than those of non-glycosylated peptides. in which the glucosamine
Peptide-N"-(N-acetyl-P- residue may be further
Separation prior to analysis by MS This additional step glucosaminyl)asparagine amidase glycosylated, to yield a
overcomes the problems raised above. Enrichment or (EC 3.5.1.52) (substituted) N-acetyl-P-D•
fractionation techniques can be used either in parallel with or glucosaminylamine and a
sequentially to direct analysis. Separation techniques such as peptide containing an aspartate
residue
liquid chromatography (LC) (2.2.29) and CE (2.2.47) are
suitable. These techniques may be interfaced with MS to Release of N-glycan chain but no
- Peptide N-glycosidase F
allow online MS measurements. release of 1V-glycan chain containing
(PNGase F)
(cxl-3)-linked core fucose
Deglycosylation of the glycopeptides Identification of
the different glycosylation sites of a glycoprotein is made - Peptide N-glycosidase A Release of N-glycan chain
possible by comparing peptide maps obtained by proteolytic (PNGaseA) containing (cxl-3)-linked core fucose
digestion of the intact glycoprotein to those obtained when Endohydrolysis of the N,N' -
the glycoprotein is deglycosylated previously or following diacetylchitobiosyl unit in high-
Mannosyl-glycoprotein endo-P-N-
proteolytic digestion. The peptide mass gives information acetylglucosaminidase (EC
mannose glycopeptides/
about the glycosylation sites and by calculating the mass glycoproteins containing
3.2.1.96)
the -[Man(GlcNAchl
difference between the intact glycopeptide and the
Asn structure
deglycosylated glycopeptide, it is possible to obtain
information about the attached glycans concerning - Endo-P-N-acetylglucosaminidase F Release of high-mannose, hybrid
composition and heterogeneity. Approaches to (endo F) and complex oligosaccharides
deglycosylation of the protein backbone are given in section - Endo-P-N-acetylglucosaminidase H Release of high-mannose, hybrid
2-3-1. A separation step can be performed after or before (endo H) oligosaccharides
deglycosylation.
0-linked glycans release
2-3 ANALYSIS OF RELEASED GLYCANS
Analysis of released glycans provides a convenient way to Glycopeptide cx-N- Hydrolysis of terminal D·
acetylgalactosaminidase (EC galactosyl-N-acetyl-cx-o-
obtain information on the various populations of glycans
3.2.1.97)* galactosaminidic residues
present on the protein (bi-, tri-, and tetra-antennary profile).
The degree of sialylation can also be addressed at this stage. * This enzyme has limited usage because of its high substrate specificity.
Depending on the chosen method, prior
derivatisation/labelling may be needed to allow the detection
Analysis of glycans provides information on the various
of the glycans. populations of glycans present on the protein (high-mannose,
Analysis of released glycans generally involves the release and hybrid, complex). Information on the relative amounts of
purification of glycans from the reaction mixture, followed by branched structures might be obtained by analysis of
the labelling/derivatisation of the glycans, where needed; the desialylated glycans.
glycans are then profiled (fractionation or separation).
A separation step may be required. It implies the use of LC
2-3-1 Release of glycans (2.2.29) and CE (2.2.47) as intermediate techniques.
The selection of the approach used for the release of glycans LC (2.2.29) can be used preparatively with individual
will depend on the glycoprotein under test. The cleavage fractions being collected (usually labelling is required) or can
agent to be employed is chosen according to the type of be directly coupled to MS (2.2.43).
cleavage needed and level of information required. Enzymatic 2-3-2-1 Analysis of unlabelled glycans
or chemical cleavage may be used. Table 2.2.59.-1 gives a
Native glycans can be analysed by high-pH anion-exchange
non-exhaustive list of enzymatic cleavage agents and their
chromatography with pulsed amperometric detection
specificity.
(HPAEC-PAD), porous graphite chromatography (PGC)
Digestion efficiency is generally dependent on the and MS (2.2.43).
accessibility of the glycans on the protein and hence the
HPAEC-PAD has high sensitivity and can also separate some
protein can be denatured to maximise glycosylation site
linkage isomers. Response factors of the different signals are
exposure, unless it is desirable to distinguish between surface
not equal for the different oligosaccharide structures.
and buried glycans.
Absolute quantification of the glycan is not possible unless an
Chemical cleavage agents might also be used, using for oligosaccharide reference library is available. Quantification
example hydrazine or alkaline borohydride for ~-elimination. can be obtained by comparison with a well-characterised
2-3-2 Analysis of glycans reference standard of the substance being tested, or by
Released glycans can be analysed or profiled by relating the peak area of each glycan to the total peak area of
chromatographic, electrophoretic and mass spectrometric all glycans in the map.
techniques, and in general by a combination of these PGC can also be used to separate native glycans because of
techniques. The choice of the method can be grouped its higher selectivity compared to the conventional non-polar
according to the nature of the glycans and level of columns. A PGC-electrospray-ionisation-MS approach can
information required. be applied for direct glycan analysis.
2-3-2-2 Analysis of labelled glycans
Labelling of glycans
V-A270 Appendix III M 2023

The type of derivatisation carried out will depend on the 3 EVALUATION AND ANALYSIS OF DATA
method used to detect glycans: UV or fluorescent. Data obtained from analytical methods for the analysis of
Derivatisation with fluorescent labels is the most commonly glycans can be analysed and evaluated for 3 different
used technique for labelling glycans at their reducing end by purposes:
reductive amination. One label can be attached to every - confirmation of identity of individual structures or
single mono- and oligo-saccharide, allowing the families of structures;
determination of molar quantities. Table 2.2.59 .-2 gives a - confirmation of compliance of the substance being tested
non-exhaustive list of commonly used fluorescent labels and with qualitative requirements;
suitable analytical techniques. - confirmation of compliance of the substance being tested
with quantitative requirements.
Table 2.2.59.-2. - Examples of fluorescent labels and suitable Specific considerations with respect to reference standards
techniques and method development of each level of analysis are set out
Name Acronym Analytical techniques in sections 4 and 5 respectively.
2-Aminobenzoic acid 2-AA LC (2.2.29), MS (2.2.43)
3-1 CONFIRMATION OF IDENTITY OF INDIVIDUAL
STRUCTURES OR FAMILIES OF STRUCTURES
2-Aminobenzamide 2-AB LC (2.2.29), MS (2.2.43) The analytical target for a glycan analysis method may be an
2-Aminopyridine 2-AP LC (2.2.29), MS (2.2.43)
individual monosaccharide (e.g. sialic acid, fucose), a defined
oligosaccharide structure (e.g. tetra-sialylated, tetra-antennary
2-Amino-9(10H)-acridinone AMAC Gel electrophoresis (2.2.23) glycan) or a family of structures sharing a common analytical
feature (e.g. tetra-sialylated glycans, tri-antennary glycans,
Trisodium 8-aminopyrene-
APTS CE (2.2.47) glycoprotein isoforms with the same charge). Confirmation of
1,3,6-trisulfonic acid
the identity of the analytical target is an essential step in the
analysis and evaluation of data, and can be achieved
Permethylation of glycans may also be used when MS absolutely, by verification of molecular structure, or
(2.2.43) is used alone for detection. It is based on the comparatively, by comparison with an appropriate reference
methylation of the oligosaccharides. standard.
Analysis of labelled glycans 3-1-1 Absolute confirmation of identity
Labelled glycans can be analysed by analytical techniques Absolute confirmation of the identity of glycan structures is
such as LC (2.2.29), CE (2.2.47) and MS (2.2.43). typically achieved during product development, and should
According to the separation properties of the glycans, glycans not necessarily be the target of routine analysis. Identity of
can be profiled and quantified by several LC (2.2.29) systems the analytical target will be assigned by reference to a known
using an appropriate label: reversed-phase (separation by molecular property of the molecule. Such absolute
hydrophobicity), normal-phase (separation by size), and identification of individual structures can require multi-step
anion-exchange (separation by charge) LC. approaches using enzymatic and chemical reactions,
2-4 MONOSACCHARIDE ANALYSIS separation techniques and online or offline detection
Monosaccharide analysis provides information on the methods, and will most commonly use the charge-to-mass
monosaccharide composition of a glycoprotein. Analysis of ratio of a molecular ion, determined using a suitable mass
monosaccharides can be performed using either colorimetric spectrometric method as the final basis for structure
or separation methods. assignment.
2-4-1 Colorimetric methods 3-1-2 Comparative confirmation of identity
The colorimetric methods, which are based on chemical During routine application of the analytical method, the
staining, provide information on the quantity of specific identity of the analytical target may be confirmed by
classes of sugars such as sialic acids, neutral sugars and comparison with process or system suitability reference
hexosamines. standards. These may be generated from known, well-
characterised glycoproteins, which may be of the same
2-4-2 Separation methods general class as the product being tested (e.g. fetuin for
The separation methods generate quantitative information on complex N-linked glycoproteins), or may be derived from a
the overall monosaccharide composition. The methods well-characterised batch of the product being tested, which
require acid hydrolysis pre-treatment of the oligosaccharide has been established as a reference standard. The following
chains of the intact glycoprotein or released glycans, prior to considerations apply to comparative assignment of structural
analysis. To release sialic acids, mild acid hydrolysis or identity:
enzymatic treatment is employed. The hydrolysis step is a - in the case of a validated high reproducibility of the
significant source of variability and may require product- retention times, the absolute retention times can be used
specific validation. for correct assignment;
Methods for separation and quantification of - alternatively, a glycan marker can be injected at the
monosaccharides include: beginning and at the end of the testing sequence and
- the use of HPAEC-PAD and PGC-MS, which allow the checked for any drifts in the retention times; based on
determination of molar quantities of native these reference chromatograms the glycans of the test
monosaccharides (sialic acids, neutral sugars and alcohol samples can be assigned;
sugars); - in cases where no standard is available to assign all glycan
- fluorophore labelling of monosaccharides followed by peaks in the test sample, absolute or normalised retention
separation methods such as reversed-phase or ion- times can be used to monitor and label unidentified
exchange chromatography, or CE. glycan peaks.
2023 Appendix III M V-A271

3-2 CONFIRMATION OF COMPLIANCE OF THE 5 POINTS TO CONSIDER IN METHOD

SUBSTANCE BEING TESTED WITH QUALITATIVE DEVELOPMENT
REQUIREMENTS This section provides means for measuring the overall
At this level of evaluation, the analytical results obtained with performance of the method during development. The extent
the product being tested are evaluated to demonstrate of method development and analytical validation is selected
compliance with specifications. Typically this is achieved by on the basis of their suitability for a specific product.
comparison with data obtained in parallel using a reference Depending on the chosen approach, several steps are
standard of the substance being tested. In evaluating the data necessary for glycan analysis, for example:
it is necessary: - isolation and purification (or desalting) of the
- to establish that the analytical result obtained using the glycoprotein;
reference standard is broadly comparable to the expected - enzymatic (or chemical) treatment of the glycoprotein to
result, to verify the suitability of the system; for example, selectively release either N- or O-linked glycans from the
in a glycan mapping procedure, this would be achieved by protein backbone;
comparison of the map obtained with the reference - isolation and purification of the released glycans;
substance with a provided specimen map obtained during - verification of released sialic acid and monosaccharide
establishment of the reference substance, and by ensuring residues;
compliance with all stated system suitability criteria; - chromophore labelling of the released glycans;
- to demonstrate similarity of the maps obtained with the - separation of the glycans, native or fluorescence labelled;
reference substance and the test substance, using any - glycan identification and quantification
specific compliance criteria given in the specific (e.g. determination of the Z number);
monograph. - determination of site occupancy based on relative
3-3 CONFIRMATION OF COMPLIANCE OF THE quantities of glycosylated and non-glycosylated peptides.
SUBSTANCE BEING TESTED WITH QUANTITATIVE Protein isolation and purification
REQUIREMENTS Isolation and purification of the glycoprotein from its matrix
3-3-1 Quantitative measurement of analyte levels and may be necessary to remove all interfering substances
expression of results (e.g. excipients, salts) and, when required, will be specified in
In some cases, e.g. measurement of sialic acid or other the specific monograph. This must be performed in a
monosaccharides, data can be expressed in order to obtain a reproducible manner in order to guarantee a quantitative
molar ratio of sialic acid to glycoprotein. Data is calculated recovery of the protein.
by reference to a reference standard for sialic acid and to a Release and isolation of oligosaccharides
validated method of protein determination. Either the The approach chosen for the release of glycans will depend
internal or external standard method may be used (see on the protein under test and will be based on the types of
general chapter 2.2.46. Chromatographic separation techniques). glycosylation, i.e. N- or O-linked glycosylation. Non-
3-3-2 Quantitative expressions of separation profile compendial approaches available for the release of glycans
Profiles or distribution patterns may be expressed numerically must be optimised in order to ascertain a quantitative
in a number of ways, including the normalisation procedure; profiling of all glycan entities. Factors that impact cleavage
the percentage content of each analytical target, e.g. glycan efficiency, such as enzyme-to-protein concentration ratio,
entity, is calculated by determining the response of the glycan temperature, reaction time course, and denaturation of
entity as a percentage of the total response of all the entities, protein prior to digestion, must be optimised.
excluding those due to solvents or any added reagents, and It is noteworthy that the enzymatic/chemical reaction must
those below the disregard limit. In addition, numerical not alter the glycan composition, e.g. not destroy sialic acid
expressions such as the Z number, which are method- and residues. Where there is more than one glycosylation site, the
product-specific and defined in specific monographs, can be enzymatic treatment should proportionally release all
used. oligosaccharide moieties attached to the protein, independent
4 REFERENCE STANDARDS
of their structure and their individual position in the protein.
Reproducible recovery of all glycan entities from the reaction
Reference standards for glycan analysis serve 2 functions: the
mixture must be confirmed.
verification of the suitability of the system and the
confirmation that the article under test complies with Derivatisation of released glycans
specified requirements. Derivatisation is usually carried out according to non-
The reference standards used for system suitability may be: compendial protocols. Therefore, the reproducible
- a reference substance for the substance being tested; derivatisation of all glycan entities must be verified. This may
- glycan moities liberated from a fully characterised be achieved through optimisation of the reaction conditions
reference standard of the substance being tested; such as amount of the derivatisation reagent, reaction
- well-characterised glycan moities liberated from temperature and time. The derivatisation reaction must not
glycoproteins (e.g. fetuin, IgG); change the glycan composition, e.g. not destroy sialic acid
- glycan markers characterised for identity and purity. residues.
The reference standard used for compliance of the Separation, identification and system suitability
glycoprotein under test is a preparation of the substances The methods employed for glycan analysis must be capable
being tested. It is noted that glycan analysis procedures of detecting and separating different glycan moieties to
described in specific monographs prescribe the use of a ascertain a reliable identification and quantification.
reference standard for the substance being tested and for The acceptance criteria for system suitability, which also
which the glycan analysis procedure has been validated. cover glycan cleavage, recovery and analysis, depend on the
critical test parameters that affect the outcome of the result.
A comparison between the glycan map of the substance
under test and that of a reference substance, being treated in
V-A272 Appendix IV A 2023

Glycan analysis necessary for the glycoprotein

Design glycan analysis methods

Small protein, limited glycosylation '>---I~ Analysis of intact glycoprotein (e.g.

and/or simple structures? mass spectrometric analysis)
Yes ' - - - - - - - . - - - - - - - - - '
No

STOP
Large molecule, complex branched glycans and/or
multiple glycosylation sites

Knowledge of site-specific
glycosylation required?

No
Protease digestion and separation of
glycopeptides
Negative charge content determination No Detailed glycan
Sialic acid and/or monosaccharides 14---< analysis required?
determination
Yes STOP

STOP Design methods for analysis of cleaved glycans (e.g. antennary

profile, identification of individual structure)

Figure 2.2.59.-2. - Guidance on methods to be used when glycan analysis is required

the same conditions, is an indicator to evaluate the

performance of the analytical procedure. In order to further
confirm the obtained results, the analyses may be repeated
Appendix IV
with an orthogonal method. The use of a reference standard A. Clarity of Solution
(e.g. reference substance of the product being examined,
system suitability glycan marker) is essential in the (Ph. Bur. method 2.2.1)
establishment of system suitability parameters and validation Opalescence is the effect of light being absorbed or scattered
of the analytical procedure. by submicroscopic particles or optical density
Reproducibility of quantitative expression (e.g. Z number inhomogeneities. The absence of any particles or
estimation) of glycan profiles must be verified. inhomogeneities in a solution results in a clear solution.
Determination of site occupancy based on relative A liquid is considered clear if its clarity is the same as that of
quantities of glycosylated and non-glycosylated water R or of the solvent used, or if its opalescence is not
peptides more pronounced than that of reference suspension I (see
Where site occupancy is estimated by comparison of Table 2.2.1.-1), when examined under the conditions
glycosylated and non-glycosylated peptides from an described below.
enzymatically digested glycoprotein, reproducible cleavage of Requirements in monographs are expressed in terms of the
both forms of the peptide must be demonstrated. visual method by comparing with the defined reference
suspensions (see Table 2.2.1.-1). However, instrumental
6 GLYCAN ANALYSIS DECISION-MAKING
methods may also be used for determining compliance with
FRAMEWORK
monograph requirements once the suitability of the
This decision-making framework is given for information and
instrument has been established as described below and
does not constitute a mandatory part of the European
calibration with reference suspensions I-N and with water R
Pharmacopoeia.
or the solvent used has been performed.
The choice of procedures used to analyse glycans is
established according to the level of information required to VISUAL METHOD
ensure the quality of the glycoprotein and is set up during Using identical test-tubes of colourless, transparent, neutral
the development phase of the product. glass with a flat base and an internal diameter of 15-25 mm,
Figure 2.2.59.-2 provides guidance in the choice of methods compare the liquid to be examined with a reference
suspension freshly prepared as described below. Ensure that
to be used when glycan analysis is required.
the depths of the layers in the 2 test-tubes are the same
(about 40 mm).
2023 Appendix IV A V-A273

Compare the liquids in diffused daylight 5 min after instrument and compared to the specifications in the
preparation of the reference suspension, viewing vertically individual monograph.
against a black background. Alternatively, the influence of the colour of the sample may
System suitability The diffusion of light must be such also be eliminated by using an infrared light-emitting diode
that reference suspension I can readily be distinguished from (IR LED) having an emission maximum at 860 nm with a
water R, and that reference suspension II can readily be 60 nm spectral bandwidth as the light source of the
distinguished from reference suspension I (see instrument.
Table 2.2.1.-1). INSTRUMENT REQUIREMENTS
INSTRUMENTAL METHOD Instruments complying with the following characteristics and
The instrumental assessment of clarity and opalescence verified using reference suspensions as described below may
provides a more discriminatory test that does not depend on be used instead of visual examination for determination of
the visual acuity of the analyst. Numerical results are more compliance with monograph requirements.
useful for process control and quality monitoring, especially - Measuring unit. NTV (nephelometric turbidity units).
in stability studies. For example, previous numerical data on NTU is based on the turbidity of a primary standard of
stability can be extrapolated to determine whether a given formazin. FTU (formazin turbidity units) or FNU
batch of a preparation will exceed shelf-life limits prior to the (formazin nephelometric units) are also used, and are
expiry date. equivalent to NTU in regions of low turbidity (up to
40 NTU). These units are used in all 3 instrumental
TURBIDIMETRY AND NEPHELOMETRY
methods (nephelometry, turbidimetry and in ratio mode).
When a suspension is viewed at right angles to the direction
- Measuring range: 0.01-1100 NTU.
of the incident light, the system appears opalescent due to
- Resolution: 0.01 NTU within the range 0-9.99 NTU;
the scattering of light by the particles of the suspension
0.1 NTU within the range 10.0-99.9 NTU; and 1 NTU
(Tyndall effect). A certain portion of the light beam entering
for the range> 100 NTU.
a turbid liquid is transmitted, another portion is absorbed
- Accuracy: ± (10 per cent of reading+ 0.01 NTU) within
and the remaining portion is scattered by the suspended
the range 0-20 NTU; ± 7.5 per cent within the range
particles. The light-scattering effect of suspended particles
20-1100 NTU.
can be measured either indirectly by observation of the
- Repeatability: ± 0.05 NTU within the range
transmitted light (turbidimetry) or directly by measuring the
0-20 NTU; ± 2 per cent of the reading within the range
scattered light (nephelometry). Turbidimetry and
20-1100 NTU.
nephelometry are more reliable in low turbidity ranges, where
there is a linear relationship between turbidity values and Instruments with measuring range or resolution, accuracy
detector signals. As the degree of turbidity increases, not all and repeatability capabilities other than those mentioned
the particles are exposed to the incident light and the above may be used provided they are sufficiently validated
scattered or the transmitted radiation of other particles is and are capable for the intended use.
hindered on its way to the detector. CONTROL OF INSTRUMENT PERFORMANCE
For quantitative measurements, the construction of - Calibration: performed with at least 4 reference
calibration curves is essential. Linearity must be based on at suspensions of formazin covering the measuring range of
least 4 levels of concentrations. Reference suspensions must interest. Reference suspensions described in this chapter
show a sufficiently stable degree of turbidity and must be or suitable reference standards calibrated against the
produced under well-defined conditions. primary reference suspensions may be used.
- Stray light: < 0.15 NTU within the range 0-10 NTU;
MEASUREMENTS IN RATIO MODE
The determination of opalescence of coloured liquids is done
< 0 .5 NTU within the range 10-1 100 NTU. Stray light
is defined as that light that reaches the nephelometric
using instruments with ratio mode, since colour provides a
detector without being a result of scatter from the sample.
negative interference, attenuating both incident and scattered
Stray light is always a positive interference and is a
light and lowering the turbidity value. The effect is so great,
significant source of error in low-range turbidity
even for moderately coloured samples, that conventional
measurements. Sources of stray light include:
nephelometers cannot be used.
imperfections in and scratches on sample cells, internal
In turbidimetry or nephelometry with ratio mode, the ratio of reflections of the optical system, contamination of the
the transmission measurement to the 90° scattered light optics or sample cell chamber with dust, and electronic
measurement is determined. This procedure compensates for noise. Instrument design can also affect stray light.
the light that is diminished by the colour of the sample. The influence of stray light becomes negligible in ratio
Instruments with ratio mode use as light source a tungsten mode measurements.
lamp with spectral sensitivity at about 550 nm operating at a
The test methodology for the specific substance/product to
filament colour temperature of 2700 K. Other suitable light
be analysed must also be verified to demonstrate its analytical
sources may also be used. Silicon photodiodes and
capability. The instrument and methodology shall be
photomultipliers are commonly used as detectors and record
consistent with the attributes of the substance to be
changes in light scattered or transmitted by the sample.
examined.
The light scattered at 90 ± 2.5° is measured by the primary
detector. Other detectors measure back and forward scatter Measurements of standards and samples should be carried
(reflected light) as well as transmitted light. The results are out under the same temperature conditions, preferably
obtained by calculating the ratio of the 90° scattered light between 20 °C and 25 °C.
measured to the sum of the components of forward scattered REFERENCE SUSPENSIONS
and transmitted light values. Formazin has several desirable characteristics that make it an
The instruments used are calibrated against standards of excellent turbidity standard. It can be reproducibly prepared
known turbidity and are capable of automatic measurement from assayed raw materials. The physical characteristics make
of turbidity. The test results are obtained directly from the it a desirable light-scatter calibration standard. The formazin
V-A274 Appendix IV B 2023

polymer consists of chains of different lengths, which fold VISUAL METHODS

into random configurations. This results in a wide variety of The examination of the degree of coloration of liquids in the
particle shapes and sizes, which allows the analysis of range brown-yellow-red is carried out using one of the
different particle sizes and shapes that are found in real 2 methods below, as prescribed in the monograph.
samples. Stabilised formazin suspensions that can be used to METHOD I
prepare stable, diluted turbidity standards are commercially Using identical tubes of colourless, transparent, neutral glass
available and may be used after comparison with the with an external diameter of 12 mm, compare 2.0 mL of the
standards prepared as described. liquid to be examined with 2.0 mL of water R, of the solvent
All steps of the preparation of reference suspensions as used for the preparation of the solution to be examined, or of
described below are carried out at 25 ± 3 °C. the reference solution (see Tables of reference solutions)
Hydrazine sulfate solution prescribed in the monograph. Compare the colours in diffuse
Dissolve 1.0 g of hydrazine sulfate R in water R and dilute to daylight, viewing horizontally against a white background.
100.0 mL with the same solvent. Allow to stand for 4-6 h. METHOD II
Primary opalescent suspension (formazin suspension) Using identical tubes of colourless, transparent, neutral glass
In a 100 mL ground-glass-stoppered flask, dissolve 2.5 g of with a flat base and an internal diameter of 15-25 mm,
hexamethylenetetramine R in 25.0 mL of water R. compare the liquid to be examined with water R, with the
Add 25.0 mL of the hydrazine sulfate solution. Mix and solvent used for the preparation of the solution to be
allow to stand for 24 h. This suspension is stable for examined, or with the reference solution (see Tables of
2 months, provided it is stored in a glass container free from reference solutions) prescribed in the monograph, the depth
surface defects. The suspension must not adhere to the glass of the layer being 40 mm. Compare the colours in diffuse
and must be mixed thoroughly before use. daylight, viewing vertically against a white background.
Standard of opalescence PREPARATION OF REFERENCE SOLUTIONS
Dilute 15.0 mL of the primary opalescent suspension to Primary solutions
1000.0 mL with water R. This suspension is freshly prepared Yellow solution Dissolve 46 g of ferric chloride R in about
and may be stored for up to 24 h. 900 mL of a mixture of 25 mL of hydrochloric acid R and
Reference suspensions 975 mL of water R and dilute to 1000.0 mL with the same
Prepare the reference suspensions according to mixture. Titrate and adjust the solution to contain 45.0 mg
Table 2.2.1.-1. Mix and shake before use. of FeC13 ,6H 2 O per millilitre by adding the same acidic
mixture. Protect the solution from light.
Table 2.2.1.-1 Titration. In a 250 mL conical flask fitted with a ground-
II ID IV glass stopper, introduce 10.0 mL of the solution, 15 mL of
Standard of opalescence 5.0 mL 10.0 mL 30.0 mL 50.0 mL water R, 5 mL of hydrochloric acid R and 4 g of potassium
WaterR 95.0 mL 90.0 mL 70.0 mL 50.0 mL iodide R, close the flask, allow to stand in the dark for 15 min
and add 100 mL of water R. Titrate the iodine released with
Measurements of reference suspensions I-IV in ratio mode 0.1 M sodium thiosulfate, using 0.5 mL of starch soluti.on R,
show a linear relationship between the concentrations and added towards the end of the titration, as indicator.
measured NTU values (see Table 2.2.1.-2). 1 mL of 0.1 M sodium thiosulfate is equivalent to 27 .03 mg of
FeC1 3,6H2O,
Table 2.2.1.-2 Red solution Dissolve 60 g of cobalt chloride R in about
F ormazin suspensions Opalescent values (NTU) 900 mL of a mixture of 25 mL of hydrochloric acid Rand
Reference suspension I 3 975 mL of water Rand dilute to 1000.0 mL with the same
Reference suspension II 6 mixture. Titrate and adjust the solution to contain 59.5 mg
Reference suspension III 18 of CoC12,6H 2 O per millilitre by adding the same acidic
Reference suspension IV 30 mixture.
Standard of opalescence 60 Titration. In a 250 mL conical flask fitted with a ground-
Primary opalescent suspension 4000 glass stopper, introduce 5.0 mL of the solution, 5 mL of
dilute hydrogen peroxide solution R and 10 mL of a 300 g/L
solution of sodium hydroxide R. Boil gently for 10 min, allow
to cool and add 60 mL of dilute suljuric acid R and 2 g of
potassium iodide R. Close the flask and dissolve the precipitate
B. Colour of Solution 1 by shaking gently. Titrate the iodine released with 0.1 M
sodium thiosulfate, using 0.5 mL of starch soluti.on R, added
(Ph. Bur. method 2.2.2)
towards the end of the titration, as indicator. The end-point
◊A solution is colourless if it has the appearance of water R or is reached when the solution turns pink.
the solvent used for the preparation of the solution to be 1 mL of 0.1 M sodium thiosulfate is equivalent to 23.79 mg of
examined, or is not more intensely coloured than reference C0Cl 2,6H2O.
solution B9 • Blue primary solution Dissolve 63 g of copper sulfate
Report the results together with the method used (method I, pentahydrate R in about 900 mL of a mixture of 25 mL of
method II or method III). hydrochloric acid Rand 975 mL of water R and dilute to
1000.0 mL with the same mixture. Titrate and adjust the
solution to contain 62.4 mg of CuSO 4,5H2 O per millilitre by
adding the same acidic mixture.
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter Titration. Into a 250 mL conical flask fitted with a ground-
5. 8. Pharmacopoeia/ harmonisation. glass stopper, introduce 10.0 mL of the solution, 50 mL of
2023 Appendix IV B V-A275

water R, 12 mL of dilute acetic acid Rand 3 g of potassium Table 2.2.2.-5. - Reference solutions GY
iodide R. Titrate the iodine released with 0.1 M sodium Volumes in millilitres
thiosulfate, using 0.5 mL of starch solution R, added towards Reference solution Hydrochloric acid
the end of the titration, as indicator. The end-point is Standard solution GY
(10 g/L HCI)
reached when the solution shows a slight pale brown colour. GY1 25.0 75.0
1 mL of 0.1 M sodium thiosulfate is equivalent to 24. 97 mg of GY 2 15.0 85.0
CuS04,SH20. GY 3 8.5 91.5
Standard solutions GY 4 5.0 95.0
Using the 3 primary solutions, prepare the 5 standard GY 5 3.0 97.0
solutions as follows. GY 6 1.5 98.5
GY 7 0.75 99.25
Table 2.2.2.-1.
Volumes in millilitres
Standard solution Table 2.2.2.-6. - Reference solutions R
Yellow Red Blue Hydrochloric
solution solution solution acid (10 g/L HCl) Volumes in millilitres
B (brown) 3.0 3.0 2.4 1.6 Reference solution Hydrochloric acid
Standard solution R
BY (brownish-yellow) 2.4 1.0 0.4 6.2 (10 g/L HCl)
Y (yellow) 2.4 0.6 0.0 7.0 R1 100.0 0.0
GY (greenish-yellow) 9.6 0.2 0.2 0.0 R2 75.0 25.0
R (red) 1.0 2.0 0.0 7.0 R3 50.0 50.0
R. 37.5 62.5
R, 25.0 75.0
Reference solutions for Methods I and II
Ru 12.5 87.5
Using the 5 standard solutions, prepare the following
R, 5.0 95.0
reference solutions.

Table 2.2.2.-2. - Reference solutions B Storage

Volumes in millilitres For Method I, the reference solutions may be stored in
Reference solution Hydrochloric acid sealed tubes of colourless, transparent, neutral glass of
Standard solution B
(10 g/L HCI) 12 mm external diameter, protected from light.
B1 75.0 25.0 For Method II, prepare the reference solutions immediately
B2 50.0 50.0 before use from the standard solutions.◊
B, 37.5 62.5
INSTRUMENTAL METHOD - METHOD III
B4 25.0 75.0
PRINCIPLE
B, 12.5 87.5
The observed colour of an object depends primarily on its
B6 5.0 95.0
light-absorbing characteristics. However, a variety of
B, 2.5 97.5
conditions such as light-source differences, spectral energy of
Bs 1.5 98.5
the illuminant, visual sensitivity of the observer, size
B9 1.0 99.0
differences, background differences and directional
differences affect the perception of colour. Hue, lightness (or
Table 2.2.2.-3. - Reference solutions BY brightness) and saturation are 3 attributes of the colour.
Instrumental measurement under defined conditions allows
Volumes in millilitres
numerical expression of a colour. The base of any
Reference solution Hydrochloric acid
Standard solution BY instrumental measurement of colour is that the human eye
(10 g/L HCl)
has been shown to detect colour via 3 types of receptors.
BY 1 100.0 0.0
Instrumental methods for measurement of colour provide
BY 2 75.0 25.0
more objective data than the subjective viewing of colours by
BY3 50.0 50.0
a small number of individuals. With adequate maintenance
BY4 25.0 75.0
and calibration, instrumental methods can provide accurate,
BY5 12.5 87.5
precise and consistent measurements of colour that do not
BY 6 5.0 95.0
drift with time. Through extensive colour-matching
BY1 2.5 97.5
experiments with human subjects having normal colour
vision, distribution coefficients (weighting factors) have been
Table 2.2.2.-4. - Reference solutions Y established for each wavelength in the visible spectrum,
giving the relative amount of stimulation of each receptor
Volumes in millilitres
type caused by the light of that wavelength.
Reference solution Hydrochloric acid
Standard solution Y The International Commission on Illumination (CIE) has
(10 g/L HCI)
Y, 100.0 0.0
developed models taking into account the light source and
75.0 25.0
the angle at which the observer is looking at the target (field
Y2
Y, 50.0 50.0
of view). In a visual test for coloration of solutions, there are
25.0 75.0
requirements that lead to the use of a 2° angle and diffuse
Y4
Y, 12.5 87.5
daylight (illuminant C). The mean sensitivity of the human
y6 5.0 95.0
eye is represented by the distribution coefficients Xi, Yi and
Zi (Figure 2.2.2.-1).
Y1 2.5 97.5
V-A276 Appendix IV B 2023

200 D x = k LT;x,S,A).
;,

y = k LT;,J;,S;AA
).

150 z =kL T;Jr,S1cf:.Jc

).

k = 100
LS;ji;f:.}.
}.

100 The tristimulus values can be used to calculate the CIE Lab
colour space co-ordinates: L * (lightness or brightness), a* (red-
green) and b*(yellow-blue); these are defined by:

L* = 116/(Y/Y,,) - 16

a*= 500[/(X/Xn) - f(Y/Yn)]

50

b* = 200[/(Y /Yn) - f(Z / Z,,)]

where X,,, Y,, and Zn are the tristimulus values of water R
and

f(X/Xn) = (X/Xn) 113 if X/Xn>(6/29) 3

400 500 600 700
otherwise f(X/Xn) = 84l/108(X/Xn) + 4/29;
Figure 2.2.2.-1. - Mean sensitivity of the human eye represented
by distribution coefficients, CIE 2° Standard Observer f(Y/Yn) = (Y/Yn)' 13 if Y/Yn>(6/29)'
= =
(D distributi.on coefficient; ). wavelength in nanometres)
otherwisef(Y /Yn) = 841/I0S(Y /Yn) + 4/29;
For any colour, the amount of stimulation of each receptor
type is defined by the set of tristimulus values (XYZ).
The relationship between the distribution coefficients and the f(Z/Zn) = (Z/Zn)'i' if Z/Zn > (6/29) 3
tristimulus values (X, Y and Z) is given by the following otherwise/(Z/Zn) = 841/I0S(Z/Zn) + 4/29.
equations, expressed in terms of integrals:

x = k f f;x;S;dA In the spectrophotometric method, transmittance values are

0 obtained at discrete wavelengths throughout the visible
spectrum. These values are then used to calculate the
7J
tristimulus values through the use of weighting factors x;,, y;_,
y =k I f;ji).S;.dA and Zic for a 2° Standard Observer and CIE standard
0 illuminant C (see the current International Commission on
Illumination publication, CIE).
SPECTROPHOTOMETRIC METHOD
Using a suitable spectrophotometer according to the
manufacturer's instructions, determine the transmittance (1)
at least over the range 400-700 nm, at intervals of not greater
than 10 nm. Express the result as a percentage. Calculate the
J
(jJ

k = 100/ y;Sicdic tristimulus values X, Y, and Zand the colour co-ordinates

L*, a* and b*.
0
DETERMINATION OF COLORATION
Calibrate the instrument according to the manufacturer's
k normalising constant characterising the recommendations. Carry out system performance tests prior
stimulation of one receptor type and the used to each measurement or at regular intervals, depending on
illumination;
the use of the apparatus. For this purpose, use certified
relative spectral power distribution of the
illuminant; reference materials within the measurement range.
X;_, ji;. and Z;_ colour matching distribution coefficients for Operate the apparatus according to the manufacturer's
CIE 2° Standard Observer;
instructions and test the sample solution and reference
spectral transmittance T, of the material;
wavelength, in nanometres. solution(s) under the same conditions (e.g. path length of the
cuvette, temperature).
In practical calculations of tristimulus values, the integration For transmittance measurements, use water R as the
is approximated by a summation, as follows: standard, assigning it a transmittance of 100.0 per cent at all
2023 Appendix VA V-A277

wavelengths in the visible spectrum. Then the weighting

factors x;, Yi and z; for CIE standard illuminant C are used
to calculate the tristimulus values corresponding to colour
Appendix V
co-ordinates L* = 100, a*= 0 and b* = 0. A. Determination of Melting Point
Reference measurements can be made using the colour
co-ordinates of water R or freshly prepared pharmacopoeia! Method I
reference solutions, or using the respective colour (Ph. Bur. method 2.2.14)
co-ordinates stored in the instrument manufacturer's The melting point determined by the capillaty method is the
database, provided the latter have been obtained under the
temperature at which the last solid particle of a compact
same testing conditions.
column of a substance in a tube passes into the liquid phase
If the test solution is turbid or hazy, it is filtered or (i.e. clear point). The melting point determined by this
centrifuged. If the test solution is not filtered or centrifuged, method is specific to the methodology (e.g. heating rate)
any haziness or turbidity is reported with the results. described in this chapter. Similarly, whenever the use of
Air bubbles are to be avoided or, where applicable, removed. certified reference materials is required, their certified values
The instrumental method is used to compare 2 solutions in refer to the described analytical procedure.
terms of their colour or colour difference, or a deviation from When prescribed in the monograph, the same apparatus and
a defined colour. Calculate the colour difference (/iE* ") method are used for the determination of other factors, such
between the test solution (t) and a reference solution (r) as meniscus formation or melting range, that characterise the
using the following equation: melting behaviour of a substance.
Equipment The equipment consists of a metal heating
block with 1 or more compartments for capillary tubes, or of
a suitable glass vessel containing a liquid bath (e.g. water,
where !iL*, !ia* and !ib* are the differences in colour
liquid paraffin or silicone oil) and fitted with a suitable means
co-ordinates.
of heating and stirring. The equipment is equipped with a
The CIE LCh colour co-ordinates may be used instead of the temperature sensor or a suitable certified thermometer
CIE Lab colour co-ordinates. allowing readings at least to the nearest 0.1 °C.
Assessment oflocation within the L*a*b* colour space Samples are introduced into the equipment in glass capillary
Instruments may provide information on the actual location tubes. The dimensions are chosen according to the
of the test solution within the L*a*b* colour space. Using manufacturer's requirements, typically with an external
appropriate algorithms, correspondence to pharmacopoeia! diameter of 1.3-1.5 mm and a wall thickness of0.1-0.3 mm.
reference solutions (such as 'test solution equals reference In some equipment glass slides are used instead of capillary
solution XY', 'test solution close to reference solution XY' or tubes.
'test solution between reference solutions XY and XZ') can The equipment is capable of heating samples at a rate of
be obtained. 1 °C/min or less. The accuracy of the equipment is at most
± 0.5 °C.
Detection can be performed either visually or instrumentally.
In the case of instrumental detection, this is generally
performed by image recording and subsequent analysis or by
a photodetector that measures the transmitted or reflected
light from the sample.
Method The substance is previously treated as described in
the monograph. Coarse crystals are to be avoided as they
might lead to false results. If necessary, samples are crushed
into a fine powder. Unless otherwise prescribed, dry the
finely powdered substance in vacuo over anhydrous silica gel R
for 24 h. Introduce a sufficient quantity into a capillary tube
to give a compact column as described by the instrument
manufacturer (e.g. 4-6 mm in height). Raise the temperature
of the apparatus to about 5 °C below the presumed melting
point. Allow the temperature to stabilise and then introduce
the capillary tube into the instrument. Finally, adjust the rate
of heating to about 1 °C/min unless otherwise prescribed.
In the case of instrumental detection, follow the instrument
manufacturer's requirements for the determination of the
melting point. For visual detection, record the temperature at
which the last particle of the substance to be examined passes
into the liquid phase.
Samples can be measured in parallel if the instrument allows
multiple sample processing.
System suitability Carry out a system suitability test
before the measurements for example by choosing a suitable
reference material with a melting point close to that expected
for the substance to be examined.
V-A278 Appendix VA 2023

Qualification I Calibration of the equipment The occurs, which is indicated by the formation of a definite
qualification / calibration is carried out periodically according meniscus or, for substances that decompose, the temperature
to the instrument manufacturer's requirements, using at least at which frothing begins. Correct the observed temperature
2 certified reference materials. These are selected to cover the for any error in the calibration of the thermometer and for
temperature range that is used on the equipment. the difference, if any, between the temperature of the
Use capillary tubes with the same dimensions as those used emergent stem of the thermometer and the temperature of
for sample measurement. the emergent stem under the conditions of standardisation of
Guidance on how to compare results obtained from certified the thermometer. The temperature of the emergent stem is
reference materials with values from the certificates can be determined by placing the bulb of a second thermometer in
found on the European Reference Materials (ERM) website contact with the emergent stem at a point approximately
(Application note 1). midway along the mercury thread in the emergent stem.
The correction to be applied is given by the following
Method II equation:
(No Ph. Bur. method)
Apparatus
(a) A glass heating vessel of suitable construction and
capacity containing one of the following, or another suitable
liquid, to a height of not less than 14 cm.
where tc = correction to be added to the
observed temperature of the
(i) A liquid paraffin of sufficiently high boiling point. melting point,
(ii) A silicone fluid of sufficiently high boiling point. fs = mean temperature of the emergent
(iii) Water. column when standardised,
(b) A suitable stirring device capable of rapidly mixing the
td = mean temperature of the emergent
column at the observed melting
liquid.
point,
(c) An accurately standardised thermometer suitable for the n = number of °C over which the
substance being examined complying with the requirements exposed column extends.
of British Standard 1365:1990 (Specification for short-range
short-stem thermometers) for thermometers designated by The corrected temperature is regarded as the melting point
one of the following Schedule Marks. of the substance. When the melting point in the monograph
is expressed as a range, the melting point of the substance
being tested must fall within that range.
Schedule Range Graduated Diameter Overall
mark of stem length Method III
at each (Ph. Bur. method 2.2.17)
oc mm mm
(max) The drop point is the temperature at which the first drop of
the melting substance to be examined falls from a cup under
defined conditions.
SA 55C/80 -10 to 55 0.50 5.5 to 8 200
SA 105Cl80 45 10 105 o.so 5.5 to 8 200 When a monograph does not specify the method to be used,
SA 155C/80 95 to 155 0.5° 5.5 to 8 200 apply method A. Any change from method A to method B is
SA 205C/80 145 to 205 0.5° 5.5 to 8 200 validated.
SA 225C/80 195 to 255 0.5° 5.5 to 8 200 METHOD A
SA 305C/80 245 to 305 0.50 5.5 to 8 200 Equipment The equipment (see Figure 2.2.17.-1) consists
SA 360C/80 295 to 360 0.5° 5.5 to 8 200 of 2 metal sheaths (A and B) screwed together. Sheath A is
fixed to a thermometer. A metal cup is loosely fixed to the
lower part of sheath B by means of 2 tightening bands. Fixed
(d) Thin-walled capillary glass tubes of hard glass, closed at supports 2 mm long determine the exact position of the cup,
one end, with a wall thickness of 0 .10 to 0 .15 mm, at least and in addition are used to centre the thermometer. A hole
12 cm in length and of internal diameter 0. 9 to 1. 1 mm. pierced in the wall of sheath B is used to balance the
The tubes should preferably be kept sealed at both ends and pressure. The draining surface of the cup must be flat and
cut as required. the edges of the outflow orifice must be at right angles to it.
Method Dry a small quantity of the finely powdered The thermometer has the form and size shown in
substance at a temperature considerably below its melting Figure 2.2.17.-1; it covers a range from 0 °C to 110 °C and
point or at a pressure of 2 kPa over a suitable desiccant, on its scale a distance of 1 mm represents a difference of
unless otherwise directed. Transfer a portion to a dry 1 °C. The thermometer bulb has a diameter of
capillary tube and pack the powder by tapping on a hard 3.5 ± 0.2 mm and a height of 6.0 ± 0.3 mm.
surface so as to form a tightly packed column 4 to 6 mm in The equipment is placed in the axis of a test-tube about
height. Heat a suitable liquid in the heating vessel and 200 mm long and with an external diameter of about
regulate the rate of rise of temperature, prior to the 40 mm. It is fixed to the test-tube by means of a laterally
introduction of the capillary tube, to 3° per minute, unless grooved stopper through which the thermometer passes.
otherwise directed, stirring constantly. When the temperature The opening of the cup is placed about 15 mm from the
reaches 10° below the lowest figure of the range for the bottom of the test-tube. The whole device is immersed in a
substance being tested, adjust the height of the thermometer beaker with a capacity of about 1 L, filled with water.
so that the immersion mark is at the level of the surface of The bottom of the test-tube is placed about 25 mm from the
the liquid and insert the capillary tube so that the closed end bottom of the beaker. The water level reaches the upper part
is near the middle of the bulb of the thermometer. Note the of sheath A. A stirrer is used to ensure that the temperature
temperature at which the liquefaction of the substance of the water remains uniform.
2023 Appendix VA V-A279

r
block at a pre-defined temperature, and of heating at a slow
and steady, pre-defined rate.

tI

~
10
~
12.5

~~

- - - ~
- - - N
-+---- A -----f1 ~

I\
l{)
: I : cci
1- -_,·._-_-_ __ B ---==1r-tt1---wr---
Jt
-- L -
1 1111 II I I i 0)
7
'Sf"
-,--1-----C---- 7.5 ± 0.05
-+----s--1_,_____ D 'Iii
-----c::-:::c-::-ccflll:t:tctclllc-
l{)
0
c:i -
~---,,."-.- 7-~ - -
l{) m E----~
t__-+-,+---=+'._~"+-1-_-_-_-_-_-_-_-__F_ _ _ _--+-1'1---H-+-----'-"+-
N
_i +I
0
~

~LI
~
I ~
- 1-_1_______
5 ---
~
6 + 0.05
3 + 0.05
J

A. upper metal sheath C. pressure-balancing E. tightening bands

A. C. light E. cartridge G. sample cup J. collector
hole
cup holder source assembly sleeve
B. lower metal sheath D. fixed supports F. metal sample cup
B. heating D. light slit F. heating H. photo-sensor K.
block element temperature
Figure 2.2.17.-1. - Equipment for the determination probe
of drop point (dimensions in millimetres)
Figure 2.2.17.-2. - Example of automated drop point equipment
Method Prepare the substance to be examined as described
in the monograph. Fill the cup to the brim with the Method Prepare the substance to be examined as described
substance to be examined. Remove the excess substance at in the monograph, then proceed as follows or according to
both ends of the cup with a spatula. When sheaths A and B the manufacturer's instructions. Remove the excess substance
have been assembled, press the cup into its housing in at both ends of the cup with a spatula. Press the cup into the
sheath B until it touches the supports. Remove with a spatula cup holder, and then press the collector sleeve onto the cup.
the substance pushed out by the thermometer. Place the Place the cartridge assembly in the heating block. Set the
equipment in the water-bath as described above. Heat the instrument to the initial isothermal conditions and the rate
water-bath and, when the temperature is at about 1O °C for subsequent heating as described in the monograph. Start
below the presumed drop point, adjust the heating rate to the temperature programme. When the first drop of molten
about 1 °C/min. Note the temperature at the fall of the first sample falls through the hole at the bottom of the sample
drop. Carry out at least 3 determinations, each time with a cup, thus interrupting the light beam, a signal from the
fresh sample of the substance. The difference between the photo-sensor causes the temperature of the heating block to
readings must not exceed 3 °C. The mean of 3 readings is be recorded automatically.
the drop point of the substance. Calibration Use the equipment according to the
METHOD B - AUTO.MATED METHOD manufacturer's instructions and carry out the prescribed
Equipment The equipment (see Figure 2.2.17.-2) consists calibrations and system performance tests at regular intervals,
of a cartridge assembly comprising a cup holder into which depending on the use of the equipment and the substances to
the sample cup containing the sample is loosely fixed, and a be examined. Benzoic acid and benzophenone are usually
collector sleeve with a horizontal light slit, which is fixed used as certified reference materials. Other materials may be
below the cup. This assembly is placed in a heating block. used provided they show no polymorphism. Proceed as
The block is a metal cylinder with a cylindrical hole along its follows or according to the manufacturer's instructions.
vertical axis into which the cartridge assembly is placed. Prepare 3 sample cups for each of 2 certified reference
There is another, narrower cylindrical vertical hole in which a materials. Place the sample cups on a clean surface. Into
temperature sensor sits. This is positioned level with the each sample cup, introduce a small quantity of the sample
sample cup. The heating block is surrounded by an electrical and press it down with a rod (diameter about 4.5 mm).
heating element. Below the heating block a lamp is mounted Check that the opening is completely blocked. Fill the
such that a beam of light shines through the light slit in the sample cup about half full and compact the sample with a
collector sleeve, and onto a photo-sensor mounted opposite. rod (diameter about 9 mm). Fill the sample cup and
The heating element is capable of maintaining the heating compact, adding more sample and compacting again if
necessary, until the sample cup is completely packed.
V-A280 Appendix V B 2023

Temperature programme for benzoic acid: start Apparatus The apparatus consists of a metal block
temperature = 118.0 °C; heating rate = 0.2 °C/min; end resistant to the substance to be examined, of good heat-
temperature = 126.0 °C. After inserting the cup at 118 °C, a conducting capacity, such as brass, with a carefully polished
waiting time of 30 s is set before heating starts. plane upper surface. The block is uniformly heated
Temperature programme for benzophenone: start throughout its mass by means of a micro-adjustable gas
temperature = 44.0 °C; heating rate = 0.2 °C/min; end heater or an electric heating device with fine adjustment.
temperature = 56.0 °C. After inserting the cup at 44 °C, a The block has a cylindrical cavity, which is wide enough to
waiting time of 30 s is set before heating starts. accomodate a thermometer that is maintained in the same
Check the 3 single results: the test is valid if the 3 results are position during the calibration of the apparatus and the
within 0.3 °C of the mean value. determination of the melting point of the substance to be
examined. The cylindrical cavity is parallel to the upper
Calculate the corrected mean temperature (T2 ) using the polished surface of the block and about 3 mm from it.
following expression: The apparatus is calibrated using appropriate substances of
known melting point.
Method Heat the block at a suitably rapid rate to a
T1 mean drop point temperature of 3 samples, in ''C; temperature about 10 °C below the presumed melting
F compensation for the difference in temperature between the temperature, then adjust the heating rate to about 1 °C/min.
sample and the point in the heating block where the At regular intervals drop a few particles of powdered and,
temperature is measured; this will vary depending upon the
design of the automatic drop point instrument and is provided where appropriate, dried substance, prepared as for the
by the manufacturer. capillary tube method, onto the block in the vicinity of the
thermometer bulb, cleaning the surface after each test.
Taking into account the drop point of the certified reference Record the temperature t 1 at which the substance melts
material (T0 ), the accuracy of the temperature scale is instantaneously for the first time in contact with the metal.
satisfactory if I T2 -T0 I is not greater than 0.3 °C. Stop the heating. During cooling drop a few particles of the
substance at regular intervals on the block, cleaning the
Method IV surface after each test. Record the temperature t2 at which
(Ph. Bur. method 2.2.15) the substance ceases to melt instantaneously when it comes
For certain substances, the following method is used to in contact with the metal.
determine the melting point (also referred to as slip point Calibration of the apparatus The apparatus may be
and rising melting point when determined by this method). calibrated using melting point reference substances such as
Use glass capillary tubes open at both ends, about 80 mm those of the World Health Organization or other appropriate
long, having an external diameter of 1.4 mm to 1.5 mm and substances.
an internal diameter of 1.0 mm to 1.2 mm.
Introduce into each of 5 capillary tubes a sufficient amount
of the substance, previously treated as described, to form in
each tube a column about 10 mm high and allow the tubes B. Determination of Freezing Point
to stand for the appropriate time and at the prescribed
(Ph. Bur. method 2.2.18)
temperature.
Unless otherwise prescribed, substances with a waxy The freezing point is the maximum temperature occurring
consistency are carefully and completely melted on a water- during the solidification of a supercooled liquid.
bath before introduction into the capillary tubes. Allow the Apparatus The apparatus (see Figure 2.2.18.-1) consists
tubes to stand at 2-8 °C for 2 h. of a test-tube about 25 mm in diameter and 150 mm long
Attach one of the tubes to a thermometer graduated in placed inside a test-tube about 40 mm in diameter and
0.5 °C so that the substance is close to the bulb of the 160 mm long. The inner tube is closed by a stopper which
thermometer. Introduce the thermometer with the attached carries a thermometer about 175 mm long and graduated in
tube into a beaker so that the distance between the bottom of 0.2 °C fixed so that the bulb is about 15 mm above the
the beaker and the lower part of the bulb of the thermometer bottom of the tube. The stopper has a hole allowing the
is 1 cm. Fill the beaker with water to a depth of 5 cm. passage of the stem of a stirrer made from a glass rod or
Increase the temperature of the water gradually at a rate of other suitable material formed at one end into a loop of
1 °C/min. about 18 mm overall diameter at right angles to the rod.
The inner tube with its jacket is supported centrally in a 1 L
The temperature at which the substance begins to rise in the
beaker containing a suitable cooling liquid to within 20 mm
capillary tube is regarded as the melting point.
of the top. A thermometer is supported in the cooling bath.
Repeat the operation with the other 4 capillary tubes and
Method Place in the inner tube sufficient quantity of the
calculate the result as the mean of the 5 readings.
liquid or previously melted substance to be examined, to
Method V cover the thermometer bulb and determine the approximate
(Ph. Bur. method 2.2.16) freezing point by cooling rapidly. Place the inner tube in a
The instantaneous melting point is calculated using the bath about 5 °C above the approximate freezing point until
following expression: all but the last traces of crystals are melted. Fill the beaker
with water or a saturated solution of sodium chloride, at a
temperature about 5 °C lower than the expected freezing
point, insert the inner tube into the outer tube, ensuring that
some seed crystals are present, and stir thoroughly until
in which t 1 is the first temperature and t2 the second solidification takes place. Note the highest temperature
temperature read under the conditions stated below. observed during solidification.
2023 Appendix V C V-A281

C. Determination of Distillation Range

(Ph. Bur. method 2.2.11)
The distillation range is the temperature interval, corrected
for a pressure of 101.3 kPa, within which a liquid or a
specified fraction of a liquid, distils under the following
t ~~
t,;
~~ conditions.
Apparatus The apparatus (see Figure 2 .2 .11.-1) consists
I
f-

r -
-
-
-
-
' of a distillation flask (A), a straight tube condenser (B) which
fits on to the side arm of the flask and a plain-bend adaptor
(C) attached to the end of the condenser. The lower end of
- -
the condenser may, alternatively, be bent to replace the
- -
adaptor. A thermometer is inserted in the neck of the flask so
-
that the upper end of the bulb is 5 mm lower than the
junction of the lower wall of the lateral tube.
-
0
The thermometer can be read to the nearest 0.2 °C and
LO
~
- - covers a range of at least 50 °C. During the determination,
the flask, including its neck, is protected from draughts by a
suitable screen.
Method Place in the flask (A) 50.0 mL of the liquid to be
-_ ----

t examined and a few pieces of porous material. Collect the

distillate in a 50 mL cylinder graduated in millilitres. Cooling
~~ by circulating water is essential for liquids distilling below
150 °C. Heat the flask so that boiling is rapidly achieved and
~ \ ..._1. . . ~8 -.1_,/ note the temperature at which the first drop of distillate falls
+I ,.._ 25 --+ into the cylinder. Adjust the heating to give a regular rate of
+- 40 _______.. distillation of 2-3 mIJmin and note the temperature when
the whole or the prescribed fraction of the liquid, measured
at 20 °C, has distilled.
Figure 2.2.18.-1. - Apparatus for the detennination of freezing
point Correct the observed temperatures for barometric pressure by
means of the formula:
Dimensions in millimetres
t1 = t2 + k(IOI.3 - b)
t1 the corrected temperature,

0
......

Figure 2.2.11.-1. - Apparatus for the detennination of distillation range

Dimensions in millimetres
V-A282 Appendix V D 2023

the observed temperature, at the barometric pressure b,

the correction factor taken from Table 2.2.11.-1 unless the F. Determination of Optical Rotation and
factor is given,
the barometric pressure, expressed in kilopascals, during the Specific Optical Rotation
distillation.
(Ph. Bur. method 2.2.7)
PRINCIPLE
Table 2.2.11.-1. - Temperature correction in relati.on to the
Optical rotation (also known as optical activity) is the
pressure
property displayed by chiral substances of rotating the plane
Distillation temperature Correction factor k of polarisation of linearly polarised light.
up to 100 'C 0.30 Optical rotation is considered to be positive (+) for
above 100 "C up to 140 'C 0.34 dextrorotatory substances (i.e. those that rotate the plane of
above 140 "C up to 190 'C 0.38 polarisation in a clockwise direction when viewed in the
above 190 "C up to 240 cc 0.41 direction facing the oncoming light beam) and negative (-)
above 240 °C 0.45 for laevorotatory substances (i.e. anticlockwise rotation).
The angle of optical rotation r.< of a liquid is the angle of
rotation of the plane of polarisation, expressed in degrees ( 0
),

at the wavelength of the D-line of sodium (11, = 589.3 nm)

measured at 20 °C through the liquid when using a path
D. Determination of Boiling Point length of 1.00 dm.
(Ph. Bur. method 2.2.12) The specific optical rotation [r.<]t0 of a substance in solution is
calculated from the angle of optical rotation, as defined
The boiling point is the corrected temperature at which the
above, with reference to a path length of 1.00 dm and a
vapour pressure of a liquid is equal to 101.3 kPa.
concentration of the substance to be examined of 1 g/mL.
Apparatus The apparatus is that used for distillation range The specific optical rotation of a substance in solution is
(2.2.11) with the exception that the thermometer is inserted always expressed with reference to a given solvent and
in the neck of the flask so that the lower end of the bulb is concentration.
level with the lower end of the neck of the distillation flask
As some equipment may not use sodium lamps, the
and that the flask is placed on a plate of isolating material
wavelength of measurement is given as 589 nm instead of
pierced by a hole 35 mm in diameter.
589.3 nm.
Method Place in the flask (A) 20 mL of the liquid to be
In certain cases specified in the monograph, the angle of
examined and a few pieces of porous material. Heat the flask
optical rotation is measured at other temperatures, other
so that boiling is rapidly achieved and record the temperature
wavelengths and/or in cells with a path length other than
at which liquid runs from the side-arm into the condenser.
1.00 dm.
Correct the observed temperature for barometric pressure by
In the conventional system adopted by the Pharmacopoeia,
means of the formula:
the specific optical rotation is expressed by its value
t1 = t2 + k(IOI.3 - b) without units; the actual units, degree millilitres per
decimetre gram [(°)·ml·dm- 1 ·g- 1] are understood.
the corrected temperature,
EQUIPMENT
the observed temperature at barometric pressure b,
the correction factor as shown in Table 2.2.11.-1 under The polarimeter typically consists of:
Distillation Range, - a light source, for example a sodium discharge lamp, a
the barometric pressure, in kilopascals, at the time of the light-emitting diode (LED) or another light source
determination.
capable of providing radiation at the desired wavelength
(589 nm unless otherwise prescribed in the monograph);
if the light source is polychromatic, a means of isolating
the required wavelength is necessary, e.g. an optical filter;
- a polariser and an analyser;
E. Determination of Refractive Index - a sample cell with a path length of 1.00 dm, unless
(Ph. Bur. method 2.2.6) otherwise specified in the monograph;
- a detection system to measure the angle of optical
The refractive index of a medium with reference to air is
rotation, which must be capable of giving readings to at
equal to the ratio of the sine of the angle of incidence of a
least the nearest 0.01 °, unless otherwise specified in the
beam of light in air to the sine of the angle of refraction of
monograph;
the refracted beam in the given medium.
- a temperature control system that indicates the
Unless otherwise prescribed, the refractive index is measured temperature with a readability of 0.1 °C; it may be
at 20 ± 0.5 °C, with reference to the wavelength of the embedded in the polarimeter (e.g. a Peltier system) or be
D-line of sodium CA = 589.3 nm); the symbol is then 0 • nt an external unit (e.g. a cycle-cryostat), and must be able
Refractometers normally determine the critical angle. In such to maintain the temperature of the liquid to within
apparatus the essential part is a prism of known refractive ± 0.5 °C of that prescribed.
index in contact with the liquid to be examined.
EQUIPMENT PERFORMANCE
Calibrate the apparatus using certified reference materials.
The accuracy of the scale is checked near the value to be
When white light is used, the refractometer is provided with measured or over an appropriate range, usually by means of
a compensating system. The apparatus gives readings certified quartz plates. Other certified reference materials may
accurate to at least the third decimal place and is provided also be suitable (e.g. sucrose solutions).
with a means of operation at the temperature prescribed.
The thermometer is graduated at intervals of 0.5 °C or less.
2023 Appendix VG V-A283

Optical rotation measurements may be used to quantify the brass weights in air of density 0.0012 g per mL is given in
amount of an enantiomer or the ratio of enantiomers present the following table. Ordinary deviations in the density of air
in a sample. For that purpose, the linearity must be checked, from the above value, here taken as the mean, do not affect
for example using sucrose solutions. the result of a determination in the significant figures
PROCEDURE prescribed for Pharmacopoeia! substances.
Determine the zero of the polarimeter and the angle of
rotation of the liquid at a wavelength of 589 nm and a Temperature Weight of a
temperature of 20 ± 0.5 °C, unless otherwise prescribed. litre of water
The zero of the polarimeter is determined with the sample oc g
cell closed. 20 997.18
For neat liquids, the zero is determined with an empty 25 996.02
sample cell. 30 994.62
For solutions, the zero is determined with the sample cell
filled with the same solvent as that used for the solution to
be examined and measured at the same temperature. Density
The sample preparation procedure is prescribed in the (No Ph. Bur. method)
monograph. The density, p 20 , of a substance is the ratio of its mass to its
Calculate the specific optical rotation at temperature t and volume at 20°. It is expressed in kg m-3 •
wavelength ). using the following formulae. The density is determined by dividing the weight in air of the
For neat liquids, the density of the liquid is taken into quantity of the liquid being examined that fills a pycnometer
account: at 20° by the weight in air of water required to fill the
pycnometer after making allowance for the thrust of the air.
The density is calculated from the expression

For solutions: 998.2(M1 + A)

P20 = M2 +A
[of= 10000!
le l. C

where weight in air (apparent mass) in grams of the

angle of rotation measured at temperature t and
substance being examined,
wavelength )., in degrees;
weight in air (apparent mass) in grams of
path length of the polarimeter sample cell, in decimetres;
water,
P, density determined at the temperature of measurement t,
A the correction factor for the thrust of the air,
in grams per cubic centimetre; for the purposes of the
0.0012M2
Pharmacopoeia, density is replaced by relative density
998.2 the density of water at 20" in kg m- 3 •
(2.2. 5);
concentration of the solution, in grams per 1itre.
In most cases, the correction for the thrust of the air may be
disregarded.
Relative density
When the limits for optical rotation or specific optical (Ph. Bur. method 2.2.5)
rotation are expressed as the dried substance, the anhydrous The relative density d{; of a substance is the ratio of the mass
substance or the solvent-free substance, the result must be of a certain volume of a substance at temperature t 1 to the
corrected for loss on drying (2.2.32), water content (2.5.12 or mass of an equal volume of water at temperature t2 •
2.5.32) or content of solvent as appropriate. Unless otherwise indicated, the relative density dig is used.
Relative density is also commonly expressed as dl 0 •
Density p20 , defined as the mass of a unit volume of the
substance at 20 °C may also be used, expressed in kilograms
G. Determination of Weight per Millilitre, per cubic metre or grams per cubic centimetre
(1 kg·m- 3 = 10-3 g-cm- 3). These quantities are related by the
Density, Relative Density and Apparent following equations where density is expressed in grams per
Density cubic centimetre:

Weight per millilitre P20 = 0.998203 x dig or dig = 1.00180 x P20

(No Ph. Bur. method)
The weight per millilitre of a liquid is the weight in g of 1 mL p20 = 0.999972 x d24° or di 0 = 1.00003 x p20
of a liquid when weighed in air at 20°, unless otherwise
specified in the monograph.
di 0 = 0.998230 x dfg
The weight per millilitre is determined by dividing the weight
in air, expressed in g, of the quantity of liquid that fills a Relative density or density is measured according to the
pycnometer at the specified temperature by the capacity, number of decimals prescribed in the monograph using a
expressed in mL, of the pycnometer at the same temperature. density bottle (solids or liquids), a hydrostatic balance
The capacity of the pycnometer is ascertained from the (solids), a hydrometer (liquids) or a digital density meter
weight in air, expressed in g, of the quantity of water required with an oscillating transducer (liquids and gases). When the
to fill the pycnometer at that temperature. The weight of a determination is made by weighing, the buoyancy of air is
litre of water at specified temperatures when weighed against disregarded, which may introduce an error of 1 unit in the
V-A284 Appendix V H 2023

3rd decimal place. When using a density meter, the buoyancy Density meters are able to achieve measurements with an
of air has no influence. error of the order of 1 x 10-3 g-cm- 3 to 1 x 10-5 g-cm- 3
Oscillating transducer density meter The apparatus and a repeatability of 1 x 10-4 g-cm- 3 to 1 x 10-6 g-cm- 3 •
consists of: Apparent density
- a U-shaped tube, usually of borosilicate glass, which
(No Ph. Bur. method)
contains the liquid to be examined;
- a magneto-electrical or piezo-electrical excitation system The term 'Apparent density' is used in the monographs for
that causes the tube to oscillate as a cantilever oscillator Dilute Ethanols, Industrial Methylated Spirit and Industrial
at a characteristic frequency depending on the density of Methylated Spirit (Ketone-free). It is defined as weight in air
the liquid to be examined; per unit volume and expressed in kg m-3 . It is named
- a means of measuring the oscillation period ('J), which 'density' in the Laboratory Alcohol Table for Laboratory Use
may be converted by the apparatus to give a direct (HM Customs and Excise 1979).
reading of density, or used to calculate density using the The apparent density is calculated from the following
constants A and B described below. expression:
The resonant frequency (f) is a function of the spring apparent density = 997 .2 x dig
constant (c) and the mass (m) of the system: where dig is the relative density of the substance being
examined and 997 .2 is the weight in air in kg of 1 cubic
f2 = _2_ = ~ X _1_ metre of water.
T2 m 4n2

Hence:

T-(M pxV) 4
- -+--
2
C C
X 1!
2
H. Viscosity
(Ph. Bur. method 2.2.8)
M mass of the tube; The dynami.c viscosity or viscosity coefficient 11 is the tangential
V inner volume of the tube. force per unit surface, known as shearing stress 1: and
expressed in pascals, necessary to move, parallel to the sliding
Introduction of 2 constants A= c/(4n 2 x V) and B = M/V, plane, a layer of liquid of 1 square metre at a rate (v) of 1
leads to the classical equation for the oscillating transducer: metre per second relative to a parallel layer at a distance (x)
of 1 metre.
p =Ax T 2 -B The ratio dv/dx is a speed gradient giving the rate of shear D
expressed in reciprocal seconds (s- 1 ), so that 11 = r/D.
The constants A and B are determined by operating the
instrument with the U-tube filled with 2 different samples of The unit of dynamic viscosity is the pascal second (Pa-s).
known density, for example, degassed water Rand air. The most commonly used submultiple is the millipascal
Control measurements are made daily using degassed second (mPa·s).
water R. The results displayed for the control measurement The kinemati.c viscosity v, expressed in square metres per
using degassed water R shall not deviate from the reference second, is obtained by dividing the dynamic viscosity 11 by
value (p 20 = 0.998203 g·cm-3, dig = 1.000000) by more the density p expressed in kilograms per cubic metre, of the
than its specified error. For example, an instrument specified liquid measured at the same temperature, i.e. v = 11/p.
to± 0.0001 g-cm-3 shall display 0.9982 ± 0.0001 g-cm-3 in The kinematic viscosity is usually expressed in square
order to be suitable for further measurement. Otherwise a millimetres per second.
re-adjustment is necessary. Calibration with certified A capillary viscometer may be used for determining the
reference materials is carried out regularly. Measurements are viscosity of Newtonian liquids and a rotating viscometer for
made using the same procedure as for calibration. The liquid determining the viscosity of Newtonian and non-Newtonian
to be examined is equilibrated in a thermostat at 20 °C liquids. Other viscometers may be used provided that the
before introduction into the tube, if necessary, to avoid the accuracy and precision are at least as satisfactory as those
formation of bubbles and to reduce the time required for obtained with the viscometers described in the related
measurement. chapters.
Factors affecting accuracy include:
Method I
- temperature uniformity throughout the tube;
(No Ph. Bur. method)
- non-linearity over a range of density;
- parasitic resonant effects; Apparatus
- viscosity, whereby solutions with a higher viscosity than The apparatus consists of a glass U-tube viscometer (Fig.SH-
the calibrant have a density that is apparently higher than I) made of clear borosilicate glass and constructed in
the true value. accordance with the dimensions shown in the figure and in
The effects of non-linearity and viscosity may be avoided by Table SH-1. The monograph states the size of viscometer to
using calibrants that have density and viscosity close to those be used.
of the liquid to be examined ( ± 5 per cent for Method
density, ± 50 per cent for viscosity). The density meter may Fill the viscometer with the liquid being examined through
have functions for automatic viscosity correction and for tube L to slightly above the mark G, using a long pipette to
correction of errors arising from temperature changes and minimise wetting the tube above the mark. Place the tube
non-linearity. vertically in a water bath and when it has attained the
Precision is a function of the repeatability and stability of the specified temperature, adjust the volume of the liquid so that
oscillator frequency, which is dependent on the stability of the bottom of the meniscus settles at the mark G. Adjust the
the volume, mass and spring constant of the cell. liquid to a point about 5 mm above the mark E. After
2023 Appendix V H V-A285

Table 5H-l U-Tube Viscometer-Dimensions

Size Nominal Kinematic Inside Outside Volume of Vertical Outside

viscometer viscosity diameter diameter of bulbC distance diameter of
constant range ofrubeR rubes 1 FtoG bulbs A and C

LandP N
mm2s-2 mm2s-l mm(±2%) mm(±5%) mm mm
mm mm
----
A2 0.003 0.9 to 3 0.50 8 to 9 6 to 7 5.0 91±4 21 to 23
B 0.01 2.0 to 10 0.71 8 to 9 6 to 7 5.0 87±4 21 to 23
C 0.03 6 to 30 0.88 8 to 9 6 to 7 5.0 83±4 21 to 23
D 0.1 20 to 100 1.40 9 to 10 7 to 8 10.0 78±4 25 to 27
E 0.3 60 to 300 2.00 9 to 10 7 to8 10.0 73±4 25 to 27
F 1.0 200 to 1000 2.50 9 to 10 7 to8 10.0 70±4 25to 27
G 3.0 600.to 3000 4.00 10 to 11 9 to 10 20.0 60±3 32 to 35
H 10.0 2000 to 10,000 6.10 10 to 11 9 to 10 20.0 50±3 32 to 35
1Use 1 to 1.25 mm wall tubing for L, N and P.

2300 s minimum flow time; 200 s minimum flow time for all other sizes.

where time in seconds for the meniscus to fall from E

to F,
p mass/volume (g cm- 3) obtained by multiplying
the relaiive density, Appendix V G, of the fluid
being examined by 0.9982.
N 75
The constant (K) of the instrument is determined using the
appropriate European Pharmacopoeia reference liquid for
viscometers.

L Method II (Capillary Viscometer Method)

C (Ph. Bur. method 2.2.9)
PRINCIPLE
F The determination of viscosity is carried out using a
25 300
suspended-level (Ubbelohde-type) capillary viscometer of
appropriate size at a temperature of 20.0 ± 0.1 °C, unless
otherwise prescribed. The time required for the level of the
G liquid to drop from one mark to the other is measured.
EQUIPMENT
R 120
The principal components of an Ubbelohde-type capillary
A
viscometer 1. are shown in Figure 2.2. 9 .-1.
PROCEDURE
Select a capillary viscometer of appropriate size to obtain a
minimum flow time of 200 s.
Calibration
Capillary viscometers are calibrated at regular intervals as
defined in the quality management system and dictated by
Fig. SH-1 the frequency of use of the equipment and the application.
U-Tube Viscometer
Calibrate the instrument at the temperature used for the
measurement by using at least 2 certified reference materials
releasing pressure or suction, measure the time taken for the matching the viscosity range of the viscometer.
bottom of the meniscus to fall from the top edge of mark E Calculate the viscometer constant (k) in square millimetres
to the top edge of mark F. per second squared, using the following expression:
Calculate, as required, either the kinematic viscosity (v) in
square millimetres per second (mm2 s- 1) from the expression:

V = Kt, dynamic viscosity of the certified reference material, in millipascal

seconds;
or the dynamic viscosity (i-/) in millipascal seconds (mPa s) p density of the certified reference material, in milligrams per cubic
from the expression: millimetre;
flow time for the certified reference material to drop from the upper
mark to the lower mark, in seconds.
1J = Kpt,

1 The European Phamiacopoeia describes the system proposed by the

International Organization for Standardization (ISO)

V-A286 Appendix V H 2023

L M N 17 = kpt

p density of the liquid to be examined at the temperarure used for

the viscosity measurement, in milligrams per cubic millimetre.

The density may be obtained by multiplying the relative

density of the liquid to be examined by 0.99820
(measurement at 20 °C) or 0.99704 (measurement at
25 °C).
Method III (Rotating Viscometer Method)
(Ph. Bur. method 2.2.10)
The principle of the method is to measure the force acting
on a rotor (torque) when it rotates at a constant angular
velocity (rotational speed) in a liquid. Rotating viscometers
are used for measuring the viscosity of Newtonian (shear-
independent viscosity) or non-Newtonian liquids (shear
dependent viscosity or apparent viscosity). Rotating
viscometers can be divided in 2 groups, namely absolute and
relative viscometers. In absolute viscometers the flow in the
measuring geometry is well defined. The measurements result
in absolute viscosity values, which can be compared with any
other absolute values. In relative viscometers the flow in the
measuring geometry is not defined. The measurements result
in relative viscosity values, which cannot be compared with
absolute values or other relative values if not determined by
the same relative viscometer method.
Different measuring systems are available for given viscosity
ranges as well as several rotational speeds.
APPARATUS
The following types of instruments are most common.
CONCENTRIC CYLINDER VISCOMETERS
(ABSOLUTE VISCOMETERS)
Figure 2.2. 9 .-1. - Suspended-level (Ubbelohde-type) capillary In the concentric cylinder viscometer (coaxial double cylinder
viscometer viscometer or simply coaxial cylinder viscometer), the
viscosity is determined by placing the liquid in the gap
Calculate the mean of the values obtained.
between the inner cylinder and the outer cylinder. Viscosity
Method measurement can be performed by rotating the inner cylinder
Charge the viscometer (Figure 2.2.9.-1) through tube L with (Searle type viscometer) or the outer cylinder (Couette type
a sufficient quantity of the liquid to be examined (previously viscometer), as shown in Figures 2.2.10.-1 and 2.2.10.-2,
brought to 20 °C unless otherwise prescribed) to fill bulb A respectively. For laminar flow, the viscosity (or apparent
while ensuring that the level of liquid in bulb B is below the viscosity) T] expressed in pascal-seconds is given by the
exit to ventilation tube M. Immerse the viscometer in the following formula:
upright position in a water-bath at 20.0 ± 0.1 °C (unless
otherwise prescribed) and allow to stand for not less than
30 min to allow the temperature to reach equilibrium. Close
tube M and draw the level of the liquid in tube N up to a
level about 8 mm above mark E. Keep the liquid at this level M torque in newton-metres acting on the cylinder surface,
by closing tube N and opening tube M. Open tube N and, m angular velocity in radians per second,
using a stopwatch, measure the time required, to at least the h height of immersion in metres of the inner cylinder in the liquid
medium,
nearest 1/5 of a second, for the level of the liquid to drop R1 radius in metres of the inner cylinder,
from mark E to mark F. R0 radius in metres of the outer cylinder,
The flow time of the liquid to be examined is the mean of k constant of the apparatus, expressed in radians per cubic metre.

3 consecutive measurements. The result is valid if the relative

standard deviation of the 3 measurements is not more than For non-Newtonian liquids it is indispensable to specify the
2.0 per cent. shear stress (,) or the shear rate (y) at which the viscosity is
measured. Under narrow gap conditions (conditions satisfied
Calculation in absolute viscometers), there is a proportional relationship
Calculate the kinematic viscosity (v) (2.2.8), in square between M and , and also between w and y:
millimetres per second, using the following expression:
,=AM r=Bw
V = kt
k viscometer constant, in square millimetres per second squared;
flow time of the liquid to be examined, in seconds.
where A and B are constants for the instrument and are
calculated from the following expressions:
Calculate the dynamic viscosity (17) (2.2.8), in millipascal - for concentric surface:
seconds, using the following expression:
2023 Appendix V H V-A287

2 2 CONE-PLATE VISCOMETERS (ABSOLUTE

I R +R
A=--i_ _O
VISCOMETERS)
4nh R~ R~ In the cone-plate viscometer, the liquid is introduced into the
gap between a flat disc and a cone forming a define angle.
- for cone-plates: Viscosity measurement can be performed by rotating the
cone or the flat disc, as shown in Figures 2.2.10.-3
I and 2.2.10.-4, respectively. For laminar flow, the viscosity (or
A=-3- B=-
2nR3 a apparent viscosity) T] expressed in pascal-seconds is given by
the following formula:

M torque in Newton-metres acting on the cone or cylinder surface,

(J) angular velocity in radians per second,
R; radius in metres of the inner cylinder,
Ro radius in metres of the outer cylinder,
R radius in metres of the cone, M torque in Newton-metres acting on the flat disc or cone surface,
h height of immersion in metres of the inner cylinder in the liquid (l) angular velocity in radians per second,
medium, a angle in radians between the flat disc and the cone,
angle in radians between the flat disk and the cone, R radius in metres of the cone,
shear stress in pascals (Pa), k constant of the apparatus, expressed in radians per cubic metre.
shear rate in reciprocal seconds (s- 1 ).
Constants A, B of the apparatus (see under concentric
cylinder viscometers).
co
co

M
M

O'.

Figure 2.2.10.-3

Figure 2.2.10.-1 M

Figure 2.2.10.-4

SPINDLE VISCOMETERS (RELATIVE

VISCOMETERS)
h In the spindle viscometer, the viscosity is determined by
rotating a spindle (for example, cylinder- or disc-shaped, as
shown in Figures 2.2.10.-5 and 2.2.10.-6, respectively)
immersed in the liquid. Relative values of viscosity (or
apparent viscosity) can be directly calculated using
conversion factors from the scale reading at a given rotational
speed.

Figure 2.2.10.-2
V-A288 Appendix V H 2023

which the measurement is made. If it is impossible to obtain

the indicated shear rate exactly, use a shear rate slightly
higher and a shear rate slightly lower and interpolate.
With relative viscometers the shear rate is not the same
throughout the sample and therefore it cannot be defined.
Under these conditions, the viscosity of non-Newtonian
liquids determined from the previous formula has a relative
character, which depends on the type of spindle and the
angular velocity as well as the dimensions of the sample
container (0 = minimum 80 mm) and the depth of
immersion of the spindle. The values obtained are
comparable only if the method is carried out under
experimental conditions that are rigorously the same.
Method IV (Falling Ball and Automatic Rolling Ball
Viscometer Methods)
(Ph. Bur. method 2.2.49)
The determination of dynamic viscosity of Newtonian liquids
using a falling ball or rolling ball viscometer is performed at
20.0 ± 0.1 °C, unless otherwise prescribed in the
monograph. The time required for a test ball to fall or roll in
the liquid to be examined from one ring mark or sensor to
the other is determined.
Figure 2.2.10.-5 METHOD A - FALLING BALL VISCOMETER
Apparatus The falling ball viscometer consists of: a glass
tube enclosed in a mantle, which allows precise control of
temperature; 6 balls made of glass, nickel-iron or steel with
different densities and diameters. The tube is fixed in such a
way that the axis is inclined by 10 ± 1° with regard to the
vertical. The tube has 2 ring marks that define the distance
the ball has to fall. Commercially available apparatuses are
supplied with tables giving the constants, the density of the
balls and the suitability of the different balls for the expected
viscosity range.
Method Fill the clean, dry tube of the viscometer,
previously brought to 20.0 ± 0.1 °C, with the liquid to be
examined, avoiding bubbles. Choose the ball that is suitable
for the viscosity range of the liquid so as to obtain a falling
time (run time) not less than 30 s. Place it in the tube, close
the tube and maintain the solution at 20.0 ± 0.1 °C for at
least 15 min. Let tl1e ball run through the liquid between the
2 ring marks once without measurement. Let it run again
and measure with a stop-watch, to at least the nearest 1/5 s,
the time required for the ball to fall from the upper to the
lower ring mark. Repeat the test run at least 3 times to
obtain a minimum of 4 run times. The result is only valid if
the relative standard deviation determined on the run times is
not greater than 2.0 per cent .
Use the mean of the run times to calculate the dynamic
viscosity (I/) in millipascal seconds using the formula:
Figure 2.2.10.-6
1/ = k(P1 - P2) X t
In a general way, the constant k of the apparatus may be
k constant, in square millimetres per second squared;
determined at various speeds of rotation using a certified
Pi density of the ball used, in grams per cubic centimetre;
viscometer calibration liquid. The viscosity ri then
corresponds to the formula: density of the liquid to be examined, in grams per cubic
centimetre, obtained by multiplying its relative density (d;g) by
M 0.9982;
1J=k-
w
mean of the run times of the ball, in seconds.
METHOD
Measure the viscosity (or apparent viscosity) according to the METHOD B - AUTOMATIC ROLLING BALL
instructions for the operation of the rotating viscometer. VISCOMETER
The temperature for measuring the viscosity is indicated in Apparatus The automatic rolling ball viscometer consists
the monograph. For non-Newtonian systems, the monograph of: several capillaries made of glass or other suitable materials
indicates the type of viscometer to be used and if absolute with different diameters enclosed in a thermostatically
viscometers are used the angular velocity or the shear rate at controlled block, which allows precise control of the
 2023 Appendix VJ V-A289

temperature; balls made of stainless steel (optionally coated) Dissymmetry factor:

or other suitable materials; a motor drive that positions the
capillary at an inclination angle from 10.0 ± 0.2c to 11E
g=-
80.0 ± 0.2° with regard to the vertical. The apparatus has at E
least 2 sensors to measure the run time of the ball over a
molar absorptivity (2. 2. 25).
predefined distance. Commercially available apparatuses are
supplied with tables giving the constants, the density of the Molar ellipticity:
balls and the suitability of the different capillaries for the
expected viscosity range. Temperature control, control of the Certain types of instruments display directly the value of
inclination angle, run-time determination, repetitions of runs ellipticity 0, expressed in degrees. When such instruments
and calculations of the mean and relative standard deviation are used, the molar ellipticity [0] may be calculated using
are performed by the apparatus software. the following equation:
Method Set the instrument software to perform a
measurement with at least 4 run times (2 forward/backward [0 ] __0_x_M_
cxlxlO
cycles) and a maximum relative standard deviation of
0.5 per cent. Choose the capillary, the ball and the [0] molar ellipticity, expressed in degrees-cm 2 -decimole- 1,
0 value of ellipticity given by the instrument,
inclination angle that are suitable for the expected viscosity
M relative molecular mass of the substance to be examined,
range of the liquid to be examined so as to obtain a rolling concentration of the solution to be examined in g/mL,
time (run time) not less than 20 s over the 100 mm distance optical path of the cell in centimetres.
or a proportional time over other distances. If no digital
density meter is connected to the viscometer, ensure that the Molar ellipticity is also related to molar circular dichroism by
density value of the liquid to be examined has been specified the following equation:
in the apparatus software. Fill the clean, dry capillary of the
viscometer with the liquid to be examined, avoiding bubbles. 4500
[0] = 2.30311£- ::::,3300.1£
Start the measurement immediately after filling the capillary. n
The instrument calculates automatically the dynamic
Molar ellipticity is often used in the analysis of proteins and
viscosity (I]) in millipascal seconds and the kinematic viscosity
nucleic acids. In this case, molar concentration is expressed
(v) in square millimetres per second.
in terms of monomeric residue, calculated using the
Additional points for monographs of the British expression:
Pharmacopoeia
The apparatus and methods described in Methods I and II molecular mass
are in agreement in all essentials with International Standards number of amino acids
ISO 3104-1994 and 3105-1994 (Methods for the
The mean relative molecular mass of the monomeric residue
determination of the viscosity ofliquids).
is 100 to 120 (generally 115) for proteins and about 330 for
nucleic acids (as the sodium salt).
Apparatus
The light source (S) is a xenon lamp (Figure 2.2.41.-1); the
J. Circular Dichroism light passes through a double monochromator (M) equipped
(Ph. Bur. method 2.2.41) with quartz prisms (Pl, P2).
The difference in absorbance of optically active substances The linear beam from the first monochromator is split into
within an absorption band for left and right circularly 2 components polarised at right angles in the second
polarised light is referred to as circular dichroism. monochromator. The exit slit of the monochromator
Direct measurement gives a mean algebraic value: eliminates the extraordinary beam.
The polarised and monochromatic light passes through a
birefringent modulator (Cr): the result is alternating circularly
polarised light.
M circular dichroic absorbance,
AL absorbance of left circularly polarised light,
The beam then passes through the sample to be examined
AR absorbance of right circularly polarised light. (C) and reaches a photomultiplier (PM) followed by an
amplifier circuit which produces 2 electrical signals: one is a
Circular dichroism is calculated using the equation: direct current Ve and the other is an alternating current at
the modulation frequency Vac characteristic of the sample to
be examined. The phase gives the sign of the circular
dichroism. The ratio VacNc is proportional to the differential
absorption M which created the signal. The region of
~, molar circular dichroism or molar differential dichroic wavelengths normally covered by a dichrograph is 170 nm to
absorptivity expressed in litre-mole - 1 -cm- i,
f:1. molar absorptivity (2.2.25) of left circularly polarised light,
800 nm.
"R molar absorptivity of right circularly polarised light, Calibration of the apparatus
concentration of the test solution in mole-litre 1,
Accuracy of absorbance scale Dissolve 10.0 mg of
optical path of the cell in centimetres.
isoandrosterone R in dioxan Rand dilute to 10.0 mL with the
The following units may also be used to characterise circular same solvent. Record the circular dichroism spectrum of the
dichroism: solution between 280 nm and 360 nm. Measured at the
maximum at 304 nm, 11£ is + 3.3.
The solution of (JS)-(+)-10-camphorsulfonic acid R may also
be used.
V-A290 Appendix V K 2023

Figure 2.2.41.-1. - Opti.cal scheme of a dichrograph

Linearity of modulation Dissolve 10.0 mg of (JS)-(+)-

10-camphorsulfoni.c acid R in water Rand dilute to 10.0 mL
L. Determination of pH Values
with the same solvent. Determine the exact concentration of (Ph. Bur. method 2.2.3)
camphorsulfonic acid in the solution by ultraviolet The pH of an aqueous solution is defined as the negative
spectrophotometry (2.2.25), taking the specific absorbance to logarithm of the activity of its hydrogen ions, expressed
be 1.49 at 285 nm. conventionally as the hydrogen ion concentration of the
Record the circular dichroism spectrum between 185 nm and solution. For practical purposes, its definition is an
340 nm. Measured at the maximum at 290.5 nm, ~e is experimental one. The pH of a solution to be examined is
+ 2.2 to + 2.5. Measured at the maximum at 192.5 nm, h.e related to that of a reference solution (pH5 ) by the following
is -4.3 to -5. equation:
(lS)-(+)- Or antipodal (JR)-(-)-ammonium
10-camphorsulfonate R can also be used. B-B,
pH=pH,--k-

in which B is the potential, expressed in volts, of the cell

containing the solution to be examined and B, is the
K. Approximate pH of Solutions potential, expressed in volts, of the cell containing the
(Ph. Bur. method 2.2.4) solution of known pH (pH,), k is the change in potential
Determine the approximate pH using a pH indicator strip R. per unit change in pH, expressed in volts and calculated
Alternatively, pH indicators such as those described in from the Nemst equation.
Table 2.2.4.-1 can be used. Table 2.2.3.-1. - Values of k at different temperatures
Table 2.2.4.-1 Temperature CC) k (V)

Reaction pH Indicator 15 0.0572

20 0.0582
Alkaline > 8 Red litmus paper R
25 0.0592
Slightly alkaline 8- 10 Phenolphthalein solution R
30 0.0601
Thymol blue solution R
35 0.0611
Strongly alkaline > 10 Phenolphthalein paper R
Thymol blue solution R
The potentiometric determination of pH is made by
Neutral 6-8 Methyl red solution R
measuring the potential difference between 2 appropriate
Phenol red solution R
electrodes immersed in the solution to be examined; 1 of
Acid < 6 Methyl red solution R these electrodes is sensitive to hydrogen ions (usually a glass
Bromothymol blue solution R 1 electrode) and the other is the reference electrode
Slightly acid 4-6 Methyl red solucion R (e.g. a silver-silver chloride electrode). They are often
Bromocresol green solution R combined as 1 compact electrode, together with a
Strongly acid <4 Congo red paper R temperature probe.
Apparatus The measuring apparatus is usually a voltmeter
with an input resistance at least 100 times that of the
electrodes used. It is normally graduated in pH units and has
a sensitivity such that discrimination of at least 0.05 pH unit
or at least 0.003 V may be achieved.
2023 Appendix V L V-A291

Table 2.2.3.-2. - pH of reference buffer solutions at various temperatures

Temperature Potassium Potassium Potassium Potassium Potassium Potassium Disoclium Sodium Calcium
CC) tetraoxalate hydrogen dihydrogen hydrogen dihydrogen clihydrogen tetraborate carbonate hydroxide,
0.05 M tartrate citrate phthalate phosphate phosphate 0.01 M 0.025 M+ saturated
saturated at 0.05 M 0.05M 0.025 M+ 0.0087 M+ Sodium at 25'C
25 'C Disoclium Disoclium bicarbonate
hydrogen hydrogen 0.025 M
phosphate phosphate
0.025 M 0.0303 M
C4H 3 KO 8 , C4H 5 KO 6 C6 H1K01 C8H 5KO 4 KH2PO4+ KH 2PO4+ Na2B 4O 7 , Na2CO 3 + Ca(OH)z
2H2O Na2HPO 4 Na2HPO4 10H2O NaHCO 3
15 1.67 3.80 4.00 6.90 7.45 9.28 10.12 12.81
20 1.68 3.79 4.00 6.88 7.43 9.23 10.06 12.63
25 1.68 3.56 3.78 4.01 6.87 7.41 9.18 10.01 12.45
30 1.68 3.55 3.77 4.02 6.85 7.40 9.14 9.97 12.29
35 1.69 3.55 3.76 4.02 6.84 7.39 9.10 9.93 12.13

!J.pHrn -0.034
+ 0.001 -0.0014 -0.0022 + 0.0012 -0.0028 -0.0028 -0.0082 -0.0096
t>.r

(1) pH variation per degree Celsius.

Recent pH meters are microprocessor-controlled and are Table 2.2.3.-2. These solutions must be traceable to primary
operated using the manufacturer's firmware or software, standards. Calibration has to be performed on a regular
according to given instructions. basis, preferably each day of use or before each series of
Management of electro~s The electrodes are stored measurements.
appropriately and according to the manufacturer's Immerse the electrodes in the solution to be examined and
recommendations (e.g. in an electrolyte solution or a suitable take the reading in the same conditions as those applied for
storage solution). Before measurement, the electrodes are the reference buffer solutions.
visually checked. Refillable electrodes are checked for the If suspensions, emulsions or solutions of non-aqueous or
absence of air bubbles in the glass bulb and to ensure that partially non-aqueous character are measured on a system
the inner electrolyte solution level is satisfactory. The filling calibrated as described above, the pH reading can only be
orifice has to remain open during the measurement. It is also considered to be an approximation of the true value. Suitable
recommended that the diaphragm of the reference electrode electrodes have to be used for pH measurements of such
is checked. Before first use, or if the electrode has been mixtures.
stored out of electrolyte solution, it is usually necessary to PREPARATION OF REFERENCE BUFFER
condition it, according to the recommendations of the SOLUTIONS
manufacturer. If pH stabilisation is too slow (i.e. a long Potassium tetraoxalate 0.05 M
response time), or a zero point shift, reduced slope or any Dissolve 12.61 g of C 4 H 3 KO 8 ,2H 2 O in carbon dioxide-free
other difficulties in calibration are observed, the electrode will water R and dilute to 1000.0 mL with the same solvent.
probably need to be cleaned or replaced. The cleaning is
performed depending on the type of sample and as Potassium hydrogen tartrate, saturated at 25 °C
prescribed in the manufacturer's manual. Regular cleaning is Shake an excess of C 4 H 5 KO 6 vigorously with carbon dioxide-
recommended. free water Rat 25 °C. Filter or decant. Prepare immediately
before use.
Calibration and measurement conditions Unless
othenvise prescribed in the monograph, all measurements are Potassium dihydrogen citrate 0.05 M
carried out at the same temperature as that used for Dissolve 11.41 g of C 6H 7 KO 7 in carbon dioxide-free water R
calibration(± 2.5 °C), usually between 20 °C and 25 °C. and dilute to 1000.0 mL with the same solvent. Prepare
Table 2.2.3.-2 shows the variation of pH with respect to immediately before use.
temperature of a number of reference buffer solutions used Potassium hydrogen phthalate 0.05 M
for calibration. Follow the manufacturer's instructions for Dissolve 10.13 g of C 8 H 5 KO 4 , previously dried for 1 hat
temperature correction. 110 ± 2 °C, in carbon dioxide-free water Rand dilute to
The calibration consists of the determination of the slope 1000.0 mL with the same solvent.
(e.g. 95-105 per cent) and the offset of the measuring Potassium dihydrogen phosphate 0.025 M + Disodium
system. Most commercial pH meters offer a "self test" or hydrogen phosphate 0.025 M
"start-up test" where, for example, the slope and asymmetry Dissolve 3.39 g of KH 2 PO 4 and 3.53 g of Na2 HPO 4 , both
potential are tested and compared to the manufacturer's previously dried for 2 hat 120 ± 2 °C, in carbon dioxide-free
specifications. The apparatus is calibrated using at least 2 water Rand dilute to 1000.0 mL with the same solvent.
buffer solutions chosen so that the expected pH value of the
Potassium dihydrogen phosphate 0.0087 M + Disodium
solution to be examined lies between the pH values of the
hydrogen phosphate 0.0303 M
buffer solutions. The range must be at least 2 pH units.
Dissolve 1.18 g ofKH2 PO 4 and 4.30 g ofNa2 HPO 4 , both
The pH of another buffer solution of intermediate pH, read
previously dried for 2 hat 120 ± 2 °C, in carbon dioxide-free
from the scale, must not differ by more than 0.05 pH units
water Rand dilute to 1000.0 mL with the same solvent.
from the value corresponding to that solution.
Disodium tetraborate 0.01 M
Reference buffer solutions are preferably commercially
Dissolve 3.80 g ofNa 2 B4 O 7 ,l0H 2 O in carbon dioxide-free
available certified reference materials. Alternatively, buffer
water Rand dilute to 1000.0 mL with the same solvent.
solutions can be prepared in-house according to
Store protected from atmospheric carbon dioxide.
V-A292 Appendix V M 2023

Sodium carbonate 0.025 M + Sodium hydrogen from left to right, and mass on the ordinate, decreasing
carbonate 0.025 M downwards. Stop the temperature increase at about 250 °C.
Dissolve 2.64 g ofNa2 CO 3 and 2.09 g ofNaHCO 3 in carbon Measure the difference on the graph between the initial and
dioxide-free water Rand dilute to 1000.0 mL with the same final mass-temperature or mass-time plateaux, which
solvent. Store protected from atmospheric carbon dioxide. corresponds to the loss of mass. The declared loss of mass
Calcium hydroxide, saturated at 25 cc for the certified reference material is stated on the label.
Shake an excess of calcium hydroxide R with carbon dioxide-free Method
water Rand decant at 25 °C. Store protected from Apply the same procedure to the substance to be examined
atmospheric carbon dioxide. using the conditions prescribed in the monograph. Calculate
STORAGE OF BUFFER SOLUTIONS the loss of mass of the substance to be examined from the
difference measured in the graph obtained. Express the loss
Store buffer solutions in suitable chemically-resistant, airtight
of mass as Amlm (per cent).
containers, such as type I glass bottles or plastic containers
suitable for aqueous solutions. If the instrument is in frequent use, carry out temperature
calibration regularly. Otherwise, calibration is carried out
Additional points for monographs of the British before each measurement.
Pharmacopoeia As the operating conditions are critical, the following
[Suitable glass electrodes and pH meters of both the parameters are noted for each measurement: pressure or flow
analogue and digital type are described in British Standards rate, gas composition, mass of the sample, heating rate,
2586:1979 and 3145:1978.] temperature range and sample pre-treatment including any
isothermal period.
DIFFERENTIAL SCANNING CALORIMETRY
Differential scanning calorimetry (DSC) is a technique that
M. Thermal Analysis 1 can be used to demonstrate the energy phenomena produced
(Ph. Bur. method 2.2.34) during heating (or cooling) of a substance (or a mixture of
Thermal analysis is a group of techniques in which the substances) and to determine the changes in enthalpy and
variation of a physical property of a substance is measured as specific heat and the temperatures at which these occur.
a function of temperature. The most commonly used The technique is used to determine the difference in heat
techniques are those which measure changes in the mass or flow (with reference to the temperature) evolved or absorbed
energy of a sample of a substance. by the test sample compared with the reference cell, as a
These techniques have different applications: function of the temperature. Two types of DSC instruments
- determination of phase changes; are available, those using power compensation to maintain a
- determination of changes in chemical composition; null temperature difference between sample and reference
- determination of purity. and those that apply a constant rate of heating and detect
temperature differential as a difference in heat flow between
THERMOGRAVIMETRY sample and reference.
Thermogravimetry (TG) or thermogravimetric analysis
Instrument
(TGA) is a technique in which the mass of a sample of a
The instrument for power compensation DSC consists of a
substance is recorded as a function of temperature according
furnace containing a sample holder with a reference cell and
to a controlled temperature programme.
a test cell. The instrument for heat flow DSC consists of a
Instrument furnace containing a single cell with a sample holder for the
The essential components of a thermobalance are a device reference crucible and the test crucible.
for heating or cooling the substance according to a given A temperature-programming device, thermal detector(s) and
temperature programme, a sample holder in a controlled a recording system which can be connected to a computer
atmosphere, an electrobalance and a means of electronic are attached. The measurements are carried out under a
signal output to a recorder or a computer. controlled atmosphere.
Temperature calibration Calibration of the instrument
The temperature sensor close to or in contact with the Calibrate the instrument for temperature and enthalpy
sample is calibrated using the Curie temperature of a change, using suitable certified materials or reference
ferromagnetic substance such as nickel. In the case of an standards.
instrument capable of simultaneously conducting TG/TGA
and differential thermal analysis (DTA) or differential
Temperature calibration It can be performed using
scanning calorimetry (DSC), the same certified reference certified reference materials having an intrinsic thermal
property, such as the melting point of pure metals or organic
materials as those for DTA and DSC may be used, for
substances, or the phase transition point of crystalline
example, indium, tin and/or zinc.
inorganic salts or oxides. Melting points of indium, tin and/or
Calibration of the electrobalance zinc are usually employed for calibration.
Place an appropriate quantity of a suitable certified reference
Heat-quantity calibration For accurate estimation of the
material (e.g. calcium oxalate monohydrate CRS) in the sample
quantity of heat change (enthalpic change) of a test sample,
holder and record the mass. Set the heating rate according to
caused by a certain physical change accompanying a
the manufacturer's instructions (e.g. 5 °C/min) and start the
temperature change, it is necessary to calibrate the
temperature increase. Record the thermogravimetric curve as
instrument using suitable certified reference materials. Similar
a graph with temperature or time on the abscissa, increasing
to temperature calibration, heat-quantity calibration may be
performed using suitable certified reference materials showing
1
a known definite enthalpic change caused by physical
This chapter has undergone pharmacopoeia/ harmonisation. See chapter
5. 8 Pharmacopoeial harmonisation.
changes, such as the melting of pure metals and/or organic
2023 Appendix V M V-A293

E
ai
.r:::.
0
"O
C
w

about 1 "C
~-----------. ---- ~ Temperature (°C)
►
Figure 2.2.34.-1. - Themzogram

substances, or the phase transition of crystalline inorganic Changes in chemical composition Measurement of heat
salts. The heats of fusion of indium, tin and/or zinc are and temperatures of reaction under given experimental
usually employed for calibration. conditions, so that, for example, the kinetics of
Operating procedure decomposition or desolvation can be determined.
Weigh in a suitable crucible an appropriate quantity of the Application to phase diagrams Establishment of phase
substance to be examined and place it in the sample holder. diagrams for solid mixtures. The establishment of a phase
Place an empty crucible in the reference holder. Set the diagram may be an important step in the preformulation and
initial and final temperatures, and the heating rate according optimisation of the freeze-drying process.
to the operating conditions prescribed in the monograph. Determination of purity The measurements of the
Begin the analysis and record the DSC curve with the fraction of substance melted at a given temperature and the
temperature or time on the abscissa (values increasing from heat of fusion by DSC enable the impurity content of a
left to right) and the energy change on the ordinate (specify substance to be determined from a single thermal diagram,
whether the change is endothermic or exothermic). requiring the use of only a few milligrams of sample with no
The temperature at which the phenomenon occurs (the onset need for repeated accurate measurements of the true
temperature) corresponds to the intersection (A) of the temperature.
extension of the baseline with the tangent at the point of In theory, the melting of an entirely crystalline, pure
greatest slope (inflexion point) of the curve (see substance at constant pressure is characterised by a heat of
Figure 2.2.34.-1). The end of the thermal phenomenon is fusion ti.H1 in an infinitely narrow range, corresponding to
indicated by the peak of the curve. the melting point T0 • A broadening of this range is a sensitive
The enthalpy of the phenomenon is proportional to the area indicator of impurities. Therefore, samples of the same
under the curve limited by the baseline; the proportionality substance, whose impurity contents vary by a few tenths of
factor is determined from the measurement of the heat of a per cent, give thermal diagrams that are visually distinct
fusion of a known substance (e.g. indium) under the same (see Figure 2.2.34.-2).
operating conditions. The determination of molar purity by DSC is based on the
Each thermogram may be accompanied by the following use of a mathematical approximation of the integrated form
data: conditions employed, record of last calibration, mass of of the van't Hoff equation applied to the concentrations (not
the sample and identification (including thermal history), the activities) in a binary system [In( 1 - x2);:::: - x 2 and
container, atmosphere (identity, flow rate, pressure), T x To ;::,; T5]. For low amounts of impurities (x2 ~ 1) and
direction and rate of temperature change, instrument and for temperatures close to the melting point T0 the equation
recorder sensitivity. can be written as follows, in which T and x 2 are variables:
Applications RT 2
T=To- 11 ~ xx2
Phase changes Determination of the temperature, heat
capacity change and enthalpy of phase changes undergone by
a substance as a function of temperature. The transitions that
may be observed include those shown in Table 2.2.34.-1. temperature of the sample, in kelvins;
melting point of the chemically pure substance, in kelvins;
Table 2.2.34.-1. gas constant for ideal gases, in joules-kelvin- 1 -mole 1;
molar heat of fusion of the pure substance, in joules-mole-1;
solid - solid transition: allotropy - polymorphism mole fraction of the impurity, i.e., the number of molecules of
desolvation the impurity divided by the total number of molecules in the
amorphous-crystalline liquid phase (or molten phase) at temperature T (expressed in
kelvins).
solid - liquid transition: melting
glass-transition
solid - gas transition: sublimation Hence, the determination of purity by DSC is limited to the
liquid - solid transition: freezing detection of impurities forming a eutectic mixture with the
recrystallisation principal compound and present at a mole fraction of
glass-transition
typically less than 2 per cent in the substance to be
liquid - gas transition: evaporation examined.
This method cannot be applied to:
- amorphous substances;
V-A294 Appendix V N 2023

about
Heat flow
(mJ/s) 1◄ 1 oc ► 1
········
,,. --;,---
--- I
/
I
/
I
I I
I
I
I EQ)
.c
I 0
-0
I w
C

I
I

.
99 50 % \ ./
►

Figure 2.2.34.-2. - Thennal diagrams according to purity

- solvates or polymorphic compounds that are unstable

within the experimental temperature range;
N. Osmolality
- impurities forming solid solutions with the principal (Ph. Bur. method 2.2.35)
substance; PRINCIPLE
- impurities that are insoluble in the liquid phase or in the
GENERAL
melt of the principal substance.
Osmolality is a measure of the total number of chemical
During the heating of the substance to be examined, the entities per kilogram of solvent, and thus provides an
impurity melts completely at the eutectic temperature. Above indication of the osmotic pressure of the solution. Osmolality
this temperature, the solid phase contains only the pure is dependent on the molal concentration of the solute(s) in
substance. As the temperature increases progressively from the solution, on their dissociation and on the deviation of the
the eutectic temperature to the melting point of the pure solution from ideal behaviour (Raoult's law).
substance, the mole fraction of the impurity in the liquid The unit of osmolality is the osmole per kilogram (osmol/kg),
phase decreases, since the quantity of liquefied pure
but the submultiple milliosmole per kilogram (mosmol/kg) is
substance increases. more commonly used.
For all temperatures above the eutectic point:
The osmolality C~m) of a solution containing i solutes is given
by the expression:
(2)

F molten fraction of the analysed sample; V; number of entities formed by the dissociation of one molecule
x2 mole fraction of the impurity in the analysed sample. of the ,,h solute; if the solute is non-ionic (non-dissociating), v,
equals I,

When the entire sample has melted, F = I and x 2 = x 2.

m, molality of the ,,h solute in the solution, in moles per kilogram
of solvent,
If equation (2) is combined with equation (1), the following molal osmotic coefficient, a dimensionless factor.
equation is obtained:
The molal osmotic coefficient is a measure of the deviation
RTJ I * of the solution from ideal behaviour. For an ideal solution,
T = To - !:..Hi x F x x2 osmolality equals molality (<I>= 1).
In the case of a real, non-ideal solution, the molal osmotic
The value of the heat of fusion of the pure substance is coefficient is influenced by the interactions occurring
obtained by integrating the melting peak. amongst the components (i.e. molecules, ions, solvent) of the
The melting point T0 of the pure substance is extrapolated solution. The more complex the composition of the solution,
from the plot of temperature T (expressed in kelvins) versus the harder it becomes to determine <I>.
I IF. The slope rt. of the curve (obtained after linearisation, if For this reason, the measurement of a colligative property
2 x'
necessary) corresponding to RT0 1,,k, allows x 2 to be such as the freezing-point depression is used as a practical
evaluated. means of determining osmolality by obtaining an overall
The fraction x; multiplied by 100 gives the mole fraction measure of the contribution of the various solutes present in
in per cent for total eutectic impurities. a solution.
PRINCIPLE OF MEASUREMENT
Unless otherwise prescribed, osmolality is determined by
measuring the freezing-point depression (!:..T_r) of a solution.
The relationship between osmolality and freezing-point
depression is given by the expression:
2023 Appendix VO V-A295

0. Conductivity1
where k1 is the molal cryoscopic constant, which is solvent- (Ph. Bur. method 2.2.38)
dependent. For water, the value of k1 is 1.86 K/osmol
(i.e. adding 1 mol of a non-dissociating solute to 1 kg of INTRODUCTION
water results in a decrease in freezing-point of 1.86 K). This general chapter provides information on how to carry
out electrical conductivity measurements (hereafter referred
EQUIPMENT to as 'conductivity') of fluids, including pure liquids. It is
An osmometer for freezing-point depression measurement intended for fluid applications when conductivity is used to
typically consists of: measure, monitor or control chemical dispensing
an appropriate sample container; (e.g. chemical purity or ionic concentration), and other
- a means of cooling the sample; applications where the ionic character of the fluid needs to be
- a temperature-sensitive resistor (thermistor), with an known or controlled.
appropriate current or potential difference measurement
Applications include, but are not limited to, solutions that
device that can indicate a temperature depression or give
may be used in clean-in-place, chromatography detection,
osmolality values directly;
ionic solution preparation, end point detection, dosing,
- a means of mixing the sample and/or inducing
fermentation and buffer production. In some cases,
solidification when supercooling occurs.
conductivity measurements can be extended to pure organic
PROCEDURE fluids such as alcohols and glycols where a weak conductivity
CALIBRATION signal exists, and the signal can be significantly increased if
Prepare reference solutions as specified in Table 2.2.35.-1, as the organic fluids become contaminated with water or salts.
necessary, using dried sodium chloride R. Commercially Conductivity is the measurement of the ability of a fluid to
available certified solutions for osmometer calibration, with conduct electricity via its ions. The ability of any ion to
osmolalities equal or similar to those listed in electrically conduct is directly related to its ion mobility.
Table 2.2.35.-1, may be used. Calibrate the equipment Conductivity is directly proportional to the concentrations of
according to the manufacturer's instructions using water R to ions in the fluid according to the following equation:
determine the zero value and at least 2 of the reference
solutions listed in Table 2.2.35.-1. Confirm the calibration all ions
using at least one additional reference solution with a known K= 1000 L CiAi
osmolality (see Table 2.2.35.-1). Select a reference solution
preferably with an osmolality within ± 50 mosmol/kg of the
conductivity, in siemens per centimetre;
expected value for the solution to be examined or close to " concentration of ion i, in moles per litre;
the centre of the expected osmolality range of the solutions to specific molar conductance of ion i, in siemens square
be examined. It is recommended that the reading is within centimetres per mole (S-cm 2 -mor 1).
± 4 mosmol/kg of the osmolality of the chosen reference
solution. Although siemens per metre is the appropriate SI unit for
conductivity, historically the unit siemens per centimetre has
Table 2.2.35.-1. - Reference solutions for osmometer calibration been selected by industry as the accepted unit.
Mass of sodium chloride R Osmolality, ~m Freezing-point Based on this equation, conductivity is not ion-selective
in waterR (mosmoUkg) depression, tiT,
because it responds to all ions. Furthermore, the specific
(g/kg) (K)
molar conductance of each ion is different. As a result, unless
3.087 100 0.186
the percentage composition of ions in the solution is limited
6.260 200 0.372
and known, the precise concentrations of ionic species cannot
9.463 300 0.558
be determined from conductivity measurements. However,
12.684 400 0.744
for examples such as a solution of a single salt, acid or base
15.916 500 0.930
(e.g. a caustic solution used in cleaning) the precise
19.147 600 1.116
concentration can be directly determined. Despite the lack of
22.380 700 1.302
ionic specificity, conductivity is a valuable laboratory and
process tool for measurement and control of total ionic
METHOD content because it is proportional to the sum of the
Rinse the sample container with the solution to be examined concentrations of all ionic species (anions and cations) for
before each measurement. Programme the device inducing diluted solutions, as described in the equation above.
solidification to start at a defined temperature below the At higher concentrations, conductivity measurements are not
expected freezing-point of the solution to be examined. perfectly linear with concentration. Conductivity
Introduce an appropriate volume of the solution to be measurements cannot be applied to solids or gases, but they
examined into the sample container according to the can be applied to the condensate of gases.
manufacturer's instructions, and start the cooling system. Another variable that influences conductivity measurements
The equipment indicates when equilibrium has been reached. is the fluid temperature. As fluid temperature increases, the
Perform the test under the conditions (cooling temperature ion conductance increases, making this physico-chernical
and volume) used to calibrate the equipment. Depending on phenomenon the predominant reason for the temperature-
the type of equipment, the osmolality can be read directly or compensation requirement when testing conductive fluids.
can be calculated from the measured freezing-point The conductivity (K) is proportional to the conductance (G),
depression. of a fluid between 2 electrodes:
The test is not valid unless the measured osmolality of the
solution to be examined lies within the calibrated osmolality
1 This general chapter has undergone phannacopoeial hannonisation.
range.
See chapter 5.8. Phannacopoeial hannonisation.
V-A296 Appendix V 0 2023

available certified and traceable standard solutions. These

recipes or certified solutions can range from 5 to
K conductivity, in siemens per centimetre;
200 000 µS-cm- 1 depending on the level of accuracy desired.
G == conductance, in siemens; Alternatively, the cell constant is determined by comparison
d distance between the electrodes, in centimetres; to other reference conductivity measuring systems (also
A area of the conducting electrodes, in square centimetres; available as an accredited calibration service). (NOTE:
K cell constant, in reciprocal centimetres, which is also equal to
the ratio of d/A.
conductivity measurements are not perfectly linear with
concentration.)
The resistivity p, expressed in ohm centimetres, of the fluid The measured cell constant of the conductivity sensor must
is, by definition, the reciprocal of the conductivity: be within 5 per cent of the nominal value indicated by the
sensor certificate, unless otherwise prescribed.
1 1 R
Modem conductivity sensors normally do not change their
p=;=GxK=K
cell constant over their lifetime. If a change in the cell
p resistivity, in ohm centimetres; constant is detected during calibration, cleaning of the sensor
K conductivity, in siemens per centimetre; according to the manufacturer's recommendations is
G conductance, in siemens;
appropriate, followed by a repeat of the calibration
R resistance, in ohms, which is the reciprocal of the conductance
(G); procedure. Sometimes 'memory effects' appear, particularly
K cell constant, in reciprocal centimetres. when changing from high to low concentrations if the sensor
is not well flushed.
EQUIPMENT CALIBRATION OF TEMPERATURE
An electrical conductivity measurement consists of the In addition to verifying the sensor's cell constant, the
determination of resistance of the fluid between and around embedded temperature device (or external temperature
the electrodes of the conductivity sensor. To achieve this device) must be appropriately calibrated for the application,
measurement, the primary instrumentation is the resistance- in order to apply the temperature compensation algorithm
measuring circuit and the conductivity sensor, and they are accurately. The temperature accuracy that is required
usually connected by a cable when the sensor and the user depends on the criticality of the temperature to the
interface are separated. application. An accuracy of ± 1 °C typically suffices.
The resistance measurement is made by applying an AC
CALIBRATION OF MEASUREMENT
(alternating current, meaning the flow of electric charge
ELECTRONICS
periodically reverses direction) voltage (or current) to the
The measurement circuit of the system is fundamentally an
electrodes, measuring the current (or voltage), and
AC resistance measuring device. Appropriate verification
calculating the resistance according to Ohm's law.
and/or calibration of the measuring circuit is required for
The alternating source is utilised to prevent polarisation
measurement systems with signal transfer via analog cable.
(collection of ions) at the electrodes. Depending on the
This is accomplished by disconnecting the measuring circuit
equipment, the frequency of the measuring system adjusts
from the sensor's electrodes, attaching traceable resistors of
automatically according to the measuring conditions of the
known value to the measuring circuit using the same
instrument, and there may be multiple resistance-measuring
measurement system cable, and verifying that the measured
circuits embedded in the measuring system. The resistance-
resistance agrees with the resistor value to an acceptable
measurement circuit may be embedded in the transmitter or
level. A typical acceptance criterion for the resistance
in the sensor.
accuracy is below 2 per cent of the reading at resistances
The conductivity sensor consists of at least 2 electrical greater than 100 Q, and increasing to 5 per cent at lower
conductors of a fixed size and geometry, separated by an resistances. However, it is recommended that the application
electrical insulator. The electrodes, insulator and any other criticality ultimately determines the desired accuracy.
wetted components are constructed of materials that are
For conductivity systems that cannot have the resistance-
unreactive to fluids with which they may come into contact.
measuring circuit disconnected from the electrodes
In addition, the sensor construction must be able to
(e.g. measurement circuit and electrodes in 1 mutual
withstand the environmental conditions (process or ambient
housing), it may be difficult to directly adjust or verify the
temperature, pressure, cleaning applications) that it would be
circuit accuracy, depending on the sensor design.
subjected to.
An alternative method of verifying the measurement system
Most conductivity sensors have temperature devices such as a integrity is a system calibration according to the procedures
platinum RTD (resistance temperature device) or NTC for cell constant determination for each measuring circuit
(negative temperature coefficient) thermistor embedded that is intended to be used.
inside the sensor, although external temperature
If verification/calibration of the sensor's cell constant,
measurement is possible. The purpose of the temperature
temperature device and measuring circuit are done at the
measurement is for temperature compensation of the
same service interval, it is recommended to verify the
conductivity measurement.
measuring circuit first, the temperature device next and the
CELL CONSTANT DETERMINATION cell constant last. Because all of these parameters are
The purpose of the sensor's cell constant is to normalise the typically very stable due to modem electronics and stable
conductance (or resistance) measurement for the geometrical sensor construction, frequent calibration (such as daily) is not
construction of the 2 electrodes. usually required. Comparison to qualified reference systems
The cell constant is determined by immersing the is also a suitable means of calibration. Calibration is
conductivity sensor in a solution of known conductivity. performed at appropriate intervals as defined in the quality
Solutions of known conductivity can be obtained by management system.
preparation of specific recipes according to national
authoritative sources, or procurement of commercially
2023 Appendix VP V-A297

TEMPERATURE COMPENSATION
As the conductivity of a fluid is temperature dependent,
P. Total Organic Carbon in Water for
temperature compensation of the conductivity measurement Pharmaceutical Use
is necessary unless otherwise prescribed (e.g. purified water, (Ph. Bur. method 2.2.44)
water for injection). An appropriate temperature
compensation algorithm will ensure that changes in the Total organic carbon (TOC) determination is an indirect
conductivity measurements can be ascribed to concentration measure of organic substances present in water for
changes and not temperature changes. Conductivity pharmaceutical use. TOC determination can also be used to
measurements are normally referenced to 25 °C. A common monitor the performance of various operations in the
form of linear temperature compensation uses a temperature preparation of medicines.
coefficient according to the following equation: A variety of acceptable methods is available for determining
TOC. Rather than prescribing a given method to be used,
Ky
this general chapter describes the procedures used to qualify
Kzs = [l + rx(T - 25)] the chosen method and the interpretation of results in limit
tests. A standard solution is analysed at suitable intervals,
,c 25 conductivity compensated to 25 'C;
KT conductivity at T;
depending on the frequency of measurements; the solution is
1- temperature coefficient of conductivity; prepared with a substance that is expected to be easily
T measured temperature. oxidisable (for example, sucrose) at a concentration adjusted
to give an instrument response corresponding to the TOC
A temperature coefficient of 2.1 per cent per degree Celsius limit to be measured. The suitability of the system is
is commonly used for many salt solutions. Most salt-based determined by analysis of a solution prepared with a
solutions have temperature coefficients of conductivity substance expected to be oxidisable with difficulty (for
ranging from 1. 9 to 2.2 per cent per degree Celsius. example, 1,4-benzoquinone).
Depending on the fluid samples, other forms of temperature The various types of apparatus used to measure TOC in
compensation may be appropriate. Non-linear temperature water for pharmaceutical use have in common the objective
compensation data for a variety of solutions is widely of completely oxidising the organic molecules in the sample
available, e.g. as described in ISO 7888 Water water to produce carbon dioxide followed by measurement of
Quality - Detennination of electrical conductivity. In cases of the amount of carbon dioxide produced, the result being
very low conductivity (below 10 µS-cm- 1), for example, used to calculate the carbon concentration in the water.
purified water used for cleaning/rinsing purposes,
The apparatus used must discriminate between organic and
two compensations need to be made. One is for the intrinsic
inorganic carbon, the latter being present as carbonate.
conductivity of water, and the other is for the other ionic
The discrimination may be effected either by measuring the
species in water. These compensations are normally
inorganic carbon and subtracting it from the total carbon, or
combined and embedded in the microprocessor-controlled
by purging inorganic carbon from the sample before
conductivity measurement systems. This is not supplied in all
oxidisation. Purging may also entrain organic molecules, but
conductivity measurement technologies.
such purgeable organic carbon is present in negligible
CONDUCTIVITY MEASUREMENT OF FLUIDS quantities in water for pharmaceutical use.
For off-line or at-line batch measurements, rinse the cleaned Apparatus Use a calibrated instrument installed either
sensor with the fluid to be measured, then perform the on-line or off-line. Verify the system suitability at suitable
measurement. Ensure that the position of the sensor in the intervals as described below. The apparatus must have a limit
container does not impact the conductivity measurement, as of detection specified by the manufacturer of 0.05 mg or less
the container walls can impact the measurement for some of carbon per litre.
electrode designs. Record the temperature and the
TOC water Use highly purified water complying with the
temperature-compensated conductivity as required.
following specifications:
For continuous on-line or in-line measurements, install the - conductivity: not greater than 1.0 µS·cm- 1 at 25 °C,
cleaned sensor into the pipe, tank or other containment - total organic carbon: not greater than 0.1 mg/L.
vessel, and flush if necessary. Make sure proper installation
Depending on the type of apparatus used, the content of
procedures are applied to prevent bubbles or particles from
heavy metals and copper may be critical. The manufacturer's
collecting between the electrodes. Ensure that the position of
instructions should be followed.
the sensor in the pipe or tank does not impact the
conductivity measurement, as the nearby surfaces can affect Glassware preparation Use glassware that has been
the measurement for some electrode designs. Record the scrupulously cleaned by a method that will remove organic
temperature and the temperature-compensated conductivity matter. Use TOG water for the final rinse of glassware.
as required. Standard solution Dissolve sucrose R, dried at 105 °C for
For all batch or continuous measurements, ensure that the 3 h in TOG water to obtain a solution containing 1. 19 mg of
wetted components of the sensor are compatible with the sucrose per litre (0.50 mg of carbon per litre).
fluid and the temperature to be measured. Test solution Using all due care to avoid contamination,
collect water to be tested in an airtight container leaving
minimal head-space. Examine the water with minimum delay
to reduce contamination from the container and its closure.
System suitability solution Dissolve 1,4-benzoquinone R
in TOG water to obtain a solution having a concentration of
0.75 mg of 1,4-benzoquinone per litre (0.50 mg of carbon
per litre).
V-A298 Appendix V Q 2023

TOC water control Use TOG water obtained at the same substance, and hence should be independent of the method
time as that used to prepare the standard solution and the of determination. The true density is determined by
system suitability solution. calculation.
Control solutions In addition to the TOG water control, It is obtained using crystallographic data (volume and
prepare suitable blank solutions or other solutions needed for composition of the unit cell) from, for example, X-ray
establishing the baseline or for calibration adjustments diffraction data, either on a single crystal or by refinement of
following the manufacturer's instructions; run the appropriate the crystalline structure from X-ray powder diffraction data.
blanks to zero the instrument. PARTICLE DENSITY
System suitability Run the following solutions and record The particle density takes into account both the true density
the responses: TOG water (rw); standard solution (r,); system and the intraparticulate porosity (sealed and/or
suitability solutwn (r,,). Calculate the percentage response experimentally non-accessible open pores). Thus, particle
efficiency using the expression: density depends on the value of the volume determined,
which in tum depends on the method of measurement.
r,, - rw X 100
The particle density can be determined using one of the 2
rs - rw
following methods.
The system is suitable if the response efficiency is not less The gas pycnometric density is determined by measuring the
than 85 per cent and not more than 115 per cent of the volume occupied by a known mass of powder, which is
theoretical response. equivalent to the volume of gas displaced by the powder
Procedure Run the test solution and record the response using a gas displacement pycnometer (2. 9. 23). In gas
(ru). The test solution complies with the test if ru is not
pycnometric density measurements, the volume determined
greater than r, - rw, excludes the volume occupied by open pores; however, it
includes the volume occupied by sealed pores or pores
The method can also be applied using on-line inaccessible to the gas. Due to the high diffusivity of helium,
instrumentation that has been adequately calibrated and which is the preferred choice of gas, most open pores are
shown to have acceptable system suitability. The location of accessible to the gas. Therefore, the gas pycnometric density
instrumentation must be chosen to ensure that the responses of a finely milled powder is generally not very different from
are representative of the water used. the true density. Hence, this density is the best estimate of
the true density of an amorphous or partially crystalline
sample and is therefore widely applicable for processed
pharmaceutical powder samples.
Q. Density of Solids The mercury porosimeter density is also called granular density.
(Ph. Bur. method 2.2.42) With this method the volume determined includes the
volume occupied by sealed pores or pores inaccessible to
The density of solids corresponds to their average mass
mercury; however, it includes the volume only from open
per unit volume and typically is expressed in grams per cubic
pores smaller than some size limit. This pore-size limit or
centimetre (gicm 3 ) although the International Unit is the
minimal access diameter depends on the maximal mercury
kilogram per cubic metre (1 gicm 3 = 1000 kgim3 ).
intrusion pressure applied during the measurement, and
Unlike gases and liquids whose density depends only on under normal operating pressures the mercury does not
temperature and pressure, the density of a solid also depends penetrate the finest pores accessible to helium. Various
on its assembly and therefore varies with the crystal structure granular densities can be obtained from one sample since, for
and degree of crystallinity. each applied mercury intrusion pressure, a density can be
When a solid is amorphous or partially amorphous, its determined that corresponds to the pore-size limit at that
density may further depend upon the history of preparation, pressure.
treatment and storage.
BULK AND TAPPED DENSITY
Therefore, unlike fluids, the densities of 2 chemically The bulk density of a powder includes the contribution of
equivalent solids may be different, and this difference reflects interparticulate void volume. Hence, the bulk density
a difference in solid-state structure. The density of depends on both the density of powder particles and the
constituent particles is an important physical characteristic of spatial arrangement of particles in the powder bed.
pharmaceutical powders.
The bulk density of a powder is often very difficult to
The density of a solid particle can assume different values measure with good reproducibility since the slightest
depending on the method used to measure the volume of the disturbance of the bed may result in a new density. Thus, it
particle. It is useful to distinguish 3 levels of expression of is essential in reporting bulk density to specify how the
density: determination was made.
- the trne density, which only includes the solid fraction of
the material; in case of crystalline material, the true The bulk density and the tapped density are determined as
mentioned in chapter 2. 9.34. Bulk density and tapped density.
density is also called crystal density;
- the particle density, which also includes the volume due to
intraparticulate pores;
- the bulk density, which further includes the interparticulate
void volume formed in the powder bed.
TRUE DENSITY
The true density of a substance is the ratio of the mass to the
volume of the unit cell, exclusive of all voids that are not a
fundamental part of the molecular packing arrangement. It is
an intrinsic property of the specified crystal structure of
2023 Appendix V R V-A299

The radioactivity of a preparation is stated at a given date.

R. Detection and Measurement of If the half-life of the radionuclide is less than 70 days, the
Radioactivity time is also indicated. This statement of the radioactive
content must be made with reference to a specified time
(Ph. Bur. method 2.2.66)
zone. The radioactivity at other times may be calculated from
INTRODUCTION the exponential decay equation or from tables.
Within the context of the European Pharmacopoeia, the term In general, a correct measurement of radioactivity requires
'radioactivity' is used both to describe the phenomenon of that consideration is given to some or all of the following:
radioactive decay and to express the physical quantity of this
Dead-time losses
phenomenon. In the monographs on radiopharmaceutical
Due to the finite resolving time (dead time) of the detector
preparations, the detection and measurement of radioactivity
and its associated electronic equipment, it may be necessary
are performed for different purposes: verification of the
to correct for losses by coincidence. The resolving time of a
characters, identification, determination of radionuclidic and
counter is the minimum time interval required by the counter
radiochemical purity, as well as determination of the
to resolve 2 single pulses. Incident radiation events at shorter
radioactivity in a substance (assay).
intervals may not be detected or may be detected as a single
Under these assumptions, the measurement can be event with the summed energy. These losses are sometimes
qualitative, quantitative or both, depending whether it is referred to as 'dead-time losses'. For a counting system with
directed to the identification of the radionuclide or the a fixed dead time r following each count, the true count rate,
determination of its activity (rate of decay) or both of them. per second, is calculated using the following expression:
Radioactive sources can produce various types of emissions,
such as alpha particles, electrons, positrons, gamma- and
X-rays, according to the radionuclidic composition.
Each radionuclide yields characteristic emissions, with the observed count rate, per second;
specific energies and relative intensities. Such radiations can the dead time, in seconds.
be detected as a result of their ionising properties in an
ionisation chamber but without further characterisation; With some equipment this correction is made automatically.
when they are detected and analysed using a spectrometer, Corrections for losses by coincidence must be made before
an energy spectrum is obtained. A detailed spectrum analysis the correction for background radiation.
is typically used to identify radionuclides present in a sample. Correction for decay during measurement
Spectrometry can also be used for quantitative determination If the time period of an individual measurement, tm, is not
of the radioactivity in sources made of a single radionuclide negligibly short compared with the half-life of the
or radionuclide mixtures or of the individual radionuclides radionuclide, T 112 , the decay during this measurement time
present. must be taken into account. For example, there is a
A measurement of radioactivity is generally performed by 5 per cent cumulative loss of counts due to decay during a
counting the number of detected decay events (emissions). counting period that is 15 per cent of the half-life of the
Therefore, the geometry of the sample during the radionuclide.
measurement of radioactivity and the acquisition time After having corrected the instrument reading (count rate,
strongly influence the result. In general, the measurement ionisation current, etc.) for background signals and, if
geometry must correspond to a calibrated geometry and the necessary, for losses due to electronic effects, the instrument
acquisition time must be long enough to reach sufficient reading corrected to the beginning of the individual
counting statistics. measurement is calculated using the following expression:
A measurement of radioactivity can be done in a stand-alone
mode (e.g. using an ionisation chamber or a spectrometer) or R()-tm)
in combination with a separation technique 1 - (e-h,,)
(e.g. radiochromatography) to account for relative
R instrument reading before decay correction, but already
contributions from different radioactive chemical species that corrected for background signal, etc.;
may be present in a mixture. i. radionuclide decay constant (In 2/T,/2);
base of natural logarithm;
MEASUREMENT OF RADIOACTIVITY measurement duration.
A direct determination of the radioactivity of a given sample,
in becquerel (Bq), may be carried out if the decay scheme of Statistics of radioactivity measurement
the radionuclide is known, but in practice many corrections The results of determinations of radioactivity show variations
are required to obtain accurate results. For this reason, it is that derive mainly from the random nature of nuclear
possible to carry out the measurement with the aid of a transformations. Counting for any finite time can yield only
primary standard source or by using measuring instruments an estimate of the true rate of nuclear transformations.
such as an ionisation chamber or a spectrometer calibrated A sufficient number of counts must be registered in order to
using suitable standards for the particular radionuclides. compensate for variations in the number of transformations
A spectrometer is used when measuring the radioactivity of per time. In the case of measurement of radioactivity, the
radionuclides in a mixture, each radionuclide being identified standard deviation of the recorded counts is the square root
by its emissions and their characteristic energies. of the counts, so at least 10 000 counts are necessary to
All measurements of radioactivity must be corrected for obtain a relative standard deviation of not more than
dead-time losses and by subtracting the background signal 1 per cent.
due to radiation in the environment and to spurious signals Linearity
generated in the equipment itself. The linearity of an instrument is the range of radioactivity for
a particular radionuclide over which its efficiency remains
constant.
V-A300 Appendix V R 2023

The linear range of a radioactivity measurement assembly Limit of quantification

can be determined by repeatedly counting a radioactive The limit of quantification (LOQ) of an individual procedure
sample in a fixed geometry as it decays from an activity level is the lowest amount of radioactivity in a sample that can be
that is above the linear range. After correction for the quantitatively determined with suitable precision and
background signal, the natural logarithm of the count rate accuracy. The LOQ is used particularly for the determination
data is plotted against the elapsed time after the first of impurities and/or degradation products. In practical terms
measurement (Figure 2.2.66.-1). the LOQ is usually considered to be 10 rimes the standard
deviation of the background signal.
16i,---------------------, ME4SUREMENT OF RADIOACTIVI1Y USING
IONISATION CHAMBERS
Apparatus
12 Ionisation chambers (including dose calibrators) are the most
y = -0.115x + 14.186
common equipment for the measurement of radioactivity in
10
the practice of radiopharmacy. It generally can measure
(/J
a.
activities from a few tens of kBq to hundreds of GBq.
0
£
It usually comprises a sealed well-type ionisation chamber
6 and built-in electronics to convert the detector signal to a
measure of radioactivity.
4
The chamber is filled with a gas across which an electrical
2 field is applied. When the gas is ionised by the radiation
emitted by the source, the resulting ionisation current is
0 measured and related to the radioactivity present in the
0 20 40 60 80 100 120
Elapsed Time (h)
ionisation chamber. The ionisation current is influenced by
the applied voltage, the energy and the intensity of the
radiation and the nature and pressure of the gas.
Figure 2.2.66.-1. - Plot showing the measured and extrapolated The instrument settings (calibration factor) may be adjusted
count rate (natural logarithm of counts per second (cps)) from a to keep a direct relationship between the ionisation produced
technetium-99m source as a function of time, starting with a level by the radiation of a specific radionuclide and the
of radioactivity above the linear range of the measuring equipment radioactivity value obtained for each measurement geometry.
Linear regression analysis of the central, linear portion of the As an ionisation chamber measures only the current resulting
data set yields a slope which is the decay constant )., which from the overall ionisation produced within the chamber, it
has a characteristic value for each radionuclide: cannot discriminate between the emissions of different
radionuclides.
In cps= -At+ c For an accurate measurement of the radioactivity of a specific
radionuclide, the measurement must be corrected for the
c represents the natural logarithm of the count rate at t = 0 contributions to the ionisation current caused by
of a perfectly linear instrument. radionuclidic impurities present in the preparation.
The resulting regression equation is used to calculate the The activity levels to be measured are limited by saturation
theoretical count rate at each time that the actual data were considerations, the range of the amplifier and the design of
recorded. Where the deviation of the measured count rate the chamber itself. The linearity range of the ionisation
from the theoretical count rate is unacceptably high, the chamber is established as described above under Linearity.
linear range of the measuring equipment has been exceeded. The ionisation chamber must be shielded to minimise
Alternatively, a series of dilutions can be made of a background signals to an acceptable level.
radioactive solution of known radioactivity concentration. Method
Equal volumes of each of the dilutions are then counted The sample is positioned inside the well of the ionisation
using standardised geometry and counter settings. The ratio chamber at a given position, using a holder. After putting the
of the count rate for each sample (after correction for sample in the ionisation chamber, the activity reading is
background signals and decay) to the calculated radioactivity made once the response is stable. Measuring the sample
of the respective sample in Bq is the counting efficiency. under exactly the same geometrical conditions as the
The range over which this ratio is constant is the useable calibration source will yield the most accurate results.
range of the measuring equipment for the radionuclide If necessary, dilute the preparation to be measured to the
concerned. same volume as that of the calibration source.
The limit of detection and the limit of quantification for Calibration The ionisation chamber is calibrated taking
equipment and procedures used for radioactivity into account the shape, dimensions, material of the
measurement must be established before their routine use. container, volume and composition of the solution, the
Limit of detection position within the chamber and the radionuclide being
The limit of detection (LOD) of an individual procedure is measured. Limits for uncertainty in calibration can be found
the lowest amount of radioactivity in a sample that can be in national and international regulations.
detected but not necessarily quantified as an exact value. Calibrate the ionisation chamber at least once a year, by
In practical terms this requires an estimate of the background using sources of radionuclides traceable to national or
signal and its standard deviation. The LOD is usually international standards in the appropriate containers (vial,
considered to be 3 times the standard deviation of the syringe) with regard to geometry. Establish and implement
background signal. subsidiary correction factors to take account of the differing
configurations of the radionuclides to be measured. Perform
2023 Appendix V R V-A301

a linearity check of the instrument's response over the radioactivity using liquid scintillation detectors see under
complete range of energies and activities for which the Beta-particle spectrometry below.
equipment is used. Calibration In order to take into account the loss of
For each setting and before each use (minimum once on counting efficiency due to quenching, the liquid scintillation
each day of use) perform a constancy check of the ionisation counter may make use of an external source, typically
chamber using standard sources of radionuclides with long barium-133 or europium- I 52, which is brought close to the
half-lives to verify its calibrated state. A check with a sample vial to release Compton electrons. The shape of the
reference source, such as caesium-137, must be performed resulting spectrum is analysed automatically to compute a
on each day of use to verify that the ionisation chamber is quench-indicating parameter. This parameter can then be
still in its calibrated state. related to the counting efficiency measuring sources of
MEASUREMENT OF RADIOACTIVI1Y USING known activity at a determined level of quenching agent.
SOLID-STATE DETECTORS The obtained quench curve allows the determination of the
Solid-state detectors include scintillating plastic fluors and activity of an unknown sample knowing the count rate and
crystals, and semiconductors. Further to their application in the value of the quenching parameter.
spectrometry (see section Spectrometry), solid-state detectors DETERMINATION OF HALF-LIFE
can be used for the measurement of radioactivity. The half-life is a characteristic of the radionuclide that may
In particular, due to their high sensitivity, plastic and crystal be used for its identification. The half-life is calculated by
scintillation detectors are used in counting low levels of measuring the variation of radioactivity of a sample to be
radioactivity. Dead-time losses must be carefully considered tested as a function of time. Perform the measurements in
with these types of detectors. Semiconductor detectors are the linearity range of a calibrated instrument.
used when a higher energy discrimination is required, for Apparatus
example in mixtures of radionuclides or when there are Half-life can be measured by using any type of quantitative
potential radionuclidic impurities with emissions of similar radioactivity detector provided it is used within a linearity
energy. range throughout the range of activities that are present
Apparatus during the measurement and the geometry is not changed
The equipment consists of a shielded detector comprising a during the measurement.
plastic or crystal scintillator coupled to a photomultiplier, or For preparations containing a radionuclide with a short half-
a semiconductor, which are connected to an amplifier and life and when stated in a monograph, determination of the
counting electronics. The system may have an adjustable approximate half-life contributes to the identification.
energy window, used for selecting a counting region of the
Method
radionuclide energy spectrum that may be adjusted by the
Half-life The preparation to be examined is used as such
operator.
or diluted or dried in a capsule after appropriate dilution.
Instruments have different properties of energy resolution The radioactive sample is prepared in a manner that will
and detection efficiency depending on the type of detector avoid loss of material during handling. If it is a liquid
and its volume and geometry. Lower efficiency requires a (solution), it is contained in a closed flask or a sealed tube.
longer counting time. If it is a residue from drying in a capsule, it is protected by a
Samples to be measured may be placed in front of the cover consisting of a sheet of adhesive cellulose acetate or of
detector or into the well of a well-type detector. Measuring some other material.
chambers may be enclosed in the detector shielding and The radioactivity of the sample must be high enough to allow
single samples may be introduced using lids or other measurements over a period corresponding to 3 estimated
positioning systems to ensure correct measurement geometry. half-lives but must be, for each measurement, within the
A scintillation detector can be used for dynamic radioactivity linearity range of the equipment. Correction for dead-time
measurement when, for example, the eluate of a liquid losses is applied if necessary.
chromatograph is directed over or through a detector, see The same source is measured in the same geometrical
section on Detection and measurement of radioactivity in conditions and at intervals usually corresponding to at least
combination with a separation technique. half of the estimated half-life. Each value is tabulated against
Method the time interval from the initial measurement. To avoid
Ensure that the sample radioactivity gives a counting rate in influence of decay during measurement, the counting time is
the linearity range of the equipment. The measurement is the same for all measurements.
started after any shielding is in place or the well cover is A graph can be drawn with time as the abscissa and the
replaced and the counting time is selected to reach sufficient logarithm of the relative instrument reading (e.g. count rate)
counts for a statistically significant value. as the ordinate. The half-life is calculated from the slope of
Calibration The detector has to be calibrated by the best linear fit of the measured values against the time
measuring its efficiency using a source of the radionuclide in corresponding to each measurement.
question traceable to national or international standards. Approximate half-life For this purpose, not fewer than
Calibration in terms of efficiency uses sources such as 3 measurements are made over a period of not less than 1/4
caesium-137, cobalt-60, barium-133 and others covering the of the estimated half-life.
desired energy range.
The sample to be examined and the instrument to be used
MEASUREMENT OF RADIOACTIVI1Y USING comply with the indications given above. The data are
LIQUID SCINTILLATION DETECTORS processed in the same way as above.
Liquid scintillation counting is commonly used for beta-
SPECTROMETRY
particle emitting samples, but is also used for alpha-particle
emitting samples. For the principles of the detection of Radionuclides can be identified by their emission spectrum.
Each type of emission (i.e. alpha particles, beta particles and
electrons, gamma- and X-rays) requires specific equipment to
V-A302 Appendix V R 2023

1000
J!l
C:
:J
0
(.)

0
~
~:J 100
C:

10

0 100 200 300 400 500 600 700 800 900 1000
Energy (keV)

Figure 2.2.66.-2. - Comparative pulse-height spectra recorded using a thallium-activated sodium iodide scintillator (upper curve) and a
high-purity germanium semiconductor detecwr (lower curve). The source was gamma- and X-ray radiation from the decay of iodine-131.

acquire an emission spectrum. Spectrometers must be Dead-time losses must be carefully considered with this type
calibrated in order to work properly and the following of detector.
sections describe the different equipment and detail the The sensitivity of a Nal(Tl) detector is higher than that of a
general procedures for a reliable measurement. germanium detector of the same size. In general, peaks in an
GAMMA-RAY SPECTROMETRY energy spectrum are identified with an uncertainty depending
General principles upon the full width of the peak at its half-maximum height
In gamma-ray spectrometry using a scintillation detector, (FWHM). The energy resolution of a solid-state scintillation
absorption of gamma- and X-rays results in production of detector is much poorer than that of a semiconductor
light, which is converted into an electrical pulse by a detector and hence peaks obtained with a semiconductor
photomultiplier. In gamma-ray spectrometry using a detector are much narrower than those obtained with a
semiconductor detector, absorption of gamma- and X-rays scintillation detector. Figure 2.2.66.-2 shows a comparison of
results in the immediate production of an electrical pulse. the spectra obtained from the same source with the 2 types
of detector.
In both cases the pulse amplitude is proportional to the
energy of the absorbed radiation. The most common The different performances of NaI(Tl) and HPGe detectors
detectors for gamma- and X-ray spectrometry are thallium- may limit their use in some spectrometric analyses.
activated sodium iodide (NaI(TI)) scintillation counters and For the identification of the radionuclide(s) in a preparation
high-purity germanium (HPGe) semiconductor detectors. and determination of radionuclidic purity, a risk assessment
A gamma-ray spectrum can be produced by collecting and on the process of radionuclide production must assess the
analysing a sufficient number of pulses. potential presence of other radionuclides with photon
energies in the same range ( ± 10 per cent) as that of the
Apparatus radionuclide(s) present in the radiopharmaceutical.
A gamma-ray spectrometer usually comprises a shielded
In case radionuclidic impurities can be present that emit
measuring chamber where the sample is positioned, a
gamma- or X-rays with an energy in the same range as that
detector, an electronic chain and a multichannel analyser.
of the photons emitted by the radionuclide in the
The shielding of the chamber must be able to reduce the preparation, a measured peak energy within a maximum
background signal to a level that allows the registration of a interval of ± 2 keV or ± 2 per cent (whichever is the larger)
correct gamma-ray spectrum. with respect to the nominal peak energy (see 5. 7. Table of
The measurement chamber has a movable cover or a drawer physical characteristics of radionuclides) is sufficient for peak
to allow the positioning of the sample. A sample holder may identification.
be present to ensure reproducible geometry between In the case where such impurities are not expected to be
measurements. present, a maximum interval of± 10 keV or ± 6 per cent
The duration of measurement is related to the radioactivity (whichever is the larger) with respect to the nominal peak
of the target radionuclide and a long period of acquisition energy is acceptable for peak identification.
may be required to achieve the necessary counting statistics.
2023 Appendix V R V-A303

Method relation to the detector. The sample/detector geometry is

Ensure that the counting rate of the sample falls within the then defined by the position of the sample relative to the
linearity range of the equipment. For liquid samples this may detector and the characteristics of the container and sample,
be achieved by appropriate dilution; for solid samples, by e.g. shape, volume and density. Figure 2.2.66.-3 shows a
increasing the source-to-detector distance or by using an typical HPGe detector efficiency curve obtained for a
attenuating material. Introduce the preparation to be cylindrical container placed on top of the detector.
examined in a container into the instrument chamber and
record the spectrum after closing the shielding. 0.035~--.-~-~-~
'' '
Ensure that the container used for quantitative measurements 0.030 · "i - · - -1- - - - t" -
1
- - 4 -
I
- - - 1- -· - - t" - - -
I I

is of the same shape, dimensions, volume and material as 0.025

!
--· )-. ·-
I
- ·-1 -
I
·-1- ., --
=
.j.. - - -

that of the calibration standard. i',' '' '' ''

c:0.020·
Ensure that the composition of the solution and the position Q)

of the container in the measuring chamber is the same for ;go.015

w ''
the container for the quantitative measurement as for the 0.010 L
'
calibration standard. 0.00
''
- - ··· - - - · r' - - -- -,' - - - -1- -
· ··· - - 1 - I'i - -,- - - I -

Radionuclide identification Calibrate the spectrometer in ' ' '' ' ''
0.00 O '
relation to energy. Determination of the correspondence of 200 400 600 800 1000 1200 1400 1600 1800 2000
the energy of the peaks detected from the sample to the Energy (keV)
energies prescribed by a monograph is a valid identification
test. Figure 2.2.66.-3. - Typical HPGe efficiency curve measured
Radionuclidic purity Calibrate the spectrometer in using a dedicated comainer set on top of the detector
relation to efficiency and energy. Determine the LOQ and
resolution of the equipment and ensure that they are in line BETA-PARTICLE SPECTROMETRY
with the limits of the radionuclides to be determined. Record In the case of a beta-particle emitter, a beta-particle
the spectrum of the preparation. spectrometer is necessary to determine the energy
distribution of the emitted beta particles. It is analogous to a
Identify the radionuclides present in the preparation to be
gamma-ray spectrometer, but frequently uses liquid
examined and determine their radioactivity with the aid of
scintillators to convert the energy of the beta particles into
chapter 5. 7. Table of physical characteristics of radionuclides.
detectable light, which can then be analysed. Beta-particle
Because the level of radionuclidic impurities, expressed as a
spectrometry is mostly achieved by dissolving or suspending
percentage of the total radioactivity, may increase or decrease
the sample in a liquid scintillation cocktail in transparent or
with time, the measured activity of each impurity must be
translucent (glass or plastic) containers and subsequent
recalculated to the activity during the shelf life of the
counting of the electrical pulses generated by a
preparation. The activities of all radionuclidic impurities need
photomultiplier from the emitted light. The pulse amplitude
to be summed (taking into account the limit of
is related to the energy of the absorbed radiation. A beta-
quantification) and related to the total radioactivity of the
particle spectrum can be produced by collecting a sufficient
preparation.
number of pulses. The liquid scintillation cocktail is chosen
The sample is placed close to the detector or within a well- in such a way that counting errors due to quenching,
type detector. All the events within a pre-set energy range are chemoluminescence, phosphorescence, etc., are minimised.
collected and displayed on a ratemeter as counts per second Coincidence counting with 2 or more photomultipliers is also
or accumulated over a pre-set period of time. If there is used to minimise counts from background radiation,
sufficient difference in photon energies emitted by the electronics, etc.
radionuclide(s), a sodium iodide detector can be suitable,
To differentiate between alpha- and beta-particle emissions,
given its high sensitivity. However, if there is a need to
pulse-shape discrimination is commonly used.
discriminate emissions of similar energy, a HPGe detector or
another semiconductor detector is needed. Radionuclide identification Determination of the
correspondence of the mean and/or maximum energies in the
Calibration Calibration in relation to energy is done by
energy spectrum from the sample to the energies prescribed
using the peaks of known sources traceable to national or
by a monograph is a valid identification test.
international standards, such as cobalt-57, caesium-137,
cobalt-60 and others covering the desired energy range. Calibration A common method of energy calibration is to
A calibration in relation to efficiency can be simultaneously use an unquenched reference sample to determine the
obtained, so that not only the energy spectrum but also the maximum energy of the beta particles emitted by the
activity of the sample and the radionuclide impurities can be radionuclide of interest.
further determined. The calibration of efficiency can be ALPHA-PARTICLE SPECTROMETRY
performed with a traceable radionuclide source with energy For the identification and assay of alpha-particle emitters,
peaks covering the desired range or with the aid of a mixed, spectrometry using liquid scintillation is mostly used.
traceable radionuclide standard with gamma-ray energies The principle is explained in the previous section on beta-
covering the desired range. particle spectrometry.
To obtain the efficiency curve, the detector response as a For the identification and determination of radionuclidic
function of the energy has to be measured using each purity of alpha-particle emitters, spectrometry using a silicon-
separate sample/detector geometry. For this reason, it is diode semiconductor detector can be used. Using this
possible to carry out the measurement with the aid of a detector, the absorption of alpha particles results in the
primary standard source. Primary standards may not be immediate production of an electrical pulse. The movement
available for radionuclides with a short half-life, e.g. some of electron-hole pairs created by the interaction of radiation
positron emitters. When measuring, the sample will mostly induces an electrical charge, which is amplified and
have to be in a container and set at a defined position in measured.
V-A304 Appendix V R 2023

The sample preparation is of crucial importance. After a products. In-line detection is usually obtained by using a
chemical separation of the radionuclide of interest, the scintillation detector connected to a ratemeter and recording
sample is electro-deposited on a stainless steel disk in the device. The scintillating material of the detector is selected
form of a very thin layer of material to minimise self- on the basis of the emission to be detected, e.g. plastic
absorption. The yield of the whole procedure can be scintillator for beta-particle emissions or scintillation crystals
determined experimentally by adding a known amount of a for gamma- and X-ray radiations. The addition of a liquid
tracer, which will take into account the chemical separation scintillation cocktail before the eluate reaches the in-line
efficiency, the electro-deposition efficiency and the counting radioactivity detector may also be used in the case of beta-
efficiency. particle-emitting radionuclides.
For both types of detectors, the pulse amplitude is related to The simultaneous use of a radioactivity detector and other
the energy of the absorbed radiation. An alpha-particle detectors (UV, refractive index, conductimetric, etc.)
spectrum can be produced by collecting a sufficient number connected in series may be used to identify the substance,
of pulses. e.g. in relation to the retention time of a known standard, to
Radionuclide identification Determination of the determine the amount of the substance using a suitable
correspondence of the energy of the peaks detected from the reference standard and to measure the radioactivity
sample to the energies prescribed by a monograph is a valid associated with such a substance. When different detectors
identification test. are coupled in series, correct the experimentally obtained
Calibration An alpha-particle spectrometer has to be retention times for the delay in time between the detectors.
calibrated in relation to energy and efficiency. This is done In liquid chromatography some radiochemical impurities,
by using the peaks from known sources covering the desired such as colloidal impurities, may be retained on the column.
energy range, such as americium-241 and plutonium-242. In such cases a separate method is required for the
Not all alpha particles emitted by the source will produce a determination of the content of the retained radiochemical
count in the system. The probability that an emitted alpha impurities and the calculation formula for the expression of
particle will interact with the detector material and produce a the total radiochemical purity takes into account the relative
count is the efficiency of the detector, which depends on the amount of the retained radiochemical impurities.
geometry. One possibility to evaluate such retention problems during
DETECTION AND MEASUREMENT OF method validation is to evaluate the radioactivity recovery
RADIOACTIVITY IN COMBINATION WITH A from the column by measuring the total radioactivity
recovered from the chromatographic equipment with and
SEPARATION TECHNIQUE
without the column.
A radioactive preparation may contain the radionuclide in
different chemical forms other than the intended one. Method
Therefore it is necessary to separate the different substances The sample is diluted if necessary and then applied to the
containing the radionuclide and determine the percentage of column in the prescribed volume and conditions. In this
radioactivity due to the radionuclide concerned associated respect it is important to demonstrate the LOD and LOQ,
with the stated chemical form and the contribution to the and the linearity of the detector throughout the range of
total radioactivity due to the radionuclide concerned coming activities to be measured.
from other substances. For this purpose, instruments for the Flow-through detector A portion of the tubing where the
detection and measurement of radioactivity are used in eluate containing the radioactive species is flowing is placed
combination with a physico-chemical separation technique. in front of or within the detector. Counting efficiency may be
In principle, any method of separation may be used. increased using a longer portion of the tube (e.g. making
Monographs for radiopharmaceutical preparations may multiple turns in front of or within the detector); however,
include the combined use of radioactivity measurement with this will reduce the ability of the system to separate 2 closely
paper chromatography (2.2.26), thin-layer chromatography eluting peaks of radioactivity.
(2.2.27), gas chromatography (2.2.28), liquid When the radiochemical purity test prescribes determination
chromatography (2.2.29), size-exclusion chromatography of the total radiochemical impurities or there is a quantitative
(2. 2.30) or electrophoresis (2. 2. 31). determination of an individual impurity, it is important to
In all cases the radioactivity of each analyte is measured after choose an appropriate threshold setting and appropriate
the separation has been achieved using the stated method. conditions for integration of the peak areas. In such tests the
Radioactivity measurement may be performed using detectors disregard limit, i.e. the limit at or below which a peak is
mounted in series with other detectors in analytical disregarded, is dependent on the method and is related to the
instruments, such as liquid chromatographs, making in-line limit of detection and limit of quantification. Thus, the
detection of analytes, or performed off-line, i.e. after the threshold setting of the data collection system corresponds to
analytical separation has been completed, by measuring the at least half of the disregard limit.
radioactivity of eluate fractions obtained after liquid Record the signal of the detectors as a function of time.
chromatographic separation of equal volume or as the Identification of peaks in the radiometric signal
distribution of radioactivity on paper chromatography or (radiochromatogram) is made on the basis of the retention
thin-layer chromatography supports. time of the analytes. The profile from other detectors may be
IN-LINE DETECTION AND MEASUREMENT OF used for this purpose.
RADIOACTIVIIY IN COMBINATION WITH LIQUID Quantification of the different components of chromatogram
CHROMATOGRAPHY and radiochromatogram profiles is made on the basis of peak
Apparatus areas. Peak areas are usually obtained by direct integration of
Liquid chromatography (see 2.2.29) may be used to separate the detector signal using commercially available software.
the principal radioactive substance of a radiopharmaceutical
preparation from radiochemical impurities or degradation
2023 Appendix V R V-A305

OFF-LINE DETECTION AND MEASUREMENT OF The detector is connected to a suitable counting device, so
RADIOACTIVI1Y that the radioactivity revealed can be measured quantitatively
Liquid chromatography (2.2.29). Provided the retention and the count rate related spatially to the surface scanned.
times of the various radiochemical species are reproducibly The radioactivity is automatically reported against the
consistent, an alternative method of radioactivity development distance and the profile describes peaks having
quantification is to collect the liquid chromatography effluent an area proportional to the number of counts per unit of
in a series of timed samples (fractions) for off-line analysis for distance.
radioactivity content. The radioactivity in the fractions
Radioactivity counter In the case where a maximum of
corresponding to the peaks can be expressed as a percentage
only 3 radiochemical components needs to be identified and
of the total of the radioactivity in all fractions, taking into
they are fully separated, the support can be cut into equal
account the limit of quantification.
strips, each having a size not more than half the length of the
Method support corresponding to the difference between the
The sample is applied on the column in the prescribed retardation factors of the 2 closest spots. Each single strip is
volume and conditions. Fractions are collected at the end of numbered starting from the origin side and counted
the chromatographic line. separately. Alternatively, for well-characterised systems the
The volume between the detector used to identify the support may be cut into 2 or more unequal portions, folded
retention time of the peaks and the collection point is if necessary to approximately equal geometry before
measured and a delay factor is calculated on the basis of the counting. An ionisation chamber or a scintillation counter
effluent flow rate and applied to each peak to estimate the can be used for this purpose, provided they are used within
time of elution of the peak at the point of collection. the instrument's linearity range and above its LOQ.
The fractions are collected on the basis of a fixed time Autoradiography This may also be used to acquire an
interval or at the time of appearance estimated from the image of the radioactivity distribution on the
delay time so that any relevant peak is collected in one or chromatographic support. In this case, the response of the
more fractions. system used for the acquisition of the image, such as a
The radioactivity of each fraction is counted using a phosphor imager or a photographic film, must be shown to
calibrated instrument such as a dose calibrator or a be linear with respect to the radioactivity in the
scintillation detector, taking into account the limit of chromatogram. Otherwise the system must be pre-calibrated
quantification and the linearity. or exposed at the same time to a series of reference
An elution profile is obtained tabulating the counts per radioactive sources, obtained by dilution from a calibrated
fraction against the elution time or volume. The activity of standard solution, covering the expected radioactivity range
fractions belonging to the same peak may be summed and that may be present on the support.
the relative percentage calculated to define radiochemical Method
purity. Deposit the required amount of sample at the origin of the
Thin-layer chromatography (2.2.27) and paper chromatographic support, with drying if necessary to avoid
chromatography (2.2.26). Provided a thin-layer spreading of the spot. Develop the chromatogram according
chromatography or a paper chromatography analytical to the prescribed method. A carrier may be added when
method has been validated for the separation of components prescribed in a particular monograph.
of a radioactive preparation, the number and relative In paper and thin-layer chromatography, it is preferable not
intensities of the separated spots can be detected and to dilute the preparation to be examined but it is important
measured using a radioactivity detector that can relate the to avoid depositing such a quantity of radioactivity that
radioactivity to a specific position on the chromatographic counting losses by coincidence (dead-time losses) occur
support. during measurement of the radioactivity.
The positions of the spots (peaks) may permit chemical After development, the support is dried and the positions of
identification by comparison with solutions of the same the radioactive areas are detected by measurement of
chemical substances (non-radioactive), using a suitable radioactivity over the length of the chromatogram, using a
detection method. suitable collimated counter, by autoradiography, or by
Apparatus cutting the strips into portions and counting each portion.
Scanning device The apparatus generally comprises a Radioactivity may be measured by integration using an
radioactivity detector, such as a position-sensitive automatic-plotting instrument or a digital counter.
proportional counter or a collimated scintillation detector The ratios of the areas under the peaks give the ratios of the
placed at a fixed distance from a scanning platform where the percentages of radioactivity due to the respective
chromatographic support to be scanned is positioned. radiochemical substances.
The radioactivity of the sample applied to the When the strips are cut into portions, the ratios of the
chromatography support must result in a counting rate in the quantities of radioactivity measured give the ratio of
linearity range of the equipment and the sample may be percentages of radioactivity due to the respective
diluted if necessary. The area to be scanned is positioned at radiochemical species.
the reference position so that the desired lane is aligned with Calibration It is important to demonstrate the limits of
the detector scanning trip. Adjust the scanning time to allow detection and quantification, and the linearity of the detector
enough counting time during the run. throughout the range of activities to be measured and in all
The detector or the platform may be moved in-plane, along positions on the support of the chromatographic system. This
the x-axis or the y-axis, so that the entire surface can be may be done by applying samples covering a range of
scanned during a single run. activities from 0.1 per cent to 100 per cent of the expected
range. Prepare the samples by dilution and apply equal
volumes of each, with drying if necessary. After examining
V-A306 Appendix V S 2023

the radioactivity profile using the equipment's standard Fe= ex !is (2)
settings, the peak areas are integrated for comparison with
the calculated amount of radioactivity applied to each spot.
c spring constant in newtons per metre;
Verify that the response of the detector over the complete As length change due to elastic deformation in metres; strain
length and width of the detector path is the same, as the gauges are used to measure the strain or elongation and convert
response may vary with the detector position. it into electrical resistance;

The peak-resolving power is influenced by the size of the

- an electromagnetic force that holds the load cell in
spot, the total radioactivity of the radionuclide and the
equilibrium. In most high-resolution balances, this is the
detector equipment. It can be checked by applying 5 µL
Lorentz force which is generated by a current inside a coil
spots separated by distances increasing from 4 mm to 20 mm
surrounding a permanent magnet.
in 2 mm increments. The approximate resolution of the
detection system can be determined from the radioactivity MECHANICAL BAI.ANCES
profile as the distance between the 2 spots where the baseline Most mechanical balances are equal-arm beam balances.
is only just clearly separated. An equal-arm beam balance performs a mass comparison
with a two-arm lever and two weighing pans. The mass of
the object being weighed is compensated for by counter
weights of known mass at the opposite end of the lever.
The counter weights are chosen in such a way that the beam
S. Balances for Analytical Purposes of the balance is in equilibrium position.
(Ph. Bur. metlwd 2.1. 7)
EQUIPMENT
The scope of this chapter is limited to balances used for Balances can be further classified according to their scale
analytical purposes. It does not cover balances used for interval, d, also known as readability. This is the smallest
manufacturing or other purposes. Any weighings performed increment of mass that can be indicated on the balance.
as part of tests prescribed to establish compliance with a
monograph of the European Pharmacopoeia must be carried Type (sub-tJ,pe) of balance Readability, d (in grams)
out according to the principles outlined in this chapter. Precision 10- 1 to 10- 3
Information about significant digits and rounding for mass Analytical :S 10-•
values prescribed in a monograph or any other chapter of the Semi-micro 10-5
European Pharmacopoeia can be found in the General Micro 10-•
Notices (under Quantities). Ultra-micro 10-7

PRINCIPLE
A balance is an instrument used to determine the mass of an Most balances in use today show the result of weighings on a
object. The SI unit for mass is the kilogram, but its digital display. Mechanical balances, e.g. beam balances,
submultiples, e.g. µg, mg or g, are often used. without such displays are rarely used.
While weighing instruments make use of different physical Balances generally have some means of showing that the
principles of mass determination, the majority are based on indication has stabilised and can be recorded or printed.
the weight, i.e. the gravitational force, FG (expressed in Balances may be connected to other equipment which
newtons), exerted by Earth on the object being weighed. documents the result of the weighing procedure, e.g. printers
Weight is defined by the following expression: or electronic systems such as laboratory information
management systems.
Fa= m-g (1) Balances may be built into analytical equipment used for
tests such as loss on drying, thermogravimetry and dynamic
vapour sorption that determine properties of a sample by
m mass of the object in kilograms;
local acceleration due to gravity in metres per square second ( a:;
measuring changes in its mass, under defined conditions.
g
9.81 m-s- 2 at sea level). Balances used for these purposes also fall within the scope of
this general chapter, but more specific requirements than
The two most common gravitational force weighing given here may be found in the corresponding general
principles are force compensation (typically used in electronic chapters.
balances) and mass comparison with a known mass (typically INSTALI.ATION AND LOCATION
used in mechanical balances). It is recommended to follow the manufacturer's instructions
Depending on the measuring principle of the balance, the when installing a balance. It is particularly important to
mass is either measured directly (e.g. beam balance) or is ensure that the installation conditions and location do not
calculated from the weight using equation (1) (e.g. balance adversely affect the performance of the balance.
using electromagnetic force compensation). The environmental parameters that influence the
ELECTRONIC BAI.ANCES performance of balances include:
Most balances used are electronic and based on force - temperature, including changes of temperature caused by
compensation. In practice, the gravitational force exerted on direct sunlight;
the object being weighed can be compensated by: - ambient humidity and pronounced changes thereof.
- elastic deformation: the object being weighed presses The optimum relative humidity (RH percentage) during a
down onto a spring element that reacts with a weighing operation is between 40 per cent and
compensation force F0 expressed in newtons, given by 60 per cent;
the following equation: - barometric pressure;
- air currents: generated by heaters, air conditioners or
devices with ventilators (e.g. computers or large
laboratory equipment), or any airflow in doorways or
2023 Appendix V S V-A307

areas of high traffic (corridors) and, in the case of toxic The weighing vessel and the sample must have the same
and other special materials, when the balance is placed temperature as their surroundings and the balance.
inside a fume cupboard; EQUIPMENT PERFORMANCE
- dust;
Weighing instruments must be periodically calibrated and
- electrostatic forces: electrostatic charging can be
checked to ensure compliance with pre-defined requirements.
significantly reduced by using metal weighing vessels or
Performance checks must be carried out between
antistatic devices. Balances must always be grounded
calibrations.
(e.g. via the electric plug). Low relative humidity
increases the risk of electrostatic charging; Minimum requirements for performance checks are given
- magnetic forces (e.g. RF generators, magnetic fields from below. The frequency of the qualification and performance
other laboratory equipment); checks is defined in each user's quality management system.
- vibrations. CALIBRATION
Irrespective of the construction materials, the weighing bench Calibration is part of balance qualification and is performed
should be stable, non-magnetic and vibration-proof. It should by the user or by a suitable competent body. Its aim is to
also be protected against electrostatic charges establish traceability of measurement results to SI units
(e.g. by grounding). (metrological traceability). The calibration results include
A balance used for analytical purposes is designed to measure measurement uncertainty and are documented in a
small masses. The weighing pan of these balances is generally calibration certificate. To further maintain such traceability, it
located inside an enclosure to reduce dust collection and the is recommended to perform calibration before any
influence of air currents on the operation of the balance. maintenance operation is carried out on the balance that
significantly alters its measurement performance. 'Significant'
It is important to level balances correctly; most balances have operations include repairs, transfer of the balance to another
a bubble level which must be brought to the centre by location or mechanical adjustment of one or more weighing
modifying the height of the feet of the instrument, others are parameters. The balance must be re-calibrated after
equipped with an electronic levelling system. Balances must significant operations. Re-calibration is not necessary after
be adjusted after levelling, using either built-in weights (if less significant operations, which include levelling the balance
available) or external calibrated weights. or adjustments using built-in weights.
Balances must be allowed to warm up after they are
PERFORMANCE CHECKS
connected to the power supply. Typically, this may take from
Performance checks are carried out to evaluate the random
a few minutes for precision balances to up to several days for
and systematic error of a balance; they consist of measuring
ultra-micro balances, depending on the model. Electronic
precision and accuracy respectively and comparing the results
balances should be left powered up, as this allows them to
obtained to pre-defined acceptance criteria. Balances are
stay in thermal equilibrium.
considered suitable if none of these errors exceeds
WEIGHING VESSELS 0.10 per cent.
Special care must be taken to ensure that the weighing vessel In practice, performance checks focus on the two weighing
and the closure are made from an inert material that is parameters that most significantly affect the performance of
compatible with the sample. The size of the weighing vessel the instrument, i.e. repeatability, for precision, and sensitivity
must not compromise the repeatability and accuracy of the as the main component of the accuracy of the balance.
weighing process. Small weighing vessels tend to give the
most accurate results. However, it may sometimes be more Accuracy is also impacted by two other parameters:
practical to use larger weighing vessels, for example, a eccentricity and linearity. A quadratic addition of the errors
volumetric flask in the case of samples that are to be diluted of these individual parameters, rather than a more
after weighing to avoid potential transfer errors. conservative linear addition, provides a more realistic
approach to the assessment of the accuracy of the balance
If considered necessary, the influence of the weighing vessel because the three individual parameters are known to be
on the repeatability and accuracy of the measurements may largely independent of each other, and it is considered
be assessed by including the vessel as a tare in the unlikely that they will occur simultaneously and have the
corresponding equipment performance checks. same algebraic sign. Therefore, the acceptance criterion for
Care should also be taken to ensure that weighing vessels each individual parameter can be set at 0.05 per cent,
composed of materials with a high degree of electrical i.e. half the overall accuracy tolerance of 0.10 per cent. While
insulation (e.g. glass and plastic) are not electrostatically accuracy is impacted by all three parameters, the impact of
charged. eccentricity and linearity is typically less than that of
Weighing vessels are made of non-magnetic materials to sensitivity. Hence, during a performance check on accuracy,
prevent magnetic interference with the components of it can be considered sufficient to investigate sensitivity only
electronic balances. (at 0.05 per cent), all the more since the three individual
Vessels suitable for weighing solid materials include weighing parameters impacting accuracy are thoroughly evaluated
paper, dishes and funnels, or sealable vessels such as bottles, during calibration.
vials, and flasks, which can also be used for liquids. Repeatability
Weighing dishes are typically made from a polymer, glass or In most cases, the net mass of the material being weighed is
a metal such as aluminium. Antistatic weighing dishes are considerably smaller than the maximum capacity of the
available for use with materials that retain static electricity. balance. When weighing such small quantities, one of the
Weighing funnels are typically made from glass or from a major contributors to measurement uncertainty is random
polymer. The design of this type of weighing vessel combines error. This is estimated by the standard deviation of the
attributes of a weighing dish and a transfer funnel, which can indications that are obtained according to the following
simplify the transfer of a weighed powder to a narrow-necked procedure.
vessel such as a volumetric flask.
V-A308 Appendix V S 2023

Use a single weight denomination preferably in the lower end sensitivity test specification (0.05 per cent). If this ratio
of the measurement range, for example not more than cannot be achieved, the conventional mass value of the test
5 per cent of the maximum capacity of the balance. load (stated on the calibration certificate of the weight) must
However, if this yields a test load below 100 mg, 100 mg be used for the assessment. In this case, the user must ensure
should be used, as smaller weights are difficult to handle. that the weight uncertainty divided by the nominal weight is
Zero the instrument, place the chosen test load on the not greater than one third of 0.05 per cent.
weighing pan and record the indication. Repeat the whole USE OF REFERENCE WEIGHTS
procedure, including zeroing, at least 10 times. The reference weights used for calibration and the sensitivity
The repeatability is satisfactory, if: test (and any other optional test for assessing the balance
accuracy) comply with either OIML R-111 or ASTM E-617
2XS (3)
standards (notably concerning metrological traceability).
- X 100 '.S: 0.10 Other weights can be used for the repeatability test, provided
msnw
their mass does not change during the test.
NOTE: ifs < 0.41 x d, where dis the readability (scale USE OF BUILT-IN WEIGHTS
interval) of the balance, replaces by 0.41 x d; In addition to testing weighing instruments with external
weights, it is accepted practice to adjust the instruments by
standard deviation of the indicated values (e.g. in grams); means of built-in weights. This makes it possible to reduce
m,,,w smallest net weight (e.g. in grams). Tus value is defined by the the frequency of sensitivity tests with external reference
user as the smallest net amount of substance that will be
weighed on the balance.
weights. For electronic balances with a built-in weight, daily
sensitivity testing with an external reference weight is not
The lower limit of "0.41 x d" for the standard deviation considered necessary, but a test with external weights should
originates from the rounding error of the balance. Given that nevertheless be conducted periodically since it allows
weighing operations comprise two readings (tare and net detection of any problems with the built-in weights
sample weight), and as the rounding error allocated to a themselves.
single reading is calculated as "0.29 x t:f', in this case the WEIGHING PROCEDURE
propagation of errors by a quadratic sum gives "0.41 x t:f'. The balance and weighing vessel must be appropriate for the
Based on the result of the repeatability test, the minimum quantity to be weighed and the expected performance. Both
weight (mmiJ of the balance can be determined. the balance and the weighing vessel must be clean and dry.
The "minimum weight" is the smallest net sample mass that The gross weight of the weighing vessel plus the material to
can be weighed on the balance, whilst continuing to comply be weighed must not exceed the maximum capacity of the
with the repeatability test criterion. It is given by the balance.
following equation: It is important that samples and weighing vessels are
equilibrated to the ambient laboratory temperature in order
lnm.in = 2000 X S (4) to avoid weighing errors. For example, a flask that is warmer
than ambient air warms up this air, which then flows upward
NOTE: ifs< 0.41 x d, replaces by 0.41 x d along the flask and reduces the apparent weight of the
Sensitivity contents by viscous friction.
The sensitivity test assesses the parameter that most Place the weighing vessel on the balance, taking care to
significantly influences the accuracy of the balance. centre it on the weighing pan. Avoid handling the vessel with
The sensitivity deviation increases approximately linearly with bare hands as this may affect the temperature and relative
the load, and thus is more significant in the upper part of the humidity. Tweezers can be used instead. After the balance
weighing range. In addition, as the influence of the random display stabilises, tare the balance, and without spilling, add
error is dominant at the lower end of the measuring range, the desired amount of material to the weighing vessel. Allow
using a test load with a mass below 5 per cent of the capacity the balance display to stabilise and record the mass. If a
of the balance to determine the error of sensitivity is not material transfer is necessary, take care to perform this
meaningful. operation quantitatively or, if it cannot be guaranteed that
the entire amount has been transferred, weigh the weighing
Sensitivity is assessed using a single test load with a mass of
vessel again and note the difference.
between 5 per cent and 100 per cent of the capacity of the
balance. Zero the instrument, place the selected test load on SAMPLES
the weighing pan and record the indication. For volatile, deliquescent or hygroscopic samples, it may be
The sensitivity is satisfactory, if: advantageous for analysts to use a vessel with a small opening
or a vessel equipped with a gas-tight closure, ensuring the
closure is put in place rapidly following weighing.
II - ml x 100 S: 0.05 (5)
The analyst can also tare both the vessel and closure prior to
m
the operation, then add the material, close the vessel and
record the indication.
m nominal weight of the test load, or its conventional mass (see ELECTROSTATIC SAMPLES
conditions below), e.g. in grams; Problems may be experienced when weighing electrostatic
I indication, e.g. in grams.
samples, since electrostatic forces may render them difficult
to handle and can lead to incorrect and non-reproducible
It is generally sufficient to use the nominal weight as the test results. In such cases, an anti-electrostatic system can be
load value for the assessment, as long as the relative helpful. Plastic and glass containers should be avoided. Metal
maximum permissible error of the test load containers usually reduce or prevent electrostatic charging.
(i.e. the maximum permissible error of the test load divided The risk of electrostatic effects is higher in working
by the nominal weight) is not more than one third of the
2023 Appendix VI V-A309

environments with low relative humidity, e.g. below

40 per cent RH. Appendix VI
MAGNETIC SAMPLES
Magnetic forces between a magnetic sample and parts of the Qualitative Reactions and Tests
balance or the environment add to gravity and may lead to (Ph. Eur. method 2.3.1)
incorrect and non-reproducible results. Special care should
therefore be taken when weighing these samples, for example, Identification reactions of ions and functional groups
by shielding them with a suitable material such as mu-metal. ACETATES
It may also be useful to increase the distance between the a) Heat the substance to be examined with an equal quantity
sample and metallic parts of the balance, as magnetic forces of oxalic acid R. Acid vapours with the characteristic odour of
decrease with increasing distance. acetic acid are liberated, showing an acid reaction (2.2.4).
b) Dissolve about 30 mg of the substance to be examined in
3 mL of water R or use 3 mL of the prescribed solution.
Add successively 0.25 mL of lanthanum nitrate solution R,
0.1 mL of 0.05 M iodine and 0.05 mL of dilute ammonia R2.
Heat carefully to boiling. Within a few minutes a blue
precipitate is formed or a dark blue colour develops.
ACETYL GROUPS
In a test-tube about 180 mm long and 18 mm in external
diameter, place about 15 mg of the substance to be
examined, or the prescribed quantity, and 0.15 mL of
phosphoric acid R. Close the tube with a stopper through
which passes a small test-tube about 100 mm long and
10 mm in external diameter containing water R to act as a
con,denser. On the outside of the smaller tube, hang a drop
of ianthq.num nitrate solution R. Except for substances
hydrolysable only with difficulty, place the apparatus in a
water-bath for 5 min, then take out the smaller tube. Remove
the drop and mix it with 0.05 mL of 0.01 M iodine on a tile.
Add at the edge 0.05 mL of dilute ammonia R2. After 1 min
to 2 min, a blue colour develops at the junction of the two
drops; the colour intensifies and persists for a short time.
For substances hydrolysable only with difficulty heat the mixture
slowly to boiling over an open flame and then proceed as
prescribed above.
ALKALOIDS
Dissolve a few milligrams of the substance to be examined,
or the prescribed quantity, in 5 mL of water R, add dilute
hydrochloric acid R until an acid reaction occurs (2.2.4), then
1 mL of potassium iodobismuthate solution R. An orange or
orange-red precipitate is formed immediately.
ALUMINIUM SALTS
Dissolve about 15 mg of the substance to be examined in
2 mL of water R or use 2 mL of the prescribed solution.
Add about 0.5 mL of dilute hydrochloric acid Rand about
0.5 mL of thioacetamide reagent R. No precipitate is formed.
Add dropwise dilute sodium hydroxide solution R. A gelatinous
white precipitate is formed which dissolves on further
addition of dilute sodium hydroxide solution R. Gradually add
ammonium chloride solution R. The gelatinous white precipitate
is re-formed.
AMINES, PRIMARY AROMATIC
Acidify the prescribed solution with dilute hydrochloric acid R
and add 0.2 mL of sodium nitrite solution R. After 1 min to
2 min, add 1 mL of fJ-naphthol solution R. An intense orange
or red colour and usually a precipitate of the same colour are
produced.
AMMONIUM SALTS
To the prescribed solution add 0.2 g of magnesium oxide R.
Pass a current of air through the mixture and direct the gas
that escapes just beneath the surface of a mixture of 1 mL of
0.1 M hydrochloric acid and 0.05 mL of methyl red solution R.
The colour of the indicator changes to yellow. On addition of
V-A310 Appendix VI 2023

1 mL of a freshly prepared 100 g!L solution of sodium which on addition of 0.05 mL to 0.1 mL of sodium sulfide
cobaltinitrite R a yellow precipitate is formed. solution R turns brown.
AMMONIUM SALTS AND SALTS OF VOLATILE b) To about 45 mg of the substance to be examined add
BASES 10 mL of dilute nitric acid R or use 10 mL of the prescribed
Dissolve about 20 mg of the substance to be examined in solution. Boil for 1 min. Allow to cool and filter if necessary.
2 mL of water R or use 2 mL of the prescribed solution. To 5 mL of the solution obtained add 2 mL of a 100 giL
Add 2 mL of dilute sodium hydroxide solution R. On heating, solution of thiourea R. A yellowish-orange colour or an
the solution gives off vapour that can be identified by its orange precipitate is formed. Add 4 mL of a 25 giL solution
odour and by its alkaline reaction (2.2.4). of sodium fluoride R. The solution is not decolorised within
30 min.
ANTIMONY COMPOUNDS
Dissolve with gentle heating about 10 mg of the substance to BROMIDES
be examined in a solution of 0.5 g of sodium potassium a) Dissolve in 2 mL of water R a quantity of the substance to
tartrate R in 10 mL of water R and allow to cool: to 2 mL of be examined equivalent to about 3 mg of bromide (Brl or
this solution, or to 2 mL of the prescribed solution, add use 2 mL of the prescribed solution. Acidify with dilute nitric
sodium sulfide solution R dropwise; an orange-red precipitate is acid R and add 0.4 mL of silver nitrate solution RI. Shake and
formed which dissolves on addition of dilute sodium hydroxide allow to stand. A curdled, pale yellow precipitate is formed.
solution R. Centrifuge and wash the precipitate with three quantities,
each of 1 mL, of water R. Carry out this operation rapidly in
ARSENIC COMPOUNDS subdued light disregarding the fact that the supernatant
Heat 5 mL of the prescribed solution on a water-bath with solution may not become perfectly clear. Suspend the
an equal volume of hypophosphorous reagent R. A brown precipitate obtained in 2 mL of water Rand add 1.5 mL of
precipitate is formed. ammonia R. The precipitate dissolves with difficulty.
BARBITURATES, NON-NITROGEN SUBSTITUTED b) Introduce into a small test-tube a quantity of the
Dissolve about 5 mg of the substance to be examined in substance to be examined equivalent to about 5 mg of
3 mL of methanol R, add 0.1 mL of a solution containing bromide (Br-) or the prescribed quantity. Add 0.25 mL of
100 g!L of cobalt nitrate R and 100 giL of calcium chloride R. water R, about 75 mg of lead dioxide R, 0.25 mL of acetic
Mix and add, with shaking, 0.1 mL of dilute sodium hydroxide acid R and shake gently. Dry the inside of the upper part of
solution R. A violet-blue colour and precipitate are formed. the test-tube with a piece of filter paper and allow to stand
for 5 min. Prepare a strip of suitable filter paper of
BENZOATES
appropriate size. Impregnate it by capillarity, by dipping the
a) To 1 mL of the prescribed solution add 0.5 mL of ferric
tip in a drop of decolorised fuchsin solution R and introduce the
chloride solution RI. A dull-yellow precipitate, soluble in
impregnated part immediately into the tube. Starting from
ether R, is formed.
the tip, a violet colour appears within 10 s that is clearly
b) Place 0.2 g of the substance to be examined, treated if distinguishable from the red colour of fuchsin, which may be
necessary as prescribed, in a test-tube. Moisten with 0.2 mL visible on a small area at the top of the impregnated part of
to 0.3 mL of sulfuric acid R. Gently warm the bottom of the the paper strip.
tube. A white sublimate is deposited on the inner wall of the
tube. CALCIUM AND CALCIUM SALTS
For monographs from the European Pharmacopoeia, use tests A
c) Dissolve 0.5 g of the substance to be examined in 10 mL
and B only.
of water R or use 10 mL of the prescribed solution.
Add 0.5 mL of hydrochloric acid R. The precipitate obtained, a) To 0.2 mL of a neutral solution containing a quantity of
after crystallisation from warm water R and drying in vacua, the substance to be examined equivalent to about 0.2 mg of
has a melting point (2. 2.14) of 120 °C to 124 °C. calcium (Ca 2 +) per millilitre or to 0.2 mL of the prescribed
solution add 0.5 mL of a 2 giL solution of
BICARBONATES glyoxalhydroxyanil R in ethanol (96 per cent) R, 0.2 mL of
a) Introduce into a test tube 0.1 g of the substance being dilute sodium hydroxide solution Rand 0.2 mL of sodium
examined suspended in 2 mL of water or use 2 mL of the carbonate solution R. Shake with 1 mL to 2 mL of
prescribed solution. Add 3 mL of 2M acetic acid, close the chloroform R and add 1 mL to 2 mL of water R.
tube immediately using a stopper fitted with a glass tube bent The chloroform layer is coloured red.
at two right angles. The solution or suspension effervesces.
b) Dissolve about 20 mg of the substance to be examined or
Heat gently and collect the gas in 5 mL of a 4.73% w/v
the prescribed quantity in 5 mL of acetic acid R. Add 0.5 mL
solution of barium hydroxide. A white precipitate is produced
of potassium ferrocyanide solution R. The solution remains
which dissolves on addition of an excess of 7M hydrochloric
clear. Add about 50 mg of ammonium chloride R. A white,
acid.
crystalline precipitate is formed.
b) Treat a solution of the substance being examined with a
c) To 5 mL of 0.4% w/v solution of the substance being
solution of magnesium sulfate; no precipitate is produced
examined add 0.2 mL of a 2% w/v solution of ammonium
(distinction from carbonates). Boil; a white precipitate is
oxalate. A white precipitate is produced which is only
produced.
sparingly soluble in 6M acetic acid but is soluble in
c) A solution liberates carbon dioxide when boiled. hydrochloric acid.
BISMUTH AND BISMUTH COMPOUNDS CARBONATES
a) To 0.5 g of the substance to be examined add 10 mL of a) Introduce into a test tube 0.1 g of the substance being
dilute hydrochloric acid R or use 10 mL of the prescribed examined suspended in 2 mL of water or use 2 mL of the
solution. Heat to boiling for 1 min. Cool and filter if prescribed solution. Add 3 mL of 2M acetic acid, close the
necessary. To 1 mL of the solution obtained add 20 mL of tube immediately using a stopper fitted with a glass tube bent
water R. A white or slightly yellow precipitate is formed at two right angles. The solution or suspension effervesces
2023 Appendix VI V-A311

evolving a colourless and odourless gas. Heat gently and solution Rl diluted ten times. A bluish-red or red colour is
collect the gas in 5 mL of 0.lM barium hydroxide. A white produced.
precipitate is produced which dissolves on addition of an IODIDES
excess of 7M hydrochloric acid.
a) Dissolve a quantity of the substance to be examined
b) Treat a solution of the substance being examined with a equivalent to about 4 mg of iodide (n in 2 mL of water R or
solution of magnesium sulfate. A white precipitate is produced use 2 mL of the prescribed solution. Acidify with dilute nitric
(distinction from bicarbonates). acid Rand add 0.4 mL of silver nitrate solution Rl. Shake and
CARBONATES AND BICARBONATES allow to stand. A curdled, pale-yellow precipitate is formed.
Introduce into a test-tube 0.1 g of the substance to be Centrifuge and wash with three quantities, each of 1 mL, of
examined and suspend in 2 mL of water R or use 2 mL of water R. Carry out this operation rapidly in subdued light
the prescribed solution. Add 3 mL of dilute acetic acid R. disregarding the fact that the supernatant solution may not
Close the tube immediately using a stopper fitted with a glass become perfectly clear. Suspend the precipitate in 2 mL of
tube bent twice at right angles. The solution or the water R and add 1.5 mL of ammonia R. The precipitate does
suspension becomes effervescent and gives off a colourless not dissolve.
and odourless gas. Heat gently and collect the gas in 5 mL of b) To 0.2 mL of a solution of the substance to be examined
barium hydroxide solution R. A white precipitate is formed that containing about 5 mg of iodide (n per millilitre, or to
dissolves on addition of an excess of hydrochloric acid Rl. 0.2 mL of the prescribed solution, add 0.5 mL of dilute
sulfuric acid R, 0.1 mL of potassium dichromate solution R,
Clll.ORIDES
2 mL of water R and 2 mL of chloroform R. Shake for a few
a) Dissolve in 2 mL of water R a quantity of the substance to
seconds and allow to stand. The chloroform layer is coloured
be examined equivalent to about 2 mg of chloride (Cll or
violet or violet-red.
use 2 mL of the prescribed solution. Acidify with dilute nitric
acid Rand add 0.4 mL of silver nitrate solution Rl. Shake and IRON AND IRON SALTS
allow to stand. A curdled, white precipitate is formed. a) Dissolve a quantity of the substance to be examined
Centrifuge and wash the precipitate with three quantities, equivalent to about 10 mg of iron (Fe2 +) in 1 mL of water R
each of 1 mL, of water R. Carry out this operation rapidly in or use 1 mL of the prescribed solution. Add 1 mL of
subdued light, disregarding the fact that the supernatant potassium ferricyanide solution R. A blue precipitate is formed
solution may not become perfectly clear. Suspend the that does not dissolve on addition of 5 mL of dilute
precipitate in 2 mL of water R and add 1.5 mL of hydrochloric acid R.
ammonia R. The precipitate dissolves easily with the possible b) Dissolve a quantity of the substance to be examined
exception of a few large particles which dissolve slowly. equivalent to about 1 mg of iron (Fe 3 +) in 30 mL of water R.
b) Introduce into a test-tube a quantity of the substance to To 3 mL of this solution or to 3 mL of the prescribed
be examined equivalent to about 15 mg of chloride (Cr) or solution, add 1 mL of dilute hydrochloric acid R and 1 mL of
the prescribed quantity. Add 0.2 g of potassium dichromate R potassium thiocyanate solution R. The solution is coloured red.
and 1 mL of sulfuric acid R. Place a filter-paper strip Take two portions, each of 1 mL, of the mixture. To one
impregnated with 0.1 mL of diphenylcarbazide solution Rover portion add 5 mL of isoamyl alcohol R or 5 mL of ether R.
the opening of the test-tube. The paper turns violet-red. Shake and allow to stand. The organic layer is coloured pink.
The impregnated paper must not come into contact with the To the other portion add 2 mL of mercuric chloride solution R.
potassium dichromate. The red colour disappears.
CITRATES c) Dissolve a quantity of the substance to be examined
For monographs from the European Pharmacopoeia, use test A equivalent to not less than 1 mg of iron (Fe3 +) in 1 mL of
only. water R or use 1 mL of the prescribed solution. Add 1 mL of
potassium f errocyanide solution R. A blue precipitate is formed
a) Dissolve in 5 mL of water R a quantity of the substance to
that does not dissolve on addition of 5 mL of dilute
be examined equivalent to about 50 mg of citric acid or use
hydrochloric acid R.
5 mL of the prescribed solution. Add 0.5 mL of sulfuric
acid Rand 1 mL of potassium permanganate solution R. Warm LACTATES
until the colour of the permanganate is discharged. Dissolve a quantity of the substance to be examined
Add 0.5 mL of a 100 g/L solution of sodium nitroprusside R in equivalent to about 5 mg of lactic acid in 5 mL of water R or
dilute suljuric acid R and 4 g of sulfamic acid R. Make alkaline use 5 mL of the prescribed solution. Add 1 mL of bromine
with concentrated ammonia R, added dropwise until all the water R and 0.5 mL of dilute suljuric acid R. Heat on a water-
sulfamic acid has dissolved. Addition of an excess of bath until the colour is discharged, stirring occasionally with
concentrated ammonia R produces a violet colour, turning to a glass rod. Add 4 g of ammonium sulfate R and mix.
violet-blue. Add dropwise and without mixing 0.2 mL of a 100 g/L
b) To a neutral solution of the substance being examined solution of sodium nitroprusside R in dilute suljuric acid R. Still
add a solution of calcium chloride; no precipitate is produced. without mixing add 1 mL of concentrated ammonia R. Allow
Boil the solution; a white precipitate is produced which is to stand for 30 min. A dark green ring appears at the
soluble in 6M acetic acid. junction of the two liquids.
ESTERS LEAD AND LEAD COMPOUNDS
To about 30 mg of the substance to be examined or the a) Dissolve 0.1 g of the substance to be examined in 1 mL of
prescribed quantity add 0.5 mL of a 70 g/L solution of acetic acid R or use 1 mL of the prescribed solution.
hydroxylamine hydrochloride R in methanol Rand 0.5 mL of a Add 2 mL of potassium chromate solution R. A yellow
100 iYL solution of potassium hydroxide R in ethanol precipitate is formed that dissolves on addition of 2 mL of
(96 per cent) R. Heat to boiling, cool, acidify with dilute strong sodium hydroxide solution R.
hydrochloric acid Rand add 0.2 mL of ferric chloride b) Dissolve 50 mg of the substance to be examined in 1 mL
of acetic acid R or use 1 mL of the prescribed solution.
V-A312 Appendix VI 2023

Add 10 mL of water R and 0 .2 mL of potassium iodide after recrystallisation from hot water R and drying in vacua,
solution R. A yellow precipitate is formed. Heat to boiling for has a melting point (2.2.14) of 156 °C to 161 °C.
1 min to 2 min. The precipitate dissolves. Allow to cool.
SILICATES
The precipitate is re-formed as glistening, yellow plates.
Mix the prescribed quantity of the substance to be examined
MAGNESIUM AND MAGNESIUM SALTS in a lead or platinum crucible by means of a copper wire
For monographs from the European Pharmacopoeia, use test A with about 10 mg of sodium fluoride R and a few drops of
only. sulfuric acid R to give a thin slurry. Cover the crucible with a
a) Dissolve about 15 mg of the substance to be examined in thin, transparent plate of plastic under which a drop of
2 mL of water R or use 2 mL of the prescribed solution. water R is suspended and warm gently. Within a short time a
Add 1 mL of dilute ammonia RJ. A white precipitate is white ring is rapidly formed around the drop of water.
formed that dissolves on addition of 1 mL of ammonium SILVER AND SILVER COMPOUNDS
chloride solution R. Add 1 mL of disodium hydrogen phosphate Dissolve about 10 mg of the substance to be examined in
solution R. A white crystalline precipitate is formed. 10 mL of water R or use 10 mL of the prescribed solution.
b) To 0.5 mL of a neutral or slightly acidic solution of the Add 0.3 mL of hydrochloric acid RJ. A curdled, white
substance being examined add 0.2 mL of a 0.1 % w/v precipitate is formed that dissolves on addition of 3 mL of
solution of titan yellow and 0.5 mL of 0.lM sodium hydroxide. dilute ammonia Rl.
A bright red turbidity is produced which gradually settles to
SODIUM AND SODIUM SALTS
give a bright red precipitate.
a) Dissolve 0.1 g of the substance to be examined in 2 mL of
MERCURY AND MERCURY COMPOUNDS water R or use 2 mL of the prescribed solution. Add 2 mL of
a) Place about 0.1 mL of a solution of the substance to be a 150 g/L solution of potassium carbonate R and heat to
examined on well-scraped copper foil. A dark-grey stain that boiling. No precipitate is formed. Add 4 mL of potassium
becomes shiny on rubbing is formed. Dry the foil and heat in pyroantimonate solution R and heat to boiling. Allow to cool in
a test-tube. The spot disappears. iced water and if necessary rub the inside of the test-tube
b) To the prescribed solution add di7ute sodium hydroxide with a glass rod. A dense white precipitate is formed.
solution R until strongly alkaline (2.2.4). A dense yellow b) Dissolve a quantity of the substance to be examined
precipitate is formed (mercuric salts). equivalent to about 2 mg of sodium (Na+) in 0.5 mL of
NITRATES water R or use 0.5 mL of the prescribed solution.
Add 1.5 mL of methoxyphenylacetic reagent R and cool in ice-
To a mixture of0.l mL ofnitrobenzene Rand 0.2 mL of
water for 30 min. A voluminous, white, crystalline precipitate
sulfuric acid R, add a quantity of the powdered substance
is formed. Place in water at 20 °C and stir for 5 min.
equivalent to about 1 mg of nitrate (NO 3l or the prescribed
The precipitate does not disappear. Add 1 mL of dilute
quantity. Allow to stand for 5 min. Cool in iced water and
add slowly and with mixing 5 mL of water R, then 5 mL of
ammonia Rl. The precipitate dissolves completely. Add 1 mL
of ammonium carbonate solution R. No precipitate is formed.
strong sodium hydroxide solution R. Add 5 mL of acetone R.
Shake and allow to stand. The upper layer is coloured deep SUi.FATES
violet. a) Dissolve about 45 mg of the substance to be examined in
PHOSPHATES (ORTHOPHOSPHATES) 5 mL of water R or use 5 mL of the prescribed solution.
Add 1 mL of dilute hydrochloric acid R and 1 mL of barium
a) To 5 mL of the prescribed solution, neutralised if
necessary, add 5 mL of silver nitrate solution Rl. A yellow
chloride solutwn RJ. A white precipitate is formed.
precipitate is formed whose colour is not changed by boiling b) To the suspension obtained during reaction (a), add
and which dissolves on addition of ammonia R. 0.1 mL of 0.05 M iodine. The suspension remains yellow
(distinction from sulfites and dithionites), but is decolorised
b) Mix 1 mL of the prescribed solution with 2 mL of
molybdovanadic reagent R. A yellow colour develops. by adding dropwise stannous chloride solution R (distinction
from iodates). Boil the mixture. No coloured precipitate is
POTASSIUM AND POTASSIUM SALTS formed (distinction from selenates and tungstates).
a) Dissolve 0 .1 g of the substance to be examined in 2 mL of
TARTRATES
water R or use 2 mL of the prescribed solution. Add 1 mL of
a) Dissolve about 15 mg of the substance to be examined in
sodium carbonate solution R and heat. No precipitate is
5 mL of water R or use 5 mL of the prescribed solution.
formed. Add to the hot solution 0.05 mL of sodium sulfide
solutwn R. No precipitate is formed. Cool in iced water and Add 0.05 mL of a 10 g/L solution of ferrous sulfate Rand
add 2 mL of a 150 g/L solution of tartaric acid R. Allow to 0.05 mL of dilute hydrogen peroxide solution R. A transient
stand. A white crystalline precipitate is formed. yellow colour is produced. After the colour has disappeared
add dilute sodium hydroxide solution R dropwise. A violet or
b) Dissolve about 40 mg of the substance to be examined in
purple colour is produced.
1 mL of water R or use 1 mL of the prescribed solution.
b) To 0.1 mL of a solution of the substance to be examined
Add 1 mL of dilute acetic acid R and 1 mL of a freshly
prepared 100 g/L solution of sodium cobaltinitrite R. A yellow containing the equivalent of about 15 mg of tartaric acid per
or orange-yellow precipitate is formed immediately. millilitre or to 0.1 mL of the prescribed solution add 0.1 mL
of a 100 g/L solution of potassium bromide R, 0 .1 mL of a
SALICYLATES 20 g/L solution of resorcinol R and 3 mL of sulfuric acid R.
a) To 1 mL of the prescribed solution add 0.5 mL of ferric Heat on a water-bath for 5 min to 10 min. A dark-blue
chloride solutwn RJ. A violet colour is produced that persists colour develops. Allow to cool and pour the solution into
after the addition of 0.1 mL of acetic acid R. water R. The colour changes to red.
b) Dissolve 0.5 g of the substance to be examined in 10 mL XANTHINES
of water R or use 10 mL of the prescribed solution. To a few milligrams of the substance to be examined or the
Add 0.5 mL of hydrochloric acid R. The precipitate obtained,
prescribed quantity add 0.1 mL of strong hydrogen peroxide
2023 Appendix VII V-A3 l 3

solution Rand 0.3 mL of dilute hydrochloru acid R. Heat to

dryness on a water-bath until a yellowish-red residue is
obtained. Add 0.1 mL of dilute ammonia R2. The colour of
Appendix VII
the residue changes to violet-red. Limit Tests
ZINC AND ZINC SALTS
Dissolve 0.1 g of the substance to be examined in 5 mL of Nessler Cylinders
water R or use 5 mL of the prescribed solution. Add 0.2 mL (No Ph. Bur. method)
of strong sodium hydroxide solution R. A white precipitate is Where the use of Nessler cylinders is prescribed in a test of the
formed. Add a further 2 mL of strong sodium hydroxide Pharmacopoeia, Nessler cylinders complying with the
solution R. The precipitate dissolves. Add 10 mL of following requirements should be used.
ammonium chloride solution R. The solution remains clear. Nessler cylinders comply with British Standard 612:1966
Add 0.1 mL of sodium sulfide solution R. A flocculent white (Specification for Nessler cylinders). They are of clear glass
precipitate is formed. with a nominal capacity of 50 mL; the overall height is about
ODOUR 15 cm, the external height to the 50-mL mark 11.0 to
(Ph. Bur. method 2.3. 4) 12.4 cm, the thickness of the wall 1.0 to 1.5 mm and the
thickness of the base 1.0 to 3.0 mm. The external heights to
On a watch-glass 6 cm to 8 cm in diameter, spread in a thin
the 50-mL mark of cylinders used for a test must not differ
layer 0.5 g to 2.0 g of the substance to be examined. After
by more than 1 mm.
15 min, determine the odour or verify the absence of odour.
Tubes for Comparative Tests
(Ph. Bur. method 2.1.5)
Tubes used for comparative tests are matched tubes of
colourless glass with a uniform internal diameter. The base is
transparent and flat.
A column of the liquid is examined down the vertical axis of
the tube against a white background, or if necessary, against
a black background. The examination is carried out in
diffused light.
It is assumed that tubes with an internal diameter of 16 mm
will be used. Tubes with a larger internal diameter may be
used instead but the volume of liquid examined must then be
increased so that the depth of liquid in the tubes is not less
than where the prescribed volume of liquid and tubes 16 mm
in internal diameter are used.
Limit Test for Aluminium
(Ph. Bur. method 2. 4.1 7)
Place the prescribed solution in a separating funnel and shake
with 2 quantities, each of 20 mL, and then with one 10 mL
quantity of a 5 g/L solution of hydroxyquinoline R in
chloroform R. Dilute the combined chloroform solutions to
50.0 mL with chloroform R (test solution).
Prepare a standard in the same manner using the prescribed
reference solution.
Prepare a blank in the same manner using the prescribed
blank solution.
Measure the intensity of the fluorescence (2.2.21) of the test
solution (11), of the standard (12 ) and of the blank (13 ) using
an excitant beam at 392 nm and a secondary filter with a
transmission band centred on 518 nm or a monochromator
set to transmit at this wavelength.
The fluorescence (1 1-13) of the test solution is not greater
than that of the standard (/rl3 ).
Limit Test for Ammonium
(Ph. Bur. method 2.4.1)
Unless otherwise prescribed, use method A.
METHOD A
Introduce the prescribed solution into a test-tube or dissolve
the prescribed quantity of the substance to be examined in
14 mL of water R in a test-tube. Make the solution alkaline if
necessary by the addition of dilute sodium hydroxide solution R,
dilute to 15 mL with water R and add 0.3 mL of alkaline
potassium tetraiodomercurate solution R. Prepare a standard by
V-A314 Appendix VII 2023

mixing 10 mL of ammonium standard solutwn (1 ppm The second tube is bent twice at right angles and the free
NH,J R, 5 mL of water Rand 0.3 mL of alkaline potassium end of the tube tapers to an internal diameter of 1 mm. This
tetrawdomercurate solutwn R. Stopper the test-tubes. end is immersed in a test-tube containing 3.0 mL of silver
After 5 min, any yellow colour in the test solution is not dwthyldithwcarbamate solutwn R. Other suitable equipment
more intense than that in the standard. may be used. Into the first tube insert 50-60 mg of lead
acetate cotton R, loosely packed, or a small plug of cotton and
METHODB a rolled piece of lead acetate paper R weighing 50-60 mg.
In a 25 mL jar fitted with a cap, place the prescribed
In the conical flask, dissolve the prescribed quantity of the
quantity of the finely powdered substance to be examined
substance to be examined in 25 mL of water R, or in the case
and dissolve or suspend in 1 mL of water R. Add 0.30 g of
of a solution adjust the prescribed volume to 25 mL with
heavy magnesium oxide R. Close immediately after placing a
water R. Add 15 mL of hydrochloric acid R, 0.1 mL of
piece of silver manganese paper R 5 mm square, wetted with a
stannous chloride solution R and 5 mL of potassium iodide
few drops of water R, under the polyethylene cap. Swirl,
solutwn R, allow to stand for 15 min and introduce 5 g of
avoiding projections of liquid, and allow to stand at 40 °C for
activated zinc R. Assemble the 2 parts of the apparatus
30 min. If the silver manganese paper shows a grey colour, it
immediately and immerse the flask in a water-bath at a
is not more intense than that of a standard prepared at the
temperature such that a uniform evolution of gas is
same time and in the same manner using the prescribed
maintained. Prepare a standard in the same manner, using
volume of ammonium standard solutwn (1 ppm NH,J R, 1 mL
1 mL of arsenic standard solution (1 ppm As) R, diluted to
of water R and 0.30 g of heavy magnesium oxide R.
25 mL with water R. If foaming occurs, 1 mL of 2--propanol R
Limit Test for Arsenic may be added to the flask.
(Ph. Eur. method 2.4.2) After at least 2 h, the colour obtained in the test-tube with
the test solution is not more intense than that obtained with
the standard.
__,_,_.._.__ 5 Suitability test The colour obtained in the test-tube with
the standard is at least as intensely coloured as 3 mL of a
mixture of 3.0 mL of yellow primacy solution, 0.6 mL of red
primary solution and 11.40 mL of a solution of hydrochloric
acid R (10 g/L HCl) (2.2.2, Method I).
METHODB
Introduce the prescribed quantity of the substance to be
A examined into a test-tube containing 4 mL of hydrochloric
acid R and about 5 mg of potassium iodide R and add 3 mL of
hypophosphorous reagent R. Heat the mixture on a water-bath
for 15 min, shaking occasionally. Prepare a standard in the
same manner, using 0.5 mL of arsenic standard solutwn
(10 ppm As) R.
After heating on the water-bath, the colour obtained in the
test-tube with the test solution is not more intense than that
0 obtained with the standard.
(')
(')
N Limit Test for Calcium
(Ph. Eur. method 2.4.3)
All solutwns used for this test are prepared with distilled water R.
To 0.2 mL of alcoholic calcium standard solutwn
(100 ppm Ca) R add 1 mL of ammonium oxalate solution R.
r--- After 1 min add a mixture of 1 mL of dilute acetic acid R and
1
I 15 mL of the prescribed solution or of a solution containing
I
the prescribed quantity of the substance to be examined, and
shake. Prepare a standard in the same manner using a
A. Lead acetate paper/cotton
mixture of 10 mL of aqueous calcium standard solution
(10 ppm Ca) R, 1 mL of dilute acetic acid Rand 5 mL of
Figure 2.4.2.-1. -Apparatus for the limit test for arsenic distilled water R.
(method A) After 15 min, any opalescence in the test solution is not
Dimensions in millimetres more intense than that in the standard.
Limit Test for Chlorides
METHOD A (Ph. Eur. method 2.4.4)
The apparatus (see Figure 2.4.2.-1) consists of a 100 mL
To 15 mL of the prescribed solution add 1 mL of dilute nitric
conical flask closed with a ground-glass stopper through
acid Rand pour the mixture as a single addition into a test-
which passes a glass tube about 200 mm long and about
tube containing 1 mL of silver nitrate solutwn R2. Prepare a
5 mm in internal diameter. The lower part of the tube tapers
standard in the same manner using 10 mL of chloride
to an internal diameter of 1 mm, and about 20 mm from its
standard solution (5 ppm Cl) R and 5 mL of water R. Examine
tip is a lateral orifice 2-3 mm in diameter. When the tube is
the tubes laterally against a black background.
in position in the stopper, the lateral orifice is at least 3 mm
below the lower surface of the stopper. A second glass tube
of the same internal diameter is connected to the first tube.
2023 Appendix VII V-A3 l 5

After standing for 5 min protected from light, any

opalescence in the test solution is not more intense than that
in the standard.
Limit Test for Fluorides
(Ph. Bur. method 2.4.5)
Introduce into the inner tube of the apparatus (see
Figure 2.4.5.-1) the prescribed quantity of the substance to
be examined, 0.1 g of acid-washed sand R and 20 mL of a
mixture of equal volumes of sulfuric acid R and water R. Heat
the jacket containing tetrachloroethane R maintained at its
boiling point (146 cq. Heat the steam generator and distil,
collecting the distillate in a 100 mL volumetric flask
containing 0.3 mL of 0.1 M sodium hydroxide and 0.1 mL of
phenolphthakin solution R. Maintain a constant volume
(20 mL) in the tube during distillation and ensure that the
distillate remains alkaline, adding 0.1 M sodium hydroxide if
necessary. Dilute the distillate to 100 mL with water R (test
solution). Prepare a standard in the same manner by
distillation, using 5 mL of fluon·de standard solution
(10 ppm F) R instead of the substance to be examined. Into
two glass-stoppered cylinders introduce 20 mL of the test
solution and 20 mL of the standard and 5 mL of
aminomethylalizan·ndiacetic acid reagent R.
After 20 min, any blue colour in the test solution (originally
red) is not more intense than that in the standard.
Limit Test for Heavy Metals
(Ph. Bur. method 2.4.8)
The methods described below require the use of thioacetamide
reagent R. As an alternative, sodium sufjide solution Rl
(0.1 mL) is usually suitable. Since tests prescribed in
monographs have been developed using thioacetamide
reagent R, if sodium sufjide solution Rl is used instead, it is
necessary to include also for methods A, B and H a monitor
solution, prepared from the quantity of the substance to be
examined prescribed for the test, to which has been added
the volume of lead standard solution prescribed for
preparation of the reference solution. The test is invalid if the
monitor solution is not at least as intense as the reference
solution.
METHOD A
Test solution 12 mL of the prescribed aqueous solution of
the substance to be examined.
Reference solution (standard) A mixture of 10 mL of
lead standard solutwn (1 ppm Pb) R or lead standard solution
(2 ppm Pb) R, as prescribed, and 2 mL of the prescribed
aqueous solution of the substance to be examined.
Blank solution A mixture of 10 mL of water R and 2 mL
of the prescribed aqueous solution of the substance to be
examined.
To each solution, add 2 mL of buffer solution pH 3.5 R. Figure 2.4.5.-1. -Apparatus for limit test for fluorides
Mix and add to 1.2 mL of thioacetamide reagent R. Dimensions in millimetres
Mix immediately. Examine the solutions after 2 min.
System suitability The reference solution shows a slight METHODB
brown colour compared to the blank solution. Test solution 12 mL of the prescribed solution of the
Result Any brown colour in the test solution is not more substance to be examined prepared using an organic solvent
intense than that in the reference solution. containing a minimum percentage of water (for example,
dioxan containing 15 per cent of water or acetone containing
If the result is difficult to judge, filter the solutions through a
15 per cent of water).
suitable membrane filter (nominal pore size 0.45 µm). Carry
out the filtration slowly and uniformly, applying moderate Reference solution (standard) A mixture of 10 mL of
and constant pressure to the piston. Compare the spots on lead standard solution (1 or 2 ppm Pb), as prescribed, and
the filters obtained with the different solutions. 2 mL of the prescribed solution of the substance to be
examined in an organic solvent. Prepare the lead standard
solution (1 or 2 ppm Pb) by dilution of lead standard solutwn
V-A316 Appendix VII 2023

(100 ppm Pb) R with the solvent used for the substance to be METHODD
examined. Test solution In a silica crucible, mix thoroughly the
Blank solution A mixture of 10 mL of the solvent used prescribed quantity of the substance to be examined with
for the substance to be examined and 2 mL of the prescribed 0.5 g of magnesium oxide Rl. Ignite to dull redness until a
solution of the substance to be examined in an organic homogeneous white or greyish-white mass is obtained.
solvent. If after 30 min of ignition the mixture remains coloured,
To each solution, add 2 mL of buffer solution pH 3.5 R. allow to cool, mix using a fine glass rod and repeat the
Mix and add to 1.2 mL of thioacetamide reagent R. ignition. If necessary repeat the operation. Heat at 800 °C for
Mix immediately. Examine the solutions after 2 min. about 1 h. Take up the residue in 2 quantities, each of 5 mL,
of a mixture of equal volumes of hydrochloric acid Rl and
System suitability The reference solution shows a slight water R. Add 0.1 mL of phenolphthalein solution Rand then
brown colour compared to the blank solution.
concentrated ammonia R until a pink colour is obtained. Cool,
Result Any brown colour in the test solution is not more add glacial acetic acid R until the solution is decolorised and
intense than that in the reference solution. add 0.5 mL in excess. Filter if necessary and wash the filter.
If the result is difficult to judge, filter the solutions through a Dilute to 20 mL with water R.
suitable membrane filter (nominal pore size 0.45 µm). Carry Reference solution (standard) Prepare as described for
out the filtration slowly and uniformly, applying moderate the test solution using the prescribed volume of lead standard
and constant pressure to the piston. Compare the spots on solutwn (10 ppm Pb) R instead of the substance to be
the filters obtained with the different solutions. examined and drying in an oven at 100-105 °C. To 10 mL
METHODC of the solution obtained add 2 mL of the test solution.
Test solution Place the prescribed quantity (not more than Monitor solution Prepare as described for the test
2 g) of the substance to be examined in a silica crucible with solution, adding to the substance to be examined the volume
4 mL of a 250 g/L solution of magnesium sulfate R in dilute of lead standard solution (10 ppm Pb) R prescribed for
sulfuric acid R. Mix using a fine glass rod. Heat cautiously. preparation of the reference solution and drying in an oven at
If the mixture is liquid, evaporate gently to dryness on a 100-105 °C. To 10 mL of the solution obtained add 2 mL of
water-bath. Progressively heat to ignition and continue the test solution.
heating until an almost white or at most greyish residue is Blank solution A mixture of 10 mL of water R and 2 mL
obtained. Carry out the ignition at a temperature not of the test solution.
exceeding 800 °C. Allow to cool. Moisten the residue with a To 12 mL of each solution, add 2 mL of buffer solutwn
few drops of dilute sulfuric acid R. Evaporate, ignite again and pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R.
allow to cool. The total period of ignition must not exceed Mix immediately. Examine the solutions after 2 min.
2 h. Take up the residue in 2 quantities, each of 5 mL, of
System suitability:
dilute hydrochloric acid R. Add 0 .1 mL of phenolphthalein
- the reference solution shows a slight brown colour
solution R, then concentrated ammonia R until a pink colour is
compared to the blank solution,
obtained. Cool, add glacial acetic acid R until the solution is
- the monitor solution is at least as intense as the reference
decolorised and add 0.5 mL in excess. Filter if necessary and
wash the filter. Dilute to 20 mL with water R. solution.
Reference solution (standard) Prepare as described for Result Any brown colour in the test solution is not more
the test solution, using the prescribed volume of lead standard intense than that in the reference solution.
solutwn (10 ppm Pb) R instead of the substance to be If the result is difficult to judge, filter the solutions through a
examined. To 10 mL of the solution obtained add 2 mL of suitable membrane filter (nominal pore size 0.45 µm). Carry
the test solution. out the filtration slowly and uniformly, applying moderate
Monitor solution Prepare as described for the test and constant pressure to the piston. Compare the spots on
solution, adding to the substance to be examined the volume the filters obtained with the different solutions.
of lead standard solution (10 ppm Pb) R prescribed for METHODE
preparation of the reference solution. To 10 mL of the Test solution Dissolve the prescribed quantity of the
solution obtained add 2 mL of the test solution. substance to be examined in 30 mL of water R or the
Blank solution A mixture of 10 mL of water R and 2 mL prescribed volume.
of the test solution. Reference solution (standard) Unless otherwise
To 12 mL of each solution, add 2 mL of buffer solutwn prescribed, dilute the prescribed volume of lead standard
pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. solutwn (1 ppm Pb) R to the same volume as the test solution.
Mix immediately. Examine the solutions after 2 min. Prepare the filtration apparatus by adapting the barrel of a
System suitability: 50 mL syringe without its piston to a support containing, on
- the reference solution shows a slight brown colour the plate, a membrane filter (nominal pore size 3 µm) and
compared to the blank solution, above it a prefilter (Figure 2.4.8.-1).
- the monitor solution is at least as intense as the reference Transfer the test solution into the syringe barrel, put the
solution. piston in place and then apply an even pressure on it until
Result Any brown colour in the test solution is not more the whole of the liquid has been filtered. In opening the
intense than that in the reference solution. support and removing the prefilter, check that the membrane
If the result is difficult to judge, filter the solutions through a filter remains uncontaminated with impurities. If this is not
suitable membrane filter (nominal pore size 0.45 µm). Carry the case replace it with another membrane filter and repeat
out the filtration slowly and uniformly, applying moderate the operation under the same conditions.
and constant pressure to the piston. Compare the spots on To the prefiltrate or to the prescribed volume of the
the filters obtained with the different solutions. prefiltrate add 2 mL of buffer solutwn pH 3.5 R. Mix and add
to 1.2 mL of thioacetamide reagent R. Mix immediately and
2023 Appendix VII V-A31 7

C"')
Membrane
co Prefilter
T""" filter
i:-.-i·.· :. ·-,:-.-/·.-:. ·. ,:-.-] r:•::,{'' :;·:.,- ._,. ::·:.,
Membrane Prefilter
filter

Prefiltration Filtration of the

of the solution solution after
(Method E) addition of the
reagents
(Method E)

<II(
13 >
15.7
<II(
►
17.4
<II(
►
Figure 2.4.8.-1. -Apparatus for the test for heavy metals
Dimensions in mz7limetres

allow to stand for 10 min and again filter as described above, reduce to 2-3 mL. Cool, cautiously add 5 mL of water R and
but inverting the order of the filters, the liquid passing first examine the colour of the solution. If the colour is yellow,
through the membrane filter before passing through the cautiously add 1 mL of strong hydrogen peroxide solution R and
prefilter (Figure 2.4.8.-1). The filtration must be carried out again evaporate until dense, white fumes are produced and
slowly and uniformly by applying moderate and constant reduce to a volume of 2-3 mL. If the solution is still yellow
pressure to the piston of the syringe. After complete in colour, repeat the addition of 5 mL of water R and 1 mL
filtration, open the support, remove the membrane filter, and of strong hydrogen peroxide solution R until the solution is
dry using filter paper. colourless. Cool, dilute cautiously with water R and rinse into
In parallel, treat the reference solution in the same manner as a 50 mL colour comparison tube, ensuring that the total
the test solution. volume does not exceed 25 mL. Adjust the solution to
Result The colour of the spot obtained with the test pH 3.0-4.0, using short range pH indicator paper as external
solution is not more intense than that obtained with the indicator, with concentrated ammonia Rl (dilute ammonia Rl
reference solution. may be used, if desired, as the specified range is
approached), dilute with water R to 40 mL and mix.
METHODF Add 2 mL of buffer solution pH 3. 5 R. Mix and add to
Test solution Place the prescribed quantity or volume of 1.2 mL of thioacetamide reagent R. Mix immediately. Dilute
the substance to be examined in a clean, dry, 100 mL long- to 50 mL with water R and mix.
necked combustion flask (a 300 mL flask may be used if the Reference solution (standard) Prepare at the same time
reaction foams excessively). Clamp the flask at an angle of and in the same manner as the test solution, using the
45°. If the substance to be examined is a solid, add a prescribed volume of lead standard solution (10 ppm Pb) R.
sufficient volume of a mixture of 8 mL of sulfuric acid R and
Monitor solution Prepare as described for the test
10 mL of nitric acid R to moisten the substance thoroughly;
solution, adding to the substance to be examined the volume
if the substance to be examined is a liquid, add a few
of lead standard solution (10 ppm Pb) R prescribed for the
millilitres of a mixture of 8 mL of sulfuric acid R and 10 mL
preparation of the reference solution.
of nitric acid R. Warm gently until the reaction commences,
allow the reaction to subside and add additional portions of Blank solution Prepare as described for the test solution,
the same acid mixture, heating after each addition, until a omitting the substance to be examined.
total of 18 mL of the acid mixture has been added. Increase Examine the solutions vertically against a white background
the amount of heat and boil gently until the solution darkens. after 2 min.
Cool, add 2 mL of nitric acid R and heat again until the System suitability:
solution darkens. Continue the heating, followed by the - the reference solution shows a brown colour compared to
addition of nitric acid R until no further darkening occurs, the blank solution,
then heat strongly until dense, white fumes are produced. - the monitor solution is at least as intense as the reference
Cool, cautiously add 5 mL of water R, boil gently until solution.
dense, white fumes are produced and continue heating to
V-A318 Appendix VII 2023

Result Any brown colour in the test solution is not more System suitabi7ity:
intense than that in the reference solution. - the spot obtained with the reference solution shows a
If the result is difficult to judge, filter the solutions through a brown colour compared to the spot obtained with the
suitable membrane filter (nominal pore size 0.45 µm). Carry blank solution,
out the filtration slowly and uniformly, applying moderate - the spot obtained with the monitor solution is at least as
and constant pressure to the piston. Compare the spots on intense as the spot obtained with the reference solution.
the filters obtained with the different solutions. Result The brown colour of the spot obtained with the test
solution is not more intense than that of the spot obtained
METHODG
with the reference solution.
CAUTION: when using high-pressure digestion vessels the safety
precautions and operating instructions given by the manufacturer METHODH
must be followed. The digestion cycles have to be elaborated Test solution Dissolve the prescribed quantity of the
depending on the type of microwave oven to be used (for example, substance to be examined in 20 mL of the solvent or solvent
energy-controlled microwave ovens, temperature-controlled mixture prescribed.
microwave ovens or high-pressure ovens). The cycle must conform Reference solution Dilute the prescribed volume of lead
to the manufacturer's instructions. The digestion cycle is suitable if standard solution (10 ppm Pb) R to 20 mL with the solvent or
a clear solution is obtained. solvent mixture prescribed.
Test solution Place the prescribed amount of the Blank solution 20 mL of the solvent or solvent mixture
substance to be examined (not more than 0.5 g) in a prescribed.
suitable, clean beaker. Add successively 2.7 mL of sulfuric To each solution, add 2 mL of buffer solution pH 3.5 R. Mix.
acid R, 3.3 mL of nitric acid Rand 2.0 mL of strong hydrogen (In some cases precipitation occurs, in which case the specific
peroxide solution R using a magnetic stirrer. Allow the monograph would describe re-dissolution in a defined volume of a
substance to react with a reagent before adding the next one. given solvent.) Add to 1.2 mL of thioacetamide reagent R.
Transfer the mixture to a dry high-pressure-resistant Mix immediately and allow to stand for 2 min. Filter the
digestion vessel (fluoropolymer or quartz glass). solutions through a suitable membrane filter (nominal pore
Reference solution (standard) Prepare as described for size 0.45 µm). Compare the spots on the filters obtained with
the test solution, using the prescribed volume of lead standard the different solutions.
solution (10 ppm Pb) R instead of the substance to be System suitability The spot obtained with the reference
examined. solution shows a brownish-black colour compared to the spot
Monitor solution Prepare as prescribed for the test obtained with the blank solution.
solution, adding to the substance to be examined the volume Result The brownish-black colour of the spot obtained
of lead standard solution (10 ppm Pb) R prescribed for the with the test solution is not more intense than that of the
preparation of the reference solution. spot obtained with the reference solution.
Blank solution Prepare as described for the test solution,
omitting the substance to be examined. Limit Test for Iron
Close the vessels and place in a laboratory microwave oven. (Ph. Bur. method 2.4.9)
Digest using a sequence of 2 separate suitable programmes. Dissolve the prescribed quantity of the substance to be
Design the programmes in several steps in order to control examined in water R and dilute to I 0 mL with the same
the reaction, monitoring pressure, temperature or energy solvent or use I 0 mL of the prescribed solution. Add 2 mL
depending on the type of microwave oven available. After the of a 200 g/L solution of citric acid monohydrate Rand 0.1 mL
first programme allow the digestion vessels to cool before of thioglycollic acid R. Mix, make alkaline with ammonia R
opening. Add to each vessel 2.0 mL of strong hydrogen and dilute to 20 mL with water R. Prepare a standard in the
peroxide solution R and digest using the second programme. same manner, using 10 mL of iron standard solution
After the second programme allow the digestion vessels to (1 ppm Fe) R.
cool before opening. If necessary to obtain a clear solution, After 5 min, any pink colour in the test solution is not more
repeat the addition of strong hydrogen peroxide solution R and intense than that in the standard.
the second digestion programme.
Limit Test for Lead in Sugars
Cool, dilute cautiously with water R and rinse into a flask,
(Ph. Bur. method 2.4. 10)
ensuring that the total volume does not exceed 25 mL.
Determine the lead by atomic absorption spectrometry
Using short-range pH indicator paper as external indicator,
(2.2.23, Method II).
adjust the solutions to pH 3.0-4.0 with concentrated
ammonia Rl (dilute ammonia Rl may be used as the specified Test solution Dissolve 20.0 g of the substance to be
range is approached). To avoid heating of the solutions use examined in a mixture of equal volumes of dilute acetic acid R
an ice-bath and a magnetic stirrer. Dilute to 40 mL with and water Rand dilute to 100.0 mL with the same mixture
water R and mix. Add 2 mL of buffer solution pH 3.5 R. of solvents. Add 2.0 mL of a clear 10 g/L solution of
Mix and add to 1.2 mL of thioacetamide reagent R. ammonium pyrrolidinedithiocarbamate Rand 10.0 mL of methyl
Mix immediately. Dilute to 50 mL with water R, mix and isobutyl ketone R and then shake for 30 s protected from
allow to stand for 2 min. bright light. Allow the layers to separate and use the methyl
isobutyl ketone layer.
Filter the solutions through a suitable membrane filter
(nominal pore size 0.45 µm). Carry out the filtration slowly Reference solutions Prepare 3 reference solutions in the
and uniformly, applying moderate and constant pressure to same manner as the test solution but adding 0.5 mL, 1.0 mL
the piston. Compare the spots on the filters obtained with and 1.5 mL respectively of lead standard solution
the different solutions. (10 ppm Pb) R in addition to the 20. 0 g of the substance to
be examined.
2023 Appendix VII V-A319

Set the zero of the instrument using methyl isobutyl ketone R (2.2.57), or an inductively coupled plasma-mass
treated as described for the test solution without the spectrometer (2.2.58).
substance to be examined. Measure the absorbance at
METHOD
283.3 nm using a lead hollow-cathode lamp as source of
Examine by atomic absorption spectrometry (AAS) (2.2.23),
radiation and an air-acetylene flame.
inductively coupled plasma-atomic emission spectrometry
The substance to be examined contains not more than (ICP-AES) (2.2.57), or inductively coupled plasma-mass
0.5 ppm of lead, unless otherwise prescribed. spectrometry (ICP-MS) (2.2.58).
Limit Test for Magnesium Deviations from the experimental parameters of the sample
(Ph. Eur. method 2.4.6) preparation procedure and the method description below are
To 10 mL of the prescribed solution add 0.1 g of disodium acceptable provided that the validation requirements are met
tetraborate R. Adjust the solution, if necessary, to pH 8.8 to and the system suitability test is fulfilled on the day of the
pH 9.2 using dilute hydrochloric acid R or dilute sodium analysis.
hydroxide solution R. Shake with 2 quantities, each of 5 mL, Sample preparation
of a 1 g/L solution of hydroxyquinoline R in chloroform R, for Clean all the glassware and laboratory equipment with a
1 min each time. Allow to stand. Separate and discard the 10 g/L solution of nitric acid R before use.
organic layer. To the aqueous solution add 0.4 mL of Test solution In a digestion flask, place the prescribed
butylamine Rand 0.1 mL of triethanolamine R. Adjust the quantity of the substance to be examined (about 0.50 g of
solution, if necessary, to pH 10.5 to pH 11.5. Add 4 mL of powdered herbal drug (1400) (2.9.12)). Add 4 mL of heavy
the solution of hydroxyquinoline in chloroform, shake for metal-free hydrochloric acid R and 6 mL of heavy metal-free
1 min, allow to stand and separate. Use the lower layer for nitric acid R. Make the flask airtight.
comparison. Prepare a standard in the same manner using a Place the digestion flasks in the microwave oven. Carry out
mixture of 1 mL of magnesium standard solutwn the digestion in 3 steps according to the following
(10 ppm Mg) R and 9 mL of water R. programme, used for 7 flasks each containing the test
Any colour in the solution obtained from the substance to be solution: 80 per cent power for 15 min, 100 per cent power
examined is not more intense than that in the standard. for 5 min, 80 per cent power for 20 min.
Limit Test for Magnesium and Alkaline-earth At the end of the cycle, allow the flasks to cool in air or
Metals water. After cooling, open each digestion flask and introduce
(Ph. Eur. method 2.4. 7) the clear, colourless solution obtained into a 50 mL
volumetric flask. Rinse each digestion flask with 2 quantities,
To 200 mL of water R add 0.1 g of hydroxylamine
each of 15 mL, of heavy metal-free dilute nitric acid R, collect
hydrochloride R, l O mL of ammonium chloride buffer solution
the rinsings in the volumetric flask and dilute to 50.0 mL
pH 10.0 R, 1 mL of 0.1 M zinc sulfate and about 15 mg of
with water R. Modifiers (e.g. in the case of AAS with
mordant black 11 triturate R. Heat to about 40 °C. Titrate
electrothermal atomisation, 1.0 mL of a 10 g/L solution of
with 0.01 M sodium edetate until the violet colour changes to
magnesium nitrate R and 1. 0 mL of a 100 g/L solution of
full blue. To the solution add the prescribed quantity of the
ammonium dihydrogen phosphate R) and stabilising agents may
substance to be examined dissolved in 100 mL of water R or
be used, if necessary.
use the prescribed solution. If the colour of the solution
changes to violet, titrate with 0.01 M sodium edetate until the Blank solution Mix 4 mL of heavy metal-free hydrochloric
full blue colour is again obtained. acid R and 6 mL of heavy metal-free nitric acid R in a
digestion flask. Carry out the digestion in the same manner
The volume of 0.01 M sodium edetate used in the second
as for the test solution.
titration does not exceed the prescribed quantity.
DETERMINATION OF ARSENIC, CADMIUM,
Limit Test for Heavy Metals in Herbal Drugs and COPPER, NICKEL AND LEAD USING AAS (2.2.23)
Herbal Drug Preparations WITH ELECTROTHERMAL ATOMISATION
(Ph. Eur. method 2.4.27) Measure the content of arsenic, cadmium, copper, nickel and
CAUTION: when using closed high-pressure digestion vessels and lead by direct calibration (2.2.23, Method I) or by the
microwave laboratory equipment, be familiar with the safety and standard additions method (2.2.23, Method 11), using
operating instructions given by the manufacturer. reference solutions of each heavy metal and the instrumental
parameters recommended in Table 2.4.27.-1.
APPARATUS
The absorbance value of the blank solution is subtracted
The apparatus typically consists of the following:
from the value obtained with the test solution.
- as digestion flasks, polytetrafluoroethylene,
perfluoroalkoxy polymer, quartz or glass flasks with a Table 2.4.27.-1. - Instrumental parameters for AAS with
volume of 20-150 mL, fitted with an airtight closure, a electrothermal atomisation
valve to adjust the pressure inside the container and a
As Cd Cu Ni Pb
polytetrafluoroethylene tube to allow release of gas;
- a system to make flasks airtight, using the same torsional Wavelength nm 193.7 228.8 324.8 232 283.5
force for each of them; Slit width nm 0.5 0.5 0.5 0.2 0.5
- a programmable microwave oven (e.g. with a magnetron Lamp current mA 10 6 7 10 5
frequency of 2450 MHz, with a selectable output from Ignition
'C 1400 800 800 800 800
temperature
0 to 1500 ± 70 Win 1 per cent increments), a
Atomisation
polytetrafluoroethylene-coated microwave cavity with a 'C 2600 1800 2300 2500 2200
temperature
variable speed exhaust fan, a rotating turntable drive Gas flow rate Umin 3 3 3 3 3
system and exhaust tubing to vent fumes;
- an atomic absorption spectrometer (2.2.23), an
inductively coupled plasma-atomic emission spectrometer
V-A320 Appendix VII 2023

DETERMINATION OF ARSENIC AND MERCURY The emission value of the blank solution is subtracted from
USING AAS (2.2.23) WITH COLD-VAPOUR OR the value obtained with the test solution.
HYDRIDE ATOMISATION DETERMINATION OF ARSENIC, CADMIUM,
Measure the content of arsenic and mercury by direct COPPER, MERCURY, NICKEL AND LEAD USING
calibration (2.2.23, Method I) or by the standard additions ICP-MS (2.2.58)
method (2.2.23, Method II), using reference solutions of Measure the content of arsenic, cadmium, copper, mercury,
arsenic or mercury and an automated continuous-flow nickel and lead by direct calibration (2.2.23, Method I) using
hydride vapour generation system. reference solutions of each heavy metal and the analytical
The absorbance value of the blank solution is subtracted isotopes and additional masses recommended in
from the value obtained with the test solution. Table 2.4.27 .-4.
Arsenic The signal intensity of the blank solution is subtracted from
Sample solution To 19.0 mL of the test solution or of the the value obtained with the test solution.
blank solution as prescribed above, add 1 mL of a 200 g/L SYSTEM SUITABILITY
solution of potassium iodide R. Allow the test solution to stand
A system suitability test must be carried out on the day of
at room temperature for about 50 min or at 70 °C for about
the analysis to ensure that the sample preparation and
4 min.
measurement system are appropriate.
Acid reagent Heavy metal-free hydrochloric acid R.
Acceptance criterion for preparation of sample
Reducing reagent 6 g/L solution of sodium solution A clear solution is obtained.
tetrahydroborate R in a 5 g/L solution of sodium hydroxide R.
Acceptance criterion for measurement system The
The recommended instrumental parameters in measured concentration of a standard solution of the metal at
Table 2.4.27 .-2 may be used. a concentration within the range of the used calibration curve
Mercury does not differ from the actual concentration by more than
Sample solution Test solution or blank solution, as 20 per cent.
prescribed above.
Table 2.4.27.-4. - Recommended analytical isotopes and
Acid reagent 515 g/L solution of heavy metal-free
additional masses for !GP-MS
hydrochloric acid R.
Isotope Element of Interest
Reducing reagent l 0 g/L solution of stannous chloride R in
heavy metal-free dilute hydrochloric acid R. 75 Arsenic
106,108, Ill, 114 Cadmium
The recommended instrumental parameters in
63, 65 Copper
Table 2.4.27.-2 may be used.
202 Mercury
Table 2.4.27 .-2. - Instrumental parameters for AAS with cold- 60, 62 Nickel
vapour or hydride atomisation 206, 207, 208 Lead

As Hg
Wavelength nm 193.7 253.7 VALIDATION REQUIREMENTS
Slit width nm 0.2 0.5 The analytical procedures used must be validated in
Lamp current mA 10 4 accordance with the relevant general methods AAS (2.2.23),
Acid reagent flow rate mUmin 1.0 1.0 ICP-AES (2.2.57) and ICP-MS (2.2.58). Additionally, the
Reducing reagent flow rate mUmin 1.0 1.0 following criteria must be fulfilled.
Sample solution flow rate mUmin 7.0 7.0 SPECIFICITY
Absorption cell Quartz Quartz Specificity is the ability to ensure that the analytical
(heated) (unheated)
procedures for sample preparation and measurement allow a
Nitrogen flow rate Umin 0.1 0.1
reliable determination of the metal(s) in the presence of
components (e.g. carrier gas, impurities, matrix) that may be
DETERMINATION OF ARSENIC, CADMIUM, expected to be present.
COPPER, MERCURY, NICKEL AND LEAD USING Acceptance criteria The procedure must be able to assess
ICP-AES (2.2.57) unequivocally each heavy metal to be determined with this
Measure the content of arsenic, cadmium, copper, mercury, procedure in the presence of components that may be
nickel and lead by direct calibration (2.2.23, Method I), using expected to be present, including other heavy metals, matrix
reference solutions of each heavy metal or a mixture of all components and other sources of interference; specificity is
measured metals, and the instrumental parameters demonstrated by complying with the accuracy requirement
recommended in Table 2.4.27.-3. for the metal(s) to be determined.

Table 2.4.27.-3. - Instrumental parameters for !GP-AES

As Cd Cu Hg Ni Pb
Wavelength nm 193.696/ 214.438/ 324.754/ 184.950/ 231.604/ 220.351/
197.197/ 226.502/ 327.396/ 253.652/ 231.997/ 283.306/
189.042 228.802 224.700 435.835 352.454 168.215
Ar, Monitorline nm 430.010 430.010 430.010 430.010 430.010 430.010
Plasma energy w 1200 1200 1200 1200 1200 1200
Peak algorithm with background
yes yes yes yes yes yes
correction
2023 Appendix VII V-A321

RANGE of solvents. Add 2.0 mL of a saturated solution of ammonium

The calibration range of each metal is within the linear range pyrrolidinedithiocarbamate R (about 10 g/L) and 10.0 mL of
of the method; test solutions containing residues at a level methyl isobutyl ketone R and then shake for 30 s protected
outside the calibration range may be diluted to from bright light. Allow the layers to separate and use the
concentrations within the calibration range. methyl isobutyl ketone layer.
Acceptance criterion Range is demonstrated by Reference solutions Prepare 3 reference solutions in the
complying with the recovery requirement. same manner as the test solution but adding 0.5 mL, 1.0 mL
ACCURACY and 1.5 mL respectively of nickel standard solution (10 ppm
Verify the accuracy using a certified reference material Ni) R in addition to the 20.0 g of the substance to be
(CRM) or by performing a test for recovery. examined.
Recovery. Recovery may be determined on a sample of the Set the zero of the instrument using methyl isobutyl ketone R
substance to be examined, spiked with a known quantity of a treated as described for preparation of the test solution
reference standard of the metal (3 concentration levels in the omitting the substance to be examined. Measure the
range of 50-150 per cent of the intended specification limit, absorbance at 232.0 nm using a nickel hollow-cathode lamp
even if the original concentration of the reference standard is as source of radiation and an air-acetylene flame.
at the specified value), in triplicate. The substance to be examined contains not more than
Acceptance criterion Spike recovery is within 70 per cent 1 ppm of nickel, unless otherwise prescribed.
and 150 per cent for the mean of 3 replicates at each Limit Test for Phosphates
concentration. (Ph. Bur. method 2.4.11)
REPEATABILIIY To 100 mL of the solution prepared and, if necessary,
Test samples Either 6 independent samples of the neutralised as prescribed add 4 mL of sulfomolybdic
substance to be examined spiked with a suitable reference reagent R3. Shake and add 0.1 mL of stannous chloride
standard at the specified concentration level, or 3 solution Rl. Prepare a standard in the same manner using
concentration levels prepared in triplicate. 2 mL of phosphate standard solution (5 ppm PO,,) R and
Acceptance criterion The relative standard deviation is in 98 mL of water R. After 10 min, compare the colours using
both cases not greater than the value indicated in 20 mL of each solution.
Table 2.4.27.-5. Any colour in the test solution is not more intense than that
INTERMEDIATE PRECISION in the standard.
The effect of random events (intra-laboratory variations) on
the analytical precision of the method must be established. Limit Test for Potassium
Acceptable experiments for establishing intermediate (Ph. Bur. method 2. 4.12)
precision include performing the repeatability analysis on To 10 mL of the prescribed solution add 2 mL of a freshly
different days, or with different instrumentation, or with prepared 10 g/L solution of sodium tetraphenylborate R.
different analysts. Only 1 of the 3 experiments is required to Prepare a standard in the same manner using a mixture of
demonstrate intermediate precision. 5 mL of potassium standard solution (20 ppm K) R and 5 mL
Acceptance criterion The relative standard deviation is of water R.
not greater than the value indicated in Table 2.4.27.-5. After 5 min, any opalescence in the test solution is not more
LIMIT OF QUANTIFICATION intense than that in the standard.
Determine the lowest concentration meeting the acceptance Limit Test for Sulfates
criterion. Use the results from the accuracy study. (Ph. Bur. method 2. 4.13)
Acceptance criterion The limit of quantification is below All solutions used for this test must be prepared with distilled
the specification limit. water R.
Table 2.4.27.-5 Add 3 mL of a 250 g/L solution of ban·um chloride R to
4.5 mL of sulfate standard solution (10 ppm SO,J Rl. Shake
Concentration Intermediate
Repeatability and allow to stand for 1 min. To 2.5 mL of this suspension
range of the metal precision (RSD)
(RSD) (per cent) add 15 mL of the prescribed solution and 0.5 mL of acetic
(mg/kg) (per cent)
0.01 - l 20 32 acid R. Prepare a standard in the same manner using 15 mL
> l 10 16 of sulfate standard solution (10 ppm SO,,) R instead of the
prescribed solution.
LIMIT OF DETECTION (ONLY APPUCABLE TO After 5 min, any opalescence in the test solution is not more
LIMIT TESTS) intense than that in the standard.
Determine the lowest concentration giving a signal clearly
distinct from that obtained with a blank solution.
Acceptance criterion The limit of detection is not more
than 0.1 times the concentration of the specification limit.
Limit Test for Nickel in Polyols
(Ph. Bur. method 2.4.15)
Determine the nickel by atomic absorption spectrometry
(2.2.23, Method 11).
Test solution Dissolve 20.0 g of the substance to be
examined in a mixture of equal volumes of dilute acetic acid R
and water Rand dilute to 100.0 mL with the same mixture
V-A322 Appendix VIII A 2023

electrode) and a reference electrode (for example, a silver-

Appendix VIII silver chloride electrode).
A three-electrode apparatus is sometimes used, consisting of
A. Non-aqueous Titration an indicator electrode, a reference electrode and a polarised
auxiliary electrode.
(No Ph. Bur. method)
Method Set the potential of the indicator electrode as
Method I prescribed and plot a graph of the initial current and the
Dissolve the prescribed quantity of the substance being values obtained during the titration as functions of the
examined in a suitable volume of anhydrous acetic acid quantity of titrant added. Add the titrant in not fewer than
previously neutralised using the indicator specified in the 3 successive quantities equal to a total of about 80 per cent
monograph, warming and cooling if necessary, or prepare a of the theoretical volume corresponding to the presumed
solution as directed. When the substance is a salt of equivalence point. The 3 values must fall on a straight line.
hydrochloric or hydrobromic acid, add 15 mL of mercury(!!) Continue adding the titrant beyond the presumed
acetate solution before neutralising the solvent, unless equivalence point in not fewer than 3 successive quantities.
otherwise directed in the monograph. Titrate with 0. lM The values obtained must fall on a straight line. The point of
perchloric acid VS to the colour change of the indicator that intersection of the 2 lines represents the end-point of the
corresponds to the maximum absolute value of dE/dV (where titration.
B is the electromotive force and Vis the volume of titrant) in
For amperometric titrations with 2 indicator electrodes, the
a potentiometric titration, Appendix VIII B, of the substance
whole titration curve is recorded and used to determine the
being examined. The indicator specified in the monograph is
end-point.
also used for the neutralisation of the mercury(IIJ acetate
solutwn and the standardisation of the titrant. Potentiometric Titration
When the temperature (t2) of the titrant at the time of the (Ph. Bur. method 2.2.20)
assay differs from the temperature (t 1) of the titrant when it In a potentiometric titration (volumetric titration with
was standardised, multiply the volume of the titrant required potentiometric end-point determination) the end-point is
by [l+0.00ll(t1 - t2 )] and calculate the result of the assay determined by recording the variation of the potential
from the corrected volume. difference between 2 electrodes (either 1 indicator electrode
Carry out a blank titration when necessary. and 1 reference electrode, or a combined electrode)
Method II immersed in the solution to be examined as a function of the
The titrant, solvent and, where necessary, the indicator to be volume of titrant added.
used are stated in the monograph. Apparatus The apparatus used comprises a millivoltmeter.
Protect the solution and titrant from atmospheric carbon Commercial autotitrator instruments may be used and are
dioxide and moisture throughout the determination. operated in accordance with the manufacturer's instructions,
using electrodes recommended for the type of titration
Dissolve the substance being examined in a suitable volume
described.
of the solvent previously neutralised to the indicator,
warming and cooling if necessary, or prepare a solution as The indicator electrode to be used depends on the substance
directed. Titrate to the colour change of the indicator that to be determined and may be a glass or metal electrode
corresponds to the maximum absolute value of dE/d V (where (e.g. platinum, gold or silver).
B is the electromotive force and Vis the volume of titrant) in For acid-base titrations, a glass-silver-silver chloride electrode
a potentiometric titration, Appendix VIII B, of the substance combination is generally used.
under examination. The titrant is standardised using the Method Prepare the sample solution as described. Add the
same solvent and indicator as specified for the substance. titrant in suitable aliquots paying particular attention to the
Carry out a blank titration when necessary. rate of addition and the volume increments near the
end-point. Continue the titration beyond this point to allow a
clear detection of the end-point.
The end-point of the titration is reached when the maximum
B. Amperometric, Potentiometric and change in potential occurs in a plot of potential versus
volume of titrant, and is expressed as the corresponding
Voltametric Titrations volume of titrant. Recording the first or second derivative
curve can facilitate the determination of the end-point.
Amperometric Titration
In potentiometric titrations of weak acids or bases using non-
(Ph. Bur. method 2.2.19) aqueous solvents, if necessary, either carry out a blank
In an amperometric titration, the end-point is determined by determination or pre-neutralise the solvent mixture. Where it
following the variation of the current measured between is impracticable to use potentiometric detection for this
2 electrodes (either one indicator electrode and one reference purpose, the solvent mixture can be pre-neutralised by
electrode or 2 indicator electrodes) immersed in the solution titration using a suitable indicator. Some examples are given
to be examined and maintained at a constant potential below:
difference as a function of the quantity of titrant added.
The potential of the measuring electrode is sufficient to Titrant Indicator
ensure a diffusion current for the electroactive substance. Perchloric acid Crystal vwler solution R

Apparatus The apparatus comprises an adjustable voltage 3 g/L solution of thymol blue R in
Tetrabutylammonium hydroxide
methanol R
source and a sensitive microammeter; the detection system
Ethanolic sodium hydroxide Thymolphthalein solution R
generally consists of an indicator electrode (for example, a
platinum electrode, a rotating-disc electrode or a carbon
2023 Appendix VIII D V-A323

Voltametric Titrations vigorously to completely dissolve the combustion products.

(Ph. Bur. method 2.2.65) Cool and after about 5 min, unless otherwise prescribed,
In voltametric titration the end-point of the titration is carefully unstopper the flask. Wash the ground parts and the
determined by following the variation of the voltage walls of the flask, as well as the sample carrier, with water R.
measured between 2 electrodes (either 1 indicator electrode Combine the combustion products and the washings and
and 1 reference electrode or 2 indicator electrodes) immersed proceed as prescribed in the monograph.
in the solution to be examined and maintained at a constant For Iodine
current as a function of the quantity of titrant added.
(No Ph. Bur. method)
Apparatus The apparatus comprises an adjustable current
Burn the specified quantity of the substance being examined
source and a voltmeter; the detection system generally
in the prescribed manner using a mixture of 10 mL of water
consists of an indicator electrode (for example, a platinum
and 2 mL of lM sodium hydroxide as the absorbing liquid.
electrode, a rotating-disc electrode or a carbon electrode) and
When the process is complete, add to the flask an excess of
a 2 nd electrode (for example, a platinum electrode, a rotating-
acetic bromine solution (between 5 and 10 mL) and allow to
disc electrode or a carbon electrode).
stand for 2 minutes. Remove the excess of bromine by the
Method Set the current to the indicator electrode as addition of formic acid (about 0.5 to 1 mL), rinse the sides of
prescribed in the monograph and plot a graph of the initial the flask with water and displace any bromine vapour above
voltage and the values obtained during the titration as the liquid with a current of air. Add 1 g of potassium iodide
functions of the quantity of titrant added. Add the titrant in and titrate with 0.02M sodium thiosuifate VS using starch
not fewer than 3 successive quantities equal to a total of mucilage, added towards the end of the titration, as indicator.
about 80 per cent of the theoretical volume corresponding to Each mL of 0.02M sodium thiosulfate VS is equivalent to
the presumed equivalence point. The 3 values must fall on a 0.4230 mg of I.
straight line. Continue adding the titrant beyond the
presumed equivalence point in not fewer than 3 successive
quantities. The values obtained must fall on another straight
line. The point of intersection of the 2 lines represents the
end-point of the titration.
D. Complexometric Titrations
Using titration systems for voltametric titration with (Ph. Bur. method 2.5.11)
2 indicator electrodes, the whole titration curve is recorded ALUMINIUM
and used to determine the end-point. Introduce 20.0 mL of the prescribed solution into a 500 mL
conical flask, add 25.0 mL of 0.1 M sodium edetate and
Determination of Primary Aromatic Amino-
10 mL of a mixture of equal volumes of a 155 g/L solution
nitrogen
of ammonium acetate R and dilute acetic acid R. Boil for 2 min,
(Ph. Bur. method 2.5.8)
then cool. Add 50 mL of ethanol R and 3 mL of a freshly
Dissolve the prescribed quantity of the substance to be prepared 0.25 g/L solution of dithizone R in ethanol R. Titrate
examined in 50 mL of dilute hydrochloric acid R or in another the excess of sodium edetate with 0.1 M zinc sulfate until the
prescribed solvent and add 3 g of potassium bromide R. Cool colour changes from greenish-blue to reddish-violet.
in ice-water and titrate by slowly adding 0.1 M sodium nitrite
1 mL of 0.1 M sodium edetate is equivalent to 2.698 mg of
with constant stirring.
Al.
Determine the end-point electrometrically or by the use of
the prescribed indicator. BISMUTH
Introduce the prescribed solution into a 500 mL conical
flask. Dilute to 250 mL with water R and then, unless
otherwise prescribed, add dropwise, with shaking, concentrated
ammonia R until the mixture becomes cloudy. Add 0.5 mL
C. Oxygen-flask Combustion of nitric acid R. Heat to about 70 °C until the cloudiness
(Ph. Bur. method 2.5.10) disappears completely. Add about 50 mg of xylenol orange
Unless otherwise prescribed the combustion flask is a conical triturate R and titrate with 0. 1 M sodium edetate until the
flask of at least 500 mL capacity of borosilicate glass with a colour changes from pinkish-violet to yellow.
ground-glass stopper fitted with a suitable carrier for the 1 mL of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi.
sample, for example in platinum or platinum-iridium. CALCIUM
Finely grind the substance to be examined, place the Introduce the prescribed solution into a 500 mL conical
prescribed quantity in the centre of a piece of filter paper flask, and dilute to 300 mL with water R. Add 6.0 mL of
measuring about 30 mm by 40 mm provided with a small strong sodium hydroxide solution Rand about 200 mg of
strip about 10 mm wide and 30 mm Jong. If paper calconecarboxylic acid triturate R. Titrate with 0. 1 M sodium
impregnated with lithium carbonate is prescribed, moisten edetate until the colour changes from violet to full blue.
the centre of the paper with a saturated solution of lithium 1 mL of 0.1 M sodium edetate is equivalent to 4.008 mg of
carbonate R and dry in an oven before use. Envelop the Ca.
substance to be examined in the paper and place it in the
sample carrier. Introduce into the flask water R or the MAGNESIUM
prescribed solution designed to absorb the combustion Introduce the prescribed solution into a 500 mL conical flask
products, displace the air with oxygen by means of a tube and dilute to 300 mL with water R. Add 10 mL of
having its end just above the liquid, moisten the neck of the ammonium chloride buffer solution pH 10. 0 R and about 50 mg
flask with water R and close with its stopper. Ignite the paper of mordant black 11 triturate R. Heat to about 40 °C then
strip by suitable means with the usual precautions. Keep the titrate at this temperature with 0.1 M sodium edetate until the
flask firmly closed during the combustion. Shake the flask colour changes from violet to full blue.
V-A324 Appendix VIII E 2023

1 mL of 0.1 M sodium edetate is equivalent to 2.431 mg of example, glass electrodes), or electrodes with charged
Mg. (positive or negative) or uncharged mobile carriers, or
sensitised electrodes (enzymatic-substrate electrodes, gas-
LEAD
indicator electrodes). The reference electrode is generally a
Introduce the prescribed solution into a 500 mL conical flask
silver-silver chloride electrode with suitable junction liquids
and dilute to 200 mL with water R. Add about 50 mg of
producing no interference.
xylenol orange triturate R and hexamethylenetetramine R until
the solution becomes violet-pink. Titrate with 0.1 M sodium Procedure Carry out each measurement at a temperature
edetate until the violet-pink colour changes to yellow. constant to ± 0.5 °C, taking into account the variation of
the slope of the electrode with temperature (see
1 mL of 0.1 M sodium edetate is equivalent to 20. 72 mg of
Table 2.2.36.-1). Adjust the ionic strength and possibly the
Pb.
pH of the solution to be analysed using the buffer reagent
ZINC described in the monograph and equilibrate the electrode by
Introduce the prescribed solution into a 500 mL conical flask immersing it in the solution to be analysed, under slow and
and dilute to 200 mL with water R. Add about 50 mg of uniform stirring, until a constant reading is obtained.
xylenol orange triturate R and hexamethylenetetramine R until
the solution becomes violet-pink. Add 2 g of Table 2.2.36.-1. - Values of k at different temperatures
hexamethylenetetramine R in excess. Titrate with 0.1 M sodium Temperature CC) k
edetate until the violet-pink colour changes to yellow. 20 0.0582
1 mL of 0.1 M sodium edetate is equivalent to 6.54 mg of Zn. 25 0.0592
30 0.0602

If the electrode system is used frequently, check regularly the

E. Potentiometric Determination of Ionic repeatability and the stability of responses, and the linearity
of the calibration curve or the calculation algorithm in the
Concentration Using Ion-selective range of concentrations of the test solution; if not, carry out
Electrodes the test before each set of measurements. The response of
the electrode may be regarded as linear if the slope S of the
(Ph. Bur. method 2.2.36)
calibration curve is approximately equal to klz;, per unit of
Ideally, the potential E of an ion-selective electrode varies pC;.
linearly with the logarithm of the activity a; of a given ion, as
METHOD I (DIRECT CALIBRATION)
expressed by the Nemst equation:
Measure at least three times in succession the potential of at
least three reference solutions spanning the expected
concentration of the test solution. Calculate the calibration
curve, or plot on a chart the mean potential E obtained
E0 part of the constant potential due to the apparatus used,
against the concentration of the ion to be determined
R gas constant,
T absolute temperature, expressed as -log 10 C; or pC;.
F Faraday's number, Prepare the test solution as prescribed in the monograph;
z, charge nwnber of the ion including its sign. measure the potential three times and, from the mean
potential, calculate the concentration of the ion to be
At a constant ionic strength, the following holds: determined using the calibration curve.
k METHOD II (MULTIPLE STANDARD ADDITIONS)
E = Eo + - log1of Ci
Zi Prepare the test solution as prescribed in the monograph.
C, molar concentration of the ion,
Measure the potential at equilibrium ET of a volume VT of
f the activity coefficient (a; = JC;), this solution of unknown concentration GT of the ion to be
RT determined. Make at least three consecutive additions of a
k
F volume Vs negligible compared to VT (Vs :S: 0.01 VT) of a
reference solution of a concentration Cs known to be within
k , k the linear part of the calibration curve. After each addition,
If: E0 +- log 10 f = E O and S = - measure the potential and calculate the difference of potential
Zi Zi
t:.E between the measured potential and ET. t:.E is related to
s slope of the calibration curve of the electrode,
the concentration of the ion to be determined by the
the following holds: E = E~ + Slog 10 q equation:
and for -log 10 Ci = pCi: E = E~ - SpC;.
Cs Vs)
The potentiometric determination of the ion concentration is t:.E = Slog 10 ( I + GT VT
carried out by measuring the potential difference between
two suitable electrodes immersed in the solution to be or
examined; the indicator electrode is selective for the ion to be
determined and the other is a reference electrode.
Apparatus Use a voltmeter allowing measurements at least
to the nearest 0.1 millivolt and whose input impedance is at
least one hundred times greater than that of the electrodes VT volwne of the test solution,
GT concentration of the ion to be determined in the test solution,
used. Vs added volume of the reference solution,
Ion-selective electrodes may be primary electrodes with a Cs concentration of the ion to be determined in the reference
crystal or non-crystal membrane or with a rigid matrix (for solution,
2023 Appendix VIII F V-A325

s slope of the electrode determined experimentally, at constant Method II

temperature, by measuring the difference between the potentials
obtained with two reference solutions whose concentrations (No Ph. Eur. method)
differ by a factor of ten and are situated within the range where For preparations in which, in accordance with the authority
the calibration curve is linear. given in the monographs, Industrial Methylated Spirit has
been used, determine the content of ethanol as described in
Plot on a graph 10'¥ (y-axis) against Vs (x-axis) and Method I but using as solution (2) a volume of the
extrapolate the line obtained until it intersects the x-axis. preparation being examined diluted with water to contain
At the intersection, the concentration CT of the ion to be between 4.0 and 6.0% v/v of total ethanol and methanol.
determined in the test solution is given by the equation:
Determine the concentration of methanol in the following
manner. Carry out the chromatographic procedure described
under Method I but using the following solutions. Solution
(1) contains 0.25% v/v of methanol and 0.25% v/v of propan-
METHOD III (SINGLE STANDARD ADDITION) 1-ol (internal standard). For solution (2) dilute a volume of
the preparation being examined with water to contain
To a volume VT of the test solution prepared as prescribed
between 0.2% and 0.3% v/v of methanol. Prepare solution
in the monograph, add a volume Vs of a reference solution
(3) in the same manner as solution (2) but adding sufficient
containing an amount of the ion to be determined known to
of the internal standard to produce a final concentration of
give a response situated in the linear part of the calibration
curve. Prepare a blank solution in the same conditions. 0.25% v/v.
Measure at least three times the potentials of the test solution The sum of the contents of ethanol and methanol is within
and the blank solution, before and after adding the reference the range specified in the monograph and the ratio of the
solution. Calculate the concentration CT of the ion to be content of methanol to that of ethanol is commensurate with
analysed using the following equation and making the Industrial Methylated Spirit having been used.
necessary corrections for the blank: Method III
(Ph. Eur. method 2. 9.10)
C _ CsVs
T - c\E, These methods are intended for the examination of liquid
10s( Vr + Vs) - Vr
pharmaceutical preparations and their ingredients that contain
VT volume of the test solution or the blank, ethanol.
GT concentration of the ion to be determined in the test solution, The ethanol content of a liquid is expressed as the number of
Vs added volume of the reference solution,
volumes of ethanol contained in 100 volumes of the liquid, the
Cs concentration of the ion to be determined in the reference
solution, volumes being measured at 20 ± 0.1 °C. This is known as the
!J.E difference between the average potentials measured before and 'percentage of ethanol by volume' (per cent VIV). The content
after adding Vs, may also be expressed in grams of ethanol per 100 g of the liquid.
S slope of the electrode determined experimentally, at constant This is known as the 'percentage of ethanol by mass'
temperature, by measuring the difference between the potentials
obtained from two reference solutions whose concentrations
(per cent m/m).
differ by a factor of ten and are situated within the range where METHOD A
the calibration curve is linear.
Where preparations contain dissolved substances, the
dissolved substances must be separated from the ethanol that
is to be determined by distillation. Where distillation would
distil volatile substances other than ethanol and water, the
F. Determination of Ethanol appropriate precautions are stated in the monograph.
The relation between the density at 20 ± 0.1 °C, the relative
Use Method I or, where appropriate, Method II unless density (corrected to vacuum) and the ethanol content of a
otherwise prescribed in the monograph. mixture of water and ethanol is given in the tables of the
Method I International Organisation for Legal Metrology (1972),
(No Ph. Eur. method) International Recommendation No. 22.
Carry out the method for gas chromatography, Apparatus
Appendix III B, using the following solutions. Solution (1) The apparatus (see Figure 2.9.10.-1) consists of a round-
contains 5.0% v/v of absolute ethanol and 5.0% v/v of propan- bottomed flask (A) fitted with a distillation head (B) with a
1-ol (internal standard). For solution (2) dilute a volume of steam trap and attached to a vertical condenser (C).
the preparation being examined with water to contain The latter is fitted at its lower part with a tube (D), which
between 4.0 and 6.0% v/v of ethanol. Prepare solution (3) in carries the distillate into the lower part of a 100 mL or
the same manner as solution (2) but adding sufficient of the 250 mL volumetric flask. The volumetric flask is immersed
internal standard to produce a final concentration of in a mixture of ice and water (E) during the distillation.
5.0% v/v. A disc having a circular aperture 6 cm in diameter is placed
under the flask (A) to reduce the risk of charring any
The chromatographic procedure may be carried out using a
dissolved substances.
column (1.5 m x 4 mm) packed with porous polymer beads
(100 to 120 mesh) (Porapak Q and Chromosorb 101 are Method
suitable) and maintained at 150° with both the inlet port and Pycnometer method/oscillating transducer density
the detector at 170°. meter method Transfer 25.0 mL of the preparation to be
Calculate the percentage content of ethanol from the areas of examined, measured at 20 ± 0.1 °C, to the distillation flask.
the peaks due to ethanol in the chromatograms obtained with Dilute with 100-150 mL of distilled water R and add a few
solutions (1) and (3). pieces of pumice. Attach the distillation head and condenser.
Distil and collect not less than 90 mL of distillate in a
V-A326 Appendix VIII F 2023

100 mL volumetric flask. Adjust the temperature to Table 2.9.10.-1. - Relationship between densiry, relative densiry
20 ± 0.1 °C and dilute to 100.0 mL with distilled water Rat and ethanol content
20 ± 0.1 °C. Determine the relative density at 20 ± 0.1 °C P20 Relative density of the Ethanol content in
using a pycnometer or an oscillating transducer density (kg-m-') distillate measured in air per cent V/V
a20 at 20 "C
meter. 20

The values indicated in Table 2.9.10.-1, column 3, are 968.0 0.9697 25.09
multiplied by 4 to obtain the percentage of ethanol by 968.5 0.9702 24.64
volume (V/V) contained in the preparation. After calculation 969.0 0.9707 24.19
of the ethanol content using the table, round off the result to 969.5 0.9712 23.74
1 decimal place. 970.0 0.9717 23.29
970.5 0.9722 22.83
971.0 0.9727 22.37
971.5 0.9733 21.91
972.0 0.9738 21.45
972.5 0.9743 20.98
973.0 0.9748 20.52
973.5 0.9753 20.05
974.0 0.9758 19.59
974.5 0.9763 19.12
975.0 0.9768 18.66
975.5 0.9773 18.19
976.0 0.9778 17.73
A
976.5 0.9783 17.25
977.0 0.9788 16.80
977.5 0.9793 16.34
978.0 0.9798 15.88
978.5 0.9803 15.43
979.0 0.9808 14.97
979.5 0.9813 14.52
980.0 0.9818 14.07
980.5 0.9823 13.63
981.0 0.9828 13.18
981.5 0.9833 12.74
982.0 0.9838 12.31
982.5 0.9843 11.87
983.0 0.9848 11.44
983.5 0.9853 11.02
984.0 0.9858 10.60
984.5 0.9863 10.18
985.0 0.9868 9.76
985.5 0.9873 9.35
986.0 0.9878 8.94
986.5 0.9883 8.53
987.0 0.9888 8.13
987.5 0.9893 7.73
Figure 2.9.10.-1. -Apparatus for the determination of ethanol 988.0 0.9898 7.34
content 988.5 0.9903 6.95
Dimensions in millimetres 989.0 0.9908 6.56
989.5 0.9913 6.17
Hydrometer method Transfer 50.0 mL of the preparation 990.0 0.9918 5.79
to be examined, measured at 20 ± 0.1 °C, to the distillation 990.5 0.9923 5.42
flask, add 200-300 mL of distilled water R and distil, as 991.0 0.9928 5.04
described above, into a volumetric flask until at least 180 mL 991.5 0.9933 4.67
has been collected. Adjust the temperature to 20 ± 0.1 °C 992.0 0.9938 4.30
and dilute to 250.0 mL with distilled water Rat 20 ± 0.1 °C. 992.5 0.9943 3.94
Transfer the distillate to a cylinder whose diameter is at least 993.0 0.9948 3.58
6 mm wider than the bulb of the hydrometer. If the volume 993.5 0.9953 3.22
is insufficient, double the quantity of the sample and dilute 994.0 0.9958 2.86
the distillate to 500.0 mL with distilled water Rat 994.5 0.9963 2.51
20 ± 0.1 °C. 995.0 0.9968 2.16
Multiply the strength by 5 to allow for the dilution during 995.5 0.9973 1.82
the determination. After calculation of the ethanol content 996.0 0.9978 1.47
using Table 2.9. 10.-1, round off the result to 1 decimal 996.5 0.9983 1.13
place. 997.0 0.9988 0.80
997.5 0.9993 0.46
998.0 0.9998 0.13
 2023 Appendix VIII F V-A327

METHODB Detection Flame ionisation.

Head-space gas chromatography (2.2.28). Injection 1.0 mL of the gaseous phase of the test solution
Internal standard solution Dilute 1.0 mL of propanol Rl and reference solutions (b), (c), (d) and (f), at least 3 times.
to 100.0 mL with water R. Dilute 1.0 mL of the solution to Elution order Methanol, ethanol, propanol.
20.0 mL with water R. Relative retention With reference to ethanol (retention
Test solution Dilute a volume of the preparation to be time= about 5.3 min): methanol= about 0.8;
examined corresponding to 0.4 g of ethanol to 50.0 mL with propanol = about 1.6.
water R. Dilute 1.0 mL of the solution to 20.0 mL with System suitability Reference solution (f):
water R. To 2.0 mL ofthis solution add 1.0 mL of the - resolution: minimum 5 between the peaks due to methanol
internal standard solution and dilute to 20.0 mL with and ethanol.
water R. Transfer 2.0 mL of this solution into an injection Establish a calibration curve with the concentration of
vial. ethanol in reference solutions (b), (c), (d) and (f) as the
Reference solution (a) Dilute 5.0 mL of anhydrous abscissa and the mean ratio of the peak area of ethanol to the
ethanol R to 100.0 mL with water R. Dilute 25.0 mL of the peak area of the internal standard in the corresponding
solution to 100.0 mL with water R. Dilute 1.0 mL of this chromatograms as the ordinate.
solution to 20.0 mL with water R. Calculate the percentage content of ethanol in the
Reference solution (b) Mix 0.5 mL of reference preparation to be examined.
solution (a) and 1.0 mL of the internal standard solution and
METHODC
dilute to 20.0 mL with water R. Transfer 2.0 mL of this
solution into an injection vial. Gas chromatography (2.2.28).
Reference solution (c) Mix 1.0 mL of reference Internal standard solution Dilute 1.0 mL of propanol Rl
solution (a) and 1.0 mL of the internal standard solution and to 100.0 mL with water R.
dilute to 20.0 mL with water R. Transfer 2.0 mL of this Test solution Dilute a volume of the preparation to be
solution into an injection vial. examined corresponding to 1 g of ethanol to 50.0 mL with
Reference solution (d) Mix 1.5 mL of reference water R. To 1.0 mL of this solution add 1.0 mL of the
solution (a) and 1.0 mL of the internal standard solution and internal standard solution and dilute to 20.0 mL with
dilute to 20.0 mL with water R. Transfer 2.0 mL of this water R.
solution into an injection vial. Reference solution (a) Dilute 1.0 mL of anhydrous
Reference solution (e) Dilute 1.0 mL of methanol R2 to ethanol R to 50.0 mL with water R.
100.0 mL with water R. Dilute 1.0 mL of the solution to Reference solution (b) Dilute 1.0 mL of methanol R2 to
20.0 mL with water R. 100.0 mL with water R. Dilute 1.0 mL of the solution to
Reference solution (j) Mix 1.0 mL of the internal 20.0 mL with water R.
standard solution, 2.0 mL of reference solution (a) and Reference solution (c) Mix 1.0 mL of the internal
2.0 mL of reference solution (e) and dilute to 20.0 mL with standard solution, 1.0 mL of reference solution (a) and
water R. Transfer 2.0 mL of this solution into an injection 2.0 mL of reference solution (b) and dilute to 20.0 mL with
vial. water R.
Cl,ose the vials immediately with a tight rubber membrane stopper Column:
coated with polytetrafiuoroethylene and secure with an aluminium - material: fused silica;
crimp cap. =
- size: l 30 m, 0 = 0.53 mm;
- stationary phase: cyanopropyl(3)phenyl(3)methyl(94)
Column:
polysiloxane R (film thickness 3 µm).
- material: fused silica;
- size: l = 30 m, 0 = 0.53 mm; Carrier gas helium for chromatography R.
- stationary phase: cyanopropyl(3)phenyl(3)methyl(94) Flow rate 3 mUmin.
polysiloxane R (film thickness 3 µm). Split ratio 1:50.
Carrier gas helium for chromatography R. Temperature:
Flow rate 3 mUmin.
Split ratio 1:50. Time Temperature
(min) CC)
Static head-space conditions that may be used:
Column 0 - 1.6 40
- equilibration temperature: 85 °C;
1.6 - 9.9 40--, 65
- equilibration time: 20 min.
9.9 - 13.6 65 --, 175
Temperature: 13.6 - 20 175
Injection port 200
Time Temperature
Detector 200
(min) (C)
Column 0 - 1.6 40
1.6 - 9.9 40--, 65 Detection Flame ionisation.
9.9 - 13.6 65 --, 175 Injection 1.0 µL of the test solution and reference
13.6-20 175 solution (c), at least 3 times.
Injection port 200 Elution order Methanol, ethanol, propanol.
Detector 200 Relative retention With reference to ethanol (retention
time = about 5.3 min): methanol = about 0.8;
propanol = about 1.6.
System suitability Reference solution (c):
V-A328 Appendix VIII G 2023

- resolution: minimum 5 between the peaks due to methanol Temperature:

and ethanol.
Calculate the ethanol content in per cent V/V using the Time Temperature
following expression: (min) CC)
Column 0 - 1.6 40
A1xl2xl00 1.6 - 9.9 40 ➔ 65
9.9 - 13.6 65 ➔ 175
A2 X 11 X Vi
13.6 - 20 175
area of the peak due to ethanol in the chromatogram obtained Injection port 200
with the test solution; Detector 200
area of the peak due to ethanol in the chromatogram obtained
with reference solution ( c);
area of the peak due to the internal standard in the Detection Flame ionisation.
chromatogram obtained with the test solution;
area of the peak due to the internal standard in the
Injection 1.0 mL of the gaseous phase of the test solution
chromatogram obtained with reference solution (c); and reference solution (c), at least 3 times.
Vi volume of the preparation to be examined in the test solution, Elution order Methanol, ethanol, 2-propanol, 1-propanol.
in millilitres.
Relative retention With reference to ethanol (retention
time = about 5.3 min): methanol = about 0.8;
=
2-propanol about 1.2; 1-propanol about 1.6. =
System suitability Reference solution (c):
- resolution: minimum 5 between the peaks due to methanol
G. Determination of Methanol and and ethanol.
Propan-2-ol Calculate the methanol content in per cent VIV using the
following expression:
(Ph. Bur. method 2. 9.11)
METHOD A
Head-space gas chromatography (2.2.28). A2 X Ii X 40
Internal standard solution Dilute 1.0 mL of propanol Rl A1 area of the peak due to methanol in the chromatogram obtained
to 100.0 mL with water R. Dilute 1.0 mL of the solution to with the test solution;
20.0 mL with water R. A2 area of the peak due to methanol in the chromatogram obtained
with reference solution (c);
Test solution Mix 1. 0 mL of the internal standard 11 area of the peak due to the internal standard in the
solution and 4.0 mL of the preparation to be examined and chromatogram obtained with the test solution;
dilute to 20.0 mL with water R. Transfer 2.0 mL of this 12 area of the peak due to the internal standard in the
solution into an injection vial. chromatogram obtained with reference solution (c).

Reference solution (a) Mix 1. 0 mL of methanol R2 and Calculate the 2-propanol content in per cent V/V using the
1.0 mL of 2-propanol R2 and dilute to 100.0 mL with following expression:
water R. Dilute 1.0 mL of the solution to 20.0 mL with
water R.
Reference solution (b) Dilute 5.0 mL of anhydrous
ethanol R to 100.0 mL with water R. Dilute 25.0 mL of the
solution to 100.0 mL with water R. Dilute 1.0 mL of this area of the peak due to 2-propanol in the chromatogram
obtained with the test solution;
solution to 20.0 mL with water R.
area of the peak due to 2-propanol in the chromatogram
Reference solution (c) Mix 1.0 mL of the internal obtained with reference solution (c);
standard solution, 2.0 mL of reference solution (a) and area of the peak due to the internal standard in the
2.0 mL of reference solution (b) and dilute to 20.0 mL with chromatogram obtained with the test solution;
area of the peak due to the internal standard in the
water R. Transfer 2.0 mL of this solution into an injection chromatogram obtained with reference solution (c).
vial.
Close the vials immediately with a tight rubber membrane stopper METHODB
coated with polytetrafiuoroethylene and secure with an aluminium Gas chromatography (2.2.28).
crimp cap.
Internal standard solution Dilute 1.0 mL of propanol Rl
Column: to 100.0 mL with water R.
- material: fused silica;
Test solution Mix 1.0 mL of the internal standard
- size: l =30 m, 0 =0.53 mm;
solution and 4.0 mL of the preparation to be examined and
- stationary phase: cyanopropyl(3)phenyl(3)methyl(94)
dilute to 20.0 mL with water R.
polysiloxane R (film thickness 3 µm).
Reference solution (a) Mix 1.0 mL of methanol R2 and
Carrier gas helium for chromatography R.
1.0 mL of 2-propanol R2 and dilute to 100.0 mL with
Flow rate 3 mIJmin. water R. Dilute 1.0 mL of the solution to 20.0 mL with
Split ratio 1:50. water R.
Static head-space conditions that may be used: Reference solution (b) Dilute 1.0 mL of anhydrous
- equilibration temperature: 85 °C; ethanol R to 50.0 mL with water R.
- equilibration time: 20 min. Reference solution (c) Mix 1.0 mL of the internal
standard solution, 1.0 mL of reference solution (b) and
2.0 mL of reference solution (a) and dilute to 20.0 mL with
water R.
2023 Appendix VIII H V-A329

Column:
- material: fused silica;
H. Determination of Nitrogen
- size: l = 30 m, 0 = 0.53 mm;
Method I
- stationary phase: cyanopropyl(3)phenyl(3)methyl(94)
(Ph. Eur. method 2.5.9)
polysuoxane R (film thickness 3 µm).
Carrier gas helium for chromatography R. SEMI-MICRO METHOD
Place a quantity of the substance to be examined (m g)
Flow rate 3 mUmin.
containing about 2 mg of nitrogen in a combustion flask, add
Split ratio 1:50. 4 g of a powdered mixture of 100 g of dipotassium sulfate R,
Temperature: 5 g of copper sulfate pentahydrate R and 2.5 g of selenium R,
and three glass beads. Wash any adhering particles from the
Time Temperature neck into the flask with 5 mL of suljuric acid R, allowing it to
(min) CC) run down the sides of the flask, and mix the contents by
Column 0 - 1.6 40 rotation. Close the mouth of the flask loosely, for example by
1.6 - 9.9 40---+ 65 means of a glass bulb with a short stem, to avoid excessive
9.9 - 13.6 65 ➔ 175 loss of sulfuric acid. Heat gradually at first, then increase the
13.6 - 20 175 temperature until there is vigorous boiling with condensation
Injection port 200 of sulfuric acid in the neck of the flask; precautions should be
Detector 200 taken to prevent the upper part of the flask from becoming
overheated. Continue the heating for 30 min, unless
Detection Flame ionisation. otherwise prescribed. Cool, dissolve the solid material by
Injection 1.0 µL of the test solution and reference cautiously adding to the mixture 25 mL of water R, cool
solution (c), at least 3 times. again and place in a steam-distillation apparatus. Add 30 mL
of strong sodium hydroxide solution R and distil immediately by
Elution order Methanol, ethanol, 2-propanol, 1-propanol.
passing steam through the mixture. Collect about 40 mL of
Relative retention With reference to ethanol (retention distillate in 20.0 mL of 0.01 M hydrochloric acid and enough
time= about 5.3 min): methanol= about 0.8; water R to cover the tip of the condenser. Towards the end
2-propanol = about 1.2; 1-propanol = about 1.6. of the distillation, lower the receiver so that the tip of the
System suitability Reference solution (c): condenser is above the surface of the acid. Take precautions
- resolution: minimum 5 between the peaks due to methanol to prevent any water on the outer surface of the condenser
and ethanol. from reaching the contents of the receiver. Titrate the
Calculate the methanol content in per cent V/V using the distillate with 0.01 M sodium hydroxide, using methyl red mixed
following expression: solutwn Ras indicator (n 1 mL of 0.01 M sodium hydroxide).
Repeat the test using about 50 mg of glucose R in place of the
substance to be examined (n 2 mL of 0.01 M sodium
hydroxide).

area of the peak due to methanol in the chromatogram obtained

. 0.01401 (n2 - ni)
with the test solution; Content of mtrogen =- ------ per cent
area of the peak due to methanol in the chromatogram obtained m
with reference solution (c);
area of the peak due to the internal standard in the Method II (Determination of Protein in Blood
chromatogram obtained with the test solution; Products)
I, area of the peak due to the internal standard in the
chromatogram obtained with reference solution (c).
(No Ph. Eur. method)
For dried blood products prepare a solution of the
preparation as directed in the monograph.
Calculate the 2-propanol content in per cent V/V using the To a volume expected to contain about 0.1 g of protein add
following expression: sufficient saline solution to produce 20 mL. To 2 mL of the
resulting solution, in a 75-mL boiling tube, add 2 mL of a
solution containing 75% v/v of nitrogen-free suljuric acid,
4.5% w/v of potassium sulfate and 0.5% w/v of copper(II)
A3 area of the peak due to 2-propanol in the chromatogram
sulfate, mix and loosely stopper the tube. Heat gradually to
obtained with the test solution; boiling, boil vigorously for 1.5 hours and cool. If the solution
A4 area of the peak due to 2-propanol in the chromatogram is not clear add 0.25 mL of hydrogen peroxide solution
obtained with reference solution (c); (20 vol), continue heating until a clear solution is produced
JI area of the peak due to the internal standard in the
chromatogram obtained with the test solution;
and cool. During heating, take precautions to ensure that the
12 area of the peak due to the internal standard in the upper part of the tube is not overheated.
chromatogram obtained with reference solution (c). Transfer the solution to a distillation apparatus using three
3-mL quantities of water, add 10 mL of 10M sodium hydroxide
and distil rapidly for 4 minutes, collecting the distillate in a
mixture of 5 mL of a saturated solution of boric acid and
5 mL of water and keeping the tip of the condenser below
the level of the acid. Lower the collection flask so that the
condenser can drain freely and continue the distillation for a
further 1 minute. Titrate with 0.02M hydrochlonc acid VS
using methyl red mixed solutwn as indicator (Vi mL).
V-A330 Appendix VIII J 2023

To a further volume of the preparation being examined, or of same solvent. Dilute 1.0 mL of this solution to 20.0 mL with
the solution prepared from it, expected to contain about acetone R.
0.1 g of protein, add 12 mL of saline solution, 2 mL of a Test solution Dissolve 0.500 g of the substance to be
7.5% w/v solution of sodium molybdate and 2 mL of a mixture examined in the internal standard solution and dilute to
of 1 volume of nitrogen-free su!juric acid and 30 volumes of 10.0 mL with the same solution.
water. Shake, allow to stand for 15 minutes, add sufficient Reference solution (a) Mix 30.0 mg of ethylene glycol R
water to produce 20 mL, shake again and centrifuge. Using with acetone Rand dilute to 100.0 mL with the same solvent.
2 mL of the resulting clear supernatant liquid repeat the Dilute 1.0 mL to 10.0 mL with the internal standard
procedure described above beginning at the words 'in a solution.
75-mL boiling tube ... ' (V2 mL). Calculate the protein
content in mg per mL of the preparation being examined, Reference solution (b) Prepare a solution of diethylene
using the expression 6.25 x 0.280(Vi-V2 ) and taking into glycol R with a concentration corresponding to the prescribed
account the initial dilution. limit and using the same solvents as for the preparation of
reference solution (a).
Column:
- material: fused silica,
- size: l = 30 m, 0 = 0.53 mm,
J. Tetrazolium Assay of Steroids - stationary phase: macrogol 20 000 R (film thickness 1 µm).
(No Ph. Bur. method) Carrier gas helium for chromatography R.
The coloured reaction products tend to adsorb onto the surface of Flow rate 10 mL/min.
the glassware. To avoid low results, the glassware should be treated Split ratio 1:3.
with the coloured reaction products before use. The treated
Temperature:
glassware should be reserved for this assay and should be washed
only with water between assays.
Time Temperature
Carry out the following procedure protected from light. (min) CC)
Dissolve a quantity of the substance being examined in Column 0 - 40 80 ➔ 200
sufficient aldehyde-free absolute ethanol to produce a solution 40 - 45 200 ➔ 230
containing 300 to 350 µgin 10 mL unless otherwise 45 - 65 230
specified in the monograph. Transfer 10 mL to a 25-mL Injection port 250
graduated flask, add 2 mL of triphenyltetrazolium chloride Detector 250
solution, displace the air in the flask with oxygen-free nitrogen,
immediately add 2 mL of dilute tetramethylammonium
Detection Flame ionisation.
hydroxi.de solution and again displace the air with oxygen-free
nitrogen. Stopper the flask, mix the contents by gently Injection 2 µL.
swirling and allow to stand in a water bath at 30° for 1 hour Relative retention With reference to 1,2-pentaned.iol
unless otherwise specified in the monograph. Cool rapidly, (retention time= about 19 min): ethylene glycol= about 0.7;
add sufficient aldehyde-free absolute ethanol to produce 25 mL, diethylene glycol = about 1.3.
mix and immediately determine the absorbance of the
resulting solution in a stoppered cell at the maximum at
485 nm, Appendix II B, using in the reference cell a solution
prepared at the same time and in the same manner using L. Residual Solvents
10 mL of aldehyde-free absolute ethanol. Repeat the operation
using the specified BPCRS or EPCRS in place of the (Ph. Bur. method 2.4.24)
substance being examined ensuring that the period of time The test procedures described in this general method may be
that elapses between the addition of the used:
tetramethylammonium hydroxide solution and the i. for the identification of the majority of Class 1 and Class 2
measurement of the absorbance is the same as for the test residual solvents in an active substance, excipient or
solution. medicinal product when the residual solvents are unknown;
ii. as a limit test for Class 1 and Class 2 solvents when
present in an active substance, excipient or medicinal
product;
K. Ethylene Glycol and Diethylene Glycol iii. for the quantification of Class 2 solvents when the limits
in Ethoxylated Substances are greater than 1000 ppm (0.1 per cent) or for the
quantification of Class 3 solvents when required.
(Ph. Bur. method 2.4.30)
Class 1, Class 2 and Class 3 residual solvents are listed in
Bthoxylated substances may contain varied amounts of ethylene general chapter 5. 4. Residual solvents.
glycol and diethylene glycol, as a result of the manufacturing
Three diluents are described for sample preparation and the
process. The following method may be used for the quantitative
conditions to be applied for head-space injection of the
determination of these substances, in particular in the case of the
gaseous sample onto the chromatographic system.
following surfactants: macrogolglycerol ricinoleate, macrogolglycerol
Two chromatographic systems are prescribed but System A is
hydroxystearate, macrogol 15 hydroxystearate, nonoxinol 9 and
preferred whilst System B is employed normally for
macrogol cetostearyl ether.
confirmation of identity. The choice of sample preparation
Gas chromatography (2.2.28). procedure depends on the solubility of the substance to be
Internal standard solution Dissolve 30.0 mg of examined and in certain cases the residual solvents to be
1,2-pentanediol R in acetone Rand dilute to 30.0 mL with the controlled.
2023 Appendix VIII L V-A331

The following residual solvents are not readily detected by Reference solution (a) (Class 1) Introduce 1.0 mL of
the head-space injection conditions described: formamide, solvent solution (a) and 5.0 mL of the appropriate diluent
2-ethoxyethanol, 2-methoxyethanol, ethylene glycol, into an injection vial.
N-methylpyrrolidone and sulfolane. Other appropriate Reference solution (ai) (Class 1) Introduce 5.0 mL of
procedures should be employed for the control of these the sample solution and 1.0 mL of solvent solution (a) into
residual solvents. an injection vial.
When the test procedure is applied quantitatively to control Reference solution (b) (Class 2) Introduce 1.0 mL of
residual solvents in a substance, then it must be validated. solvent solution (b) and 5.0 mL of the appropriate diluent
PROCEDURE into an injection vial.
Examine by gas chromatography with static head-space Reference solution (c) Introduce 5.0 mL of the sample
injection (2.2.28). solution and 1.0 mL of solvent solution (c) into an injection
Sample preparation 1 vial.
This is intended for the control of residual solvents in water- Reference solution (d) Introduce 1.0 mL of the blank
soluble substances. solution and 5.0 mL of the appropriate diluent into an
Sample solution (1) Dissolve 0.200 g of the substance to injection vial.
be examined in water Rand dilute to 20.0 mL with the same Close the vials with a tight rubber membrane stopper coated with
solvent. polytetrafiuoroethylene and secure with an aluminium crimp cap.
Shake to obtain a homogeneous solution.
Sample preparation 2
This is intended for the control of residual solvents in water- The following static head-space injection conditions may be
insoluble substances. used:
Sample solution (2) Dissolve 0.200 g of the substance to
Sample preparation
be examined in dimethylformamide R (DMF) and dilute to procedure
20.0 mL with the same solvent. Operating parameters 2 3
Sample preparation 3 Equilibration temperature CC) 80 105 80
This is intended for the control of N,N-dimethylacetamide Equilibration time (min) 60 45 45
and/or N,N-dimethylformarnide, when it is known or Transfer-line temperature ('CJ 85 110 105
suspected that one or both of these substances are present in Carrier gas: nitrogen for chromatography R or helium for chromatography R at an
the substance to be examined. appropriate pressure
Sample solution (3) Dissolve 0.200 g of the substance to Pressurisation time (s) 30 30 30
be examined in 1,3-dimethyl-2-imidazolidinone R (DMI) and Injection volume (m.L)
dilute to 20.0 mL with the same solvent.
In some cases none of the above sample preparation The chromatographic procedure may be carried out using:
procedures are appropriate, in which case the diluent to be
used for the preparation of the sample solution and the static SYSTEM A
head-space conditions to be employed must be demonstrated - a fused-silica capillary or wide-bore column 30 m long
to be suitable. and 0.32 mm or 0.53 mm in internal diameter coated
with cyanopropyl(3)phenyl(3)methyl(94)polysiloxane R (film
Solvent solution (a) To 1.0 mL of Class 1 residual solvent
thickness 1.8 µm or 3 µm);
solution CRS, add 9 mL of dimethyl sulfoxide R and dilute to
- nitrogen for chromatography R or helium for
100.0 mL with water R. Dilute 1.0 mL of this solution to
chromatography R as the carrier gas, split ratio 1:5 with a
100 mL with water R. Dilute 1.0 mL of this solution to
linear velocity of about 35 cm/s;
10.0 mL with water R.
- a flame-ionisation detector (a mass spectrometer may also
The reference solutions correspond to the following limits: be used or an electron-capture detector for the
- benzene: 2 ppm; chlorinated residual solvents of Class 1);
- carbon tetrachloride: 4 ppm;
maintaining the temperature of the column at 40 °C for
1,2-dichloroethane: 5 ppm;
20 min, then raising the temperature at a rate of 10 °C
- 1,1-dichloroethene: 8 ppm;
per min to 240 °C and maintaining it at 240 °C for 20 min
- 1, I, I -trichloroethane: I O ppm.
and maintaining the temperature of the injection port at
Solvent solution (b) Dissolve appropriate quantities of the 140 °C and that of the detector at 250 °C; or, where there is
Class 2 residual solvents in dimethyl sulfoxide R and dilute to interference from the matrix, use:
100.0 mL with the same solvent. Dilute in water R to give a
SYSTEMB
concentration of 1/20 of the limits stated in Table 2 (see 5.4.
- a fused-silica capillary or wide-bore column 30 m long
Residual solvents).
and 0.32 mm or 0.53 mm in internal diameter coated
Solvent solution (c) Dissolve 1.00 g of the solvent or with macrogol 20 000 R (film thickness 0.25 µm);
solvents present in the substance to be examined in dimethyl - nitrogen for chromatography R or helium for
sulfoxide R or water R, if appropriate, and dilute to 100.0 mL chromatography Ras the carrier gas, split ratio 1:5 with a
with water R. Dilute to give a concentration of 1/20 of the linear velocity of about 35 cm/s;
limit(s) stated in Table I or 2 (see 5.4. Residual solvents). - a flame-ionisation detector (a mass spectrophotometer
Blank solution Prepare as described for solvent may also be used or an electron-capture detector for the
solution (c) but without the addition of solvent(s) (used to chlorinated residual solvents of Class l);
verify the absence of interfering peaks). maintaining the temperature of the column at 50 °C for
Test solution Introduce 5.0 mL of the sample solution 20 min, then raising the temperature at a rate of 6 °C
and 1.0 mL of the blank solution into an injection vial. per min to 165 °C and maintaining it at 165 °C for 20 min
V-A332 Appendix VIII L 2023

2 4/5

------------,-
0 2 3 4 5 6 7 8 9 10 11 12 13 14 min

1. 1, 1-dichloroethene 2. 1,1,1-trichloroethane 3. carbon tetrachloride 4. benzene 5. 1,2-dichloroethanc

Figure 2.4.24.-1. - Typical chromatogram of Class 1 solvents using the conditions described for System A and Procedure 1. Flame-
ionisation detector
19
I I \
3 4 5/6 7 9 12 14/15 16 18 ab/cd 20 21

11

17
2
8

10
'~.,.,,__ __, ~
13
\._}I,__ _ _ _ _ _ __.,

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 11ioo 18.00 20.00 22.00 2400 26.00 2&00 30.00 32.00 34.00
Minutes

I. methanol 5. 1,2-dichloroethene 9. cyclohexane 13. 1,4-dioxane 17. methylbutylketone 20. curnene

2. acetonitrile 6. nitromethane 10. 1,2-dimethoxyethane 14. pyridine 18. chlorobenzene 21. tetralin
3. dichlorornethane 7. tetrahydrofuran 11. 1,1,2-trichloroethene 15. methylisobutylketone 19. xylene mix
a. ethylbenzene
b. p-xylene
c. m-xylene
d. o-xylene
4. hexane 8. chloroform 12. rnethylcyclohexane 16. toluene

Figure 2.4.24.-2. - Chromatogram of Class 2 solvents (solvent solution (b)) using the conditions described for System A and Procedure
1. Flame-ionisation detector
2023 Appendix VIII L V-A333

2/3

2 3 min

1. I, 1-dichloroethene 2. 1,1,1-trichloroethane 3. carbon tetrachloride 4. benzene 5. 1,2-dichloroethane

Figure 2.4.24.-3. - Chromatogram of Class 1 residual solvents using the conditions described for System Band Procedure 1. Flame-
ionisation detector
19
4 9/12 5/11 2/15 16 a" b 'c 19d

18

3/10
7

20

1
17
\_ Jl 8
~ UL/\ 6
'-'
14

0.00 O.flJ 1.00 1.flJ 200 2.flJ 3.00 3.flJ 4.00 4.flJ 5.00 5.flJ 6.00 liflJ 7.00 7flJ 8.00
Mirutes

I. methanol 5. 1,2-dichloroethcne 9. cyclohexane 13. 1,4-dioxane 17. methylbutylketone 20. cumene

2. acetonitrile 6. nitromethane JO. 1,2-dimethoxyethane 14. pyridine 18. chlorobenzene 21. tetralin (tR = 27 min)
3. dichloromethane 7. tetrahydrofuran 11. 1,1,2-trichloroethene 15. methylisobutylketone 19. xylene mix
a. ethylbenzene
b. p-xylene
c. m-xylene
d. o-xylene
4. hexane 8. chloroform 12. methylcyclohexane 16. toluene

Figure 2.4.24.-4. - Typical chromatogram of Class 2 residual solvents (solvent solutwn (b)) using the conditions described for System B
and Procedure 1. Flame-ionisation detector
V-A334 Appendix VIII L 2023

Test solution

No Passes test
No further action

No Passes test
No further action

Yes

Preparation of test
and reference solutions

less than half the area of

the peak obtained with
the reference solution
Passes test

greater than half the area

of the peak obtained with
the reference solution

Fails test

Figure 2.4.24.-5. - Diagram relating to the identification of residual solvents and the application of limit tests
2023 Appendix VIII M V-A335

and maintaining the temperature of the injection port at Inject 1 mL of the gaseous phase of reference solution (d)
140 °C and that of the detector at 250 °C. onto the column. No interfering peaks should be observed.
Inject 1 mL of the gaseous phase of reference solution (a) Inject 1 mL of the gaseous phase of the test solution and
onto the column described in System A and record the 1 mL of the gaseous phase of reference solution (c) on to the
chromatogram under such conditions that the signal-to-noise column. Repeat these injections twice more.
ratio for 1,1,1-trichloroethane can be measured. The signal- The mean area of the peak of the residual solvent(s) in the
to-noise ratio must be at least 5. A typical chromatogram is chromatograms obtained with the test solution is not greater
shown in Figure 2.4.24.-1. than half the mean area of the peak of the corresponding
Inject 1 mL of the gaseous phase of reference solution (a 1) residual solvent(s) in the chromatograms obtained with
onto the column described in System A. The peaks due to reference solution (c). The test is not valid unless the relative
the Class 1 residual solvents are still detectable. standard deviation of the differences in areas between the
Inject 1 mL of the gaseous phase of reference solution (b) analyte peaks obtained from 3 replicate paired injections of
onto the column described in System A and record the reference solution (c) and the test solution, is at most
chromatogram under such conditions that the resolution 15 per cent.
between acetonitrile and dichloromethane can be determined. A flow diagram of the procedure is shown in
The system is suitable if the chromatogram obtained Figure 2.4.24.-5.
resembles the chromatogram shown in Figure 2.4.24.-2 and When a residual solvent (Class 2 or Class 3) is present at a
the resolution between acetonitrile and dichloromethane is at level of 0.1 per cent or greater then the content may be
least 1.0. quantitatively determined by the method of standard
Inject 1 mL of the gaseous phase of the test solution onto the additions.
column described in System A. If in the chromatogram
obtained, there is no peak which corresponds to one of the
residual solvent peaks in the chromatograms obtained with
reference solution (a) or (b), then the substance to be M. Residual Ethylene Oxide and Dioxan
examined meets the requirements of the test. If any peak in
the chromatogram obtained with the test solution (Ph. Bur. method 2.4.25)
corresponds to any of the residual solvent peaks obtained The test is intended for the determination of residual
with reference solution (a) or (b) then System B is to be ethylene oxide and dioxan in samples soluble in water or
employed. dimethylacetamide. For substances that are insoluble or
Inject 1 mL of the gaseous phase ofreference solution (a) insufficiently soluble in these solvents, the preparation of the
onto the column described in System B and record the sample solution and the head-space conditions to be
chromatogram under such conditions that the signal-to-noise employed are given in the individual monograph.
ratio for benzene can be measured. The signal-to-noise ratio Head-space gas chromatography (2.2.28).
must be at least 5. A typical chromatogram is shown in A. For samples soluble in or miscible with water, the
Figure 2.4.24.-3. following procedure may be used.
Inject 1 mL of the gaseous phase of reference solution (a 1) Test solution Weigh 1.00 g (My) of the substance to be
onto the column described in System B. The peaks due to examined in a 10 mL vial (other sizes may be used
the Class 1 residual solvents are still detectable. depending on the operating conditions) and add 1.0 mL of
Inject 1 mL of the gaseous phase of reference solution (b) water R. Close and mix to obtain a homogeneous solution.
onto the column described in System B and record the Allow to stand at 70 °C for 45 min.
chromatogram under such conditions that the resolution Reference solution (a) Weigh 1.00 g (MR) of the
between acetonitrile and 1,1,2-trichloroethene can be substance to be examined into an identical I 0 mL vial, add
determined. The system is suitable if the chromatogram 0.50 mL of dwxan solution R2 and 0.50 mL of ethylene oxide
obtained resembles the chromatogram shown in solutwn R3. Close and mix to obtain a homogeneous solution.
Figure 2.4.24.-4 and the resolution between acetonitrile and Allow to stand at 70 °C for 45 min.
1,1,2-trichloroethene is at least 1.0.
Reference solution (b) To 0.50 mL of ethylene oxide
Inject 1 mL of the gaseous phase of the test solution onto the solution R3 in a 10 mL vial add 0.1 mL of a freshly prepared
column described in System B. If in the chromatogram 10 mg/L solution of acetaldehyde Rand 0.10 mL of dioxan
obtained, there is no peak which corresponds to any of the solution Rl. Close and mix to obtain a homogeneous solution.
residual solvent peaks in the chromatogram obtained with the Allow to stand at 70 °C for 45 min.
reference solution (a) or (b), then the substance to be
B. For samples soluble in or miscible with
examined meets the requirements of the test. If any peak in
dimethylacetamide, the following procedure may be used.
the chromatogram obtained with the test solution
corresponds to any of the residual solvent peaks obtained Test solution Weigh 1.00 g (My) of the substance to be
with reference solution (a) or (b) and confirms the examined in a 10 mL vial (other sizes may be used
correspondence obtained when using System A, then proceed depending on the operating conditions) and add 0.20 mL of
as follows. water Rand 1.0 mL of dimethylacetamide R. Close and mix to
obtain a homogeneous solution. Allow to stand at 90 °C for
Inject 1 mL of the gaseous phase of reference solution (c)
45 min.
onto the column described for System A or System B.
If necessary, adjust the sensitivity of the system so that the Reference solution (a) Weigh 1.00 g (MR) of the
height of the peak corresponding to the identified residual substance to be examined into an identical 10 mL vial, add
solvent(s) is at least 50 per cent of the full scale of the 0.10 mL of dioxan solution Rl, 0.10 mL of ethylene oxide
recorder. solutwn R2 and 1.0 mL of dimethylacetamide R. Close and
mix to obtain a homogeneous solution. Allow to stand at
90 °C for 45 min.
V-A336 Appendix VIII N 2023

Reference solution (b) To 0.10 mL of ethylene oxide mass of the substance to be examined in reference solution (a),
in grams;
solution R2 in a 10 mL vial, add 0.1 mL of a freshly prepared
C amount of ethylene oxide added to reference solution (a), in
10 mg/L solution of acetaldehyde Rand 0.10 mL of dioxan micrograms.
solution Rl. Close and mix to obtain a homogeneous solution.
Allow to stand at 70 °C for 45 min.
Column: DrxC
- material: glass or fused silica;
- size: l =30 m, 0 = 0.32 mm;
area of the peak due to dioxan in the chromatogram obtained
- stationary phase: methylpolysiloxane R (film thickness with the test solution;
1.0 µm). area of the peak due to dioxan in the chromatogram obtained
Carrier gas helium for chromatography R or nitrogen for with reference solution (a);
C amount of dioxan added to reference solution (a) in
chromatography R. micrograms.
Linear velocity 20 emfs.
Split ratio 1:20.
Static head-space conditions that may be used:
- equilibration temperature: 70 °C (90 °C for solutions in
dimethylacetamide); N. N,J\LDimethylaniline
- equz7ibration time: 45 min; (Ph. Bur. method 2.4.26)
- transfer-line temperature: 75 °C (150 °C for solutions in
dimethylacetamide);
METHOD A
- carrier gas: helium for chromatography R; Examine by gas chromatography (2.2.28), using N,N-
- pressurisation time: I min; diethylaniline R as the internal standard.
- injection time: 12 s. Internal standard solution Dissolve 50 mg of N,N-
Temperature: diethylaniline R in 4 mL of 0.1 M hydrochloric acid and dilute
to 50 mL with water R. Dilute 1 mL of this solution to
Time Temperature 100 mL with water R.
(min) (°C) Test solution Dissolve in a ground-glass-stoppered tube
Colunm 0-5 50 0.50 g of the substance to be examined in 30.0 mL of
5 - 31 50-, 180 water R. Add 1.0 mL of the internal standard solution.
31 - 32.5 180 ➔ 230 Adjust the solution to a temperature of 26-28 °C.
32.5 - 37.5 230 Add 1.0 mL of strong sodium hydroxide solution Rand mix
Injection port 150 until completely dissolved. Add 2.0 mL of trimethylpentane R.
Detector 250 Shake for 2 min and allow the phases to separate. Use the
upper layer.
Detection Flame ionisation. Reference solution Dissolve 50.0 mg of N,N-
Injection A suitable volume, for example 1.0 mL, of the dimethylaniline R in 4.0 mL of 0.1 M hydrochloric acid and
gaseous phase of the test solution and of reference dilute to 50.0 mL with water R. Dilute 1.0 mL of this
solutions (a) and (b). Repeat the procedure twice more. solution to 100.0 mL with water R. Dilute 1.0 mL of this
solution to 30.0 mL with water R. Add 1.0 mL of the
System suitability Reference solution (b): internal standard solution and 1.0 mL of strong sodium
- resolution: minimum 2.0 between the peaks due to
hydroxide solution R. Add 2.0 mL of trimethylpentane R. Shake
acetaldehyde and ethylene oxide;
for 2 min and allow the phases to separate. Use the upper
- signal-to-noise ratio: minimum 5 for the peaks due to
layer.
ethylene oxide and dioxan.
The chromatographic procedure may be carried out using:
Verification of precision
- a fused-silica capillary column 25 m long and 0.32 mm in
For each pair of injections, calculate for ethylene oxide and internal diameter coated with phenyl(S0)methyl(S0)
for dioxan the difference in area between the peaks obtained polysiloxane R (film thickness 0.52 µm),
with the test solution and reference solution (a). The test is - helium for chromatography R as the carrier gas with a split
not valid unless the relative standard deviation of the 3 values ratio 1:20, a column head pressure of 50 kPa and a split
obtained for ethylene oxide is not greater than 15 per cent vent of 20 mUmin,
and the relative standard deviation of the 3 values obtained - a flame-ionisation detector,
for dioxan is not greater than 15 per cent. If the weighings - a split-liner consisting of a column about 1 cm long
used for the test solution and reference solution (a) differ packed with diatomaceous earth for gas chromatography R
from 1.00 g by more than 0.5 per cent, the appropriate impregnated with 10 per cent mlm of methylpolysiloxane R,
corrections must be made.
maintaining the temperature of the column at 150 °C for
Calculate the content of ethylene oxide or dioxan in parts per 5 min, then raising the temperature at a rate of 20 °C
million from the following expressions: per min to 275 °C and maintaining it at 275 °C for 3 min
and maintaining the temperature of the detector at 300 °C
and that of the injection port at 220 °C.
The retention times are: N,N-dimethylaniline about 3.6 min,
Ar area of the peak due to ethylene oxide in the chromatogram N,N-diethylaniline about 5.0 min.
obtained with the test solution;
Inject 1 µL of the test solution and 1 µL of the reference
AR area of the peak due to ethylene oxide in the chromatogram
obtained with reference solution (a); solution.
Mr mass of the substance to be examined in the test solution, in
grams;
2023 Appendix VIII P V-A337

METHODB with the following temperature programme:

Examined by gas chromatography (2.2.28), using
naphthalene R as the internal standard. Time Temperature Rate Comment
(min) CC) ('C/min)
Internal standard solution Dissolve 50 mg of
Column 0-2 40 isothermal
naphthalene R in cyclohexane R and dilute to 50 mL with the
2 - 7.3 40 ➔ 200 30 linear gradient
same solvent. Dilute 5 mL of this solution to 100 mL with
7.3 - 10.3 200 isothermal
cyclohexane R.
Injection port 200
Test solution To 1.00 g of the substance to be examined
Detector 300
in a ground-glass-stoppered tube add 5 mL of 1 M sodium
hydroxide and 1.0 mL of the internal standard solution.
Stopper the tube and shake vigorously for 1 min. Centrifuge Inject I µL of the test solution and 1 µL of the reference
if necessary and use the upper layer. solution.
Reference solution To 50.0 mg of N,N-dimethylaniline R The test is not valid unless the resolution between the peaks
add 2 mL of hydrochloric acid R and 20 mL of water R, shake due to 2-ethylhexanoic acid (first peak) and the internal
to dissolve and dilute to 50.0 mL with water R. Dilute standard is at least 2.0.
5.0 mL of this solution to 250.0 mL with water R. Calculate the percentage content of 2-ethylhexanoic acid
To 1.0 mL of the latter solution in a ground-glass-stoppered from the expression:
tube add 5 mL of 1 M sodium hydroxide and 1.0 mL of the
internal standard solution. Stopper the tube and shake
AT x IR x mR x 2
vigorously for 1 min. Centrifuge if necessary and use the ARxhxmT
upper layer. AT area of the peak due to 2-ethylhexanoic acid in the
The chromatographic procedure may be carried out using: chromatogram obtained with the test solution,
AR area of the peak due to 2-ethylhexanoic acid in the
- a glass column 2 m long and 2 mm in internal diameter
chromatogram obtained with the reference solution,
packed with silanised diatomaceous eanh for gas IT area of the peak due to the internal standard in the
chromatography R impregnated with 3 per cent mlm of chromatogram obtained with the test solution,
phenyl(50)methyl(50)polysiloxane R, IR area of the peak due to the internal standard in the
chromatogram obtained with the reference solution,
- nitrogen for chromatography R as the carrier gas at a flow
m,, mass of the substance to be examined in the test solution, in
rate of 30 mUmin, grams,
- a flame-ionisation detector, mR mass of 2-ethylhexanoic acid in the reference solution, in grams.
maintaining the temperature of the column at 120 °C and
that of the injection port and of the detector at 150 °C.
Inject 1 µL of the test solution and 1 µL of the reference
solution.
P. Total Protein
(Ph. Bur. method 2.5.33)
Many of the assay methods described in this chapter can be
0. 2-Ethylhexanoic Acid performed using kits from commercial sources.
(Ph. Bur. method 2.4.28) METHOD 1
Examine by gas chromatography (2.2.28), using Protein in solution absorbs ultraviolet light at a wavelength of
3-cyclohexylpropionic acid R as the internal standard. 280 nm, due to the presence of aromatic amino acids, mainly
Internal standard solution Dissolve I 00 mg of tyrosine and tryptophan, in the protein structure. This
3-cyclohexylpropionic acid R in cyclohexane R and dilute to property can be used for assay purposes. If the buffer used to
I 00 mL with the same solvent. dissolve the protein has a high absorbance relative to that of
water, an interfering substance is present. This interference
Test solution To 0.300 g of the substance to be examined,
may be obviated by using the buffer as compensation liquid
add 4.0 mL of a 33 per cent V/V solution of hydrochloric
but if the interfering substance produces a high absorbance,
acid R. Shake vigorously for I min with 1.0 mL of the
the results may nevertheless be compromised. At low
internal standard solution. Allow the phases to separate (if
concentrations, protein adsorbed onto the cell may
necessary, centrifuge for a better separation). Use the upper
significantly reduce the content in solution. This can be
layer.
prevented by preparing samples at higher concentration or by
Reference solution Dissolve 75.0 mg of 2-ethylhexanoic using a non-ionic detergent in the preparation.
acid R in the internal standard solution and dilute to
Test solution Dissolve a suitable quantity of the substance
50.0 mL with the same solution. To 1.0 mL of the solution
to be examined in the prescribed buffer to obtain a solution
add 4.0 mL of a 33 per cent VIV solution of hydrochloric
having a protein concentration between 0.2 mgimL and
acid R. Shake vigorously for I min. Allow the phases to
2 mgimL.
separate (if necessary, centrifuge for a better separation).
Use the upper layer. Reference solution Prepare a solution of a suitable
reference substance for the protein to be determined, in the
The chromatographic procedure may be carried out using:
same buffer and at the same protein concentration as the test
- a wide-bore fused-silica column 10 m long and 0.53 mm
solution.
in internal diameter coated with macrogol
20 000 2-nitroterephthalate R (film thickness 1.0 µm), Procedure
- helium for chromatography R as the carrier gas at a flow Keep the test solution, the reference solution and the
rate of 10 mUmin, compensation liquid at the same temperature during the
- a flame-ionisation detector, performance of this test. Determine the absorbances (2. 2. 25)
of the test solution and the reference solution in quartz cells
V-A338 Appendix VIII P 2023

at 280 nm, using the prescribed buffer as the compensation Reference solutions Dissolve the reference substance for
liquid. The response must be linear in the range of protein the protein to be determined in the prescribed buffer. Dilute
concentrations to be assayed to obtain accurate results. portions of this solution with the same buffer to obtain not
Light scattering fewer than five reference solutions having protein
The accuracy of the determination of protein can be concentrations evenly spaced over a suitable range situated
diminished by the scattering of light by the test sample. If the between 5 µgimL and 100 µgimL.
proteins in solution exist as particles comparable in size to Blank Use the buffer used to prepare the test solution and
the wavelength of the measuring light (250 nm to 300 nm), the reference solutions.
scattering of the light beam results in an apparent increase in Copper sulfate reagent Dissolve 100 mg of copper sulfate
absorbance of the test sample. To calculate the absorbance at pentahydrate Rand 0.2 g of sodium tartrate R in distilled
280 nm due to light scattering, determine the absorbances of water R and dilute to 50 mL with the same solvent. Dissolve
the test solution at wavelengths of 320 nm, 325 nm, 330 nm, 10 g of anhydrous sodium carbonate R in distilled water R and
335 nm, 340 nm, 345 nm and 350 nm. Plot the logarithm of dilute to 50 mL with the same solvent. Slowly pour the
the observed absorbance against the logarithm of the sodium carbonate solution into the copper sulfate solution
wavelength and determine the standard curve best fitting the with mixing. Use within 24 h.
plotted points by linear regression. Extrapolate the curve to Alkaline copper reagent Mix 1 volume of copper sulfate
determine the logarithm of the absorbance at 280 nm. reagent, 2 volumes of a 50 giL solution of sodium dodecyl
The antilogarithm of this value is the absorbance attributed sulfate R and 1 volume of a 32 giL solution of sodium
to light scattering. Correct the observed values by subtracting hydroxide R. Store at room temperature and use within
the absorbance attributed to light scattering from the total 2 weeks.
absorbance at 280 nm to obtain the absorbance value of the
Diluted phosphomolybdotungstic reagent Mix 5 mL of
protein in solution. Filtration with a 0.2 µm filter that does
phosphomolybdotungstic reagent R with 55 mL of distilled
not adsorb protein or clarification by centrifugation may be
water R. Store in an amber bottle, at room temperature.
performed to reduce the effect of light scattering, especially if
the solution is noticeably turbid. Procedure
To 1.0 mL of each reference solution, of the test solution
Calculations
and of the blank, add 1.0 mL of alkaline copper reagent and
Use corrected values for the calculations. Calculate the
mix. Allow to stand for 10 min. Add 0.5 mL of the diluted
concentration of protein in the test solution (Cu) from the
phosphomolybdotungstic reagent, mix and allow to stand at
following equation:
room temperature for 30 min. Determine the absorbances
(2.2.25) of the solutions at 750 nm, using the solution from
Cu= Cs(Au/As)
the blank as compensation liquid.
where Cs is the concentration of protein in the reference Calculations
solution and Au and As are the corrected absorbances of the The relationship of absorbance to protein concentration is
test solution and the reference solution, respectively. non-linear; however, if the range of concentrations used to
prepare the standard curve is sufficiently small, the latter will
METH0D2
approach linearity. Plot the absorbances of the reference
This method (commonly referred to as the Lowry assay) is
solutions against the protein concentrations and use linear
based on the reduction by protein of the
regression to establish the standard curve. From the standard
phosphomolybdotungstic mixed acid chromogen in the
curve and the absorbance of the test solution, determine the
phosphomolybdotungstic reagent, which results in an
concentration of protein in the test solution.
absorbance maximum at 750 nm.
The phosphomolybdotungstic reagent reacts primarily with Interfering substances
tyrosine residues in the protein. Colour development reaches In the following procedure, deoxycholate-trichloroacetic acid
a maximum in 20 min to 30 min at room temperature, after is added to a test sample to remove interfering substances by
which there is a gradual loss of colour. Because the method precipitation of proteins before determination; this technique
is sensitive to interfering substances, a procedure for can also be used to concentrate proteins from a dilute
precipitation of the protein from the test sample may be solution.
used. Most interfering substances cause a lower colour yield; Add 0.1 mL of a 1.5 giL solution of sodium deoxycholate R to
however, some detergents cause a slight increase in colour. 1 mL of a solution of the substance to be examined.
A high salt concentration may cause a precipitate to form. Mix using a vortex mixer and allow to stand at room
Because different protein species may give different colour temperature for 10 min. Add 0.1 mL of a 720 giL solution
response intensities, the reference substance and test protein of trichk!roacetic acid Rand mix using a vortex mixer.
must be the same. Where separation of interfering substances Centrifuge at 3000 g for 30 min, decant the liquid and
from the protein in the test sample is necessary, proceed as remove any residual liquid with a pipette. Redissolve the
directed below for interfering substances prior to preparation protein pellet in 1 mL of alkaline copper reagent.
of the test solution. The effect of interfering substances may METHOD3
be minimised by dilution, provided the concentration of the This method (commonly referred to as the Bradford assay) is
test protein remains sufficient for accurate measurement.
based on the absorption shift from 4 70 nm to 595 nm
Use distilled water R to prepare all buffers and reagents used observed when the acid blue 90 dye binds to protein.
for this method. The acid blue 90 dye binds most readily to arginine and
Test solution Dissolve a suitable quantity of the substance lysine residues in the protein which can lead to variation in
to be examined in the prescribed buffer to obtain a solution the response of the assay to different proteins. The protein
having a concentration within the range of the standard used as reference substance must therefore be the same as
curve. A suitable buffer will produce a solution of pH 10.0 to the protein to be determined. There are relatively few
10.5. interfering substances, but it is preferable to avoid detergents
2023 Appendix VIII P V-A339

and ampholytes in the test sample. Highly alkaline samples concentrations evenly spaced over a suitable range situated
may interfere with the acidic reagent. between 10 µg/mL and 1200 µg/mL.
Use distilled water R to prepare all buffers and reagents used Blank Use the buffer used to prepare the test solution and
for this method. the reference solutions.
Test solution Dissolve a suitable quantity of the substance BCA reagent Dissolve 10 g of disodium buinchoninate R,
to be examined in the prescribed buffer to obtain a solution 20 g of sodium carbonate monohydrate R, 1.6 g of sodium
having a concentration within the range of the standard tartrate R, 4 g of sodium hydroxide R, and 9.5 g of sodium
curve. hydrogen carbonate R in distilled water R. Adjust, if necessary,
Reference solutions Dissolve the reference substance for to pH 11.25 with a solution of sodium hydroxide R or a
the protein to be determined in the prescribed buffer. Dilute solution of sodium hydrogen carbonate R. Dilute to 1000 mL
portions of this solution with the same buffer to obtain not with distilled water R and mix.
fewer than five reference solutions having protein Copper-BCA reagent Mix 1 mL of a 40 g/L solution of
concentrations evenly spaced over a suitable range situated copper sulfate pentahydrate R and 50 mL of BCA reagent.
between 0.1 mg/mL and 1 mg/mL. Procedure
Blank Use the buffer used to prepare the test solution and Mix 0.1 mL of each reference solution, of the test solution
the reference solutions. and of the blank with 2 mL of the copper-BCA reagent.
Acid blue 90 reagent Dissolve 0.10 g of acid blue 90 R in Incubate the solutions at 37 °C for 30 min, note the time
50 mL of alcohol R. Add 100 mL of phosphoric acid R, dilute and allow the mixtures to cool to room temperature. Within
to 1000 mL with distilled water R and mix. Filter the solution 60 min of the end of incubation, determine the absorbances
and store in an amber bottle at room temperature. Slow (2. 2. 25) of the reference solutions and of the test solution in
precipitation of the dye occurs during storage. Filter the quartz cells at 562 nm, using the blank as compensation
reagent before using. liquid. After the solutions have cooled to room temperature,
the colour intensity continues to increase gradually.
Procedure
Add 5 mL of acid blue 90 reagent to 0.100 mL of each Calculations
reference solution, of the test solution and of the blank. The relationship of absorbance to protein concentration is
Mix by inversion. Avoid foaming, which will lead to poor non-linear; however, if the range of concentrations used to
reproducibility. Determine the absorbances (2.2.25) of the prepare the standard curve is sufficiently small, the latter will
standard solutions and of the test solution at 595 nm, using approach linearity. Plot the absorbances of the reference
the blank as compensation liquid. Do not use quartz (silica) solutions against protein concentrations and use linear
spectrophotometer cells because the dye binds to this regression to establish the standard curve. From the standard
material. curve and the absorbance of the test solution, determine the
concentration of protein in the test solution.
Calculations
The relationship of absorbance to protein concentration is METHOD 5
non-linear; however, if the range of concentrations used to This method (commonly referred to as the biuret assay) is
prepare the standard curve is sufficiently small, the latter will based on the interaction of cupric (Cu2+) ion with protein in
approach linearity. Plot the absorbances of the reference alkaline solution and resultant development of absorbance at
solutions against protein concentrations and use linear 545 nm. This test shows minimal difference between
regression to establish the standard curve. From the standard equivalent IgG and albumin samples. Addition of the sodium
curve and the absorbance of the test solution, determine the hydroxide and the biuret reagent as a combined reagent,
concentration of protein in the test solution. insufficient mixing after the addition of the sodium
METHOD4 hydroxide, or an extended time between the addition of the
This method (commonly referred to as the bicinchoninic acid sodium hydroxide solution and the addition of the biuret
or BCA assay) is based on reduction of the cupric (Cu 2 +) ion reagent will give IgG samples a higher response than albumin
to cuprous (Cu 1+) ion by protein. The bicinchoninic acid
samples. The trichloroacetic acid method used to minimise
reagent is used to detect the cuprous ion. Few substances the effects of interfering substances also can be used to
interfere with the reaction. When interfering substances are determine the protein content in test samples at
present their effect may be minimised by dilution, provided concentrations below 500 µg/mL.
that the concentration of the protein to be determined Use distilled water R to prepare all buffers and reagents used
remains sufficient for accurate measurement. Alternatively, for this method.
the protein precipitation procedure given in Method 2 may Test solution Dissolve a suitable quantity of the substance
be used to remove interfering substances. Because different to be examined in a 9 g/L solution of sodium chloride R to
protein species may give different colour response intensities, obtain a solution having a concentration within the range of
the reference protein and protein to be determined must be the concentrations of the reference solutions.
the same. Reference solutions Dissolve the reference substance for
Use distilled water R to prepare all buffers and reagents used the protein to be determined in a 9 g/L solution of sodium
for this method. chloride R. Dilute portions of this solution with a 9 g/L
Test solution Dissolve a suitable quantity of the substance solution of sodium chloride R to obtain not fewer than three
to be examined in the prescribed buffer to obtain a solution reference solutions having protein concentrations evenly
having a concentration within the range of the concentrations spaced over a suitable range situated between 0.5 mg/mL
of the reference solutions. and 10 mg/mL.
Reference solutions Dissolve the reference substance for Blank Use a 9 g/L solution of sodium chloride R.
the protein to be determined in the prescribed buffer. Dilute Biuret reagent Dissolve 3.46 g of copper sulfate
portions of this solution with the same buffer to obtain not pentahydrate R in 10 mL of hot distilled water R, and allow to
fewer than five reference solutions having protein cool (Solution A). Dissolve 34.6 g of sodium citrate R and
V-A340 Appendix VIII P 2023

20.0 g of anhydrous sodium carbonate R in 80 mL of hot solution of sodium chloride R to obtain not fewer than five
distilled water R, and allow to cool (Solution B). reference solutions having protein concentrations evenly
Mix solutions A and B and dilute to 200 mL with distilled spaced over a suitable range situated between 10 µg/mL and
water R. Use within 6 months. Do not use the reagent if it 200 µg/mL. Adjust the solutions to pH 8 to 10.5 before
develops turbidity or contains any precipitate. addition of the phthalaldehyde reagent.
Procedure Blank solution Use a 9 g/L solution of sodium chloride R.
To one volume of the test solution add an equal volume of a Borate buffer solution Dissolve 61.83 g of boric acid R in
60 g/L solution of sodium hydroxide R and mix. Immediately distilled water Rand adjust to pH 10.4 with a solution of
add biuret reagent equivalent to 0.4 volumes of the test potassium hydroxide R. Dilute to 1000 mL with distilled
solution and mix rapidly. Allow to stand at a temperature water R and mix.
between 15 "C and 25 °C for not less than 15 min. Within Phthalaldehyde stock solution Dissolve 1.20 g of
90 min of addition of the biuret reagent, determine the phthalaldehyde R in 1.5 mL of methanol R, add 100 mL of
absorbances (2.2.2S) of the reference solutions and of the test borate buffer solution and mix. Add 0.6 mL of a 300 g/L
solution at the maximum at 545 nm, using the blank as solution of macrogol 23 lauryl ether R and mix. Store at room
compensation liquid. Any solution that develops turbidity or temperature and use within 3 weeks.
a precipitate is not acceptable for calculation of protein
Phthalaldehyde reagent To 5 mL of phthalaldehyde
concentration.
stock solution add 15 µL of 2-mercaptoethanol R. Prepare at
Calculations least 30 min before use. Use within 24 h.
The relationship of absorbance to protein concentration is
Procedure
approximately linear within the indicated range of protein
Mix 10 µL of the test solution and of each of the reference
concentrations for the reference solutions. Plot the
solutions with 0.1 mL ofphthalaldehyde reagent and allow to
absorbances of the reference solutions against protein
stand at room temperature for 15 min. Add 3 mL of 0.5 M
concentrations and use linear regression to establish the
sodium hydroxide and mix. Determine the fluorescent
standard curve. Calculate the correlation coefficient for the
intensities (2.2.21) of solutions from the reference solutions
standard curve. A suitable system is one that yields a line
and from the test solution at an excitation wavelength of
having a correlation coefficient not less than 0.99. From the
340 nm and an emission wavelength between 440 and
standard curve and the absorbance of the test solution,
455 nm. Measure the fluorescent intensity of a given sample
determine the concentration of protein in the test solution.
only once, since irradiation decreases the fluorescence
Interfering substances intensity.
To minimise the effect of interfering substances, the protein
Calculations
can be precipitated from the test sample as follows: add
The relationship of fluorescence to protein concentration is
0.1 volumes of a 500 g/L solution of trichloroacetic acid R to
linear. Plot the fluorescent intensities of the reference
1 volume of a solution of the test sample, withdraw the
solutions against protein concentrations and use linear
supernatant layer and dissolve the precipitate in a small
regression to establish the standard curve. From the standard
volume of 0.5 M sodium hydroxide. Use the solution obtained
curve and the fluorescent intensity of the test solution,
to prepare the test solution.
determine the concentration of protein in the test solution.
METHOD6
METHOD 7
This fluorimetric method is based on the derivatisation of the
This method is based on nitrogen analysis as a means of
protein with o-phthalaldehyde, which reacts with the primary
protein determination. Interference caused by the presence of
amines of the protein (N-terminal amino acid and the
other nitrogen-containing substances in the test sample can
i:-amino group of lysine residues). The sensitivity of the assay
affect the determination of protein by this method. Nitrogen
can be increased by hydrolysing the protein before adding
analysis techniques destroy the test sample during the
o-phthalaldehyde. Hydrolysis makes the IX-amino group of the
analysis but are not limited to protein presentation in an
constituent amino acids available for reaction with the
aqueous environment.
phthalaldehyde reagent. The method requires very small
quantities of the protein. Primary amines, such as tris Procedure A
(hydroxymethyl)aminomethane and amino acid buffers, react Proceed as prescribed for the determination of nitrogen by
with phthalaldehyde and must be avoided or removed. sulfuric acid digestion (2.5.9) or use commercial
Ammonia at high concentrations reacts with phthalaldehyde. instrumentation for Kjeldahl nitrogen assay.
The fluorescence obtained when amine reacts with Procedure B
phthalaldehyde can be unstable. The use of automated Commercial instrumentation is available for nitrogen analysis.
procedures to standardise this procedure may improve the Most nitrogen analysis instruments use pyrolysis
accuracy and precision of the test. (i.e. combustion of the sample in oxygen at temperatures
Use distilled water R to prepare all buffers and reagents used approaching 1000 °C), which produces nitric oxide (NO)
for this method. and other oxides of nitrogen (NOx) from the nitrogen present
Test solution Dissolve a suitable quantity of the substance in the substance to be examined. Some instruments convert
to be examined in a 9 g/L solution of sodium chloride R to the nitric oxides to nitrogen gas, which is quantified using a
obtain a solution having a concentration within the range of thermal-conductivity detector. Other instruments mix nitric
the concentrations of the reference solutions. Adjust the oxide (NO) with ozone (0 3) to produce excited nitrogen
solution to pH 8 to 10.5 before addition of the dioxide (N0 2 *), which emits light when it decays and can be
phthalaldehyde reagent. quantified with a chemiluminescence detector. A protein
reference material that is relatively pure and is similar in
Reference solutions Dissolve the reference substance for
composition to the test proteins is used to optimise the
the protein to be determined in a 9 g/L solution of sodium
injection and pyrolysis parameters and to evaluate
chloride R. Dilute portions of this solution with a 9 g/L
consistency in the analysis.
2023 Appendix VIII SI V-A341

Calculations 40 min. Allow the digestion vessels to cool before opening.

The protein concentration is calculated by dividing the Add 2.0 mL of strong hydrogen peroxide solutwn R and repeat
nitrogen content of the sample by the known nitrogen the digestion step. Allow the digestion vessels to cool before
content of the protein. The known nitrogen content of the opening. Quantitatively transfer to a 25 mL flask, add
protein can be determined from the chemical composition of 0.5 mL of a 10 g!L solution of magnesium nitrate Rand
the protein or by comparison with a suitable reference 0.5 mL of a 100 glL solution of ammonium dihydrogen
substance. phosphate R, dilute to 25.0 mL with water for
chromatography R and mix.
Reference solutions Into 4 volumetric flasks, introduce
25 µL, 50 µL, 75 µLand 100 µL of nickel standard solutwn
a. Acetic Acid in Synthetic Peptides (5 ppm Ni) R. To each flask, add 0.5 mL of a 10 glL
solution of magnesium nitrate R, 0.5 mL of a 100 glL solution
(Ph. Eur. method 2.5.34)
of ammonium dihydrogen phosphate Rand 6.0 mL of nickel-free
Examine by liquid chromatography (2.2.29). nitric acid Rand dilute to 25.0 mL with water for
Test solution Prepare as described in the monograph. chromatography R. Mix to obtain reference solutions
The concentration of peptide in the solution may be adapted, containing respectively 5 nglmL, 10 nglmL, 15 nglmL and
depending on the expected amount of acetic acid in the 20 nglmL (ppb) of nickel.
sample. Zero solution In a volumetric flask, introduce 1.0 mL of a
Reference solution Prepare a 0.10 g!L solution of glacial 10 glL solution of magnesium nitrate R, 1.0 mL of a 100 glL
acetic acid R in a mixture of 5 volumes of mobile phase B solution of ammonium dihydrogen phosphate R and 12. 0 mL of
and 95 volumes of mobile phase A. nickel-free nitric acid R. Dilute to 50.0 mL with water for
The chromatographic procedure may be carried out using: chromatography R and mix.
- a stainless steel column 0.25 m long and 4.6 mm in Method Determine the absorbance of each solution at
internal diameter packed with octadecylsuyl silica gel for 232.0 nm using a suitable graphite furnace atomic absorption
chromatography R (5 µm), (GFAA) spectrometer equipped with a background
- as mobile phase at a flow rate of 1.2 mIJmin: compensation system, a pyrolytically-coated tube and a nickel
Mobile phase A Dilute 0. 7 mL of phosphoric acid R to hollow-cathode lamp. The optimal temperature programme
1000 mL with water R; adjust the pH to 3.0 with strong may be different for each instrument, so the programme
sodium hydroxide solution R, recommended by the spectrometer manufacturer may be
used. The following temperature parameters for GFAA
Mobile phase B Methanol R2,
analysis are given as an example: maintain the drying
temperature of the furnace at 120 °C for 35 s after a 5 s
Time Mobile phase A Mobile phase B
(min) (per cent VIV) (per cent V/V) ramp, the ashing temperature at 1100 °C for 10 s after a 30 s
ramp, the cooling temperature at 800 °C for 5 s after a 5 s
0-5 95 5
95--> 50 5--, 50
decrease, and the atomisation temperature at 2600 °C for
5 - 10
7 s. Use the zero solution to set the instrument to zero.
10 - 20 50 50
50 __, 95 50--, 5
Using the calibration curve, determine the concentrations of
20 - 22
the test solution and the blank solution from the
22 - 30 95 5
corresponding absorptions. If necessary, dilute with the zero
solution to obtain a reading within the calibrated absorbance
- as detector a spectrophotometer set at 210 nm.
range.
Inject 10 µL of the reference solution and 10 µL of the test
Calculate the content of Ni in micrograms per gram (ppm)
solution. In the chromatograms obtained, the peak
using the following expression:
corresponding to acetic acid has a retention time of 3-4 min.
The baseline presents a steep rise after the start of the linear cxf
gradient, which corresponds to the elution of the peptide
mx40
from the column. Determine the content of acetic acid in the
peptide. measured concentration of Ni, in nanograms per millilitre;
f dilution factor of the test solution;
m mass of the substance to be examined, in grams.

R. Nickel in Hydrogenated Vegetable Oils

(Ph. Eur. method 2.4.31)
Atomic absorption spectrometry (2.2.23, Method 1).
S1. Methyl, Ethyl and lsopropyl
The reagents magnesium nitrate R and ammonium dihydrogen Methanesulfonate in Methanesulfonic
phosphate R must be controlled for nickel before use. The actual Acid
nickel content is taken into account in the calculation of the nickel
content of the sample. (Ph. Eur. method 2.5.37)
Test solution Weigh 0.250 g (m) of the substance to be The following method has been validated for the methyl,
examined into a suitable high-pressure-resistant digestion ethyl and isopropyl esters of methanesulfonic acid at
vessel (fluoropolymer or quartz glass), add 6.0 mL of nickel- concentrations in the range of 0.5 ppm to 100 ppm.
free nitric acid Rand 2.0 mL of strong hydrogen peroxide If it is intended to be used to determine levels of
solution R. Prepare a blank solution in the same manner. methanesulfonic acid esters outside this validated range, for
Place the closed vessels in a laboratory microwave oven and example in early steps of the synthesis prior to their removal,
digest with an appropriate programme, e.g. 1000 W for
V-A342 Appendix VIII S2 2023

the concentration of the test solution has to be adjusted Relative retention With reference to the internal
accordingly. standard (BMS) (retention time = about 7.6 min):
Gas chromatography (2.2.28) coupled with mass MMS = about 0.3; EMS = about 0.5; IMS = about 0.6.
spectrometry (2.2.43). System suitability:
Internal standard solution Dilute 7 µL of butyl - resolution: minimum 3.0 between the peaks due to EMS
methanesulfonate CRS (BMS) to 10.0 mL with methylene and IMS in the chromatogram obtained with reference
chloride R. Dilute 10 µL of the solution to 100.0 mL with solution (a);
methylene chloride R. - signal-to-noise ratio: minimum 10 for the peaks due to
MMS, EMS and IMS in the chromatogram obtained
Test solution Add 0.74 g of the substance to be examined
with reference solution (b).
to 10.0 mL of water Rand extract with 10.0 mL of the
internal standard solution. Allow to separate and transfer the Calculate the content of MMS, EMS or IMS in parts per
organic layer to a vial containing anhydrous sodium suljau R. million using the following expression:
Shake and filter.
A2 xii x W1 xCx0.148
Reference solution (a) Dissolve 50 mg each of methyl
A 1 x]zxW2
methanesuifonate R (MMS), ethyl methanesuifonate R (EMS)
and isopropyl methanesulfonate R (IMS) in the internal area of the peak due to MMS, EMS or IMS in the
standard solution and dilute to 50.0 mL with the same chromatogram obtained with reference solution (a);
solution. Dilute 7 4 µL of the solution to 10.0 mL with the area of the peak due to MMS, EMS or IMS in the
internal standard solution. Dilute 100 µL of this solution to chromatogram obtained with the test solution;
C percentage content of MMS, EMS or IMS;
10.0 mL with the internal standard solution. area of the peak due to the internal standard in the
Ii
Reference solution (b) Dilute 3.0 mL of reference chromatogram obtained with reference solution (a);
solution (a) to 10.0 mL with the internal standard solution. area of the peak due to the internal standard in the
chromatogram obtained with the test solution;
Column: mass of MMS, EMS or IMS used to prepare reference
- material: fused silica; solution (a), in milligrams;
=
- size: l 15 m, 0 0.25 mm; = mass of the substance to be examined in the test solution, in
milligrams;
- stationary phase: methylpolysiloxane R (film thickness 0.148 dilution factor.
1 µm).
Carrier gas helium for chromatography R.
Flow rate 1 mIJmin.
Pulsed splitless 250 kPa, 0.25 min.
Temperature:
S2. Methyl, Ethyl and lsopropyl
Methanesulfonate in Active Substances
Time Temperature
(min) CC)
(Ph. Bur. method 2.5.38)
Column 0-1 55 The following general method has been validated for the
1- 9 55-, 135 determination of methyl, ethyl and isopropyl esters of
Injection port 240 methanesulfonic acid (in concentrations between 0.2 ppm
Detector: transfer line 280 and 5 ppm) in betahistine mesilate.
source 230 If it is intended to use the method for other active
analyser 150 substances, particularly those that contain different
concentrations of the methanesulfonic acid esters, the
Detection Mass spectrometer as described below; adjust concentrations of the test solution and reference solutions
the detector settings so as to comply with the system must be adjusted accordingly and the method must be
suitability criteria: suitably validated.
- quadrupole mass spectrometer equipped with an electron METHOD
impact ionisation mode (70 eV); Head-space gas chromatography (2.2.28) coupled with mass
- mass spectrometer parameters for the fragrnentometric spectrometry (2.2.43). Prepare the test solution and reference
mode (single-ion monitoring (SIM)) set as follows: solutions immediately before use.
Solvent mixture water R, acetonitrile R (20:80 V/V). The
Duration of
Substance mlz
monitoring
use of acetonitrile of appropriate purity is essential.
Solution A Dissolve with the aid of ultrasound 30 mg of
tR between 7 .0 min and anhydrous sodium thiosulfate Rand 60.0 g of sodium iodide R in
Butyl methanesulfonate (BMS) 56
9.0 min
water R and dilute to 50.0 mL with the same solvent.
Methyl mcthanesulfonatc (MMS) 80
tR between 2.0 min and Internal standard solution Dilute 10 µL of butyl
3.5 min methanesulfonate CRS (BMS) to 10.0 mL with the solvent
tR between 4.0 min and mixture. Dilute 20 µL of the solution to 100.0 mL with the
Ethyl methanesulfonate (EMS) 79
4.7 min solvent mixture.
tR between 4. 7 min and
Blank solution Introduce 0.50 mL of solution A and
Isopropyl methanesulfonate (IMS) 123 0.50 mL of the internal standard solution into a headspace
5.5 min
vial and seal the vial immediately with a
polytetrafluoroethylene-coated silicon membrane and an
Injection 2 µL.
aluminium cap.
2023 Appendix VIII S3 V-A343

Test solution Weigh 25.0 mg of the substance to be Quantitation ion Qualification ion
Substance
examined into a 20 mL headspace vial. Add 0.50 mL of (m/z) (mlz)
solution A and 0.50 mL of the internal standard solution and Butyl iodide (Bul)* 184 127
seal the vial immediately with a polytetrafluoroethylene-
coated silicon membrane and an aluminium cap. Methyl iodide (Mel)* 142 127
Following the derivatisation reaction, a precipitate may be Ethyl iodide (EtI)* 156 127
observed, however this does not affect the validity of the
quantification. lsopropyl iodide (iPrl)* 170 127

Reference solution (a) Dissolve 25.0 mg each of methyl * formed from BMS, MMS, EMS and IMS in the derivatisation reaction.
methanesulfonate R (J\11viS), ethyl methanesulfonate R (EMS)
and isopropyl methanesulfonate R (IMS) in toluene R and dilute
Injection 1 mL of the gas phase of the test solution,
to 5.0 mL with the same solvent. Dilute 50 µL of the
reference solutions (b) and (c) and the blank solution.
solution to 25.0 mL with the internal standard solution.
Relative retention With reference to the internal standard
Reference solution (b) Dilute 20 µL of reference
(BuI) (retention time= about 8.5 min): Mel= about 0.51;
solution (a) to 20.0 mL with the internal standard solution.
Etl = about 0.63; iPrI = about 0.68.
Introduce 0.50 mL of this solution and 0.50 mL of
solution A into a 20 mL headspace vial and seal the vial System suitability:
immediately with a polytetrafluoroethylene-coated silicon - resolution: minimum 1.5 between the peaks due to Etl and
membrane and an aluminium cap. iPrI in the chromatogram obtained with reference
solution (c);
Reference solution (c) Dilute 500 µL of reference
- signal-to-noise ratio: minimum 10 for the peak due to each
solution (a) to 20.0 mL with the internal standard solution.
alkyl iodide in the chromatogram obtained with reference
Introduce 0.50 mL of this solution and 0.50 mL of
solution (b).
solution A into a 20 mL headspace vial and seal the vial
immediately with a polytetrafluoroethylene-coated silicon Calculate the content in parts per million of each alkyl
membrane and an aluminium cap. methanesulfonate using the following expression:
Column: A2 X 11 X W1 X C X 0.05
- material: fused silica;
A1xI2xW2
- size: l = 30 m, 0 = 0.25 mm;
- stationary phase: polar-deactivated macrogol R (film A1 area of the peak due to each alkyl iodide in the chromatogram
thickness 1 µm). obtained with reference solution (c);
A2 area of the peak due to each alkyl iodide in the chromatogram
Carrier gas helium for chromatography R.
obtained with the test solution;
The use of an inen inlet liner without glass wool significantly C percentage content of each ester;
reduces the effect of carry-over between the injections. 11 area of the peak due to the internal standard in the
chromatogram obtained with reference solution (c);
Flow rate 0.5 mIJmin. 12 area of the peak due to the internal standard in the
Split ratio 1:20. chromatogram obtained with the test solution;
W1 mass of each ester used to prepare reference solution (a), in
Static head-space conditions that may be used: milligrams;
- equilibration temperature: 60 °C; W2 mass of the substance to be examined in the test solution, in
- equilibration time: 30 min; milligrams;
- transfer-line temperature: 120 °C. 0.05 dilution factor.

Temperature:

Time Temperature
(min) CC)
Column 0- 1 40
S3. Methanesulfonyl Chloride in
1 - 10 40 ➔ 130 Methanesulfonic Acid
Injection port 220
(Ph. Bur. method 2.5.39)
Detector transfer line 280
source 250 The following method has been validated for the
analyser 200 determination of methanesulfonyl chloride in
methanesulfonic acid at concentrations in the range of
At the end of analysis, the temperature of the column is 0.05 ppm to 50 ppm.
raised to 240 °C and maintained at this temperature for Gas chromatography (2.2.28) coupled with mass
7 min. spectrometry (2.2. 43).
Detection Mass spectrometer as described below; adjust Internal standard solution Dissolve 7 µL of butyl
the detector settings so as to comply with the system methanesulfonate CRS (BMS) in methylene chloride R and
suitability criteria; alternatively a suitable electron-capture dilute to 10.0 mL with the same solvent. Dilute 5.0 mL of
detector may be used: this solution to 50.0 mL with methylene chloride R.
- quadrupole mass spectrometer equipped with an electron Test solution To 5 mL of water R, add 7.4 g of the
impact ionisation mode (70 eV); substance to be examined and mix slowly. After cooling, add
- mass spectrometer parameters for the fragmentometric 5. 0 mL of methylene chloride R and 100 µL of the internal
mode (single-ion monitoring (SIM)) set as follows: standard solution and shake. Allow to separate and transfer
the organic layer to a vial containing 1 g of anhydrous sodium
sulfate R. Repeat the extraction twice with 5.0 mL of
V-A344 Appendix VIII S4 2023

methylene chloride R each time, combine the organic layers - signal-to-noise ratio: minimum 10 for the peak due to
and filter. methanesulfonyl chloride in the chromatogram obtained
Reference solution (a) Dissolve 50.0 mg of with reference solution (c).
methanesulfonyl chloride R in methylene chloride R and dilute to Calculate the content of methanesulfonyl chloride in parts
10.0 mL with the same solvent. Dilute 1.0 mL of the per million using the following expression:
solution to 10.0 mL with methylene chloride R. Dilute 300 µL
of this solution to 10.0 mL with methylene chloride R. A2 X 11 X W1 X C X 1.5
Reference solution (b) Dilute 500 µL of reference A 1 x12xW2
solution (a) and 100 µL of the internal standard solution to area of the peak due to methanesulfonyl chloride in the
15,0 mL with methylene chloride R. chromatogram obtained with reference solution (b);
Reference solution (c) Dilute 25 µL of reference area of the peak due to methanesulfonyl chloride in the
chromatogram obtained with the test solution;
solution (a) and I 00 µL of the internal standard solution to
percentage content of methanesulfonyl chloride;
15.0 mL with methylene chloride R. area of the peak due to BMS in the chromatogram obtained
Column: with reference solution (b);
area of the peak due to BMS in the chromatogram obtained
- material: fused silica;
with the test solution;
- size: l = 15 m, 0 = 0.25 mm; mass of methanesulfonyl chloride used to prepare reference
- stationary phase: methylpol,ysiloxane R (film thickness solution (a), in milligrams;
1 µm). W2 mass of the sample in the test solution, in milligrams;
1.5 dilution factor.
Carrier gas helium for chromatography R.
Flow rate 1 mUmin.
Pulsed splitless 60 kPa, 0.1 min.
Temperature:
S4. Methyl, Ethyl and lsopropyl
Time
(min)
Temperature
CC)
Toluenesulfonate in Active Substances
Column 0-4 40 (Ph. Bur. method 2.5.40)
4-8 40--> 200 The following general method has been validated for the
Injection port 240 determination of methyl, ethyl and isopropyl esters of
Detector: transfer line 280 toluenesulfonic acid (in concentrations between 0.2 ppm and
source 230 5 ppm) in sultamicillin tosilate dihydrate.
analyser 150
If it is intended to use the method for other active
substances, particularly those that contain different
At the end of analysis the temperature of the column is concentrations of the toluenesulfonic acid esters, the
raised to 270 °C and maintained at this temperature for concentrations of the test solution and reference solutions
8 min. must be adjusted accordingly and the method must be
Detection Mass spectrometer as described below; adjust suitably validated.
the detector settings so as to comply with the system
METHOD
suitability criteria:
Head-space gas chromatography (2.2.28) coupled with mass
- quadrupole mass spectrometer equipped with an electron
spectrometry (2.2.43). Prepare the test solution and reference
impact ionisation mode (70 eV);
solutions immediately before use.
- mass spectrometer parameters for the fragmentometric
mode (single-ion monitoring (SIM)) set as follows: Solvent mixture water R, acetonitrile R (20:80 V/V). The
use of acetonitrile of appropriate purity is essential.
Substance mlz Duration of monitoring Solution A Dissolve 30 mg of anhydrous sodium
thiosulfate Rand 60.0 g of sodium iodide R in water R using
Methanesulfonyl sonication and dilute to 50.0 mL with the same solvent.
79 tR between 3.3 min and 6.0 min
chloride
Internal standard solution Dilute 10 µL of butyl
Butyl methanesulfonate CRS (BMS) to 10.0 mL with the solvent
methanesulfonate 56 tR between 6.0 min and 8.0 min mixture. Dilute 20 µL of the solution to 100.0 mL with the
(BMS)
solvent mixture.
Blank solution Introduce 0.5 mL of solution A and
Injection 5 µL of the test solution, reference solutions (b) 0.5 mL of the internal standard solution into a headspace vial
and (c), the internal standard solution and methylene and seal the vial immediately with a polytetrafluoroethylene-
chloride R. coated silicon membrane and an aluminium cap.
Relative retention With reference to the internal standard Test solution Weigh 25.0 mg of the substance to be
(BMS) (retention time= about 7.2 min): methanesulfonyl examined into a 20 mL headspace vial. Add 0.50 mL of
chloride = about 0.68. solution A and 0.50 mL of the internal standard solution and
System suitability: seal the vial immediately with a polytetrafluoroethylene-
- in the chromatogram obtained with the internal standard coated silicon membrane and an aluminium cap.
solution, there is no peak with the same retention time as Following the derivatisation reaction, a precipitate may be
methanesulfonyl chloride; observed, however this does not affect the validity of the
- resolution: minimum 5.0 between the peaks due to quantification.
methanesulfonyl chloride and EMS in the chromatogram Reference solution (a) Dissolve 25.0 mg each of methyl
obtained with reference solution (b); toluenesulfonate R (MTS), ethyl toluenesulfonate R (ETS) and
2023 Appendix VIII S5 V-A345

isopropyl toluenesulfonate R (ITS) in toluene R and dilute to Relative retention With reference to the internal standard
5.0 mL with the same solvent. Dilute 50 µL of the solution (Bui) (retention time = about 8.5 min): Mel = about 0.51;
to 25.0 mL with the internal standard solution. = =
Etl about 0.63; iPrl about 0.68.
Reference solution (b) Dilute 40 µL of reference System suitability:
solution (a) to 20.0 mL with the internal standard solution. - resolution: minimum 1.5 between the peaks due to Etl and
Introduce 0.50 mL of this solution and 0.50 mL of iPrl in the chromatogram obtained with reference
solution A into a 20 mL headspace vial and seal the vial solution (c);
immediately with a polytetrafluoroethylene-coated silicon signal-to-noise ratio: minimum 10 for the peak due to each
membrane and an aluminium cap. alkyl iodide in the chromatogram obtained with reference
Reference solution (c) Dilute 500 µL of reference solution (b).
solution (a) to 20.0 mL with the internal standard solution. Calculate the content in parts per million of each alkyl
Introduce 0.50 mL of this solution and 0.50 mL of toluenesulfonate using the following expression:
solution A into a 20 mL headspace vial and seal the vial
immediately with a polytetrafluoroethylene-coated silicon A2 X Ii X W1 X C X 0.05
membrane and an aluminium cap. A1 X lz X W2
Column:
A, area of the peak due to each alkyl iodide in the chromatogram
- material: fused silica; obtained with reference solution (c);
- size: l = 30 m, 0 =0.25 mm; area of the peak due to each alkyl iodide in the chromatogram
- stationary phase: polar-deactivated macrogol R (film obtained with the test solution;
thickness 1 µm). C percentage content of each ester;
I, area of the peak due to the internal standard in the
Carrier gas helium for chromatography R. chromatogram obtained with reference solution (c);
The use of an inert inlet liner without glass wool significantly area of the peak due to the internal standard in the
chromatogram obtained with the test solution;
reduces the effect of carry-over between the injections. w, mass of each ester used to prepare reference solution (a), in
Flow rate 0.5 mUmin. milligrams;
mass of the substance to be examined in the test solution, in
Split ratio 1:20. milligrams;
Static head-space conditions that may be used: 0.05 dilution factor.
equilibration temperature: 60 °C;
- equilibration time: 30 min;
- transfer-line temperature: 120 °C.
Temperature:
S5. Methyl, Ethyl and lsopropyl
Time
(min)
Temperature
CC)
Benzenesulfonate in Active Substances
Column 0- I 40 (Ph. Eur. method 2.5.41)
I - 10 40 ➔ 130 The following general method has been validated for the
Injection port 220 determination of methyl, ethyl and isopropyl esters of
Detector transfer line 280 benzenesulfonic acid (in concentrations between 2.5 ppm
source 250 and 40 ppm) in amlodipine besilate.
analyser 200
If it is intended to use the method for other active
substances, particularly those that contain different
At the end of analysis the temperature of the column is concentrations of the benzenesulfonic acid esters, the
raised to 240 °C and maintained at this temperature for concentrations of the test solution and reference solutions
7 min. must be adjusted accordingly and the method must be
Detection Mass spectrometer as described below; adjust suitably validated.
the detector settings so as to comply with the system This method is not suitable for clopidogrel besilate as it was
suitability criteria: observed that methyl benzenesulfonate was obtained during
- quadrupole mass spectrometer equipped with an electron the gas chromatographic analysis as an artefact originating
impact ionisation mode (70 eV); from degradation.
- mass spectrometer parameters for the fragmentometric
METHOD
mode (single-ion monitoring (SIM)) set as follows:
Head-space gas chromatography (2.2.28) coupled with mass
spectrometry (2.2.43). Prepare the test solution and reference
Substance Quantitation ion Qualification ion
(mlz) (mlz)
solutions immediately before use.
Solvent mixture water R, acetonitrile R (20:80 V/V). The
Butyl iodide (Bul)* 184 127 use of acetonitrile of appropriate purity is essential.
Methyl iodide (Mel)* 142 127 Solution A Dissolve with the aid of ultrasound 30 mg of
anhydrous sodium thiosulfate Rand 60.0 g of sodium iodide R in
Ethyl iodide (Etl)* 156 127
water R and dilute to 50.0 mL with the same solvent.
Isopropyl iodide (iPrI)* 170 127 Internal standard solution Dilute 10 µL of butyl
* formed from BMS, MTS, ETS and ITS in the derivatisation reaction.
methanesulfonate CRS (BMS) to 10.0 mL with the solvent
mixture. Dilute 20 µL of the solution to 100.0 mL with the
solvent mixture.
Injection 1 mL of the gas phase of the test solution, Blank solution Introduce 0.50 mL of solution A and
reference solutions (b) and (c) and of the blank solution. 0.50 mL of the internal standard solution into a headspace
V-A346 Appendix VIII T 2023

vial and seal the vial immediately with a Substance

Quantitation ion Qualification ion
polytetrafluoroethylene-coated silicon membrane and an (mlz) (mlz)
aluminium cap. Butyl iodide (Bui)* 184 127
Test solution Weigh 25.0 mg of the substance to be
Methyl iodide (Mel)* 142 127
examined into a 20 mL headspace vial. Add 0.50 mL of
solution A and 0.50 mL of the internal standard solution and Ethyl iodide (EtI)* 156 127
seal the vial immediately with a polytetrafluoroethylene-
coated silicon membrane and an aluminium cap. Isopropyl iodide (iPrl)* 170 127

Following the derivatisation reaction, a precipitate may be * formed from BMS, MBS, EBS and IMS in the derivatisation reaction.
observed, however this does not affect the validity of the
quantification.
Injection I mL of the gas phase of the test solution,
Reference solution (a) Dissolve 25.0 mg each of methyl reference solutions (b) and (c) and the blank solution.
benzenesulfonate R (MES), ethyl benzenesulfonate R (EBS) and
Relative retention With reference to the internal standard
isopropyl methanesulfonate R (IMS) in toluene R and dilute to
(Bui) (retention time= about 8.5 min): Mel= about 0.51;
5.0 mL with the same solvent. Dilute 50 µL of the solution
Etl = about 0.63; iPrl = about 0.68.
to 25.0 mL with the internal standard solution.
System suitability:
Reference solution (b) Dilute 40 µL of reference
resolution: minimum 1.5 between the peaks due to Etl and
solution (a) to 20.0 mL with the internal standard solution.
iPrl in the chromatogram obtained with reference
Introduce 0.50 mL of this solution and 0.50 mL of
solution (c);
solution A into a 20 mL headspace vial and seal the vial
- signal-to-noise ratio: minimum 10 for the peak due to each
immediately with a polytetrafluoroethylene-coated silicon
alkyl iodide in the chromatogram obtained with reference
membrane and an aluminium cap.
solution (b).
Reference solution (c) Dilute 500 µL of reference
Calculate the content in parts per million of each alkyl
solution (a) to 20.0 mL with the internal standard solution.
benzenesulfonate using the following expression:
Introduce 0.50 mL of this solution and 0.50 mL of
solution A into a 20 mL headspace vial and seal the vial A2xl1 x W1 xCx0.05
immediately with a polytetrafluoroethylene-coated silicon
A 1 xl2 x W2
membrane and an aluminium cap.
Column: area of the peak due to each alkyl iodide in the chromatogram
- material: fused silica; obtained with reference solution (c);
area of the peak due to each alkyl iodide in the chromatogram
- size: l = 30 m, 0 = 0.25 mm; obtained with the test solution;
- stationary phase: polar-deactivated macrogol R (film percentage content of each ester;
thickness 1 µm). area of the peak due to the internal standard in the
chromatogram obtained with reference solution (c);
Carrier gas helium for chromatography R.
= area of the peak due to the internal standard in the
The use of an inert inlet liner without glass wool significantly chromatogram obtained with the test solution;
reduces the effect of carry-over between the injections. mass of each ester used to prepare reference solution (a), in
milligrams;
Flow rate 0.5 mUmin. == mass of the substance to be examined in the test solution, in
Split ratio I :20. milligrams;
0.05 dilution factor.
Static head-space conditions that may be used:
- equz7ibration temperature: 60 °C;
- equi7ibration time: 30 min;
- transfer-line temperature: 120 °C.
Temperature: T. Determination of Elemental Impurities
Time Temperature (Ph. Eur. method 2.4.20)
(min) CC) INTRODUCTION
Column 0- 1 40 This chapter describes the general approach for the
1 - 10 40--> 130 determination of elemental impurities in medicinal products
Injection port 220 or substances for pharmaceutical use. As the chemical
Detector transfer line 280 composition of the considered samples and the specification
source 250 limits for the element(s) of interest vary considerably, it is not
analyser 200 possible to describe all suitable sample preparation and
measurement methods. Therefore, any method that fulfils the
At the end of analysis, the temperature of the column is requirements described in this chapter may be used.
raised to 240 °C and maintained at this temperature for The results of the analysis are acceptable only if the system
7 min. suitability has been demonstrated by a suitable test. Before
Detection Mass spectrometer as described below; adjust the initial use of a method, the analyst must ensure that the
the detector settings so as to comply with the system method is appropriate for the samples and instruments used.
suitability criteria: This is accomplished by applying a validation procedure to
- quadrupole mass spectrometer equipped with an electron methods not described in the individual monograph or by a
impact ionisation mode (70 eV); system suitability test for methods which are described in the
- mass spectrometer parameters for the fragmentometric monograph. Decision trees for the choice of the sample
mode (single-ion monitoring (SIM)) set as follows: preparation and the measurement procedures are presented
in Figures 2.4.20.-1 and 2.4.20.-2.
2023 Appendix VIII T V-A34 7

No Yes

direct analysis?

No

No
Yes
an aqueous
medium?

No

Yes Yes
Yes

No

Yes Perform closed- Verify procedure

vessel digestion and/or instrument

No Perform open- or closed-

vessel digestion or
incineration

No

Figure 2.4.20.-2

Figure 2.4.20.-1. - Elemental impurities decision tree: sample preparation

PROCEDURES define the measurement method. The sample preparation

As a reference procedure is not provided for each element, method should yield a sufficient quantity of sample to allow
matrix and concentration, the choice of procedure according quantification of each element at the specified limit stated in
to Figures 2.4.20.-1 and 2.4.20.-2, including sample the individual monograph or the general chapter.
preparation, detection technique and instrument parameters, All suitable sample preparation methods and measurement
is the responsibility of the user. techniques (e.g. 2.2. 22. Atomic emission spectrometry (AES),
Use the flow chart in Figure 2.4.20.-1 to define the sample 2.2.23. Atomic absorption spectrometry (MS), 2.2.37. X-ray
preparation method and the flow chart in Figure 2.4.20.-2 to fluorescence spectrometry (XRFS), 2.2.57. Inductively coupled
V-A348 Appendix VIII T 2023

Figure 2.4.20.-1:
prepare the sample

No

Develop a
test procedure

Yes
No
Validate the
test procedure

Perform the Verify the

analysis using measurement
monograph procedure system

Perform analysis using

the validated procedure

Yes Is the recovery No

~---<of standard solution>-------------'
acceptable?

Verify the Yes

measurement
system

Report result

Figure 2.4.20.-2. - Elemental impurities decision tree: measurement

plasma-atomic emission spectrometry (ICP-AES), The choice of solvents also includes, but is not limited to,
2.2.58. Inductively coupled plasma-mass spectrometry (ICP-MS), the use of dilute bases, straight or diluted organic solvents,
2.4.2. Arsenic, 2.4.8. Heavy metals, 2.4.9. Iron, 2.4.10. Lead in combinations of acids or bases, and combinations of organic
sugars, 2.4.15. Nickel in polyols, 2.4.31. Nickel in hydrogenated solvents.
vegetable oils) can be used for the determination of elemental Acids, bases, and hydrogen peroxide of high purity must be
impurities, if the method has been verified before the initial used, especially when ICP-MS is employed. For aqueous
use by a system suitability test or a validation procedure solutions, use deionised distilled water R. Diluents must be
according to this chapter. checked for interference if they are used in an analysis.
If no sample preparation and/or measurement method is Because it is not always possible to obtain organic solvents
described in the individual monograph, a suitable sample that are free from elemental impurities, organic solvents of
preparation and/or measurement method must be developed the highest purity possible with regard to these contaminants
and validated (see Figures 2.4.20.-1 and 2.4.20.-2). must be used. Specifically for ICP techniques, where samples
SAMPLE PREPARATION are introduced into the plasma via solution nebulisation, it is
Sample preparation is critical to the success of elemental important to consider the potential matrix effects and
analysis. Many techniques not using direct measurement are interferences that might arise from the solvent. The use of an
heavily dependent on sample transport. appropriate internal standard and/or matching the standard
matrix with samples should be applied for ICP-AES and
If an atomisation system is used, the most conventional
ICP-MS analyses in cases where accuracy and precision are
means by which samples are introduced into the atomisation
not sufficient. In any case, the selection of an appropriate
system is by solution nebulisation. In this case, solid samples
internal standard should take into account the element(s) of
must be dissolved in order to be introduced into the
interest, ionisation energy, wavelengths or masses, and the
atomisation system. Samples may be dissolved in any
nature of the sample matrix.
appropriate solvent. The use of aqueous or dilute nitric acid
solutions is strongly recommended, due to minimal Where a sample is found not to be soluble in any acceptable
interference with these solvents compared to other solvents. solvent, a variety of digestion or incineration techniques can
Hydrochloric acid, hydrofluoric acid, perchloric acid, sulfuric be employed. These include hot-plate digestion, incineration
acid and hydrogen peroxide, at various concentrations, can and microwave-assisted digestions, using an open- or closed-
be used to dissolve the samples. The viscosity of sulfuric acid vessel.
is greater than that of the other acids and is to be taken into The decision regarding the type of digestion technique to be
account as it can affect the overall fluidity of the solution. used depends on the nature of the sample being digested, as
2023 Appendix VIII T V-A349

well as on the element(s) of interest and the concentration m mass of the sample in the initial sample solutionj in grams;
range of the elements to be quantified. Open-vessel digestion Vi volume of the initial sample preparation, in millilitres;
V2 total volume of any dilution performed, in millilitres;
is not recommended for the analysis of volatile elements. V3 volume of initial sample preparation used in any dilution
The suitability of a digestion technique, whether open- or performed, in millilitres.
closed-vessel, should be supported by spike recovery
experiments in order to verify that, within an acceptable VALIDATION REQUIREMENTS
tolerance, volatile elements have not been lost during sample Some validation requirements provided below may differ
preparation. The digestion cycle is suitable if a clear solution from those provided in general chapters of the Ph. Eur.
is obtained. (e.g. 2.2.22 (AES), 2.2.23 (AAS), 2.2.57 (ICP-AES), 2.2.58
It is important to consider the selection of the type, the (ICP-,l\1S)).
material of construction, the pretreatment, and the cleaning Before the initial use of the selected procedure, the analyst
of analytical labware used in elemental analyses. The material must ensure that the sample preparation and measurement
must be inert and, depending on the specific application, method are appropriate for the element(s) of interest, sample
resistant to caustics, acids, and/or organic solvents. For some matrix and instrument used. This is accomplished by
analyses, care must be exercised to prevent the adsorption of following the validation procedure before the initial use and
elemental impurities onto the surface of a vessel, particularly the system suitability test on the day of the analysis.
in ultra-trace analyses. Contamination of sample solutions by
For elemental impurities, validation of a limit test must
elemental impurities and ions present in the container can
include specificity and limit of detection.
also lead to inaccurate results.
The following section defines the characteristics for the
The use of volumetric glassware that does not comply with
acceptability of a quantitative procedure. It must be
Class A requirements of the appropriate International
demonstrated experimentally that such a procedure complies
Standard of the International Organization for
with the validation requirements, with an appropriate system
Standardization (ISO) is acceptable if the validation or the
suitability test using material spiked with a suitable reference
system suitability test of the method using such glassware
material. The test materials must be spiked before any
have experimentally demonstrated that the method is suitable
sample preparation steps. For example, if a test material is to
for the intended purpose.
be digested, the material must be spiked at the beginning of
CAUTION: when using high-pressure digestion vessels and the digestion procedure.
microwave laboratory equipment, the safety precautions and
SPECIFICI1Y
operating instructions given by the manufacturer must be follOVJed.
Specificity is the ability to ensure that the analytical
MEASUREMENT procedure (sample preparation and measurement) allows a
Method reliable determination of the element(s) of interest in the
The choice of the techniques depends mainly on the sample presence of components (e.g. carrier gas, impurities, matrix)
matrix and the characteristics and specification limits of the that may be expected to be present.
element(s) of interest. Analyse according to the instructions Acceptance criteria The procedure must be able to assess
of the manufacturer of the equipment regarding program and unequivocally each elemental impurity to be determined with
wavelength. this procedure in the presence of components that may be
System suitability expected to be present, including other elemental impurities,
A system suitability test must be carried out on the day of matrix components and other sources of interference;
the analysis to ensure that the sample preparation and specificity is demonstrated by complying with the accuracy
measurement system are appropriate. requirement for the element(s) to be determined.
Acceptance criterion for preparation of sample RANGE
solution A clear solution is obtained. Acceptance criterion Range is demonstrated by
Acceptance criterion for measurement system The complying with the recovery requirement.
measured concentration of a standard solution of the element ACCURACY
at a concentration within the range of the used calibration Verify the accuracy using a certified reference material or by
curve does not differ from the actual concentration by more performing a test for recovery. Elemental impurity
than 20 per cent. solutions CRS may be used.
Calculation The recovery may be determined on a sample of the
The blank value of reagents must be taken into account for substance to be examined, spiked with a known quantity of a
the calculation of the content. Upon completion of the reference standard of the element of interest (3 concentration
analysis, the concentration of a given element in the sample levels in the range of 50-150 per cent of the intended
is calculated by the software of the instrument from the specification limit, even if the original concentration of the
concentration of the element in the test solution. If no reference standard is at the specified value), in triplicate.
calculation software is available or no indication for Acceptance criterion Spike recovery is within 70 per cent
calculation is given in the general chapter corresponding to and 150 per cent for the mean of 3 replicates at each
the method used, the concentration of a given element in the concentration.
sample can be calculated from the concentration of the
REPEATABILI1Y
element in the solution using the following expression:
Test samples Either 6 independent samples of the
substance to be examined spiked with a suitable reference
standard at the specified concentration level, or 3
concentration levels prepared in triplicate.
C concentration of element in the analysed samp1e, in micrograms
per gram;
Acceptance criterion The relative standard deviation is in
A instrument reading of the concentration of the element in the both cases not more than 20 per cent.
sample solution, in micrograms per millilitre;
V-A350 Appendix VIII U 2023

INTERMEDIATE PRECISION Limit:

The effect of random events (intra-laboratory variations) on - tetraburylammonium: any spot due to tetrabutylammonium
the analytical precision of the method must be established. is not more intense than the corresponding spot in the
Acceptable experiments for establishing intermediate chromatogram obtained with the reference solution
precision include performing the repeatability analysis on (2.6mg/V).
different days, or with different instrumentation, or by
different analysts. Only 1 of the 3 experiments is required to
demonstrate intermediate precision.
Acceptance criterion The relative standard deviation is V. ALNitrosamines in Active Substances
not more than 25 per cent.
(Ph. Bur. method 2.5.42)
LIMIT OF QUANTIFICATION
This chapter describes analytical procedures for the detection
Use the results from the accuracy study. Determine the
of various N-nitrosamines in particular active substances.
lowest concentration meeting the acceptance criterion.
Procedures A and B have been validated as limit tests
Acceptance criterion The limit of quantification is below (30 ppb) and procedure C has been validated as a
the specification limit. quantitative test. The scope of each procedure is defined in
LIMIT OF DETECTION (ONLY APPUCABLE TO Table 2.5.42.-1. With these three procedures, it is possible to
LIMIT TESTS) analyse the following N-nitrosamines: N-nitroso-
Determine the lowest concentration giving a signal clearly dimethylamine (NDMA); N-nitroso-diethylamine (NDEA);
distinct from that obtained with a blank solution. N-nitroso-dibutylamine (NDBA); N-nitroso-N-methyl-4-
Acceptance criterion The limit of detection is not more aminobutyric acid (NMBA); N-nitroso-diisopropylamine
than O.5 times the concentration of the specification limit. (NDiPA); N-nitroso-ethyl-isopropylamine (NEiPA) and
N-nitroso-dipropylamine (NDPA).
Procedure A uses deuterated N-nitroso-diethylamine
(NDEA-d10) as internal standard. Procedures Band C use
U. Tetrabutylammonium in N-nitroso-ethylmethylamine (NEMA) as internal standard.
When a procedure is applied to substances outside of the
Radiopharmaceutical Preparations scope covered by the initial validation (see Table 2.5.42.-1)
(Ph. Bur. method 2.4.33 Tetraburylammonium in or to medicinal products or if procedure A or B is used
Radwpharmaceutical Preparations) quantitatively, then it must be validated.
Thin-layer chromatography (2.2.27).
Table 2.5.42.-1. - Scope of the validatwn
Solvent mixture anhydrous ethanol R, water R
Active NOMA NDEA
(10:90 V/V). substance
Test solution The preparation to be examined. (monograph
number)
Reference solution Dissolve 0.86 g of tetrabutylammonium
NDBA NMBA NDiPA
hydroxide R in the solvent mixture and dilute to 100 mL with
the solvent mixture. Dilute 1 mL of the solution to V with NEPA NDPA
the solvent mixture, V being the maximum recommended Candesartan A*BC ABC C A AC AC C
dose in millilitres. cilexetil (2573)
Irbesartan A*BC ABC C A AC AC C
Plate TLC silica gel plate R; use a polyester plate. (2465)
Mobile phase: Losartan A*BC ABC C A AC AC C
- mobile phase A: concentrated ammonia R, methanol R potassium
(10:90 V/V); (2232)

- mobile phase B: concentrated ammonia R, methanol R O/mesartan A*BC ABC C A AC AC C

medoxomil
(0.5: 100 V/V). (2600)
Use either mobile phase A or mobile phase B. The choice of Valsartan A*BC ABC C A AC AC C
mobile phase depends on the matrix (for example, mobile (2423)
phase Bis suitable for preparations stabilised with ascorbate). * In procedure A, the presence of dimethylforrnarnide (DMF) in the substance
Spiking experiments can be done to verify the suitability of to be examined may interfere with the detection of NDMA.
the mobile phase and to show that the sample matrix has no
influence on the results.
PROCEDURE A (LC-MS/MS)
Application 2 µL. Liquid chromatography (2.2.29) coupled with mass
Development Over 4/5 of the plate. spectrometry (2.2.43).
Drying At 50 °C for maximum 30 s. Internal standard solution 9.0 ng/mL solution of
Detection Place 0.5 g of iodine R at the bottom of a deuterated N-nitroso-diethylamine R (NDEA-d10) in
chromatographic tank; close the tank and heat it with a methanol R3.
stream of hot air (about 60-80 °C) until it is filled with pink N-Nitrosamines spiking solution For each
vapour; place the plate in the tank; leave the plate in contact N-nitrosamine concerned, use the corresponding CRS (N-
with the iodine vapour for 1 min; withdraw the plate. nitroso-dimethylamine CRS, N-nitroso-diethylamine CRS, N-
System suitability: nitroso-N-methyl-4-aminobutyric acid CRS, N-nitroso-
- the chromatogram obtained with the reference solution diisopropylamine CRS and N-nitroso-ethyl-isopropylamine CRS).
shows a clearly visible spot. In a single volumetric flask, dilute 300 µL of each of
2023 Appendix VIII V V-A351

these CRS to 50.0 mL with methanol R3. Dilute 300 µL of Substance MRM Collision energy Fragmentor
transitions (m/z) (V) voltage (V)
this solution to 100.0 mL with methanol R3.
principal
Test solution Suspend 150.0 mg of the substance to be (qualifier)
examined in 0.5 mL of methanol R3. Add 0.5 mL of the NDMA 75---> 58 1I 20
internal standard solution. Mix thoroughly for 5 min and (75---> 43) 15 15
sonicate for 15 min. Add 4. 0 mL of water for NMBA 147 ---> 117 2 41
chromatography R. Mix thoroughly for 5 min and sonicate for (147---> 87) 6 46
15 min. Centrifuge at about 3000 g for 5 min. Filter the NDEA 103 __. 75 9 25
supernatant through a membrane filter (nominal pore size (103 ---> 47) 17 20
0.20 µm). Use the filtrate. NEiPA 117 ➔ 75 7 54
Spiked solution Suspend 150.0 mg of the substance to be (117--->47) 15 59
examined in 0.5 mL of the N-nitrosamines spiking solution. NDiPA 131 ---> 89 2 20
Add 0.5 mL of the internal standard solution. (131 ---> 47) 10 25
Mix thoroughly for 5 min and sonicate for 15 min. NDEA-d 10 113 ➔ 81 6 76
Add 4.0 mL of water for chromatography R. Mix thoroughly (113 ➔ 34) 14 81
for 5 min and sonicate for 15 min. Centrifuge at about
3000 g for 5 min. Filter the supernatant through a membrane NOTE: acquisition can be started at 3.0 min and stopped at
filter (nominal pore size 0.20 µm). Use the filtrate. 15. 5 min before elution of the active substances; during non-
Reference solution Dilute 0.5 mL of the N-nitrosamines acquisition the eluent is directed to waste. Time segments can be
spiking solution with 0.5 mL of the internal standard defined accordingly.
solution. Mix thoroughly for 5 min and sonicate for 15 min. Autosampler Set at 5 °C.
Add 4.0 mL of water for chromatography R. Mix thoroughly
Injection 20 µL of the spiked solution, the test solution
for 5 min and sonicate for 15 min. Centrifuge at about
and the reference solution.
3000 g for 5 min. Filter through a membrane filter (nominal
pore size 0.20 µm). Use the filtrate. Relative retention With reference to NDEA-d 10 (retention
time = about 8.6 min): NDMA = about 0.6;
Column:
- size: l = 0.15 m, 0 = 4.6 mm;
= =
NMBA* about 0.7; NDEA about 1.0; NEiPA* = about
1.3; NDiPA = about 1.6.
- stationary phase: end-capped octadecylsilyl silica gel for
chromatography R (3 µm); *Exist as conformers that can be separated under the specified
- temperature: 40 °C. chromatographic conditions. Integrate both peaks or include the
tailing, as appropriate.
Mobile phase:
- mobile phase A: 0.1 per cent V/V solution offonnic acid R System suitability For each N-nitrosamine:
in water for chromatography R; - repeatability: use the principal MRM transitions,
- mobile phase B: methanol R3; maximum relative standard deviation of 20 per cent for
the ratio between the area of the peak due to the
Time Mobile phase A Mobile phase B N-nitrosamine and the area of the peak due to the
(min) (per cent VIP) (per cent VIP) internal standard, determined on 6 injections of the
0- 1 80 20 reference solution;
1- 6 80---> 55 20 ➔ 45 - signal-to-noise ratio 1; for the spiked solution, minimum 5
6 - 14 55 45 for the peak corresponding to the principal transition of
14 - 16 55 _, 5 45---> 95 NDMA and minimum 10 for the peak corresponding to
16 - 35 5 95 the principal transition of other N-nitrosamines;
- signal-to-noise ratio 2: for the spiked solution, minimum 3
for the peak corresponding to the qualifier transition.
Flow rate 0.5 mUmin.
Limit Use the principal MRM transitions.
Detection Triple-quadrupole mass spectrometer in
multiple reaction monitoring (MRM) mode. The following For the test solution, calculate the ratio between the area of
settings have been found to be suitable and are given as the peak due to each N-nitrosamine detected and the area of
examples; the settings may be adjusted so as to comply with the peak due to the internal standard (R,).
the system suitability criteria: For the spiked solution, calculate the ratio between the area
- ionisation mode: APCI-positive; of the peak due to each N-nitrosamine and the area of the
- heater temperature: 350 °C; peak due to the internal standard (R,).
- nebuliser pressure: 310 kPa; For each N-nitrosamine detected, the ratio of R, to Rr must
- gas temperature: 300 °C; be smaller than 0.50.
- drying gas flow: 5 Umin; The test is not valid unless the ratio between the area of the
- corona current: 6 µA; peak corresponding to the principal transition and the area of
- dwell time: 200 ms; the peak corresponding to the qualifier transition for the test
- capillary voltage (Vcap): 1.5 kV; solution is within 20 per cent of the same ratio calculated for
- cell acceleration voltage: 3 V; the spiked solution.
- MRM mode parameters:
PROCEDURE B (GC-MS)
Gas chromatography (2.2.28) coupled with mass
spectrometry (2. 2. 43).
Sample preparation 1
(valsartan, losartan potassium and olmesartan medoxomil).
V-A352 Appendix VIII V 2023

Internal standard solution (1) Dissolve 5 mg of N- pore size 0.45 µm) to obtain a clear solution. Use the clear
nitroso-ethylmethylamine R (NEMA) in methanol R3 and dilute solution.
to 10.0 mL with the same solvent. Dilute 500 µL of the Reference solution (2) Add I 00 µL of the N-nitrosamines
solution to 10.0 mL with water for chromatography R. spiking solution (2) to 5.0 mL of the extraction solution.
Extraction mixture Dissolve 40.0 g of sodium hydroxide R Shake well for at least 5 min, then centrifuge at about 4000 g
in about 800 mL of water for chromatography R. Add 100 µL for 5 min. Collect the supernatant solution. If necessary,
of the internal standard solution (1) and dilute to 1000 mL filter the supernatant through a membrane filter (nominal
with water for chromatography R. pore size 0.45 µm) to obtain a clear solution. Use the clear
N-Nitrosamines spiking solution (1) For each solution.
N-nitrosamine concerned, use the corresponding CRS (N- Column:
nitroso-dimethylamine CRS and N-nitroso-diethylamine CRS). - material: fused silica;
In a single volumetric flask, dilute 200 µL of each of =
- size: l 30 m, 0 = 0.25 mm;
these CRS to 20.0 mL with water for chromatography R. - stationary phase: cyanopropylphenylene(6)methyl(94)
Dilute 300 µL of this solution to 20.0 mL with water for polyst?oxane R (film thickness 1.4 µm).
chromatography R. Carrier gas helium for chromatography R.
Test solution (1) Suspend 250.0 mg of the substance to Flow rate 1.0 mUmin
be examined in 10.0 mL of the extraction mixture. Vortex Injection mode: pulsed splitless 172 kPa, 0.5 min.
and shake well for 5 min. Extract with 2.0 mL of methylene
Temperature:
chloride Rl. Shake well for at least 5 min then centrifuge at
about 4000 g for 5 min. Collect the lower layer (organic).
Time Temperature
If necessary, centrifuge to obtain a clear solution. Use the (min) CC)
clear solution.
Column 0 - 0.5 40
Spiked solution (1) Suspend 250.0 mg of the substance 0.5 - 7.0 40 ➔ 170
to be examined in 10.0 mL of the extraction mixture. 7.0 - 10.7 170 ➔ 280
Add 100 µL of the N-nitrosamines spiking solution (1). 10.7 - 17.0 280
Vortex and shake well for 5 min. Extract with 2.0 mL of Injection pon 250
methylene chloride Rl. Shake well for at least 5 min then Transfer line 240
centrifuge at about 4000 g for 5 min. Collect the lower layer
(organic). If necessary, centrifuge to obtain a clear solution.
Use the clear solution. Detection Single quadrupole mass spectrometer in single
ion monitoring (SIM) mode. The following settings have
Reference solution (1) Add 100 µL of the N-nitrosamines been found to be suitable and are given as examples; the
spiking solution ( 1) to 10. 0 mL of the extraction mixture. settings may be adjusted so as to comply with the system
Vortex and shake well for 5 min. Extract with 2.0 mL of suitability criteria:
methylene chloride Rl. Shake well for at least 5 min then - electron impact ionisation mode: 70 eV;
centrifuge at about 4000 g for 5 min. Use the lower layer - ion source temperature: 230 °C;
(organic). - analyser temperature: 150 °C;
Sample preparation 2 - dwell time: 300 ms;
(candesartan cilexetil and irbesartan). - gain factor. 5;
Internal standard solution (2) Dissolve 5 mg of N- - SIM mode parameters:
nitroso-ethylmethylamine R (NEMA) in methanol R3 and dilute
to 10.0 mL with the same solvent. Dilute 100 µL of the Substance m/z Start of monitoring
solution to 10.0 mL with methanol R3. (rnin)
NOMA 74 5.0
Extraction solution Dilute 100 µL of internal standard
NEMA 88 6.3
solution (2) to 100.0 mL with methylene chloride Rl.
NDEA 102 7.0
N-Nitrosamines spiking solution (2) For each
N-nitrosamine concerned, use the corresponding CRS (N-
nitroso-dimethylamine CRS and N-nitroso-diethylamine CRS). NOTE: acquisition can be started at 5 min; during non-
In a single volumetric flask, dilute 200 µL of each of acquisition the eluent is directed to waste. Time segments can be
these CRS to 10.0 mL with methanol R3. Dilute 300 µL of defined accordingly.
this solution to 20.0 mL with methanol R3. Injection 2.5 µL of the spiked solution, the test solution
Test solution (2) Suspend 500.0 mg of the substance to and reference solution, (1) or (2) as appropriate.
be examined in 5.0 mL of the extraction solution. Relative retention With reference to the internal standard
Add 100 µL of methanol R3. Shake well for at least 5 min, (NEMA) (retention time= about 6.7 min): NDMA = about
then centrifuge at about 4000 g for 5 min. Collect the 0.9; NDEA = about 1.1.
supernatant solution. If necessary, filter the supernatant System suitability:
through a membrane filter (nominal pore size 0.45 µm) to - repeatability; maximum relative standard deviation of
obtain a clear solution. Use the clear solution. 20 per cent for the ratio between the area of the peak due
Spiked solution (2) Suspend 500.0 mg of the substance to each N-nitrosamine and the area of the peak due to
to be examined in 5.0 mL of the extraction solution. the internal standard, determined on 6 injections of the
Add 100 µL of the N-nitrosamines spiking solution (2). reference solution.
Shake well for at least 5 min, then centrifuge at about 4000 g - signal-to-noise ratio: for the spiked solution, minimum 10
for 5 min. Collect the supernatant solution. If necessary, for the peak due to each N-nitrosamine.
filter the supernatant through a membrane filter (nominal Limit Use the stated m/z.
2023 Appendix VIII V V-A353

For the test solution, calculate the ratio between the area of Temperature:
the peak due to each N-nitrosamine detected and the area of
the peak due to the internal standard (R,). Time Temperature
(min) (C)
For the spiked solution, calculate the ratio between the area
of the peak due to each N-nitrosamine and the area of the Column 0 - 0.5 40
peak due to the internal standard (R,). 0.5 - 2.2 40 ➔ 140
2.2 - 4.2 140
For each N-nitrosamine detected, the ratio of R, to Rr must
4.2 - 6.2 140 ➔ 180
be smaller than 0.50.
6.2 - 6.7 180
PROCEDURE C (GC-MS/MS) 6.7 - 8.7 180 ➔ 240
Gas chromatography (2.2.28) coupled with mass 8.7 - 10.5 240
spectrometry (2.2. 43). 10.5 - 11.5 240 ➔ 280
Internal standard solution Dissolve 5 mg of N-nitroso- 11.5 - 14.0 280
ethylmethylamine R (NE.MA) in methanol RJ and dilute to Injection port 250
10.0 mL with the same solvent. Dilute 500 µL of the Transfer line 240
solution to 10.0 mL with water for chromatography R.
Extraction mixture Dissolve 40.0 g of sodium hydroxide R Detection Triple quadrupole mass spectrometer in multiple
in 500 mL of water for chromatography R. Add 100 µL of the reaction monitoring (MRM) mode. The following settings
internal standard solution, then 50 mL of acetonitrile Rl, and have been found to be suitable and are given as examples;
dilute to 1000 rnL with water for chromatography R. the settings may be adjusted so as to comply with the system
N-Nitrosamines spiking solution For each suitability criteria:
N-nitrosamine concerned, use the corresponding CRS (N- - electron impact ionisati.on mode: 40 eV;
nitroso-dimethylamine CRS, N-nitroso-diethylamine CRS, N- - ion source temperature: 230 °C;
nitroso-dibutylamine CRS, N-nitroso-diisopropylamine CRS, N- - analyser temperature: 150 °C (both);
nitroso-ethyl-zsopropylamine CRS and N-nitroso- - dwell time: 200 ms;
dipropylamine CRS). In a single volumetric flask, dilute - gainfactor. 15;
100 µL of each of these CRS to 10. 0 mL with water for - collision gas: nitrogen for chromatography R at 1. 5 mIJmin;
chromatography R. Dilute 300 µL of this solution to 20.0 mL - MRM mode parameters:
with water for chromatography R.
Substance MRM. transitions Collision energy (V)
Test solution Suspend 250.0 mg of the substance to be (m/z)
examined in 10.0 mL of the extraction mixture. Vortex and principal
shake well for 5 min. Extract with 2.0 mL of methylene (qualifier)
chloride Rl. Shake well for at least 5 min, then centrifuge at NDMA 74 ➔ 44
about 10 000 g for 5 min. Use the lower layer (organic). (74 ➔ 42) 22
Spiked solution Suspend 250.0 mg of the substance to be NEMA 88 ➔ 71 5
examined in 10.0 mL of the extraction mixture. Add 100 µL (88 ➔ 42) 22
of the N-nitrosamines spiking solution. Vortex and shake well NDEA 102---> 85 3
for 5 min. Extract with 2.0 mL of methylene chloride Rl. (102 ➔ 56) 19
Shake well for at least 5 min, then centrifuge at about NEiPA 116---> 99 5
10 000 g for 5 min. Use the lower layer (organic). (116---> 44) 14
NDiPA 130 ➔ 88 5
Reference solution (a) Add 50 µL of the N-nitrosamines
(/30 ➔ 71) 14
spiking solution to 10.0 mL of the extraction mixture. Vortex
NDPA 130--->113 1
and shake well for 5 min. Extract with 2.0 mL of methylene
(130 ➔ 88)
chloride RJ. Shake well for at least 5 min, then centrifuge at
NDBA 158 ---> 141
about 10 000 g for 5 min. Use the lower layer (organic).
(158 ➔ 99) 7
Reference solution (b) Add 100 µL of the
N-nitrosamines spiking solution to 10.0 mL of the extraction
mixture. Vortex and shake well for 5 min. Extract with NOTE: acquisition can be started at 4.5 min; during non-
2.0 mL of methylene chloride Rl. Shake well for at least acquisition the eluent is directed to waste. Time segments can be
5 min, then centrifuge at about 10 000 g for 5 min. Use the defined accordingly.
lower layer (organic). Injection 3 µL of the spiked solution, the test solution and
Reference solution (c) Add 200 µL of the N-nitrosamines reference solution (b).
spiking solution to 10.0 mL of the extraction mixture. Vortex Relative retention With reference to the internal standard
and shake well for 5 min. Extract with 2.0 mL of methylene (NE.MA) (retention time = about 5.5 min):
chloride Rl. Shake well for at least 5 min, then centrifuge at ND.MA= about 0.9; NDEA = about 1.1; NEiPA = about
about 10 000 g for 5 min. Use the lower layer (organic). 1.3; NDiPA = about 1.4; NDPA = about 1.5;
Column: NDBA = about 1.8.
- material: fused silica; System suitability For each N-nitrosamine:
- size: l = 30 m, 0 = 0.25 mm; - repeatability: use the principal MRM transitions,
- stationary phase: cyanopropylphenylene(6)methyl(94) maximum relative standard deviation of 20 per cent for
polysz7oxane R (film thickness 1.4 µm). the ratio between the area of the peak due to the
Carrier gas helium for chromatography R. N-nitrosamine and the area of the peak due to the
internal standard, determined on 6 injections of reference
Flow rate 1.3 mIJmin.
solution (b);
Injection mode: pulsed splitless 276 kPa, 0.5 min.
V-A354 Appendix IX A 2023

- signal-to-noise ratio 1: for the spiked solution, minimum 10

for the peak corresponding to the principal transition;
- signal-to-noise ratio 2: for the spiked solution, minimum 3
Appendix IX
for the peak corresponding to the qualifier transition. A. Determination of Sulfated Ash
Limit Use the principal MRM transitions.
Use Method I unless otherwise directed.
For the test solution, calculate the ratio between the area of
the peak due to each N-nitrosamine detected and the area of Method I
the peak due to the internal standard (R,). (No Ph. Bur. method)
For the spiked solution, calculate the ratio between the area Heat a platinum dish to redness for 10 minutes, allow to cool
of the peak due to each N-nitrosamine and the area of the in a desiccator and weigh. Unless otherwise specified in the
peak due to the internal standard (Rr). monograph, place 1 g of the substance being examined in the
For each N-nitrosamine detected, the ratio of R, to Rr must dish, moisten with sulfuric acid, ignite gently, again moisten
be smaller than 0.50. with sulfuric acid and ignite at about 800°. Cool, weigh again,
ignite for 15 minutes and repeat this procedure until two
The test is not valid unless the ratio between the area of the
successive weighings do not differ by more than 0.5 mg.
peak corresponding to the principal transition and the area of
the peak corresponding to the qualifier transition for the test Methodll 1
solution is within 20 per cent of the same ratio calculated for (Ph. Bur. method 2.4.14)
the spiked solution.
Ignite a suitable crucible (for example, silica, platinum,
When procedure C is used as a quantitative test Inject porcelain or quartz) at 600 ± 50 °C for 30 min, allow to
3 µL of the spiked solution, the test solution and reference cool in a desiccator over silica gel or other suitable desiccant
solutions (a), (b) and (c). and weigh. Place the prescribed amount of the substance to
System suitability For each N-nitrosamine: be examined in the crucible and weigh. Moisten the
- repeatability: use the principal MRM transitions, substance to be examined with a small amount of sulfuric
maximum relative standard deviation of 20 per cent for acid R (usually 1 mL) and heat gently at as low a
the ratio between the area of the peak corresponding to temperature as practicable until the sample is thoroughly
the N-nitrosamine and the area of the peak due to the charred. After cooling, moisten the residue with a small
internal standard, determined on 6 injections of reference amount of sulfuric acid R (usually 1 mL), heat gently until
solution (b); white fumes are no longer evolved and ignite at 600 ± 50 °C
- signal-to-noise ratio 1: for reference solution (a), minimum until the residue is completely incinerated. Ensure that flames
10 for the peak corresponding to the principal transition; are not produced at any time during the procedure. Allow
- signal-to-noise ratio 2: for reference solution (a), minimum the crucible to cool in a desiccator over silica gel or other
3 for the peak corresponding to the qualifier transition. suitable desiccant, weigh it again and calculate the percentage
Calculation Use the principal MRM transitions. of residue.
Use reference solutions (a), (b) and (c) to establish a If the amount of the residue so obtained exceeds the
calibration curve using the ratio between the area of the peak prescribed limit, repeat the moistening with sulfuric acid R
due to each N-nitrosamine detected and the area of the peak and ignition, as previously, for 30 min periods until
due to the internal standard versus the concentration of the 2 consecutive weighings do not differ by more than 0.5 mg
N-nitrosamines. The concentration of any N-nitrosamines in or until the percentage of residue complies with the
the test solution is interpolated from the calibration curve. prescribed limit.
The test is not valid unless: The amount of substance used for the test (usually 1-2 g) is
- the ratio between the area of the peak corresponding to chosen so that at the prescribed limit the mass of the residue
the principal transition and the area of the peak (usually about 1 mg) can be measured with sufficient
corresponding to the qualifier transition for the test accuracy.
solution is within 20 per cent of the same ratio calculated
for the spiked solution;
- the calculated recovery in all spiked solutions is between
70-130 per cent. B. Determination of Sulfur Dioxide
Method I
(No Ph. Bur. method)
Apparatus A round-bottomed flask of 1000- to 1500-mL
capacity is fitted with a water-cooled reflux condenser the
upper end of which is connected to two absorption tubes in
series. The flask is fitted with a gas inlet tube which reaches
nearly to the bottom of the flask. Each absorption tube
contains 10 mL of hydrogen peroxide solution (20 vol)
previously neutralised with 0 .1 M sodium hydroxide VS using
bromophenol blue solution as indicator.
Method Place in the flask 500 mL of water and 20 mL of
hydrochloric acid. Pass through the flask a steady current of
nitrogen or carbon dioxide that has been bubbled through dilute

1 This chapter has undergone pharmacopoeia! harmonisation. See chapter

5. 8 Pharmacopoeia/ harmonisation.
 2023 Appendix IX C V-A355

sodium carbonate solution and gradually heat the liquid until it To 10 mL of dilute hydrogen peroxide solution R, add O.15 mL
boils. Maintain the current of nitrogen or carbon dioxide, allow of a 1 g/L solution of bromophenol blue R in ethanol
the solution to boil for about 10 minutes and cool the flask (20 per cent V/V) R. Add 0.1 M sodium hydroxide until a
by gradual immersion in water. Introduce, while momentarily violet-blue colour is obtained, without exceeding the
removing the stopper of the flask, a weighed quantity of end-point. Pour the solution into the receiving tube (D) and
50 to 100 g of the substance being examined, heat gently and mount the tube on the apparatus as shown in Figure
boil for 45 minutes. Disconnect the absorption tubes before 2.5.29.-1.
turning off the current of nitrogen or carbon dioxide and titrate Without interrupting the stream of carbon dioxide, remove
the combined contents with O.lM sodium hydroxide VS. the dropping funnel (B) and introduce into the flask (A)
Each mL of O.1 M sodium hydroxide VS is equivalent to 25.0 g (m ) of the substance to be examined, rinsing with
3.203 mg of sulfur dioxide. 100 mL of water R. Replace the dropping funnel, close the
Repeat the operation without the substance being examined. tap and pour 80 mL of dilute hydrochloric acid R into the
The solution in the absorption tubes remains neutral. funnel. Open the tap to allow the hydrochloric acid solution
to flow into the flask. Make sure that no sulfur dioxide
Method II escapes by closing the tap before the last few millilitres of
(Ph. Bur. method 2.5.29) hydrochloric acid solution drain out. Boil for 1 h.
EQUIPMENT Open the tap of the dropping funnel then stop the flow of
The apparatus as shown in Figure 2.5.29.-1 comprises: carbon dioxide . Transfer the contents of the receivingtube
- a ground-glass 3-neck round-bottomed flask (A); (D) to a 200 mL conical flask, rinsing the tube with a little
- a dropping funnel (B); water R. Heat on a water-bath for 15 min and allow to cool.
- a reflux condenser (C); Add 0.1 mL of a 1 giL solution of bromophenol blue R in
- a receiving tube (D); ethanol (20 per cent V/V) R and titrate with 0.1 M sodium
- a transfer tube (E); hydroxide until the colour changes from yellow to violet-
- a gas port. blue (Vi). Carry out a blank titration (V2 ).
PROCEDURE Results
Method Calculate the content of sulfur dioxide, in parts per million,
Introduce 150 mL of water R into the flask (A) and using the following expression:
equilibrate the whole system by passing carbon dioxide R for n
15 min at a rate of about 100 mIJmin. 32 030x (Vi - Vi) X -
m
Vi volwne of titrant used in the titration, in millilitres;
E Ve volume of titrant used in the blank titration, in millilitres;
n molarity of the sodium hydroxide solution used as titrant, in
moles per litre;
m mass of the sample, in grams.

D
C. Determination of Water
Use Method IA unless otherwise directed.
Method I Semi-micro Determination of Water
(Ph. Bur. method 2.5.12)
The semi-micro determination of water is based upon the
quantitative reaction of water with sulfur dioxide and iodine
in a suitable anhydrous medium in the presence of a base
with sufficient buffering capacity.
APPARATUS
The apparatus consists of a titration vessel with:
- 2 identical platinum electrodes;
- tight inlets for introduction of solvent and titrant;
- an inlet for introduction of air via a desiccant;
- a sample inlet fitted with a stopper or, for liquids, a
septum.
Inlet systems for introduction of dry nitrogen or for
aspiration of solvents may also be fitted.
The titration is carried out according to the instrument
supplier's instructions. Care is taken throughout the
determination to avoid exposure of reagents and solvents to
atmospheric moisture. The end-point is determined using 2
identical indicator electrodes connected to an electrical
source that maintains between the electrodes either a
Figure 2.5.29.-1.-Apparatus for the determination of sulfur constant current (2.2.65. Voltametric titration) or a constant
dio:;,dde content voltage (2.2.19. Amperometric titration). Where direct titration
is used (method A), addition of titrant causes either a
V-A356 Appendix IX C 2023

decrease in voltage where constant current is maintained or Calculate the mean percentage recovery (r).
an increase in current where constant voltage is maintained, The reagent/solvent system is considered to be acceptable if r is
until the end-point is reached. Instruments with automatic between 97.5 per cent and 102.5 per cent.
end-point detection are commonly used. Instrument Calculate the regression line. The x-axis represents the
qualification is carried out according to established quality cumulative water added whereas the y-axis represents the
system procedures, for example using a suitable certified sum of the initial water content determined for the substance
reference material (sodium amirwsalicylate dihydrate for (M) and the cumulative water determined after each
equipment qualification CRS may be used). addition. Calculate the slope (b), the intercept with the y-axis
STANDARDISATION (a) and the intercept of the extrapolated calibration line with
To the titration vessel, add methanol R, dried if necessary, or the x-axis (cl).
the solvent recommended by the supplier of the titrant. Calculate the percentage errors (e 1 and e2 ) using the
Where applicable for the apparatus used, eliminate residual following expressions:
water from the measurement cell or carry out a pre-titration.
Introduce a suitable amount of water in an appropriate form a-M
e1 =100~
(water R or a certified reference material) and carry out the
titration, stirring for the necessary time. The water equivalent
is not less than 80 per cent of that indicated by the supplier.
Standardise the titrant before the first use and at suitable e2 = 100 ldl -M
intervals thereafter. M
Unless otherwise prescribed, use Method A. a the y-axis intercept, in milligrams of water;
d the x-axis intercept, in milligrams of water;
METHOD A M water content of the substance, in milligrams of water.
Introduce into the titration vessel methanol R, or the solvent
indicated in the monograph or recommended by the supplier The reagent/solvent system is considered to be acceptable if:
of the titrant. Where applicable for the apparatus used, - le, I and le2 I are not greater than 2.5 per cent;
eliminate residual water from the measurement cell or carry - b is between 0.975 and 1.025.
out a pre-titration. Introduce the substance to be examined
rapidly and carry out the titration, stirring for the necessary Method II Determination of Water by Distillation
extraction time. (Ph. Bur. method 2.2.13)
METHODB The apparatus (see Figure 2.2.13.-1) consists of a glass
Introduce into the titration vessel methanol R, or the solvent flask (A) connected by a tube (D) to a cylindrical tube (B)
indicated in the monograph or recommended by the supplier fitted with a graduated receiving tube (E) and reflux
of the titrant. Where applicable for the apparatus used, condenser (C). The receiving tube (E) is graduated in
eliminate residual water from the measurement cell or carry 0.1 mL. The source of heat is preferably an electric heater
out a pre-titration. Introduce the substance to be examined with rheostat control or an oil bath. The upper portion of the
rapidly and in a suitable state of division. Add an accurately flask and the connecting tube may be insulated.
measured volume of the titrant, sufficient to give an excess of Method Clean the receiving tube and the condenser of the
about 1 mL or the prescribed volume. Allow to stand apparatus, thoroughly rinse with water, and dry.
protected from light for 1 min or the prescribed time, with Introduce 200 mL of toluene R and about 2 mL of water R
stirring. Titrate the excess of reagent using methanol R or the into the dry flask. Distil for 2 h, then allow to cool for about
prescribed solvent, containing an accurately known quantity 30 min and read the water volume to the nearest 0.05 mL.
of water. Place in the flask a quantity of the substance, weighed with
SUITABILITY an accuracy of 1 per cent, expected to give about 2 mL to
3 mL of water. If the substance has a pasty consistency,
The accuracy of the determination with the chosen titrant
weigh it in a boat of metal foil. Add a few pieces of porous
must be verified for each combination of substance, titrant
material and heat the flask gently for 15 min. When the
and solvent to be examined. The following procedure, given
toluene begins to boil, distil at the rate of about two drops
as an example, is suitable for samples containing 2.5-25 mg
per second until most of the water has distilled over, then
of water.
increase the rate of distillation to about four drops per
The water content of the substance to be examined is second. When the water has all distilled over, rinse the inside
determined using the reagent/solvent system chosen. of the condenser tube with toluene R. Continue the
Thereafter, in the same titration vessel, sequential known distillation for 5 min, remove the heat, allow the receiving
amounts of water, corresponding to about 50-100 per cent of tube to cool to room temperature and dislodge any droplets
the amount found in the substance to be examined, are of water which adhere to the walls of the receiving tube.
added in an appropriate form (at least 5 additions) and the When the water and toluene have completely separated, read
water content is determined after each addition. Calculate the volume of water and calculate the content present in the
the percentage recovery (r) after each addition using the substance as millilitres per kilogram, using the formula:
following expression:
1000(n2 - n,)
W2
r= 100w, m

the mass in grams of the substance to be examined,

amount of water added, in milligrams; the number of millilitres of water obtained in the first
amount of water found, in milligrams. distillation,
n2 the total number of millilitres of water obtained in the 2
distillations.
2023 Appendix IX C V-A357

the anode reacts immediately with the water and the sulfur
dioxide contained in the reaction cell. The quantity of water
in the substance is directly proportional to the quantity of
I electricity (in coulombs), corresponding to electric current (in
amperes) multiplied by time (in seconds), used for iodine
generation up until the titration end-point. When all of the
water in the reaction cell has been consumed, the end-point
is reached and thus an excess of iodine appears. 1 mole of
iodine corresponds to 1 mole of water, an amount of
I electricity of 10.71 C corresponds to 1 mg of water.
I 0
Moisture is eliminated from the reaction cell by pre-titration,
0 i.e. the electrolyte reagent is titrated to dryness before starting
I C N
Ill the sample analysis. Individual determinations can be carried
I out successively in the same reagent solution, under the
following conditions:
I - each component of the test mixture is compatible with
the other components;
- no other reactions take place;
- the volume and the water capacity of the electrolyte
I reagent are sufficient.
Coulometric titration is intended for the quantitative
I determination of small quantities of water (from 10 µg),
however a working range of 100 µg to 10 mg of water is
recommended for reproducibility reasons.
Accuracy and precision of the method are predominantly
governed by the sample preparation and the extent to which
B
atmospheric moisture is excluded from the system. Control
of the system must be monitored by measuring the amount
of baseline drift.
APPARATUS
The apparatus consists of a reaction cell, electrodes and a
magnetic stirrer. The reaction cell consists of a large anode
compartment and a smaller cathode compartment.
E LO
I'--
Depending on the design of the electrode, both
compartments can be separated by a diaphragm. Each
compartment contains a platinum electrode. Liquid or
solubilised samples are introduced through a septum, using a
syringe. Alternatively, an evaporation technique may be used
in which the sample is heated in an oven and the water is
evaporated and carried into the cell by means of a stream of
dry inert gas. The introduction of solid samples into the cell
should in general be avoided. However, if it has to be done it
is effected through a sealable port; appropriate precautions
I must be taken to avoid the introduction of moisture from air,

A
I such as working in a glove box in an atmosphere of dry inert
gas. The analytical procedure is controlled by a suitable
I electronic device, which also displays the results.
I Instrument qualification is carried out according to
165
,I established quality system procedures, for example using a
suitable certified reference material. Sodium aminosalicylate
Figure 2.2.13.-1. -Apparatus for the determination of water by dihydrate for equipment qualification CRS may be used when
distillation proceeding by direct or liquid sample introduction, whereas
amoxicillin trihydrate for peiformance verification CRS may be
Dimensions in millimetres used with the evaporation technique.

Method III Coulometric Titration METHOD

(Ph. Bur. method 2.5.32) Fill the compartments of the reaction cell with electrolyte
reagent for the micro determination of water R according to the
PRINCIPLE manufacturer's instructions and perform the coulometric pre-
The coulometric titration of water is based upon the titration to a stable end-point. Introduce the prescribed
quantitative reaction of water with sulfur dioxide and iodine quantity of the substance to be examined into the reaction
in an anhydrous medium in the presence of a base with cell and titrate again to a stable end-point, stirring for at least
sufficient buffering capacity. In contrast to the volumetric 30 s, unless otherwise indicated in the monograph. If an oven
method described in general chapter 2.5.12. Water: semi-micro is used, the prescribed quantity of sample is introduced into
determination, iodine is produced electrochemically in the the oven and heated. After evaporation of the water from the
reaction cell by oxidation of iodide. The iodine produced at sample into the reaction cell, the titration is started.
V-A358 Appendix IX D 2023

Alternatively, the evaporated moisture is immediately titrated temperature ± 2 °C. Use one of the following procedures,
while heating the sample in the oven to avoid loss of unless otherwise prescribed in the monograph.
evaporated water already collected in the reagent solution - In a desiccator: the drying is carried out over about 100 g
during prolonged heating. Read the value from the of molecular sieve R at atmospheric pressure and at room
instrument's output and calculate if necessary the percentage temperature.
or quantity of water that is present in the substance. When - In vacua: the drying is carried out over about 100 g of
appropriate to the type of sample and the sample molecular sieve R at a pressure not exceeding 2.5 kPa, at
preparation, perform a blank titration. room temperature or at the temperature prescribed in the
VERIFICATION OF ACCURACY monograph.
- In an oven at a specified temperature: the drying is
At appropriate intervals, such as at least at the beginning and
carried out at atmospheric pressure in an oven at the
the end of a series of sample titrations, introduce a defined
temperature prescribed in the monograph.
quantity of water, in the same order of magnitude as the
quantity of water in the sample, using a suitable certified After drying in an oven, allow the weighing bottle and the
reference material and perform the coulometric titration. sample to cool to room temperature in a desiccator and
The recovery is within the range of 97.5 per cent to weigh the weighing bottle containing the dried sample.
102.5 per cent for an addition of 1000 µg of H 2 O and within The mass of the sample is the difference between the mass of
the range of 90.0 per cent to 110.0 per cent for the addition the filled weighing bottle and the mass of the dried empty
of 100 µg ofH 2 O. weighing bottle.
The loss on drying is the difference in the mass of the sample
before and after drying, expressed as a percentage, mlm being
implicit.
D. Determination of Loss on Drying
(Ph. Bur. method 2.2.32)
PRINCIPLE E. Limit Test for Carbon Monoxide in
Loss on drying is the loss of mass after drying under
specified conditions, calculated as a percentage (mlm). Medicinal Gases
Drying to constant mass means that 2 consecutive weighings (Ph. Bur. method 2.5.25)
do not differ by more than 0.5 mg, the 2nd weighing
METHOD I
following an additional period of at least 30 min of drying
Apparatus The apparatus (Figure 2.5.25.-1) consists of
under the conditions prescribed for the substance to be
the following parts connected in series:
examined.
- a U-rube (U1 ) containing anhydrous silica gel R
EQUWMENT impregnated with chromium trioxide R;
The equipment typically consists of: - a wash bottle (F1 ) containing 100 mL of a 400 g/L
- weighing bottles that are made of suitable inert material solution of potassium hydroxide R;
and can easily be dried to constant mass; their diameter is - a U-tube (U2) containing pellets of potassium hydroxide R;
large enough so that the layer of the substance to be - a U-tube (U3 ) containing diphosphorus pentoxide R
examined does not exceed about 5 mm; dispersed on previously granulated, fused pumice;
- an analytical balance by which it is possible to determine - a U-tube (U4) containing 30 g of recrystallised iodine
a change in mass of 0 .1 mg; pentoxide R in granules, previously dried at 200 °C and
- depending on the procedure to be applied, a desiccator, a kept at a temperature of 120 °C (1) during the test; the
vacuum cabinet, a vacuum oven or an ordinary laboratory iodine pentoxide is packed in the tube in 1 cm columns
oven; in any case, the temperature of ovens is adjustable separated by 1 cm columns of glass wool to give an
to the specified temperature ± 2 °C; vacuum ovens in effective length of 5 cm;
which the pressure can at least be reduced to about 2 kPa a reaction tube (F2) containing 2.0 mL of potassium iodide
are suitable; ovens are qualified according to established solution Rand 0.15 mL of starch solution R.
quality system procedures, for example by using a suitable Method Flush the apparatus with 5.0 L of argon R and, if
certified reference material (sodium aminosalicylate necessary, discharge the blue colour in the iodide solution by
dihydrate for equipment qualification CRS may be used). adding the smallest necessary quantity of freshly prepared
Equipment using other means of drying such as microwaves, 0.002 M sodium thiosulfate. Continue flushing until not more
halogen lamps, infrared lamps or mixed technologies may be than 0.045 mL of 0.002 M sodium thiosulfate is required after
used provided they are demonstrated to be fit for purpose. passage of 5.0 L of argon R. Pass the gas to be examined
PROCEDURE from the cylinder through the apparatus, using the prescribed
volume and the flow rate. Flush the last traces of liberated
It is recommended to perform the test in an environment
iodine into the reaction tube by passing through the
that has minimal impact on sample measurement
apparatus 1.0 L of argon R. Titrate the liberated iodine with
(e.g. humidity).
0.002 M sodium thiosulfate. Carry out a blank test, using the
Weigh an empty weighing bottle that has been previously prescribed volume of argon R. The difference between the
dried under the conditions prescribed for the substance to be volumes of 0. 002 M sodium thiosulfate used in the titrations is
examined for at least 30 min, then weigh the weighing bottle not greater than the prescribed limit.
filled with the prescribed quantity of substance to be
examined. Dry to constant mass or for the prescribed time. METHOD II
Where the drying temperature is indicated by a single value Gases absorb light at one or more specific wavelengths. This
rather than a range, drying is carried out at the prescribed property is widely used to allow highly selective measurement
of their concentrations.
2023 Appendix IX F V-A359

U1 F1 U2 U3 U4 F2

0
0
,....
I I
I I
I I

I 1-
-1 1--
:. I I-_
100 m
-: I j--
_ 11-
-1 -
- I I -
_:-1 I -
1-

Figure 2.5.25.-1. - Apparatus for the detennination of carbon monoxide

Dimensions in millimetres

Description and principle of measurement

The concentration of carbon monoxide in other gases can be
F. Determination of Carbon Dioxide in
determined using an infrared analyser. Medicinal Gases
The infrared analyser generally consists of a light source (Ph. Bur. method 2.5.24)
emitting broadband infrared radiation, an optical device, a
sample cell and a detector. The optical device may be Gases absorb light at one or more specific wavelengths. This
positioned either before or after the sample cell; it consists of property is widely used to allow highly selective measurement
one or several optical filters, through which the broadband of their concentrations.
radiation is passed. The optical device in this case is selected Description and principle of measurement
for carbon monoxide. The measurement light beam passes The concentration of carbon dioxide in other gases can be
through the sample cell and may also pass through a determined using an infrared analyser.
reference cell if the analyser integrates such a feature (some The infrared analyser generally consists of a light source
use an electronic system instead of a reference cell). emitting broadband infrared radiation, an optical device, a
When carbon monoxide is present in the sample cell, sample cell and a detector. The optical device may be
absorption of energy in the measurement light beam will positioned either before or after the sample cell and it
occur according to the Beer-Lambert law and this produces a consists of one or several optical filters, through which the
change in the detector signal. This measurement signal is broadband radiation is passed. The optical device in this case
compared to a reference signal to generate an output related is selected for carbon dioxide. The measurement light beam
to the concentration of carbon monoxide. The generated passes through the sample cell and may also pass through a
signal is linearised in order to obtain the carbon monoxide reference cell if the analyser integrates such a feature (some
concentration. To prevent the entry of particles into the use an electronic system instead of a reference cell).
sensors, which could cause stray-light phenomena, the When carbon dioxide is present in the sample cell,
apparatus is fitted with a suitable filter. absorption of energy in the measurement light beam will
Required technical specifications occur according to the Beer-Lambert law and this produces a
When used for a limit test, the carbon monoxide infrared change in the detector signal. This measurement signal is
analyser meets the following technical specifications: compared to a reference signal to generate an output related
- limit of detection: (generally defined as a signal-to-noise to the concentration of carbon dioxide. The generated signal
ratio of 2) maximum 20 per cent of the maximum is linearised in order to obtain the carbon dioxide
admissible concentration; concentration. To prevent the entry of particles into the
- repeatability: maximum relative standard deviation of sensors, which could cause stray-light phenomena, the
10 per cent of the maximum admissible concentration, apparatus is fitted with a suitable filter.
determined on 6 measurements; Required technical specifications
- linearity: maximum 10 per cent of the maximum When used for a limit test, the infrared analyser meets the
admissible concentration. following technical specifications:
The technical specifications must be met in the presence of - limit of detection: (generally defined as a signal-to-noise
the other gas impurities in the sample. ratio of 2) maximum 20 per cent of the maximum
admissible concentration;
V-A360 Appendix IX G 2023

- repeatability: maximum relative standard deviation of

10 per cent of the maximum admissible concentration,
H. Determination of Oxygen in Medicinal
determined on 6 measurements; Gases
- linearity: maximum 10 per cent of the maximum
(Ph. Bur. method 2.5.27)
admissible concentration.
The technical specifications must be met in the presence of Oxygen in gases is determined using a paramagnetic analyser.
the other gas impurities in the sample. The principle of the method is based on the high
paramagnetic sensitivity of the oxygen molecule. Oxygen
exerts a strong interaction on magnetic fields, which is
measured electronically, amplified and converted to a reading
G. Determination of Nitrogen Monoxide of oxygen concentration. The measurement of oxygen
concentration is dependent upon the pressure and
and Nitrogen Dioxide in Medicinal Gases temperature and, if the analyser is not automatically
(Ph. Bur. metlwd 2.5.26) compensated for variations in temperature and pressure, it
must be calibrated immediately prior to use. As the
Nitrogen monoxide and nitrogen dioxide in gases are paramagnetic effect of oxygen is linear, the instrument must
determined using a chemiluminescence analyser have a suitable range with a readability of 0.1 per cent or
(Figure 2.5.26.-1). better.
The apparatus consists of the following: Calibration of the instrument Make the setting in the
- a device for filtering, checking and controlling the flow of following manner:
the gas to be examined, - set the zero by passing nitrogen Rl through the instrument
- a converter that reduces nitrogen dioxide to nitrogen until a constant reading is obtained;
monoxide, to determine the combined content of nitrogen - set the scale to 100 per cent by passing oxygen R through
monoxide and nitrogen dioxide. The efficiency of the the instrument at the same flow rate as for nitrogen Rl
converter has to be verified prior to use, until a constant reading is obtained.
- a controlled-flow-rate ozone generator; the ozone is
Assay Pass the gas to be examined through the instrument
produced by high-voltage electric discharges across two
at a constant flow rate until a constant reading is obtained.
electrodes; the ozone generator is supplied with pure
Record the concentration of oxygen in the gas to be
oxygen or with dehydrated ambient air and the
examined.
concentration of ozone obtained must greatly exceed the
maximum content of any detectable nitrogen oxides,
- a chamber in which nitrogen monoxide and ozone can
react,
- a system for detecting light radiation emitted at a J. Determination of Water in Medicinal
wavelength of 1.2 µm, consisting of a selective optical Gases
filter and a photomultiplier tube.
(Ph. Bur. method 2.5.28)
Water in gases is determined using an electrolytic
hygrometer, described below.
The measuring cell consists of a thin film of diphosphorus
pentoxide, between 2 coiled platinum wires which act as
electrodes. The water vapour in the gas to be examined is
absorbed by the diphosphorus pentoxide, which is

Sample flow control

Filter for entry

of sample Reaction chamber
Converter Optical filter
N0 2 -+ NO
/Refrigerated chamber

Filter to eliminate
ozone

Ozone generator 1-------,

system

- Controls
- NO - (NO+N0 2) cycle

NO (NO+NO) N02

Figure 2.5.26.-1. - Chemiluminescence analyser

2023 Appendix IX K V-A361

transformed to phosphoric acid, an electrical conductor. Arsine detector tube

A continuous voltage applied across the electrodes produces Sealed glass tube containing adsorbent filters and suitable
electrolysis of the water and the regeneration of the supports for the gold salt or other appropriate indicator.
diphosphorus pentoxide. The resulting electric current, which The minimum value indicated is 0.25 ppm or less, with a
is proportional to the water content in the gas to be relative standard deviation of at most 20 per cent.
examined, is measured. This system is self-calibrating since it Carbon dioxide detector tube
obeys Faraday's law. Sealed glass tube containing adsorbent filters and suitable
Take a sample of the gas to be examined. Allow the gas to supports for hydrazine and crystal violet indicators.
stabilise at room temperature. Purge the cell continuously The minimum value indicated is 100 ppm with a relative
until a stable reading is obtained. Measure the water content standard deviation of at most 15 per cent.
in the gas to be examined, making sure that the temperature Carbon monoxide detector tube
is constant throughout the device used to introduce the gas Sealed glass tube containing adsorbent filters and suitable
into the apparatus. supports for di-iodine pentoxide, selenium dioxide and
The electrolytic hygrometer achieves accurate sample flows fuming sulfuric acid indicators. The minimum value
by using a mass flow controller to deliver a constant indicated is 5 ppm or less, with a relative standard deviation
volumetric flow rate to ensure that the water content is of at most 15 per cent.
determined accurately. The calibration of the mass flow Hydrogen sulfide detector tube
controller is normally performed using nitrogen. When using Sealed glass tube containing adsorbent filters and suitable
gases other than nitrogen for calibration, consult the supports for an appropriate lead salt indicator. The minimum
manufacturer's instructions for the appropriate conversion value indicated is 0.2 ppm or less, with a relative standard
factors and ensure that the correct cell is used for the type of deviation of at most 10 per cent.
gas to be examined. Nitrogen monoxide and nitrogen dioxide detector tube
Sealed glass tube containing adsorbent filters and suitable
supports for an oxidising layer (Cr(VI) salt) and the
K. Gas Detector Tubes diphenylbenzidine indicator. The minimum value indicated is
0.5 ppm with a relative standard deviation of at most
(Ph. Bur. method 2.1. 6)
15 per cent.
Gas detector tubes are cylindrical, sealed tubes consisting of an Oil detector tube
inert transparent material and are constructed to allow the Sealed glass tube containing adsorbent filters and suitable
passage of gas. They contain reagents adsorbed onto inert supports for the sulfuric acid indicator. The minimum value
substrates that are suitable for the visualisation of the substance indicated is 0 .1 mg/m 3 with a relative standard deviation of at
to be detected and, if necessary, they also contain preliminary most 30 per cent.
layers and/or adsorbent filters to eliminate substances that Phosphine detector tube
interfere with the substance to be detected. The layer of Sealed glass tube containing adsorbent filters and suitable
indicator contains either a single reagent for the detection of a supports for the gold salt or other appropriate indicator.
given impurity or several reagents for the detection of several The minimum value indicated is 0.2 ppm or less, with a
substances (monolayer tube or multilayer tube). relative standard deviation of at most 20 per cent.
The test is carried out by passing the required volume of the Sulfur dioxide detector tube
gas to be examined through the indicator tube. The length of Sealed glass tube containing adsorbent filters and suitable
the coloured layer or the intensity of a colour change on a supports for the iodine and starch indicator. The minimum
graduated scale gives an indication of the impurities present. value indicated is 0.5 ppm with a relative standard deviation
The calibration of the detector tubes is verified according to of at most 15 per cent.
the manufacturer's instructions. Water vapour detector tube
Operating conditions Examine according to the Sealed glass tube containing adsorbent filters and suitable
manufacturer's instructions or proceed as follows. supports for the magnesium perchlorate indicator.
The gas supply is connected to a suitable pressure regulator The minimum value indicated is 67 ppm or less, with a
and needle valve. Connect the flexible tubing fitted with a relative standard deviation of at most 20 per cent.
Y-piece to the valve and adjust the flow of gas to be
2 3 4
examined to purge the tubing in order to obtain an
appropriate flow (Figure 2.1.6.-1). Prepare the indicator tube 1---------,r-------- 7
and fit to the metering pump, following the manufacturer's
instructions. Connect the open end of the indicator tube to
the short leg of the tubing and operate the pump by the 5
appropriate number of strokes to pass a suitable volume of
gas to be examined through the tube. Read the value
corresponding to the length of the coloured layer or the
intensity of the colour on the graduated scale. If a negative 6
result is achieved, indicator tubes can be verified with a
calibration gas containing the appropriate impurity.
In view of the wide variety of available compressor oils, it is
I. Gas supply 5. Indicator tube
necessary to verify the reactivity of the oil detector tubes for
2. Pressure regulator 6. Indicator tube pump
the oil used. Information on the reactivity for various oils is 3. Needle valve 7. End open to atmosphere
given in the leaflet supplied with the tube. If the oil used is 4. Y-piece
not cited in the leaflet, the tube manufacturer must verify the
reactivity and if necessary provide a tube specific for this oil. Figure 2.1.6.-1. -Apparatus for gas detector tubes
V-A362 Appendix IX L 2023

It is essential for both reasons, therefore, that as much as

L. Determination of Nitrous Oxide in possible is known about the effects of moisture on solids
Gases before strategies are developed for their handling, storage and
use.
(Ph. Bur. method 2.5.35)
Some of the more critical pieces of required information
Gases absorb light at one or more specific wavelengths. This concerning water-solid interactions are:
property is widely used to allow highly selective measurement - total amount of water present;
of their concentrations. - the extent to which adsorption and absorption occur;
Description and principle of measurement - whether or not hydrates form;
The concentration of nitrous oxide in other gases can be - specific surface area of the solid, as well as such
determined using an infrared analyser. properties as degree of crystallinity, degree of porosity,
The infrared analyser generally consists of a light source and glass transition and melting temperature;
emitting broadband infrared radiation, an optical device, a - site of water interaction, the extent of binding, and the
sample cell and a detector. The optical device may be degree of molecular mobility;
positioned either before or after the sample cell and it - effects of temperature and relative humidity;
consists of one or several optical filters, through which the - essentially irreversible hydration;
broadband radiation is passed. The optical device in this case - kinetics of moisture uptake;
is selected for nitrous oxide. The measurement light beam - various factors that might influence the rate at which
passes through the sample cell and may also pass through a water vapour can be taken up by a solid;
reference cell if the analyser integrates such a feature (some - for water-soluble solids capable of being dissolved by the
use an electronic system instead of a reference cell). sorbed water, under which conditions dissolution will take
When nitrous oxide is present in the sample cell, absorption place.
of energy in the measurement light beam will occur PHYSICAL STATES OF SORBED WATER
according to the Beer-Lambert law and this produces a Water can physically interact with solids in different ways.
change in the detector signal. This measurement signal is It can interact at the surface (adsorption) or it can penetrate
compared to a reference signal to generate an output related the bulk solid structure (absorption). When both adsorption
to the concentration of nitrous oxide. The generated signal is and absorption occur, the term sorption is often used.
linearised in order to obtain the nitrous oxide concentration. Adsorption is particularly critical in affecting the properties of
To prevent the entry of particles into the sensors, which solids when the specific surface area is large. Large values of
could cause stray-light phenomena, the apparatus is fitted specific surface area are seen with solids having very small
with a suitable filter. particles, as well as with solids having a high degree of
intraparticle porosity. Absorption is characterised by an
association of water per gram of solid that is much greater
than that which can form a monomolecular layer on the
M. Water-Solid Interactions: available surface, and an amount that is generally
independent of the specific surface area.
Determination of Sorption-Desorption Most crystalline solids will not absorb water into their bulk
Isotherms and of Water Activity1 structures because of the close packing and high degree of
(Ph. Bur. method 2.9.39) order of the crystal lattice. Indeed, it has been shown that the
degree of absorption into solids exhibiting partial crystallinity
INTRODUCTION and partial amorphous structure is often inversely
Pharmaceutical solids as raw materials or as constituents of proportional to the degree of crystallinity. With some
dosage forms most often come in contact with water during crystalline solids, however, crystal hydrates may form. These
processing and storage. This may occur (a) during hydrates may exhibit a stoichiometric relationship, in terms of
crystallisation, lyophilisation, wet granulation, or spray water molecules bound per solid molecule, or they may be
drying; and (b) because of exposure upon handling and non-stoichiometric. Upon dehydration, crystal hydrates may
storage to an atmosphere containing water vapour or either retain their original crystal structure, or lose their
exposure to other materials in a dosage form that contain crystallinity and become amorphous, or transform into a new
water capable of distributing it to other ingredients. Some anhydrous or less-hydrated crystal form.
properties known to be altered by the association of solids Amorphous or partially amorphous solids are capable of
with water include rates of chemical degradation in the taking up significant amounts of water because there is
"solid-state", crystal growth and dissolution, dispersibility sufficient molecular disorder in the solid to permit
and wetting, powder flow, lubricity, powder compactibility, penetration, swelling or dissolution. Such behaviour is
compact hardness and microbial contamination. observed with most amorphous polymers and with small-
Although precautions can be taken when water is perceived molecular-mass solids rendered amorphous during
to be a problem, i.e. eliminating all moisture, reducing preparation, e.g. by lyophilisation, or after milling.
contact with the atmosphere, or controlling the relative The introduction of defects into highly crystalline solids will
humidity of the atmosphere, such precautions generally add also produce this behaviour. The greater the chemical affinity
expense to the process with no guarantee that during the life of water for the solid, the greater the total amount that can
of the product further problems associated with moisture will be absorbed. When water is absorbed by amorphous solids,
be avoided. It is also important to recognise that there are the bulk properties of the solid can be significantly altered.
many situations where a certain level of water in a solid is It is well established, for example, that amorphous solids,
required for proper performance, e.g. powder compaction. depending on the temperature, can exist in at least one of
2 states, "glassy" or "fluid"; the temperature at which one
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter

5. 8. Pharmacopoeia/ harmonisation.
 2023 Appendix IX M V-A363

state transforms into the other is the glass transition Rates of water uptake
temperature, Tg. The rate and extent to which solids exposed to the
Water absorbed into the bulk solid structure, by virtue of its atmosphere might either sorb or desorb water vapour can be
effect on the free volume of the solid, can act as an efficient a critical factor in the handling of solids. Even the simple act
plasticiser and reduce the value of Tg. Since the rheological of weighing out samples of solid on an analytical balance and
properties of "fluid" and "glassy" states are quite different, the exposure, therefore, of a thin layer of powder to the
i.e. the "fluid" state exhibits much less viscosity as one goes atmosphere for a few minutes can lead to significant error in,
increasingly above the glass transition temperature, it is not for example, the estimation of loss on drying values. It is well
surprising that a number of important bulk properties established that water-soluble solids exposed to relative
dependent on the rheology of the solid are affected by humidities above that exhibited by a saturated solution of
moisture content. Since amorphous solids are metastable that solid will spontaneously dissolve via deliquescence and
relative to the crystalline form of the material, with small- continue to dissolve over a long time period. The rate of
molecular-mass materials, it is possible for absorbed moisture water uptake in general depends on a number of parameters
to initiate reversion of the solid to the crystalline form, not found to be critical in equilibrium measurements because
particularly if the solid is transformed by the sorbed water to rates of sorption are primarily mass-transfer controlled with
a "fluid" state. This is the basis of "cake collapse" often some contributions from heat-transfer mechanisms. Thus,
observed during the lyophilisation process. An additional factors such as vapour diffusion coefficients in air and in the
phenomenon noted specifically with water-soluble solids is solid, convective airflow, and the surface area and geometry
their tendency to deliquesce, i.e. to dissolve in their own of the solid bed and surrounding environment, can play an
sorbed water, at relative humidities, RH,, in excess of the important role. Indeed, the method used to make
relative humidity of a saturated solution of the solid, RH0 • measurements can often be the rate-determining factor
Deliquescence arises because of the high water solubility of because of these environmental and geometric factors.
the solid and the significant effect it has on the colligative DETERMINATION OF SORPTION-DESORPTION
properties of water. It is a dynamic process that continues to ISOTHERMS
occur as long as RH; is greater than RH0 • Principle
The key to understanding the effects water can have on the The tendency to take up water vapour is best assessed by
properties of solids, and vice versa, rests with an measuring sorption or desorption as a function of relative
understanding of the location of the water molecule and its humidity, at constant temperature, and under conditions
physical state. More specifically, water associated with solids where sorption or desorption is essentially occurring
can exist in a state that is directly bound to the solid, as well independently of time, i.e. equilibrium. Relative humidity,
as in a state of mobility approaching that of bulk water. This RH, is defined by the following expression:
difference in mobility has been observed through such
measurements as heats of sorption, freezing point, nuclear Pc X 100
magnetic resonance, dielectric properties and diffusion. Such Po
changes in mobility have been interpreted as arising because P, pressure of water vapour in the system;
of changes in the thermodynamic state of water as more and P0 saturation pressure of water vapour under the same conditions.
more water is sorbed. Thus, water bound directly to a solid
is often thought as unavailable to affect the properties of the The ratio P jP0 is referred to as the relative pressure.
solid, whereas larger amounts of sorbed water may become Sorption or water uptake is best assessed starting with dried
more clustered and form water more like that exhibiting samples and subjecting them to a known relative humidity.
solvent properties. In the case of crystal hydrates, the Desorption is studied by beginning with a system already
combination of intermolecular forces (hydrogen bonding) containing sorbed water and reducing the relative humidity.
and crystal packing can produce very strong water-solid As the name indicates, the sorption-desorption isotherm is
interactions. Recognising that the presence of water in an valid only for the reference temperature, hence a special
amorphous solid can affect the glass transition temperature isotherm exists for each temperature. Ordinarily, at
and hence the physical state of the solid, at low levels of equilibrium, moisture content at a particular relative
water, most polar amorphous solids are in a highly viscous humidity must be the same, whether determined from
glassy state because of their high values of Tg, Hence, water sorption or desorption measurements. However, it is
is "frozen" into the solid structure and is rendered immobile common to see sorption-desorption hysteresis.
by the high viscosity, e.g. 10 13 Pa·s. As the amount of water Methods
sorbed increases and Tg decreases, approaching ambient Samples may be stored in chambers at various relative
temperatures, the glassy state approaches that of a "fluid" humidities (Figure 2.9.39.-1). The mass gained or lost for
state and water mobility along with the mobility of the solid each sample is then measured. The major advantage of this
itself increases significantly. At high RH, the degree of water method is convenience, while the major disadvantages are the
plasticisation of the solid can be sufficiently high so that slow rate of reaching constant mass, particularly at high
water and the solid can now achieve significant amounts of relative humidities, and the error introduced in opening and
mobility. In general, therefore, this picture of the nature of closing the chamber for weighing.
sorbed water helps to explain the rather significant effect
Dynamic gravimetric water sorption systems allow the on-line
moisture can have on a number of bulk properties of solids
weighing of a sample in a controlled system to assess the
such as chemical reactivity and mechanical deformation.
interaction of the material with moisture at various
It suggests strongly that methods of evaluating chemical and
programmable levels of relative humidity at a constant
physical stability of solids and solid dosage forms take into
temperature. The major benefit of a controlled system is that
account the effects water can have on the solid when it is
isothermal conditions can be more reliably established and
sorbed, particularly when it enters the solid structure and
that the dynamic response of the sample to changing
acts as a plasticiser.
conditions can be monitored. Data points for the
determination of the sorption isotherm (e.g. from O per cent
V-A364 Appendix IX M 2023

A B

I D
-U:

H G F

A. Humidity controller D. Humidity regulated module G. Vapour humidifier

B. Temperature controlled chamber E. Reference H. Flow control module
C. Balance module F. Sample I. Dry gas

Figure 2.9.39.-1. - Example of an apparatus for the detenninatwn of the water sorptwn (other designs are possible)

to approximately 95 per cent RH, non condensing) are only dehydration, chemical degradation or sublimation. Using
taken after a sufficiently constant signal indicates that the higher temperatures to induce desorption, as in a
sample has reached equilibrium at a given level of humidity. thermogravimetric apparatus, likewise must be carefully
In some cases (e.g. deliquescence), the maximum time may carried out because of these possible pitfalls.
be restricted although the equilibrium level is not reached. Report and interpretation of the data
The apparatus must adequately control the temperature to Sorption data are usually reported as a graph of the apparent
ensure a good baseline stability as well as accurate control of mass change in per cent of the mass of the dry sample as a
the relative humidity generation. The required relative function of relative humidity or time. Sorption isotherms are
humidities can be generated, e.g. by accurately mixing dry reported both in tabular form and as a graph.
and saturated vapour gas with flow controllers. The measurement method must be traceable with the data.
The electrostatic behaviour of the powder must also be
Adsorption-desorption hysteresis can be interpreted, for
considered. The verification of the temperature and the
example, in terms of the porosity of the sample, its state of
relative humidity (controlled with, for example, a certified
agglomeration (capillary condensation), the formation of
hygrometer, certified salt solutions or deliquescence points of
hydrates, polymorphic change, or liquefying of the sample.
certified salts over an adequate range), must be consistent
Certain types of systems, particularly those with microporous
with the instrument specification. The balance must provide
solids and amorphous solids, are capable of sorbing large
a sufficient mass resolution and long term stability.
amounts of water vapour. Here, the amount of water
It is also possible to measure amounts of water uptake not associated with the solid as relative humidity is decreased, is
detectable gravimetrically using volumetric techniques. greater than the amount that originally sorbed as the relative
In some cases, direct analysis of water content by different humidity was increased. For microporous solids, vapour
methods such as determination of the boiling point, adsorption-desorption hysteresis is an equilibrium
determination of water by distillation, loss on drying or gas phenomenon associated with the process of capillary
chromatography may be advantageous. In the case of condensation. This takes place because of the high degree of
adsorption, to improve sensitivity, one can increase the irregular curvature of the micropores and the fact that they
specific surface area of the sample by reducing particle size or "fill" (adsorption) and "empty" (desorption) under different
by using larger samples to increase the total area. It is equilibrium conditions. For non-porous solids capable of
important, however, that such comminution of the solid does absorbing water, hysteresis occurs because of a change in the
not alter the surface structure of the solid or render it more degree of vapour-solid interaction due to a change in the
amorphous or otherwise less ordered in crystallinity. equilibrium state of the solid, e.g. conformation of polymer
For absorption, where water uptake is independent of specific chains, or because the time scale for structural equilibrium is
surface area, only increasing sample size will help. Increasing longer than the time scale for water desorption. In measuring
sample size, however, will increase the time to establish some sorption-desorption isotherms, it is therefore important to
type of equilibrium. To establish accurate values, it is establish that something close to an equilibrium state has
important to get desolvation of the sample as thoroughly as been reached. Particularly with hydrophilic polymers at high
possible. Higher temperatures and lower pressures (vacuum) relative humidities, the establishment of water sorption or
facilitate this process; however, one must be aware of any desorption values independent of time is quite difficult, since
adverse effects this might have on the solid such as
2023 Appendix IX M V-A365

one is usually dealing with a polymer plasticised into its component. Theoretically, all types of hygrometers can be
"fluid" state, where the solid is undergoing significant used, but for analytical purposes miniaturisation and
change. robustness are a precondition. The Aw measurement may be
In the case of crystal hydrate formation, the plot of water conducted using the dew point/chilled mirror method 2 •
uptake versus pressure or relative humidity will in these cases A polished, chilled mirror is used as a condensing surface.
exhibit a sharp increase in uptake at a particular pressure and The cooling system is electronically linked to a photoelectric
the amount of water taken up will usually exhibit a cell into which light is reflected from the condensing mirror.
stoichiometric mole:mole ratio of water to solid. In some An air stream, in equilibrium with the test sample, is directed
cases, however, crystal hydrates will not appear to undergo a at the mirror, which cools until condensation occurs on the
phase change or the anhydrous form will appear amorphous. mirror. The temperature at which this condensation begins is
Consequently, water sorption or desorption may appear more the dew point from which the ERH is determined.
like that seen with adsorption processes. X-ray Commercially available instruments using the dew
crystallographic analysis and thermal analysis are particularly point/chilled mirror method or other technologies need to be
useful for the study of such systems. evaluated for suitability, qualified, and calibrated when used
For situations where water vapour adsorption occurs to make water activity determinations. These instruments are
predominantly, it is very helpful to measure the specific typically calibrated over an adequate range, for example,
surface area of the solid by an independent method and to using some saturated salt solutions at 25 °C such as those
express adsorption as mass of water sorbed per unit area of listed in Table 2.9.39.-1.
solid surface. This can be very useful in assessing the possible Table 2.9.39.-1. - Standard saturated salt solutions
importance of water sorption in affecting solid properties.
For example, 0.5 per cent mlm uptake of water could hardly Saturated salts solutions ERH
at 25 "'C (per cent)
Aw
cover the bare surface of 100 m 2/g, while for 1.0 m 2/g this
amounts to 100 times more surface coverage. In the case of Potassium sulfate
97.3 0.973
pharmaceutical solids which have a specific surface area in (K2SO4)
the range of0.01 m 2/g to 10 m 2/g, what appears to be low
Barium chloride
water content could represent a significant amount of water 90.2 0.902
(BaCl 2 )
for the available surface. Since the "dry surface area" is not a
factor in absorption, sorption of water with amorphous or Sodium chloride
75.3 0.753
(NaCl)
partially amorphous solids can be expressed on the basis
of unit mass corrected for crystallinity, when the crystal form Magnesium nitrate
52.9 0.529
does not sorb significant amounts of water relative to the (Mg(NO 3h)
amorphous regions.
i\1.agnesium chloride
32.8 0.328
DETERMINATION OF THE WATER ACTIVITY (MgC[z)
Principle Lithium chloride
Water activity, Aw, is the ratio of vapour pressure of water in 11.2 0.112
(LlCI)
the product (P) to saturation pressure of water vapour (P0 ) at
the same temperature. It is numerically equal to 1/100 of the
relative humidity (RH) generated by the product in a closed
system. RH can be calculated from direct measurements of
partial vapour pressure or dew point, or from indirect
measurement by sensors whose physical or electric
characteristics are altered by the RH to which they are
exposed. Ignoring activity coefficients, the relationship
between Aw and equilibrium relative humidity (ERH) are
represented by the following equations:
p
Aw=-
Po

ERH (per cent)= Aw x 100

Method
The water activity is determined by placing the sample in a
small airtight cup inside which the equilibrium between the
water in the solid and the headspace can be established.
The volume of the headspace must be small in relation to the
sample volume in order not to change the sorption state of
sample during the test. The equilibration as a
thermodynamic process takes time but may be accelerated by
forced circulation within the cell. The acquired water activity
value is only valid for the simultaneously determined
temperature. This requires a precise temperature-measuring
device as part of the equipment. Furthermore, the probe
must be thermally insulated to guarantee a constant
temperature during the test. The sensor measuring the
humidity of the headspace air above the sample is a key 2 AOAC International Official Method 978.18.
V-A366 Appendix X A 2023

D. Hydroxyl Value
Appendix X (Ph. Bur. method 2.5.3)
A. Acetyl Value The hydroxyl value Iott is the number that expresses in
(No Ph. Bur. method) milligrams the quantity of potassium hydroxide required to
neutralise the acid combined by acylation in 1 g of the
The acetyl value of a substance is the number of mg of
substance.
potassium hydroxide required to neutralise the acetic acid
liberated by the hydrolysis of 1 g of the acetylated substance. METHOD A
Determine the saponificatwn value, Appendix X G. Introduce the quantity of the substance to be examined
Acetylate by the following method. To 10 gin a 200-mL shown in Table 2.5.3.-1 (mg) into a 150 mL acetylation
Kjeldahl flask add 20 mL of acetic anhydride. Support the flask fitted with a reflux condenser, unless another quantity is
flask on a sheet of heat resistant material in which a hole prescribed in the monograph. Add the quantity of acetic
about 4 = in diameter has been cut and heat with a small, anhydride solution Rl stated in Table 2.5.3.-1 and attach the
reflux condenser.
naked flame, not more than 25 mm in height and which does
not impinge on the bottom of the flask. Boil gently under a Table 2.5.3.-1
reflux air condenser for 2 hours, allow to cool, pour into
Presumed value /oH Quantity of sample Volume of
600 mL of water contained in a large beaker, add 0.2 g of
(g) acetylating reagent (mL)
pumice powder and boil for 30 minutes. Cool, transfer to a
10 - 100 2.0 5.0
separating funnel and discard the lower layer. Wash the
JOO - 150 1.5 5.0
acetylated product with three or more 50-mL quantities of a
150-200 1.0 5.0
warm, saturated solution of sodium chloride until the washings
200 - 250 0.75 5.0
are no longer acidic to litmus paper. Finally shake with 20 mL
250 - 300 0.60 or 1.20 5.0 or 10.0
of warm water and remove the aqueous layer as completely as
300 - 350 1.0 10.0
possible. Pour the acetylated substance into a small dish, add
350 - 700 0.75 15.0
1 g of powdered anhydrous sodium sulfate, stir thoroughly and
700 - 950 0.5 15.0
filter through a dry, pleated filter paper. Determine the
saponification val,ue of the acetylated substance.
Calculate the acetyl value from the expression 1335(b--- Heat the flask in a water-bath for 1 h keeping the level of the
a)/(1335-a) where a is the saponification value of the water about 2.5 cm above the level of the liquid in the flask.
substance and b is the saponification value of the acetylated Withdraw the flask and allow to cool. Add 5 mL of water R
substance. through the upper end of the condenser. If a cloudiness
appears add sufficient pyridine R to clear it. Shake the flask
and replace in the water-bath for 10 min. Withdraw the flask
and allow to cool. Rinse the condenser and the walls of the
B. Acid Value flask with 5 mL of ethanol (96 per cent) R, previously
(Ph. Bur. method 2.5.1) neutralised in the presence of phenolphthal,ein solution Rl.
The acid value h is the number that expresses, in milligrams Titrate with 0.5 M alcoholic potassium hydroxide using 0.2 mL
the quantity of potassium hydroxide required to neutralise of phenolphthalein solution Rl as indicator (n 1 mL of 0.5 M
the free acids present in 1 g of the substance. alcoholic potassium hydroxide). Carry out a blank test under
the same conditions (n 2 mL of 0.5 M alcoholic potassium
Dissolve 10.00 g of the substance to be examined, or the
hydroxide).
quantity prescribed, (mg), in 50 mL of a mixture of equal
volumes of ethanol (96 per cent) R and light petroleum R3, 1 28.05(n 2 - ni)
previously neutralised with 0.1 M potassium hydroxide or lQH =---'--------'-+IA
m
0.1 M sodium hydroxide, unless otherwise specified, using
0.5 mL of phenolphthalein solution Rl as indicator. METHODB
If necessary, heat to about 90 °C to dissolve the substance to Introduce the prescribed quantity of the substance to be
be examined. When the substance to be examined has examined (m g) into a perfectly dry 5 mL conical flask fitted
dissolved, titrate with 0.1 M potassium hydroxide or with a ground-glass or suitable plastic stopper and add
0.1 M sodium hydroxide until the pink colour persists for at 2.0 mL of propionic anhydride reagent R. Close the flask and
least 15 s (n mL of titrant). When heating has been applied shake gently to dissolve the substance. Allow to stand for 2 h
to aid dissolution, maintain the temperature at about 90 °C unless otherwise prescribed. Remove the stopper and transfer
during the titration. the flask and its contents into a wide-mouthed 500 mL
conical flask containing 25.0 mL of a 9 g/L solution of
aniline R in cyclohexane R and 30 mL of glacial acetic acid R.
Swirl the contents of the flask, allow to stand for 5 min, add
0.05 mL of crystal violet solution R and titrate with 0.1 M
C. Ester Value perchloric acid until an emerald-green colour is obtained
(n 1 mL of 0.1 M perchloric acid). Carry out a blank test under
(Ph. Bur. method 2.5.2) the same conditions (n 2 mL of 0.1 M perchloric acid).
The ester value h is the number that expresses in milligrams
the quantity of potassium hydroxide required to saponify the IoH = 5.610(n 1 - n2 )
esters present in 1 g of the substance. It is calculated from m
the saponification value Is and the acid value JA:
To take account of any water present, determine this
(y per cent) by the semi-micro determination of water
h =ls-h (2.5.12).
2023 Appendix X F V-A367

The hydroxyl value is then given by the equation: The mass of the sample is such that there will be an excess
of iodine chlon·de solution R of 50 per cent to 60 per cent of
IoH = (hydroxyl value as determined) - 31.ly the amount added, i.e. 100 per cent to 150 per cent of the
amount absorbed.
E. Iodine Value Introduce the prescribed quantity of the substance to be
(Ph. Bur. method 2.5.4) examined (m g) into a 250 mL flask fitted with a ground-
glass stopper and previously rinsed with glacial acetic acid R
The iodine value 11 is the number that expresses in grams the or dried, and dissolve it in 15 mL of a mixture of equal
quantity of halogen, calculated as iodine, that can be fixed in volumes of cydohexane R and glacial acetic acid R, unless
the prescribed conditions by 100 g of the substance. otherwise prescribed. If necessary, melt the substance before
When the monograph does not specify the method to be used, dissolution (melting point greater than 50 "C). Add very
method A is applied. Any change from method A to method B is slowly the volume of iodine chloride solution R stated in
validated. Table 2.5.4.-2. Close the flask and keep it in the dark for
METHOD A 30 min, unless otherwise prescribed, shaking frequently.
Unless otherwise prescribed, use the following quantities Add 10 mL of a 100 g/L solution of potassium iodide R and
(Table 2.5.4.-1) for the determination. 100 mL of water R. Titrate with 0.1 M sodium thiosulfate,
shaking vigorously until the yellow colour is almost
Table 2.5.4.-1 discharged. Add 5 mL of starch solution R and continue the
Presumed value 11 Quantity of sample (g) titration adding the 0.1 M sodium thiosulfate dropwise until
the colour is discharged (n 1 mL of 0.1 M sodium thiosulfate).
less than 20 1.0
20 - 60
Carry out a blank test under the same conditions (n 2 mL of
0.5 - 0.25
0.1 M sodium thiosulfate).
60 - 100 0.25 - 0.15
more than I 00 0.15 - 0.10
I,= 1.269 (n2 - n1)
m
Introduce the prescribed quantity of the substance to be
examined (m g) into a 250 mL flask fitted with a ground- Iodine Monochloride Method
glass stopper and previously dried or rinsed with glacial acetic (No Ph. Bur. method)
acid R, and dissolve it in 15 mL of chloroform R unless
When the use of iodine flasks is prescribed, use flasks with a
otherwise prescribed. Add very slowly 25.0 mL of iodine nominal capacity of 250 mL and complying with British
bromide solution R. Close the flask and keep it in the dark for
Standard 2735: 1956 (Specification for iodine flasks), unless
30 min unless otherwise prescribed, shaking frequently. otherwise specified.
Add 10 mL of a 100 g/L solution of potassium iodide R and
100 mL of water R. Titrate with 0. 1 M sodium thiosulfate, Dissolve the specified quantity of the substance being
shaking vigorously until the yellow colour is almost examined in 10 mL of dichloromethane in a dry iodine flask.
discharged. Add 5 mL of starch solution R and continue the Add 20 mL of iodine monochloride solution, insert the stopper,
titration adding the 0.1 M sodium thiosulfate dropwise until previously moistened with dilute potassium iodide soluti.on, and
the colour is discharged (n 1 mL of 0.1 M sodium thiosulfate). allow to stand in the dark at 15° to 25c for 30 minutes. Place
Carry out a blank test under the same conditions (n 2 mL of 15 mL of dilute potassium iodide solution in the top cup,
0. 1 M sodium thiosulfate). carefully remove the stopper, rinse the stopper and the sides
of the flask with 100 mL of water, shake and titrate with
I1_- l.269 (n2 - ni) 0.lM sodium thiosulfate VS using starch mucilage, added
m towards the end of the titration, as indicator. At the same
time carry out the operation in exactly the same manner, but
METHODB without the substance being examined.
Unless otherwise prescribed, use the following quantities Calculate the iodine value from the expression 1.269 vlw
(Table 2.5.4.-2) for the determination. where v is the difference, in mL, between the titrations and
w is the weight, in g, of the substance taken.
Table 2.5.4.-2
The approximate weight, in g, of the substance to be taken
Presumed value Mass (g) Mass (g) Iodine chloride
(corresponding ( corresponding to solution (mL)
may be calculated by dividing 20 by the highest expected
I,
to an excess of an excess of iodine value. If more than half of the available halogen is
150 per cent (Cl) 100 per cent IC!) absorbed, the test must be repeated, using a smaller quantity
<3 10 10 25 of the substance.
3 8.4613 10.5760 25
5.0770 6.3460 25
10 2.5384 3.1730 20
20 0.8461 1.5865 20 F. Peroxide Value
40 0.6346 0.7935 20
(Ph. Bur. method 2.5.5)
60 0.4321 0.5288 20
80 0.3173 0.3966 20 The peroxide value lp is the number that expresses in
100 0.2538 0.3173 20 milliequivalents of active oxygen the quantity of peroxide
120 0.2115 0.2644 20 contained in 1000 g of the substance, as determined by the
140 0.1813 0.2266 20 methods described below.
160 0.1587 0.1983 20 When the monograph does not specify the method to be used,
180 0.1410 0.1762 20 method A is applied. Any change from method A to method B is
200 0.1269 0.1586 20 validated.
V-A368 Appendix X G 2023

METHOD A Ip= l000(Vi - Vo)c

Place 5.00 g of the substance to be examined (m g) in a m
250 mL conical flask fitted with a ground-glass stopper. concentration of the sodium thiosulfate solution, in moles per
Add 30 mL of a mixture of 2 volumes of chloroform R and litre.
3 volumes of glacial acetic acid R. Shake to dissolve the
substance and add 0.5 mL of saturated potassium iodide
solution R. Shake for exactly 1 min then add 30 mL of
water R. Titrate with 0.01 M sodium thiosulfate, adding the
titrant slowly with continuous vigorous shaking, until the G. Saponification Value
yellow colour is almost discharged. Add 5 mL of starch
The saponification value is the number of mg of potassium
solution R and continue the titration, shaking vigorously, until
hydroxide required to neutralise the free acids and to
the colour is discharged (n 1 mL of 0. 01 M sodium thiosulfate).
saponify the esters in 1 g of the substance.
Carry out a blank test under the same conditions (n 2 mL of
0.01 M sodium thiosulfate). The volume of 0.01 M sodium Use Method I unless otherwise specified in the monograph.
thiosulfate used in the blank titration must not exceed Method I
0.1 mL. (No Ph. Bur. method)
Dissolve 35 to 40 g of potassium hydroxide in 20 mL of water
and add sufficient ethanol (96%) to produce 1000 mL. Allow
to stand overnight and pour off the clear liquid.
METHODB Weigh 2 g of the substance into a 200-mL flask, add
Cany out the operations avoiding exposure to actinic light. 25.0 mL of the ethanolic solution of potassium hydroxide
Place 50 mL of a mixture of 2 volumes of trimethylpentane R and boil under a reflux condenser for 1 hour, rotating the
and 3 volumes of glacial acetic acid R in a conical flask and contents frequently. While the solution is still hot, titrate the
replace the stopper. Swirl the flask until the substance to be excess of alkali with 0.5M hydrochloric acid VS using 1 mL of
examined (mg; see Table 2.5.5.-1) has dissolved. Using a phenolphthalein solution Rl as indicator. Repeat the operation
suitable volumetric pipette, add 0.5 mL of saturated potassium without the substance being examined.
iodide solution Rand replace the stopper. Allow the solution Calculate the saponification value from the expression 28.05
to stand for 60 ± 1 s, thoroughly shaking the solution v/w where v is the difference, in mL, between the titrations
continuously, then add 30 mL of water R. and w is the weight, in g, of substance taken.

Table 2.5.5.-1 Method II

Expected peroxide Mass of substance
(Ph. Bur. method 2. 5. 6)
value Ip to be examined (g) The saponification value ls is the number that expresses in
0 to 12 5.00 to 2.00 milligrams the quantity of potassium hydroxide required to
12 to 20 2.00 to 1.20 neutralise the free acids and to saponify the esters present in
20 to 30 1.20 to 0.80 1 g of the substance.
30 to 50 0.800 to 0.500 Unless otherwise prescribed, use the quantities indicated in
50 to 90 0.500 to 0.300 Table 2.5.6.-1 for the determination.

Table 2.5.6.-1
Titrate the solution with 0.01 M sodium thiosulfate (Vi mL),
adding it gradually and with constant, vigorous shaking, until Presumed value ls Quantity of sample (g)
the yellow iodine colour has almost disappeared. Add about <3 20
0.5 mL of starch solution Rl and continue the titration, with 3 to 10 12 to 15
constant shaking especially near the end-point, to liberate all 10 to 40 8 to 12
of the iodine from the solvent layer. Add the sodium 40 to 60 5 to 8
thiosulfate solution dropwise until the blue colour just 60 to 100 3 to 5
disappears. 100 to 200 2.5 to 3
Depending on the volume of 0.01 M sodium thiosulfate used, 200 to 300 1 to 2

it may be necessary to titrate with 0.1 M sodium thiosulfate. 300 to 400 0.5 to 1

NOTE There is a 15 s to 30 s delay in neutralising the

starch indicator for peroxide values of 70 and greater, due to Introduce the prescribed quantity of the substance to be
the tendency of trimethylpentane to float on the surface of examined (mg) into a 250 mL borosilicate glass flask fitted
the aqueous medium and the time necessary to adequately with a reflux condenser. Add 25.0 mL of 0.5 M alcoholic
mix the solvent and the aqueous titrant, thus liberating the potassium hydroxide and a few glass beads. Attach the
last traces of iodine. It is recommended to use 0.1 M sodium condenser and heat under reflux for 30 min, unless otherwise
thiosulfate for peroxide values greater than 150. A small prescribed. Add 1 mL of phenolphthalein solution Rl and
amount (0.5 per cent to 1.0 per cent m/m) of high HLB titrate immediately (while still hot) with 0.5 M hydrochloric
emulsifier (for example polysorbate 60) may be added to the acid (n 1 mL of 0.5 M hydrochlori.c acid). Carry out a blank
mixture to retard the phase separation and decrease the time test under the same conditions (n 2 mL of 0.5 M hydrochlori.c
lag in the liberation of iodine. acid).
Carry out a blank determination ( V0 mL). If the result of the
blank determination exceeds 0.1 mL of titration reagent, ls = 28.05(n2 - n1)
replace the reagents and repeat the determination. m
2023 Appendix X J V-A369

flask to a separating funnel with the aid of 100 mL of

H. Unsaponifiable Matter water R. Shake the liquid carefully with 3 quantities, each of
The unsaponifiable matter is the percentage content, w/w, of 100 mL, of peroxide-free ether R. Combine the ether layers in
material not volatile at 100° to 105° that is obtained by another separating funnel containing 40 mL of water R, shake
extraction with an organic solvent from the saponified gently for a few minutes, allow to separate and reject the
substance being examined. aqueous phase. Wash the ether phase with 2 quantities, each
Use Method I unless otherwise specified in the monograph. of 40 mL, of water R then wash successively with 40 mL of a
Use ungreased ground-glass glassware for each method. 30 g/L solution of potassium hydroxide R and 40 mL of
water R; repeat this procedure 3 times. Wash the ether phase
Method I several times, each with 40 mL of water R, until the aqueous
(No Ph. Bur. method) phase is no longer alkaline to phenolphthalein. Transfer the
To 2.0 to 2.5 g of the substance being examined in a ether phase to a tared flask, washing the separating funnel
250-mL flask add 25 mL of 0.5M ethanolic potassium with peroxide-free ether R.
hydroxide and boil under a reflux condenser in a water-bath Distil off the ether with suitable precautions and add 6 mL
for 1 hour, swirling the contents frequently. Wash the of acetone R to the residue. Carefully remove the solvent in a
contents of the flask into a separating funnel with the aid of current of air. Dry to constant mass at 100-105 cc. Allow to
50 mL of water and, while the liquid is still slightly warm, cool in a desiccator and weigh (a g).
extract by shaking vigorously with three 50-mL quantities of
peroxide-free ether, rinsing the flask with the first quantity of . 100a
Unsapomfiable matter = - - per cent
ether. Mix the ether solutions in a separating funnel m
containing 20 mL of water. (If the ether solutions contain
solid suspended matter, filter them into the separating funnel Dissolve the residue in 20 mL of alcohol R, previously
through a fat-free filter paper and wash the filter paper with neutralised to phenolphthalein solution R and titrate with 0.1 M
peroxide-free ether.) Gently rotate the separating funnel for a ethanolic sodium hydroxide. If the volume of 0.1 M ethanolic
few minutes without violent shaking, allow the liquids to sodium hydroxide used is greater than 0.2 mL, the separation
separate and discard the aqueous layer. Wash the ether of the layers has been incomplete; the residue weighed
solution by shaking vigorously with two 20-mL quantities of cannot be considered as "unsaponifiable matter". In case of
water and then treat with three 20-mL quantities of doubt, the test must be repeated.
0.5M potassium hydroxide, shaking vigorously on each
occasion, each treatment being followed by washing with
20 mL of water. Finally wash with successive 20-mL
quantities of water until the aqueous layer is no longer J. Determination of Cineole
alkaline to phenolphthalein solution Rl. Transfer the ether
(Ph. Bur. method 2.8.11)
extract to a weighed flask, rinsing the separating funnel with
peroxide-free ether, distil the ether and add 3 mL of acetone to Weigh 3. 00 g of the oil, recently dried with anhydrous sodium
the flask. With the aid of a gentle current of air, remove the sulfate R, into a dry test-tube and add 2.10 g of melted
solvent completely from the flask, which is almost entirely cresol R. Place the tube in the apparatus for the determination
immersed in boiling water and preferably held obliquely and of freezing point (2. 2.18) and allow to cool, stirring
rotated. Dry to constant weight at a temperature not continuously. When crystallisation takes place there is a small
exceeding 80° and dissolve the contents of the flask in 10 mL rise in temperature. Note the highest temperature reached
of freshly boiled ethanol (96%), previously neutralised to (t1)-
phenolphthalein solution Rl. Titrate with 0.lM ethanolic sodium Remelt the mixture on a water-bath at a temperature that
hydroxide VS using phenolphthalein solution Rl as indicator. does not exceed t 1 by more than 5 °C and place the tube in
If the volume of 0.lM ethanolic sodium hydroxide VS required the apparatus, maintained at a temperature 5 °C below t 1 •
does not exceed 0 .1 mL, the amount of residue weighed is to When crystallisation takes place, or when the temperature of
be taken as the unsaponifiable matter. Calculate the the mixture has fallen 3 °C below t 1, stir continuously. Note
unsaponifiable matter as a percentage of the substance being the highest temperature at which the mixture crystallises (t2).
examined. Repeat the operation until 2 highest values obtained for t2 do
If the volume of 0 .1 M ethanolic sodium hydroxide VS required not differ by more than 0.2 °C. If supercooling occurs,
exceeds 0.1 mL, the amount ofresidue weighed cannot be induce crystallisation by adding a small crystal of the
taken as the unsaponifiable matter and the test must be complex consisting of 3.00 g of cineole Rand 2.10 g of
repeated. melted cresol R. If t2 is below 27.4 °C, repeat the
determination after the addition of 5.10 g of the complex.
Method II The content of cineole corresponding to the highest
(Ph. Bur. method 2.5. 7) temperature observed (t2) is given in Table 2.8.11.-1.
The term "unsaponifiable matter" is applied to the If 5 .10 g of the complex has been added, calculate the
substances non-volatile at 100-105 cc obtained by extraction cineole content per cent m/m from the expression:
with an organic solvent from the substance to be examined
after it has been saponified. The result is calculated 2(A - 50)
as per cent mlm.
Use ungreased ground-glass glassware. where A is the value found in Table 2.8.11.-1.
Introduce the prescribed quantity of the substance to be The content of cineole, corresponding to the highest
examined (m g) into a 250 mL flask fitted with a reflux temperature observed (t2), is obtained, where necessary, by
condenser. Add 50 mL of 2 M alcoholic potassium hydroxide R interpolation.
and heat on a water-bath for 1 h, swirling frequently. Cool to
a temperature below 25 "C and transfer the contents of the
V-A370 Appendix X K 2023

Table 2.8.11.-1 Foreign Esters in Essential Oils

t, cineole t, cineole t, cineole t, cineole (Ph. Bur. method 2.8.6)
'C per cent "C per cent "C per cent 'C per cent Heat 1 mL of the essential oil for 2 min on a water-bath with
mlm mlm mlm mlm
3.0 mL of a freshly prepared 100 g/L solution of potassium
24 45.5 32 56.0 40 67.0 48 82.0 hydroxide R in alcohol R. No crystals are formed within
25 47.0 33 57.0 41 68.5 49 84.0 30 min, even after cooling.
26 48.5 34 58.5 42 70.0 50 86.0
27 49.5 35 60.0 43 72.5 51 88.5 Odour and Taste of Essential Oils
28 50.5 36 61.0 44 74.0 52 91.0 (Ph. Bur. method 2.8.8)
29 52.0 37 62.5 45 76.0 53 93.5 Mix 3 drops of the essential oil with 5 mL of alcohol
30 53.5 38 63.5 46 78.0 54 96.0 (90 per cent V/V) R and stir in 10 g of powdered sucrose R.
31 54.5 39 65.0 47 80.0 55 99.0 The odour and taste are similar to that of the plant or parts
of the plant from which the essential oil has been obtained.
Residue on Evaporation of Essential Oils
(Ph. Bur. method 2.8. 9)
K. Determination of Aldehydes The residue on evaporation of an essential oil is the
(No Ph. Bur. method) percentage by mass of the oil which remains after evaporation
on a water-bath under the conditions specified below.
To 1 g of the oil in a glass-stoppered tube (approximately
Apparatus The apparatus (see Figure 2.8.9.-1) consists of:
150 mm x 25 mm) add 5 mL of toluene and 15 mL of
- water-bath with a cover having holes of 70 mm diameter;
alcoholic hydroxylamine solution, shake vigorously and titrate
- evaporating dish of heat-resistant glass which is inert to
immediately with 0.5M potassium hydroxide in ethanol (60%)
the contents;
VS until the red colour changes to yellow. Continue shaking
- desiccator.
and neutralising until the full yellow colour of the indicator is
permanent in the lower layer after shaking vigorously for
2 minutes and allowing to separate; the reaction is complete 86
in about 15 minutes. This procedure gives an approximate
value for the aldehyde content of the oil.
Repeat this procedure, using as the colour standard for the
end point of the titration the titrated liquid of the first
determination with the addition of 0.5 mL of 0.5M potassium
hydroxide in ethanol (60%) VS. Calculate the content of
aldehydes from the second determination, using the
equivalent given in the monograph.
0
I!)

70
L. Oxidising Substances
(Ph. Bur. method 2.5.30)
Transfer 4.0 g to a glass-stoppered, 125 mL conical flask and
add 50.0 mL of water R. Insert the stopper and swirl for
5 min. Transfer to a glass-stoppered 50 mL centrifuge tube Figure 2.8.9.-1
and centrifuge. Transfer 30.0 mL of the clear supernatant to Dimensions in millimetres
a glass-stoppered 125 mL conical flask. Add 1 mL of glacial
acetic acid R and 0. 5 g to 1. 0 g of potassium iodide R. Insert
Method Weigh the evaporating dish after having heated it
on the water-bath for 1 h and cooled it in the desiccator.
the stopper, swirl, and allow to stand for 25 min to 30 min
in the dark. Add 1 mL of starch solution R and titrate with Weigh into the evaporating dish 5.00 g of the essential oil,
0.002 M sodium thiosulfate until the starch-iodine colour
unless otherwise prescribed. Evaporate the oil by heating on
disappears. Carry out a blank determination. Not more than a water-bath in a draught-free atmosphere for the prescribed
1.4 mL of 0. 002 M sodium thiosulfate is required time. Allow to cool in the desiccator and weigh.
(0.002 per cent, calculated as H 2 0 2). During the test, the level of water in the bath is maintained
1 mL of 0. 002 M sodium thiosuifate is equivalent to 34 µg of about 50 mm beneath the level of the cover.
oxidising substances, calculated as hydrogen peroxide. Solubility in Alcohol of Essential Oils
(Ph. Bur. method 2.8.10)
Place 1.0 mL of the essential oil in a 25 mL or 30 mL glass-
stoppered cylinder. Place in a constant temperature device,
M. Essential Oils maintained at a temperature of 20 ± 0.2 °C. Using a burette
of at least 20 mL capacity, add the alcohol of the strength
Fatty Oils and Resinified Essential Oils in Essential prescribed in the monograph by increments of 0.1 mL until
Oils solution is complete and then continue adding by increments
(Ph. Bur. method 2. 8. 7) of 0.5 mL to a total of 20 mL, shaking frequently and
Allow 1 drop of the essential oil to fall onto filter paper. vigorously. Record the volume of alcohol added when a clear
The drop evaporates completely within 24 h without leaving solution has been obtained and, if the solution becomes
any translucent or greasy spot. cloudy or opalescent before 20 mL of alcohol has been
2023 Appendix X N V-A371

added, record the volume added when the cloudiness or Plate A suitable octadecylsilyl silica gel for high-
opalescence appears and, where applicable, the volume added performance thin-layer chromatography as the coating
when the cloudiness or opalescence disappears. substance.
If a clear solution has not been obtained when 20 mL of Mobile phase:
alcohol of the prescribed strength has been added, repeat the - mobile phase A: ether R;
test using the next highest concentration of alcohol. - mobile phase B: methylene chloride R, glacial acetic acid R,
An essential oil is said to be "soluble in n volumes and more acetone R (20:40:50 VIVIV).
of alcohol of given strength t" when the clear solution in n Application 1 µL.
volumes remains clear when compared with the undiluted oil Development Twice over a path of 0.5 cm with mobile
after further addition of alcohol of the same strength up to a phase A, then twice over a path of 8 cm with mobile
total of 20 volumes of alcohol. phase B.
An essential oil is said to be "soluble in n volumes of alcohol Drying In air.
of given strength t, becoming cloudy when diluted" when the
Detection Spray with a 100 g/L solution of phosphomolybdic
clear solution in n volumes becomes cloudy in n 1 volumes (n 1 acid R in ethanol (96 per cent) R. Heat the plate at 120 °C for
less than 20) and stays so after further gradual addition of about 3 min and examine in daylight.
alcohol of the same strength up to a total of 20 volumes of
alcohol. The chromatogram obtained typically shows spots
comparable to those in Figure 2.3.2.-1.
An essential oil is said to be "soluble in n volumes of alcohol
of given strength t with cloudiness between n 1 and METHODB
n2 volumes" when the clear solution in n volumes becomes Thin-layer chromatography (2. 2. 2 7).
cloudy in n 1 volumes (n 1 less than 20) and stays so after Test solution Unless otherwise prescribed, dissolve about
further gradual addition of alcohol of the same strength up to 20 mg (1 drop) of the fatty oil in 3 mL of methylene
a total of n2 volumes of alcohol and then becomes clear (n 2 chloride R.
less than 20). Reference solution Dissolve about 20 mg (1 drop) of
An essential oil is said to be "soluble with opalescence" when maize oil R in 3 mL of methylene chloride R.
the alcoholic solution shows a bluish tinge, similar to that of Plate A suitable octadecylsilyl silica gel for high-
a standard of opalescence freshly prepared as follows: mix performance thin-layer chromatography as the coating
0.5 mL of silver nitrate solution R2 and 0.05 mL of nitric substance.
acid R; add 50 mL of a 12 mg/L solution of sodium
Mobile phase methylene chloride R, glacial acetic acid R,
chloride R; mix and allow to stand protected from light for
acetone R (20:40:50 VIVIV).
5 min.
Application l µL as bands of 8 mm. A suitable automated
Water in Essential Oils apparatus may be used.
(Ph. Bur. method 2.8.5) Development Over a path of 7 cm.
Mix 10 drops of the essential oil with 1 mL of carbon Drying In air.
disulfide R. The solution remains clear on standing.
Detection Treat with a 100 g/L solution of phosphomolybdic
acid R in ethanol (96 per cent) R. Heat the plate at 120 °C for
3 min and examine in daylight.
The chromatogram obtained typically shows zones
N. Fixed Oils comparable to those in Figure 2.3.2.-2.
AJkaline Impurities in Fatty Oils Test for Foreign Oils by Thin-layer
(Ph. Bur. method 2.4. 19) Chromatography
In a test-tube mix 10 mL ofrecently distilled acetone Rand (Ph. Bur. method 2.4.21)
0.3 mL of water R and add 0.05 mL of a 0.4 g/L solution of Examine by thin-layer chromatography (2.2.27) using
bromophenol blue R in alcohol R. Neutralise the solution if kieselguhr G R as the coating substance. Impregnate a plate
necessary with 0.01 M hydrochloric acid or 0.01 M sodium by placing it in a chromatographic tank containing the
hydroxide. Add 10 mL of the oil to be examined, shake and necessary quantity of a mixture of 10 volumes of hquid
allow to stand. Not more than 0.1 mL of 0.01 M hydrochloric paraffin R and 90 volumes of light petroleum R so that the
acid is required to change the colour of the upper layer to plate dips about 5 mm beneath the surface of the liquid.
yellow. When the impregnation mixture has risen by at least 12 cm
from the lower edge of the plate, remove the plate and allow
Identification of Fixed Oils by Thin-layer the solvent to evaporate for 5 min. Carry out the
Chromatography chromatography in the same direction as the impregnation.
(Ph. Bur. method 2.3.2)
Preparation of the mixture offatty acids Heat 2 g of
METHOD A the oil with 30 mL of 0.5 M alcoholic potassium hydroxide
Thin-layer chromatography (2.2.27). under a reflux condenser for 45 min. Add 50 mL of water R,
Test solution Unless otherwise prescribed, dissolve about allow to cool, transfer to a separating funnel and extract with
20 mg ( 1 drop) of the fatty oil in 3 mL of methylene three quantities, each of 50 mL, of ether R. Discard the ether
chloride R. extracts, acidify the aqueous layer with hydrochloric acid R
and extract with three quantities, each of 50 mL, of ether R.
Reference solution Dissolve about 20 mg (1 drop) of
Combine the ether extracts and wash with three quantities,
maize oz7 R in 3 mL of methylene chloride R.
each of 10 mL, of water R; discard the washings, dry the
ether over anhydrous sodium sulfate R and filter. Evaporate the
ether on a water-bath. Use the residue to prepare the test
V-A372 Appendix X N 2023

16

I. arachis oil 5. soya-bean oil 9. sunflower oil I 3. evening primrose oil

2. sesame oil 6. rapeseed oil (erucic acid-free) 10. almond oil 14. safflower oil (type I)
3. maize oil 7. linseed oil 11. wheat-germ oil I 5. safflower oil (type II)
4. rapeseed oil 8. olive oil 12. borage oil I 6. hydrogenated arachis oil

Figure 2.3.2.-1. - Chromatograms for the identification of fatty oils (method A)

I. arachis oil 5. soya-bean oil 9. sunflower oil 13. evening primrose oil
2. sesame oil 6. rapeseed oil (erucic acid-free) 10. almond oil 14. safflower oil (type I)
3. maize oil 7. linseed oil 11. wheat-germ oil 15. safflower oil (type II)
4. rapeseed oil 8. olive oil 12. borage oil 16. hydrogenated arachis oil

Figure 2.3.2.-2. - Chromatograms for the identification of fatty oils (method B)

solution. The fatty acids may also be obtained from the soap bottom of the chamber. After some time brown or yellowish-
solution prepared during the determination of the brown spots become visible. Remove the plate and allow to
unsaponifiable matter. stand for a few minutes. When the brown background colour
Test solution Dissolve 40 mg of the mixture of fatty acids has disappeared, spray with starch solution R. Blue spots
obtained from the substance to be examined in 4 mL of appear which may become brown on drying and again
chloroform R. become blue after spraying with water R. The chromatogram
Reference solution Dissolve 40 mg of the mixture of fatty obtained with the test solution always shows a spot with an
acids obtained from a mixture of 19 volumes of maize oil R Rp of about 0.5 (oleic acid) and a spot with an Rp of about
and 1 volume of rapeseed oil R in 4 mL of chloroform R. 0.65 (linoleic acid) corresponding to the spots in the
chromatogram obtained with the reference solution. With
Apply to the plate 3 µL of each solution. Develop over a
some oils a spot with an Rp of about 0.75 may be present
path of 8 cm using a mixture of 10 volumes of water R and
(linolenic acid). By comparison with the spot in the
90 volumes of glacial acetic acid R. Dry the plate at 110 °C
chromatogram obtained with the reference solution, verify
for 10 min. Allow to cool and, unless otherwise prescribed,
the absence in the chromatogram obtained with the test
place the plate in a chromatographic chamber, with a tightly
solution of a spot with an Rp of about 0.25 (erucic acid).
fitting lid, that has previously been saturated with iodine
vapour by placing iodine R in an evaporating dish at the
2023 Appendix X N V-A373

Test for Foreign Oils by Gas Chromatography System suitability When using the mixture of calibrating
(Ph. Bur. method 2.4.22) substances in Table 2.4.22.-1 or Table 2.4.22.-3:
The test for foreign oils is carried out on the methyl esters of - resolution: minimum 1.8 between the peaks due to methyl
the fatty acids contained in the oil to be examined by gas oleate and methyl stearate in the chromatogram obtained
chromatography (2.2.28). with reference solution (a);
- signal-to-noise ratio: minimum 5 for the peak due to
METHOD A methyl myristate in the chromatogram obtained with
This method is not applicable to oils that contain glycerides offatty reference solution (b);
acids with an epoxy-, hydroepoxy-, hydroperoxy-, cyclopropyl or - number of theoretical plates: minimum 30 000, calculated
cyclopropenyl group, or those that contain a large proportion of for the peak due to methyl stearate in the chromatogram
fatty acids of chain length less than 8 carbon atoms or to oils with obtained with reference solution (a).
an acid value greater than 2. 0.
System suitability When using the mixture of calibrating
Test solution When prescribed in the monograph, dry the substances in Table 2.4.22.-2:
oil to be examined before the methylation step. Weigh 1.0 g - resolution: minimum 4.0 between the peaks due to methyl
of the oil into a 25 mL round-bottomed flask with a ground- caprylate and methyl decanoate in the chromatogram
glass neck fitted with a reflux condenser and a gas port into obtained with reference solution (a);
the flask. Add 10 mL of anhydrous methanol Rand 0.2 mL of - signal-to-noise ratio: minimum 5 for the peak due to
a 60 g/L solution of potassium hydroxide R in methanol R. methyl caproate in the chromatogram obtained with
Attach the reflux condenser, pass nitrogen R through the reference solution (b);
mixture at a rate of about 50 mUmin, shake and heat to - number of theoretical plates: minimum 15 000, calculated
boiling. When the solution is clear (usually after about for the peak due to methyl decanoate in the
10 min), continue heating for a further 5 min. Cool the flask chromatogram obtained with reference solution (a).
under running water and transfer the contents to a separating
funnel. Rinse the flask with 5 mL of heptane R and transfer ASSESSMENT OF CHROMATOGRAMS
the rinsings to the separating funnel and shake. Add 10 mL Avoid working conditions tending to give masked peaks
of a 200 g/L solution of sodium chloride R and shake (presence of constituents with small differences between
vigorously. Allow to separate and transfer the organic layer to retention times, for example linolenic acid and arachidic
a vial containing anhydrous sodium sulfate R. Allow to stand, acid).
then filter. Qualitative analysis
Reference solution (a) Prepare 0.50 g of the mixture of Identify the peaks in the chromatogram obtained with
calibrating substances with the composition described in one reference solution (c) (isothermal operating conditions or
of the 2.4.22 tables, as prescribed in the individual linear temperature programming).
monograph (if the monograph does not mention a specific When using isothermal operating conditions, the peaks may
solution, use the composition described in Table 2.4.22.-1). also be identified by drawing calibration curves using the
Dissolve in heptane R and dilute to 50.0 mL with the same chromatogram obtained with reference solution (a) and the
solvent. information given in Tables 2.4.22.-1, 2.4.22.-2 or 2.4.22.-3.
Reference solution (b) Dilute 1.0 mL of reference
Table 2.4.22.-1. - Mixture of calibrating substances (for gas
solution (a) to 10.0 mL with heptane R.
chromatography with capillary column and split inlet system, it is
Reference solution (c) Prepare 0.50 g of a mixture of recommended that the component with the longest chain length of
fatty acid methyl esters that corresponds in composition to the mixture to be examined be added to the calibration mixture,
the mixture of fatty acids indicated in the monograph of the when the qualitative analysis is done using calibration curves)
substance to be examined. Dissolve in heptane R and dilute
Mixture of the following substances Composition (per cent m/m)
to 50.0 mL with the same solvent. Commercially available
Methyl laurate R 5
mixtures of fatty acid methyl esters may also be used.
Methyl myristate R 5
Column:
Methyl palmitate R 10
- material: fused silica, glass or quartz;
Methyl stearate R 20
=
- size: l 10-30 m, 0 =0.2-0.8 mm;
Methyl arachidate R 40
- stationary phase: macrogol 20 000 R (film thickness
Methyl oleate R 20
0.1-0.5 µm) or another suitable stationary phase.
Carrier gas helium for chromatography R or hydrogen for
chromatography R. Table 2.4.22.-2. - Mixture of calibrating substances (for gas
Flow rate 1.3 mUmin (for a column 0 = 0.32 mm). chromatography with capillary column and split inlet system, it is
Split ratio 1: 100 or less, according to the internal diameter recommended that the component with the longest chain length of
of the column used (1:50 when 0 = 0.32 mm). the mixture to be examined be added to the calibration mixture,
when the qualitative analysis is done using calibration curves)
Temperature:
Mixture of the following substances Composition
- column: in isothermal conditions, 160-200 °C, according
(per cent m/m)
to the length and type of column used (200 °C for a
Methyl caproate R 10
column 30 m long and coated with a layer of macrogol
Methyl caprylate R 10
20 000 R); if a linear temperature programming is
necessary, raise the temperature of the column at a rate of Methyl decanoate R 20
Methyl laurate R 20
3 °C/min from 170 °C to 230 °C, for example;
- injection port: 250 "C; 1'...fethyl myn'state R 40
- detector. 250 °C.
Detection Flame ionisation.
Injection 1 µL.
V-A374 Appendix X N 2023

Table 2.4.22.-3. - Mixture of calibrating substances (for gas that of the solvent, is set at 100 per cent. The content of a
chromatography with capillary column and split inlet system, it is constituent is calculated by determining the area of the
recommended that the component with the longest chain length of corresponding peak as a percentage of the sum of the areas
the mixture to be examined be added to the calibration mixture, of all the peaks. Disregard any peak with an area less than
when the qualitative analysis is done using calibration curves) 0.05 per cent of the total area.
Mixture of the following substances Composition (per cent m/m) In certain cases, for example in the presence of fatty acids
Methyl myristate R 5 with 12 or less carbon atoms, correction factors can be
Methyl palmitate R 10 prescribed in the individual monograph to convert peak areas
Methyl stearate R 15 in per cent m/m.
Methyl arachidate R 20 METHODB
Methyl oleate R 20 This method is not applicable to oils that contain glycerides offatty
Methyl eicosenoate R 10 acids with an epoxy-, hydroepoxy-, hydroperoxy-, cyclopropyl or
Methyl behenate R 10 cyclopropenyl group or to oils with an acid value greater than 2. 0.
Methyl lignocerate R 10
Test solution Introduce 0.100 g of the substance to be
examined into a 10 mL centrifuge tube with a screw cap.
Measure the reduced retention time (t' iJ of each peak in the Dissolve with 1 mL of heptane R and 1 mL of dimethyl
chromatogram obtained with reference solution (a). t' R is the carbonate R and mix vigorously under gentle heating
retention time measured from the solvent peak and not from (50-60 °C). Add, while still warm, 1 mL of a 12 g/L solution
the time of injection. Plot the straight line: of sodium R in anhydrous methanol R, prepared with the
necessary precautions, and mix vigorously for about 5 min.
log 10 (t~) = f( equivalent chain length)
Add 3 mL of distilled water R and mix vigorously for about
The logarithms of t' R of unsaturated acids are situated on 30 s. Centrifuge for 15 min at 1500 g. Inject 1 µL of the
this line at points corresponding to non-integer values of organic phase.
carbon atoms known as 'equivalent chain lengths'; the Reference solutions and assessment of
equivalent chain length is the length of the theoretical chromatograms Where there is no specific prescription in
saturated chain that would have the same t' R as the fatty acid the individual monograph, proceed as described under
to be identified. For example, linoleic acid has the same t' R Method A.
as the theoretical saturated fatty acid having 18.8 carbon Column:
atoms. - material: fused silica;
Identify the peaks in the chromatogram obtained with the = =
- size: l 30 m, 0 0.25 mm;
test solution by means of the straight line and the reduced - stationary phase: macrogol 20 000 R (film thickness
retention times. Equivalent chain lengths are given in 0.25 µm).
Table 2.4.22.-4. Carrier gas helium for chromatography R.
Flow rate 0.9 mIJmin.
Table 2.4.22.-4. - Equivalent chain lengths (this value, which is
to be calculated using calibration curves, is given as an example Split ratio 1:100.
for a column of macrogol 20 000 R) Temperature:
Fatty acid Equivalent chain length
Time Temperature
Caproic acid 6.0
(min) CC)
Caprylic acid 8.0
Column 0 - 15 100
Capric acid 10.0
15 - 36 100 ➔ 225
Laurie acid 12.0
36 - 61 225
Myristic acid 14.0
Injection port 250
Palmitic acid 16.0
Detector 250
Palmitoleic acid 16.3
Margaric acid 17.0
Stearic acid 18.0 Detection Flame ionisation.
Oleic acid 18.3 Injection 1 µL.
Linoleic acid 18.8
METHODC
Gamma-linolenic acid 19.0
This method is not applicable to oils that contain glycerides offatty
Alpha-linolenic acid 19.2
acids with epoxy-, hydroepoxy-, hydroperoxy-, aldehyde, ketone,
Arachidic acid 20.0
cyclopropyl and cyclopropenyl groups, and conjugated
Eicosenoic acid (gondoic acid) 20.2
polyunsaturated and acetylenic compounds because of partial or
Arachidonic acid 21.2
complete destruction of these groups.
Behenic acid 22.0
Erucic acid 22.2
Test solution Dissolve 0 .10 g of the substance to be
12-0xostearic acid 22.7
examined in 2 mL of a 20 g/L solution of sodium hydroxide R
in methanol Rina 25 mL conical flask and boil under a
Ricinoleic acid 23.9
12-Hydroxystearic acid 23.9
reflux condenser for 30 min. Add 2.0 mL of boron trifiuoride-
Lignoceric acid 24.0
methanol solution R through the condenser and boil for
Nervonic acid 24.2
30 min. Add 4 mL of heptane R through the condenser and
boil for 5 min. Cool and add 10.0 mL of saturated sodium
chloride solution R, shake for about 15 sand add a quantity of
Quantitative analysis saturated sodium chloride solution R such that the upper phase
In general, the normalisation procedure is used in which the is brought into the neck of the flask. Collect 2 mL of the
sum of the areas of the peaks in the chromatogram, except
 2023 Appendix X P V-A375

upper phase, wash with 3 quantities, each of 2 mL, of Internal standard methyl tricosanoate R.
water R and dry over anhydrous sodium sulfate R. Test solutions Prepare all test solutions in duplicate.
Reference solutions, chromatographic procedure and Step A
assessment of chromatograms Where there is no - Test solution (a). Dissolve a quantity of the sample to be
specific prescription in the individual monograph, proceed as examined according to Table 2.4.29.-1 and 70.0 mg of
described under Method A. the internal standard in a 50 mivL solution of
butylhydroxytoluene R in trimethylpentane R and dilute to
10.0 mL with the same solution. Gentle heating (up to
60 °C) may be applied to dissolve the internal standard.
O. Anisidine Value Ethyl esters are now ready for analysis. For trig]ycerides
(Ph. Eur. method 2.5.36) continue as described in step B.

The anisidine value is defined as 100 times the optical Table 2.4.29.-1
density measured in a 1 cm cell of a solution containing 1 g Approximate sum EPA+ DHA Mass of sample to be examined
of the substance to be examined in 100 mL of a mixture of (per cent) (g)
solvents and reagents according to the following method. 30 - 50 0.4 - 0.5
Cany out the operations as rapidly as possible, avoiding exposure 50 - 70 0.3
to actinic light. 70 - 90 0.25

Test solution (a) Dissolve 0.500 g of the substance to be

examined in trimethylpentane Rand dilute to 25.0 mL with Test solution (b). Dissolve a quantity of the sample to be
the same solvent. examined according to Table 2.4.29.-1 in a 50 mivL
Test solution (b) To 5.0 mL of test solution (a) add solution of butylhydroxytoluene R in trimethylpentane R and
1.0 mL of a 2.5 g/L solution of p-anisidine R in glacial acetic dilute to 10.0 mL with the same solution. Ethyl esters are
acid R, shake and store protected from light. now ready for analysis. For trig]ycerides continue as
described in step B.
Reference solution To 5.0 mL of trimethylpentane R add
1.0 mL of a 2.5 g/L solution of p-anisidine R in glacial acetic StepB
acid R, shake and store protected from light. Introduce 2.0 mL of test solutions (a) and (b) obtained in
step A into separate quartz tubes and evaporate the solvent
Measure the absorbance (2.2.25) of test solution (a) at the
with a gentle current of nitrogen R. Add 1.5 mL of a 20 g/L
maximum at 350 nm using trimethylpentane Ras the
solution of sodium hydroxide R in methanol R, cover with
compensation liquid. Measure the absorbance of test
nitrogen R, cap tightly with a polytetrafluoroethylene-lined
solution (b) at 350 nm exactly 10 min after its preparation,
cap, mix and heat on a water-bath for 7 min. Allow to cool.
using the reference solution as the compensation liquid.
Add 2 mL of boron trichloride-methanol solution R, cover with
Calculate the anisidine value from the expression: nitrogen R, cap tightly, mix and heat on a water-bath for
30 min. Cool to 40-50 °C, add 1 mL of trimethylpentane R,
25 x (l.2A1 -A2) cap and shake vigorously for at least 30 s. Immediately add
m 5 mL of a saturated sodium chloride solution R, cover with
A, absorbance of test solution (b) at 350 nm,
nitrogen R, cap and shake thoroughly for at least 15 s.
A2 absorbance of test solution (a) at 350 nm, Transfer the upper layer to a separate tube. Shake the
m mass of the substance to be examined in test solution (a), in methanol layer once more with 1 mL of trimethylpentane R.
grams. The combined trimethylpentane extracts may be washed
using 2 quantities, each of 1 mL, of water R and dried over
anhydrous sodium sulfate R.
Reference solutions Prepare reference solutions (a 1 ) and
(a 2) in duplicate; reference solution (c) only has to be
P. Oils Rich in Omega-3-acids prepared for triglycerides, and only if tetracos-15-enoic acid
1. Composition of Fatty Acids
methyl ester is not clearly observed in the chromatogram
obtained with test solution (a).
(Ph. Bur. method 2.4.29)
- Reference solution (aJ. Dissolve 70.0 mg of the internal
The assay may be used for quantitative determination of the standard and 90.0 mg of eicosapentaenoic acid ethyl
eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and ester CRS in a 50 mivL solution of butylhydroxytoluene R
total omega-3-acids content in products from fish oil containing in trimethylpentane Rand dilute to 10.0 mL with the same
omega-3 acids in different concentrations. The method is applicable solution. Gentle heating (up to 60 DC) may be applied to
to triglycerides or ethyl esters. The former includes fish oils/fish-liver dissolve the internal standard.
oils and omega-3-concentrates in triglyceride form. The results are - Reference solution (az). Dissolve 60.0 mg of docosahexaenoic
expressed as tnglycerides or ethyl esters, respectively. acid ethyl ester CRS and 70.0 mg of the internal standard
EPAANDDHA in a 50 mivL solution of butylhydroxytoluene R in
Gas chromatography (2.2.28). Cany out the operations as trimethylpentane Rand dilute to 10.0 mL with the same
rapidly as possible, avoiding exposure to actinic light, oxidising solution. Gentle heating (up to 60 °C) may be applied to
agents, oxidation catalysts (for example, copper and iron) and air. dissolve the internal standard.
The assay is carried out on the methyl esters (after For ethyl ester samples, reference solutions (a 1) and (a 2 ) are
derivatisation of trig]ycerides - see step B below) or ethyl ready for analysis. For analysis of triglycerides, continue with
esters of (all-Z)-eicosa-5,8, 11, 14, 17-pentaenoic acid (EPA; step B in the same manner as for test solutions (a) and (b).
20:5 n-3) and (all-Z)-docosa-4,7,10,13,16,19-hexaenoic acid - Reference solution (b).Dissolve 0.300 g of methyl
(DHA; 22:6 n-3) in the substance to be examined. arachidate R, 0.300 g of methyl behenate R, 0.300 g of
V-A376 Appendix X P 2023

methyl palmitate Rand 0.300 g of methyl stearate Rina Table 2.4.29.-2

50 mg/L solution of butylhydroxytoluene R in Fatty acid methyl ester Theoretical response factor
trimethylpentane Rand dilute to 10.0 mL with the same Methyl palmitate 1.049
solution. Methyl stearate 1.029
- Reference solution (c). Dissolve a mixture containing Methyl arachidate 1.013
55.0 mg of docosahexaenoic acid methyl ester R and 5.0 mg Methyl behenate 1.000
of tetracos-15-enoic acid methyl ester Rina 50 mg/L
solution of butylhydroxytoluene R in trimethylpentane R and
resolution:
dilute to 10.0 mL with the same solution.
- ethyl esters: minimum 1.2 between the peaks due to
Column: methyl tricosanoate and heneicosapentaenoic acid
- material: fused silica; ethyl ester in the chromatogram obtained with test
- dimensions: l = at least 25 m, 0 = 0.25 mm; solution (a);
- stationary phase: macrogol 20 000 R (film thickness - triglycerides: minimum 1.2 between the peaks due to
0.2 µm). docosahexaenoic acid methyl ester and tetracos-15-
Carrier gas hydrogen for chromatography R or helium for enoic acid methyl ester in the chromatogram obtained
chromatography R. with test solution (a) or with reference solution (c);
Flow rate 1 mUmin. in the chromatogram obtained with test solution (a), the
Split ratio 1:200, alternatively splitless with temperature peaks due to methyl tricosanoate and any
control (sample solutions need to be diluted 1/200 with a heneicosapentaenoic acid methyl ester present when
50 mg/L solution of butylhydroxytoluene R in compared with the chromatogram obtained with test
m·methylpentane R before injection). solution (b) are clearly separated.
If necessary, adapt the split ratio and/or sample dilution to Calculate the percentage content of EPA and DHA using the
obtain a symmetry factor of 0.8-1.5 for the peaks due to the following expression and taking into account the assigned
methyl or ethyl esters of eicosapentaenoic acid and value of the reference substances:
docosahexaenoic acid, while at the same time observing that
for test solution (b) any peaks due to the corresponding
esters of linolenic acid (Cl 8:3; n-3), stearidonic acid (Cl 8:4;
n-3), eicosatetraenoic acid (C20:4; n-3), heneicosapentaenoic Rf response factor for EPA and DHA as given by the expression:
acid (C21:5; n-3), and docosapentaenoic acid (C22:5; n-3)
are clearly detectable. If both requirements are unachievable, Ax,3 X mx,r
then the clear detection of the corresponding esters
Ax,r X mx,3
mentioned above for test solution (b) takes precedence.
If necessary, adapt the split ratio and/or sample dilution to
m, mass of the internal standard in test solution (a), in
obtain a symmetry factor of 0.8-1.5 for the peaks due to the milligrams;
components of reference solution (b). mass of the sample to be examined in test solution (a), in
Temperature: milligrams;
mass of the internal standard in reference solution (a 1) (EPA
determination), or in reference solution (a 2 ) (DHA
Split injection Splitless injection determination), in milligrams;
Time Temperature Time Temperature mx,r mass of eicosapentaenoic acid ethyl ester CRS in reference
(min) ( C) 0
(min) (C) solution (a 1) or docosahexaenoic acid ethyl ester CRS in
reference solution (a2 ), in milligrams;
Column 0-2 170 0-2 90 area of the peak due to eicosapentaenoic acid ester or
Ax
2 - 25.7 170 .... 240 2 - 4.7 90 .... 170 docosahexaenoic acid ester in the chromatogram obtained
25.7 - 28 240 4.7 - 28 170 ➔ 240 with test solution (a);
28 - 30 240 Ax,r area of the peak due to eicosapentaenoic acid ester in the
chromatogram obtained with reference solution (a 1 ) or
Injection port 250 90 - 250*
docosahexaenoic acid ester in the chromatogram obtained
Detector 270 270 with reference solution (a 2 );
*90 · C for on-column injection.
A, area of the peak due to the internal standard in the
chromatogram obtained with test solution (a);
Ax,3 area of the peak due to the internal standard in the
Detection Flame ionisation. chromatogram obtained with reference solution (a 1 ) (EPA
Injection 1 ~LL determination) or with reference solution (a2) (DHA
determination);
System suitability: C conversion factor between ethyl ester and triglycerides:
- in the chromatogram obtained with reference solution (b), Ethyl esters: C = 1.00;
multiply the area of the peaks due to methyl palmitate, Triglycerides: C = 0.954 for EPA;
C = 0.957 for DHA.
methyl stearate, methyl arachidate and methyl behenate
by the corresponding response factors in Table 2.4.29.-2;
normalise the corrected areas of the peaks of the fatty TOTAL OMEGA-3 ACIDS
acid methyl esters to a sum of 100 per cent; the From the assay for EPA and DHA, calculate the percentage
normalised area percentage of each fatty acid methyl ester content of the total omega-3 acids using the following
is to be within ± 1.0 percentage units of the expression and identifying the peaks from the
corresponding weight percentage; chromatograms:

EPA+ DHA + An-3 (EPA+ DHA)

AEPA +AoHA
EPA percentage content of EPA;
DHA percentage content of DHA;
2023 Appendix X P V-A377

Table 2.4.32.-1. - Preparation of calibration solutions

Calibration solutions
Cholesterol stock Cholesterol working Internal standard
Cholesterol concentration Cholesterol concentration solution (mL) solution (mL) working solution (mL)
(mg/mL) (mg/g)(a)

0.1 20.0 4.0 1.0

0.05 10.0 2.0 1.0
0.015 3.0 0.60 1.0
0.005 1.0 2.0 1.0
0.001 0.2 0.40 1.0

(a)Based on a sample weight of0.100 g of the substance to be examined.

An-J sum of the areas of the peaks due to linolenic acid (C18:3; middle layer of the oil for weighing). Add 1.0 mL of the
n-3), stearidonic acid (C18:4; n-3), eicosatetraenoic acid
(C20:4; n-3), heneicosapentaenoic acid (C21:5; n-3), and
internal standard working solution. Evaporate the solvent on
docosapentaenoic acid (C22:5; n-3) in the chromatogram a heating block at 50 °C under a gentle stream of nitrogen R.
obtained with test solution (b); Add 0.5 mL of a 50 per cent m/m solution of potassium
AEPA area of the peak due to EPA ester in the chromatogram hydroxide R and 3.0 mL of ethanol (96 per cent) R. Fill the
obtained with test solution (b); tube with nitrogen R, cap and homogenise. Heat on the
AnHA area of the peak due to DHA ester in the chromatogram
obtained with test solution (b). heating block at 100 °C for 1 h. Cool for about 10 min, add
6.0 mL of distilled water R and homogenise. Condition a
20 mL solid phase extraction (SPE) column containing 1 g
2. Total Cholesterol in Oils Rich in Omega-3-Acids of end-capped octadecylsilyl silica gel for chromatography R
(Ph. Bur. method 2.4.32) (particles with a diameter of 55 µm and a pore size of 7 nm)
This method may be used for the quantitative determination of the with 5 mL of a 50 per cent V/V solution of ethanol
sum of free and esterified cholesterol in products of fish oils rich in (96 per cent) R in distilled water R. Transfer 5.0 mL of the
omega-3 acids ( as ethyl esters or triglycerides). saponified sample to the SPE column ensuring that the
Gas chromatography (2.2.28). column does not dry out. Wash the column with 5.0 mL of a
Internal standard stock solution Dissolve 0 .15 g of 50 per cent V/V solution of ethanol (96 per cent) R in distilled
(Sa)-cholestane R in heptane Rand dilute to 50.0 mL with the water R. Elute the column using 20.0 mL of a
same solvent. The solution may be stored in a deep-freeze for 10 per cent V/V solution of ethyl acetate R in heptane R.
up to 6 months. Collect the eluate and use it as the test solution.
Internal standard working solution. Prepare the Column:
solution immediately before use Dilute 1.0 mL of the - material: fused silica;
internal standard stock solution to 10.0 mL with heptane R. - size: l = 15 m, 0 = 0.25 mm;
- stationary phase: phenyl(5)methyl(95)polysiloxane R (film
Cholesterol stock solution Dissolve 50.0 mg of
thickness 0.25 µm).
cholesterol R in heptane Rand dilute to 100.0 mL with the
same solvent. The solution may be stored in a deep-freeze for Carrier gas helium for chromatography R.
up to 6 months. Pressure 48 kPa (corresponding to a flow rate of about
Cholesterol working solution. Prepare the solution 0.6 mUmin at 200 °C and about 0.4 mUmin at 330 °C).
immediately before use Dilute 1.0 mL of the cholesterol Split ratio 1:5.
stock solution to 10.0 mL with heptane R. Temperature:
Cholesterol and cr-tocopherol stock solution Dissolve
50.0 mg of cholesterol Rand 50.0 mg of a-tocopherol R in Time Temperature
heptane Rand dilute to 100.0 mL with the same solvent. (min) CC)
The solution may be stored at room temperature for up to Column 0- 1 200
3 months. 1 - 7.5 200 __. 330
7.5 - 10 330
Reference solution. Prepare the solution on the day of
Injection port 250
use Dilute 1.0 mL of the cholesterol and cr-tocopherol
stock solution to 100.0 mL with heptane R. Detector 340

Calibration solutions See Table 2.4.32.-1. Prepare the

solutions on the day of use. Dilute each solution to 20.0 mL Prior to analysis, heat the column at 340 °C for at least
with a 10 per cent V/V solution of ethyl acetate R in 30 min using the indicated pressure.
heptane R. Detection Flame ionisation.
For high levels of cholesterol (3.0-20.0 mg/g), use all Injection 1 µL.
5 calibration solutions. Retention time (5cr)-cholestane = about 7.5 min;
For low levels of cholesterol (0.2-3.0 mg/g), use the following cholesterol = about 9 min.
calibration solutions: 0.2 mg/g, 1.0 mg/g and 3.0 mg/g. Plot the calibration curve. The x-axis represents the nominal
Test solution Weigh 0 .100 g of the substance to be concentration of cholesterol in milligrams per gram of the
examined into a 15 mL quartz tube (for fish oils and cod- substance to be examined in each calibration solution.
liver oils, shake the oil to be examined vigorously in a The y-axis represents the ratio of the area of the peak due to
suitable container, allow to stand for 10-15 min and while cholesterol to the area of the peak due to (5cx)-cholestane in
maintaining the container upright, remove a sample from the
V-A378 Appendix X Q 2023

the chromatogram obtained with each calibration solution. volume of about 1 mL. Depending on the unsaponifiable
Calculate the slope (S) and the intercept with the y-axis (Y). content of the oil, adapt the final concentration of the
System suitability: solution to 25-50 mg/mL.
- resolution: minimum 1.5 between the peaks due to Test solution (b) Treat 5.00 g of rapeseed oil Ras
cholesterol and C(-tocopherol in the chromatogram prescribed for the substance to be examined, beginning at the
obtained with the reference solution; words "Add 50 mL of 2 M alcoholic potassium hydroxide R".
- the coefficient of determination (r2) of the calibration Test solution (c) Treat 5.00 g of sunflower oil R as
curve is not less than 0.995. prescribed for the substance to be examined, beginning at the
Calculate the content of total cholesterol, expressed in words "Add 50 mL of 2 M alcoholic potassium hydroxide R".
milligrams of cholesterol per gram of substance to be Reference solution Dissolve 25 mg of cholesterol R and
examined, using the following expression: 10 mg of betulin R in 1 mL of methykne chwride R.
&__y Use a separate plate for each test solution. Apply as a band
~s x0.100 of 10 mm, at 20 mm from the base and 10 mm from the left
m1 X edge, 10 µL of the reference solution and as bands of
150 mm, at 20 mm from the base, 0.5 mL of test
area of the peak due to cholesterol in the chromatogram
obtained with the test solution;
solutions (a), (b) or (c). Develop over a path of 17 cm using
area of the peak due to (5<>)-cholestane in the chromatogram a mixture of 35 volumes of ether R and 65 volumes of
obtained with the test solution; hexane R. Dry the plates in a current of nitrogen R. Spray the
mass of the substance to be examined in the test solution, in plates with a 2 g/L solution of dichk!rofiuorescein R in
grams;
y anhydrous ethanol R and examine in ultraviolet light at
intercept of the calibration curve with the y-axis;
s slope of the calibration curve, in grams per milligram. 254 nm. The chromatogram obtained with the reference
solution shows bands due to cholesterol and betulin.
The chromatograms obtained with the test solutions show
bands with similar Rp values due to sterols. From each of the
chromatograms, remove an area of coating corresponding to
Q. Sterols in Fatty Oils the area occupied by the sterol bands and additionally the
area of the zones 2-3 mm above and below the visible zones
(Ph. Bur. method 2.4.23) corresponding to the reference solution. Place separately
When the monograph does not specify the method to be used, in three 50 mL flasks. To each flask add 15 mL of methylene
method A is applied. Any change from method A to method B chloride Rand heat under reflux with stirring, for 15 min.
must be validated. Filter each solution through a sintered-glass filter (40) (2. 1. 2)
or suitable filter paper and wash each filter with 3 quantities,
METHOD A
each of 15 mL, of methylene chloride R. Place the combined
Separation of the sterol fraction (TLC)
filtrate and washings from each filter separately in 3 flasks,
Prepare the unsaponifiable matter and then isolate the sterol
evaporate under a stream of nitrogen R to 5-10 mL. Transfer
fraction of the fatty oil by thin-layer chromatography
to a small test tube and evaporate to dryness under a stream
(2.2.27), using a TLC silica gel plate R with a 0.2 mm to
of nitrogen R.
0.5 mm layer.
Determination of the sterols (GC)
Test solution (a) In a 150 mL flask fitted with a reflux
Gas chromatography (2.2.28). Carry out the operations
condenser, place a volume of a 2 g/L solution of betulin R in
protected from humidity and prepare the solutions immediately
methylene chloride R containing betulin corresponding to
before use.
about 10 per cent of the sterol content of the sample used for
the determination (e.g. in the case of olive oil add 500 µL, in Test solution To the sterols separated from the substance
the case of other vegetable oils add 1500 µL of the betulin to be examined by thin-layer chromatography add a freshly
solution). If the monograph requires the percentage content prepared mixture of 0.04 mL of chlorotrimethylsilane R,
of the individual sterols in the sterol fraction, the addition of 0.1 mL of hexamethyldisilazane Rand 0.5 mL of anhydrous
betulin may be omitted. Evaporate to dryness under a pyridine R. Allow to stand for at least 5 min and use the
current of nitrogen R. Add 5.00 g (m) of the substance to be liquid phase.
examined. Add 50 mL of 2 M alcoholic potassium hydroxide R Reference solution ( a) To 9 parts of the sterols separated
and heat on a water-bath for 1 h, swirling frequently. Cool to from rapeseed oil R by thin-layer chromatography add 1 part
a temperature below 25 °C and transfer the contents of the of choksterol R. To the mixture add a freshly prepared
flask to a separating funnel with 100 mL of water R. Shake mixture of 0.04 mL of chlorotrimethylsilane R, 0.1 mL of
the liquid carefully with 3 quantities, each of 100 mL, of hexamethyldisilazane Rand 0.5 mL of anhydrous pyridine R.
peroxide-free ether R. Combine the ether layers in another Allow to stand for at least 5 min and use the liquid phase.
separating funnel containing 40 mL of water R, shake gently Reference solution (b) To the sterols separated from
for a few minutes, allow to separate and reject the aqueous sunflower oil R by thin-layer chromatography add a freshly
phase. Wash the ether phase with several quantities, each of prepared mixture of 0.04 mL of chlorotrimethylsilane R,
40 mL, of water R, until the aqueous phase is no longer 0.1 mL of hexamethyldisilazane Rand 0.5 mL of anhydrous
alkaline to phenolphthalein. Transfer the ether phase to a pyridine R. Allow to stand for at least 5 min and use the
tared flask, washing the separating funnel with peroxide-free liquid phase.
ether R. Distil off the ether with suitable precautions and add Column:
6 mL of acetone R to the residue. Carefully remove the - material: fused silica;
solvent in a current of nitrogen R. Dry to constant mass at - size: l = 20-30 m, 0 = 0.25-0.32 mm;
100-105 °C. Allow to cool in a desiccator and weigh.
Transfer the residue to a small test tube with methylene
chloride R. Evaporate under a stream of nitrogen R to a
2023 Appendix X Q V-A379

- stationary phase: phenyl(5)methyl(95)polysiloxane R or Ax m' x 100

cyanopropyl(7)phenyl(7)methyl(86)pol.ysiloxane R (film A'xm
thickness 0.25 µm).
A area of the peak due to the component to be determined;
Carrier gas hydrogen for chromatography R or helium for AI area of the peak due to betulin;
chromatography R. m mass of the sample of the substance to be examined, in grams;
rn' mass of betulin R added, in milligrams.
Linear velocity 30-50 emfs (hydrogen) or 20-35 emfs
(helium).
METHODB
Split ratio 1:50 (hydrogen) or 1:100 (helium). Preparation of the unsaponifiable matter
Temperature: Prepare the unsaponifiable matter according to the method
- column: 260 °C; stated in the test for unsaponifiable matter of the monograph
- injection port: 280 °C; on the substance to be examined. Failing this, prepare the
- detector: 290 °C. unsaponifiable matter according to the method described in
Detection Flame ionisation. chapter 2.5. 7. Unsaponifiable matter. After the final
Injection 1 µL. neutralisation step, evaporate the ethanol, then add 6 mL of
Identification of peaks The chromatogram obtained with acetone R and evaporate the solvent. Dry the residue at
100-105 °C. It is not necessary to dry to constant mass.
reference solution (a) shows 4 principal peaks corresponding
to cholesterol, brassicasterol, campesterol and ~-sitosterol and Simultaneously prepare under the same conditions the
the chromatogram obtained with reference solution (b) shows unsaponifiable matter of sunflower oil R. This will in
4 principal peaks corresponding to campesterol, stigmasterol, particular serve to locate the sterol fraction to be collected.
~-sitosterol and Li 7-stigmastenol. The relative retentions of Separation of the sterol fraction (LC)
the sterols with reference to ~-sitosterol (main peak) are Liquid chromatography (2.2.29).
given in Table 2.4.23.-1. Test solution Take up the residue with 3 quantities, each
of 4 mL, of the solvent used during the preparation of the
Table 2.4.23.-1. - Relative retentions of sterols with reference to
unsaponifiable matter (generally ether R or light petroleum R)
/3-sitosterol for 2 different columns
and transfer to a 15 mL tube. Evaporate to dryness under a
Cyanopropyl Phenyl(S)methyl current of nitrogen R. Dissolve the residue in a volume of
(7) (phenyl) (95)polysiloxane
(7) (methyl)(86) mobile phase sufficient to obtain a solution with an
polysiloxane approximate concentration of 40 mg/mL. Add a few drops of
Cholesterol 0.64 0.63 2-propanol Rl to improve the solubility (3 drops are normally
Brassicasterol 0.70 0.71 sufficient to ensure complete solubilisation). Filter through a
24-Methylenecholesterol 0.79 0.80 membrane filter (nominal pore size 0.45 µm).
Campesterol 0.82 0.81 Reference solution Proceed as described for the test
Campestanol 0.83 0.82 solution with the unsaponifiable matter obtained with
Stigmasterol 0.87 0.87 sunflower oil R.
I'.7-Campesterol 0.93 0.92 Precolumn:
i'.5,23-Stigmastadienol 0.95 0.95 - size: l= 5 mm, 0 = 4.6 mm;
Clerosterol 0.96 0.96 - stationary phase: silica gel for chromatography R (5 µm) with
~-Sitosterol I I a pore size of 6 nm.
Sitostanol I.OJ 1.02 Column:
i'.5-Avenasterol 1.03 1.03
- size: l = 0.25 m, 0 = 4.6 mm;
i'.5,24-Stigmastadienol 1.09 I.OS - stationary phase: silica gel for chromatography R (5 µm) with
I'.7-Stigmastenol r1> 1.13 1.12
a pore size of 6 nm.
I'.7-Avenasterol 1.18 1.16
Mobile phase 2-propanol Rl, hexane R (1:99 V!V).
Betulin 1.4 1.4
Flow rate 1 mUmin.
(I) This sterol may also be referred to as i'.7-stigmasterol in literature.
Detection Spectrophotometer at 210 nm.
The peak due to the internal standard (betulin) must be Injection 50 µL.
clearly separated from the peaks due to the sterols to be Identification of the peaks due to sterols The sterol
determined. fraction elutes at the end of the chromatogram. Locate the
For the chromatogram obtained with the test solution, fraction to be collected using the chromatogram obtained
identify the peaks and calculate the percentage content of with the reference solution, which shows 2 principal peaks
each sterol in the sterol fraction of the substance to be eluting approximately between 23 min and 32 min. Collect
examined using the following expression: the fraction at the detector outlet in a 15 mL tube with a
screw cap. Evaporate the solvent under a current of
A nitrogen R.
S X 100
Detennination of the sterols (GC)
A area of the peak due to the component to be determined;
Gas chromatography (2.2.28).
s sum of the areas of the peaks due to the components indicated Test solution Dissolve the residue of the sterol fraction
in Table 2.4.23.-1; disregard the peak due to betulin. obtained with the test solution in the previous LC step in
0.2 mL of anhydrous pyridine Rand 0.2 mL of a mixture of
If required in the monograph, calculate the content of each 1 volume of chlorotrimethylsilane R and 99 volumes of N, O-bis
sterol in milligrams per 100 grams of the substance to be (trimethylsilyl)trifiuoroacetamide R. Stopper the tube tightly
examined using the following expression: and heat at 80 °C for 20 min. Allow to cool and use the
liquid phase.
V-A380 Appendix XIA 2023

Reference solution Dissolve the residue of the sterol

fraction obtained with the reference solution in the previous
LC step in 0.2 mL of anhydrous pyridine Rand 0.2 mL of a
Appendix XI
mixture of 1 volume of chlorotrimethylsilane R and 99 volumes A. Total Solids
of N,O-bis(trimethylsilyl)trifiuoroacetamide R. Stopper the tube
tightly and heat at 80 °C for 20 min. Allow to cool and use (No Ph. Eur. method)
the liquid phase. The term 'total solids' is applied to the residue obtained
A standard of cholesterol (cholesterol R) may also be used, when the prescribed amount of the preparation is dried to
alone or as a mixture with the sterol fraction of sunflower oil. constant weight under the conditions specified below.
Proceed with derivatisation as described for the test solution. APPARATUS
Column: A shallow, flat-bottomed, flanged dish, about 75 mm in
- material: fused silica; diameter and about 25 mm deep, made of nickel or other
- size: l =30 m, 0 = 0.25 mm; suitable metal of high heat conductivity and low specific heat
- stationary phase: phenyl(5)methyl(95)polysiloxane R (film and which is not affected by boiling water. Suitable dishes
thickness 0.25 µm). are described in British Standard 1742: 1951 (Methods for
Carrier gas helium for chromatography R. the chemical analysis of condensed milk).
Flow rate 2.6 mlJmin. METHOD

Split ratio 1:25. Place the quantity stated in the monograph in a tared dish,
evaporate at as low a temperature as possible until the
Temperature: ethanol is removed and heat on a water-bath until the residue
is apparently dry. Transfer to an oven operating without a
Time Temperature
(min) ('C)
fan and dry to constant weight at 105° unless otherwise
stated in the monograph. It may be necessary, for residues of
Column 0 - 38 260
38 - 44 260 ➔ 290
a hygroscopic nature, to use a dish provided with a well-
44 - 49 290 fitting cover and to cool in a desiccator.
Injection port 290
Detector 290

Detection Flame ionisation. 81. Ethanol-soluble Extractive

Injection 1-3 µL (depending on the expected amount of (No Ph. Eur. method)
sterols in the substance to be examined). Macerate 5 g of the air-dried drug, coarsely powdered, with
Identification of peaks Use the chromatogram obtained 100 mL of ethanol of the specified strength in a closed flask
with the reference solution to identify the peaks due to for 24 hours, shaking frequently during the first 6 hours and
campesterol, stigmasterol, B-sitosterol and ~7-stigmastenol. then allowing to stand for 18 hours. Filter rapidly, taking
Identify the peaks due to the sterols in the chromatogram precautions against loss of ethanol, evaporate 20 mL of the
obtained with the test solution using the chromatogram filtrate to dryness in a tared, flat-bottomed, shallow dish and
obtained with the reference solution and the relative dry at 105° to constant weight. Calculate the percentage of
retentions with reference to B-sitosterol (main peak) given in ethanol-soluble extractive with reference to the air-dried
Table 2.4.23.-1. drug.
System suitability Reference solution:
- resolution: minimum 4.0 between the peaks due to
campesterol and stigmasterol.
Calculate the percentage content of each sterol in the sterol 82. Water-soluble Extractive
fraction of the substance to be examined using the following (No Ph. Eur. method)
expression: To 5 g of the powdered drug (710) add 200 mL of boiling
A water. Allow to stand for 10 minutes, shaking occasionally.
S X 100 Allow to cool, dilute to 200 mL with water and filter.
Evaporate 20 mL of the filtrate to dryness on a water-bath.
A area of the peak due to the component to be determined;
S sum of the areas of the peaks due to the components indicated
Dry the residue in an oven at 100° to 105° for 15 minutes.
in Table 2.4.23.-1, except betulin.

C. Swelling Index
(Ph. Eur. method 2.8.4)
The swelling index is the volume in millilitres occupied by
1 gram of a drug, including any adhering mucilage, after it
has swollen in an aqueous liquid for 4 h.
In a 25 mL ground-glass stoppered cylinder graduated over a
height of 125 ± 5 mm in 0.5 mL divisions, place 1.0 g of
the drug, whole or of the degree of comminution prescribed
in the monograph. Unless otherwise prescribed, moisten the
drug with 1.0 mL of alcohol R, add 25 mL of water Rand
close the cylinder. Shake vigorously every 10 min for 1 h.
2023 Appendix XIE V-A381

Allow to stand for 3 h. At 90 min after the beginning of the

test, release any large volumes of liquid retained in the layer
of the drug and any particles of the drug floating at the
surface of the liquid by rotating the cylinder about a vertical
axis. Measure the volume occupied by the drug, including
any adhering mucilage. Carry out 3 tests at the same time.
The swelling index is given by the mean of the 3 tests.

D. Foreign Matter
(Ph. Eur. method 2. 8. 2)
Herbal drugs should be free from moulds, insects and other
animal contamination.
Foreign matter is material consisting of any or all of the
following:
I) foreign organs: matter coming from the source plant but
not defined as the herbal drug;
2) foreign elements: matter not coming from the source plant
and of either vegetable or mineral origin.
DRIED PLANTS
Sampling and sample preparation
Apply general chapter 2.8.20. Herbal drugs: sampling and
sample preparation.
Determination of foreign matter
Weigh 100-500 g of the sample, or the minimum quantity
prescribed in the individual monograph, and spread it out in
a thin layer. Examine for foreign matter by inspection with
the unaided eye or by use of a lens (6 x ). Separate foreign
matter, weigh it and calculate the percentage present.
FRESH PLANTS
Where general chapter 2.8.20. Herbal drugs: sampling and Figure 2.8.12.-1. -Apparatus for the determination of essential
sample preparation cannot be applied, use one of the following oils in herbal drugs
methods, as appropriate: use method A when the test can be Dimensions in millimetres
carried out on the whole batch; use method B when the test
cannot be carried on the whole batch.
METHOD A
Sampling and sample preparation E. Essential Oils in Herbal Drugs
Carry out the test on the whole batch.
(Ph. Eur. method 2. 8.12)
Determination of foreign matter
Spread the batch out in a thin layer and examine for foreign PRINCWLE
matter by inspection with the unaided eye or by use of a lens The determination of essential oils in herbal drugs is carried
(6 x ). Separate foreign matter, weigh it and calculate the out by steam distillation in a special apparatus under the
percentage present. conditions described below. The distillate is collected in the
graduated tube using the solvent prescribed in the
METHODB monograph (usually xylene R, trimethylpentane R or 1,2,4-
Sampling and sample preparation trimethylbenzene R) to take up the essential oil; the aqueous
If it is not possible to inspect the whole batch, proceed as phase is automatically returned to the distillation flask.
follows.
EQUWMENT
Bulk sample Prepare the bulk sample as described in The equipment typically consists of:
general chapter 2.8.20. Herbal drugs: sampling and sample
- a suitable round-bottomed flask with a short, ground-glass
preparation. neck that has an internal diameter of about 29 mm at the
Test sample Use the bulk sample or, where the bulk wide end;
sample is greater than 1 kg, reduce it to a mass of - a condenser assembly (see Figure 2.8.12.-1) that closely
500-1000 g by a suitable method which retains the fits the flask, the different parts being fused into 1 piece
representative nature of the bulk sample. and made of glass with a low coefficient of thermal
Determination of foreign matter expansion:
Use the sample, or the minimum quantity prescribed in the - a vented stopper (K') with an opening about 1 mm in
individual monograph, and spread it out in a thin layer. diameter, and a tube (KJ, the wide end of which is
Examine for foreign matter by inspection with the unaided made of ground-glass and has an internal diameter of
eye or by use of a lens (6 x ). Separate foreign matter, weigh 10mm;
it and calculate the percentage present. - a pear-shaped swelling Cl) with a capacity of 3 mL;
V-A382 Appendix XI F 2023

- a tube (JL) graduated in 0.01 mL; Recovery of the mixture of solvent and essential oil
- a bulb-shaped swelling (L) with a capacity of about When the essential oil is to be used for other analytical
2mL; purposes, the water-free mixture of solvent and essential oil
- a 3-way tap (M); may be recovered as follows: remove the stopper (K') and
- a junction (B) situated at a level 20 mm higher than introduce 0.1 mL of a 1 g/L solution of sodium
the uppermost graduation of tube JL; fiuoresceinate Rand 0.5 mL of water R. Run the mixture of
- a suitable heating device, allowing fine temperature solvent and essential oil into the bulb-shaped swelling (L) by
control; opening tap M, allow to stand for 5 min and lower the level
- a vertical support with a horizontal ring covered with of the mixture slowly until it just reaches the level of tap M.
insulating material. Open tap M clockwise so that the water flows out of the
connecting tube (BM). Wash the tube with acetone R
PROCEDURE
introduced through the filling funnel (N). Tum tap M anti-
Use a thoroughly cleaned apparatus. Carry out the
clockwise in order to recover the mixture of solvent and
determination according to the nature of the herbal drug to
essential oil in an appropriate flask.
be examined. Place the prescribed volume of distillation
liquid in the flask, add a few pieces of porous porcelain and
attach the condenser assembly. Introduce water R through
the filling funnel (N) until it reaches level B. Remove the
stopper (K') and introduce the prescribed quantity of the F. Continuous Extraction of Drugs
solvent indicated in the monograph using a pipette with its (No Ph. Bur. method)
tip placed at the bottom of tube K. Re-insert the
stopper (K') and ensure that the vent is unobstructed. Heat Where the process of maceration or percolation is specified in a
the liquid in the flask to boiling and adjust the distillation monograph, carry out the following procedures with any
rate to 2-3 mIJmin, unless otherwise prescribed. modification indicated in the monograph.
MACERATION
Detennination of the rate of distillation
During distillation, determine the rate of distillation by Place the solid materials with the whole of the menstruum in
lowering the level of the water by means of tap M until the a closed vessel and allow to stand for 7 days, shaking
meniscus reaches the level of the lower mark (a) (see Figure occasionally. Strain, press the mare and mix the liquids
2.8.12.-2). Close tap Mand measure the time taken for the obtained. Clarify by subsidence or filtration.
liquid to reach the upper mark (b). Modify the heat to obtain PERCOIATION
the target distillation rate. If the distillation rate is still not Moisten the solid materials with a sufficient quantity of the
within the prescribed range, repeat the operation. If the menstruum, allow to stand for 4 hours in a well-closed
distillation conditions are not changed, it is sufficient to vessel, pack in a percolator and add sufficient of the
determine the distillation rate at regular intervals rather than menstruum to saturate the materials. When the liquid begins
before each test. to drop from the percolator, close the outlet, add sufficient of
the menstruum to leave a layer above the drug and allow to
macerate for 24 hours. Allow percolation to proceed slowly
Mark b
until the percolate measures about three-quarters of the
-t-- required volume. Press the mare, mix the expressed liquid
with the percolate and add sufficient of the menstruum to
"'3ml
produce the required volume. Clarify by subsidence or
t filtration.
Mark a
Continuous extraction of a drug for the purpose of an assay
consists of percolating the drug with the solvent stated in the
monograph at a temperature approximately that of the
boiling point of the solvent.
Figure 2.8.12.-2
The apparatus described below, or any similar apparatus,
Detennination of the solvent volume after blank may be used, provided that it permits the uniform
distillation percolation of the drug and the regular flow of the vapour of
If using xylene R or trimethylpentane R, distil for 30 min. the solvent around the percolator.
Ensure that tubes BM and JM are connected via tap M The apparatus is shown in Fig. llF-1. A is an outer tube of
during distillation. Stop heating and wait at least 10 min stout glass; the wider part is about 18 cm long and has an
before reading the volume of solvent in the graduated tube. internal diameter of 4.8 to 5 cm; the lower end C is about
If using 1,2,4-trimethylbenzene R, the 30 min blank distillation 5 cm long and has an external diameter of about 1.6 cm. B
step is not necessary. Stop heating after adjusting the is a straight glass tube open at both ends, about 9 cm long
distillation rate and wait at least 10 min before reading the and with an external diameter of about 3.8 cm; over its
volume of solvent in the graduated tube. lower, flanged end is tied firmly a piece of calico or other
suitable material. D is a glass coil which supports the margin
Detennination of the essential oil in the herbal drug of the tube B and prevents it from resting in contact with the
Introduce the prescribed quantity of the herbal drug into the outer tube A. The lower end C of the outer tube A is fitted
flask and continue distillation as described above for the time by a cork or ground-glass joint to the distillation flask E, in
and at the rate prescribed. Stop heating, read the volume of which a suitable quantity of the solvent has been placed.
liquid collected in the graduated tube after 10 min and The drug to be extracted, previously moistened with the
subtract the volume of the solvent previously noted. solvent or subjected to any preliminary treatment required, is
The difference represents the quantity of essential oil in the introduced into the inner tube B, which is supported so that
sample. Calculate the result in millilitres per kilogram of the percolate drops into the outer tube. A pad of absorbent
herbal drug.
2023 Appendix XI J V-A383

cotton G is placed on the top of the drug, the inner tube is

lowered into position and the outer tube connected by means
H. Stomata
of a suitable cork or ground-glass joint with the tube of a (Ph. Bur. method 2.8.3)
reflux condenser F. The flask is heated and the extraction STOMATA
continued as directed. There are several types of stomata (see Figure 2.8.3.-1),
distinguished by the form and arrangement of the
surrounding cells:
(1) The anomocytic (irregular-celled) type: the stoma is
surrounded by a varying number of cells in no way differing
from those of the epidermis generally,
(2) The anisocytic (unequal-celled) type: the stoma is usually
surrounded by 3 subsidiary cells, of which one is markedly
smaller than the others,
(3) The diacytic (cross-celled) type: the stoma is accompanied
by 2 subsidiary cells, whose common wall is at right angles to
the guard cells,
( 4) The paracytic (parallel-celled) type: the stoma has on each
side one or more subsidiary cells parallel to the long axis of
the pore and guard cells.
STOMATAL INDEX
l00x S
Stomata! Index = --S-
E+
s the number of stomata in a given area of leaf,
E the number of epidermal cells (including trichomes) in the same
area of leaf.

For each sample of leaf, make not fewer than

10 determinations and calculate the mean.
Fig. llF-1 Apparatus for the Continuous Extraction of Drugs

G. Complete Extraction of Alkaloids 2

(No Ph. Bur. method)
Complete extraction is indicated by the following tests.
EXTRACTION WITH AN AQUEOUS OR ALCOHOLIC LIQUID
After extracting at least three times with the liquid, add to
0 .1 to 0 .2 mL of the next portion, after acidifying with
2M hydrochloric acid if necessary, 0.05 mL of potassium
tetrawdomercurate solution or, for solanaceous alkaloids,
0.05 mL of potassium iodobismuthate solutwn RJ. 3 4
No precipitate or turbidity is produced.
EXTRACTION WITH AN IMMISCIBLE SOLVENT
After extracting at least three times with the solvent, add to
1 to 2 mL of the next portion 1 to 2 mL of 0 .1 M hydrochl,oric
acid, remove the organic solvent by evaporation, transfer the
aqueous residue to a test tube and add 0.05 mL of potassium
Figure 2.8.3.-1
tetrawdomercurate solution or, for solanaceous alkaloids,
0.05 mL of potassium iodobismuthate solutwn RJ or, for
emetine, 0.05 mL of iodinated potassium iodide solutwn.
Not more than a very faint opalescence is produced.
CONTINUOUS EXTRACTION J. Ash
After percolating for at least 2 hours, collect 1 to 2 mL of the Use Method I unless otherwise directed in the monograph.
effluent and carry out the procedure described under
'Extraction with an aqueous or alcoholic liquid' or Method I
'Extraction with an immiscible solvent', as appropriate. (No Ph. Bur. method)
FOR HERBAL DRUGS
Incinerate 2 to 3 g of the ground drug in a tared platinum or
silica dish at a temperature not exceeding 450° until free
from carbon, cool and weigh. If a carbon-free ash cannot be
obtained in this way, exhaust the charred mass with hot
V-A384 Appendix XI K 2023

water, collect the residue on an ashless filter paper, incinerate protect the commodity from deterioration during storage and
the residue and filter paper, add the filtrate, evaporate to transport. Pesticide residues can be present and are
dryness and ignite at a temperature not exceeding 450°. controlled in herbal drugs and herbal drug preparations.
Calculate the percentage of ash with reference to the air- Limits
dried drug. Unless otherwise indicated in the monograph, the herbal
FOR OTHER SUBSTANCES drug to be examined at least complies with the limits
Carry out the above method using 1 g, unless otherwise indicated in Table 2.8.13.-1. The limits applying to
stated. Calculate the percentage of ash. pesticides that are not listed in Table 2.8.13.-1 and whose
presence is suspected for any reason comply with the limits
Method II (levels) cross-referred to by Regulation (EC) No. 396/2005,
(Ph. Bur. method 2. 4.16) including annexes and successive updates. Limits for
Heat a silica or platinum crucible to redness for 30 min, pesticides that are not listed in Table 2.8.13.-1 or in
allow to cool in a desiccator and weigh. Unless otherwise European Union texts are calculated using the following
prescribed, evenly distribute 1.00 g of the substance or the expression:
powdered herbal drug to be examined in the crucible. Dry at
100 °C to 105 °C for 1 h and ignite to constant mass in a ADixM
muffle furnace at 600 °C ± 25 °C, allowing the crucible to MDDHnx 100
cool in a desiccator after each ignition. Flames should not be
produced at any time during the procedure. If after ADI acceptable daily intake, as published by FAO-WHO, in
milligrams per kilogram of body mass;
prolonged ignition the ash still contains black particles, take body mass in kilograms (60 kg);
up with hot water, filter through an ashless filter paper and daily dose of the herbal drug, in kilograms.
ignite the residue and the filter paper. Combine the filtrate
with the ash, carefully evaporate to dryness and ignite to The limits for pesticides in herbal drug preparations are
constant mass. calculated using the following expressions:

If DER :S 10: MRLHDxDER

ADixM
K. Acid-insoluble Ash If DER> 10: MDDHP X 100

Use Method I unless otherwise directed in the monograph.

maximum residue limit of the pesticide in the herbal drug
Method I as given in Table 2.8.13.-1 or in EU texts or calculated
using the expression mentioned above;
(No Ph. Bur. method)
DER drug/extract ratio, i.e. the ratio between the quantity of
Boil the ash for 5 minutes with 25 ml of 2M hydrochloric acid, herbal drug used in the manufacture of a herbal drug
collect the insoluble matter in a sintered-glass crucible or on preparation and the quantity of herbal drug preparation
an ashless filter paper, wash with hot water and ignite. obtained;
daily dose of the herbal drug preparation, in kilograms.
Calculate the percentage of acid-insoluble ash with reference
to the air-dried drug.
The competent authority may grant total or partial
Method II exemption from the test when the complete history (nature
(Ph. Bur. method 2.8.1) and quantity of the pesticides used, date of each treatment
during cultivation and after the harvest) of the treatment of
Ash insoluble in hydrochloric acid is the residue obtained
the batch is known and can be checked precisely according
after extracting the sulfated or total ash with hydrochloric
to good agricultural and collection practice (GACP).
acid, calculated with reference to 100 g of drug.
To the crucible containing the residue from the Table 2.8.13.-1.
determination of sulfated or total ash, add 15 mL of water R Substance Limit
and 10 mL of hydrochloric acid R, cover with a watch-glass, (mg/kg)
boil the mixture gently for 10 min and allow to cool. Filter Acephate 0.1
through an ashless filter, wash the residue with hot water R Alachlor 0.05
until the filtrate is neutral, dry, ignite to dull redness, allow Aldrin and dieldrin (sum of) 0.05
to cool in a desiccator and weigh. Reheat until the difference Azinphos-ethyl 0.1
between 2 consecutive weighings is not more than 1 mg. Azinphos-methyl 1
Bromophos-ethyl 0.05
Bromophos-methyl 0.05
Brompropylate 3
L. Pesticide Residues Chlordane (sum of cis-, tram - and oxychlordane) 0.05
Chlorfenvinphos 0.5
(Ph. Bur. method 2.8.13)
Chlorpyriphos-ethyl 0.2
Definition Chlorpyriphos-methyl 0.1
For the purposes of the Pharmacopoeia, a pesticide is any Chlorthal-dimethyl 0.01
substance or mixture of substances intended for preventing, Cyfluthrin (sum of) 0.1
destroying or controlling any pest, unwanted species of plants 1'-Cyhalothrin 1
or animals causing harm during or otherwise interfering with Cypermethrin and isomers (sum of)
the production, processing, storage, transport or marketing of DDT (sum of o,p'-DDE, p,p'-DDE, o,p'-DDT, p,p'-DDT, o,
herbal drugs. The item includes substances intended for use p'-TDE and p,p'-TDE)
as growth-regulators, defoliants or desiccants and any Deltamethrin 0.5
substance applied to crops, either before or after harvest, to Diazinon 0.5
2023 Appendix XI M V-A385

Substance Limit revisions of this document). In particular, they satisfy the

(mg/kg)
following criteria:
Dichlofluanid 0.1 - the chosen method, especially the purification steps, is
Dichlorvos suitable for the combination pesticide residue/substance to
Dicofol 0.5 be examined, and not susceptible to interference from
Dimethoate and omethoate (sum of) 0.1 co-extractives;
Dithiocarbamates (expressed as CS2) 2 - natural occurrence of some constituents is considered in
Endosulfan (sum of isomers and endosulfan sulfate) 3 the interpretation of results (e.g. disulfide from crucifers
Endrin 0.05 or bromide from seaweed);
Ethion 2 - the concentration of test and reference solutions and the
Etrimphos 0.05 setting of the apparatus are such that the responses used
Fenchlorophos (sum of fenchlorophos and fenchlorophos- 0.1 for quantification of the pesticide residues are within the
oxon)
dynamic range of the detector; test solutions containing
Fenitrothion 0.5
pesticide residues at a level outside the dynamic range,
Fenpropathrin 0.03
may be diluted within the calibration range, provided that
Fensulfothion (sum of fensulfothion, fensulfothion-oxon, 0.05
fensulfothion-oxonsulfon and fensulfothion-sulfon)
the concentration of the matrix in the solution is adjusted
Fenthion (sum of fenthion, fenthion-oxon, fenthion-oxon- 0.05
in the case where the calibration solutions must be
sulfon, fenthion-oxon-sulfoxid, fenthion-sulfon and fenthion- matrix-matched;
sulfoxid) - between 70 per cent to 110 per cent of each pesticide is
Fenvalerare 1.5 recovered;
Flucytrinate 0.05 - repeatability of the method: RSD is not greater than the
r-Fluvalinate 0.05 values indicated in Table 2.8.13.-2;
Fonophos 0.05 - reproducibility of the method: RSD is not greater than
Heptachlor (sum of heptachlor, cis-heptachlorepoxide and 0.05 the values indicated in Table 2.8.13.-2.
rrans-heptachlorepoxide)
Hexachlorbenzene 0.1 Table 2.8.13.-2.
Hexachlorocyclohexane (sum of isomers a-, P-, 8- and e) 0.3 Concentration range of Repeatability (RSD) Reproducibility (RSD)
Lindan (y-hexachlorocyclohexane) 0.6 the pesticide (per cent) (per cent)
Malathion and malaoxon (sum of) 1 (mg/kg)
Mecarbam 0.05 0.001 - 0.01 30 60
Methacriphos 0.05 > 0.01 - 0.1 20 40
Methamidophos 0.05 > 0.1 - 1 15 30
Methidathion 0.2 > I 10 20
Methoxychlor 0.05
Mircx 0.01
Monocrotophos 0.1
Parathion-ethyl and paraoxon-ethyl (sum of) 0.5
Parathion-methyl and paraoxon-methyl (sum of) 0.2 M. Tannins in Herbal Drugs
Pendimcthalin 0.5
Pentachloranisol 0.01
(Ph. Bur. method 2. 8.14)
Permethrin and isomers (sum of) 1 Carry out all the extraction and dilution operations protected from
Phosalonc 0.1 light.
Phosmet 0.05 In the case of a herbal drug or a dry extract, to the stated
Piperonyl butoxidc 3 amount of the powdered drug (180) (2.9.12) or the extract in
Pirimiphos-ethyl 0.05 a 250 mL round-bottomed flask add 150 mL of water R.
Pirimiphos-methyl (sum of pirimiphos-methyl and N-desethyl- 4 Heat on a water-bath for 30 min. Cool under running water
pirimiphos-methyl)
and transfer quantitatively to a 250 mL volumetric flask.
Procymidone 0.1
Rinse the round-bottomed flask and collect the washings in
Profenophos 0.1
the volumetric flask, then dilute to 250.0 mL with water R.
Prothiophos 0.05
Allow the solids to settle and filter the liquid through a filter
Pyrethrum (sum of cinerin I, cinerin II, jasmolin I, 3
jasmolin II, pyrethrin I and pyrethrin II)
paper 125 mm in diameter. Discard the first 50 mL of the
Quinalphos 0.05
filtrate.
Quintozene (sum of quintozene, pentachloraniline and methyl In the case of a liquid extract or a tincture, dilute the stated
penthachlorphenyl sulfide) amount of the liquid extract or tincture to 250.0 mL with
S-421 0.02 water R. Filter the mixture through a filter paper 125 mm in
Tecnazene 0.05 diameter. Discard the first 50 mL of the filtrate.
Tetradifon 0.3 Total polyphenols Dilute 5.0 mL of the filtrate to
Vinclozolin 0.4 25.0 mL with water R. Mix 2.0 mL of this solution with
1.0 mL of phosphomolybdotungstic reagent Rand 10.0 mL of
Sampling of herbal drugs water R and dilute to 25.0 mL with a 290 wL solution of
Sampling is done according to general chapter 2.8.20. Herbal sodium carbonate R. After 30 min measure the absorbance
drngs: sampling and sample preparation. (2.2.25) at 760 nm (A 1), using water Ras the compensation
Qualitative and quantitative analysis of pesticide liquid.
residues Polyphenols not adsorbed by hide powder To 10.0 mL
The analytical procedures used are validated (e.g. according of the filtrate, add 0.10 g of hide powder CRS and shake
to Document No. SANCO/10232/2006 or any subsequent vigorously for 60 min. Filter and dilute 5.0 mL of the filtrate
V-A386 Appendix XI N 2023

to 25.0 mL with water R. Mix 2.0 mL of this solution with Sample preparation
1.0 mL of phosplwmolybdotungstic reagent R and 10.0 mL of If necessary, reduce the sample to a powder (710) (2.9.12).
water R and dilute to 25.0 mL with a 290 g/L solution of To 1.0 g of sample add 100 mL of boiling water R. Heat on
sodium carbonate R. After 30 min measure the absorbance a water-bath for 30 min, stirring continuously. Allow to cool
(2.2.25) at 760 nm (A 2 ), using water Ras the compensation and dilute to 100 mL with water R. Shake vigorously and
liquid. filter, discarding the first 2 mL of the filtrate. The filtrate is
Standard Dissolve immediately before use 50.0 mg of labelled C-1 and has a dilution factor (DF) of 100.
pyrogallol R in water Rand dilute to 100.0 mL with the same If liquids have to be examined, 1 mL of the liquid is diluted
solvent. Dilute 5.0 mL of the solution to 100.0 mL with with a suitable solvent to 100 mL and designated C-1.
water R. Mix 2.0 mL of this solution with 1.0 mL of Determination of the bitterness value
plwsplwmolybdotungstic reagent Rand 10.0 mL of water Rand
dilute to 25.0 mL with a 290 g/L solution of sodium Test solutions:
carbonate R. After 30 min measure the absorbance (2.2.25) at 10.0 mL of C-1 is diluted with water R to 100 mL: C-2 (DF = 1000)
760 nm (A 3), using water Ras the compensation liquid. 10.0 mL of C-2 is diluted with water R to 100 mL: C-3 (DF = 10 000)
20.0 mL of C-3 is diluted with water R to 100 mL: C-3A (DF = 50 000)
Calculate the percentage content of tannins expressed as 10.0 mL of C-3 is diluted with water R to 100 mL: C-4 (DF = 100 000)
pyrogallol from the expression:
Starting with dilution C-4 each panel member determines the
62.5(A1 -A2)m2 dilution which still has a bitter taste. This solution is
A3 xm1 designated D. Note the DF of solution D is Y.
mass of the sample to be examined, in grams; Starting with solution D prepare the following sequence of
mass of pyrogallol, in grams. dilutions:

Solution D (mL) 1.2 1.5 2.0 3.0 6.0 8.0

water R (mL) 8.8 8.5 8.0 7.0 4.0 2.0

N. Bitterness Value
(Ph. Bur. metlwd 2.8.15) Determine the number of millilitres of solution D which,
when diluted to 10.0 mL with water R, still has a bitter
The bitterness value is the reciprocal of the dilution of a taste (X).
compound, a liquid or an extract that still has a bitter taste.
Calculate the bitterness value for each panel member from
It is determined by comparison with quinine hydrochloride,
the expression:
the bitterness value of which is set at 200 000.
Determination of the correction factor
A taste panel comprising at least 6 persons is recommended.
Yxk)
(XxO.l
The mouth must be rinsed with water R before tasting.
To correct for individual differences in tasting bitterness Calculate the bitterness value of the sample to be examined
amongst the panel members it is necessary to determine a as the average value for all panel members.
correction factor for each panel member.
Stock solution Dissolve 0.100 g of quinine hydrochloride R
in water Rand dilute to 100.0 mL with the same solvent.
Dilute 1.0 mL of this solution to 100.0 mL with water R. 0. Foam Index
Reference solutions Prepare a series of dilutions by (Ph. Bur. method 2.8.24)
placing in a first tube 3.6 mL of the stock solution and
increasing the volume by 0.2 mL in each subsequent tube to
PRINCIPLE
a total of 5 .8 ml.; dilute the contents of each tube to The foam index is determined by measuring the height of the
10.0 mL with water R. foam produced by the equivalent of 1 g of herbal drug or
herbal drug preparation under the stated test conditions.
Determine as follows the dilution with the lowest
concentration that still has a bitter taste. Take 10.0 mL of EQUIPMENT
the weakest solution into the mouth and pass it from side to The equipment (see Figure 2.8.24.-1) typically consists of:
side over the back of the tongue for 30 s. If the solution is - a pipette stand;
not found to be bitter, spit it out and wait for 1 min. Rinse - a 50 mL class A pipette with a rubber filler bulb;
the mouth with water R. After 10 min, use the next dilution - a 250 mL glass measuring cylinder with 2 mL
in order of increasing concentration. graduations and an internal diameter of 38 ± 3 mm.
Calculate the correction factor k for each panel member from Mount the pipette on its stand so that the tip will be located
the expression: 45 cm from the base of the cylinder placed under it. Position
the cylinder so that the pipette tip is aligned with its centre.
k=-n- Before performing the test, fill the pipette and cylinder with
5.00 water to check that the liquid dispensed from the pipette
runs into the centre of the measuring cylinder. Mark the
n number of millilitres of the stock solution in the dilution of
lowest concentration that is judged to be bitter. location of the measuring cylinder.

Persons who are unable to taste any bitterness when using

the reference solution prepared from 5.8 mL of stock
solution have to be excluded from the panel.
2023 Appendix XI R V-A387

volume of test solution, taking care not to produce any foam.

Position the measuring cylinder on its mark under the
pipette. Open pipette filler valve E to allow the solution to
run from the pipette into the measuring cylinder. Record the
maximum height of the foam before it starts to collapse.
Repeat the test twice, carefully rinsing the pipette and the
measuring cylinder with distilled water R between tests.
Results
Determine the foam index (IF) using the following equation:

H
h=-
m
H height of the resulting foam, in centimetres;
m mass of the herbal drug or herbal drug preparation used to
prepare the test solution, in grams.

B-
Record the average of 3 determinations as the result.
This result represents the height of the foam for a test
solution with a theoretical concentration of 1 g in 100 mL.

P. Dry Residue of Extracts

(Ph. Bur. method 2.8.16)
In a flat-bottomed dish about 50 mm in diameter and about
30 mm in height, introduce rapidly 2.00 g or 2.0 mL of the
extract to be examined. Evaporate to dryness on a water-bath
and dry in an oven at 100-105 °C for 3 h. Allow to cool in a
desiccator over diphosphorus pentoxide R or anhydrous silica
gel R and weigh. Calculate the result as a mass percentage or
c---- in grams per litre.
45 cm

Q. Loss on Drying of Extracts

(Ph. Bur. method 2. 8.1 7)
In a flat-bottomed dish about 50 mm in diameter and about
30 mm in height, weigh rapidly 0.50 g of the extract to be
examined, finely powdered. Dry in an oven at 100-105 °C
for 3 h. Allow to cool in a desiccator over diphosphorus
A. rubber pipette filler
B. 50 mL class A pipette
penwxide R or anhydrous silica gel R and weigh. Calculate the
C. 250 mL glass measuring cylinder with 2 mL graduations and an internal result as a mass percentage.
diameter of 38 ± 3 mm

Figure 2.8.24.-1. -Apparatus suitable for measuring the foam

index R. Test for Aristolochic Acids in Herbal
PROCEDURE Drugs
Preparation of the test solution Use Method Rl unless otherwise directed in the monograph.
Introduce the quantity of powdered herbal drug (355)
(2. 9.12) or herbal drug preparation prescribed in the Rl. Test for Aristolochic Acids in Herbal Drugs
monograph into a 250 mL beaker. Add 50 mL of distilled (Ph. Bur. method 2.8.21)
water R and allow to stand for 30 min, mixing 3-5 times with CAUTION: aristolochic acids are very toxic and carcinogenic.
a spatula during this period to disperse the powder without Perfonn manipulations in a fume cupboard whenever possible.
producing any foam. Rinse the spatula and the internal walls Take particular precautions, such as use of a glove box, when the
of the beaker with a further 50 mL of distilled water R to substance is in dry fonn because of its electrostatic properties and
ensure that as much of the herbal drug or herbal drug the tendency to disperse through the working areas.
preparation as possible is contained within the liquid. Allow Methods A and B are intended to be cross-referenced in
to stand undisturbed for 30 min. Filter through a filter paper monographs on herbal drugs that, according to
125 mm in diameter. Use the filtrate as the test solution. chemotaxonomic knowledge, are expected to be free from
Method aristolochic acids, but that may be subject to adulteration or
Mount the pipette on the stand. Take up 50.0 mL of the test substitution with plant material containing aristolochic acids.
solution using the pipette. Rinse the measuring cylinder with Methods A and B are intended to be used in the screening of
distilled water R to wet its walls, then introduce the remaining herbal drugs for aristolochic acids at the stated limits and will
V-A388 Appendix XI R 2023

usually be complemented by macroscopic and/or microscopic Reference solution (a) Dissolve the contents of a Vial of
tests to exclude plant material containing aristolochic acids. aristolochic acid I CRS in the solvent mixture to obtain a
Method C will not be used in specific monographs but is concentration of 0.04 µg/mL of aristolochic acid I.
provided as a method to confirm the presence of aristolochic Reference solution (b) Dissolve the contents of a vial of
acid I at levels equal to or greater than 2 ppm. It may be aristolochic acid for system suitability CRS (containing
applied if chromatographic data suggests the presence of aristolochic acids I and II) in the solvent mixture and dilute
aristolochic acid I. to 20.0 mL with the solvent mixture.
These methods are not designed for inclusion as assay Column:
methods in monographs on those drugs that produce - size: l = 0.15 m, 0 = 2.1 mm;
aristolochic acids as secondary metabolites; for these, a more - stationary phase: octadecylsilyl silica gel for chromatography R
sensitive, validated method is required. (3.5 µm);
- temperature: 40 °C.
METHOD A: SCREENING TEST FOR
ARISTOLOCHIC ACIDS Mobile phase:
Thin-layer chromatography (2.2.27). - mobile phase A: trifluoroacetic acid R, water R
(0.1:99.9 V/V);
Solvent mixture anhydrous formic acid R, water R,
- mobile phase B: trifluoroacetic acid R, acetonitrile R
methanol R (1:9:40 VIVIV).
(0.1:99.9 VIV);
Test solution To 1.0 g of the powdered herbal drug (710)
(2. 9.12) add 10.0 mL of the solvent mixture, sonicate for Time Mobile phase A Mobile phase B
10 min and centrifuge. (min) (per cent V/V) (per cent V/V)
Reference solution (a) Disperse an amount of 0 - 25 85 ➔ 35 15 ➔ 65
aristolochia HRS corresponding to 0.10 mg of aristolochic 25 - 30 35 ➔ 0 65 ➔ 100
acid I in 20.0 mL of the solvent mixture, sonicate for 10 min 30 - 31 0 ➔ 85 100 ➔ 15
and centrifuge.
Reference solution (b) Dilute 1.0 mL of reference Flow rate 0.3 mIJmin.
solution (a) to 25.0 mL with methanol R. Detection Spectrophotometer at 390 nm.
Plate TLC silica gel F254 plate R (2-10 µm). Injection 25 µL.
Mobile phase anhydrous formic acid R, water R, ethyl System suitability:
acetate R, toluene R (3:3:30:60 V/V/V/V); use the upper layer. - resolution: minimum 3.0 between the peaks due to
Application 20 µL as bands of 8 mm. aristolochic acids I and II in the chromatogram obtained
Development Over a path of 6 cm. with reference solution (b);
Drying In a current of cold air for 5 min. - signal-to-noise ratio: minimum 10 for the peak due to
aristolochic acid I in the chromatogram obtained with
Detection Spray with a 100 g/L solution of stannous
reference solution (a).
chloride R in dilute hydrochlon·c acid R until the plate is slightly
wet, heat at 100 °C for 1 min and examine in ultraviolet light Limit:
at 365 nm. - the sample complies with the test if the chromatogram
obtained with the test solution shows no peak with the
System suitability:
same retention time as the peak due to aristolochic acid I
- the chromatogram obtained with reference solution (a)
in the chromatogram obtained with reference solution (a)
shows 2 greenish-blue zones due to aristolochic acids I
(2 ppm).
= =
and II between Rp 0.35 and RF 0.55, which may not
be completely separated; METHOD C: CONFIRMATORY TEST FOR
- the chromatogram obtained with reference solution (b) ARISTOLOCHIC ACID I
shows at least 1 of these zones (corresponding to 2 ppm Liquid chromatography (2.2.29) coupled with mass
of aristolochic acid I). spectrometry (2.2.43).
Results In the chromatogram obtained with the test Solvent mixture acetonitrile R, water R (50:50 V/V).
solution no zone is similar in position and fluorescence to Test solution Weigh 2.0 g of the powdered herbal
any of the zones due to aristolochic acids in the drug (710) (2. 9.12) into a 250 mL, brown, screw-cap bottle
chromatogram obtained with reference solution (a). and add 100.0 mL of the solvent mixture. Sonicate for
If the chromatogram obtained with the test solution shows 30 min and filter through a membrane filter (nominal pore
any zones similar in position and fluorescence to any of the size 0.45 µm).
zones due to aristolochic acids I and II in the chromatogram Reference solution (a) Dissolve the contents of a vial of
obtained with reference solution (a), apply method B. aristolochic acid I CRS in the solvent mixture to obtain a
METHOD B: LIMIT TEST FOR ARISTOLOCHIC concentration of 0.04 µg/mL of aristolochic acid I.
ACIDI Reference solution (b) Prepare a solution according to
Liquid chromatography (2.2.29). the instructions supplied with aristolochic acid I CRS to obtain
Solvent mixture acetonitrile R, water R (50:50 VIV). a concentration of 0.45 µg of aristolochic acid I in 10.0 mL
of the test solution.
Test solution Weigh 2.0 g of the powdered herbal drug
(710) (2.9.12) into a 250 mL, brown, screw-cap bottle and Column:
add 100.0 mL of the solvent mixture. Stir for 30 min at = =
- size: l 0.15 m, 0 2.1 mm;
about 300 r/min and filter through a membrane filter - stationary phase: octadecylsilyl silica gel for chromatography R
(nominal pore size 0.45 µm). (3.5 µm);
- temperature: 40 °C.
2023 Appendix XI R V-A389

Mobile phase: interval of the average of the 2 ratios of reference

mobile phase A: anhydrous formic acid R, 1 g/L solution of solution (a); otherwise it is necessary to adjust the
ammonium acetate R in water R (0.1:99.9 V/V); detector settings.
mobile phase B: anhydrous formic acid R, 1 g/L solution of Results Evaluate the average ratios (265/281 and 296/281)
ammonium acetate R in methanol R (0.1:99.9 V/V); of the relative intensity of the 3 product ions of aristolochic
acid I in the test solution; evaluate the average of the 2 ratios
Time Mobile phase A Mobile phase B of the signals at the retention time of aristolochic acid I in
(min) (per cent V/J/) (per cent V/J/)
reference solution (a); if the average of the 2 ratios of the test
0 - 15 70----> 0 30--, 100
solution is within the ± 40 per cent interval of the average of
15 - 16 0 100 the 2 ratios of reference solution (a), aristolochic acid I is
16 - 17 0----, 70 100 ----> 30 present in the test solution.

Flow rate 0.4 mUmin. R2. Test for Aristolochic Acids I and II in Herbal
Drugs
Injection 20 µL; inject reference solution (a) twice, the test
(No Ph. Bur. Method)
solution twice, reference solution (a) twice, then reference
solution (b) twice. CAUTION Aristolochic acids have been shown to be highly toxic
and carcinogenic. Extraordinary care should be taken in artv
Detection Mass detector as described below under A or B.
procedure in which they are used.
Adjust the flow rate, the temperature and the detector
settings so as to comply with the system suitability criterion. In line with the prohibition of the use of Aristolochia species
in unlicensed herbal medicines in the United Kingdom, a test
A. Ion-trap mass spectrometer equipped with an
for absence of aristolochic acids I and II in herbal drugs has
electrospray ionisation (ESI) interface and MS0 analyser.
been included in the British Pharmacopoeia. The limit of
Set the mass spectrometer parameters for the MS 3 mode as detection has been shown to be 0.00078 mg/mL
follows: (approximately 1 ppm of aristolochic acids I and II). It is
advised that the limit of detection for the system-in-use is
Mode Parent (mlz)
Isolation Relative collision energy determined by the analyst. Aristolochic acids I and II are not
width (mlz) (per cent) confined to the genus Aristolochia. The acids are also
359 reported as present in certain species of Asarum.
MS 2 2.0 30
[M + NH4]+ Sample preparation
MS 3 298 2.0 35 Unless otherwise specified in the monograph, weigh 2 g of
the ground herbal drug into a centrifuge tube. Add 10 mL of
0.lM sodium hydroxide, shake for at least 2 hours and
- full scan of product ions: from mlz 80 to mlz 370;
centrifuge the mixture for 10 minutes at approximately
- product ions to be monitored: mlz 252, mlz 268 and
4000 revolutions per minute. Filter the supernatant layer if
mlz 281.
visible particles remain in the suspension. Pass a 1.0 mL
System suitability: portion of the sample solution with the aid of vacuum
- signal-to-noise ratio: minimum 100 for the monitored through a solid-phase extraction column of 1 mL capacity
product ions in the chromatogram obtained with and containing 30 mg of divinylbenzene and vinylpyrrolidone
reference solution (a); copolymer for chromatography (30 µm) (Waters Oasis HLB,
- matrix interference test: the average of the 2 ratios of 30 mg/mL or Phenomenex StrataX, 30 mg/mL is suitable)
reference solution (b) is inside the ± 40 per cent previously washed with 1 mL of methanol, followed by 1 mL
interval of the average of the 2 ratios of reference of water. Wash the column with 1 mL of 0.lM sodium
solution (a); otherwise it is necessary to adjust the hydroxide followed by 1 mL of a mixture containing
detector settings. 2 volumes of glacial acetic acid, 28 volumes of water and
Results Evaluate the average ratios (252/268 and 281/268) 70 volumes of methanol. Elute the sample with 0.25 mL of a
of the relative intensity of the 3 product ions of aristolochic mixture containing 98% of methanol and 2% of concentrated
acid I in the test solution; evaluate the average of the 2 ratios ammonia. Evaporate the extract to dryness at 40° under a
of the signals at the retention time of aristolochic acid I in stream of nitrogen and dissolve in 0.25 mL of methanol. If a
reference solution (a); if the average of the 2 ratios of the test larger sample volume is required then a larger capacity solid-
solution is within the ± 40 per cent interval of the average of phase extraction column may be used.
the 2 ratios of reference solution (a), aristolochic acid I is Use this as solution (1) for the identification method
present in the test solution. described below.
B. Triple-quadrupole mass spectrometer equipped with an Identification
ESI interface and MS 0 analyser. Carry out the method for liquid chromatography,
Set the mass spectrometer parameters for the MS 2 mode as Appendix III D, using the following solutions. Prepare
follows: solution (1) as described above under Sample preparation.
- precursor ion: mlz 359 [M + NH4 ]+; Solution (2) contains 0.01 % w/v of aristolochic acid BPCRS in
- product ions to be monitored: mlz 265, mlz 281 and methanol.
mlz 296. The chromatographic procedure may be carried out using
System suitability: (a) a stainless steel column (25 cm x 4.6 mm) packed with
- signal-to-noise ratio: minimum 100 for the monitored base-deactivated octadecylsilyl silica gel for chromatography
product ions in the chromatogram obtained with (4 µm) (Genesis C18 is suitable) maintaining the column
reference solution (a); temperature at 30C, (b) a mixture of 45 volumes of 0.1 % v/v
- matnx interference test: the average of the 2 ratios of of orthophosphoric acid and 55 volumes of acetonitrile as the
reference solution (b) is inside the ± 40 per cent mobile phase with a flow rate of 1.3 mL per minute and (c)
V-A390 Appendix XI S 2023

a detection wavelength of 225 run. The identity of any peaks Test solution Use an immunoaffinity column containing
suspected to be due to arisotolochic acids I and II may be antibodies against aflatoxin B 1 with a capacity of not less
clarified by use of the UV spectrum recorded with a diode than 100 ng of aflatoxin B 1 and which gives a recovery of not
array detector. less than 80 per cent when a solution of 5 ng of aflatoxin B1
Inject 10 µL of solution (2) and allow the chromatography to in a mixture of 12.5 mL of methanol Rand 87.5 mL of
proceed for 10 minutes. The test is not valid unless the water R is passed through. Allow the immunoaffinity column
resolution factor between the peaks corresponding to to reach room temperature. To 5.00 g of the powdered drug
aristolochic acid II (retention time about 6 minutes) and (500) (2. 9.12) add 100 mL of a mixture of 30 volumes of
aristolochic acid I (retention time about 7 minutes) is at least water Rand 70 volumes of methanol R and extract by
3.0. Inject solution (2) six times. The relative standard sonication for 30 min. Filter through folded filter paper.
deviation of the areas of the peaks is at most 1.5%. Pipette 10.0 mL of the clear filtrate into a 150 mL conical
Inject 10 µL of solution (1) and allow the chromatography to flask. Add 70 mL of water R. Pass 40 mL through the
proceed for 30 minutes. In the chromatogram obtained with immunoaffinity column at a flow rate of 3 mUmin (not
solution (1) the peaks due to aristolochic acid I and exceeding 5 mUmin). Wash the column with 2 volumes,
aristolochic acid II are absent. each of 10 mL, of water R at a flow rate not exceeding
5 mUmin and dry by applying a slight vacuum for 5-10 s or
by passing air through the immunoaffinity column by means
of a syringe for 10 s. Apply 0.5 mL of methanol R to the
column and allow to pass through by gravity. Collect the
S. Determination of Mycotoxins in Herbal eluate in a 5 mL volumetric flask. After 1 min, apply a 2nd
Drugs portion of 0.5 mL of methanol R. After a further 1 min, apply
a 3rd portion of 0.5 mL of methanol R. Collect most of the
1. Determination of Aflatoxin B 1 in Herbal Drugs applied elution solvent by pressing air through or applying
(Ph. Bur. method 2.8.18) vacuum to the column. Dilute to 5 mL with water R and
shake well. If the solution is clear it can be used directly for
CAUTION: aftatoxins are very toxic and carcinogenic. Perform
analysis. Otherwise, pass it through a disposable filter unit
manipulations in a fume cupboard whenever possible. Take
prior to injection. Use a disposable filter unit (e.g. 0.45 µm
particular precautions, such as use of a glove box, when toxins are
pore size polytetrafluoroethylene filter) that has been shown
in dry form because of their electrostatic properties and the
not to cause loss of aflatoxin by retention.
tendency to disperse through the working areas. Decontamination
procedures for laboratory wastes of aftatoxins were developed by Aflatoxin B 1 primary stock solution Dissolve aftatoxin
the International Agency for Research on Cancer (!ARC). B 1 R in a mixture of 2 volumes of acetonitrile R and
98 volumes of toluene R to give a 10 µg/mL solution.
Aflatoxins are naturally occurring mycotoxins produced
To determine the exact concentration of aflatoxin B 1 in the
mainly by Aspergillus ftavus and Aspergillus parasiticus. These
stock solution, record the absorption curve (2.2.25) between
fungi are common and widespread in nature and are most
330 nm and 370 nm in quartz cells.
often found when certain grains are grown under conditions
of stress such as drought. The mould occurs in soil, decaying Calculate the aflatoxin B 1 mass concentration, in micrograms
vegetation, hay, and grains undergoing microbial spoilage, per millilitre, using the following expression:
and invades all types of organic substrates whenever and
AxMxlO0
wherever the conditions are favourable for its growth.
Favourable conditions include high moisture content and sxl
high temperature. At least 13 different types of aflatoxin are A absorbance determined at the maximum of the absorption
produced in nature and most of these are known to be highly curve;
toxic and carcinogenic. Aflatoxin B1 is considered the most M molar mass of aflatoxin B1 (312 g/mol);
molar absorptivity of aflatoxin B1 in the toluene-acetonitrile
toxic. Herbal drugs that are subject to contamination by
mixture (1930 m 2/mol);
aflatoxins are tested by a validated method. optical path length of the cell (I cm).
Unless otherwise indicated in the monograph, herbal drugs
contain not more than 2 µg/kg of aflatoxin B 1 • Aflatoxin B 1 secondary stock solution Prepare a
The competent authority may also require compliance with a secondary stock solution containing 100 ng/mL aflatoxin B 1
limit of 4 µg/kg for the sum of aflatoxins B 1, B2 , G 1 and G 2 • by diluting aflatoxin B 1 primary stock solution with a mixture
The method described below is cited as an example of a of 2 volumes of acetonitrile R and 98 volumes of toluene R.
method that has been shown to be suitable for devil's claw Wrap the flask tightly in aluminium foil and store it below
root, ginger and senna pods. Its suitability for other herbal 4 "C. Before use, do not remove the aluminium foil until the
drugs must be demonstrated or another validated method contents have reached room temperature. If the solution has
used. to be stored for a long period (for example, 1 month), weigh
the flask and record the mass before and after each use of the
METHOD solution.
Liquid chromatography (2.2.29).
Aflatoxin B 1 standard solutions Place the volumes of
Aftatoxins are subject to light degradation. Carry out the aflatoxin secondary stock solution indicated in
determination protected from daylight by using UV-absorbing foil Table 2.8.18.-1 in separate 250 mL volumetric flasks. Pass a
on windows in combination with subdued light, or curtains or stream of nitrogen through at room temperature until the
blinds in combination with artificial light (fluorescent tubes are solvent has just evaporated. To each flask, add 75 mL of
acceptable). Protect aftatoxin-containing solutions from daylight. methanol R, allow the aflatoxin B 1 to dissolve and dilute to
Rinse glassware before use with a 10 per cent V/V solution of 250 mL with water R.
suljuric acid R and then rinse carefully with distilled water R
until no more acid is present.
2023 Appendix XI S V-A391

Table 2.8.18.-1. -Afiatoxin B 1 standard solutions - polytetrafluoroethylene reaction tube, l = 0.12 m,

Final concentration of 0 = 0.25 mm;
Volwne of secondary
Standard solution standard solution - mobile phase B.
stock solution (µL)
(ng/mL)
Elution order Aflatoxin G 2 , aflatoxin G 1, aflatoxin B2 ,
125 0.05 aflatoxin B,.
2 250 0.1
Calculation Calculate the calibration curve y = ax + b,
3 500 0.2
with aflatoxin B 1 concentration (ng/mL) on the x-axis and
4 750 0.3
the signal (S) on the y-axis. The aflatoxin B1 concentration
5 1000 0.4
(C) in a measured solution is equal to S~b.
Calculate the aflatoxin B 1 content of the herbal drug, in
Calibration curve Prepare the calibration curve using nanograms per gram, using the following expression:
aflatoxin B 1 standard solutions 1 to 5, which cover a range
equivalent to 1-8 µg/kg of aflatoxin B1 in the herbal drug. VixVixC
Check the plot for linearity. If the content of aflatoxin B 1 in mx V,
the sample to be examined is outside of the calibration range,
the test solution must be diluted to an aflatoxin content that m mass of the herbal drug taken for analysis, in grams;
is appropriate for the established calibration curve. Vi volume of the solvent used for extraction, in millilitres;
Vi aliquot taken for immunoaffinity clean-up, in millilitres;
Column: V2 final volume of solution after elution from the immunoaffinity
- size: l =0.25 m, 0 = 4.6 mm; column and dilution, in millilitres;
- stationary phase: octadecylsilyl silica gel for chromatography R C measured aflatoxin B1 concentration of the test solution,
(5 µm). in nanograms per millilitre.

Mobile phase:
The presence of aflatoxin B I may be confirmed by recording
- mobile phase A (for post-column derivatisation with
the chromatogram without post-column derivatisation, which
photochemical reactor or pyridinium bromide):
leads to a large decrease (greater than 10-fold) in the
acetonitrile R, methanol R, water R (2:3:6 VIVIV);
response due to aflatoxin B 1 .
- mobile phase B (for post-column derivatisation with
electrochemically derived bromine): add 0.12 g of 2. Determination of Ochratoxin A in Herbal Drugs
potassium bromide R and 350 µL of dilute nitric (Ph. Bur. method 2.8.22)
acid Rl per litre of mobile phase A. CAUTION: ochratoxin A is nephrotoxic and nephrocarcinogenic.
Flow rate 1 mUmin. Peiform manipulations in a fume cupboard. Take particular
Detection Fluorescence detector with a 360 nm excitation precautions, such as use of a glove box, when toxins are in dry
filter and a 420 nm cut-off emission filter, or equivalent. form because of their electrostatic properties and the tendency to
Recommended settings for adjustable detectors are 365 nm disperse through the working areas. Decontamination procedures
(excitation wavelength) and 435 nm (emission wavelength). for laboratory glassware containing ochratoxin A are necessary
Injection 500 µL. (see appendix).
Post-column derivatisation with pyridinium hydrobromide Herbal drugs that are subject to contamination by
perbromide (PBPB): ochratoxin A are tested by a validated method.
- pulseless pump; The method described below is cited as an example of a
- T-piece with zero dead volume; method that has been shown to be suitable for liquorice
- polytetrafluoroethylene reaction tube, l = 0.45 m, extract and liquorice root. Its suitability for other herbal
0 = 0.5 mm; drugs must be demonstrated or another validated method
- mobile phase A; used.
- post-column derivatisation reagent: dissolve 50 mg of METHOD
pyridinium hydrobromide perbromide R in 1000 mL of Liquid chromatography (2.2.29).
water R (store protected from light and use within
Use brown glassware that is free from detergent residues.
4 days);
If necessary rinse glassware before use with a 10 per cent V/V
- flow rate of the derivatisation reagent: 0.4 mIJmin.
solution of sulfuric acid R and then rinse carefully with distilled
Post-column derivatisation with photochemical reactor (PHRED) water R until no more acid is present.
- reactor unit with one 254 nm low pressure mercury UV
bulb (minimum 8 W);
Solution A Mix 80 volumes of water R, previously adjusted
- polished support plate; to pH 2.3 with anhydrous formic acid R, and 20 volumes of
acetonitrile R.
- knitted reactor coil: polytetrafluoroethylene tubing knitted
tightly around the UV bulb, l = 25 m, 0 = 0.25 mm, Test solution Use an immunoaffinity column containing
nominal void volume 1.25 mL; antibodies against ochratoxin A with a capacity of not less
- exposure time: 2 min; than 100 ng of ochratoxin A and which gives a recovery of
- mobile phase A. not less than 70 per cent. Allow the immunoaffinity column
to reach room temperature.
Post-column derivatisation with electrochemically generated
bromine (KOBRA): To 2.00 g of the powdered drug (250) (2.9.12) add 80 mL
- KOBRA-cell: electrochemical cell that generates a reactive of a 30 g/L solution of sodium hydrogen carbonate R and
form of bromine for derivatisation of aflatoxins, resulting extract by sonication for 30 min (change water of ultrasonic
in enhanced fluorescence; available from various bath after 15 min). Cool to room temperature and dilute to
commercial suppliers; 100.0 mL (Vi) with the same solution. Centrifuge.
- Derivation direct-current supply in series with the Mix thoroughly 5.0 mL ( Vi) of the clear supernatant with
KOBRA-cell, providing a constant current of about 30 mL buffer solution pH 7.4 Rand pass the whole solution
100 µA; through the immunoaffinity column at a flow rate of
V-A392 Appendix XI T 2023

3 m!Jmin (do not exceed 5 m!Jmin). Wash the column first Time Mobile phase A Mobile phase B
(min) (per cent V/JI) (per cent V/JI)
with 10 mL buffer solution pH 7. 4 R then with 2 quantities,
each of 10 mL, of water R at a flow rate not exceeding 0 - 30 80--+ 40 20--+ 60
5 m!Jmin and dry by applying a slight vacuum for 5-10 s or 30 - 35 40--+ 20 60--+ 80
by passing air through the immunoaffinity column by means 35 - 37 20 80
of a syringe for 10 s. Apply 0.5 mL of methanol R to the 37 -40 20--+ 80 80--+ 20
column and allow to pass through by gravity.
Collect the eluate in a 4 mL glass vial. After 30 s, apply a 2nd Flow rate 0.8 m!Jmin.
quantity of 0.5 mL of methanol R and allow to pass through Detection Fluorescence detector; recommended settings
the column by gravity into the same glass vial. After a further for adjustable detectors are 330 nm (excitation wavelength)
30 s, repeat with a 3rd portion of 0.5 mL of methanol R. and 460 nm (emission wavelength).
Collect any solvent retained on the column by pressing air Injection 20 µL.
through or applying vacuum to the column. Evaporate the
Calculation Calculate the calibration curve y = ax + b,
combined eluates completely to dryness using a thermal
with ochratoxin A concentration (in nanograms per millilitre)
block with a nitrogen blanket (40 °C). Dissolve the residue in
on the x-axis and the signal (S) on the y-axis. The ochratoxin
0.5 mL (Vi) of solution A. If the solution is clear it can be
A concentration (C) in a measured solution is equal to S~b.
used directly for analysis. Otherwise, pass it through a
disposable filter unit prior to injection. Use a disposable Calculate the ochratoxin A content of the herbal drug, in
filter unit (e.g. 0.45 µm pore size polytetrafluoroethylene nanograms per gram, using the following expression:
filter) that has been shown not to cause loss of ochratoxin A
VixVixC
by retention.
mx Vi
Ochratoxin A primary stock solution Dilute 1.0 mL of
ochratoxin A solution R to 100.0 mL with methanol Rand m mass of the herbal drug used to prepare the test solution, in
shake thoroughly. grams;
Ochratoxin A secondary stock solution Dilute 10.0 mL Vi volume of dilution, in millilitres;
Vi aliquot taken for immunoaffinity clean-up, in millilitres;
of ochratoxin A primary stock solution to 100.0 mL with Vi volume in which the residue is taken up, in millilitres;
methanol R and shake thoroughly. C measured ochratoxin A concentration of the test solution, in
nanograms per millilitre.
Ochratoxin A standard solutions Place the volumes of
ochratoxin A primary stock solution or ochratoxin A
secondary stock solution indicated in Table 2.8.22.-1 into
separate flasks and make up to 50.0 mL with solution A.
APPENDIX: DECONTAMINATION
PROCEDURES FOR LABORATORY
Table 2.8.22.-1. - Ochratoxin A standard solutions
GLASSWARE
Standard solution Volume of Final concentration
ochratoxin A primary of ochratoxin A in Rinse glassware with methanol R and decontaminate by
stock solution (µL) standard solution immersion in strong sodium hypochlorite solution R for at least
(ng/mL) 2 h, then wash thoroughly with water.
1 5000 50
2 2500 25
3 1000 10
4 500 5 T. Herbal Drugs: Sampling and Sample
5 250 2.5

Standard solution Volume of Final concentration

Preparation
ochratoxin A secondary of ochratoxin A in (Ph. Bur. method 2.8.20)
stock solution (µL) standard solution
(ng/mL) In order to reduce the effect of sampling in qualitative and
quantitative analysis, it is necessary to ensure that the
6 500 0.5
composition of the sample used is representative of the batch
7 100 0.1
of material being examined. The following procedures are the
minimum considered applicable for herbal drugs. NOTE:
Calibration curve Prepare the calibration curve using other procedures may be used if they can be demonstrated to
ochratoxin A standard solutions 1 to 7, which cover a range produce representative batch samples.
equivalent to 0.5-250 µg/kg of ochratoxin A in the herbal
drug. Check the plot for linearity. If the content of BULK SAMPLE
ochratoxin A in the sample to be examined is outside of the Where external examination of containers, markings and
calibration range, the test solution must be diluted to an labels of a batch indicate that it can be considered to be
ochratoxin A content that is appropriate for the established homogeneous, sample the number of randomly selected
calibration curve. containers indicated below. Where a batch cannot be
Column: considered to be homogeneous, divide it into sub-batches
= =
- size: l 0.15 m, 0 4.6 mm; that are as homogeneous as possible, then sample each sub-
batch as a homogeneous batch using, as a minimum, the
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 µm); number of randomly selected containers indicated below.
- temperature: 45 °C.
Mobile phase:
- mobile phase A: water R adjusted to pH 2.3 with phosphoric
acid R;
- mobile phase B: acetonitrile R;
2023 Appendix XI T V-A393

Table 2.8.20.-1. - Operation of the sampling procedure in order to obtain the prescribed bulk sample
Mass of herbal
drug in 0.5 l 5
container (kg)

Total mass of No. of No. of Total No. of No. of Total No. of No. of Total
herbal drug in containers containers mass of containers containers mass of containers containers mass of
the batch (kg) in batch to be samples in batch to be samples in batch to be samples
sampled (g) sampled (g) sampled (g)

0.5 I I 125 - - - - - -

I 2 2 125 1 I 125 - - -

5 10 5 125 5 4 125 I I 125

10 20 6 125 10 5 125 2 2 125

25 - - - 25 6 250 5 4 250

100 - - - 100 II 500 20 6 500

250 - - - - - - 50 9 625

500 - - - - - - 100 11 1000

Mass of herbal
drug in 25 125 500
container (kg)

Total mass of No. of No. of Total No, of No. of Total No. of No. of Total
herbal drug in containers containers mass of containers containers mass of containers containers mass of
the batch (kg) in batch to be samples in batch to be samples in batch to be samples
sampled (g) sampled (g) sampled (g)

25 I I 250 - - - - - -

100 4 3 500 - - - - - -

250 10 5 625 2 2 625 - - -

500 20 6 1000 4 3 1000 I I 1000

1000 40 8 1800 8 4 1800 2 2 1800

2000 80 10 3000 16 5 3000 4 3 3000

3000 120 12 3000 24 6 3000 6 4 3000

5000 200 16 5000 40 8 5000 10 5 5000

10 000 400 21 8000 80 10 8000 20 6 8000

25 000 800 30 12 500 160 14 12 500 40 8 12 500

Number of containers to Minimum mass of samples as a

Number of containers in batch (N) Mass of herbal drug in the batch
be sampled (n) percentage of the mass of the
(kg)
batch of herbal drug
I -3 all
< 50 1.00*
> 3 n*= ,IN +I
50 - JOO 0.50
* round n up to the next integer > 100 - 250 0.25
> 250 - 500 0.20
Take one sample from each container to be sampled. > 500 - 1000 0.18
The sample is taken from the upper, middle or lower section > 1000 - 2500 0.15
of the container, such that the samples taken are > 2500 - 5000 0.10
representative of different parts of the containers. In the case > 5000 - 10 000 0.08
of large bales or bags, samples must be taken from a depth of > I O 000 - 25 000 0.05
at least 10 cm. The mass of the material taken from each NOTE: if the mass of the batch is greater than 25 000 kg, it is divided into
container is such that the total mass of the bulk sample sub-batches, and the procedure is applied to each sub-batch as though it were
complies with the following values. a homogeneous batch.
* subject to a minimum total mass of 125 g for the bulk sample; if this
minimum requirement represents more than 10.0 per cent of the mass of
herbal drug in the batch, the whole batch may be used as the sample.

Prepare the bulk sample by combining and thoroughly

mixing the samples taken from each of the randomly selected
containers (see Table 2.8.20.-1).
V-A394 Appendix XI U 2023

TEST SAMPLE Examination under polarised light (between crossed nicol

Unless otherwise prescribed in the monograph, prepare the prisms) is used to identify starch granules (black cross
test sample as follows. phenomenon), calcium oxalate crystals (refringence) or
Reduce the size of the bulk sample by quartering (see Note lignified structures.
below) or by any other method that produces a MOUNTING IN CHLORAL HYDRATE SOLUTION
homogeneous sample, making sure that each retained portion Place 2-3 drops of chloral hydrate solution R on a glass
remains representative of the whole, until the minimum microscope slide. Disperse a very small quantity of the
retained quantity complies with the following conditions. powdered drug in the liquid and cover the preparation with a
cover slip. Heat the preparation very gently to boiling on a
Type of herbal drug Minimum weight of test sample hot plate or a micro gas burner. Maintain gentle boiling for a
Roots, rhizomes, bark, herbs 500 g or mass of whole sample if bulk short time. Make sure that the quantity of mounting fluid is
sample is less than 500 g sufficient. If necessary, add more fluid using a tapered glass
Leaves, flowers, seeds, fruits 250 g or mass of whole sample if bulk pipette. Allow to cool and then examine under a microscope.
sample is less than 250 g
Repeat the heating until the starch granules and the water-
Broken or fragmented drugs (where 125 g
average mass of the pieces is less than
soluble contents of the cells are no longer visible. Examine
0.5 g) under a microscope.
Chloral hydrate tends to crystallise as long needles. To avoid
NOTE: quartering consists of placing the bulk sample, thoroughly this, proceed as follows: after heating, remove the cover slip;
mixed, as a level and square-shaped heap and dividing it to the preparation add 1 drop of a 10 per cent V/V mixture
diagonally into 4 equal parts. 2 opposite quarters are retained and of chloral hydrate solution R in glycerol R; place a clean cover
carefully remixed. The process is repeated as necessary until the slip on the preparation; examine under a microscope.
required minimum mass is obtained for the test sample. MOUNTING IN A 50 PER CENT V/V SOLUTION OF
Mill the test sample in a single pass through a 1 mm screen GLYCEROL
or the screen size specified in the monograph. The use of a Place 2 drops of a 50 per cent V/V solution of glycerol R on a
milling machine is recommended. glass microscope slide. Disperse a very small quantity of the
Pass the milled sample through a 1 mm standard sieve or the powdered drug in the liquid and cover the preparation with a
sieve specified in the monograph. The residue retained on cover slip. Examine under a microscope.
the sieve must not be more than 10 per cent of the total MOUNTING IN A 10 PER CENT V/V ALCOHOLIC
mass of the milled sample, of which not more than SOLUTION OF PHLOROGLUCINOL AND
2 per cent of the total mass of the milled sample may be of a HYDROCHLORIC ACID
particle size greater than 1.5 mm or 1.5 times the specified Place a very small quantity of the powdered drug on a glass
particle size in the monograph. If these conditions are met, microscope slide. Add 1-2 drops of a 10 per cent V/V
the sample and residue are to be well mixed to form the test alcoholic solution of phloroglucinol R. Mix and allow the
sample for analysis. solvent to evaporate almost completely. Add 1-2 drops of
In those cases where these requirements are not met, the test hydrochloru acid R and cover the preparation with a cover
sample for analysis is composed of the 2 parts measured slip. Examine immediately under a microscope. The red
separately. Therefore, the quantity required for each analysis colour indicates the presence of lignin.
is derived by weighing proportional quantities of the powder
MOUNTING IN LACTIC REAGENT
and the residue.
Place 2-3 drops of lactic reagent R on a glass microscope
NOTE: for determination of muroscopic characters, a portion of slide. Disperse a very small quantity of the powdered drug in
the milled test sample is re-milled through a 0.355 mm screen. the liquid and cover the preparation with a cover slip. Heat
the preparation very gently to boiling. Maintain gentle boiling
for a short time. Make sure that the quantity of mounting
fluid is sufficient. If necessary, add more fluid using a tapered
U. Microscopic Examination of Herbal glass pipette. Allow to cool and then examine under a
Drugs microscope. Lignified structures stain bright yellow;
structures containing cellulose remain colourless. Starch
(Ph. Bur. method 2.8.23) granules stain more or less violet; certain secretions (e.g.,
The microscopic examination of herbal drugs is carried out essential oils, resins, oleoresins) stain orange and cork stains
on the powdered drug (355) (2.9.12) unless otherwise red.
prescribed in the monograph. MOUNTING IN RUTHENIUM RED SOLUTION
Chloral hydrate solution R is the most commonly prescribed Place 2 drops of ruthenium red solution R on a glass
reagent. However, certain features are not visible or not easily microscope slide. Disperse a very small quantity of the
seen after mounting in this reagent. In this case, other powdered drug in the liquid and cover the preparation with a
reagents are used, for example, a 50 per cent V/V solution of cover slip. After about 1 minute, allow a drop of distilled
glycerol R, which makes it possible to visualise starch water R to be taken up between the slide and the cover slip.
granules. It may also be necessary to prescribe specific Examine under a microscope. The mucilage stains violet red.
reagents in a monograph, for example: lactic reagent R which
is used to show the presence of various features, 10 per cent
V/V alcoholic solution of phloroglucinol R and hydrochloru
acid R, which are used to identify the presence of lignin in
cells or tissues, ruthenium red solution R, which is used to
show the presence of mucilage in cells or glycerol R used to
show the presence of starch and inulin.
2023 Appendix XIV V-A395

- one primer is used (this may be one used in the original

V. Deoxyribonucleic Acid (DNA) Based PCR) and binds specifically to one end of the single
Identification Techniques for Herbal stranded amplicons;
- a thermo-stable polymerase enzyme constructs the
Drugs complementary DNA sequence, starting from the primer
1. INTRODUCTION binding position;
Deoxyribonucleic Acid (DNA) barcoding has been widely - at random positions the construction of the
used as a molecular method for the identification of herbal complementary DNA sequence is halted due to the
drugs. This method uses short regions of DNA with species incorporation of a fluorescently labelled
specific sequences as barcodes for recognition. Within the di-deoxynucleotide (ddNTP);
British Pharmacopoeia, selected barcode sequences will be - amplicons are formed of every possible length, each with
the basis for any molecular identification technique. Where a a fluorescently labelled ddNTP terminal end;
DNA-based method is specified within a monograph as an - amplicons are separated by capillary electrophoresis
identification method, the identified region and its species according to their length, and the fluorescent label
specific sequence will be published as part of the monograph. recorded using a laser;
An example of a barcoding protocol is given below and - the nucleotide identity at each base position of the
contains the methodology and the sequence of the region amplicon is ascertained;
identified as specific to Ocimum tenuiflornm. - this process is repeated with another primer (this may be
DNA barcoding of plant material is achieved by a multistage one used in the original PCR) that binds specifically to
procedure involving DNA extraction, polymerase chain the opposite end of the single stranded amplicons.
reactions (PCRs) and Sanger sequencing. DNA-based 4. LABORATORY SET-UP REQUIREMENTS
identification techniques for herbal drugs utilise PCR in the Because of the high risk of contamination of the working
following two applications: materials with environmental, user or different sample DNA,
(a) to sequence a target region of DNA that contains key segregation of working areas by time and/or space is an
characters for species identification, effective method of reducing this risk. The movement of
(b) the use of a species specific PCR for a target DNA people and materials between working areas should be kept
sequence and subsequent detection of the amplicon. to a minimum and be monitored. All areas should be suitably
The principles and quality requirements for these two decontaminated before and after each use and safeguards
applications are described. Alternate methods may be used if introduced to prevent cross contamination of samples.
they comply with the quality requirements described below. Recommended working areas include:
- DNA extraction for the use of reagents and equipment
2. SCOPE OF METHOD used to extract DNA from plant materials;
To establish the requirements for DNA extraction from - Pre-PCR exclusively for the use of reagents, equipment
herbal drug material and the use of this extracted DNA for and materials that have not encountered amplicons;
either: - PCR equipment positioned in a dedicated area for the
(a) amplification and sequencing of a target region of the amplification process which occurs in a closed system;
extracted DNA for the purposes of species identification; or, - post-PCR dedicated to amplicon detection methods, as
(b) amplicon production and detection techniques to for instance gel electrophoresis and UV transillumination.
demonstrate the presence of a defined DNA sequence as 5. DNA EXTRACTION
indicated in (a) and (b) under INTRODUCTION. Pure DNA must be isolated from the herbal drug to be
3. PRINCIPLE OF THE METHOD tested. Several methods are available for this purpose,
DNA sequences for the identification of herbal drugs can be including those based on commercial kits. Representative
detected by either direct DNA sequencing or species specific samples must be taken from each batch to be tested to
amplification. Both techniques rely on the complementary ensure consistency, Appendix XI T. Herbal Drngs: Sampling
base pairing of DNA. PCR must be utilised to amplify the and Sample Preparations. DNA extraction using 200 mg of
target region. herbal drug as a superfine powder is suitable for the test.
DNA extraction should also be performed on herbal drug
KEY STAGES OF PCR ARE:
mixed with tmH-psbA BP Nucleic Acid Reference Material
- double stranded DNA is denatured at high temperature
(BPNARM). The efficiency of the extraction shall be
to form single stranded DNA;
confirmed by PCR amplification of the tmH-psbA BPNARM
- two primers bind specifically to regions flanking the
and herbal drug DNA.
sequence to be amplified, one on either strand of DNA;
- a thermo-stable polymerase enzyme constructs the Many constituents present in herbal drugs are inhibitory to
complementary DNA sequence starting from the primer enzyme driven reactions such as PCR. Care must be taken to
binding positions; ensure that DNA used for testing is pure and sufficiently free
- the three processes described above are repeated as from inhibitors. Inhibitors extracted with DNA can be
required by the protocol, (typically between 30 and removed by the use of purification procedures, an example is
40 times) to result in the formation of millions of copies given under: DNA-BASED IDENTIFICATION OF
of the sequence between the primer binding positions; OCIMUM TENUIFLORUM LINN (Holy Basil); 2.
this is the amplicon; DNA PURIFICATION.
- the amplicon is detected by various means and can be 6. AMPLIFICATION
used as the template for Sanger sequencing. PCR amplification is conducted using defined cycling
KEY STAGES OF SANGER SEQUENCING ARE: parameters, including denaturation and binding
- the amplicons are denatured at high temperature to form temperatures. Many different commercial enzymes and kits
single stranded DNA amplicons; are available which can be used once validated for the
intended purpose. When commercial kits are used the
V-A396 Appendix XIV 2023

manufacturers' guidelines for optimisation should be - re-suspend the pellet in 200 µL of ethanol (70%) at
followed. ambient temperature;
The concentrations shown below may be used as a generic - centrifuge at 14,000 revolutions per minute for
starting point for method optimisation: Polymerase buffer 10 minutes at ambient temperature;
(lx), MgC12 (2.5 mM), DNA polymerase (1 Unit), Primers - remove the supernatant liquid taking care not to disturb
(0.1 µMeach), dNTPs (0.1 µMeach), DNA (10 ng may be the pellet, allow any excess ethanol to evaporate at
suitable) and water MB. ambient temperature for at least 25 minutes or until dry.
- dissolve the pellet in 50 µL of tris-EDTA buffer pH 8.0;
To conduct a PCR the number of reactions required must be
- dilute 1 volume of the DNA solution with 9 volumes of
known. From each DNA extraction, 2 PCRs are required
tris-EDTA buffer pH 8.0 immediately prior to testing.
together with a positive and a negative control reaction.
All common components should be measured and combined 3. AMPLIFICATION
together to create a master mix, usually this will contain all Prepare as described in Appendix XIV, 6.
solutions except the DNA. The master mix is divided equally AMPIJFICATION.
between each reaction tube and the DNA is added Amplification of the trnH-psbA region of the plastid genome
individually. is achieved using the following primers and cycling
All PCR tests should include relevant positive and negative programme.
controls. To the positive control reaction validated DNA
should be added. A solution of trnH-psbA BPNARM is Primer name Sequence (5' to 3 ')
recommended. To the negative control, water MB should be tmH CGCGCATGGTGGATICACAATCC
psbA GTIATGCATGAACGTAATGCTC
added in place of the DNA.
7. DETECTION Cycling programme:
Specific amplicons may be detected based on their size or initial denaturation step at 95° for 5 minutes;
composition.
35 cycles consisting of
Detection by size can be achieved by agarose gel - 95° for 1 minute;
electrophoresis or capillary electrophoresis. - 30 seconds at touchdown temperature;
Detection by sequence can be achieved by probe - 72° for 1 minute;
hybridisation or enzyme cleavage. Amplicon size and final extension period at 72° for 7 minutes;
sequence both contribute to melt-curve analyses.
The touchdown temperature begins at 58° and is reduced by
A valid test is only achieved if the positive control reaction(s) 1° per cycle until 48°, then continued at 48° for the
gives an unambiguously positive result and the negative remainder of the program.
control reaction gives an unambiguously negative result.
If amplification is unsuccessful after purification and dilution
A positive result can be shown by the presence of a band of
of the DNA sample, secondary PCR using 1 µL of the initial
the expected size using agarose gel electrophoresis.
amplification as the template for a second is acceptable using
A negative result will be the absence of a band.
the same parameters.
8. SEQUENCING
4. AMPIJCON DETECTION
Sanger sequencing can be conducted using various
Any validated method may be used for this purpose; agarose
commercially available kits and/or service providers.
gel electrophoresis incorporating a DNA specific dye is
Any validated protocol may be followed and manufacturers'
suitable. Recommended parameters are a 1.0% w/v gel made
guidelines may be used with commercial kits. A minimum of
with 0.5x TEE buffer MB, run at 60V for 1 hour and
two reads must be produced for each DNA amplification,
subsequently visualised in an appropriate system such as a
with at least one in either direction, and assembled into a
UV transilluminator.
contig. The contig should have an overall Phred score of at
least 20; a value of 30 and over is preferable. A positive result is shown by the presence of a band of the
expected size (approximately 400 bp); a DNA ladder should
The text below is provided for information and describes the
be run adjacent to the samples to show size separation.
application of DNA-Based Identificatwn techniques to a herbal
A negative result is shown by the absence of a band at this
drug.
position on the gel.
DNA-BASED IDENTIFICATION OF OCIMUM
5. SEQUENCING
TENUIFLORUM LINN. (HOLY BASIL)
The amplicon is sequenced using the amplification primers
1. DNA EXTRACTION
and is conducted as described under Appendix XI V, 8.
Follow the procedure described in Appendix XIV, 5. SEQUENCING
DNA EXTRACTION.
6. OCIMUM TENUIFLORUM TRNH-PSBA REGION
2. DNA PURIFICATION SEQUENCE
Care should be taken to ensure that inhibitory substances The sequence of the trnH-psbA spacer region for Ocimum
commonly found in Ocimum tenuiflorum herbal drug are tenuiflorum is given below, with the bases shown in lower case
removed from DNA subsequent to extraction. Many text being the key bases for identification. These must be
methods are available for this, as for instance the method checked against all contigs produced using multiple
using propan-2-ol MB described below: alignment sofrware (Clustal Omega may be suitable).
- to 50 µL of DNA extraction solution add 35 µL of
propan-2-ol MB at 4°, mix by pipetting;
- centrifuge at 14,000 revolutions per minute for
30 minutes at 4°;
- remove the supernatant, taking care not to disturb the
pellet which may not be visible;
2023 Appendix XI V V-A397

>Ocimum_trnH-psbA_reference_sequence against all contigs produced using multiple alignment

GTTATGCATGAACGTAATGCTCATAACTTCCCTCTAGACCTAGCTGCTAT software (Clustal Omega may be suitable).
TGAAGCTCCAACAAATGGCTAAGacttgttCTTGGTGTGTAGAAGTTTTT
GAAATATAGATAAATATAAGGAGCAATAAACCCTTTCTTGTTCTATCAAA
>Anethum_graveolens_Sowa_ITS2_reference_sequence
AGAGGGTTTATTGCTCCTTTCTTTTCTTTTCAATTATGAGTcctttTCTA
TTTGCTTGCCCCAACCACTCACTCCTTGATGAGATGTGCTGGTTTTTGGG
TTTTTCTagtAGTATTGGACTTACCTAGACTTTTCTTTCCATAAAAGAAA
CGGAAATTGGCCTCCCGTGCCTTGTTGTGCGGTTGGTGCAAAAGCGAGTC
GAAGATAAAAAAAATGATTGAAATTTTATCTTTTGTTTTACAATTTTGCA
TCCGGCGTTGGACGTCGTGACATCGGTGGTTGTAAAAGACCCTCTTGACT
AAAAAAATTCAAATTGAAAAAGTGAATTCGTaaataAATAGAAAATTTTC TGTCGCACGAATCCTCGTCATCTAAGTGAGCTCTAGGACCCTTgggcacc
TTTTTAGATTTTTAGAGTAGAGGGGCGGATGTAGCCAAGTGGATCAAGGC acACAATCTGTTTGCCCTAACTGTGACCCCAGGTCAGGCG
AGTGGattgtGAATCCACCCATGCGCG

7. SEQUENCE MATCHING 7. SEQUENCE MATCHING

trnH-psbA sequences for the identification of Ocimum ITS2 sequences for the identification of Anethum graveolens
tenuijlorum samples must be a minimum of 300 bp in length, Sowa samples must be a minimum of 240 bp in length, and
and must cover the key bases for identification. Overall must cover the key bases for identification. Overall matching
matching of the sequences should be above 95%, and the key of the sequences should be above 95%, and the key bases
bases must match 100% with no gaps. must match I 00% with no gaps.
DNA-BASED IDENTIFICATION OF ANETHUM DNA-BASED IDENTIFICATION OF TRIBULUS
GRAVEOLENS SOWA TERRESTRIS FRUIT
1. DNA EXTRACTION 1. DNA EXTRACTION
Follow the procedure described in Appendix XIV, 5. Follow the procedure described in Appendix XI V, 5.
DNA EXTRACTION. DNA EXTRACTION.
2. DNA PURIFICATION 2. DNA PURIFICATION
DNA extracted from Anethum graveolens Sowa does not DNA extracted from Tribulus terrestis Fruit does not require
require further purification; however, a I: I O dilution in Tris- further purification.
EDTA buffer pH 8. 0 can enhance results. 3. AMPLIFICATION
3. AMPLIFICATION Prepare as described in Appendix XI V, 6.
Prepare as described in Appendix XI V, 6. AMPLlFICATION.
AMPLlFICATION. Amplification of the ITS2 region of the nuclear genome is
Amplification of the ITS2 region of the nuclear genome is achieved using the following primers and cycling programme.
achieved using the following primers and cycling programme.
Primer name Sequence (5' to 3 ')
Primer name Sequence (5 1 to 3 1)
ITS! Forward TCCGTAGGTGMCCTGCGG
ITS2 Forward ATGCGATACTTGGTGTGMT ITS4 Reverse TCCTCCGCTTATTGATATGC
ITS2 Reverse GACGCTTCTCCAGACTACMT
Cycling programme:
Cycling programme: - initial denaturation step at 95° for 5 minutes;
- initial denaturation step at 95° for 5 minutes; - 35 cycles consisting of
- 40 cycles consisting of - 95° for 60 seconds;
- 95° for 30 seconds; - 60° for 30 seconds;
- 56° for 30 seconds; - 72° for 60 seconds;
- 72° for 45 seconds; - final extension period at 72° for 7 minutes.
- final extension period at 72° for 10 minutes. 4. AMPLICON DETECTION
4. AMPLICON DETECTION Any validated method, which incorporates a DNA ladder,
Any validated method, which incorporates a DNA ladder, may be used for this purpose; agarose gel electrophoresis
may be used for this purpose; agarose gel electrophoresis incorporating a DNA specific dye is suitable. Recommended
incorporating a DNA specific dye is suitable. Recommended parameters are a 2.0% w/v gel made with 0.5x TBE buffer
parameters are a 2.0% w/v gel made with 0.5x TBE buffer MB, run at 60 V for I hour and subsequently visualised in
MB, run at 60 V for 1 hour and subsequently visualised in an appropriate system such as a UV transilluminator.
an appropriate system such as a UV transiltuminator. A positive result is shown by the presence of a band of the
A positive result is shown by the presence of a band of the expected size (approximately 700 bp); a DNA ladder should
expected size (approximately 480 bp); a DNA ladder should be run adjacent to the samples to show size separation.
be run adjacent to the samples to show size separation. A negative result is shown by the absence of a band at this
A negative result is shown by the absence of a band at this position on the gel.
position on the gel. 5. SEQUENCING
5. SEQUENCING The amplicon is sequenced using the amplification primers
The amplicon is sequenced using the amplification primers and is conducted as described under Appendix XI V, 8.
and is conducted as described under Appendix XIV, 8. SEQUENCING
SEQUENCING 6. TRIBULUS TERRESTRIS FRUIT ITS REGION
6. ANETHUM GRAVEOLENS SOWA ITS2 REGION SEQUENCE
SEQUENCE The sequence of the ITS region for Tribulus terrestris Fruit is
The sequence of the ITS2 region for Anethum graveolens given below, with the bases shown in lower case text being
Sowa is given below, with the bases shown in lower case text the key bases for identification. These must be checked
being the key bases for identification. These must be checked against all contigs produced using multiple alignment
software (Clustal Omega may be suitable).
V-A398 Appendix XIV 2023

>Tribulus_terrestris_ITS_reference_sequence
CACtcgygcgATGCGTTCCACGctctccaCGGGGACTTGgccaccgcgcg
tTGCTTTATCGGATCATAACAAACCCCGGCGCGGAATGCGTCAAGGAATC
TtwaaATGCGTCGGCACGGCCTTGTGACCCTATCGCAGGGCGTCAGTGCC
AGTGCACTATTactACACGAACGACTCTCGGCAACGGATATCTCGGCTCT
CGCATCGATGAAGAACGTAGCGAAATGCGATACTT

7. SEQUENCE MATCHING
Amplification of the ITS region for the identification of
Tribulus terrestris samples yields a product of approximately
700 bp, within the amplification product lies the reference
sequence which must be a minimum of 235 bp in length,
and must cover the key bases for identification. Overall
matching of the sequences should be above 95%, and the key
bases must match 100% with no gaps.
2023 Appendix XIV V-A399

GLOSSARY
This glossary of terms relating to Appendix XI V is published for information only
Table XI V-1
Term Definition
Amplicon The DNA product of a PCR.
Amplification The copying of DNA during a PCR.
Base call The identification of a DNA base by sequencing software.
Base pair (bp) The complementary pairing of two nucleotides, A&T or G&C, which forms the
unit of measurement for the length of a DNA molecule.
BPNARM British Pharmacopoeia Nucleic Acid Reference Material.
Consensus sequence The product of the combining of several individual DNA sequencing reads,
providing a consensus of the correct sequence.
Contig A set of overlapping DNA sequencing reads from one sample which can be used
to produce a consensus sequence.
Deoxynucleotide (dNTP) The monomer or individual unit of DNA; Adenine (A), Cytosine (C), Guanine
(G) and Thymine (T).
di-deoxynucleotide (ddNTP) A modified form of the DNA monomer without an -OH group present on the 3'
carbon of the deoxyribose sugar which is required to bind a subsequent
nucleotide.
DNA Deoxyribonucleic Acid, a double stranded, helical molecule.
DNA ladder Mixture of DNA molecules of known base pair length. These provide a measure
of how far a DNA molecule travels during gel electrophoresis.
Internal transcribed spacer (ITS) The spacer DNA situated between the small-subunit ribosomal RNA (rRNA)
and large-subunit rRNA genes in the chromosome or the corresponding
transcribed region in the polycistronic rRNA precursor transcript
Mix by pipetting Drawing up and expelling a substance up to ten times using an automatic
pipette, with the aim of mixing the solutions.
Master mix A mixture containing the common components for several PCRs, this is made in
a large batch or master mix which is then divided between individual reactions.
Master mixes contain enough reagents for the required number of tests, typically
plus one to allow for pipetting errors.
Mix by pipetting Drawing up and expelling a substance up to ten times using an automatic
pipette, with the aim of mixing the solutions.
PCR Polymerase Chain Reaction - an enzyme driven reaction where DNA molecules
are replicated.
Phred score The likelihood that a base call in a DNA sequence is incorrect, a score of 20 has
a 1 in 100 probability of being an incorrect call, 30 is 1 in 1000 etc.
Positive control A reaction comprising all common PCR components and a known DNA sample,
thereby proving the suitability of all reagents.
Primer (oligonucleotide) A short single stranded DNA molecule which binds to the DNA to be amplified
in a PCR. This enables the enzyme to commence replication, and therefore the
binding positions define the start and finish point of the PCR.
Probe hybridisation The complementary binding of an oligonucleotide to a target DNA molecule
causing a measurable response.
Sanger sequencing The method by which a DNA sequence is resolved, developed by Frederick
Sanger and colleagues.
Sequencing Identifying the order of the nucleotide sequence of DNA.
TBE buffer MB Tris-borate-EDTA Buffer Solution pH 8.4 MB
Thermal cycler The machine that performs the cycling of temperatures required for a PCR.
Water MB Deionised, filtered and autoclaved water.
V-A400 Appendix XI W 2023

different diluted reference solution, e.g. 'Rl/20', may be

W. High-Performance Thin-Layer prescribed instead). Both solutions are used as intensity
Chromatography of Herbal Drugs and references.
Herbal Drug Preparations Intensity marker
Use one or more of the substances in the reference solution
(Ph. Bur. method 2.8.25) and in the diluted reference solution as intensity marker(s)
High-performance thin-layer chromatography (HPTLC) is for the evaluation of the chromatogram.
used for qualitative analysis of herbal drugs and herbal drug Preparation of system suitability test solution
preparations. It is a thin-layer chromatographic technique Prepare the system suitability test (SST) solution as stated in
(2.2.27) that usually uses a glass plate coated with a uniform, the individual monograph.
porous layer (average pore size 6 nm), typically 200 µm Sample application and plate layout
thick, of irregular particles of silica gel between 2 µm and Samples are usually applied as narrow bands 8 mm in length
10 µm in size and with an average size of 5 µm, a polymeric at a distance of 8 mm from the lower edge of the plate.
binder and a fluorescence indicator (F254). The results are The centre of the first track, which is used for the system
qualified using a system suitability test. suitability test solution, is positioned 20 mm from the left
EQUIPMENT edge of the plate. The minimum distance between tracks
The equipment used for qualitative HPTLC typically consists (centre to centre) is 11 mm. A maximum of 15 tracks are
of: applied onto a standard plate. If no electronic solvent front
- glass plates, as described above, usually 20 x 10 cm in detection device is used, the development distance is marked
size; with a pencil close to the right or left edge of the plate
- devices suitable for the application of specified volumes of Conditioning of the plate
solutions as bands and allowing control of the dimensions Following sample application and unless otherwise stated in
and position of application; the individual monograph, expose the plate to air with a
- a device suitable for conditioning the stationary phase at suitable relative humidity obtained using a saturated solution
the prescribed relative humidity; of magnesium chloride R (for example, by allowing the plate to
- a suitable chromatographic tank (for example, a twin stand in a closed chamber containing such a solution for 1 h
trough chamber); or by using preconditioned air).
- a device suitable for the reproducible drying of the
Preparation of the tank and development of the plate
developed plate;
Unless otherwise stated in the individual monograph, the
- devices suitable for the application of reagents to, and
chromatographic separation is performed in a saturated tank.
heating of, the plate as part of the derivatisation
Where a twin trough chamber is used, place a piece of filter
procedure;
paper in the rear trough. Load the tank with a sufficient
- a system suitable for the electronic documentation of
quantity of mobile phase to wet the filter paper completely
chromatograms under 254 nm UV, 366 nm UV and
and achieve a level of 5 mm in both troughs. With the lid
white light.
closed, leave the tank for 20 min for saturation. Introduce
NOTE Normal thin-layer chromatographic methods using the plate in a vertical position into the front trough of the
glass plates or sheets coated with particles of 5-40 µm or tank so that the coating layer faces the filter paper. When the
HPTLC aluminium-backed sheets may be used, provided mobile phase has reached the prescribed distance (usually
that the results obtained fulfil the general system suitability 70 mm from the lower edge of the plate), remove the plate
criteria that the bands develop perpendicular to the lower from the tank and dry in a vertical position in a stream of air
edge of the plate and the solvent front is parallel to the upper at room temperature. Other tank configurations and
edge of the plate, and satisfy the system suitability test stated development distances may be specified in an individual
in the individual monograph. monograph.
METHOD NOTE Other tanks may be employed if the results
Preparation of test solution obtained fulfil all of the system suitability criteria.
The test solution is prescribed in the individual monograph Visualisation
and is usually prepared as follows. Chromatograms on the plate are visualised as stated in the
For dry herbal drugs or dry herbal extracts, mix 0.5 g of the individual monograph under Detection. Where derivatisation
powdered herbal drug or 0.1 g of the dry herbal extract with reagents are used, typically 3.5 mL of reagent solution is
5 .0 mL of methanol R and sonicate for 15 min; filter or homogenously sprayed onto a plate of size 20 x I O cm, or
centrifuge and use the filtrate or supernatant as the test the plate is immersed into the reagent solution, typically at a
solution. speed of 5 mm/s for a dwell time of I s. Observation may be
For essential oils, dissolve 50 µL of the essential oil in performed under 254 nm UV, 366 nm UV or white light
1.0 mL of toluene R and use this solution as the test solution. prior to and/or after derivatisation. When pictures are
Preparation of reference solutions digitally recorded, exposure time should be adjusted based on
Reference solutions are prescribed in the individual the track used for the system suitability test solution.
monograph and are usually prepared as follows. Prepare a System suitability test
1 mglmL solution (identified as 'R' in the chromatograms This test is based on the separation of 2 substances that have
associated with the monograph) of suitable reagent(s) or similar retardation factors (RF values) but that are barely
reference standard(s) in methanol R or, for essential oils, in separable under the specified chromatographic conditions
toluene R. Prepare a second reference solution (diluted (for example, chlorogenic acid and hyperoside in
reference solution, identified as 'Rl/4' in the chromatograms chromatographic systems used for flavonoids). The results for
associated with the monograph) by mixing I volume of this the test and reference solutions are only valid when the
solution and 3 volumes of the same solvent (in some cases a
2023 Appendix XI X V-A401

system suitability test solution satisfies the separation considered as a unit because of common characteristics
requirement stated in the individual monograph. relevant for the intended analysis, as demonstrated by
Visual evaluation performing the analytical procedure used.
The chromatograms obtained with the test and reference Once an analytical procedure has been shown to meet the
solutions are compared with the descriptions in the results validation requirements for a representative matrix
section of the individual monograph, with respect to zone (e.g. peppermint leaf), the procedure may be assumed to be
position and colour, as well as intensity for the test solution. valid for any other matrix belonging to the corresponding
Zones of the test solution described as 'equivalent' or without matrix group (e.g. leaf). To allocate a new sample to a
an indication of intensity have intensities similar to the zone validated matrix group, the analyst must perform an
of the intensity marker in the reference solution (R). Zones assessment to decide, subject to approval by the competent
described as 'intense' are visually more intense than the zone authority, whether confirmation of validity of the analytical
of the intensity marker in the reference solution; zones procedure for the new sample is necessary. The validity is
described as 'faint' are visually less intense than the zone of confirmed in routine analysis by performing additional
the intensity marker in the reference solution, but equal to or measurements to demonstrate that the verification
more intense than the zone of the intensity marker in the requirements specified in Table 2.8.26.-3 are met. This
diluted reference solution (Rl/4, Rl/20, etc.); zones applies in particular to potentially interfering samples, which
described as 'very faint' are visually less intense than the zone are identified on the basis of sound scientific judgement,
of the intensity marker in the diluted reference solution. i.e. analytical judgement with a consideration of the
interferences that could occur.
For instance, following the above example, an analytical
procedure may be assumed to be valid for any 'leaf sample
X. Contaminant Pyrrolizidine Alkaloids after it has been shown to meet the validation requirements
for a 'peppermint leaf sample. Then, to allocate new 'leaf
(Ph. Bur. method 2.8.26)
samples that are not expected to contain interfering
INTRODUCTION compounds (e.g. polyphenols) to the validated 'leaf matrix
Pyrrolizidine alkaloids (PAs) are nitrogen-containing group, the analyst does not need to confirm (subject to
compounds that occur naturally in plants. Several hundred approval by the competent authority) in routine analysis that
structurally distinct PAs have been found in several thousand the verification requirements are met with these samples.
different plant species. Many of these plants are common Conversely, to allocate 'green tea leaf samples (which
weeds, which can contaminate raw plant materials used for contain polyphenols) and 'peppermint leaf dry extract'
the production of medicinal products. This results in samples (which contain the matrix at a higher concentration
contamination of raw plant materials by PAs, usually at very producing interfering matrix effects) to the validated 'leaf
low levels. This general chapter covers trace analysis of target matrix group, the analyst needs to confirm in routine analysis
PAs in herbal drugs, preparations thereof and medicinal that the verification requirements are met with these samples.
products. It does not cover the determination of PAs that
SAMPLE PREPARATION
occur naturally in uncontaminated plant materials.
The sample to be analysed must be representative of the
Target PAs The target PAs are echimidine, echimidine batch of material being examined. Due to problems of
N-oxide, erucifoline, erucifoline N-oxide, europine, europine inhomogeneity caused by spot contamination, it is important
N-oxide, heliotrine, heliotrine N-oxide, intermedine, to ensure that the sample has a uniform particle size and a
intermedine N-oxide, jacobine, jacobine N-oxide, homogeneous distribution of PAs or PA-containing material.
lasiocarpine, lasiocarpine N-oxide, lycopsamine, lycopsamine The selection of the appropriate sample preparation depends
N-oxide, monocrotaline, monocrotaline N-oxide, retrorsine, on the material being examined and is the responsibility of
retrorsine N-oxide, senecionine, senecionine N-oxide, the analyst, who may use any appropriately validated sample
seneciphylline, seneciphylline N-oxide, senecivernine, preparation procedure, including, but not limited to, the
senecivernine N-oxide, senkirkine and trichodesmine. following procedures.
Limits Patient exposure to PAs from medicinal products Herbal drugs and herbal teas Grind at least 20 g to a
should be as low as possible and must not exceed the particle size of about 0.25 mm or less, using a centrifugal
maximum daily intake agreed by the competent authority. mill, and homogenise using an overhead shaker. If the test
PRINCIPLE material cannot be ground in this way, it can be mixed with
As there is considerable variation in the composition of the dry ice (ratio 2:1) or liquid nitrogen, or the sample amount
herbal drugs, preparations thereof and medicinal products may be increased, and ground to a particle size of about
concerned, and in the applicable limits, it is difficult to 0.5 mm or less.
describe all the methods suitable for quantitative analysis of Dry extracts Dry extracts may be examined as such.
the target PAs. This general chapter therefore permits the use If necessary, comminute to a suitable particle size.
of any procedure consisting of chromatography coupled with Powdered herbal drugs compressed into tablets
MS/MS (see Glossary) or high-resolution MS (see Glossary) Comminute to a suitable particle size.
that meets the validation requirements specified in
Liquid products Mix homogeneously and filter or
Table 2.8.26.-2. As an example, it describes an analytical centrifuge if necessary.
procedure that has been shown to be suitable for the
determination of target PAs in a number of matrices. PROCEDURE
Whatever the analytical procedure selected, including the The fallowing procedure is given as an example.
procedure given as an example, the analyst must first confirm Liquid chromatography (2.2.29) coupled with mass
that these validation requirements are met, using at least one spectrometry (2. 2.43).
representative matrix from each matrix group analysed. This procedure has been validated for the fallowing matrices:
A matrix group comprises a number of matrices that are chamomile flower, melissa tea (containing melissa leaf, aniseed
V-A402 Appendix XI X 2023

fruit and peppermint leaf), rooibos tea (containing rooibos leaf, Standard solution (g) Mix 280 µL of the matrix blank
vanilla fruit and vanilla extract) and another mi:xed herbal tea solution and 20 µL of standard solution (a).
(containing rooibos leaf, blackberry leaf, lemon verbena leaf, Standard solution (h) Mix 280 µL of the matrix blank
peppermint leaf, chamomile flower, fennel fruit, liquorice root and solution and 20 µL of standard solution (d).
cinnamon bark). The working range must be appropriate to the Column:
levels of interest; the working range here is given as an example. - size: l = 0.15 m, 0 = 2.1 mm;
Test solution Proceed as directed under Sample - stationary phase: end-capped octadecylsi[yl silica gel for
preparation. To 2.0 g of the sample add 20.0 mL of a chromatography R (1.9 µm);
0.28 per cent V/V solution of sulfuric acid R. The sample - temperature: 40 °C.
must be wetted completely. Extract in an ultrasonic bath for Mobile phase:
15 min and centrifuge at 3800 g for 10 min. Decant the - mobile phase A: dissolve 315 mg of ammonium formate R in
supernatant. Repeat the extraction once, using the residue about 5 mL of water for chromatography R, mix with 1 mL
obtained in the centrifugation step. Combine the of formic acid R and dilute to 1000 mL with water for
supematants and neutralise to pH 7 .0 with a 20 per cent V/V chromatography R;
solution of concentrated ammonia R. Filter. In a vacuum - mobile phase B: dissolve 315 mg of ammonium formate R in
chamber, condition a 6 mL solid phase extraction (SPE) about 5 mL of water for chromatography R, mix with 1 mL
column containing 0.5 g of end-capped octadecylsi[yl silica gel of formic acid R and dilute to 1000 mL with methanol R;
for chromatography R (20 µm) with 5 mL of methanol R and
then with 5 mL of water R. Transfer 10.0 mL of the filtrate Time Mobile phase A Mobile phase B
to the SPE column. Wash the column with 2 quantities, each (min) (per cent V/V) (per cent V/V)
of 5 mL, of water R and dry the cartridge in the vacuum 0 - 0.5 95 5
chamber. Elute using 10 mL of methanol R. Evaporate the 0.5 - 7 95 ➔ 50 5--> 50
eluate to dryness in a water-bath at 50 ± 5 °C under a 7 - 7.5 50 ➔ 20 50--> 80
stream of nitrogen R. Dissolve the residue in 1.0 mL of a 7.5 - 7.6 20--> 0 80 --> 100
5 per cent V/V solution of methanol R with shaking. Filter 7.6 - 10.1 0 100
through a membrane filter (nominal pore size 0.20 µm).
Stock solution (a) Dissolve 1.0 mg of echimidine R, Flow rate 0.3 mUmin.
1.0 mg of echimidine N-oxide R, 1.0 mg of erucifoline R,
1.0 mg of erucifoline N-oxide R, 1.0 mg of europine Detection Triple-quadrupole mass spectrometer in selected
hydrochloride R, 1.0 mg of europine N-oxide R, 1.0 mg of reaction monitoring (SRM) mode. For PA identification and
heliotrine R, 1.0 mg of heliotrine N-oxide R, 1.0 mg of quantification, at least 2 specific transitions are necessary.
intermedine N-oxide R, 1.0 mg ofJacobine R, 1.0 mg of The relevant transitions, the collision energies (CEs) and the
Jacobine N-oxide R, 1.0 mg of lasiocarpi,ne R, 1.0 mg of retention times obtained with the described LC-MS/MS
lasiocarpine N-oxide R, 1.0 mg of {vcopsamine N-oxide R, conditions are given in Table 2.8.26.-1 for all target PAs.
1.0 mg of monocrotaline R, 1.0 mg of monocrotaline N-oxide R, The following settings have been found to be suitable and are
1.0 mg of retrorsine R, 1.0 mg of retrorsine N-oxide R, 1.0 mg given as examples; if the detector has different setting
of senecionine N-oxide R, 1.0 mg of seneciphylline N-oxide R, parameters, adjust the detector settings so as to comply with
1.0 mg of senecivernine R, 1.0 mg of senecivernine N-oxide R, the system suitability criterion:
1.0 mg of senkirkine R and 1.0 mg of trichodesmine R in - ionisation: ESl-positive;
methanol R and dilute to 10.0 mL with the same solvent. - ion spray voltage: 3.5 kV;
- capillary temperature: 270 °C;
Stock solution (b) Dissolve 1.0 mg of senecionine Rand - nebuliser temperature: 300 °C;
1.0 mg of seneciphylline R in about 8 mL of methanol R in a - sheath gas pressure: 310 kPa;
water-bath at about 55 °C. Allow to cool and dilute to - ion sweep gas pressure: 14 kPa;
10.0 mL with methanol R. - auxiliary gas pressure: 69 kPa.
Stock solution (c) Dissolve 1. 0 mg of intermedine R and Injection I O µL of the test solution and standard
1. 0 mg of lycopsamine R in acetonitrile R and dilute to solutions (a), (b), (c), (d), (e), (t), (g) and (h).
10.0 mL with the same solvent.
System suitability Standard solution (h):
Stock solution (d) Mix 1.0 mL of stock solution (a), - signal-to-noise ratio: minimum 10 for each peak due to
1.0 mL of stock solution (b) and 1.0 mL of stock each target PA in the corresponding extracted ion
solution (c) and dilute to 100.0 mL with methanol R. chromatogram (total ion current of the SRM transition
Matrix blank solution See Glossary. used for quantification).
Standard solution ( a) Mix 340 µL of the matrix blank Calibration function For each target PA, determine the
solution and 60 µL of stock solution (d). values of a and b using a least-squares fit:
Standard solution (b) Mix 175 µL of the matrix blank
solution and 25 µL of stock solution (d). y=ax+b
Standard solution (c) Mix 180 µL of the matrix blank
y peak areas of the corresponding target PA in the extracted ion
solution and 20 µL of stock solution (d). chromatograms (total ion current of the corresponding SRM
Standard solution (d) Mix 185 µL of the matrix blank transition used for quantification), obtained with standard
solutions (a), (b), (c), (d), (e), (f), (g) and (h);
solution and 15 µL of stock solution (d).
a slope of the resulting calibration function;
Standard solution (e) Mix 100 µL of the matrix blank X concentrations of the corresponding target PA in standard
solution and 10 µL of stock solution (d). solutions (a), (b), (c), (d), (e), (f), (g) and (h), in micrograms
per litre;
Standard solution (j) Mix 100 µL of the matrix blank b y-intercept of the resulting calibration function.
solution and 20 µL of standard solution (a).
2023 Appendix XI X V-A403

Table 2.8.26.-1. - Relevant transitions, collision energies and retention times for the target PAs
Target PA Precursor ion (Da) S-Lens (V) Product ion (Da) CE (V) Retention time (min)

120.1 36

Monocrotaline 326.2 129 194.1 29 4.25

280.1 23

120.3 32

Erucifoline 350.2 110 138.1 30 4.87

322.2 23

118.3 37

Monocrotaline N-oxide 342.1 90 120.2 34 4.99

137.2 37

136.1 30

Erucifoline N-oxide 366.1 129 120.1 33 5.16

118.1 34

120.1 36

Jacobine 352.1 120 155.2 29 5.25

280.1 22

138.1 20

Europine 330.1 89 156.2 28 5.34

254.1 16

120.2 24

Intermedine 300.1 112 138.3 18 5.40

156.3 28

120.2 18

Lycopsamine 300.1 112 138.2 28 5.50

156.3 28

120.1 32

Jacobine N-oxide 368.1 110 296.1 23 5.55

324.0 26

172.1 32

Europine N-oxide 346.1 91 270.1 35 5.63

328.1 23

111.2 40

Intermedine N-oxide 316.1 95 138.1 21 5.91

172.1 29

111.2 40

Lycopsamine N-oxide 316.1 95 138.1 21 6.02

172.1 29

120.3 31

Retrorsine 352.2 120 138.3 30 6.32

324.2 28

120.3 39

Trichodesmine 354.2 109 222.2 30 6.37

308.2 24
V-A404 Appendix XI X 2023

Target PA Precursor ion (Da) S-Lens (V) Product ion (Da) CE (V) Retention time (min)

136.2 36

Retrorsine N-oxide 368.2 110 118.2 33 6.41

120.1 36

120.3 29

Seneciphylline 334.2 100 138.4 29 6.56

306.2 26

138.3 22

Heliotrine 314.2 91 156.3 28 6.72

120.3 34

118.2 32

Seneciphylline N-oxide 350.2 110 136.3 33 6.79

120.3 33

136.1 32

Heliotrine N-oxide 330.2 110 138.1 35 7.03

172.2 29

138.2 30

Senecivemine 336.2 135 120.2 30 7.26

308.2 27

138.2 30

Senecionine 336.2 135 120.2 30 7.33

308.2 27

118.1 32

Senecivemine N-oxide 352.1 110 120.1 35 7.42

136.3 34

118.1 32

Senecionine N-oxide 352.2 110 120.1 35 7.54

136.3 34

254.1 32

Echimidine N-oxide 414.2 129 352.1 24 8.01

396.2 25

120.3 26

Echimidine 398.2 112 220.3 18 8.02

238.2 19

122.3 32

Senkirkine 366.2 106 150.3 27 8.19

168.2 30

120.2 28

Lasiocarpine 412.2 94 336.3 19 8.99

220.2 19

136.1 32

Lasiocarpine l\l-oxide 428.1 110 254.1 30 9.33

352.3 25
 2023 Appendix XI X V-A405

Table 2.8.26.-2. - Validation requirements

Validation parameter (to be assessed for each target PA in the corresponding extracted ion chromatograms) Requirement

Position of the peaks due to at least 2 product ions acquired in SRM or

MRM mode and obtained with a spiked matrix sampleC 1l at least at the fully overlap
limit of quantitarion (LOQ)
MS/MS
Difference in ion ratioCI) between a spiked matrix sampleCll and a reference
solution, both at least at the LOQ
maximum ± 30 per cent

Position of the peaks due to at least 2 ions(2 ) obtained with a spiked matrix
fully overlap
sample<') at least at the LOQ
ldentificatzon
maximum S ppm for ions with
Mass accuracy(!) of each of at least 2 ionsC 2l
obtained with a spiked matrix masses 2- 200 Da
High-resolution MS
sample(!) at least at the LOQ
maximum 1 mDa for ions with
masses < 200 Da

Signal-to-noise ratio of each of at least 2 ions(Z) obtained with a spiked

minimum 3( 3 )
matrix sample(!) at least at the LOQ

Difference in response between reference solutions and matrix-matched standard solutions within
Mairix effect
the working rangeC 1l, at one or more concentration points chosen by the analyst
maximum ± 20 per cent

Difference in retention time between spiked matrix samples(l) and reference solutions within the
working range(!) (applicable if the identification criteria in this table are met), at one or more maximum ± 0.1 min
concentration points chosen by the analyst
Specificity
maximum 30 per cent of the
Difference in response of each interfering peak between matrix blank solution and solvent blank(!)
LOQ

Deviation of the concentration of the calibration standards (reference solutions or matrix-matched

Linearity standard solutions) calculated by the calibration function, from the true concentration, for at least maximum ± 20 per cent
5 concentrations covering the working rangeCIJ

Percentage recovery obtained with spiked matrix samples Cl) for a minimum of 3 concentrations
Accuracy within the working rangeCIJ (the lowest representing the LOQ) and with at least 3 determinations at 70-120 per centC4 J
each of these concentrations

Relative standard deviation (RSD), obtained with spiked matrix samples 0 l, for a minimum of
Repeatability 3 concentrations within the working range(!) (the lowest representing the LOQ) and at least maximum 20 per cent
3 determinations at each of these concentrations

Limit of Signal-to-noise ratio, obtained with a spiked matrix sample(]) at the lowest concentration in the
minimum 10
quantitatwn (LOQ/5! working rangeC 1 l (applicable if the accuracy and repeatability criteria in this table are met)

(IJ See Glossary.

C2J At least 2 ions, preferably including the protonated molecule, detected in full scan mode or full scan mode (MS I data) in combination with fragmentation
modes (MS 2 data). Satisfactory measurement accuracy can be obtained in both cases by operating over an mlz range of 150-600.
(,) If noise is absent, a signal should be present in at least 5 subsequent scans.
C4J In exceptional cases, mean recovery rates outside of 70-120 per cent can be accepted if they are consistent (i.e. RSD :S 20 per cent) and the basis for this is
well established (e.g. due to analyte distribution in a partitioning step), but the mean recovery must not be lower than 30 per cent or above 140 per cent.
Howeveri in these cases the correction for recovery is required.
l 5J Alternative methods to check the LOQ may be used, if scientifically justified and approved by the competent authority.

Calculate the sum of target PAs in the sample to be matched standard solutions (see Glossary) or with the
examined, in micrograms per kilogram (ppb): standard addition method.
Method reproducibility is demonstrated according to the
relevant quality systems of the analyst's laboratory, subject to
approval by the competent authority.
VERIFICATION REQUIREMENTS
A,, peak area of the nm target PA in the extracted ion If PA-free samples are not available and therefore spiked
chromatogram (total ion current of the corresponding SRM matrix samples (see Glossary) cannot be prepared, then
transition used for quantification), obtained \vith the test
solution;
verification can be carried out with spiked samples (see
b., y-intercept of the corresponding calibration function; Glossary), correcting for the corresponding PA content.
a,, slope of the corresponding calibration function; In addition, for the second specificity parameter in the table,
m mass of the sample to be analysed used to prepare the test a 'non-PA-free matrix blank solution' can be used instead of
solution, in grams.
a matrix blank solution (except for those target PAs that are
If stereoisomeric pairs co-elute, calculate their content as a present and identified in the 'non-PA-free matrix blank
sum. solution'). The suitability of the spiked samples and 'non-PA-
free matrix blank solutions' (depending on the content level
VALIDATION REQUIREMENTS
of target PAs) used is the responsibility of the analyst.
Matrix effects may be reduced by diluting test solutions. If an
analytical procedure using calibration with external standards GLOSSARY
(i.e. using reference solutions (see Glossary)) fails the matrix High-resolution MS
effect criterion, the analyst may address signal suppression or For example, Q-TOF. All ion fragmentation (AIF), variable
enhanced matrix effects by using calibration with matrix- data-independent acquisition (vDWSWATH), collision cross
V-A406 Appendix XI X 2023

Table 2.8.26.-3. - Verification requirements

Verification parameter (to be assessed for each target PA in the corresponding extracted ion chromatograms) Requirement

Position of the peaks due to at least 2 product ions acquired in SRM or

fully overlap
MRM mode and obtained with a spiked matrix sample at least at the LOQ
MS/MS
Difference in ion ratio between a spiked matrix sample and a reference
maximum ± 30 per cent
solution, both at least at the LOQ

Position of the peaks due to at least 2 ions(I) obtained with a spiked matrix
fully overlap
sample at least at the LOQ
ldemification
maximum 5 ppm for ions with
masses 2: 200 Da
Mass accuracy of each of at least 2 ions(IJ obtained with a spiked matrix
High-resolution MS
sample at least at the LOQ
maximum 1 mDa for ions with
masses < 200 Da

Signal-to-noise ratio of each of at least 2 ions(IJ obtained with a spiked

minimum 3C 2l
matrix sample at least at the LOQ

Difference in retention time between spiked matrix samples and reference solutions within the
working range (applicable if the identification criteria in this table are met), at one or more maximum ± 0.1 min
concentration points chosen by the analyst
Specificity
maximum 30 per cent of the
Difference in response of each interfering peak between matrix blank solution and solvent blank
LOQ

Percentage recovery, obtained with a spiked matrix sample, at the specified limit, or at 10 times
Accuracy the lowest concentration in the working range (i.e. 10 times the LOQ), with at least 60-140 per cent(3 )
3 determinations

Relative standard deviation, obtained with a spiked matrix sample, at the specified limit, or at
Repeatability 10 times the lowest concentration in the working range (i.e. 10 times the LOQ), with at least maximum 20 per cent
3 determinations

(JJ At least 2 ions, preferably including the protonated molecule, detected in full scan mode or full scan mode (MS 1 data) in combination with fragmentation
modes (MS 2 data). Satisfactory measurement accuracy can be obtained in both cases by operating over an mlz range of 150-600.
2
C l If noise is absent, a signal should be present in at least 5 subsequent scans.
C3l In exceptional cases, mean recovery rates outside of 60-140 per cent can be accepted if they are consistent (i.e. RSD ~ 20 per cent) and the basis for this is
well established (e.g. due to analyte distribution in a partitioning step), but the mean recovery must not be lower than 30 per cent or above 140 per cent.
However, in these cases the correction for recovery is required.

section (CCS/IMS) or high-energy tracking (MSE/HD- primary ionisation process is isolated, fragmented usually by
MRM) can be used. collision and defined product ions are detected.
Ion ratio PA-free matrix
Intensity (or peak area) ratio of the less-intense ion selected A matrix where the content of each target PA is less than the
to that of the more-intense ion selected. corresponding limit of detection.
Mass accuracy Reference solution
Deviation of the measured mass from the theoretical mass of PA reference standard(s) dissolved in an appropriate solvent.
an ion (true value). It can be expressed: Solvent blank
as an absolute value (in mDa): measured mass - theoretical The solvent used to prepare the standard/reference solutions.
mass;
Spiked matrix sample
as a relative value (in ppm):
An appropriate PA-free matrix spiked with PA reference
measured mass - theoretical mass solution, then treated in the same manner as the sample to
, X 106 be examined, e.g. extracted and dissolved in the same solvent
theoretical mass
as that used to prepare the test solution.
Matrix blank solution Spiked sample
An appropriate PA-free representative matrix that has been An appropriate non-PA-free matrix spiked with PA reference
treated in the same manner as the sample to be examined, solution, then treated in the same manner as the sample to
e.g. extracted and dissolved in the same solvent as that used be examined, e.g. extracted and dissolved in the same solvent
to prepare the test solution. as that used to prepare the test solution.
Matrix-matched standard solution Working range
An appropriate PA-free representative matrix that has been The interval between the upper and lower concentrations
treated in the same manner as the sample to be examined (both included) of the analytes (i.e. PAs) in the sample for
then spiked with PA reference solution. which it has been demonstrated that the analytical procedure
MS/MS has a suitable level of repeatability, accuracy and linearity.
For example, tandem mass spectrometer/triple quadrupole, The working range must at least cover the range from the
ion trap or Q-trap. Tandem mass spectrometry (MS/MS or reporting threshold to 5 times this threshold. The reporting
MS2) in selected or multiple reaction monitoring (SRM, threshold depends on the maximum daily intakes of total
MRM) mode. An MS procedure in which a defined P As agreed by the competent authority and the maximum
precursor ion (in most cases the molecular ion) from the daily dose of the material analysed.
2023 Appendix XII A V-A407

Discs The use of discs is permitted only where specified or

Appendix XII allowed. Each tube is provided with a cylindrical disc
9.35-9.65 mm thick and 20.55-20.85 mm in diameter.
A. Disintegration The disc is made of a suitable, transparent plastic material
1. Disintegration of Tablets and Capsules 1 having a specific gravity of 1.18-1.20. 5 parallel 1.9-2.1 mm
holes extend between the ends of the cylinder. One of the
(Ph. Bur. method 2. 9.1)
holes is centred on the cylindrical axis. The other holes are
This test is provided to determine whether tablets or capsules parallel to the cylindrical axis and centred 5.8-6.2 mm from
disintegrate within the prescribed time when placed in a the axis on imaginary lines perpendicular to the axis and to
liquid medium under the experimental conditions presented each other, as defined in Figure 2.9.1.-1. 4 identical
below. trapezoidal-shaped planes are cut into the wall of the
For the purposes ofthis test, disintegration does not imply cylinder, nearly perpendicular to the ends of the cylinder.
complete dissolution of the unit or even of its active The trapezoidal shape is symmetrical; its parallel sides
constituent. Complete disintegration is defined as that state coincide with the ends of the cylinder and are parallel to an
in which any residue of the unit, except fragments of imaginary line connecting the centres of 2 adjacent holes
insoluble coating or capsule shell, remaining on the screen of 6 mm from the cylindrical axis. The parallel side of the
the test apparatus or adhering to the lower surface of the trapezoid on the bottom of the cylinder has a length of
discs, if used, is a soft mass having no palpably firm core. 1.5-1.7 mm and its bottom edges lie at a depth of
♦Use Test A for tablets and capsules that are not greater 1.5-1.8 mm from the cylinder's circumference. The parallel
than 18 mm long. For larger tablets or capsules use Test B. ♦ side of the trapezoid on the top of the cylinder has a length
of9.2-9.6 mm and its centre lies at a depth of2.5-2.7 mm
TEST A - TABLETS AND CAPSULES OF NORMAL from the cylinder's circumference. All surfaces of the disc are
SIZE smooth.
Apparatus
If the use of discs is specified, add a disc to each tube and
The apparatus consists of a basket-rack assembly, a 1 L, low-
operate the apparatus as directed under Procedure. The discs
form beaker, 138-160 mm in height and having an inside
conform to the dimensions shown in Figure 2.9.1.-1.
diameter of 97-115 mm for the immersion fluid, a
thermostatic arrangement for heating the fluid at 35-39 °C, The use of automatic detection employing modified discs is
and a device for raising and lowering the basket in the permitted where the use of discs is specified or allowed. Such
immersion fluid at a constant frequency rate of 29-32 cycles discs must comply with the requirements of density and
per minute, through a distance of 53-57 mm. The volume of dimension given in this chapter.
the fluid in the vessel is such that at the highest point of the Procedure
upward stroke the wire mesh remains at least 15 mm below Place 1 dosage unit in each of the 6 tubes of the basket and,
the surface of the fluid, and descends to not less than 25 mm if prescribed, add a disc. Operate the apparatus using the
from the bottom of the vessel on the downward stroke. At no specified medium as the immersion fluid, maintained at
time should the top of the basket-rack assembly become 37 ± 2 °C. At the end of the specified time, lift the basket-
submerged. The time required for the upward stroke is equal rack assembly from the fluid and observe the dosage units: all
to the time required for the downward stroke, and the of the dosage units have disintegrated completely. If 1 or
change in stroke direction is a smooth transition, rather than 2 dosage units fail to disintegrate, repeat the test on
an abrupt reversal of motion. The basket-rack assembly 12 additional dosage units. The requirements of the test are
moves vertically along its axis. There is no appreciable met if not fewer than 16 of the 18 dosage units tested have
horizontal motion or movement of the axis from the vertical. disintegrated.
Basket-rack assembly The basket-rack assembly consists ♦TEST B - LARGE TABLETS AND LARGE
of 6 open-ended transparent tubes, each 75.0-80.0 mm long CAPSULES
and having an inside diameter of20.7-23.0 mm and a wall Apparatus
1.0-2.8 mm thick; the tubes are held in a vertical position by The apparatus consists of a basket-rack assembly, a 1 L, low-
2 plates, each 88-92 mm in diameter and 5.0-8.5 mm in form beaker, 138-160 mm in height with an inside diameter
thickness, with 6 holes, each 22-26 mm in diameter, of 97-115 mm for the immersion fluid, a thermostatic
equidistant from the centre of the plate and equally spaced arrangement for heating the fluid at 35-39 °C, and a device
from one another. Attached to the under surface of the lower for raising and lowering the basket in the immersion fluid at
plate is a woven stainless steel wire cloth, which has a plain a constant frequency rate of 29-32 cycles per minute,
square weave with 1.8-2.2 mm mesh apertures and with a through a distance of 53-57 mm. The volume of the fluid in
wire diameter of 0.57-0.66 mm. The parts of the apparatus the beaker is such that at the highest point of the upward
are assembled and rigidly held by means of 3 bolts passing stroke the wire mesh remains at least 15 mm below the
through the 2 plates. A suitable means is provided to surface of the fluid, and descends to not less than 25 mm
suspend the basket-rack assembly from the raising and from the bottom of the beaker on the downward stroke.
lowering device using a point on its axis. At no time should the top of the basket-rack assembly
The design of the basket-rack assembly may be varied become submerged. The time required for the upward stroke
somewhat provided the specifications for the glass tubes and is equal to the time required for the downward stroke, and
the screen mesh size are maintained. The basket-rack the change in stroke direction is a smooth transition, rather
assembly conforms to the dimensions shown in than an abrupt reversal of motion. The basket-rack assembly
Figure 2.9.1.-1. moves vertically along its axis. There is no appreciable
horizontal motion or movement of the axis from the vertical.
Basket-rack assembly The basket-rack assembly consists
of 3 open-ended transparent tubes, each 75.0-80.0 mm long
1 This chapter has undergone pharmacopoeia! harmonisation. See chapter
with an inside diameter of 32.5-33.5 mm and a wall
5. 8 Pharmacopoeia/ harmonisation.
V-A408 Appendix XII A 2023

Basket-rack
assembly

Disc

2.6 ± 0.1
Top
view
I/)
N
+I
~
r--
r--

1---9_0±_2 --im 11 mHi ''lci

Side
view

+I
0
I/)
a;

c:i

~
N
+I Bottom
'SI" view
N

Figure 2.9.1.-1. -Apparatus for Disintegration Test A

Dimensions in millimetres

2.0-3.0 mm thick. The tubes are held in a vertical position upper plate to enable the assembly to be attached to a
by 2 separate and superimposed rigid plastic plates, each mechanical device.
95-99 mm in diameter and 7.5-10.5 mm in thickness, with 3 The design of the basket-rack assembly may be varied
holes. The holes are equidistant from the centre of the plate somewhat provided the specifications for the glass tubes and
and equally spaced from one another. Attached to the under the screen mesh size are maintained. The basket-rack
surface of the lower plate is a woven stainless steel wire cloth, assembly conforms to the dimensions shown in
which has a plain square weave with mesh apertures of Figure 2.9.1.-2.
1.8-2.2 mm and with a wire diameter of 0.60-0.66 mm.
Discs The use of discs is permitted only where specified or
The plates are held firmly in position by vertical metal rods
allowed. Each tube is provided with a cylindrical disc
at the periphery. A metal rod is also fixed to the centre of the
15.15-15.45 mm thick and 31.27-31.53 mm in diameter.
2023 Appendix XII A V-A409

2.5 ± 0.5 33.0 ± 0.5

U"l
c:i
+I
~ 3.15 ± 0.10
N

U"l
c:i
+I
a
C'1
C'")

31.40±0.13

I
I
■ D U"l

-- - c:i
a
+I
C'")

ILi

Figure 2.9.1.-2. -Apparatus for Disintegration Test B

Dimensions in millimetres

The disc is made of suitable transparent plastic material the test are met if not fewer than 16 of the 18 dosage units
having a specific gravity of 1.18-1.20. Each disc is pierced by tested have disintegrated. ♦
7 parallel holes, each 3.05-3.25 mm in diameter. One of the
holes is centred on the cylindrical axis. The other holes are 2. Disintegration Test for Suppositories and
parallel to the cylindrical axis and spaced equally on a circle Pessaries
with a diameter of 8.3-8.5 mm centred on the axis. (Ph. Bur. method 2.9.2)
All surfaces of the disc are smooth. The disintegration test determines whether the suppositories
or pessaries soften or disintegrate within the prescribed time
Procedure
Test 6 dosage units either by using 2 basket-rack assemblies when placed in a liquid medium in the experimental
in parallel or by repeating the procedure. Place 1 dosage unit conditions described below.
in each of the 3 tubes and, if prescribed, add a disc. Operate Disintegration is considered to be achieved when:
the apparatus using the specified medium as the immersion a) dissolution is complete,
fluid, maintained at 37 ± 2 cc. At the end of the specified b) the components of the suppository or pessary have
time, lift the basket-rack assembly from the fluid and observe separated: melted fatty substances collect on the surface of
the dosage units: all of the dosage units have disintegrated the liquid, insoluble powders fall to the bottom and soluble
completely. If 1 or 2 dosage units fail to disintegrate, repeat components dissolve, depending on the type of preparation,
the test on 12 additional dosage units. The requirements of the components may be distributed in one or more of these
ways,
V-A410 Appendix XII A 2023

The test is carried out using three such apparatuses each

containing a single sample. Each apparatus is placed in a
beaker with a capacity of at least 4 L filled with water
maintained at 36-37 °C, unless otherwise prescribed.
The apparatuses may also be placed together in a vessel with
a capacity of at least 12 L. The beaker is fitted with a slow
stirrer and a device that will hold the cylinders vertically not
less than 90 mm below the surface of the water and allow
them to be inverted without emerging from the water.
Method Use three suppositories or pessaries. Place each
one on the lower disc of a device, place the latter in the
sleeve and secure. Invert the apparatuses every 10 min.
Examine the samples after the period prescribed in the
monograph. To pass the test all the samples must have
disintegrated.
METHOD OF OPERATION FOR VAGINAL
TABLETS
I
50 1 Use the apparatus described above, arranged so as to rest on
the hooks (see Figure 2.9.2.-2). Place it in a beaker of
suitable diameter containing water maintained at 36-37 °C
with the level just below the upper perforated disc. Using a
pipette, adjust the level with water at 36-37 °C until a
uniform film covers the perforations of the disc. Use three
vaginal tablets. Place each one on the upper plate of an
apparatus and cover the latter with a glass plate to maintain
appropriate conditions of humidity. Examine the state of the
samples after the period prescribed in the monograph.
To pass the test all the samples must have disintegrated.

t
C
- /

I - '
- '
-
/
, I I ,,,,
- / /

.,. ,' - /
24 / - I
~:11~1,
36
I ; /./~/--_I

\ \ _.., I .,.,
I ' -

Figure 2. 9 .2.-1. - Apparatus for disintegration of suppositories

and pessaries
iE
Dimensions in millimetres

c) there is softening of the sample that may be accompanied

A. glass plate D. water
by appreciable change of shape without complete separation B. vaginal tablet E. dish, beaker
of the components, the softening is such that the suppository C. water surface
or pessary no longer has a solid core offering resistance to
pressure of a glass rod, Figure 2.9.2.-2.
d) rupture of the gelatin shell of rectal or vaginal capsules
occurs allowing release of the contents, Monographs of the British Pharmacopoeia
The following additional points apply to monographs of the
e) no residue remains on the perforated disc or if a residue
British Pharmacopoeia.
remains, it consists only of a soft or frothy mass having no
solid core offering resistance to pressure of a glass rod ACCEPTANCE CRITERIA
(vaginal tablets). For moulded suppositories, disintegration occurs in not more
Apparatus The apparatus (Figure 2.9.2.-1) consists of a than 30 minutes for fat-based suppositories and in not more
sleeve of glass or suitable transparent plastic, of appropriate than 60 minutes for water-soluble suppositories, unless
thickness, to the interior of which is attached by means of otherwise justified and authorised.
three hooks a metal device consisting of two perforated For moulded pessaries, disintegration occurs in not more
stainless metal discs each containing 39 holes 4 mm in than 60 minutes unless otherwise justified and authorised.
diameter; the diameter of the discs is similar to that of the For rectal capsules and vaginal tablets and capsules,
interior of the sleeve; the discs are about 30 mm apart. disintegration occurs in not more than 30 minutes.
2023 Appendix XII B V-A4 l 1

necessary to avoid the formation of gas bubbles in the flow-

Recommendations on Dissolution through cell. A suitable method of de-aeration is as follows:
Testing heat the medium while stirring gently to about 41 °C,
immediately filter under vacuum using a filter with a porosity
(Ph. Bur. general texts 5.17.1)
of 0.45 µm or less, with vigorous stirring, and continue
This general chapter is non-mandatory; it provides information on stirring under vacuum for about 5 min. Other de-aeration
dissolution testing, on recommended dissolution media and on the techniques for removal of dissolved gases may be used.
expression of dissolution specifications for oral dosage forms (see Using the paddle or basket apparatus, the volume of
general chapter 2. 9.3. Dissolution test for solid dosage forms). This dissolution medium is normally 500-1000 mL. A stirring
information represents generally accepted parameters used in the speed of between 50 r/min and 100 r/min is normally chosen;
field of dissolution. it must not exceed 150 r/min.
In the determination of the dissolution rate of the active For the flow-through apparatus, the liquid flow rate is
substance(s) of a solid dosage form, the following are to be normally set between 4 mUmin and 50 mUmin.
specified:
- the apparatus to be used, and in cases where the flow- RECOMMENDED DISSOLUTION MEDIA
through apparatus is specified, which flow-through cell is The following dissolution media may be used.
to be used;
Table 5.17.1.-1. - Examples of dissolution media
- the composition, the volume and the temperature of the
dissolution medium; pH Dissolution media
- the rotation speed or the flow rate of the dissolution
pH 1.0 HCI
medium;
- the time, the method and the amount for sampling of the pH 1.2 NaCl,HCI
test solution or the conditions for continuous monitoring;
pH 1.5 NaCl,HCI
- the method of analysis;
- the acceptance criteria. pH 4.5 Phosphate or acetate buffer
The choice of apparatus to be used depends on the physico- pH 5.5 and pH 5.8 Phosphate or acetate buffer
chemical characteristics of the dosage form. When a large
quantity of dissolution medium is required to ensure sink pH6.8 Phosphate buffer
conditions, or when a change of pH is necessary, the flow- pH 7.2 and pH 7.5 Phosphate buffer
through apparatus may be preferred.
EXPERIMENTAL TESTING CONDITIONS The composition and preparation of these various media are
The use of the basket and the paddle apparatus and the indicated below.
reciprocating cylinder apparatus is generally based on the
principle of operating under sink conditions, i.e. in such a Hydrochloric acid media
manner that the material already in solution does not exert a - 0.2 M hydrochloric acid.
significant modifying effect on the rate of dissolution of the - 0.2 M sodium chloride. Dissolve 11.69 g of sodium
remainder. Sink conditions normally occur in a volume of chloride R in water Rand dilute to 1000.0 mL with the
dissolution medium that is at least 3-10 times the saturation same solvent.
volume. For preparing media with the pH values indicated in
In general, an aqueous medium is used. The composition of Table 5.17.1.-2, mix 250.0 mL of 0.2 M sodium chloride
the medium is chosen on the basis of the physico-chemical and the specified volume of 0.2 M hydrochloric acid, and
characteristics of the active substance(s) and excipient(s) dilute to 1000.0 mL with water R.
within the range of conditions to which the dosage form is Table 5.17.1.-2. - Hydrochloric acid media
likely to be exposed after its administration. This applies in
particular to the pH and the ionic strength of the dissolution pH HCI (mL)
medium. 1.2 425.0
The pH of the dissolution medium is usually set between
pH 1 and pH 8. In justified cases, a higher pH may be 1.3 336.0

needed. For the lower pH values in the acidic range, 0.1 M 1.4 266.0
hydrochloric acid is normally used. Recommended dissolution
media are described hereafter. 1.5 207.0

Water is recommended as a dissolution medium only when it 1.6 162.0

is proven that the pH variations do not have an influence on
1.7 130.0
the dissolution characteristics.
In specific cases, and subject to approval by the competent 1.8 102.0
authority, dissolution media may contain enzymes,
1.9 81.0
surfactants, further inorganic substances and organic
substances. For the testing of preparations containing poorly 2.0 65.0
aqueous-soluble active substances, modification of the
2.1 51.0
medium may be necessary. In such circumstances, a low
concentration of surfactant is recommended; it is 2.2 39.0
recommended to avoid the use of organic solvents.
Gases dissolved in the dissolution medium can affect the The hydrochloric acid media may also be prepared by
results of the dissolution test. This is true in particular for the replacing sodium chloride with potassium chloride.
flow-through apparatus, where de-aeration of the medium is
V-A412 Appendix XII B 2023

Acetate buffer solutions - to remove only half of the medium each time (half change
- 2 M acetic acid. Dilute 120.0 g of glacial acetic acid R to method) and replace it with a buffer solution of
1000.0 mL with water R. higher pH: the initial pH is 1.2 and the second solution is
- Acetate buffer solution pH 4.5. Dissolve 2.99 g of sodium phosphate buffer solution pH 7.5; or,
acetate R in water R. Add 14.0 mL of 2 M acetic acid and - to an initial solution at pH 1.5, to add a dose of a powder
dilute to 1000.0 mL with water R. mixture containing tris(hydroxymethyl)aminomethane Rand
- Acetate buffer solution pH 5.5. Dissolve 5.98 g of sodium anhydrous sodium acetate R to obtain pH 4.5 and a second
acetate R in water R. Add 3.0 mL of 2 M acetic acid and dose to obtain pH 7 .2, as described below:
dilute to 1000.0 mL with water R. - hydrochloric acid pH 1. 5: dissolve 2 g of sodium
- Acetate buffer solution pH 5.8. Dissolve 6.23 g of sodium chloride R in water R, add 31.6 mL of 1 M hydrochloric
acetate R in water R. Add 2.1 mL of 2 M acetic acid and acid and dilute to 1000.0 mL with water R;
dilute to 1000.0 mL with water R. - buffer solution pH 4.5: mix 2.28 g of tris(hydroxymethyl)
Phosphate buffer solutions aminomethane R with 1.77 g of anhydrous sodium
For preparing buffers with the pH values indicated in acetate R; dissolve this mixture in the hydrochloric
Table 5.17.1.-3, mix 250.0 mL of 0.2 M potassium dihydrogen acid pH 1.5 described above;
phosphate R and the specified volume of 0.2 M sodium - buffer solution pH 7.2: mix 2.28 g of tris(hydroxymethyl)
hydroxide, and dilute to 1000.0 mL with water R. aminomethane R with 1. 77 g of anhydrous sodium
acetate R; dissolve this mixture in the buffer solution
Table 5.17.1.-3. - Phosphate buffer solutions pH 4.5 described above.
pH 5.8 6.0 6.2 6.4 6.6 6.8 The flow-through cell may be used for the continuous
change of pH.
NaOH
18.0 28.0 40.5 58.0 82.0 112.0 QUALIFICATION AND VALIDATION
(mL)
Due to the nature of the test method, quality by design is an
pH 7.0 7.2 7.4 7.6 7.8 8.0
important qualification aspect for in vitro dissolution test
NaOH equipment. Any irregularities such as vibration or undesired
145.5 173.5 195.5 212.0 222.5 230.5
(mL) agitation by mechanical imperfections are to be avoided.
Qualification of the dissolution test equipment has to
Other phosphate buffer solutions consider the dimensions and tolerances of the apparatus.
- Phosphate buffer solution pH 4.5. Dissolve 13.61 g of Critical test parameters, such as temperature and volume of
potassium dihydrogen phosphate R in 750 mL of water R. dissolution medium, rotation speed or liquid flow rate,
Adjust the pH if necessary with 0.1 M sodium hydroxide or sampling probes and procedures, have to be monitored
with 0.1 M hydrochloric acid. Dilute to 1000.0 mL with periodically during the periods of use.
water R. The performance of the dissolution test equipment may be
- Phosphate buffer solution pH 5. 5 R. monitored by testing a reference product that is sensitive to
- Phosphate buffer solution pH 6.8 Rl. hydrodynamic conditions. Such tests may be performed
- Buffer solution pH 7.2 R. periodically or continuously for comparative reasons with
- 0.33 M phosphate buffer solution pH 7.5 R. other laboratories.
Simulated intestinal fluid pH 6.8 During testing, critical inspection and observation are
Mix 77.0 mL of 0.2 M sodium hydroxide, 250.0 mL of a required. This approach is especially important to explain
solution containing 6.8 g of potassium dihydrogen phosphate R, any outlying results.
and 500 mL of water R. Add 10.0 g of pancreas powder R, Validation of automated systems, whether concerning the
mix and adjust the pH if necessary. Dilute to 1000.0 mL sampling and analytical part or the dissolution media
with water R. preparation and test performance, has to consider accuracy,
Simulated gastric fluid precision, and the avoidance of contamination by any
Dissolve 2.0 g of sodium chloride R and 3.2 g of pepsin dilutions, transfers, cleaning and sample or solvent
powder R in water R, add 80 mL of 1 M hydrochloric acid and preparation procedures.
dilute to 1000. 0 mL with water R. If required, pepsin powder EXPRESSION OF DISSOLUTION SPECIFICATIONS
may be omitted. FOR ORAL DOSAGE FORMS
Increasing pH The dissolution specification is expressed in terms of the
For a test involving increasing pH, one of the following quantity (Q) of active substance dissolved in a specified time,
sequences may be used: expressed as a percentage of the content stated on the
product label.
Time (h) 0- 1 1-2 I 2-3 I 3-414-515-616-7 I 7 Conventional-release dosage forms
In most cases, when tested under reasonable and justified test
pH 1.0
conditions, the acceptance criteria at level S 1 are that at least
pH 1.2 6.8 80 per cent of the active substance is released within a
specified time, typically 45 min or less. This corresponds to a
pH 1.2 2.5 I 4.5
I 7.0
I 7.5
Q value of 75 per cent, since, as referred to in
pH 1.5 4.5
I 7.2 Table 2.9.3.-1, for level S 1 the individual value of each of the
6 units tested is not less than Q + 5 per cent, i.e. not less
than 80 per cent.
To achieve this pH variation, it is possible either:
- to substitute one buffer solution for another (whole Typically, a single-point acceptance criterion is sufficient to
substitution); demonstrate that the majority of the active substance has
been released, although in certain circumstances it may be
2023 Appendix XII B V-A413

necessary to test at additional time point( s), in order to preparation and stirring element during the test is preferable.
demonstrate adequate dissolution. The vessel is cylindrical, with a hemispherical bottom and a
Prolonged-release dosage forms capacity of 1 L. Its height is 160-210 mm and its inside
The dissolution test acceptance criteria for prolonged-release diameter is 98-106 mm. Its sides are flanged at the top.
dosage forms is normally expected to consist of 3 or more A fitted cover may be used to retard evaporation3 • The shaft
points. The 1st specification point is intended to prevent is positioned so that its axis is not more than 2 mm at any
unintended rapid release of the active substance ('dose point from the vertical axis of the vessel and rotates smoothly
dumping'). It is therefore set after a testing period and without significant wobble that could affect the results.
corresponding to a dissolved amount typically of 20 per cent A speed-regulating device is used that allows the shaft
to 30 per cent. The 2nd specification point defines the rotation speed to be selected and maintained at a specified
dissolution pattern and so is set at around 50 per cent rate, within ± 4 per cent.
release. The final specification point is intended to ensure Shaft and basket components of the stirring element are
almost complete release, which is generally understood as fabricated of stainless steel, type 316 or equivalent, to the
more than 80 per cent release. specifications shown in Figure 2.9.3.-1.
Delayed-release dosage forms A basket having a gold coating of about 2.5 µm
A delayed-release dosage form may release the active (0.0001 inch) thick may be used. The dosage unit is placed
substance(s) fractionally or totally according to the in a dry basket at the beginning of each test. The distance
formulation design when tested in different dissolution between the inside bottom of the vessel and the bottom of
media, e.g. in increasing pH conditions. Dissolution the basket is maintained at 25 ± 2 mm during the test.
specifications therefore have to be decided on a case-by-case Apparatus 2 (Paddle apparatus)
basis. Use the assembly from Apparatus 1, except that a paddle
Gastro-resistant dosage forms require at least 2 specification formed from a blade and a shaft is used as the stirring
points in a sequential test and 2 different specifications in a element. The shaft is positioned so that its axis is not more
parallel test. In a sequential test, the I st specification point than 2 mm from the vertical axis of the vessel, at any point,
represents an upper limit and is set after I h or 2 h in acidic and rotates smoothly without significant wobble that could
medium, and the 2nd after a pre-set time period of testing in affect the results. The vertical center line of the blade passes
an adequate buffer solution (preferably pH 6.8). through the axis of the shaft so that the bottom of the blade
In most cases the acceptance criteria at level B 1 are that at is flush with the bottom of the shaft. The paddle conforms to
least 80 per cent of the active substance is released. This the specifications shown in Figure 2.9.3.-2. The distance of
corresponds to a Q value of 75 per cent, since, as referred to 25 ± 2 mm between the bottom of the blade and the inside
in Table 2.9.3.-4, for level B 1 the individual value of each of bottom of the vessel is maintained during the test.
the 6 units tested is not less than Q + 5 per cent, i.e. not less The metallic or suitably inert, rigid blade and shaft comprise
than 80 per cent. a single entity. A suitable two-part detachable design may be
used provided the assembly remains firmly engaged during
the test. The paddle blade and shaft may be coated with a
suitable coating so as to make them inert. The dosage unit is
allowed to sink to the bottom of the vessel before rotation of
B. Dissolution the blade is started. A small, loose piece of non-reactive
1. Dissolution Test for Tablets and Capsules material, such as not more than a few turns of wire helix,
(Dissolution Test for Solid Dosage Fonns) 1 may be attached to dosage units that would otherwise float.
(Ph. Bur. method 2.9.3) An alternative sinker device is shown in Figure 2.9.3.-3.
This test is provided to determine compliance with the Other validated sinker devices may be used.
dissolution requirements for solid dosage forms administered Apparatus 3 (Reciprocating cylinder)
orally. In this chapter, a dosage unit is defined as I tablet or The assembly consists of a set of cylindrical, flat-bottomed
1 capsule or the amount specified. glass vessels; a set of glass reciprocating cylinders; inert
APPARATUS fittings (stainless steel type 316 or other suitable material)
Apparatus 1 (Basket apparatus) and screens that are made of suitable nonsorbing and
The assembly consists of the following: a vessel, which may nonreactive material, and that are designed to fit the tops
be covered, made of glass or other inert, transparent and bottoms of the reciprocating cylinders; a motor and drive
materia!2; a motor; a drive shaft; and a cylindrical basket assembly to reciprocate the cylinders vertically inside the
(stirring element). The vessel is partially immersed in a vessels, and if desired, index the reciprocating cylinders
suitable water-bath of any convenient size or heated by a horizontally to a different row of vessels. The vessels are
suitable device such as a heating jacket. The water-bath or partially immersed in a suitable water-bath of any convenient
heating device permits maintaining the temperature inside the size that permits holding the temperature at 37 ± 0.5 °c
vessel at 37 ± 0.5 °C during the test and keeping the during the test. No part of the assembly, including the
dissolution medium in constant, smooth motion. No part of environment in which the assembly is placed, contributes
the assembly, including the environment in which the significant motion, agitation, or vibration beyond that due to
assembly is placed, contributes significant motion, agitation, the smooth, vertically reciprocating cylinder. A device is used
or vibration beyond that due to the smoothly rotating stirring that allows the reciprocation rate to be selected and
element. Apparatus that permits observation of the maintained at the specified dip rate, within ± 5 per cent.
An apparatus that permits observation of the preparations
and reciprocating cylinders is preferable. The vessels are
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter provided with an evaporation cap that remains in place for
5. 8 Pharmacopoeia/ harmonisation.
2 The materials must not sorb, react, or interfere with the preparation to be 3 If a cover is used, it provides sufficient openings to allow ready insertion of

tested. the themzometer and withdrawal of samples.

V-A414 Appendix XII B 2023

constant flow ( ± 5 per cent of the nominal flow rate); the

flow profile is sinusoidal with a pulsation of
120 ± 10 pulses/min. A pump without pulsation may also be
used. Dissolution test procedures using the flow-through cell
must be characterised with respect to rate and any pulsation.
The flow-through cell (see Figures 2.9.3.-5 and 2.9.3.-6) of
6.3 to 6.5 transparent and inert material is mounted vertically, with a
or 9.4 to 10.1 filter system that prevents escape of undissolved particles
from the top of the cell; standard cell diameters are 12 mm
and 22.6 mm; the bottom cone is usually filled with small
LO
0
glass beads of about 1 mm diameter, with 1 bead of about
Vent hole +I 5 mm positioned at the apex to protect the fluid entry tube;
2.0 ± 0.5 ..... a tablet holder (see Figures 2.9.3.-5 and 2.9.3.-6) is available
ll')
for positioning of special dosage forms. The cell is immersed
Retention spring in a water-bath, and the temperature is maintained at
with 3 tangs 37 ± 0.5 °C.
on 120° centers The apparatus uses a clamp mechanism and 2 O-rings for
Clear opening the fixation of the cell assembly. The pump is separated from
20.2 ± 1.0 the dissolution unit in order to shield the latter against any
vibrations originating from the pump. The position of the
pump must not be on a level higher than the reservoir flasks.
Screen O.D
Tube connections are as short as possible. Use suitably inert
22.2 ± 1.0 tubing, such as polytetrafluoroethylene, with a 1.6 mm inner
diameter and inert flanged-end connections.
0
('") Apparatus suitability The determination of suitability of
+I +I
0 0 the apparatus to perform dissolution testing must include
t--'. t--'. conformance to the dimensions and tolerances of the
N C')
apparatus as given above. In addition, critical test parameters
that have to be monitored periodically during use include
volume and temperature of the dissolution medium, rotation
A speed (Apparatus 1 and 2), dip rate (Apparatus 3), and flow
rate of medium (Apparatus 4).
Determine the acceptable performance of the dissolution test
assembly periodically.
PROCEDURE
0 APPARATUS 1 AND 2
~ ('")
.....
+I +I Conventional-release solid dosage forms
N 0
0 ll') Procedure Place the stated volume of the dissolution
N N
medium (± 1 per cent) in the vessel of the specified
apparatus. Assemble the apparatus, equilibrate the dissolution
medium to 37 ± 0.5 °C, and remove the thermometer. ◊The
test may also be carried out with the thermometer in place,
provided it is shown that results equivalent to those obtained
1) Screen with welded seam: 0.22-0.31 mm wire without the thermometer are obtained.◊
diameter with wire opening of 0.36-0.44 mm. Place 1 dosage unit in the apparatus, taking care to exclude
After welding the screen may be slighty altered.
air bubbles from the surface of the dosage unit. Operate the
2) Maximum allowable nmout at "A" is 1.0 mm
when the part is rotated on center line axis with apparatus at the specified rate. Within the time interval
basket mounted. specified, or at each of the times stated, withdraw a specimen
from a zone midway between the surface of the dissolution
Figure 2.9.3.-1. -Apparatus 1, Basket stirring element medium and the top of the rotating basket or blade, not less
Dimensions in millimetres than 1 cm from the vessel wall. Where multiple sampling
times are specified, replace the aliquots withdrawn for
the duration of the test. The components conform to the analysis with equal volumes of fresh dissolution medium at
dimensions shown in Figure 2.9.3.-4 unless otherwise 37 °C or, where it can be shown that replacement of the
specified. medium is not necessary, correct for the volume change in
Apparatus 4 (Flow-through cell) the calculation. Keep the vessel covered for the duration of
The assembly consists of a reservoir and a pump for the the test and verify the temperature of the medium at suitable
dissolution medium; a flow-through cell; a water-bath that times. Perform the analysis using a suitable assay method 4 •
maintains the dissolution medium at 37 ± 0.5 °C. Use the Repeat the test with additional dosage units.
specified cell size.
The pump forces the dissolution medium upwards through
the flow-through cell. The pump has a delivery range 4 Test specimens are filtered immediately upon sampling unless filrration is

between 240 mUh and 960 mlJh, with standard flow rates demonsrrated to be unnecessary. Use an inert filter that does not cause
of 4 mIJmin, 8 mUmin, and 16 mUmin. It must deliver a adsorption of the active substance or contain extractable substances that
would inteifere with the analysis.
2023 Appendix XII B V-A415

9.4 to 10.1

0:,

'°
C"")
I{)
c:i
+I
0
cri

A and B dimensions do not vary more than 0.5 mm when part is rotated on center line axis. Tolerances
are ± 1.0 mm unless otherwise stated.

Figure 2.9.3.-2. -ApparatuS 2, Paddle stining element

Dimensions in millimetres

If automated equipment is used for sampling or the In such cases, dissolved gases must be removed prior to
apparatus is otherwise modified, verification that the testing5 •
modified apparatus will produce results equivalent to those Time Where a single time specification is given, the test
obtained with the apparatus described in this chapter, is may be concluded in a shorter period if the requirement for
necessary. minimum amount dissolved is met. Samples are to be
Dissolution medium A suitable dissolution medium is withdrawn only at the stated times, within a tolerance of
used. The volume specified refers to measurements made ± 2 per cent.
between 20 °C and 25 °C. If the dissolution medium is a
buffered solution, adjust the solution so that its pH is within
0.05 units of the specified pH. Dissolved gases can cause 5 A method of deaeration is as follows: heat the medium, while stirring
bubbles to form, which may change the results of the test. gendy, w about 41 °C, immediately filter under vacuum using a filter having
a porosity of 0. 45 µm or less, with vigorous stirring, and continue stirring
under vacuum for about 5 min. Other validated deaeration techniques for
removal of dissolved gases may be used.
V-A416 Appendix XII B 2023

3.5-4.0 3.0-3.5 3.5-4.0

·,J 1·
A B

n n I I n n n
IA IA 'A lh 'A rn
3.5-4.0 II 3.5-4.0
11/ II ,II ,II II II

J I.
~

. lJ lJ lJ II II II

-
25-26 12.0 ± 0.2

A: acid-resistant wire clasp B: acid-resistant wire support

Figure 2.9.3.-3. -Alternative sinker

Dimensions in millimetres

Prolonged-release solid dosage forms 0.20 M solution of trisodium phosphate dodecahydrate R

Procedure Proceed as described for conventional-release and adjusting, if necessary, with 2 M hydrochloric acid R or
dosage forms. 2 M sodium hydroxide R to a pH of 6.8 ± 0.05. This may
Dissolution medium Proceed as described for also be accomplished by removing from the apparatus the
conventional-release dosage forms. vessel containing the acid and replacing it with another
Time The test-time points, generally 3, are expressed in vessel, containing the buffer and transferring the
dosage unit to the vessel containing the buffer. Continue
hours.
to operate the apparatus for 45 min, or for the specified
Delayed-release solid dosage forms time. At the end of the time period, withdraw an aliquot
Procedure Use Method A or Method B. of the fluid and perform the analysis using a suitable assay
Method A method.
- Acid stage. Place 750 mL of 0.1 M hydrochloric acid in the Time All test times stated are to be observed within a
vessel, and assemble the apparatus. Allow the medium to tolerance of ± 2 per cent, unless otherwise specified.
equilibrate to a temperature of 37 ± 0.5 °C. Place
1 dosage unit in the apparatus, cover the vessel and APPARATUS3
operate the apparatus at the specified rate. After 2 h of Conventional-release solid dosage forms
operation in 0.1 M hydrochloric acid, withdraw an aliquot Procedure Place the stated volume of the dissolution
of the fluid and proceed immediately as directed under medium ( ± 1 per cent) in each vessel of the apparatus.
Buffer stage. Perform an analysis of the aliquot using a Assemble the apparatus, equilibrate the dissolution medium
suitable assay method. to 37 ± 0.5 °C, and remove the thermometer. Place 1
- Buffer stage. Complete the operations of adding the buffer dosage unit in each of the reciprocating cylinders, taking care
and adjusting the pH within 5 min. With the apparatus to exclude air bubbles from the surface of each dosage unit,
operating at the rate specified, add to the fluid in the and immediately operate the apparatus as specified. During
vessel 250 mL of a 0.20 M solution of trisodium phosphate the upward and downward stroke, the reciprocating cylinder
dodecahydrate R that has been equilibrated to moves through a total distance of9.9-10.1 cm. Within the
37 ± 0.5 °C. Adjust, if necessary, with 2 M hydrochloric time interval specified, or at each of the times stated, raise
acid R or 2 M sodium hydroxide R to a pH of 6.8 ± 0.05. the reciprocating cylinders and withdraw a portion of the
Continue to operate the apparatus for 45 min, or for the medium from a zone midway between the surface of the
specified time. At the end of the time period, withdraw an dissolution medium and the bottom of each vessel. Perform
aliquot of the fluid and perform the analysis using a the analysis as directed. If necessary, repeat the test with
suitable assay method. additional dosage units.
Method B Replace the aliquot withdrawn for analysis with equal
- Acid Stage. Place 1000 mL of 0.1 M hydrochloric acid in volumes of fresh dissolution medium at 37 °C or, where it
the vessel and assemble the apparatus. Allow the medium can be shown that replacement of the medium is not
to equilibrate to a temperature of 37 ± 0.5 °C. Place necessary, correct for the volume change in the calculation.
1 dosage unit in the apparatus, cover the vessel, and Keep the vessel covered with the evaporation cap for the
operate the apparatus at the specified rate. After 2 h of duration of the test and verify the temperature of the
operation in 0.1 M hydrochloric acid, withdraw an aliquot medium at suitable times.
of the fluid, and proceed immediately as directed under Dissolution medium Proceed as described for
Buffer stage. Perform an analysis of the aliquot using a conventional-release dosage forms under Apparatus 1 and 2.
suitable assay method. Time Proceed as described for conventional-release dosage
- Buffer stage. For this stage of the procedure use buffer forms under Apparatus 1 and 2.
that has previously been equilibrated to a temperature of
37 ± 0.5 °C. Drain the acid from the vessel and add Prolonged-release dosage forms
1000 mL of pH 6.8 phosphate buffer, prepared by mixing Procedure Proceed as described for conventional-release
dosage forms under Apparatus 3.
3 volumes of 0.1 M hydrochloric acid with 1 volume of a
2023 Appendix XII B V-A41 7

Dissolution medium Proceed as described for prolonged- .. 50.8 ± 1 --1

release dosage forms under Apparatus 1 and 2. I I • 38.1 ± 1 • I Air holes
Time Proceed as described for prolonged-release dosage ~03.9±0.1
forms under Apparatus 1 and 2. .----rl-r-+-'rl-r-----,
Delayed-release dosage forms 't'- 7
Procedure Proceed as described for delayed-release dosage
forms, Method B, under Apparatus 1 and 2, using one row
I
of vessels for the acid stage media and the following row of +I I Evaporation cap
co
vessels for the buffer stage media, and using the volume of
'°
<O
I
medium specified (usually 300 mL). I
Time Proceed as directed for delayed-release dosage forms
under Apparatus 1 and 2.
APPARATVS4
Conventional-release dosage forms
Procedure Place the glass beads into the cell specified.
Place 1 dosage unit on top of the beads or, if specified, on a Mesh screen
wire carrier. Assemble the filter head and fix the parts
together by means of a suitable clamping device. Introduce
by the pump the dissolution medium warmed to
37 ± 0.5 °C through the bottom of the cell to obtain the
flow rate specified and measured with an accuracy of
5 per cent. Collect the eluate by fractions at each of the I
I
times stated. Perform the analysis as directed. Repeat the test I
with additional dosage units. I
I
Dissolution medium Proceed as described for +I I
I
I i-->---- Glass reciprocating
conventional-release dosage forms under Apparatus 1 and 2. 0
0 I I
cylinder
I
Time Proceed as described for conventional-release dosage I
forms under Apparatus 1 and 2. I
I I
Prolonged-release dosage forms I
I
I
Procedure Proceed as described for conventional-release I
dosage forms under Apparatus 4.
Dissolution medium Proceed as described for !1
~
I I - - - - Mesh screen
conventional-release dosage forms under Apparatus 4. I

Time Proceed as described for conventional-release dosage

forms under Apparatus 4.
I
47 ± 1.4
Delayed-release dosage forms
Procedure Proceed as described for delayed-release dosage
forms under Apparatus 1 and 2, using the specified media.
Time Proceed as described for delayed-release dosage
forms under Apparatus 1 and 2.
INTERPRETATION
- - - Glass vessel
Conventional-release solid dosage forms
Unless otherwise specified, the requirements are met if the
quantities of active substance dissolved from the dosage units +I
0
tested conform to Table 2.9.3.-1. Continue testing through co
the 3 levels unless the results conform at either S 1 or S 2 .
The quantity Q, is the specified amount of dissolved active
substance, expressed as a percentage of the labelled content;
the 5 per cent, 15 per cent, and 25 per cent values in the
Table are percentages of the labelled content so that these
values and Q are in the same terms.

Table 2.9.3.-1
Level Number Acceptance criteria
tested I
L ___ _j_ ___ _J
6 Each unit is not less than Q + 5 per cent.
6 Average of 12 units (S 1 + S 2 ) is equal to or greater
than Q, and no unit is less than Q -15 per cent. I
12 Average of 24 units (S 1 + S2 -,- S,) is equal to or greater
than Q, not more than 2 units arc less than Figure 2.9.3.-4. -Apparatus 3, glass vessel and reciprocating
Q -15 per cent, and no is less than Q -25 per cent. cylinder
Dimensions in millimetres
V-A418 Appendix XII B 2023

ll)

ll)
0
+I
ll)
LO
C') - - Score for the holder

ll)

03

C')

C:
.E

·r 00.8 ±0.05

ll)
(0
ll)
0

ll)
....:

ll)
N
0
+I
ll)
24.0 + 8· 5
C\i

Figure 2.9.3.-5. -Apparatus 4, large cell for tablets and capsules (top), tablet holder for the large cell (bottom)
Dimensions in millimetres unless otherwise specified
2023 Appendix XII B V-A419

LO
c:i
+I
LO
Ii)

so·

LO 012 ± 0.2
c:i
+I
0
LO

- - - - - Score for the holder

LO

~---------
"E---i:--------===.i:~-----
I 00.8 ± 0.05
------+-------03

<D

+I
~
LO + 0.5
c-,i 13.5 0

Figure 2.9.3.-6. -Apparatus 4, small cell for tablets and capsules (top), tablet holder for the small cell (bottom)
Dimensions in millimetres unless otherwise specified

Prolonged-release dosage forms The limits embrace each value of Q,, the amount dissolved at
Unless otherwise specified, the requirements are met if the each specified fractional dosing interval. Where more than
quantities of active substance dissolved from the dosage units one range is specified, the acceptance criteria apply
tested conform to Table 2.9.3.-2. Continue testing through individually to each range.
the 3 levels unless the results conform at either L 1 or L 2 •
Limits on the amounts of active substance dissolved are
expressed in terms of the percentage of labelled content.
V-A420 Appendix XII B 2023

Table 2.9.3.-2 2. Dissolution Test for Patches

Level Number Acceptance criteria (Ph. Bur. method 2.9.4)
tested This test is used to determine the dissolution rate of the
6 No individual value lies outside each of the stated ranges active substance(s) of patches.
and no individual value is less than the stated amount at
the final test time.
The disk assembly method, the cell method or the rotating
6 The average value of the 12 units (L 1 + L 2 ) lies within
cylinder method may be used, as suitable, according to the
each of the stated ranges and is not less than the stated composition, dimensions and shape of the patch.
amount at the final test time; none is more than A membrane may be used for the purpose of the test.
10 per cent of labelled content outside each of the stated The membrane material (inert porous cellulose, silicone, etc.)
ranges; and none is more than I O per cent of labelled
content below the stated amount at the final test time.
must not affect the release kinetics of the active substance(s)
12 The average value of the 24 units (L 1 + L 2 + L 3 ) lies
from the patch. It must also be free of substances that may
within each of the stated ranges, and is not less than the interfere with the performance of the patch (e.g. grease).
stated amount at the final test time; not more than 2 of The membrane may be suitably treated before the tests, for
the 24 units are more than 10 per cent of labelled example by maintaining it for 24 h in the medium to be used
content outside each of the stated ranges; not more
in the test. The membrane is applied over the release surface
than 2 of the 24 units are more than 10 per cent of
labelled content below the stated amount at the final test of the patch in such a way as to avoid the formation of air
time; and none of the units is more than 20 per cent of bubbles.
labelled content outside each of the stated ranges or
more than 20 per cent of labelled content below the 1. DISK ASSEMBLY METHOD
stated amount at the final test time. Equipment
Use the paddle and vessel assembly from the paddle
Delayed-release dosage forms apparatus described in the dissolution test for solid oral
Acid stage Unless otherwise specified, the requirements of dosage forms (2. 9. 3) with the addition of a stainless steel disk
this portion of the test are met if the quantities, based on the assembly (SSDA) in the form of a net with an aperture of
percentage of the labelled content of active substance 125 µm (see Figure 2.9.4.-1).
dissolved from the units tested conform to Table 2.9.3.-3. The SSDA holds the system at the bottom of the vessel and
Continue testing through the 3 levels unless the results of is designed to minimise any dead volume between the SSDA
both acid and buffer stages conform at an earlier level. and the bottom of the vessel. The SSDA holds the patch flat,
with the release surface facing upwards and parallel to the
Table 2.9.3.-3 bottom of the paddle blade. A distance of 25 ± 2 mm
Level Number Acceptance criteria between the bottom of the paddle blade and the surface of
tested the SSDA is maintained during the test (see Figure 2.9.4.-2).
Ai 6 No individual value exceeds I O per cent dissolved. The temperature is maintained at 32 ± 0.5 °C. The vessel
A2 6 The average value of the 12 units (A 1 + A 2 ) is not more may be covered during the test to minimise evaporation.
than 10 per cent dissolved, and no individual unit is
greater than 25 per cent dissolved.
A, 12 The average value of the 24 units (A 1 + A 2 + A 3 ) is not
more than I O per cent dissolved, and no individual unit
is greater than 25 per cent dissolved.

Buffer stage Unless otherwise specified, the requirements

___,___ Dissolution vessel
are met if the quantities of active substance dissolved from
the units tested conform to Table 2.9.3.-4. Continue testing
through the 3 levels unless the results of both stages conform
at an earlier level. The value of Qin Table 2.9.3.-4 is
75 per cent dissolved unless otherwise specified. _,__ _ _ _.....__ Paddle
The quantity, Q, is the specified total amount of active
substance dissolved in both the acid and buffer stages,
expressed as a percentage of the labelled content.
The 5 per cent, 15 per cent and 25 per cent values in the
Table are percentages of the labelled content so that these
values and Q are in the same terms.

Table 2.9.3.-4
Level Number Acceptance criteria
tested
6 No unit is less than Q + 5 per cent. Disk assembly
6 The average value of the 12 units (B 1 + B,) is equal to
or greater than Q, and no unit is less than
Q-15 per cent. Figure 2.9.4.-2. - Paddle and disk
12 The average value of the 24 units (B 1 + B 2 + B3 ) is
equal to or greater than Q, not more than 2 units are less Procedure
than Q - I 5 per cent, and no unit is less than Place the prescribed volume of the dissolution medium in the
Q -25 per cent. vessel and equilibrate the medium to the prescribed
temperature. Apply the patch to the SSDA, ensuring that the
Recommendations on dissolution testing are given in general release surface is as flat as possible. The patch may be
chapter 5.17.1. attached to the SSDA by a prescribed adhesive or by a strip
of a double-sided adhesive tape. The adhesive or tape are
2023 Appendix XII B V-A421

125 µm mesh
stainless steel net 4.5mm

D t 3.3 mm

41.2 mm

Figure 2.9.4.-1. - Disk assembly

previously tested for the absence of interference with the 27 mm, 38 mm, 45 mm, 52 mm, corresponding to volumes
assay and of adsorption of the active substance(s). Press the of 1.49 mL, 2.95 mL, 4.14 mL, 5.52 mL, respectively.
patch, release surface facing upwards, onto the side of the Cover The cover has a central opening with a diameter
SSDA made adhesive. The applied patch must not overlap selected according to the size of the patch to be examined.
the borders of the SSDA. For this purpose and provided that The patch can thus be precisely centred, and its release
the preparation is homogeneous and uniformly spread on the surface limited. The following diameters may be used:
backing layer, an appropriate and exactly measured piece of 20 mm, 32 mm, 40 mm, 50 mm corresponding to areas of
the patch may be cut and used to test the dissolution rate. 3.14 cm2 , 8.04 cm2 , 12.57 cm2 , 19.63 cm 2 , respectively.
This procedure may also be necessary to achieve appropriate The cover is held in place by nuts screwed onto bolts
sink conditions. This procedure must not be applied to projecting from the support. The cover is sealed to the
membrane-type patches. Place the patch mounted on the support by a rubber ring set on the reservoir.
SSDA flat at the bottom of the vessel with the release surface Extraction cell The cell holds the patch flat, with the
facing upwards. Immediately rotate the paddle, at 100 r/min release surface facing upwards and parallel to the bottom of
for example. At predetermined intervals, withdraw a sample the paddle blade. A distance of 25 ± 2 mm is maintained
from the zone midway between the surface of the dissolution between the lower surface of the paddle blade and the upper
medium and the top of the paddle blade, not less than 1 cm surface of the cell (see Figure 2.9.4.-4). The temperature is
from the vessel wall. maintained at 32 ± 0.5 °C. The vessel may be covered
Perform the assay on each sample, correcting for any volume during the test to minimise evaporation.
losses, as necessary. Repeat the test with additional patches.
Procedure
2. CELL METHOD Place the prescribed volume of the dissolution medium in the
Equipment vessel and equilibrate the medium to the prescribed
Use the paddle and vessel assembly from the paddle temperature. Precisely centre the patch in the cell with the
apparatus described in the dissolution test for solid oral release surface facing upwards. Close the cell, if necessary
dosage forms (2.9.3) with the addition of the extraction cell applying a hydrophobic substance (for example, petrolatum)
(eel[). to the flat surfaces to ensure the seal, and ensure that the
The cell is made of chemically inert materials and consists of patch stays in place. Introduce the cell flat into the bottom of
a support, a cover and, if necessary, a membrane placed on the the vessel with the cover facing upwards. Immediately rotate
patch to isolate it from the medium that may modify or the paddle, at 100 r/min for example. At predetermined
adversely affect the physico-chemical properties of the patch intervals, withdraw a sample from tl1e zone midway between
(see Figure 2.9.4.-3). the surface of the dissolution medium and the top of the
Support The central part of the support forms a cavity paddle blade, not less than 1 cm from the vessel wall.
intended to hold the patch. The cavity has a depth of Perform the assay on each sample, correcting for any volume
2.6 mm and a diameter that is appropriate for the size of the losses, as necessary. Repeat the test with additional patches.
patch to be examined. The following diameters can be used:
V-A422 Appendix XII B 2023

20mm
32mm@

50mm

~---Cover

,_____ Membrane

2.6 mm ~==~~~~~~~:: : :~~~[:====--

•t Bolts
Rubber
ring
..r ◄---- Reservoir

52mm
21.23 cm 2

70.5 mm
27mm
f-----3Bmm
'------45mm
1----------- 52 mm

Figure 2.9.4.-3. - Extraction cell

the paddle and shaft with a stainless steel cylinder stirring

element (cylinder) (see Figure 2.9.4.-5). The patch is placed
on the cylinder at the beginning of each test. The distance
between the inside bottom of the vessel and the cylinder is
maintained at 25 ± 2 mm during the test. The temperature
-i-------1-- Paddle
is maintained at 32 ± 0.5 °C. The vessel is covered during
the test to minimise evaporation.
Procedure
Place the prescribed volume of the dissolution medium in the
~-.._- Dissolution vessel vessel and equilibrate the medium to the prescribed
temperature. Remove the protective liner from the patch and
place the adhesive side on a piece of suitable inert porous
membrane that is at least 1 cm larger on all sides than the
patch. Place the patch on a clean surface with the membrane
in contact with this surface. Two systems for adhesion to the
cylinder may be used:
25±2 mm
- apply a suitable adhesive to the exposed membrane
borders and, if necessary, to the back of the patch;
- apply a double-sided adhesive tape to the external wall of
the cylinder.
Using gentle pressure, carefully apply the non-adhesive side
Figure 2.9.4.-4. - Paddle over extraction cell of the patch to the cylinder, so that the release surface is in
contact with the dissolution medium and the long axis of the
3. ROTATING CYLINDER METHOD patch fits around the circumference of the cylinder.
Equipment The system for adhesion used is previously tested for absence
Use the assembly of the paddle apparatus described in the of interference with the assay and of adsorption of the active
dissolution test for solid oral dosage forms (2.9.3). Replace substance(s).
2023 Appendix XII B V-A423

A A
tI_______ ~ ___.______._,_.___,___
t
1.270
/' +
4 holes at 1.111 dia. equally
spaced on 2.540 dia. b.c.
at 63.4 ± 0.5° angle to surface Interference fit
i-- 2.222
0.94-1.01 --1 ' 1--

•
1.112
t
l
40.640
5.079

TOLERANCES:± 0.0127 I
3.670
l This adaptor
section is to
be used for
1-- 2.045- larger systems

FINISH: Degrease before final

assembly or rod and cylinder 9.383

MATERIAL: Stainless steel =-~m~====~t====:;1i=- s.rz j

·-- 2.222-I
Figure 2.9.4.-5. - Cylinder stirring element
Dimensions in centimetres

Place the cylinder in the apparatus and immediately rotate the 3. Dissolution Test for Lipophilic Solid Doseage
cylinder, at I 00 r/min for example. At predetermined Forms
intervals, withdraw a sample from the zone midway between (Ph, Bur. method 2. 9.42)
the surface of the dissolution medium and the top of the
APPARATUS
rotating cylinder, not less than 1 cm from the vessel wall.
The apparatus (see Figure 2.9,42,-1) consists of:
Perform the assay on each sample, correcting for any volume - A reservoir for the dissolution medium.
withdrawn, as necessary. Repeat the test with additional - A pump that forces the dissolution medium upwards
patches. through the flow-through cell.
4. INTERPRETATION - A flow-through cell shown in Figure 2.9.42.-2 specifically
The requirements are met if the quantity of active intended for lipophilic solid dosage forms such as
substance(s) released from the patch, expressed as the suppositories and soft capsules. It consists of
amount per surface area per time unit, is within the 3 transparent parts which fit into each other. The lower
prescribed limits at the defined sampling times.
V-A424 Appendix XII B 2023

part (1) is made up of 2 adjacent chambers connected to ,,,,,,,,,,

,,,,,,,,,,,
an overflow device. ,,,,,,,,,,
,,,,,
The dissolution medium passes through chamber A and is
subjected to an upwards flow. The flow in chamber B is (3)
downwards directed to a small-size bore exit which leads
upwards to a filter assembly. The middle part (2) of the cell
Capillary tube
has a cavity designed to collect lipophilic excipients which
float on the dissolution medium. A metal grill serves as a (2)
rough filter. The upper part (3) holds a filter unit for paper,
glass fibre or cellulose filters.
- A water-bath that will maintain the dissolution medium at 15°
37 ± 0.5 °C.

"'
"' C")

"' co
C")

Reservoir for Pump Thermostatically

bd:
,:,,'.

Collecting
•.
............
Sieve with
dissolution controlled flow-through receptacles point
medium cell and filter for analysis

Figure 2.9.42.-1. - Flow-through apparatus

Dissolution medium Figure 2.9.42.-2. - Flow-through cell

If the dissolution medium is buffered, adjust its pH to within
Dimensions in millimetres
± 0.05 units of the prescribed value. Remove any dissolved
gases from the dissolution medium before the test since they 4. Dissolution Test for Medicated Chewing Gum
can cause the formation of bubbles that significantly affect (Ph. Bur. method 2.9.25)
the results.
PRINCIPLE
METHOD
The test is used to determine the dissolution rate of active
Place 1 unit of the preparation to be examined in substances in medicated chewing gums. This is done by
chamber A. Close the cell with the prepared filter assembly. applying a mechanical kneading procedure to a piece of gum
At the beginning of the test, chamber A requires air removal placed in a small chamber designed to simulate the process
via a small orifice connected to the filter assembly. Heat the of chewing.
dissolution medium to an appropriate temperature taking the
melting point of the preparation into consideration. Using a
056
suitable pump, introduce the warmed dissolution medium
through the bottom of the cell to obtain a suitable
continuous flow through an open or closed circuit at the
prescribed rate (± 5 per cent). When the dissolution
-----,-----
medium reaches the overflow, air starts to escape through the
capillary and chamber B fills with the dissolution medium.
1
The preparation spreads through the dissolution medium
y -a------i----- I
according to its physico-chemical properties.
In justified and authorised cases, representative fractions of
large volume suppositories may be tested. I
I
SAMPLING AND EVALUATION
Samples are always collected at the outlet of the cell,
irrespective of whether the circuit is opened or closed. --t---
Filter the liquid removed using an inert filter of appropriate 1
pore size that does not cause significant adsorption of the
active substance from the solution and does not contain ----~----_
substances extractable by the dissolution medium that would
interfere with the prescribed analytical method. Proceed with
analysis of the filtrate as prescribed.
--~--
The quantity of the active substance dissolved in a specified
time is expressed as a percentage of the content stated on the
label. Figure 2.9.25.-2 - Funnel
Dimensions in millimetres
2023 Appendix XII B V-A425

0 15 M6x16

0
co
E
N

SECTION G-G
N
O'l
D

SECTION F-F

A B C

A. horizontal piston B. guide C. chewing chamber D. funnel E. vertical piston

Figure 2.9.25.-1 -Apparatus A - Chewing chamber and pistons

Dimensions in millimetres

APPARATUS A start from their outermost position, move to their innermost

Chewing apparatus A (Figure 2.9.25.-1) consists of: position then return to their outermost position. Within one
- 1 chewing chamber; cycle, the vertical piston moves from its lowest position to its
- 1 vertical piston; uppermost position and back to its lowest position.
- 2 horizontal pistons with O-rings and sealing rings. Each horizontal piston has a stroke of 25.0 mm.
The chewing chamber consists of 4 individual parts: The maximum distance between these 2 pistons is 50 mm.
- 1 central chamber; The minimum distance between the 2 horizontal pistons is
- 1 funnel (Figure 2.9.25.-2); 0.1 mm to 1.0 mm. The vertical piston has a stroke of
- 2 guides with bushes (Figure 2.9.25.-3). 22.0 mm.
The funnel and guides are mounted on the central chamber. Horizontal piston movement is controlled so that the
The O-rings are incorporated in the piston recess with the 2 pistons are at their innermost position at the same time.
sealing ring around it; the sealing rings ensure that the Vertical piston movement is controlled so that it does not
chamber is watertight. The horizontal pistons are placed in conflict with the movement of the horizontal pistons.
the chewing chamber through the guides. If necessary, the machine can be constructed so that the
The gum is artificially chewed by the horizontal pistons, and horizontal pistons rotate around their own axes in opposite
the vertical piston ensures that the gum stays in the right direction to each other by the end of the chew in order to
place between chews. obtain maximum chewing.
Machine speed is controlled to ensure a constant cycle.
One cycle (chew) is defined as follows: the horizontal pistons
V-A426 Appendix XII B 2023

036
- whether the analysis is performed on the gum residue or
024.95 on the dissolution medium;
- method of analysis.

Lt:!
.....
C')

tr

32
. . . . . . . . ::-3
I _I. I. 50
- .1_1 B
63

Figure 2.9.25.-3 - Guide (section G-G)

Dimensions in millimetres
... ,
C
L.T--r.J
All parts of the apparatus that may come into contact with I I D
I I
the preparation or the dissolution medium are chemically I I I
I I I
inert and do not adsorb, react with or interfere with the I I I
I I I E
sample. I I I
I
APPARATUSB I
I
Chewing apparatus B (Figure 2.9.25.-4) consists of:
- 1 test cell (Figure 2.9.25.-5 or 2.9.25.-6);
- 1 vertical axle with upper chewing surface (Figures F
2.9.25.-7 and 2.9.25.-8); G
- 1 base chamber with lower chewing surface (Figures
2.9.25.-9 and 2.9.25.-10);
- 1 device for up-and-down chewing motion;
- 1 revolving device for the vertical axle.
The gum is artificially chewed by the lower and upper
chewing surfaces. Machine speed is controlled to ensure a
constant cycle. The distance between the lower and upper
chewing surfaces may be set up to 5 mm. The turning angle
of the revolving device is about 20°.
The test cells may also be equipped with 1 or 2 glass
sampling tubes, coming through the thermostatic double H
wall. These tubes also make it possible to have an external
sink, which may be necessary to achieve sink conditions for
sparingly soluble substances.
The gum is usually sandwiched between 2 circular plastic
A. revolving device for the upper E. upper chewing surface
nets to prevent disintegration. chewing surface
Nets made from nylon (PA6) with an aperture of 1.4 mm B. stand F. lower chewing surface
and a wire diameter of 0.405 mm may be used. C. test cell G. base chamber
D. axle H. device for up-and-down chewing
All parts of the apparatus that may come into contact with motion
the preparation or the dissolution medium are chemically
inert and do not adsorb, react with or interfere with the Figure 2.9.25.-4 -Apparatus B
sample.
PROCEDURE Place the prescribed volume of dissolution medium in the
For each determination, the following information is needed: chewing chamber, usually 20 mL of phosphate buffer solution
- apparatus used (type A or type B); pH 6. 0 R2. Maintain the medium temperature at
- composition, volume and temperature of the dissolution 37 ± 0.5 °C using an electrical device with external control
medium; (apparatus A) or with a thermostat (apparatus B). Set the
- number of chews per minute; machine speed at the prescribed number of chews per minute
- time and sampling method; (typically up to 60). Accurately weigh a portion of gum or
the whole gum, put it into the chewing chamber and start the
machine.
2023 Appendix XII B V-A427

0 59 ±2

N
Ol

\ ...)
0

0 37.3 ± 0.1
042 + 1

Figure 2.9.25.-5 - Test cell

Dimensions in millimetres
059±2

N
Ol

I. 0 37.2 ± 0.1

Figure 2.9.25.-6 - Test cell (straight)

Dimensions in millimetres
V-A428 Appendix XII B 2023

SAMPLING AND EVALUATION

Stop the apparatus at the prescribed time. Remove the gum
residue and take a sample of the dissolution medium.
Determine the content of active substance(s) by a suitable
method. Medium replacement may be made after each
sampling procedure; compensation by calculation of medium
volume change or sample dilution is needed. Alternatively,
determine the content of active substance(s) remaining in the
gum residue. Carry out the test successively on 6 medicated
chewing gums.
The quantity of active substance(s) dissolved in a specified
time is expressed as a percentage of the content stated on the
label.
028

M16x1

N
0
0 I Blasted surface RA 1.5-2.1 c:i
N
+I

I \ N

I
I
0
0) Figure 2.9.25.-8 - Upper chewing surface
I Dimensions in millimetres
I

61.6
I 014
I

N
c--i
1
r-<--"'I
I I
M8

,,/ ·~-
""
co .:...-:!

LO
ci
M8

08.5

027

Figure 2.9.25.-7 -Axle

Dimensions in millimetres

8.8 2x N <'>
N

co

Figure 2.9.25.-9 - Base chamber

Dimensions in millimetres
2023 Appendix XII B V-A429

M8
typically in milligrams per minute per square centimetre
(mg-min- 1-cm- 2).
APPARATUS
A typical apparatus consists of a punch and die fabricated
out of hardened steel. The base of the die has 3 threaded
holes for the attachment of a surface plate made of polished
steel, providing a mirror-smooth base for the compact.
The die has a 0.1-1.0 cm diameter cavity into which a
measured amount of the powder to be tested is placed.
The punch is then inserted in the die cavity and the material
is compressed, generally using a benchtop hydraulic press.
A hole through the head of the punch allows insertion of a
I
029 metal rod to facilitate removal from the die after the test.
037
A compact is formed in the cavity with a single face of
defined area exposed on the bottom of the die
Blasted surface RA 1.5-2.1 (Figure 2. 9 .29 .-1). The bottom of the die cavity is threaded
so that at least 50-75 per cent of the compact can dissolve
without falling out of the die. The top of the die has a
threaded shoulder that allows it to be attached to a holder.
The holder is mounted on a laboratory stirring device, and
the entire die, with the compact still in place, is immersed in
N
the dissolution medium and rotated by the stirring device.
0
ci PROCEDURE
+I
N Weigh the material onto a piece of weighing paper. Attach
the surface plate to the underside of the die, and secure it
with the 3 provided screws. Transfer the sample of powder
tested into the die cavity. Place the punch into the chamber,
Figure 2.9.25.-10 - Lower chewing surface
and secure the metal plate on the top of the assembly.
Dimensions in millimetres Compress the powder using a hydraulic press by applying a
suitable pressure for a sufficient dwell time to ensure a stable
5. Intrinsic Dissolution compact with minimal porosity; the disintegration of the
(Ph. Bur. method 2.9.29) compact has to be prevented as far as possible, since it would
The test is intended to determine the intrinsic dissolution cause an increase in surface area and hence in dissolution
rate of pure solid substances following compaction. It is rate. Detach the surface plate, and screw the die with punch
carried out under specified experimental conditions such that still in place into the holder. Tighten securely. Remove all
a practical measure of the intrinsic dissolution rate is loose powder from the surface of the die by blowing
obtained. compressed air or nitrogen across the surface of the compact.
The intrinsic dissolution rate is a theoretical value referring to Slide the die-holder assembly into the dissolution test chuck
pure solid substances having null porosity, but, practically, and tighten. Position the shaft in the spindle so that when
intrinsic dissolution rate is determined on substances having the test head is lowered, the exposed surface of the compact
a minimal porosity. will be 3.8 cm from the bottom of the vessel. The disc
assembly is aligned to minimise wobble and air bubbles are
PRINCIPLE
not allowed to form as this could decrease the compact
The intrinsic dissolution rate is defined as the dissolution rate surface in contact with the dissolution medium. If possible,
of pure substances following compaction under the condition sink conditions are maintained throughout the test. However,
of constant surface area. Its assessment is useful in the in order to obtain detectable concentrations of solute, the use
characterisation of active substances and excipients. of a relatively small volume of medium may be necessary as a
The dissolution rate of pure substances can be affected by all consequence of the limited surface available for dissolution.
the solid state properties such as crystal habit, crystallinity, Warm the dissolution medium to the temperature chosen for
amorphism, polymorphism, pseudo-polymorphism, particle the test. Lower the test head into position before rotation.
size and specific surface area. In addition, it can also be Care should be taken to ensure that air bubbles are excluded
influenced by extrinsic factors (test conditions), such as from the surface of the compact as this could decrease the
hydrodynamics, temperature, viscosity, pH, buffer strength compact surface in contact with the dissolution medium.
and ionic strength of the dissolution medium. Operate the apparatus immediately at the speed of rotation
The assessment of intrinsic dissolution rate of a solid chosen for the test.
substance involves the preparation of a compact. Assurance Collect samples at fixed time intervals and assay them by
of appropriate compaction properties of the powder to be means of an analytical method of suitable sensitivity and
tested is needed prior to performing the test. accuracy.
The intrinsic dissolution rate is determined by exposing a
constant area of the compacted substance to an appropriate ASSESSMENT OF THE RESULTS
dissolution medium, while maintaining constant stirring rate, The data for the cumulative amount dissolved at each time
temperature, ionic strength and pH. point are corrected for sampling losses. To calculate the
intrinsic dissolution rate, plot the cumulative amount of
The intrinsic dissolution rate is expressed in terms of
sample dissolved per unit area of the compact against time.
dissolved mass of substance per time per exposed area,
The cumulative amount dissolved per unit area is given by
V-A430 Appendix XII B 2023

0 9.75 ± 0.35 - - - F

0 41.1 ± 0.4

0 8.0 -t----~
E
N
(0
r---.

A
101
I◄
--.------------------,

0 54.0 ± 0.4 i-,,-----+-+-1----..i B

-1f_.__ ________________
0 7.985 ± 0.005 -l 1-
L..---1---------. A

A. Surface plate B. Die C. Neoprene gasket D. Punch E. Holder and shaft assembly F. Die underside

Figure 2.9.29.-1. - Typical apparatus used to obtain the compact for the determination of the intrinsic dissolution
Dimensions in millimetres

the cumulative amount dissolved at each time point divided 6. Apparent Dissolution
by the surface area exposed. Linear regression is then (Ph. Bur. method 2.9.43)
performed on the normalised experimental data relevant to This method is mainly used to determine the apparent
an appropriate time interval preceding the possible dissolution rate of pure solid substances. It may also be used
disintegration of the compact. The intrinsic dissolution rate for the determination of the apparent dissolution rate of
of the substance tested, expressed in milligrams per minute active substances in preparations presented as powders or
per square centimetre, is determined from the slope of the granules.
regression line. The result for intrinsic dissolution rate must
be accompanied by a statement of the precise conditions of APPARATUS
compact preparation and test method (dissolution medium, All parts of the apparatus that may come into contact with
volume of medium used, stirring rate, temperature etc.). the sample or the dissolution medium are chemically inert
and do not adsorb, react with, or interfere with the test
NOTE When necessary and justified, an apparatus with a
sample. No part of the assembly or its environment
different configuration may be used, such as a die holder that
contributes significant motion, agitation or vibration beyond
holds the compact in a fixed vertical position, with agitation
that resulting from the flow-through system.
provided by a paddle positioned at a defined distance from
the surface of the compact. Apparatus that permits observation of the sample is
preferable.
2023 Appendix XII B V-A431

The apparatus (see Figure 2.9.43.-1) consists of: Immediately filter the liquid removed using an inert filter of
- a reservoir for the dissolution medium; appropriate pore size that does not cause significant
- a pump that forces the dissolution medium upwards adsorption of the substances from the solution and does not
through the flow-through cell; contain substances extractable by the dissolution medium
- a flow-through cell, preferably of transparent material, that would interfere with the prescribed analytical method.
mounted vertically with a filter system preventing escape Proceed with the analysis of the filtrate as prescribed.
of undissolved particles; ASSESSMENT OF THE RESULTS
- a water-bath that will maintain the dissolution medium at
When the test is performed for batch release purposes, an
the chosen temperature (generally 37 ± 0.5 °C).
adequate number of replicates is carried out.
The results are expressed as:
- the amount of dissolved substance by time unit (if the
dissolution is linear);
- the dissolution time of the whole sample and at
appropriate intermediate stages.

,..._ ~---F
<O
N
A B C D cri

A. reservoir for B. pump C. thermostatically D. collecting vessels ~----E

dissolution controlled flow- for analysis
medium through cell and filter
co
lO
Figure 2.9.43.-1. - Flow-through apparatus

The flow-through cell shown in Figure 2.9.43.-2 consists of

3 parts that fit into each other. The lower part supports a ~----B
system of grids and filters on which the powder is placed.
The middle part, which fits onto the lower part, contains an
insert that sieves the sample when the dissolution medium C')
flows through the cell. This insert is made up of 2 parts: a (')
conical sieve that is placed on the sample and a clip placed
midway down the middle part to hold the sieve in place
when the dissolution medium passes through. A 2nd filtration
assembly (grid and filter) is placed on top of the middle part
before fitting the upper part through which the dissolution 0.8
medium flows out of the cell.
DISSOLUTION MEDIUM
If the dissolution medium is buffered, adjust its pH to within
± 0.05 units. Remove any dissolved gases from the A. lower part C. clip E. middle part
B. sieve D. insert F. upper part
dissolution medium before the test, since they can cause the
formation of bubbles, which significantly affect the results.
Figure 2.9.43.-2. - Flow-through cell
METHOD Dimensions in millimetres
Place a bead of 5 ± 0.5 mm diameter at the bottom of the
cone of the lower part followed by glass beads of suitable
Monographs of the British Pharmacopoeia
size, preferably of 1 ± 0.1 mm diameter. Place a sieve (with
The following additional points apply to monographs of the
0.2 mm apertures), a suitable filter and a 2nd sieve on top of
the lower part. Fit the middle part onto the lower part. British Pharmacopoeia.
Weigh the assembly. Place the sample on the filtration APPARATUS
assembly and weigh the sample in the cell. Place the sieve of The choice of the apparatus to be used depends on the
the insert, cone upwards, on the sample, and position the physico-chemical characteristics of the dosage form. When
clip midway down the middle part. Place a sieve (with this Appendix is invoked in an individual tablet or capsule
0.2 mm apertures) and a suitable filter on top of the middle monograph of the British Pharmacopoeia, use Apparatus I
part. Fit the upper part. Heat the dissolution medium to the unless otherwise directed.
chosen temperature. Using a suitable pump, introduce the PROCEDURE
dissolution medium through the bottom of the cell to obtain The dissolution medium is that specified in the individual
a suitable continuous flow through an open or closed circuit monograph. Unless otherwise indicated in the monograph,
at the prescribed rate ± 5 per cent. withdraw samples at 45 minutes.
SAMPLING Where one tablet or capsule is directed to be placed in the
Samples of dissolution medium are collected at the outlet of apparatus, for each of the six tablets or capsules tested the
the cell, irrespective of whether the circuit is opened or amount of active ingredient in solution is not less than 70%
closed. of the prescribed or stated amount, unless otherwise specified
in the monograph, except that if one fails this requirement a
V-A432 Appendix XII C 2023

further six may be tested individually and all must comply. with water Rand then with alcohol Rand dry at 100-105 °C
Where two or more tablets or capsules are directed to be for 1 h, or, if the nature of the container precludes heating at
placed together in the apparatus, a total of six replicate tests this temperature, dry at a lower temperature to constant
are carried out. In each test the amount of active ingredient mass. Allow to cool in a desiccator and weigh. The mass of
in solution per tablet or capsule is not less than 70% of the the contents is the difference between the weighings. Repeat
prescribed or stated amount, unless otherwise specified in the the procedure with another 19 containers.
monograph. No retesting is permitted.
2. Uniformity and Accuracy of Delivered Doses
Where capsule shells interfere with the analysis, remove the from Multidose Containers
contents of no fewer than six capsules as completely as
(Ph. Bur. method 2.9.27)
possible and dissolve the empty capsule shells in the specified
volume of dissolution medium. Carry out the test as directed The following test is intended for dosage forms that are supplied in
in the individual monograph and make any necessary multidose containers provided at manufacture with a measuring
correction. Correction factors should not be greater than device.
25% of the labelled content. If the delivered dose is defined on the label as a mass, weigh
individually 20 doses taken at random from one or more
containers using the measuring device provided as instructed.
Determine the individual and average masses. Unless
C. Consistency of Formulated otherwise specified, not more than 2 of the individual masses
deviate from the average mass by more than 10 per cent and
Preparations none deviates by more than 20 per cent.
1. Uniformity of Weight (Mass) If the monograph specifies an accuracy requirement for the
(Ph. Bur. method 2.9.5) delivered dose, the average mass does not differ from the
Weigh individually 20 units taken at random or, for single- nominal mass by more than the percentage specified, unless
dose preparations presented in individual containers, the otherwise justified and authorised.
contents of 20 units, and determine the average mass. If the delivered dose is defined on the label as a volume,
Not more than 2 of the individual masses deviate from the measure individually the volume of 20 doses taken at random
average mass by more than the percentage deviation shown from one or more containers using the measuring device
in Table 2.9.5.-1 and none deviates by more than twice that provided as instructed. Determine the individual and average
percentage. volumes. Unless otherwise specified, not more than 2 of the
For capsules and powders for parenteral administration, individual volumes deviate from the average volume by more
proceed as described below. than 10 per cent and none deviates by more than
20 per cent. Alternatively, the volume can be determined by
CAPSULES measuring the mass and calculating the volume using the
Weigh an intact capsule. Open the capsule without losing any density.
part of the shell and remove the contents as completely as If the monograph specifies an accuracy requirement for the
possible. For soft shell capsules, wash the shell with a delivered dose, the average volume does not differ from the
suitable solvent and allow to stand until the odour of the nominal volume by more than the percentage specified,
solvent is no longer perceptible. Weigh the shell. The mass of unless otherwise justified and authorised.
the contents is the difference between the weighings. Repeat
the procedure with another 19 capsules. 3. Uniformity of Content
(Ph. Bur. method 2. 9. 6)
Table 2.9.5.-1
The test for uniformity of content of single-dose preparations
Pharmaceutical Form Average Mass Percentage is based on the assay of the individual contents of active
deviation
substance(s) of a number of single-dose units to determine
Tablets (uncoated and film- 80 mg or less 10
whether the individual contents are within limits set with
coated) More than 80 mg and less
7.5 reference to the average content of the sample.
than 250 mg
250 mg or more 5 The test is not required for multivitamin and trace-element
preparations and in other justified and authorised
Capsules, granules (uncoated, Less than 300 mg 10
single-dose) and powders
circumstances.
300 mg or more 7.5
(single-dose) Method Using a suitable analytical method, determine the
Powders for parenteral More than 40 mg 10
individual contents of active substance(s) of 10 dosage units
administration* (single-dose) taken at random.
Suppositories and pessaries All masses 5 Apply the criteria of test A, test B or test C as specified in
the monograph for the dosage form in question.
Powders for eye-drops and Less than 300 mg 10
powders for eye lotions (single- 300 mg or more 7.5 TESTA
dose) The preparation complies with the test if each individual
* When the average mass is equal to or below 40 mg, the preparation is not content is between 85 per cent and 115 per cent of the
submitted to the test for uniformity of mass but to the test for uniformity of average content. The preparation fails to comply with the test
content of single-dose preparations (2. 9. 6). if more than one individual content is outside these limits or
if one individual content is outside the limits of 75 per cent
POWDERS FOR PARENTERAL ADMINISTRATION to 125 per cent of the average content.
Remove any paper labels from a container and wash and dry If one individual content is outside the limits of 85 per cent
the outside. Open the container and without delay weigh the to 115 per cent but within the limits of 75 per cent to
container and its contents. Empty the container as 125 per cent, determine the individual contents of another 20
completely as possible by gentle tapping, rinse it if necessary dosage units taken at random. The preparation complies with
 2023 Appendix XII C V-A433

the test if not more than one of the individual contents of the ( 1) solutions enclosed in single-dose containers and in soft
30 units is outside 85 per cent to 115 per cent of the average capsules;
content and none is outside the limits of 7 5 per cent to (2) solids (including powders, granules and sterile solids) that
125 per cent of the average content. are packaged in single-dose containers and contain no added
TESTB active or inactive substances;
The preparation complies with the test if not more than one (3) solids (including sterile solids) that are packaged in
individual content is outside the limits of 85 per cent to single-dose containers, with or without added active or
115 per cent of the average content and none is outside the inactive substances, that have been prepared from true
limits of 75 per cent to 125 per cent of the average content. solutions and freeze-dried in the final containers and are
The preparation fails to comply with the test if more than labelled to indicate this method of preparation;
3 individual contents are outside the limits of 85 per cent to (4) hard capsules, uncoated tablets, or film-coated tablets,
115 per cent of the average content or if one or more containing 25 mg or more of an active substance comprising
individual contents are outside the limits of 75 per cent to 25 per cent or more, by mass, of the dosage unit or, in the
125 per cent of the average content. case of hard capsules, the capsule contents, except that
If 2 or 3 individual contents are outside the limits of uniformity of other active substances present in lesser
85 per cent to 115 per cent but within the limits of proportions is demonstrated by meeting content uniformity
75 per cent to 125 per cent, determine the individual requirements.
contents of another 20 dosage units taken at random. The test for content uniformity is required for all dosage
The preparation complies with the test if not more than forms not meeting the above conditions for the mass
3 individual contents of the 30 units are outside the limits of variation test. ♦Alternatively, products that do not meet the
85 per cent to 115 per cent of the average content and none 25 mg/25 per cent threshold limit may be tested for
is outside the limits of 75 per cent to 125 per cent of the uniformity of dosage units by mass variation instead of the
average content. content uniformity test on the following condition: the
TESTC concentration Relative Standard Deviation (RSD) of the
The preparation complies with the test if the average content active substance in the final dosage units is not more than
of the 10 dosage units is between 90 per cent and 2 per cent, based on process validation data and development
110 per cent of the content stated on the label and if the data, and if there has been regulatory approval of such a
individual content of each dosage unit is between 75 per cent change. The concentration RSD is the RSD of the
and 125 per cent of the average content. concentration per dosage unit (mlm or m!V), where
concentration per dosage unit equals the assay result per
4. Uniformity of Dosage Units 1 dosage unit divided by the individual dosage unit mass.
(Ph. Bur. method 2.9.40) See the RSD formula in Table 2.9.40.-2. ♦
To ensure the consistency of dosage units, each unit in a CONTENT UNIFORMITY
batch should have an active substance content within a Select not fewer than 30 units, and proceed as follows for the
narrow range around the label claim. Dosage units are dosage form designated. Where different procedures are used
defined as dosage forms containing a single dose or a part of for assay of the preparation and for the content uniformity
a dose of an active substance in each dosage unit. ◊Unless test, it may be necessary to establish a correction factor to be
otherwise stated,◊ the uniformity of dosage units specification applied to the results of the latter.
is not intended to apply to solutions, suspensions, emulsions
or gels in single-dose containers intended for local action Solid dosage forms
following cutaneous administration. ◊The test for content Assay I 0 units individually using an appropriate analytical
method. Calculate the acceptance value (see
uniformity is not required for multivitamin, single-vitamin
and trace-element preparations.◊ Table 2.9.40.-2).
The term 'uniformity of dosage unit' is defined as the degree Liquid or semi-solid dosage forms
of uniformity in the amount of the active substance among Assay 10 units individually using an appropriate analytical
dosage units. Therefore, the requirements of this chapter method. Carry out the assay on the amount of well-mixed
apply to each active substance being comprised in material that is removed from an individual container in
dosage units containing 1 or more active substances, unless conditions of normal use. Express the results as delivered
otherwise specified elsewere in this Pharmacopoeia. dose. Calculate the acceptance value (see Table 2.9.40.-2).
The uniformity of dosage units can be demonstrated by Calculation of Acceptance Value
either of 2 methods: content uniformity or mass variation Calculate the Acceptance Value (AV) using the formula:
(see Table 2.9.40.-1).
The test for content uniformity of preparations presented in IM-Xj +ks
dosage units is based on the assay of the individual contents
for which the terms are as defined in Table 2.9.40.-2.
of active substance(s) of a number of dosage units to
determine whether the individual contents are within the MASS VARIATION
limits set. The content uniformity method may be applied in Carry out an assay for the active substance(s) on a
all cases. representative sample of the batch using an appropriate
The test for mass variation is applicable for the following analytical method. This value is result A, expressed as
dosage forms: percentage of label claim (see Calculation of Acceptance
Value). Assume that the concentration (mass of active
substance per mass of dosage unit) is uniform. Select not
fewer than 30 dosage units, and proceed as follows for the
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter
dosage form designated.
5. 8 Pharmacopoeia/ harmonisation.
V-A434 Appendix XII C 2023

Table 2.9.40.-1. -Application of Content Uniformity (CU) and Mass Variation (MV) test for dosage forms
Dosage forms Type Sub-Type Dose and ratio of active substance

2:: 25 mg and 2:: 25 per cent < 25 mg or < 25 per cent

Tablets uncoated MV CU

coated film-coated MV CU

others CU CU

Capsules hard MV CU

soft suspensions, emulsions, gels CU CU

solutions MV MV
Solids in single-dose
single component MV MV
containers

solution freeze-dried in final

multiple components MV MV
container

others CU CU

Solutions enclosed in MV MV
single-dose containers

Others: dosage forms not CU CU

addressed by the other
categories in this table
including but not limited
to suppositories,
transdermal patches and
semi-solid preparations
applied cutaneously and
intended for systemic
distribution of the active
substance

Uncoated or film-coated tablets Liquid ◊or semi-solid◊ dosage forms

Accurately weigh 10 tablets individually. Calculate the active Accurately weigh the amount of liquid or semi-solid that is
substance content, expressed as percentage of label claim, of removed from each of 10 individual containers in conditions
each tablet from the mass of the individual tablets and the of normal use. If necessary, compute the equivalent volume
result of the assay. Calculate the acceptance value. after determining the density. Calculate the active substance
Hard capsules content in each container from the mass of product removed
Accurately weigh 10 capsules individually, taking care to from the individual containers and the result of the assay.
preserve the identity of each capsule. Remove the contents of Calculate the acceptance value.
each capsule by suitable means. Accurately weigh the Calculation of Acceptance Value
emptied shells individually, and calculate for each capsule the Calculate the acceptance value (AV) as shown in content
net mass of its contents by subtracting the mass of the shell uniformity, except that the individual contents of the units
from the respective gross mass. Calculate the active substance are replaced with the individual estimated contents defined
content in each capsule from the mass of product removed below.
from the individual capsules and the result of the assay.
Calculate the acceptance value. individual estimated contents of the dosage units
tested;
Soft capsules
Accurately weigh 10 intact capsules individually to obtain where
their gross masses, taking care to preserve the identity of each
capsule. Then cut open the capsules by means of a suitable A
clean, dry cutting instrument such as scissors or a sharp open x,=w,x W
blade, and remove the contents by washing with a suitable
individual masses of the dosage units tested;
solvent. Allow the occluded solvent to evaporate from the content of active substance (percentage of label claim)
shells at room temperature over a period of about 30 min, obtained using an appropriate analytical method
taking precautions to avoid uptake or loss of moisture. Weigh (assay);
the individual shells, and calculate the net contents. Calculate w mean of individual masses (w 1, w2 , ... , wi-J.
the active substance content in each capsule from the mass of
product removed from the individual capsules and the result
of the assay. Calculate the acceptance value. CRITERIA
Apply the following criteria, unless otherwise specified.
Solid dosage forms other than tablets and capsules
Proceed as directed for hard capsules, treating each unit as Solid, semi-solid and liquid dosage forms
described therein. Calculate the acceptance value. The requirements for dosage uniformity are met if the
acceptance value of the first 10 dosage units is less than or
equal to Ll per cent. If the acceptance value is greater
2023 Appendix XII C V-A435

Table 2.9.40.-2.
Variable Definition Conditions Value
x Mean of individual contents (x 1 , Xz, ... ,
Xn), expressed as a percentage of the
label claim

xi, x 2 , ... , x 11 Individual contents of the

dosage units tested, expressed as a
percentage of the label claim

n Sample size (number of dosage units

in a sample)

k Acceptability constant If n = 10, then 2.4

If n = 30, then 2.0

1/2
s Sample standard deviation I:" (x;-X)
-,
i=l
n-1

IOOs
RSD Relative standard deviation
x

-
M (case I) Reference value If M=X
To be applied when T <; 101.5 98.5 per cent <; X <; 101.5 per cent, (AV=ks)
then

If X < 98. 5 per cent, then M = 98.5 per cent

(AV= 98.5 -X + ks)

IfX> 101.5 per cent, then M = 101.5 per cent

(AV= X -101.5 + ks)

- -
M (case 2) Reference value If 98.5 per cent <; X <; T, then M=X
To be applied when T > 101.5 (AV= ks)

If X < 98.5 per cent, then M = 98. 5 per cent

(AV= 98.5 -X + ks)

lfX > T, then M = Tper cent

(AV=X-T+ks)

Acceptance value (AV) General formula:

IM -XI +ks
Calculations are specified above for
the different cases.

LI Maximum allowed acceptance value LI = 15.0 unless otherwise specified

L2 Maximum allowed range for deviation On the low side, no dosage unit result L2 = 25.0 unless otherwise specified
of each dosage unit tested from the can be less than 0. 75 M while on the
calculated value of M high side, no dosage unit result can
be greater than 1.25 M (This is based
on L2 value of 25.0)

T Target content per dosage unit at

time of manufacture, expressed as a
percentage of the label claim. Unless
otherwise stated, T is equal to
100 per cent or T is the
manufacturer's approved target
content per dosage unit
V-A436 Appendix XII C 2023

than LI per cent, test the next 20 dosage units and calculate emptying the needle into a dry tared beaker by slowly and
the acceptance value. The requirements are met if the final constantly depressing the piston. Determine the volume in
acceptance value of the 30 dosage units is less than or equal millilitres calculated as the mass in grams divided by the
to LI per cent and no individual content of the dosage unit density.
is less than (1 ~L2 x 0.0l)M or more than (1 + L2 x 0.01) The volume measured for each of the containers is not less
M in calculation of acceptance value under content than the nominal volume.
uniformity or under mass variation. Unless otherwise
specified, LI is 15.0 and L2 is 25.0. PARENTERAL INFUSIONS
Select one container. Transfer the contents into a dry
5. Extractable Volume of Parenteral Preparations 2 measuring cylinder of such a capacity that the volume to be
(Test for Extractable Volume of Parenteral Preparations, Ph. Bur. determined occupies at least 40 per cent of the nominal
method 2. 9.17) volume of the cylinder. Measure the volume transferred.
Suspensions and emulsions are shaken before withdrawal of The volume is not less than the nominal volume.
the contents and before the determination of the density.
Oily and viscous preparations may be warmed according to 7. Preparations for Inhalation: Aerodynamic
the instructions on the label, if necessary, and thoroughly Assessment of Fine Particles
shaken immediately before removing the contents. (Ph. Bur. method 2.9.18)
The contents are then cooled to 20-25 °C before measuring This test is used to determine the fine particle characteristics
the volume. of the aerosol clouds generated by preparations for
inhalation.
SINGLE-DOSE CONTAINERS
Select 1 container if the nominal volume is 10 mL or more, Unless otherwise justified and authorised, one of the
3 containers if the nominal volume is more than 3 mL and following apparatus and test procedures is used.
less than 10 mL, or 5 containers if the nominal volume is Stage mensuration ls performed periodically together
3 mL or less. Take up individually the total contents of each with confirmation of other dimensions critical to the effective
container selected into a dry syringe of a capacity not operation of the impactor.
exceeding 3 times the volume to be measured, and fitted Re-entrainment (for apparatus D and E) To ensure
with a 21-gauge needle not less than 2.5 cm in length. Expel efficient particle capture, coat each plate with glycerol,
any air bubbles from the syringe and needle, then discharge silicone oil or similar high viscosity liquid, typically deposited
the contents of the syringe without emptying the needle into from a volatile solvent. Plate coating must be part of method
a standardised dry cylinder (graduated to contain rather than validation and may be omitted where justified and
to deliver the designated volumes) of such size that the authorised.
volume to be measured occupies at least 40 per cent of its Mass balance The total mass of the active substance is
graduated volume. Alternatively, the volume of the contents not less than 75 per cent and not more than 125 per cent of
in millilitres may be calculated as the mass in grams divided the average delivered dose determined during testing for
by the density. uniformity of delivered dose. This is not a test of the inhaler
For containers with a nominal volume of 2 mL or less, the but it serves to ensure that the results are valid.
contents of a sufficient number of containers may be pooled
APPARATUS A - GLASS IMPINGER
to obtain the volume required for the measurement provided
The apparatus is shown in Figure 2.9.18.-1 (see also
that a separate, dry syringe assembly is used for each
Table 2.9.18.-1).
container. The contents of containers holding 10 mL or
more may be determined by opening them and emptying the Procedure for nebulisers
contents directly into the graduated cylinder or tared beaker. Introduce 7 mL and 30 mL of a suitable solvent into the
upper and lower impingement chambers, respectively.
The volume is not less than the nominal volume in case of
containers examined individually, or, in case of containers Connect all the component parts. Ensure that the assembly is
with a nominal volume of 2 mL or less, is not less than the vertical and adequately supported and that the jet spacer peg
sum of the nominal volumes of the containers taken of the lower jet assembly just touches the bottom of the
collectively. lower impingement chamber. Connect a suitable pump fitted
with a filter (of suitable pore size) to the outlet of the
MULTIDOSE CONTAINERS apparatus. Adjust the air flow through the apparatus, as
For injections in multidose containers labelled to yield a measured at the inlet to the throat, to 60 ± 5 Umin.
specific number of doses of a stated volume, select one
Introduce the liquid preparation for inhalation into the
container and proceed as directed for single-dose containers
reservoir of the nebuliser. Fit the mouthpiece and connect it
using the same number of separate syringe assemblies as the
by means of an adapter to the device.
number of doses specified.
Switch on the pump of the apparatus and after 10 s switch
The volume is such that each syringe delivers not less than
on the nebuliser.
the stated dose.
After 60 s, unless otherwise justified, switch off the nebuliser,
CARTRIDGES AND PREFILLED SYRINGES wait for about 5 s and then switch off the pump of the
Select 1 container if the nominal volume is 10 mL or more, apparatus. Dismantle the apparatus and wash the inner
3 containers if the nominal volume is more than 3 mL and surface of the upper impingement chamber collecting the
less than 10 mL, or 5 containers if the nominal volume is washings in a volumetric flask. Wash the inner surface of the
3 mL or less. If necessary, fit the containers with the lower impingement chamber collecting the washings in a
accessories required for their use (needle, piston, syringe) and second volumetric flask. Finally, wash the filter preceding the
transfer the entire contents of each container without pump and its connections to the lower impingement chamber
and combine the washings with those obtained from the
2 This chapter has undergone pharmacopoeia/ harmonisation. See chapter lower impingement chamber. Determine the amount of
5. 8 Phannacopoeial hannonisation.
2023 Appendix XII C V-A437

active substance collected in each of the 2 flasks. Express the 95

results for each of the 2 parts of the apparatus as a •I
percentage of the total amount of active substance.
Procedure for pressurised inhalers
Place the actuator adapter in position at the end of the throat
so that the mouthpiece end of the actuator, when inserted to 107
a depth of about 10 mm, lines up along the horizontal axis of
the throat and the open end of the actuator, which accepts
the pressurised container, is uppermost and in the same
vertical plane as the rest of the apparatus. 45° 40
,j,
Introduce 7 mL and 30 mL of a suitable solvent into the
upper and lower impingement chambers, respectively.

I
Connect all the component parts. Ensure that the assembly is
vertical and adequately supported and that the lower jet- 63
spacer peg of the lower jet assembly just touches the bottom
of the lower impingement chamber. Connect a suitable pump 15
to the outlet of the apparatus. Adjust the air flow through the
apparatus, as measured at the inlet to the throat, to
60 ± 5 IJmin.
Prime the metering valve by shaking for 5 s and discharging
once to waste; after not less than 5 s, shake and discharge
again to waste. Repeat a further 3 times.
Shake for about 5 s, switch on the pump to the apparatus
and locate the mouthpiece end of the actuator in the adapter, -· +- 0 5.3
discharge once immediately. Remove the assembled inhaler
from the adapter, shake for not less than 5 s, relocate the
mouthpiece end of the actuator in the adapter and discharge
again. Repeat the discharge sequence. The number of
discharges should be minimised and typically would not be 0 1.85 ± 0.125
greater than 10. After the final discharge wait for not less
than 5 s and then switch off the pump. Dismantle the
apparatus. Figure 2.9.18.-1. -Apparatus A: glass impinger
Wash the inner surface of the inlet tube to the lower Dimensions in millimetres (wlerances ± 1 mm unless otherwise
impingement chamber and its outer surface that projects into prescribed)
the chamber with a suitable solvent, collecting the washings
in the lower impingement chamber. Determine the content
of active substance in this solution. Calculate the amount of FINE PARTICLE DOSE AND PARTICLE
active substance collected in the lower impingement chamber SIZE DISTRIBUTION
per discharge and express the results as a percentage of the APPARATUS C - MULTI-STAGE LIQUID
dose stated on the label. IMPINGER
Procedure for powder inhalers The multi-stage liquid impinger consists of impaction
Introduce 7 mL and 30 mL of a suitable solvent into the stages 1 (pre-separator), 2, 3 and 4 and an integral filter
upper and lower impingement chambers, respectively. stage (stage 5), see Figures 2.9.18.-4/6. An impaction stage
Connect all the component parts. Ensure that the assembly is comprises an upper horizontal metal partition wall (B)
vertical and adequately supported and that the jet-spacer peg through which a metal inlet jet tube (A) with its impaction
of the lower jet assembly just touches the bottom of the plate (D) is protruding. A glass cylinder (E) with sampling
lower impingement chamber. Without the inhaler in place, port (F) forms the vertical wall of the stage, and a lower
connect a suitable pump to the outlet of the apparatus. horizontal metal partition wall (G) through which the
Adjust the air flow through the apparatus, as measured at the tube (H) connects to the next lower stage. The tube into
inlet to the throat, to 60 ± 5 IJmin. stage 4 (U) ends in a multi-jet arrangement. The impaction
Prepare the inhaler for use and locate the mouthpiece in the plate (D) is secured in a metal frame 0) which is fastened by
apparatus by means of a suitable adapter. Switch on the 2 wires (K) to a sleeve (L) secured on the jet tube.
pump for 5 s. Switch off the pump and remove the inhaler. The horizontal face of the collection plate is perpendicular to
Repeat the discharge sequence. The number of discharges the axis of the jet tube and centrally aligned. The upper
should be minimised and typically would not be greater surface of the impaction plate is slightly raised above the
than 10. Dismantle the apparatus. edge of the metal frame. A recess around the perimeter of
the horizontal partition wall guides the position of the glass
Wash the inner surface of the inlet tube to the lower cylinder. The glass cylinders are sealed against the horizontal
impingement chamber and its outer surface that projects into
partition walls with gaskets (M) and clamped together by
the chamber with a suitable solvent, collecting the washings
6 bolts (N). The sampling ports are sealed by stoppers.
in the lower impingement chamber. Determine the content The bottom-side of the lower partition wall of stage 4 has a
of active substance in this solution. Calculate the amount of concentrical protrusion fitted with a rubber O-ring (P) which
active substance collected in the lower impingement chamber
seals against the edge of a filter placed in the filter holder.
per discharge and express the results as a percentage of the The filter holder (R) is constructed as a basin with a
dose stated on the label.
concentrical recess in which a perforated filter support (S) is
V-A438 Appendix XII C 2023

Table 2.9.18.-1. - Component specification for apparatus A in

Figure 2.9.18.-1
Code Item Description Dimen-
sions*

A Mouthpiece Moulded rubber adapter for

adaptor actuator mouthpiece. Stage 1

(pre-separator)
B Throat Modified round-bottomed flask: 50mL
- ground-glass inlet socket 29/32
- ground-glass oudet cone 24/29

C Neck Modified glass adapter:

Stage 2
- ground-glass inlet sacker 24129
- ground-glass outlet cone 24/29
Lower outlet section of precision-
bore glass tubing:
- bore diameter 14 Stage 3

Selected bore light-wall glass

tubing:
- external diameter 17

D Upper Modified round-bottomed flask 100 mL

impingement - ground-glass inlet socket 24/29 Stage4

chamber - ground-glass outlet cone 24/29

E Coupling tube Medium-wall glass tubing:

- ground-glass cone 14/23
Stage 5 (filter)

Bent section and upper vertical

section: Outlet
- external diameter 13

Lower vertical section:

- external diameter 8

F Screwthread, Plastic screw cap 28/13

side-arm Silicone rubber ring 28/11
adaptor PTFE washer 28/11
Figure 2.9.18.-4. -Apparatus C: multi-stage liquid impinger
Glass screwthread: Procedure for pressurised inhalers
- thread size 28 Dispense 20 mL of a solvent, capable of dissolving the active
Side-arm outlet to vacuum pump: substance into each of stages 1 to 4 and replace the stoppers.
- minimum bore diameter 5
Tilt the apparatus to wet the stoppers, thereby neutralising
electrostatic charge. Place a suitable filter capable of
G Lower jct Modified polypropylene filter holder see quantitatively collecting the active substance in stage 5 and
assembly connected to lower vertical section Figure
assemble the apparatus. Place a suitable mouthpiece adapter
of coupling tube by PTFE tubing. 2.9.18.-1
in position at the end of the induction port so that the
Acetal circular disc with the centres
mouthpiece end of the actuator, when inserted, lines up
of four jets arranged on a projected
circle of diameter 5.3 mm with an along the horizontal axis of the induction port and the
integral jet spacer peg: 10 inhaler is positioned in the same orientation as intended for
- peg diameter 2 use. Connect a suitable vacuum pump to the outlet of the
-
apparatus and adjust the air flow through the apparatus, as
peg protrusion 2
measured at the inlet to the induction port, to
H Lmver Conical flask 250mL 30 Umin (± 5 per cent). Switch off the pump.
impingement - ground-glass in/er socket 24/29 Unless otherwise prescribed in the patient instructions, shake
chamber the inhaler for 5 s and discharge 1 delivery to waste. Switch
* Dimensions in millimetres, unless otheraise stated. on the pump to the apparatus, locate the mouthpiece end of
the actuator in the adapter and discharge the inhaler into the
flush-fitted. The filter holder is dimensioned for 76 mm apparatus, depressing the valve for a sufficient time to ensure
diameter filters. The assembly of impaction stages is clamped complete discharge. Wait for 5 s before removing the
onto the filter holder by 2 snap-locks (D. Connect an assembled inhaler from the adapter. Repeat the procedure.
induction port (see Figure 2.9.18.-7) onto the stage 1 inlet The number of discharges should be minimised and typically
jet tube of the impinger. A rubber O-ring on the jet tube would not be greater than 10. The number of discharges is
provides an airtight connection to the induction port. sufficient to ensure an accurate and precise determination of
A suitable mouthpiece adapter is used to provide an airtight the fine particle dose. After the final discharge, wait for 5 s
seal between the inhaler and the induction port. The front and then switch off the pump.
face of the inhaler mouthpiece must be flush with the front
face of the induction port.
2023 Appendix XII C V-A439

stage center

'
I.
wl
(6x)

V H

I;, ;'(

I O~
........,
\ I
k '.) ,.. co

,' \ \.
\
I

'
'\
0\ '

II
5

' I++-++ ;(:,:

4
B
- h(?x)
multi-jet pattern K N

multi-jet U only

J
C

e
D

Figure 2.9.18.-5. - Apparatus C: details of jet tube and impaction plate. Inserts show end of multi-jet tube U leading to stage 4.
(Numbers and lowercase letters refer to Table 2.9.18.-3 and uppercase letters refer to Figure 2.9.18.-4).

Dismantle the filter stage of the apparatus. Carefully remove Procedure for powder inhalers
the filter and extract the active substance into an aliquot of Place a suitable low resistance filter capable of quantitatively
the solvent. Remove the induction port and mouthpiece collecting the active substance in stage 5 and assemble the
adapter from the apparatus and extract the active substance apparatus. Connect the apparatus to a flow system according
into an aliquot of the solvent. If necessary, rinse the inside of to the scheme specified in Figure 2.9.18.-8 and
the inlet jet tube to stage 1 with solvent, allowing the solvent Table 2.9.18.-4. Unless otherwise defined, conduct the test
to flow into the stage. Extract the active substance from the at the flow rate, Q u,, used in the test for uniformity of
0

inner walls and the collection plate of each of the 4 upper delivered dose, drawing 4 L of air from the mouthpiece of
stages of the apparatus into the solution in the respective the inhaler and through the apparatus.
stage by carefully tilting and rotating the apparatus, observing Connect a flowmeter to the induction port. Use a flowmeter
that no liquid transfer occurs between the stages. calibrated for the volumetric flow leaving the meter, or
Using a suitable method of analysis, determine the quantity calculate the volumetric flow leaving the meter (Q,ui) using
of active substance contained in each of the aliquots of
solvent.
Calculate the fine particle dose (see Calculations).
V-A440 Appendix XII C 2023

Table 2.9.18.-2. - Component specificatwnfor apparatus C in Table 2.9.18.-3. - Dimension/1J ofjet tube with impaction plate
Figures 2. 9.18.-4/6 of apparatus C
Code* Item Description Dimen- Type Code<2) Stage I Stage 2 Stage 3 Stage 4 Filter
sions** (stage 5)

A,H Jet tube Metal tube screwed onto partition wall see Figure Distance 1 9.5 5.5 4.0 6.0 n.a.
sealed by gasket (C), polished inner 2.9.18.-5 (-.0+.5) (-.0+.5) (-.0+.5) (-.0+.5)
surface
Distance 2 26 31 33 30.5 0
B,G Partition wall Circular metal plate Distance 3 8 5 5 5 5
- diameter 120 Distance 4 3 3 3 3 n.a.
- thickness see Figure Distance 5 0 3 3 3 3
2.9.18.-5 6 (3)
Distance 20 25 25 25 25
C Gasket e.g. PTFE to fit jet tube Distance 7 n.a. n.a. n.a. 8.5 n.a.

D Impaction Porosity O sintered-glass disk Diameter C 25 14 8.0 21 14

(± .1)
plate - diameter see Figure
2.9.18.-5 Diameter d so 30 20 30 n.a.

E Glass cylinder Plane polished cut glass tube Diameter e 27.9 16.5 10.5 23.9 n.a.

- height, including gaskets 46 Diameter f 31.75 22 14 31 22

(-.0+.5)
- omer diameter 100
Diameter g 25.4 21 13 30 21
- wall thickness 3.5
Diameter h n.a. n.a. n.a. 2.70 n.a.
- sampling port (F) diameter 18 (± .5)
- stopper in sampling port ISO 24/25 Diameter j n.a. n.a. n.a. 6.3 n.a.

Metal frame Diameter k n.a. n.a. n.a. 12.6 n.a.

J L-profiled circular frame with slit

- inner diameter to fit Radius(4) r 16 22 27 28.5 0

impaction
plate Radius s 46 46 46 46 n.a.

- height 4 Radius t n.a. 50 50 50 50

- thickness of horizontal, section 0.5 Angle w 10' 53' 53' 53' 53'
- thickness of vertical. section 2 Angle u n.a. n.a. n.a. 45' n.a.

Wire Steel wire interconnecting metal frame Angle V n.a. n.a. n.a. 60' n.a.
K
and sleeve (2 for each frame)
(1) Measures in millimetres with tolerances according to ISO 2768-m unless otherwise
- diameter 1 stated
(2) Refer to Figure 2.9.18.-5
L Sleeve Metal sleeve secured on jet tube by (3) Including gasket
screw (4) Relative centreline of stage compartment
- n.a. = not applicable
inner diameter to fit jet tube
- height 6
- thickness 5
081
M Gasket e.g. silicone to fit glass
cylinder

N Bolt Metal bolt with nut (6 pairs)

·- length 205
- diameter 4

p O-ring Rubber O-ring

- diameter X thickness 66.34 X 2.62

Q O-ring Rubber O-ring

- diameter x thickness 29.1 X 1.6

R Filter holder Metal housing with stand and outlet see Figure
2.9.18.-6

s Filter support Perforated sheet metal

- diameter 65 Figure 2.9.18.-6. -Apparatus C: details of the filter stage
- hole diameter 3 (stage 5). Numbers refer to dimensions (0 = diameter).
- distance between holes (centre-points) 4 Uppercase letters refer to Table 2.9.18.-2.
T Snap-locks Dimensions in millimetres unless otherwise stated
u Multi-jet tube Jet tube (H) ending in multi-jet see inserts the ideal gas law. For a meter calibrated for the entering
arrangement. Figure
2.9.18.-5
volumetric flow (Q;n), use the following expression:

* Refers to Figure 2.9.18,-4. n _ Q;n xPo

** Measures in millimetres with tolerances according to iso 2768-m unless otherwise
~ut - Po -!!i.P
stated.

atmospheric pressure,
pressure drop over the meter.
2023 Appendix XII C V-A441

Drill, counter-bore and tap

for an M-4 cap screw. 19.0
Note: use minimum clearance
for screw in the lower part to aid 97 31.8
in precise alignment.
79 38
/
/
-,J{J - - - - -1 - - I- -
J c..,
:'_ - - - ----r - --+ -
- - - - - ,_ - L
+---- -,----+---
i I ;
0
I+
0

/, i I
' l
/ I
/ :I 1
'
l
I()
0
r-
14.3 , I- r· J 45 degree
8.6 / I I
chamfer
/, I' I
, I '
I
'17$0 I_ '
.:f- 70
25.4
38

Do not break edge

100 ± 10

Joint must be leak tight

M-4 socket
head cap screw

Isometric view of induction port

1. Material may be aluminium, stainless steel or other suitable material.

2. Machine from 38 mm bar stock.
3. Bore 19 mm hole through bar.
4. Cut tube to exact 45' as shown.
5. The inner bores and tapers should be smooth - surface roughness Ra approx. 0.4 µm.
6. Mill joining cads of stock to provide a liquid tight leak-free seal.
7. Set up a holding fixture for aligning the inner 19 mm bore and for drilling and tapping M4 x 0.7 threads. There must be virtually no mismatch of the inner
bores in the miter joint.

Figure 2.9.18.-7. - Induction port

Dimensions in millimetres unless otherwise stated

Adjust the flow control valve to achieve steady flow through Dispense 20 mL of a solvent, capable of dissolving the active
the system at the required rate, Q,u, (± 5 per cent). Switch substance into each of the 4 upper stages of the apparatus
off the pump. Ensure that critical flow occurs in the flow and replace the stoppers. Tilt the apparatus to wet the
control valve by the following procedure. stoppers, thereby neutralising electrostatic charge. Place a
With the inhaler in place and the test flow rate established, suitable mouthpiece adapter in position at the end of the
measure the absolute pressure on both sides of the control induction port.
valve (pressure reading points P2 and P3 in
Figure 2.9.18.-8). A ratio P3/P2 of less than or equal to 0.5
indicates critical flow. Switch to a more powerful pump and
re-measure the test flow rate if critical flow is not indicated.
V-A442 Appendix XII C 2023

D Impactor

Vacuum
pump
Two-way
Solenoid
Valve
i iFlow
Control
Valve
C F
Connector Vacuum Tubing
8
A

Figure 2.9.18.-8. - Experimental set-up for testing pcnvder

inhalers

Table 2.9.18.-4. - Component specification for Figure 2. 9.18.-8

Code Item Description

A Connector ID 2 8 mm, e.g., short metal coupling, with

low-diameter branch to P3.

B Vacuum tubing A length of suitable tubing having an

ID 2 8 mm and an internal volume of
25 ± 5 mL.

C 2-way solenoid A 2-way, 2-port solenoid valve having a

valve minimum airflow resistance orifice with
ID 2 8 mm and an opening time :S I 00 ms.
(e.g. type 256-A0S, Burkert GmbH,
D-74653 Ingelfingen), or equivalent.

D Vacuum pump Pump must be capable of drawing the required

flow rate through the assembled apparatus with
the powder inhaler in the mouthpiece adapter
(e.g. product type 1023, 1423 or 2565, Gast
Manufacturing Inc., Benton Harbor, MI
49022), or equivalent. Connect the pump to
the 2-way solenoid valve using short and/or
wide (ID 2 10 mm) vacuum tubing and
connectors to minimise pump capacity
requirements.

E Timer Timer capable to drive the 2-way solenoid

valve for the required duration (e.g. type G814,
RS Components International, Corby,
NN! 7 9RS, UK), or equivalent.

P2 P3 Pressure Determine under steady-state flow condition Figure 2. 9 .18. -9. - Apparatus D: Andersen cascade impactor
measurements with an absolute pressure transducer. used for pressurised inhalers
F Flow control Adjustable regulating valve with maximum
valve Cv 2 I, (e.g. type 8FV12LNSS, Parker to flow into the stage. Extract the active substance from the
Hannifin pie., Barnstaple, EX3 I !NP, UK), or inner walls and the collection plate of each of the 4 upper
equivalent. stages of the apparatus into the solution in the respective
stage by carefully tilting and rotating the apparatus, observing
Prepare the powder inhaler for use according to patient that no liquid transfer occurs between the stages.
instructions. With the pump running and the 2-way solenoid Using a suitable method of analysis, determine the amount of
valve closed, locate the mouthpiece of the inhaler in the active substance contained in each of the aliquots of solvent.
mouthpiece adapter. Discharge the powder into the Calculate the fine particle dose (see Calculations).
apparatus by opening the valve for the required time, T
APPARATUS D - ANDERSEN CASCADE
(± 5 per cent). Repeat the procedure. The number of
IMPACTOR
discharges should be minimised and typically would not be
The Andersen 1 ACFM non-viable cascade impactor consists
greater than 10. The number of discharges is sufficient to
of 8 stages together with a final filter. Material of
ensure an accurate and precise determination of fine particle
construction may be aluminium, stainless steel or other
dose.
suitable material. The stages are clamped together and sealed
Dismantle the filter stage of the apparatus. Carefully remove with O-rings. Critical dimensions applied by the
the filter and extract the active substance into an aliquot of manufacturer of apparatus D are provided in
the solvent. Remove the induction port and mouthpiece Table 2.9.18.-5. In use, some occlusion and wear of holes
adapter from the apparatus and extract the active substance will occur. In-use mensuration tolerances need to be justified.
into an aliquot of the solvent. If necessary, rinse the inside of In the configuration used for pressurised inhalers
the inlet jet tube to stage 1 with solvent, allowing the solvent
2023 Appendix XII C V-A443

Cross-section
'
15 -------,
R 12. 7 ------I
~:.47 R15

8~
' R15.87 ~ ~005
Top view ' R14.4
Groove for O-ring
R12.7

R44

R6.70±0.03 10°±1° R8 - - - - R38.3: g 2

107
R6.70 ± 0.03
Do not break edge ~106
450±30 V 102

~100
94
Material: aluminium, stainless steel
or other suitable material.

Side view Except where noted, all edges to be broken

and burrs removed.

Surface to be clean machine - tooled finish.

_/27 Interior bore surface roughness Ra
-22 approx. 0.4 µm.
-14
0-ring: nominal dimensions: ID 29 mm,
OD 32 mm, width 1.8 mm.
-------0

~ Do not break edge

Figure 2.9.18.-10. - Connection of the induction port to the preseparator of the Andersen cascade impactor
Dimensions in millimetres unless otherwise stated

(Figure 2.9.18.-9) the entry cone of the impactor is Table 2.9.18.-5. - Critical dimensions for apparatus D
connected to an induction port (see Figure 2.9.18.-7).
Description Number Dimension (mm)
A suitable mouthpiece adapter is used to provide an airtight
seal between the inhaler and the induction port. The front Stage 0 nozzle diameter 96 2.55 ± 0.025
face of the inhaler mouthpiece must be flush with the front Stage 1 nozzle diameter 96 1.89 ± 0.025
face of the induction port. Stage 2 nozzle diameter 400 0.914 ± 0.0127
In the configuration for powder inhalers, a pre-separator is Stage 3 nozzle diameter 400 0.711 ± 0.0127
placed above the top stage to collect large masses of non-
respirable powder. It is connected to the induction port as
Stage 4 nozzle diameter 400 0.533 ± 0.0127
shown in Figure 2.9.18.-10. To accommodate high flow rates Stage 5 nozzle diameter 400 0.343 ± 0.0127
through the impactor, the outlet nipple, used to connect the Stage 6 nozzle diameter 400 0.254 1: 0.0127
impactor to the vacuum system is enlarged to have an Stage 7 nozzle diameter 201 0.254 ± 0.0127
internal diameter of greater than or equal to 8 mm.
Procedure for pressurised inhalers
Assemble the Andersen impactor with a suitable filter in
place. Ensure that the system is airtight. In that respect,
follow the manufacturer's instructions. Place a suitable
V-A444 Appendix XII C 2023

mouthpiece adapter in position at the end of the induction Prepare the powder inhaler for use according to the patient
port so that the mouthpiece end of the actuator, when instructions. With the pump running and the 2-way solenoid
inserted, lines up along the horizontal axis of the induction valve closed, locate the mouthpiece of the inhaler in the
port and the inhaler unit is positioned in the same orientation mouthpiece adapter. Discharge the powder into the
as the intended use. Connect a suitable pump to the outlet of apparatus by opening the valve for the required time,
the apparatus and adjust the air flow through the apparatus, T (± 5 per cent). Repeat the discharge sequence.
as measured at the inlet to the induction port, to The number of discharges should be minimised and typically
28.3 Umin(± 5 per cent). Switch off the pump. would not be greater than 10. The number of discharges is
Unless otherwise prescribed in the patient instructions, shake sufficient to ensure an accurate and precise determination of
the inhaler for 5 s and discharge one delivery to waste. fine particle dose.
Switch on the pump to the apparatus, locate the mouthpiece Dismantle the apparatus. Carefully remove the filter and
end of the actuator in the adapter and discharge the inverted extract the active substance into an aliquot of the solvent.
inhaler into the apparatus, depressing the valve for a Remove the pre-separator, induction port and moutl1piece
sufficient time to ensure complete discharge. Wait for 5 s adapter from the apparatus and extract the active substance
before removing the assembled inhaler from the adapter. into an aliquot of the solvent. Extract the active substance
Repeat the procedure. The number of discharges should be from the inner walls and the collection plate of each of the
minimised and typically would not be greater than 10. stages of the apparatus into aliquots of solvent.
The number of discharges is sufficient to ensure an accurate Using a suitable method of analysis, determine the quantity
and precise determination of the fine particle dose. After the of active substance contained in each of the aliquots of
final discharge, wait for 5 s and then switch off the pump. solvent.
Dismantle the apparatus. Carefully remove the filter and Calculate the fine particle dose (see Calculations).
extract the active substance into an aliquot of the solvent.
Remove the induction port and mouthpiece adapter from the APPARATUSE
apparatus and extract the active substance into an aliquot of Apparatus E is a cascade impactor with 7 stages and a micro-
the solvent. Extract the active substance from the inner walls orifice collector (MOC). Over the flow rate range of
and the collection plate of each of the stages of the apparatus 30 Umin to 100 Umin the 50 per cent-efficiency cut-off
into aliquots of solvent. diameters (D 50 values) range between 0.24 µm to 11. 7 µm,
evenly spaced on a logarithmic scale. In this flow range, there
Using a suitable method of analysis, determine the quantity
are always at least 5 stages with D 50 values between 0.5 µm
of active substance contained in each of the aliquots of
and 6.5 µm. The collection efficiency curves for each stage
solvent.
are sharp and minimise overlap between stages.
Calculate the fine particle dose (see Calculations).
Material of construction may be aluminium, stainless steel or
Procedure for powder inhalers other suitable material.
The aerodynamic cut-off diameters of the individual stages of this
apparatus are currently not well-established at flow rates other Table 2.9.18.-6. - Critical dimensions for apparatus E
than 28.3 L!min. Users must justify and validate the use of Description Dimension
the impactor in the chosen conditions, when flow rates (nun)
different from 28.3 Umin are selected.
Pre-separator (dimension a - see Figure 2.9.18.-15) 12.8 ± 0.05
Assemble the Andersen impactor with the pre-separator and
a suitable filter in place and ensure that the system is airtight. Stage 1* Nozzle diameter 14.3 ± 0.05
Depending on the product characteristics, the pre-separator Stage 2* Nozzle diameter 4.88 ± 0.04
may be omitted, where justified and authorised. Stages 6 Stage 3* Nozzle diameter 2.185 ± 0.02
and 7 may also be omitted at high flow rates, if justified. Stage 4* Nozzle diameter 1.207 ± 0.01
The pre-separator may be coated in the same way as the
Stage 5* Nozzle diameter 0.608 ± 0.01
plates or may contain 10 mL of a suitable solvent. Connect
Stage 6* Nozzle diameter 0.323 ± 0.01
the apparatus to a flow system according to the scheme
specified in Figure 2.9.18.-8 and Table 2.9.18.-4. Stage 7* Nozzle diameter 0.206 ± 0.01

Unless otherwise defined, conduct the test at the flow rate, MOC* approx. 0.070
Q0 u,, used in the test for uniformity of delivered dose drawing Cup depth (dimension b - see Figure 2.9.18.-14) 14.625 ± 0.10
4 L of air from the mouthpiece of the inhaler and through Collection cup surface roughness (Ra) 0.5 - 2 µm
the apparatus. Stage 1 nozzle to seal body distance** - dimension c 0 ± 1.18
Connect a flowmeter to the induction port. Use a flowmeter Stage 2 nozzle to seal body distance** - dimension c 5.236 ± 0.736
calibrated for the volumetric flow leaving the meter, or
Stage 3 nozzle to seal body distance** - dimension c 8.445 ± 0.410
calculate the volumetric flow leaving the meter (Q,mt) using
the ideal gas law. For a meter calibrated for the entering Stage 4 nozzle to seal body distance** - dimension c 11.379 ± 0.237
volumetric flow (Q;n), use the following expression: Stage 5 nozzle to seal body distance** - dimension c 13.176 ± 0.341
Stage 6 nozzle to seal body distance** - dimension c 13.999 ± 0.071
Stage 7 nozzle to seal body distance** - dimension c 14.000 ± 0.071
MOC nozzle to seal body distance** - dimension c 14.429 to
Pu atmospheric pressure, 14.571
/J.P ::;; pressure drop over the meter.
* See Figure 2.9.18.-13
** See Figure 2.9.18.-14
Adjust the flow control valve to achieve steady flow through
the system at the required rate, Q,,ur (± 5 per cent). Ensure
that critical flow occurs in the flow control valve by the The impactor configuration has removable impaction cups
procedure described for Apparatus C. Switch off the pump. with all the cups in one plane (Figures 2.9.18.-11/14). There
2023 Appendix XII C V-A445

Induction port

Pre-separator

Impactor body

Airflow outlet

Clamping mechanism

Figure 2.9.18.-11. -Apparatus E (shown with the pre-separator in place)

are 3 main sections to the impactor; the bottom frame that cup surface below the MOC. For impactors operated at
holds the impaction cups, the seal body that holds the jets 60 Umin, the MOC is capable of collecting 80 per cent of
and the lid that contains the interstage passageways 0.14 µm particles. For formulations with a significant fraction
(Figures 2.9.18.-11/12). Multiple nozzles are used at all but of particles not captured by the MOC, there is an optional
the first stage (Figure 2.9.18.-13). The flow passes through filter holder that can replace the MOC or be placed
the impactor in a saw-tooth pattern. downstream of the MOC (a glass fibre filter is suitable).
Critical dimensions are provided in Table 2.9.18.-6. Procedure for pressurised inhalers
In routine operation, the seal body and lid are held together Place cups into the apertures in the cup tray. Insert the cup
as a single assembly. The impaction cups are accessible when tray into the bottom frame, and lower into place. Close the
this assembly is opened at the end of an inhaler test. impactor lid with the seal body attached and operate the
The cups are held in a support tray, so that all cups can be handle to lock the impactor together so that the system is
removed from the impactor simultaneously by lifting out the airtight.
tray. Connect an induction port with internal dimensions defined
An induction port with internal dimensions (relevant to the in Figure 2.9.18.-7 to the impactor inlet. Place a suitable
airflow path) defined in Figure 2.9.18.-7 connects to the mouthpiece adapter in position at the end of the induction
impactor inlet. A pre-separator can be added when required, port so that the mouthpiece end of the actuator, when
typically with powder inhalers, and connects between the inserted, lines up along the horizontal axis of the induction
induction port and the impactor. A suitable mouthpiece port. The front face of the inhaler mouthpiece must be flush
adapter is used to provide an airtight seal between the inhaler with the front face of the induction port. When attached to
and the induction port. the mouthpiece adapter, the inhaler is positioned in the same
Apparatus E contains a terminal Micro-Orifice orientation as intended for use. Connect a suitable pump to
Collector (MOC) that for most formulations will eliminate the outlet of the apparatus and adjust the air flow through
the need for a final filter as determined by method validation. the apparatus, as measured at the inlet to the induction port,
The MOC is an impactor plate with nominally 4032 holes, to 30 Umin ( ± 5 per cent). Switch off the pump.
each approximately 70 µm in diameter. Most particles not Unless otherwise prescribed in the patient instructions, shake
captured on stage 7 of the impactor will be captured on the the inhaler for 5 s and discharge 1 delivery to waste. Switch
V-A446 Appendix XII C 2023

Stage 1 nozzle Interstage passageway

Micro-orifice
collector (MOC)

Lid with seal

body attached

Location pin

cup tray in place

Figure 2.9.18.-12. -Apparatus E showing component parts

on the pump to the apparatus. Prepare the inhaler for use separator base. Fit the pre-separator base to the impactor
according to the patient instructions, locate the mouthpiece inlet. Add 15 mL of the solvent used for sample recovery to
end of the actuator in the adapter and discharge the inhaler the central cup of the pre-separator insert. Place the pre-
into the apparatus, depressing the valve for a sufficient time separator body on top of this assembly and close the
to ensure a complete discharge. Wait for 5 s before removing 2 catches.
the assembled inhaler from the adapter. Repeat the Connect an induction port with internal dimensions defined
procedure. The number of discharges should be minimised, in Figure 2.9.18.-7 to the impactor inlet or pre-separator
and typically would not be greater than 10. The number of inlet. Place a suitable mouthpiece adapter in position at the
discharges is sufficient to ensure an accurate and precise end of the induction port so that the mouthpiece end of the
determination of the fine particle dose. After the final inhaler, when inserted, lines up along the horizontal axis of
discharge, wait for 5 s and then switch off the pump. the induction port. The front face of the inhaler mouthpiece
Dismantle the apparatus and recover the active substance as must be flush with the front face of the induction port. When
follows: remove the induction port and mouthpiece adapter attached to the mouthpiece adapter, the inhaler is positioned
from the apparatus and recover the deposited active in the same orientation as intended for use. Connect the
substance into an aliquot of solvent. Open the impactor by apparatus to a flow system according to the scheme specified
releasing the handle and lifting the lid. Remove the cup tray, in Figure 2.9.18.-8 and Table 2.9.18.-4.
with the collection cups, and recover the active substance in Unless otherwise prescribed, conduct the test at the flow
each cup into an aliquot of solvent. rate, Q,u,, used in the test for uniformity of delivered dose
Using a suitable method of analysis, determine the quantity drawing 4 L of air from the mouthpiece of the inhaler and
of active substance contained in each of the aliquots of through the apparatus. Connect a flowmeter to the induction
solvent. port. Use a flowmeter calibrated for the volumetric flow
Calculate the fine particle dose (see Calculations). leaving the meter, or calculate the volumetric flow leaving the
meter (Q,,a) using the ideal gas law. For a meter calibrated
Procedure for powder inhalers
for the entering volumetric flow (Q;n), use the following
Assemble the apparatus with the pre-separator
expression:
(Figure 2.9.18.-15). Depending on the product
characteristics, the pre-separator may be omitted, where
justified.
Place cups into the apertures in the cup tray. Insert the cup
tray into the bottom frame, and lower into place. Close the P0 atmospheric pressure,
l!.P pressure drop over the meter.
impactor lid with the seal body attached and operate the
handle to lock the impactor together so that the system is
Adjust the flow control valve to achieve steady flow through
airtight.
the system at the required rate, Q,u, ( ± 5 per cent). Ensure
When used, the pre-separator should be assembled as that critical flow occurs in the flow control valve by the
follows: assemble the pre-separator insert into the pre- procedure described for Apparatus C. Switch off the pump.
2023 Appendix XII C V-A447

Stage 2 Stage 4 Stage 6 MOC

6 holes 52 holes 396 holes 4032 holes

Stage 1 Stage 3 Stage 5 Stage 7

1 hole 24 holes 152 holes 630 holes
Figure 2.9.18.-13. -Apparatus E: nozzle configuration
Interstage passage Interstage passage
to next stage from previous stage

Lid

:::;:a====.~ Seal body

,,..--'-----,~Cup tray

Bottom
~ frame

Collection cup J Multi-nozzle stage

Figure 2.9.18.-14. -Apparatus E: configuration of interstage passageways

Prepare the powder inhaler for use according to the patient CALCULATIONS
instructions. With the pump running and the 2-way solenoid From the analysis of the solutions, calculate the mass of
valve closed, locate the mouthpiece of the inhaler in the active substance deposited on each stage per discharge and
mouthpiece adapter. Discharge the powder into the the mass of active substance per discharge deposited in the
apparatus by opening the valve for the required time, induction port, mouthpiece adapter and when used, the pre-
T ( ± 5 per cent). Repeat the discharge sequence. separator.
The number of discharges should be minimised and typically Starting at the final collection site (filter or MOC), derive a
would not be greater than 10. The number of discharges is table of cumulative mass versus cut-off diameter of the
sufficient to ensure an accurate and precise determination of respective stage (see Tables 2.9.18.-7 for
fine particle dose. Apparatus C, 2.9.18.-8 for Apparatus D, 2.9.18.-9 for
Dismantle the apparatus and recover the active substance as Apparatus E). Calculate by interpolation the mass of the
follows: remove the induction port and mouthpiece adapter active substance less than 5 µm. This is the Fine Particle
from the pre-separator, when used, and recover the deposited Dose (FPD).
active substance into an aliquot of solvent. When used, If necessary, and where appropriate (e.g., where there is a
remove the pre-separator from the impactor, being careful to log-normal distribution), plot the cumulative fraction of
avoid spilling the cup liquid into the impactor. Recover the active substance versus cut-off diameter (see
active substance from the pre-separator. Tables 2.9.18.-7/9) on log probability paper, and use this
Open the impactor by releasing the handle and lifting the lid. plot to determine values for the Mass Median Aerodynamic
Remove the cup tray, with the collection cups, and recover Diameter (MMAD) and Geometric Standard
the active substance in each cup into an aliquot of solvent. Deviation (GSD) as appropriate. Appropriate computational
Using a suitable method of analysis, determine the quantity methods may also be used.
of active substance contained in each of the aliquots of
solvent.
Calculate the fine particle dose (see Calculations).
V-A448 Appendix XII C 2023

Pre-separator body

Nozzle diameter,
dimension a
Catch

/ Central cup

t
( )
u c::J u ►
t
Pre-separator insert

Pre-separator base

Figure 2. 9 .18. -15. - Apparatus E: pre-separator configuration

8. Preparations for Nebulisation: Characterisation measure of the mass of active substance that would be
(Ph. Bur. method 2. 9. 44) delivered to patients.
Products used for nebulisation and intended for pulmonary Active substance delivery rate and total active substance
delivery are characterised using the following tests: delivered are appropriate characteristics because they allow
- Active substance delivery rate and total active substance the mass delivered to be characterised in a standard way
delivered; regardless of the nebuliser used. Accordingly, the test
- Aerodynamic assessment of nebulised aerosols. methodology described below allows that the mass of active
These tests standardise the approach given to the assessment substance delivered in the 1st period (typically 1 min) is
of the dose that would be delivered to a patient but are not measured (consequently giving an assessment of active
intended to provide assessment of the nebuliser device itself, substance delivery rate) as well as capturing the total mass of
which is described in the European standard active substance delivered.
EN 13544-1:2007+Al:2009, Respiratory therapy APPARATUS
equipment - Part 1: Nebulizing systems and their Breathing simulator
components. A commercially available breathing simulator, which is able
The mass- rather than the number-weighted size distribution to generate the breathing profiles specified in
is more appropriate to evaluate product performance. Indeed, Table 2.9.44.-1, is used for the test. The breathing profile
active substance mass as a function of aerodynamic diameter indicated for adults is used unless the medicinal product is
is more indicative of therapeutic effect within the respiratory specifically intended for use in paediatrics, where alternate
tract. patterns should be used, as indicated in Table 2.9.44.-1.
ACTIVE SUBSTANCE DELIVERY RATE AND Table 2.9.44.-1. - Breathing simulator specijicatwns
TOTAL ACTIVE SUBSTANCE DELIVERED
Item Specification
These tests are performed to assess the rate of delivery to the
patient and the total active substance delivered to the patient, Adult Neonate Infant Child

using standardised conditions of volumetric flow rate. It is Tidal volume 500mL 25 mL 50 mL 155 mL
essential that breath-enhanced and breath-actuated nebulisers Frequency 15 cycles/min 40 cycles/min 30 cycles/min 2 5 cycles/min
be evaluated by a breathing simulator, as the output of these Waveform sinusoidal sinusoidal sinusoidal sinusoidal
types of device is highly dependent on inhalation flow rate. Inhalation/ 1:1 1:3 1:3 1:2
The methodology below describes the use of a standard exhalation
breathing pattern defined for adults. Should a particular ratio

product for nebulisation only be indicated for paediatric

(i.e. neonate, infant or child) use, then paediatric breathing Filter system
pattern(s) must be used. Breathing patterns are used, rather A suitably validated low-resistance filter, capable of
than continuous flow rates, to provide a more appropriate quantitatively collecting the aerosol and enabling recovery of
2023 Appendix XII C V-A449

Table 2.9.18.-7. - Calculations for Apparatus C. Use q = ✓ (60/Q) , where Q is the test flow rate in litres per minute (~ 1,,for powder
inhalers)
Cut-off di:nneter Mass of active substance deposited Cumulative mass of active substance Cumulative fraction of active substance
(,tm) per discharge deposited per discharge (per cent)

d, = 1.7 x q mass from stage 5, m 5 * C4 = IIl5 f4 = (c4 /c) x 100

d3 = 3.1 X q mass from stage 4, m 4 C3 = C4 + ffi4 f3 = (c 3 ic) x 100
d2 = 6.8 X q mass from stage 3, m 3 C2 = C3 + ffi3 f2 = (c 2 /c) x 100
mass from stage 2, m 2 C = C2 + illz 100

* Stage 5 is the filter stage

Table 2.9.18.-8. - Calculations for Apparatus D when used at a flow rate of 28.3 Llmin
Cut-off diameter Mass of active substance deposited Cumulative mass of active substance Cumulative fraction of active substance
(,,m) per discharge deposited per discharge (per cent)

d1 = 0.4 mass from stage 8, m 8 f7 = (cic) x 100

d 6 = 0.7 mass from stage 7, m 7 f6 = (c 6/c) x 100
d, = 1.1 mass from stage 6, m 6 f5 = (c 5/c) x 100
d, = 2.1 mass from stage S, m 5 f4 = (c 4 /c) x 100
d3 = 3.3 mass from stage 4, m 4 [3 = (c)c) x 100
d 2 = 4.7 mass from stage 3, m 3 f2 = (c 2 /c) x 100

d, = 5.8 mass from stage 2, m 2 f 1 = (c 1/c) x 100

d 0 = 9.0 mass from stage 1, m 1 co = c1 + m1 fo = (c0 /c) x 100
mass from stage 01 m 0 c=c 0 +m 0 100

Table 2. 9 .18.-9. - Calculations for Apparatus E. Use q = (60/QY, where Q is the test flow rate in litres per minute, and x is listed in
the table
Cut-off di:nneter X Mass of active substance Cumulative mass of active substance Cumulative fraction of active
(µm) deposited per discharge deposited per discharge substance (per cent)

d, = 0.34 x q 0.67 mass from MOC or terminal C7 = ms F 7 = (c7 /c) x 100

filter, m 8
d 6 = 0.55 x q 0.60 mass from stage 7, rn 7 C6 = C7 + ffi7 F 6 = (c6 /c) x 100
d, = 0.94 x q 0.53 mass from stage 6, m 6 c5 = c6 + m6 F 5 = (c5 /c) x 100
d4 = 1.66 X q 0.47 mass from stage 5, m 5 C4 = C5 + ID5 F 4 = (c4/c) x 100
d3 = 2.82 X q 0.50 mass from stage 4, m 4 C3 = C4 + ffi4 F 3 = (c3/c) x 100
d2 = 4.46 X q 0.52 mass from stage 3., m 3 C2 = C3 + ffi3 F 2 = (c 2/c) x 100
d, = 8.06 x q 0.54 mass from stage 2, m 2 C1 = C2 + fil2 F1 = (c1/c) x 100
mass from stage I, m 1 C = C1 + ffi1 100

the active substance with an appropriate solvent, is used for in 60 s is insufficient for this analysis, the length of the time
the test. The dead volume of the filter casing does not exceed interval for aerosol collection can be increased. If the filter is
10 per cent of the tidal volume used in the breath simulation. soaked with the preparation, this time can be decreased.
METHOD At the end of this initial period, stop the nebuliser.
Attach the filter (contained in the filter holder) (A) to the Place a fresh filter and filter holder in position and continue
breath simulator (B) according to Figure 2.9.44.-1. Fill the until nebulisation ceases. Interrupt nebulisation and exchange
nebuliser (C) with the volume of the medicinal product as filters if necessary, to avoid filter saturation.
specified in the patient instructions. Attach the mouthpiece RESULTS
of the nebuliser to the inhalation filter using a mouthpiece Using a suitable method of analysis, determine the mass of
adapter if required, ensuring that connections are airtight. active substance collected on the filters and filter holders
Make sure the nebuliser is positioned in the same orientation during each time interval. Determine the active substance
as intended for use; this may require tilting the breathing delivery rate by dividing the mass of active substance
simulator and filter holder. Set the breathing simulator to collected on the first inhalation filter by the time interval
generate the specified breathing pattern. used for collection. Determine the total mass of active
Start the breathing simulator then, at the beginning of an substance delivered by summing the mass of active substance
inhalation cycle, start the nebuliser. Operate the nebuliser for collected on all inhalation filters and filter holders.
a defined initial time period. The time chosen, usually
AERODYNAMIC ASSESSMENT OF NEBULISED
60 ± 1 s, must allow sufficient active substance deposition
AEROSOLS
on the inhalation filter to allow quantitative analysis. If the
Nebulised products need to be size-characterised at flow rates
quantity of active substance deposited on the inhalation filter
lower than the range that is normally used for powder
V-A450 Appendix XII C 2023

A B

A. inhalation filter and filter holder B. breathing simulator C. nebuliser

Figure 2.9.44.-1. - Experimental set-up for breathing simulator testing

inhalers and metered-dose inhalers. A flow rate of 15 Umin to the impactor, is used to capture any fine droplets that pass
is recommended in the European standard because this value beyond the last size fractionating stage.
represents a good approximation to the mid-inhalation flow A pre-separator is not used for testing nebuliser-generated
rate achievable by a tidally breathing healthy adult (500 mL aerosols.
tidal volume).
METHOD VALIDATION
Although low-angle laser light scattering instruments (laser
Impactor stage overloading
diffractometers) can provide rapid size-distribution
measurements of nebuliser-generated aerosols, these During method development and validation, it is important
to confirm that the volume of liquid sampled from the
techniques do not detect the active substance; rather they
nebuliser does not overload the impactor. Visual inspection
measure the size distribution of the droplets irrespective of
their content. This may not be a problem with homogeneous of the collection surfaces on stages collecting most of the
droplets may reveal streaking if overloading has occurred.
solutions, but can result in significant error if the product to
be nebulised is a suspension, or if droplet evaporation is This phenomenon is usually also associated with an increase
in mass of active substance collected on the final stage and
significant as can be the case with certain nebuliser types.
back-up filter. Reducing the sampling period (T0) is the most
Cascade impactors enable the aerosol to be characterised
effective way to avoid overloading in any given system,
unambiguously in terms of the mass of active substance as a
balancing overloading with analytical sensitivity.
function of aerodynamic diameter. Laser diffraction may be
used if validated against a cascade impaction method. Re-entrainment
Apparatus E (see below under Apparatus), a cascade Droplet bounce and re-entrainment are less likely with
impactor, has been calibrated at 15 Umin specifically to nebuliser-produced droplets than with solid particles from
meet the recommendation of the European standard, and is inhalers and for that reason coating would not normally be
therefore used for this test. Determining mass balance in the required.
same way as for powder inhalers and metered-dose inhalers is METHOD
not straightforward, in that the dose is being captured as a Pre-cool the assembled impactor and induction port in a
continuous output, and hence is not included. As part of refrigerator (set at about 5 °C) for not less than 90 min and
method development, recovery experiments must be start the determination within about 5 min of removal of the
performed to validate the method. impactor from the refrigerator. Other methods that maintain
It is also recognised that the control of evaporation of the impactor at a constant temperature (for example, use of a
droplets produced by nebulisers may be critical to avoid bias cooling cabinet) can also be employed when validated.
in the droplet size assessment process. Evaporation can be Set up the nebuliser with a supply of driving gas (usually air
minimised by cooling the impactor to a temperature of about or oxygen), or use a compressor, at the pressure and flow
5 °C, typically achieved by cooling the impactor in a rate specified by the manufacturer of the nebuliser. Take
refrigerator for about 90 min. Typically, at least after each precautions to ensure that the gas supply line does not
day of use, the apparatus must be fully cleaned, including the become detached from the nebuliser when under pressure.
inter-stage passageways, in view of the greater risk of Fill the nebuliser with the volume of the medicinal product
corrosion caused by the condensation/accumulation of saline- as specified in the patient instructions.
containing droplets on inter-stage metalwork associated with Remove the impactor from the refrigerator. Attach the
cooling the impactor. It is recommended to dry all surfaces induction port to the impactor, and connect the outlet of the
of the apparatus after each test, for example with compressed impactor/external filter to a vacuum source that is capable of
air. Note: the micro-orifice collector (MOC) should not be drawing air through the system at 15 Umin as specified in
dried with compressed air. Figure 2.9.44.-2. Turn on the flow through the impactor.
APPARATUS Connect a flow meter, calibrated for the volumetric flow
A detailed description of Apparatus E and the induction port leaving the meter, to the induction port. Adjust the flow
is contained in general chapter 2. 9.18, and includes details of control valve located between the impactor and the vacuum
critical dimensions and the qualification process for the source to achieve a steady flow through the system at
impactor (stage mensuration). 15 Umin(± 5 per cent). Remove the flow meter.
A back-up filter in addition to the micro-orifice Make sure the nebuliser is positioned in the same orientation
collector (MOC) must be used to ensure quantitative as intended for use then attach the mouthpiece of the
recovery of active substance from the nebulised aerosol at the nebuliser to the induction port, using a mouthpiece adapter if
specified flow rate of 15 Umin. The filter is located below required, ensuring that connections are airtight. Switch on
the MOC (internal filter option) or a filter in holder, external the flow/compressor for the nebuliser. Sample for a
2023 Appendix XII C V-A451

C D

A. nebuliser B. induction port C. impactor (apparatus E) D. flow control valve E. vacuum source

Figure 2.9.44.-2. - Apparatus E for measuring the size distribution of preparations for nebulisation

predetermined time (T0 ). Once determined, this time (T0 ) Table 2.9.44.-2. - Cut-off sizes for Apparatus Eat 15 Umin
must be defined and used in the analytical method for a Stage Cut-off diameter (µm)
particular medicinal product to ensure that mass fraction 14.1
data can be compared. At the end of the sampling period, 2 8.61
switch off the driving gas flow/compressor to the nebuliser, 3 5.39
remove the nebuliser from the induction port and switch off 4 3.30
the flow from the vacuum source to the impactor. 5 2.08
Dismantle the impactor and, using a suitable method of 6 1.36
analysis, determine the mass of active substance collected in 7 0.98
the induction port, on each stage and on the back-up
filter/external filter as described for Apparatus E in general If necessary, and where appropriate, determine values for the
chapter 2. 9.18. Add the mass of active substance collected in mass median aerodynamic diameter (MMAD) and the
the MOC to that deposited on the back-up filter/external geometric standard deviation (GSD), as appropriate.
filter and treat as a single sample for the purpose of
subsequent calculations. 9. Demonstration of Uniformity of Dosage Units
Calculate the mass fraction (Fm,comp) of the active substance Using Large Samples Sizes
deposited on each component of the impactor, commencing (Ph. Bur. method 2.9.47)
with the induction port and proceeding in order through the The procedure is intended for, but not limited to, the evaluation of
impactor, using the following expression: medicinal products that are manufactured using process analytical
technology (PAI) methodology.
mcomp
Pm.comp=~ Compliance with general chapter 2. 9. 40. Uniformity of
dosage units can be demonstrated by the following procedure,
mcomp mass associated with the component under evaluation; when large samples (sample size n?:: 100) are evaluated.
M total mass collected by the system.
Application of this chapter does not constitute a mandatory
requirement. It presents 2 alternative tests (Alternative 1 and
Present Fm,comp in order of location within the measurement Alternative 2). Fulfilling the requirements of either of the
equipment, beginning at the induction port and ending with 2 alternatives is considered as evidence that the medicinal
the back-up filter of the impactor (see Figure 2.9.44.-3). product tested complies with general chapter 2. 9. 40.
Where appropriate, Fm,comp for adjacent stages of the The 2 alternatives are considered equivalent in their
impactor may be combined in order to report the mass demonstration of compliance with general chapter 2. 9. 40.
fraction collected on a group of stages as a single value.
Determine the cumulative mass-weighted particle-size ALTERNATIVE 1 (PARAMETRIC)
distribution of the aerosol size-fractionated by the impactor Select not fewer than 100 units according to a predefined
in accordance with the procedure given in general chapter sampling plan.
2. 9.18. Starting at the filter, derive a cumulative mass versus The consistency of dosage units is evaluated by content
effective cut-off diameter of the respective stages (see uniformity or mass variation as prescribed in Table 2.9.40.-1.
Table 2.9.44.-2 for the appropriate cut-off diameters at Calculate the acceptance value (A V) using the following
15 Umin). Plot the cumulative fraction of active substance expression:
versus cut-off diameter in a suitable format, for example
logarithmic or log-probability format. Where appropriate, IM-XI +ks
determine by interpolation the fraction either below a given
size or between an upper and a lower size limit. for which the terms are defined in Table 2.9.40.-2, but using
the sample size-dependent value for k defined in
Table 2.9.47.-1.
V-A452 Appendix XII C 2023

0.0
t:: N ("') 'SI' I{) (0 I'- ()
0 (I) (I) (I) (I) (I) (I) (I) 0
Cl. C) C) C) C) C) C) C)
co co co co co ~
C .l9 .l9 ';:::
0 u5 (/) (/) u5 u5 u5 u5
:g ~
:J u:::
-0
.E:

Figure 2.9.44.-3. - Example of mass fraction of droplets presented in terms of location within the sampling system

CRITERIA Table 2.9.47.-2 is to be interpreted as follows:

Apply the following criteria, unless otherwise specified. - for a sample size of n = 400, enter the table at n 2 394:
The requirements for dosage form uniformity are met if: cl = 11 and c2 = 3;
- for a sample size of n = 450, enter the table at n 2 434:
1. the acceptance value (AV) is less than or equal to Ll; and cl =12 and c2 3; =
2. in the calculation of acceptance value (AV) under content uniformity or - for a sample size of n = 500, enter the table at n 2 490:
under mass variation, the number of individual dosage units outside
(1 ± L2 x 0.0l)M is less than or equal to c2 as defined for a given
=
cl 13 and c2 4. =
sample size n in Table 2.9.47.-1. 10. Content of active ingredient on actuation of the
valve test
Unless otherwise specified, L1 is 15.0 and L2 is 25.0. The foUowing test conditions are for use in Preparations for
Table 2.9.47.-1. is to be interpreted as follows: Inhalation of the British Pharmacopoeia. Specifically the
- for a sample size of n = 400, enter the table at n 2 385: methodology should be applied to Pressurised Inhalation products.
k = 2.23 and c2 = 3;
Content of active ingredient delivered by actuation of
- for a sample size of n = 450, enter the table at n 2 407:
the valve
k =2.24 and c2 = 3;
Remove the pressurised container from the actuator and
- for a sample size of n = 500, enter the table at n 2 490:
remove all labels and markings which may be present on the
=
k 2.24 and c2 4. = container with a suitable solvent. Dry the container, replace
ALTERNATIVE 2 (NON-PARAMETRIC) in its actuator, shake for about 30 seconds and prime the
Select not fewer than 100 units according to a predefined metering valve as follows. Discharge once to waste, wait for
sampling plan. not less than 5 seconds and discharge again to waste.
The consistency of dosage units is evaluated by content Remove the pressurised container from its actuator, clean the
uniformity or mass variation as prescribed in Table 2.9.40.-1. valve stem (internally and externally) and the valve ferrule by
Assay individually or weigh the units and calculate individual washing with a suitable solvent. Dry the complete valve
contents as prescribed in general chapter 2. 9.40. Count the assembly using an air line fitted with an appropriate narrow
number of individual dosage units with a content outside jet to ensure that all solvent is removed from the inside of the
( 1 ± L1 x 0. 01) T and the number of individual valve stem.
dosage units with a content outside (1 ± L2 x 0.0l)T. Place a stainless steel base plate that has three legs and a
Evaluate if the values are within the limits defined in central circular indentation with a hole about 1.5 mm in
Table 2.9.47.-2. diameter in a small vessel suitable for shaking and add the
CRITERIA volume of solvent specified in the monograph. The size of
Apply the following criteria, unless otherwise specified. the vessel is such that when the pressurised inhalation is
discharged into the specified volume of solvent as described
The requirements for dosage form uniformity are met if:
below the discharge takes place not less than 25 mm below
the surface of the solvent.
1. the number of individual dosage units outside (1 ± L1 x O.OJ)T is less
than or equal to cl; and Shake the pressurised container for about 30 seconds and
2. the number of individual dosage units outside (1 ± L2 x 0.0l)T is less place it inverted in the vessel. Discharge 10 deliveries below
than or equal to c2. the surface of the solvent actuating the valve at intervals of
not less than 5 seconds, maintaining the pressurised
cl and c2 for a given sample size n are defined in container in the vertical plane and discharging the pressurised
Table 2.9.47 .-2. Unless otherwise specified, Ll is 15.0 and inhalation through the hole in the centre of the base plate. (It
L2 is 25.0. may be necessary because of the nature of the formulation to
shake the pressurised container between each actuation of the
2023 Appendix XII C V-A453

Table 2.9.47.-1. -Acceptability constant (k) and acceptable number of dosage units with a content outside (1 ± L2 x 0.0l)M (= c2)
for a given sample size n

n(2) k c2 n (2) k c2 n (2) k c2 n (2) k c2 n (2) k c2 n (2) k c2

100 2.15 804 2.26 2480 2.29 23 4366 2.30 41 6252 2.31 59 8243 2.31 78
7
105 2.16 905 2.27 2585 2.29 24 4471 2.30 42 6357 2.31 60 8347 2.31 79

908 2.27 8 2690 2.29 25 4576 2.30 43 6462 2.31 61

120 2.17 0 8452 2.31 80
1013 2.27 9 2794 2.29 26 4680 2.30 44 6566 2.31 62
139 2.18 8557 2.31 81
1118 2.27 10 2899 2.29 27 4785 2.30 45 6671 2.31 63
161 2.19
8662 2.31 82
1223 2.27 3004 2.29 28 4890 2.30 46 6776 2.31 64
176 2.19 11 8767 2.31 83
1276 2.28 3109 2.29 4995 2.30 47 6881 2.31 65
189 2.20 29
1 8871 2.31 84
1328 2.28 12 3171 2.30 5099 2.30 48 6985 2.31 66
224 2.21
1432 2.28 13 3213 2.30 30 5204 2.30 49 7090 2.31 67 8976 2.31 85
270 2.22
1537 2.28 14 3318 2.30 31 5309 2.30 50 7195 2.31 68 9081 2.31 86
280 2.22
2 1642 2.28 15 3423 2.30 32 5414 2.30 51 7300 2.31 69 9186 2.31 87
328 2.23
1747 2.28 16 3528 2.30 33 5519 2.30 52 7404 2.31 70
9290 2.31 88
385 2.23
1851 2.28 3633 2.30 34 5623 2.30 53 7509 2.31 71
3 9395 2.31 89
17
407 2.24
1918 2.29 3737 2.30 35 5728 2.30 54 7614 2.31 72
9500 2.31 90
490 2.24
7719 2.31 73
1956 2.29 18 3842 2.30 36 5833 2.30 55
4
9605 2.31 91
516 2.25 2061 2.29 19 3947 2.30 37 5938 2.30 56 7824 2.31 74
9710 2.31 92
594 2.25 2166 2.29 20 4052 2.30 38 6042 2.30 7928 2.31 75
5 57
672 2.26 8033 2.31 76 9814 2.31 93
2270 2.29 21 4156 2.30 39 6136 2.31

699 2.26 6 2375 2.29 22 4261 2.30 40 6147 2.31 58 8138 2.31 77 9919 2.31 94

valve; where this is the case shaking should be carried out

without removing the pressurised container from its inverted
position in the vessel.) Remove the pressurised container,
wash it with the specified solvent and dilute the combined
solution and washings to the volume specified in the
monograph. Determine the amount of active ingredient by
the method described under the Assay and calculate the
amount delivered from each actuation of the valve.
The result lies within the range for the content of active
ingredient stated in the monograph.
V-A454 Appendix XII C 2023

Table 2. 9 .4 7 .-2. - Acceptable number of individual dosage units with a content outside (1 ± L1 x 0. 01) T (= cl) and
(1 ± L2 x 0.01) T (= c2) respectively, for a given sample size n

n~) c1 c2 n~) c1 c2 n~) c1 c2 n~) c1 c2 n~) c1 c2 n~) c1 c2 n~) c1 c2

100 3 1432 35 2899 67 4366 98 5833 129 7300 160 8767 191
123 4 0 1476 36 13 2935 68 27 4377 99 41 5835 130 7304 161 8780 192 83
55 69
159 5 1521 37 2981 69 4424 100 5883 131 7351 162
8828 193
176 5 1537 37 3004 69 4471 101 5930 132 7399 163
8871 193
196 6 1566 38 14 3027 70 28 4518 102 42 5938 132 7404 163
1 8875 194
234 7 1611 39 3073 71 4565 103 5977 133 56 7447 164 70 84
1642 39 8923 195
273 8 3109 71 4576 103 6024 134 7494 165
1656 40 3120 72 6042 134 7509 165 8971 196
280 8 15 4612 104 43
29
1701 41 3166 73 6072 135 57 7542 166 71 8976 196
313 9 2 4658 105
1746 42 3212 74 6119 136 7589 167 9019 197 85
353 10 4680 105
1747 42 3213 74 6147 136 7614 167
385 10 4705 106 44 9066 198
1791 43 16 3259 75 30 6166 137 58 7637 168 72
394 11 4752 107 9081 198
3 1836 44 3305 76 6214 138 7684 169
434 12 4785 107 9114 199 86
1851 44
3318 76 6252 138 7719 169
476 13 4799 108 45 9162 200
1882 45 17
3351 77 31 6261 139 7732 170 73
490 13 4846 109 59
1927 46 9186 200
3398 78 6308 140 7779 171
517 14 4 4890 109
1956 46 9210 201 87
3423 78 6355 141 7824 171
559 15 18 4893 110
1972 47 46
3444 79 32 6357 141 7827 172 9257 202
594 15 2018 48 4940 111 74
3491 80 6403 142 60 7875 173 9290 202
601 16 2061 48 4987 112
5 3528 80 6450 143 7922 174 9305 203 88
644 17 2063 49 4995 112
19 3537 81 6462 143 7928 174
686 18 2109 50 33 5034 113 47 9353 204
3584 82 6498 144 61 7970 175 75
699 18 2154 51 5081 114 9395 204
3630 83 6545 145 8017 176
729 19 6 2166 51 5099 114 9401 205
3633 83 6566 145 8033 176 89
772 20 2200 52 20 5128 115 48 9449 206
3677 84 34 6592 146 62 8065 177 76
804 20 2246 53 5175 116
3723 85 6640 147 8113 178 9496 207
815 21 2270 53 5204 116
7 3737 85 6671 147 8138 178 9500 207
858 22 2291 54 21 5222 117 49
3770 86 35 6687 148 63 8160 179 77 9544 208 90
23 2337 55 5269 118
902
3817 87 6734 149 8208 180 9592 209
2375 55 5309 118
908 23
3842 87 6776 149 8243 180
2383 56 9605 209
945 24 8 22 5317 119
3863 88 36 50 6782 150 8256 181 78
2429 57 64 9640 210 91
989 25 5364 120
3910 89 6829 151 8303 182
2475 58 9688 211
1013 25 5411 121
2480 58 3947 89 6877 152 8347 182
1033 26 9 5414 121 9710 211
2520 59 23 3956 90 6881 152 8351 183
27 37 5458 122 51 79
1077 4003 91 6924 153 65 8399 184 9735 212 92
2566 60
1118 27 4050 92 5505 123 6972 154 8446 185 9783 213
2585 60
1121 28 4052 92 5519 123 6985 154 8452 185
2612 61 24 9814 213
10
1165 29 4097 93 38 5552 124 52 7019 155 66 8494 186 80
2658 62 9831 214 93
1209 30 4143 94 5599 125 7067 156 8542 187
2690 62 9879 215
1223 30 4156 94 5623 125 7090 156 8557 187
2704 63 25
9919 215
1253 31 11 2750 64 4190 95 39 5647 126 53 7114 157 67 8589 188 81
9927 216
1298 32 2794 64 4237 96 5694 127 7161 158 8637 189
5728 127 9975 217 94
1328 32 2796 65 4261 96 7195 158 8662 189
26 10023 218
1342 33 12 2843 66 4284 97 40 5741 128 54 7209 159 68 8685 190 82
1387 34 2889 67 4330 98 5788 129 7256 160 8732 191 10070 219
2023 Appendix XIII A V-A455

the equipment from top to bottom, outside and then inside,

Appendix XIII with particle-free water R.
Take care not to introduce air bubbles into the preparation
A. Particulate Contamination: Sub-visible to be examined, especially when fractions of the preparation
are being transferred to the container in which the
Particles1 determination is to be carried out.
(Ph. Bur. method 2.9.19) In order to check that the environment is suitable for the
Particulate contamination of injections and infusions consists test, that the glassware is properly cleaned and that the water
of mobile, undissolved particles, other than gas bubbles, that to be used is particle-free, the following test is carried out:
are unintentionally present. ◊Particulate contamination may determine the particulate contamination of 5 samples of
originate from various sources and is to be minimised, particle-free water R, each of 5 mL, according to the method
independent of its type. The level of particulate described below. If the number of particles of 10 µm or
contamination in parenteral preparations must be controlled. greater size exceeds 25 for the combined 25 mL, the
◊ precautions taken for the test are not sufficient.
For the determination of particulate contamination, The preparatory steps must be repeated until the
environment, glassware and water are suitable for the test.
2 procedures, Method 1 (light obscuration particle count
test) and Method 2 (microscopic particle count test), are Method
specified hereinafter. When examining injections and Mix the contents of the sample by slowly inverting the
infusions for sub-visible particles, Method 1 is preferably container 20 times successively. If necessary, cautiously
applied. However, it may be necessary to test some remove the sealing closure. Clean the outer surfaces of the
preparations by the light obscuration particle count test container opening using a jet of particle-free water R and
followed by the microscopic particle count test to reach a remove the closure, avoiding any contamination of the
conclusion on conformance to the requirements. contents. Eliminate gas bubbles by appropriate measures
Not all parenteral preparations can be examined for sub- such as allowing the sample to stand for 2 min or sonicating.
visible particles by one or both of these methods. When For large-volume parenterals, single units are tested.
Method 1 is not applicable, e.g. in case of preparations For small-volume parenterals less than 25 mL in volume, the
having reduced clarity or increased viscosity, the test is contents of 10 or more units are combined in a cleaned
carried out according to Method 2. Emulsions, colloids, and container to obtain a volume of not less than 25 mL; where
liposomal preparations are examples. Similarly, products that justified and authorised, the test solution may be prepared by
produce air or gas bubbles when drawn into the sensor may mixing the contents of a suitable number of vials and diluting
also require ◊specific precautions during sample preparation to 25 mL with particle-free water R or with an appropriate
and/or◊ microscopic particle count testing. If the viscosity of solvent without contamination of particles when particle-free
the preparation to be tested is sufficiently high so as to water R is not suitable. Small-volume parenterals having a
preclude its examination by either test method, a quantitative volume of 25 mL or more may be tested individually.
dilution with an appropriate ◊particle-free◊ diluent may be Powders for parenteral administration are reconstituted with
made to decrease viscosity, as necessary, to allow the analysis particle-free water R or with an appropriate solvent without
to be performed. contamination of particles when particle-free water R is not
The results obtained in examining a discrete unit or group suitable.
of units for particulate contamination cannot be extrapolated The number of test specimens must be adequate to provide a
with certainty to other units that remain untested. Thus, statistically sound assessment. For large-volume parenterals
statistically sound sampling plans must be developed if valid or for small-volume parenterals having a volume of 25 mL or
inferences are to be drawn from observed data to characterise more, fewer than 10 units may be tested, based on an
the level of particulate contamination in a large group appropriate sampling plan.
of units. Remove 4 portions, each of not less than 5 mL, and count
METHOD 1. LIGHT OBSCURATION PARTICLE the number of particles equal to or greater than 10 µm and
COUNT TEST 25 µm. Disregard the result obtained for the first portion,
Use a suitable apparatus based on the principle of light and calculate the mean number of particles for the
blockage which allows an automatic determination of the size preparation to be examined.
of particles and the number of particles according to size. ◊Alternative method
The apparatus is calibrated using suitable certified reference This method is intended to improve the applicability of the
materials consisting of dispersions of spherical particles of test to biological preparations. However, it can be used for
known sizes between 10 µm and 25 µm. These standard any type of preparation.
particles are dispersed in particle-free water R. Care must be Clean the outer surfaces of the container(s) using a jet of
taken to avoid aggregation of particles during dispersion. particle-free water R, avoiding any contamination of the
General precautions contents. Samples are tested under in-use conditions, as
The test is carried out under conditions limiting particulate directed in the instructions for use (e.g. expelled syringe
contamination, preferably in a laminar-flow cabinet. contents).
Very carefully wash the glassware and filtration equipment For parenteral preparations that have a sufficient volume
used, except for the membrane filters, with a warm detergent (i.e. a volume large enough to permit testing), testing of
solution and rinse with abundant amounts of water to individual units is often preferred.
remove all traces of detergent. Immediately before use, rinse If the volume is not sufficient, take a suitable number
of units, mix each one carefully and thoroughly and then
1 This chapter has undergone phannacopoeial hannonisation. See chapter
combine the contents in a separate container to obtain the
5. 8 Phamzacopoeial hamwnisation.
V-A456 Appendix XIII A 2023

Qooe

GFOVcircle

Qo oQ

• Reference
circle
Qo oQ

oo•e Cross
hairs

Linear scale
11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111~-

Figure 2.9.19.-1. - Circular diameter graticule

volume required for a single test, depending on the millilitre equal to or greater than 10 µm and does not exceed
instrument capability and the properties of the sample. 3 per millilitre equal to or greater than 25 µm.
Powders for parenteral administration are reconstituted with Test 1.B - Solutions for infusion or solutions for injection suppHed
particle-free water R or with an appropriate particle-free in containers with a nominal content of less than 100 mL
solvent when particle-free water R is not suitable. The preparation complies with the test if the average number
Eliminate gas bubbles by appropriate measures such as of particles present in the units tested does not exceed
allowing the sample to stand, applying a gentle vacuum or 6000 per container equal to or greater than 10 µm and does
sonicating. Sonication of preparations containing proteins is not exceed 600 per container equal to or greater than 25 µm.
not recommended. METHOD 2. MICROSCOPIC PARTICLE COUNT
The number of test specimens must be adequate to provide a TEST
statistically sound assessment. For large- and small-volume Use a suitable binocular microscope, filter assembly for
parenterals, an adequate volume of sample must be provided retaining particulate contamination and membrane filter for
for analysis; however, single units may be tested based on a examination.
statistically sound sampling plan.
The microscope is equipped with an ocular micrometer
Remove 4 approximately equal portions, typically 5 mL each, calibrated with an objective micrometer, a mechanical stage
and count the number of particles equal to or greater than capable of holding and traversing the entire filtration area of
10 µm and 25 µm. Disregard the result obtained for the first the membrane filter, 2 suitable illuminators to provide
portion, and calculate the mean number of particles for the episcopic illumination in addition to oblique illumination,
preparation to be examined. Volumes smaller than 5 mL can and is adjusted to 100 ± 10 magnifications.
also be tested, provided that this amount is appropriately
The ocular micrometer is a circular diameter graticule (see
justified. In general, for parenteral preparations that do not
Figure 2.9.19.-1.) and consists of a large circle divided by
have a sufficient volume (e.g. less than 25 mL), performing
crosshairs into quadrants, transparent and black reference
the test using a volume of 1 mL to 5 mL may be acceptable
circles 10 µm and 25 µm in diameter at 100 magnifications,
if permitted by the instrument.◊
and a linear scale graduated in 10 µm increments. It is
Evaluation calibrated using a stage micrometer that is certified by either
For preparations supplied in containers with a nominal a domestic or an international standard institution. A relative
volume of more than 100 mL, apply the criteria of test I.A. error of the linear scale of the graticule within ± 2 per cent
For preparations supplied in containers with a nominal is acceptable. The large circle is designated the graticule field
volume of less than 100 mL, apply the criteria of test l.B. of view (GFOV).
♦For preparations supplied in containers with a nominal 2 illuminators are required. One is an episcopic brightfield
volume of 100 mL, apply the criteria of test l.B. ♦ illuminator internal to the microscope, the other is an
If the average number of particles exceeds the limits, test the external, focusable auxiliary illuminator adjustable to give
preparation by the microscopic particle count test. reflected oblique illumination at an angle of 10-20°.
Test 1.A - Solutions for infusion or solutions for injection supplied The filter assembly for retaining particulate contamination
in containers with a nominal content of more than 100 mL consists of a filter holder made of glass or other suitable
The preparation complies with the test if the average number material, and is equipped with a vacuum source and a
of particles present in the units tested does not exceed 25 per suitable membrane filter.
2023 Appendix XIII A V-A457

The membrane filter is of suitable size, black or dark grey in and the number of particles that are equal to or greater than
colour, non-gridded or gridded, and 1.0 µm or finer in 25 µm. Alternatively, partial filter count and determination of
nominal pore size. the total filter count by calculation is allowed. Calculate the
General precautions mean number of particles for the preparation to be
The test is carried out under conditions limiting particulate examined.
contamination, preferably in a laminar-flow cabinet. The particle sizing process with the use of the circular
Very carefully wash the glassware and filter assembly used, diameter graticule is carried out by transforming mentally the
except for the membrane filter, with a warm detergent image of each particle into a circle and then comparing it to
solution and rinse with abundant amounts of water to the 10 µm and 25 µm graticule reference circles. Thereby the
remove all traces of detergent. Immediately before use, rinse particles are not moved from their initial locations within the
both sides of the membrane filter and the equipment from graticule field of view and are not superimposed on the
top to bottom, outside and then inside, with particle-free reference circles for comparison. The inner diameter of the
water R. transparent graticule reference circles is used to size white
and transparent particles, while dark particles are sized by
In order to check that the environment is suitable for the
using the outer diameter of the black opaque graticule
test, that the glassware and the membrane filter are properly
reference circles.
cleaned and that the water to be used is particle-free, the
following test is carried out: determine the particulate In performing the microscopic particle count test do not
contamination of a 50 mL volume of particle-free water R attempt to size or enumerate amorphous, semi-liquid, or
according to the method described below. If more than otherwise morphologically indistinct materials that have the
20 particles 10 µm or larger in size or if more than 5 particles appearance of a stain or discolouration on the membrane
25 µm or larger in size are present within the filtration area, filter. These materials show little or no surface relief and
the precautions taken for the test are not sufficient. present a gelatinous or film-like appearance. In such cases
The preparatory steps must be repeated until the the interpretation of enumeration may be aided by testing a
environment, glassware, membrane filter and water are sample of the solution by the light obscuration particle count
suitable for the test. test.
◊Alternative method
Method
Mix the contents of the samples by slowly inverting the This method is intended to improve the applicability of the
container 20 times successively. If necessary, cautiously test to biological preparations. However, it can be used for
remove the sealing closure. Clean the outer surfaces of the any type of preparation.
container opening using a jet of particle-free water R and Clean the outer surfaces of the container(s) using a jet of
remove the closure, avoiding any contamination of the particle-free water R, avoiding any contamination of the
contents. contents. Samples are tested under in-use conditions, as
For large-volume parenterals, single units are tested. directed in the instructions for use (e.g. expelled syringe
For small-volume parenterals less than 25 mL in volume, the contents).
contents of 10 or more units are combined in a cleaned For parenteral preparations that have a sufficient volume
container; where justified and authorised, the test solution (i.e. a volume large enough to permit testing), testing of
may be prepared by mixing the contents of a suitable number individual units is often preferred.
of vials and diluting to 25 mL with particle-free water R or If the volume is not sufficient, take a suitable number
with an appropriate solvent without contamination of of units, mix each one carefully and thoroughly and then
particles when particle-free water R is not suitable. Small- combine the contents in a separate container to obtain the
volume parenterals having a volume of 25 mL or more may volume required for a single test, depending on the
be tested individually. instrument capability and the properties of the sample.
Powders for parenteral administration are constituted with Powders for parenteral administration are reconstituted with
particle-free water R or with an appropriate solvent without particle-free water R or with an appropriate particle-free
contamination of particles when particle-free water R is not solvent when particle-free water R is not suitable.
suitable.
The number of test specimens must be adequate to provide a
The number of test specimens must be adequate to provide a statistically sound assessment. For large- and small-volume
statistically sound assessment. For large-volume parenterals parenterals, an adequate volume of sample must be provided
or for small-volume parenterals having a volume of 25 mL or for analysis; however, single units may be tested based on a
more, fewer than 10 units may be tested, based on an statistically sound sampling plan.
appropriate sampling plan.
Continue as described under Method 2 above, starting from
Wet the inside of the filter holder fitted with the membrane "Wet the inside ... ".◊
filter with several millilitres of particle-free water R. Transfer to
Evaluation
the filtration funnel the total volume of a solution pool or of
For preparations supplied in containers with a nominal
a single unit, and apply vacuum. If needed, add stepwise a
volume of more than 100 mL, apply the criteria of test 2.A.
portion of the solution until the entire volume is filtered.
After the last addition of solution, begin rinsing the inner For preparations supplied in containers with a nominal
walls of the filter holder by using a jet of particle-free water R. volume of less than 100 mL, apply the criteria of test 2.B.
Maintain the vacuum until the surface of the membrane filter ♦For preparations supplied in containers with a nominal
is free from liquid. Place the filter in a Petri dish and allow volume of 100 mL, apply the criteria of test 2.B. ♦
the filter to air-dry with the cover slightly ajar. After the filter Test 2.A - Solutions for infusion or solutions for injection supplied
has been dried, place the Petri dish on the stage of the in containers with a nominal content of more than 100 mL
microscope, scan the entire membrane filter under the The preparation complies with the test if the average number
reflected light from the illuminating device, and count the of particles present in the units tested does not exceed 12 per
number of particles that are equal to or greater than 10 µm
V-A458 Appendix XIII B 2023

millilitre equal to or greater than 10 µm and does not exceed primary container is not possible, the contents may be
2 per millilitre equal to or greater than 25 µm. transferred for inspection into a sample container that is free
Test 2.B - Solutions for infusion or solutions for inJection supplied from visible particles, taking precautions to prevent
in containers with a nominal content of less than 100 mL contamination during transfer. Repeat the inspection in front
The preparation complies with the test if the average number of the black panel (A). Record the presence of any visible
of particles present in the units tested does not exceed particles.
3000 per container equal to or greater than 10 µm and does
not exceed 300 per container equal to or greater than 25 µm.

C. Particulate Contamination: Sub-Visible

Particles in Non-Injectable Liquid
B. Particulate Contamination: Visible Preparations
Particles (Ph. Eur. method 2.9.53)
(Ph. Eur. method 2.9.20)
INTRODUCTION
Particulate contamination consists of mobile undissolved Particulate contamination in non-injectable liquid
substances, other than gas bubbles, unintentionally present in preparations consists of mobile, undissolved substances, other
liquid preparations. than gas bubbles, that may originate from various sources
The test is intended to provide a simple procedure for the and are unintentionally present in the preparation. Particulate
visual assessment of the quality of liquid preparations, if contaminsation should be minimised and controlled,
applicable after reconstitution, as regards visible particles. independent of its type.
EQUWMENT 2 procedures are described hereinafter for the determination
The equipment (see Figure 2.9.20.-1) consists of a viewing of particulate contamination: Method 1 (light obscuration
station comprising: particle count test) and Method 2 (microscopic particle
- a matt black panel (A) of appropriate size held in a count test). It is preferable to use Method 1 when examining
vertical position; non-injectable liquid preparations for sub-visible particles.
- a non-glare white panel (B) of appropriate size held in a However, not all non-injectable liquid preparations can be
vertical position next to the black panel; examined directly for sub-visible particles by one or both of
- a non-glare white panel (C) of appropriate size held in a these methods. When Method 1 is not applicable, for
horizontal position next to (A) and (B); example in the case of preparations having reduced clarity or
- a lampholder (D) fitted with a suitable, shaded, white- increased viscosity (e.g. emulsions, colloids, liposomal
light source and with a suitable light diffuser preparations), the test is carried out according to Method 2.
(e.g. a viewing illuminator containing two 13 W Similarly, preparations that produce air or gas bubbles when
fluorescent tubes, each 525 mm in length, or an drawn into the sensor may also require specific precautions
appropriate light-emitting diode (LED) light source). during sample preparation and/or microscopic particle count
The intensity of illumination at the viewing point is testing.
maintained between 2000 lux and 3750 lux, although If the viscosity of the preparation to be tested precludes its
higher values may be required for coloured glass or plastic examination by either procedure, a quantitative dilution with
containers and for coloured or turbid preparations. an appropriate particle-free solvent may be carried out to
decrease the viscosity to a value that allows the analysis to be
D
performed.
The results obtained when examining a discrete unit or
group of units cannot be extrapolated with certainty to
untested units. Thus, statistically sound sampling plans must
be developed if valid inferences are to be drawn from
observed data to characterise the level of particulate
A
contamination in a large group of units. In the case of large-
and small-volume preparations, an adequate volume of
sample must be provided for analysis; however, single units
may be tested in a statistically sound sampling plan.
METHOD 1. LIGHT OBSCURATION PARTICLE
COUNT TEST
Use a suitable apparatus based on the principle of light
Figure 2. 9 .20. -1. - Equipment for visible particles blockage that allows an automatic determination of the size
of particles and the number of particles according to size.
METHOD The apparatus is calibrated using suitable certified reference
Adequate visual inspection of the container and contents is materials consisting of dispersions of spherical particles of
necessary, which may require the removal of any adherent known sizes between 10 µm and 25 µm. These standard
labels from the container. Gently swirl or invert the particles are dispersed in particle-free water R. Care must be
container, ensuring that air bubbles are not introduced, and taken to avoid agglomeration of particles during dispersion.
observe without magnification for about 5 s in front of the General precautions
white panel (B), although longer observation times may be Carry out the test under conditions limiting particulate
required for coloured glass or plastic containers and for contamination, preferably in a laminar-flow cabinet.
coloured or turbid preparations. Where inspection in the
2023 Appendix XIII C V-A459

0<>••

GFOV circle

Qo oQ

• Reference
circle
(Jo

Cross
hairs

Linear scale
11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111m111~

Figure 2.9.53.-1. - Circular diameter graticule

Very carefully wash the glassware and any filtration Remove 4 portions, each of approximately 5 mL, and count
equipment used, except for the membrane filters, with a the number of particles equal to or larger than 10 µm and
warm detergent solution and rinse abundantly with water to 25 µm. Disregard the result obtained for the first portion
remove all traces of detergent. Immediately before use, rinse and, from the remaining portions, calculate the average
the equipment from top to bottom, outside and then inside, number of particles in the preparation to be examined.
with particle-free water R. Volumes smaller than 5 mL can also be tested, provided that
Take care not to introduce air bubbles into the preparation this amount is appropriately justified. In general, for products
to be examined, especially when fractions of the preparation that do not have a sufficient volume (e.g. less than 25 mL), it
are being transferred to the container in which the may be acceptable to carry out the test with a volume of
determination is to be carried out. 1-5 mL.
In order to check that the environment is suitable for the METHOD 2. MICROSCOPIC PARTICLE COUNT
test, that the glassware is properly cleaned and that the water TEST
to be used is particle-free, carry out the following test: Use a suitable binocular microscope, a filter assembly for
determine the particulate contamination of 5 samples of retaining particulate contamination and a membrane filter for
particle-free water R, each of 5 mL, following the procedure examination.
described below. If the number of particles of 10 µm or The microscope is equipped with an ocular micrometer
larger in size exceeds 25 for the combined 25 mL, the calibrated with an objective micrometer, a mechanical stage
precautions taken for the test are not sufficient. capable of holding and traversing the entire filtration area of
The preparatory steps must be repeated until the the membrane filter, and 2 suitable illuminators to provide
environment, glassware and water are suitable for the test. episcopic illumination in addition to oblique illumination;
Procedure it is adjusted to 100 ± 10 x magnification.
Clean the outer surfaces of the container(s) using a jet of The ocular micrometer is a circular diameter graticule (see
particle-free water R, avoiding any contamination of the Figure 2.9.53.-1) and consists of a large circle divided by
contents. Samples are tested in a manner that most closely crosshairs into quadrants, transparent and black reference
represents the product fill. circles 10 µm and 25 µm in diameter at
For preparations that have a sufficient volume for a single 100 x magnification, and a linear scale graduated in 10 µm
test, based on instrument capability and the properties of the increments. The scale is calibrated using a stage micrometer
sample, testing of individual units is often preferred to that is certified by either a national or an international
estimate the level and variation of particulate contamination standard institution. A relative error of the linear scale of the
in an entire group of units. graticule within ± 2 per cent is acceptable. The large circle
For preparations that do not have a sufficient volume, take a is designated the graticule field of view (GFOV).
suitable number of units, mix each one carefully and 2 illuminators are required. One is an episcopic brightfield
thoroughly and then combine the contents in a separate illuminator internal to the microscope, the other is an
container to obtain the volume required for a single test, external, focusable auxiliary illuminator adjustable to give
depending on the instrument capability and the properties of reflected oblique illumination at an angle of 10-20°.
the sample. The filter assembly consists of a filter holder made of glass or
Preparations presented as powders are reconstituted with other suitable material, and is equipped with a vacuum
particle-free water R or with an appropriate particle-free source and a suitable membrane filter.
solvent when particle-free water R is not suitable. The membrane filter is of suitable size, black or dark grey in
Eliminate gas bubbles by appropriate measures such as colour, non-gridded or gridded, and 1.0 µm or finer in
allowing the sample to stand, applying a gentle vacuum or nominal pore size.
sonicating. Preparations containing proteins should not be
sonicated.
V-A460 Appendix XIII C 2023

General precautions transparent graticule reference circles is used to size white

Carry out the test under conditions limiting particulate and transparent particles, while dark particles are sized using
contamination, preferably in a laminar-flow cabinet. the outer diameter of the black opaque graticule reference
Very carefully wash the glassware and filter assembly used, circles.
except for the membrane filter, with a warm detergent In performing the microscopic particle count test do not
solution and rinse abundantly with water to remove all traces attempt to size or enumerate amorphous, semi-liquid, or
of detergent. Immediately before use, rinse both sides of the otherwise morphologically indistinct materials that have the
membrane filter and the equipment from top to bottom, appearance of a stain or discoloration on the membrane
outside and then inside, with particle-free water R. filter. These materials show little or no surface relief and
In order to check that the environment is suitable for the present a gelatinous or film-like appearance. In such cases
test, that the glassware and the membrane filter are properly the interpretation of enumeration may be aided by testing a
cleaned and that the water to be used is particle-free, carry sample of the preparation using the light obscuration particle
out the following test: determine the particulate count test.
contamination of a 50 mL volume of particle-free water R,
following the procedure described below. If more than
20 particles of 10 µm or larger in size or if more than
5 particles of 25 µm or larger in size are present within the
filtration area, the precautions taken for the test are not
sufficient. The preparatory steps must be repeated until the
environment, glassware, membrane filter and water are
suitable for the test.
Procedure
Clean the outer surfaces of the container(s) using a jet of
particle-free water R, avoiding any contamination of the
contents. Samples are tested in a manner that most closely
represents the product fill.
For preparations that have a sufficient volume for a single
test, based on instrument capability and properties of the
sample, testing of individual units is often preferred to
estimate the level and variation of particulate contamination
in an entire group of units.
For preparations that do not have a sufficient volume, take a
suitable number of units, mix each one carefully and
thoroughly and then combine the contents in a separate
container to obtain the volume required for a single test,
depending on the instrument capability and the properties of
the sample.
Preparations presented as powders are reconstituted with
particle-free water R or with an appropriate particle-free
solvent when particle-free water R is not suitable.
Wet the inside of the filter holder fitted with the membrane
filter with several millilitres of particle-free water R. Transfer to
the filtration funnel the total volume of a sample pool or of a
single unit, and apply vacuum. Alternatively and if necessary,
filter the sample portion by portion until the entire volume is
filtered. After adding the last portion, rinse the inner walls of
the filter holder using a jet of particle-free water R. Maintain
the vacuum until the surface of the membrane filter is free
from liquid. Place the filter in a Petri dish and allow the filter
to air-dry with the cover slightly ajar. After the filter has been
dried, place the Petri dish on the stage of the microscope,
scan the entire membrane filter under the reflected light from
the illuminating device, and count the number of particles
that are 10 µm or larger in size and the number of particles
that are 25 µm or larger in size. Alternatively, partial filter
count and determination of the total filter count by
calculation is allowed. Calculate the average number of
particles for the preparation to be examined.
The particle sizing process with the use of the circular
diameter graticule is carried out by transforming mentally the
image of each particle into a circle and then comparing it to
the 10 µm and 25 µm graticule reference circles. Thereby the
particles are not moved from their initial locations within the
graticule field of view and are not superimposed on the
reference circles for comparison. The inner diameter of the
2023 Appendix XIV A V-A461

In order to assess the validity of the assay, use not fewer than
Appendix XIV 3 doses of the reference substance and 3 doses of the
antibiotic to be examined having the same presumed activity
Biological Assays and Tests as the doses of the reference substance. It is preferable to use
a series of doses in geometric progression. In routine assays
General guidance concerning biological assays and tests is provided when the linearity of the system has been demonstrated over
in Supplementary Chapter I H. an adequate number of experiments using a three-point
assay, a two-point assay may be sufficient, subject to
agreement by the competent authority. However, in all cases
of dispute, a three-point assay as described above must be
A. Microbiological Assay of Antibiotics applied.
(Ph. Bur. method 2.7.2) Arrange the solutions on each Petri dish or on each
rectangular dish according to a statistically suitable design,
The potency of an antibiotic is estimated by comparing the
except for small Petri dishes that cannot accommodate more
inhibition of growth of sensitive micro-organisms produced
than 6 solutions, arrange the solutions of the antibiotic to be
by known concentrations of the antibiotic to be examined
examined and the solutions of the reference substance in an
and a reference substance.
alternate manner to avoid interaction of the more
The reference substances used in the assays are substances concentrated solutions.
whose activity has been precisely determined with reference
Incubate at a suitable temperature for about 18 h. A period
to the corresponding international standard or international
of diffusion prior to incubation, usually 1-4 h, at room
reference preparation.
temperature or at about 4 °C, as appropriate, may be used to
The assay must be designed in a way that will permit minimise the effects of the variation in time between the
examination of the validity of the mathematical model on application of the solutions and to improve the regression
which the potency equation is based. If a parallel-line model slope.
is chosen, the 2 log dose-response (or transformed response)
Measure the diameters to the nearest 0.1 mm or the areas of
lines of the preparation to be examined and the reference
the circular inhibition zones to the nearest 0.01 and calculate
preparation must be parallel; they must be linear over the
the potency using appropriate statistical methods.
range of doses used in the calculation. These conditions must
be verified by validity tests for a given probability, usually Use in each assay the number of replications per dose
P = 0.05. Other mathematical models, such as the slope ratio sufficient to ensure the required accuracy and precision.
model, may be used provided that proof of validity is The assay may be repeated and the results combined
demonstrated. statistically to obtain the required accuracy and precision and
to ascertain whether the potency of the antibiotic to be
Unless otherwise stated in the monograph, the confidence
examined is not less than the minimum required.
limits (P = 0. 95) of the assay for potency are not less than
95 per cent and not more than 105 per cent of the estimated B. TURBIDIMETRIC METHOD
potency. Inoculate a suitable medium with a suspension of the chosen
Carry out the assay by method A or method B. micro-organism having a sensitivity to the antibiotic to be
examined such that a sufficiently large inhibition of microbial
A. DIFFUSION METHOD growth occurs in the conditions of the test. Use a known
Liquefy a medium suitable for the conditions of the assay quantity of the suspension chosen so as to obtain a readily
and inoculate it at a suitable temperature, for example 48 °c measurable opacity after an incubation period of about 4 h.
to 50 °C for vegetative forms, with a known quantity of a
Use the inoculated medium immediately after its preparation.
suspension of micro-organisms sensitive to the antibiotic to
be examined, such that clearly defined zones of inhibition of Using the solvent and the buffer solution indicated in
suitable diameter are produced with the concentrations of the Table 2. 7 .2.-2 prepare solutions of the reference substance
antibiotic used for the assay. Immediately pour into Petri and of the antibiotic to be examined having known
dishes or large rectangular dishes a quantity of the inoculated concentrations presumed to be of equal activity.
medium to form a uniform layer 2-5 mm thick. Alternatively, In order that the validity of the assay may be assessed, use
the medium may consist of 2 layers, only the upper layer not fewer than 3 doses of the reference substance and
being inoculated. 3 doses of the antibiotic to be examined having the same
Store the dishes so that no appreciable growth or death of presumed activity as the doses of the reference substance.
the micro-organisms occurs before the dishes are used and so It is preferable to use a series of doses in geometric
that the surface of the medium is dry at the time of use. progression. In order to obtain the required linearity, it may
be necessary to select from a large number 3 consecutive
Using the solvent and the buffer solution indicated in
doses, using corresponding doses for the reference substance
Table 2. 7.2.-1, prepare solutions of the reference substance
and the antibiotic to be examined.
and of the antibiotic to be examined having known
concentrations and presumed to be of equal activity. Apply Distribute an equal volume of each of the solutions into
the solutions to the surface of the medium, for example, in identical test-tubes and add to each tube an equal volume of
sterile cylinders of porcelain, stainless steel or other suitable inoculated medium (for example, 1 mL of the solution and
material, or in cavities prepared in the agar. The same 9 mL of the medium). For the assay of tyrothricin add
volume of solution must be added to each cylinder or cavity. 0.1 mL of the solution to 9.9 mL of inoculated medium.
Alternatively, use sterile absorbent paper discs of suitable Prepare at the same time 2 control tubes without antibiotic
quality; impregnate the discs with the solutions of the both containing the inoculated medium and to one of which
reference substance or the solutions of the antibiotic to be is added immediately 0.5 mL of formaldehyde R. These tubes
examined and place on the surface of the agar. are used to set the optical apparatus used to measure the
growth.
V-A462 Appendix XIV A 2023

Table 2.7.2.-1. - Diffusion assay

Solvent to be used in Medium and
Reference Micro- Incubation
Antibiotic preparing the stock Buffer solution (pH) final pH(± 0.1
substance organism temperature
solution pH unit)

Saccharomyces
Amp/wtericin B for
cerevisiae
Amphotericin B microbiok,gical Dimethyl sulfoxide R pH 10.5 (0.2 M) F-pH6.l 35-37 "C
ATCC 9763
assay CRS
IP 1432-83

Micrococcus luteus
0. OJ M hydrochwric NCTC 7743
Bacitracin zinc Bacitracin zinc C RS pH 7.0 (0.05 M) A-pH 7.0 35-39 'C
acid CIP 53.160
ATCC 10240

Mycobacterium
Bleomycin
Bkomycin su/fate CRS WaterR pH 6.8 (0.1 M) smegmatis G-pH7.0 35-37 'C
sulfate
ATCC 607

Bordetella B - pH 7.3 35-39 "C

bronchiseptica
NCTC 8344
CIP 53.157
Colistimethate Colistimethate ATCC 4617
WaterR pH 6.0 (0.05 M)
sodium sodium CRS
Escherichia coli B-pH7.3 35-39 "C
NCIMB 8879
CIP 54.127
ATCC 10536

Bordetella B - pH 7.3 35-39 'C

bronchiseptica
NCTC 8344
Colistin sulfate for CIP 53.157
Colistin sulfate microbiowgical Water R pH 6.0 (0.05 M) ATCC 4617
assay CRS
Escherichia coli B-pH7.3 35-39 "C
NCIMB 8879
CIP 54.127
ATCC 10536

Bacillus subtilis E-pH7.9 30-37 "C

NCTC 10400
CIP 52.62
Framycetin Framycetin ATCC 6633
WaterR pH 8.0 (0.05 M)
sulfate sulfate CRS
Bacillus pumilus E-pH7.9 30-37 "C
NCTC 8241
CIP 76.18

Bacillus pumilus A- pH 7.9 35-39 °C

NCTC 8241
CIP 76.18
Gentamicin Gentamicin
Water R pH 8.0 (0.05 M) Staphylococcus A- pH 7.9 35-39 °C
sulfate sulfate CRS
epidermidis
NCIMB 8853
CIP 68.21
ATCC 12228

Bacillus subtilis
Methanol R (see the CIP 52.62
Josamycin Josamycin CRS pH 5.6 A- pH 6.6 35-37 "C
monograph) ATCC 6633
NCTC 10400

Bacillus subtilis
Josamycin Josamycin Methanol R (see the CIP 52.62
pH 5.6 A- pH 6.6 35-37 'C
propionate propionate CRS monograph) ATCC 6633
NCTC 10400

Bacillus subtilis A- pH 7.9 30-37 "C

Kanamycin NCTC 10400
monosulfate CIP 52.62
ATCC 6633
Kanamycin
WaterR pH 8.0 (0.05 M)
monosulfate CRS Staphylococcus A-pH7.9 35-39 "C
aureus
Kanamycin acid
NCTC 7447
sulfate
CIP 53.156
ATCC 6538 P
2023 Appendix XIV A V-A463

Solvent to be used in Medium and

Reference Micro- Incubation
Antibiotic preparing the stock Buffer solution (pH) final pH(± 0.1
substance organism temperature
solution pH unit)

Bacillus pumilus E-pH7.9 30-37 C

NCTC 8241
CIP76.18
Neomycin sulfate for
Neomycin
microbiowgical Water R pH 8.0 (0.05 M)
sulfate Bac,1/us subtilis E - pH 7.9 30-37 C
assay CRS
NCTC 10400
CIP 52.62
ATCC 6633

Staphylococcus
Netilmicin aureus
Netilmicin sulfate CRS Water R pH 8.0 ± 0.1 A- pH 7.9 32-35 'C
sulfate ATCC 6538 P
ClP 53.156

Candida tropicalis F- pH 6.0 30-37 C

CIP 1433-83
NCYC 1393
pH 6.0 (0.05 M)
Nystatin Nystatin CRS Dimethy/formamide R containing 5 per cent V/V Saccharomyces F - pH 6.0 30-32 'C
of dimethy/formamide R cerevisiae
NCYC 87
CIP 1432-83
ATCC 9763

Bordetella
Po/ymyxin B sulfate bronchiseptica
Polymyxin B
for microbiological WaterR pH 6.0 (0.05 M) NCTC 8344 B - pH 7.3 35-39 ·'C
sulfate
assay CRS CIP 53.157
ATCC 4617

Micrococcus luteus
Rifamycin Rifamycin NCTC 8340
MethanolR pH 7.0 (0.05 M) A-pH 6.6 35-39 C
sodium sodium CRS CIP 53.45
ATCC 9341

Bacillus subtilis
NCTC 10400
Spiramycin Spiramycin CRS Methanol R pH 8.0 (0.05 M) A-pH7.9 30-32 'C
CIP 52.62
ATCC 6633

Bacillus subtilis A-pH7.9 30-37 ''C

NCTC 8236
CIP 1.83
Streptomycin Streptomycin
Water R pH 8.0 (0.05 M)
sulfate sulfate CRS Bacillus subtilis A-pH7.9 30-37 "C
NCTC 10400
CIP 52.62
ATCC 6633

Bacillus subtilis
NCTC 10400
Teicoplanin Teicoplanin CRS pH 6.0 (0.05 M) pH 6.0 (0.05 M) H - pH 7 .8-8.0 35-37 "C
CIP 52.62
ATCC 6633

Tylosin for
veterinary use A mixture of 40 volumes
2.5 per cent V/V Micrococcus luteus
Tylosin of methanol R and
solution of methanol R NCTC 8340
phosphate for Tylosin CRS 60 volumes of 0.1 M A- pH 8.0 32-35 "C
in 0.1 M phosphate CIP 53.45
veterinary use phosphate buffer solution
buffer solution pH 7. 0 R ATCC 9341
Tylosin tartrate pH 8.0 R
for veterinary use

Bacillus subtilis
Vancomycin Vancomycin NCTC 10400
Water R pH 8.0 A- pH 8.0 37-39 'C
hydrochloride hydrochloride CRS CIP 52.62
ATCC 6633

Place all the tubes, randomly distributed or in a Latin square treatment and measure the opacity to 3 significant figures
or randomised block arrangement, in a water-bath or other using suitable optical apparatus. Alternatively use a method
suitable apparatus fitted with a means of bringing all the which allows the opacity of each tube to be measured after
tubes rapidly to the appropriate incubation temperature and exactly the same period of incubation.
maintain them at that temperature for 3-4 h, taking Calculate the potency using appropriate statistical methods.
precautions to ensure uniformity of temperature and identical
Linearity of the dose-response relationship, transformed or
incubation time.
untransformed, is often obtained only over a very limited
After incubation, stop the growth of the micro-organisms by range. It is this range which must be used in calculating the
adding 0.5 mL of formaldehyde R to each tube or by heat activity and it must include at least 3 consecutive doses in
V-A464 Appendix XIV A 2023

Table 2. 7 .2.-2. - Turbidimetric assay

Solvent to be Medium and final
Reference Buffer solution Incubation
Antibiotic used in preparing Micro-organism pH(± 0.1
substance (pH) temperature
the stock solution pH unit)

Escherichia coli
Colistimethate Colistimethate NCIMB 8666
WaterR pH 7.0 C - pH 7.0 35-37 °C
sodium sodium CRS CIP 2.83
ATCC 9637

Escherichia coli
Colistin sulfate for
NCIMB 8666
Colistin sulfate mu:robiowgical Water R pH 7.0 C - pH 7.0 35-37 'C
CIP 2.83
assay CRS
ATCC 9637

Staphylococcus aureus
Framycetin NCTC 7447
Framycetin sulfate Water R pH 8.0 C - pH 7.0 35-37 °C
sulfate CRS CIP 53.156
ATCC 6538 P

Staphylococcus aureus
Gentamicin NCTC 7447
Gentamicin sulfate Water R pH 7.0 C-pH7.0 35-37 °C
sulfate CRS CIP 53.156
ATCC 6538 P

Enterococcus hirae
CIP 58.55
Gramicidin CRS Methanol R pH 7.0* ATCC 10541 C - pH 7.0 35-37 'C
Staphylococcus aureus
Gramicidin
ATCC 6538 P

*Addition of a detergent may be necessary to avoid adsorption on the material during the dilutions, for example 0.1 mgimL of
polysorbate 80 R

Staphylococcus aureus
Methanol R (see the CIP 53.156
Josamycin Josamycin CRS pH 5.6 C - pH 8.0 35-37 °C
monograph) ATCC 6538 P
NCTC 7447

Staphylococcus aureus
Josamycin Josamycin Methanol R (see the CIP 53.156
pH 5.6 C - pH 8.0 35-37 'C
propionate propionate CRS monograph) ATCC 6538 P
NCTC 7447

Kanamycin Staphylococcus aureus

monosulfate Kanamycin NCTC 7447
WaterR pH 8.0 C - pH 7.0 35-37 °C
Kanamycin acid monosulfate CRS CIP 53.156
sulfate ATCC 6538 P

Staphylococcus aureus
Neomycin sulfate for
NCTC 7447
Neomycin sulfate microbiological WaterR pH 8.0 C - pH 7.0 35-37 "C
CIP 53.156
assay CRS
ATCC 6538 P

Escherichia coli
Rijamycin NCIMB 8879
Rifamycin sodium Methanol R pH 7.0 C - pH 7.0 35-37 'C
sodium CRS CIP 54.127
ATCC 10536

Staphylococcus aureus
NCTC 7447
Spiramycin Spiramycin CRS Methanol R pH 7.0 C - pH 7.0 35-37 'C
CIP 53.156
ATCC 6538 P

Kl.ebsiella pneumoniae
Streptomycin Streptomycin NCTC 7427
WaterR pH 8.0 C - pH 7.0 35-37 "C
sulfate sulfate CRS CIP 53.153
ATCC 10031

Tylosin for 2.5 per cent V/V

veterinary use solution of Staphylococcus aureus
methanol R in NCTC 6571
Ty!osin CRS pH 7.0 C - pH 7.0 37 C
0. 1 M phosphate ATCC 9144
Tylosin tartrate for
buffer solution CIP 53.154
veterinary use
pH 7.0R

Enterococcus hirae
Tyrothricin Gramicidin CRS Alcohol R Alcohol R C - pH 7.0 37 'C
ATCC 10541

Staphylococcus aureus
Vancomycin Vancomycin
WaterR pH 8.0 CIP 53.156 C - pH 7.0 37-39 'C
hydrochloride hydrochwride CRS
ATCC 6538 P
 2023 Appendix XIV A V-A465

order to permit linearity to be verified. In routine assays Buffer solutions

when the linearity of the system has been demonstrated over Buffer solutions having a pH between 5.8 and 8.0 are
an adequate number of experiments using a three-point prepared by mixing 50.0 mL of 0.2 M potassium dihydrogen
assay, a two-point assay may be sufficient, subject to phosphate R with the quantity of 0. 2 M sodium hydroxide
agreement by the competent authority. However, in all cases indicated in Table 2.7.2.-3. Dilute with freshly prepared
of dispute, a three-point assay must be applied. distilled water R to produce 200.0 mL.
Use in each assay the number of replications per dose
Table 2.7.2.-3
sufficient to ensure the required accuracy and precision.
The assay may be repeated and the results combined pH 0.2 M Sodium hydroxide (mL)
statistically to obtain the required accuracy and precision and 5.8 3.72
to ascertain whether the potency of the antibiotic to be 6.0 5.70
examined is not less than the minimum required. 6.2 8.60
The following section is published for infonnation. 6.4 12.60
6.6 17.80
6.8 23.65
RECOMMENDED MICRO-ORGANISMS 7.0 29.63
The following text details the recommended micro-organisms 7.2 35.00
and the conditions of use. Other micro-organisms may be 7.4 39.50
used provided that they are shown to be sensitive to the 7.6 42.80
antibiotic to be examined and are used in appropriate media 7.8 45.20
and appropriate conditions of temperature and pH. 8.0 46.80
The concentrations of the solutions used should be chosen so
as to ensure that a linear relationship exists between the These buffer solutions are used for all microbiological assays
logarithm of the dose and the response in the conditions of shown in Table 2. 7 .2.-1 with the exception of bleomycin
the test. sulfate and amphotericin B.
Preparation of inocula For bleomycin sulfate, prepare the buffer solution pH 6.8 as
Bacillus cereus var. mycoides; Bacillus subtilis; Bacil,lus pumil,us. follows: dissolve 6.4 g of potassium dihydrogen phosphate R and
Spore suspensions of the organisms to be used as inocula are 18.9 g of disodium hydrogen phosphate dodecahydrate R in
prepared as follows. water R and dilute to 1000 mL with water R.
Grow the organism at 35-37 °C for 7 days on the surface of For amphotericin B, prepare the 0.2 M phosphate buffer
a suitable medium to which has been added 0.001 g/L of solution pH 10.5 as follows: dissolve 35 g of dipotassium
manganese sulfate R. Using sterile water R, wash off the hydrogen phosphate R in 900 mL of water R, add 20 mL of
growth, which consists mainly of spores. Heat the suspension 1 M sodium hydroxide and dilute to 1000.0 mL with water R.
at 70 °C for 30 min and dilute to give an appropriate
Culture media
concentration of spores, usually 10 x 106 to 100 x 10 6 per
The following media or equivalent media may be used.
millilitre. The spore suspensions may be stored for long
periods at a temperature not exceeding 4 °C. Medium A
Alternatively, spore suspensions may be prepared by Peptone 6g
Pancreatic digest of casein 4g
cultivating the organisms in medium C at 26 °C for 4-6 days, Beef extract 1.5 g
then adding, aseptically, sufficient manganese sulfate R to give Yeast extract 3g
a concentration of 0.001 g/L and incubating for a further Glucose monohydrate 1g
48 h. Examine the suspension microscopically to ensure that Agar 15 g
Water to 1000 mL
adequate spore formation has taken place (about 80 per cent)
and centrifuge. Re-suspend the sediment in sterile water R to
Medium B
give a concentration of 10 x 106 to 100 x 10 6 spores per
Pancreatic digest of casein 17 g
millilitre, and then heat to 70 °C for 30 min. Store the
Papaic digest of soya bean 3g
suspension at a temperature not exceeding 4 °C. Sodium chloride 5g
Bordetella bronchiseptica. Grow the test organism on Dipotassium hydrogen phosphate 2.5 g
medium Bat 35-37 °C for 16-18 h. Wash off the bacterial Glucose monohydrate 2.5 g
Agar 15 g
growth with sterile water R and dilute to a suitable opacity. Polysorbate 80 10 g
Staphylococcus aureus; Klebsiella pneumoniae; Escherichia coli; Water to 1000 mL
Micrococcus luteus; Staphylococcus epidennidis. Prepare as
described above for B. bronchiseptica but using medium A The polysorbate 80 is added to the hot solution of the other
and adjusting the opacity to one which has been shown to ingredients after boiling, and immediately before adjusting to
produce a satisfactory dose-response relationship in the volume.
turbidimetric assay, or to produce clearly defined zones of Medium C
inhibition of convenient diameter in the diffusion assay, as Peptone 6g
appropriate. Beef extract 1.5 g
Saccharomyces cerevisiae; Candida tropicalis. Grow the test Yeast extract 3g
Sodium chloride 3.5 g
organism on medium Fat 30-37 °C for 24 h. Wash off the Glucose monohydrate 1g
growth with a sterile 9 g/L solution of sodium chloride R. Dipotassium hydrogen phosphate 3.68 g
Dilute to a suitable opacity with the same solution. Potassium dihydrogen phosphate 1.32 g
Water to 1000 mL
V-A466 Appendix XIV A 2023

MediumD Culture Media The following media or equivalent media

Heart extract 1.5 g may be used.
Yeast extract 1.5 g
Peptone-casein 5g Medium I
Glucose monohydrate 1g D-Glucose 10 g
Sodium chloride 3.5 g Tryptone 6g
Dipotassium hydrogen phosphate 3.68 g Yeast extract* 2g
Potassium dihydrogen phosphate 1.32 g Water to produce 1000 mL
Potassium nitrate 2g
Water to 1000 mL
Adjust to pH 8.0 with lM sodium hydroxide or
0. lM orthophosphoric acid.
MediumE
(*Ardamine yeast extract supplied by Champlain Industries
Peptone 5g
Meat extract 3g Inc., Clifton, NJ 07012, USA is suitable.)
Disodium hydrogen phosphate,12H2 O 26.9 g
Agar 10 g
Water to 1000 mL

The disodium hydrogen phosphate is added as a sterile

solution after sterilisation of the medium.
MediumF
Peptone 9.4 g
Yeast extract 4.7 g
Beef extract 2.4 g
Sodium chloride 10.0 g
Glucose monohydrate 10.0 g
Agar 23.5 g
Water to 1000 mL

Medium G
Glycerol 10 g
Peptone 10 g
Meat extract 10 g
Sodium chloride 3g
Agar 15 g
Water to 1000 mL

pH 7 .0 ± 0.1 after sterilisation.

Medium H
Peptone 5.0 g
Agar 15.0 g
Beef extract powder 3.0 g
Water to 1000 mL

pH 7.8 - 8.0 adjusted with 0.1 M sodium hydroxide.

Monographs of the British Pharmacopoeia
The following additional information and guidance apply to
monographs of the British Pharmacopoeia.
The required minimum precision for an acceptable assay of
any particular antibiotic or preparation is defined in the
appropriate monograph in the paragraph on the Assay. This
degree of precision is the minimum acceptable for
determining that the final product complies with the official
requirements. It may be inadequate for a decision about the
potency that should be stated on the label or used as the
basis for calculating the quantity of an antibiotic to be
incorporated in a preparation. In such circumstances, assays
of greater precision may be desirable with, for instance,
fiducial limits of error of the order of 98 to 102%. With this
degree of precision, the lower fiducial limit lies close to the
estimated potency. By using this limit, instead of the
estimated potency, to assign a potency to the antibiotic either
for labelling or for calculating the quantity to be included in
a preparation, there is less likelihood of the final preparation
subsequently failing to comply with the official requirements
for potency.
2023 Appendix XIV A V-A467

Method A. Diffusion Method

Table 2.7.2.-1 -Diffusion Assay; Additional Section for
Monographs of the British Phamzacopoeia

Antibiotic Reference Solvent to Buffer Micro-organism Medium and Incubation

substance be used in solution final pH (± 0.1 temperature
preparing (pH) pH unit)
the stock
Solution

Erythromycin Bacillus pumilus A; 7.9 30 - 37°

Estolate NCTC 8241
CIP 76.18
Erythromycin Ethyl Erythromycin Methanol pH 8.0
Succinate EPCRS (0.05M) Bacillus subtilis A; 7.9 30 - 37°
NCTC 10400
Erythromycin CIP 52.62
Stearate ATCC 6633

Lymecycline Lymecycline pH5.8 Bacillus pumilus A;6.6 37 - 39°

2nd lnt. Ref., NCTC 8241
1971 CIP 76.18

Polymyxin B Polymyxin B Water pH 6.0 Bordetella B; 7.3 35 - 39°

Sulphate Sulphate EPCRS (0.05M) bronchiseptica
NCTC 8344
CIP 53.157
ATCC 4617

Method B. Turbidimetric Method

Table 2.7.2.-2 -Turbidimetric Assay; Additional Section for Monographs of the British Phamzacopoeia

Antibiotic Reference Solvent to Buffer Micro-organism Medium and Incubation

substance be used in solution final pH (± 0.1 temperature
preparing (pH) pH unit)
the stock
Solution

Apramycin Apramycin BPCRS * Salmonella I; 8.0 37°

cholerasuis

Erythromycin Klebsiella D; 7.0 35 - 37°

Estolate pneumoniae
NCTC 7427
Erythromycin Ethyl Erythromycin Methanol pH 8.0 CIP 53.153
Succinate EPCRS ATCC 10031
C; 7.0 35 - 37°
Erythromycin Staphylococcus
Stearate aureus
NCTC 7447
CIP 53.156
ATCC 6538 P

* Solution prepared by dissolving 16.73 g of dipotassium hydrogen orthophosphate and 0.523 g of potassium dihydrogen
orthophosphate in about 750 ml of water, if necessary adjusting to pH 8.0 with 0.lM sodium hydroxide or 0.lM orthophosphoric
acid, and diluting to 1000 ml with water.
V-A468 Appendix XIV B 2023

immunodiffusion methods may be performed using different

B. lmmunochemical Methods supports such as tubes, plates, slides, cells or chambers.
(Ph. Bur. method 2. 7.1) Where the immunoprecipitating system consists of one
Immunochemical methods are based on the selective, antigen combining with its corresponding antibody, the
reversible and non-covalent binding of antigens by system is referred to as simple; when it involves related but
antibodies. These methods are employed to detect or not serologically identical reactants, the system is complex and
quantify either antigens or antibodies. The formation of an where several serologically unrelated reactants are involved,
antigen-antibody complex may be detected, and the amount the system is multiple.
of complex formed may be measured by a variety of In simple diffusion methods, a concentration gradient is
techniques. The provisions of this general method apply to established for only one of the reactants diffusing from an
immunochemical methods using labelled or unlabelled external source into the gel medium containing the
reagents, as appropriate. corresponding reactant at a comparatively low concentration.
The results of immunochemical methods depend on the Single radial immunodiffusion (SRID) is a simple
experimental conditions and the nature and quality of the quantitative immunodiffusion technique. When the
reagents used. It is essential to standardise the components of equilibrium between the external and the internal reactant
an immunoassay and to use, wherever available, international has been established, the circular precipitation area,
reference preparations for immunoassays. originating from the site of the external reactant, is directly
The reagents necessary for many immunochemical methods proportional to the amount of the antigen applied and
are available as commercial assay kits, that is, a set including inversely proportional to the concentration of the antibody in
reagents (particularly the antigen or the antibody) and the gel.
materials intended for the in vitro estimation of a specified In double diffusion methods, concentration gradients are
substance as well as instructions for their proper use. established for both reactants. Both antigen and antibody
The kits are used in accordance with the manufacturers' diffuse from separate sites into an initially immunologically
instructions; it is important to ascertain that the kits are neutral gel.
suitable for the analysis of the substance to be examined, Comparative double diffusion methods Are used for
with particular reference to selectivity and sensitivity. qualitatively comparing various antigens versus a suitable
Guidance concerning immunoassay kits is provided by the antibody or vice versa. The comparison is based on the
World Health Organization, Technical Report Series 658 presence or absence of interaction between the precipitation
(1981). patterns. Reactions of identity, non-identity or partial identity
METHODS IN WHICH A LABELLED ANTIGEN OR of antigens/antibodies can be distinguished.
A LABELLED ANTIBODY IS USED Immunoelectrophoretic methods
Methods using labelled substances may employ suitable lmmunoelectrophoresis (IE) is a qualitative technique
labels such as enzymes, fluorophores, luminophores and combining 2 methods: gel electrophoresis followed by
radioisotopes. Where the label is a radioisotope, the method immunodiffusion.
is described as a "radio-immunoassay". Crossed immunoelectrophoresis Is a modification of the
The recommendations for the measurement of radioactivity IE method. It is suitable both for qualitative and quantitative
given in the monograph on Radiopharmaceutical Preparations analysis. The first part of the procedure is an ordinary gel
(0125) are applicable to immunoassays involving electrophoresis, after which a longitudinal gel strip,
radioisotopes. All work with radioactive materials must be containing the separated fractions to be determined, is cut
carried out in conformity with national legislation and out and transferred to another plate. The electrophoresis in
internationally accepted codes of practice for protection the second direction is carried out perpendicular to the
against radiation hazards. previous electrophoretic run in a gel containing a
METHODS IN WHICH AN UNLABELLED ANTIGEN comparatively low concentration of antibodies corresponding
OR ANTIBODY IS USED to the antigens. For a given antibody concentration and gel
Immunoprecipitation methods thickness, the relationship between the area of the respective
lmmunoprecipitation methods include flocculation and precipitation peaks and the amount of the corresponding
precipitation reactions. When a solution of an antigen is antigen is linear.
mixed with its corresponding antibody under suitable Electroimmunoassay Often referred to as rocket immuno-
conditions, the reactants form flocculating or precipitating electrophoresis is a rapid quantitative method for determining
aggregates. The ratio of the reactants which gives the shortest antigens with a charge differing from that of the antibodies or
flocculation time or the most marked precipitation is called vice versa. The electrophoresis of the antigen to be
the optimal ratio, and is usually produced by equivalent determined is carried out in a gel containing a comparatively
amounts of antigen and antibody. lmmunoprecipitation can lower concentration of the corresponding antibody. The test
be assessed visually or by light-scattering techniques material and dilutions of a standard antigen used for
(nephelometric or turbidimetric assay). An increase in calibration are introduced into different wells in the gel.
sensitivity can be obtained by using antigen- or antibody- During electrophoresis, migrating peak-shaped precipitation
coated particles (e.g. latex) as reactants. zones originating from the wells are developed. The front of
In flocculation methods, stepwise dilutions of one of the the precipitate becomes stationary when the antigen is no
reactants is usually used whereas, in immunodiffusion (ID) longer in excess. For a given antibody concentration, the
methods, the dilution is obtained by diffusion in a gel relationship between the distance travelled by the precipitate
medium: concentration gradients of one or both of the and the amount of antigen applied is linear.
reactants are obtained, thus creating zones in the gel medium Counter-immunoelectrophoresis Is a rapid quantitative
where the ratio of the reactants favours precipitation. While method allowing concentration gradients of external antigen
flocculation methods are performed in tubes, and external antibody to be established in an electric field
depending on the different charges. Dilutions of a standard
2023 Appendix XIV C V-A469

for calibration and dilutions of the test material are Significant non-parallelism indicates that the antibody or
introduced into a row of wells in a gel and a fixed amount of antigen discriminates between test and standard, and the
the corresponding reactant is introduced into an opposite row results are not valid.
of wells. The titre of the test material may be determined as In displacement immunoassays, the values for non-specific
the highest dilution showing a precipitation line. binding and maximum displacement at high test or standard
A number of modifications of crossed immunoelectrophoresis concentration must not be significantly different. Differences
and electroimmunoassay methods exist. may indicate effects due to the matrix, either inhibition of
Other techniques combine separation of antigens by binding or degradation of tracer.
molecular size and serological properties.
Visualisation and characterisation of
immunoprecipitation lines
These may be performed by selective or non-selective stains, C. Test for Bacterial Endotoxins
by fluorescence, by enzyme or isotope labelling or other (Bacterial Endotoxins, Ph. Bur. method 2. 6.14/
relevant techniques. Selective staining methods are usually
performed for characterisation of non-protein substances in The test for bacterial endotoxins (BET) is used to detect or
the precipitates. quantify endotoxins from gram-negative bacteria using
amoebocyte lysate from the horseshoe crab (Limulus
In translucent gels such as agar or agarose, the precipitation polyphemus or Tachypleus tridentatus). There are 3 techniques
line becomes clearly visible in the gel, provided that the for this test: the gel-clot technique, which is based on gel
concentration of each of the reactants is appropriate. formation; the turbidimetric technique, based on the
VALIDATION OF THE METHOD development of turbidity after cleavage of an endogenous
Validation criteria substrate; and the chromogenic technique, based on the
A quantitative immunochemical method is not valid unless: development of colour after cleavage of a synthetic peptide-
1) The antibody or antigen does not significantly discriminate chromogen complex.
between the test and standard. For a labelled reactant, the The following 6 methods are described in the present
corresponding reactant does not significantly discriminate chapter:
between the labelled and unlabelled compound,
2) The method is not affected by the assay matrix, that is, Method A. Gel-clot method: limit test
Method B. Gel-clot method: quantitative test
any component of the test sample or its excipients, which can Method C. Turbid imetric kinetic method
vary between samples. These may include high Method D. Chromogenic kinetic method
concentrations of other proteins, salts, preservatives or Method E. Chromogenic end-point method
contaminating proteolytic activity, Method F. Turbidimetric end-point method

3) The limit of quantitation is below the acceptance criteria

Proceed by any of the 6 methods for the test. In the event of
stated in the individual monograph,
doubt or dispute, the final decision is made based upon
4) The precision of the assay is such that the variance of the method A unless otherwise indicated in the monograph.
results meets the requirements stated in the individual
The test is carried out in a manner that avoids endotoxin
monographs,
contamination.
5) The order in which the assay is performed does not give
rise to systematic errors. 1. APPARATUS
Depyrogenate all glassware and other heat-stable apparatus in
Validation methods a hot-air oven using a validated process. A commonly used
In order to verify these criteria, the validation design includes minimum time and temperature is 30 min at 250 °C.
the following elements:
If employing plastic apparatus, such as microtitre plates and
1) The assay is performed at least in triplicate, pipette tips for automatic pipetters, use apparatus shown to
2) The assay includes at least 3 different dilutions of the be free of detectable endotoxin and which does not interfere
standard preparation and 3 dilutions of sample preparations in the test.
of presumed activity similar to the standard preparation, NOTE: in this chapter, the tenn 'tube' includes all types of
3) The assay layout is randomised, receptacles, for example microtitre plate wells.
4) If the test sample is presented in serum or formulated with 2. REAGENTS, TEST SOLUTIONS
other components, the standard is likewise prepared, (1) Amoebocyte lysate
5) The test includes the measurement of non-specific binding Amoebocyte lysate is a lyophilised product obtained from
of the labelled reactant, amoebocyte lysate from the horseshoe crab (Limulus
6) For displacement immunoassay: polyphemus or Tachypleus tridentatus). This reagent refers only
(a) maximum binding (zero displacement) is determined, to a product manufactured in accordance with the
regulations of the competent authority.
(b) dilutions cover the complete response range from values
close to non-specific binding to maximum binding, preferably NOTE: amoebocyte lysate reacts with some fJ-glucans in addition
for both standard and test preparations. to endotoxins. Amoebocyte lysate preparations which do not react
with glucans are available; they are prepared by removing from
STATISTICAL CALCULATION amoebocyte lysate the G factor, which reacts with glucans, or by
To analyse the results, response curves for test and standard inhibiting the G factor reacting system of amoebocyte lysate. These
may be analysed by the methods described in 5.3. Statistical preparations may be used for endotoxin testing in the presence of
Analysis of Results of Biological Assays and Tests. glucans.

1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter

5. 8 Pharmacopoeial harmonisation.
V-A470 Appendix XIV C 2023

(2) Lysate solution K

Dissolve amoebocyte lysate in water for BET or in a buffer, M
as recommended by the lysate manufacturer, by gentle K threshold pyrogenic dose of endotoxin per kilogram of body
stirring. Store the reconstituted lysate, refrigerated or frozen, massl
as indicated by the manufacturer. M maximum recommended bolus dose of product per kilogram of
body mass.
(3) Water for BET (water for bacterial endotoxins test)
Water for injections R or water produced by other procedures When the product is to be injected at frequent intervals or
that shows no reaction with the lysate employed at the infused continuously, M is the maximum total dose
detection limit of the reagent. administered in a single hour period.
3. PREPARATION OF THE STANDARD The endotoxin limit for active substances administered
ENDOTOXIN STOCK SOLUTION parenterally is specified in units such
The standard endotoxin stock solution is prepared from an as IU/mL, IU/mg, IU/Unit of biological activity, etc., in
endotoxin reference standard that has been calibrated against monographs.
the International Standard, for example endotoxin Concentration of test solution:
standard BRP. - mg/mL if the endotoxin limit is specified by mass
Endotoxin is expressed in International Units (IU). (IU/mg),
The equivalence in IU of the International Standard is stated - Units/mL if the endotoxin limit is specified by unit of
by the World Health Organization. biological activity (IU/Unit),
NOTE: one International, Unit (JU) of endotoxin is equal, to one - mUmL if the endotoxin limit is specified by volume
Endotoxin Unit (E. U.). (IU/mL).
Follow the specifications in the package leaflet and on the
the labelled lysate sensitivity in the gel-clot technique (IU/mL)
label for preparation and storage of the standard endotoxin
or the lowest concentration used in the standard curve of the
stock solution. turbidimetric or chromogenic techniques.
4. PREPARATION OF THE STANDARD
ENDOTOXIN SOLUTIONS 7. GEL-CLOT TECHNIQUE (METHODS A AND B)
After vigorously mixing the standard endotoxin stock The gel-clot technique allows detection or quantification of
solution, prepare appropriate serial dilutions of this solution endotoxins and is based on cloning of the lysate in the
using water for BET. presence of endotoxins. The minimum concentration of
Use the solutions as soon as possible to avoid loss of activity endotoxins required to cause the lysate to clot under
by adsorption. standard conditions is the labelled lysate sensitivity.
To ensure both the precision and validity of the test, confirm
5. PREPARATION OF THE TEST SOLUTIONS the labelled lysate sensitivity and perform the test for
Prepare the test solutions by dissolving or diluting active interfering factors as described under 1. Preparatory testing.
substances or medicinal products using water for BET. Some
substances or preparations may be more appropriately 1. PREPARATORY TESTING
dissolved or diluted in other aqueous solutions. If necessary, (i) Confirmation of the labelled lysate sensitivity
adjust the pH of the test solution (or dilution thereof) so that Confirm in 4 replicates the labelled sensitivity A, expressed
the pH of the mixture of the lysate and test solution falls in IU/mL, of the lysate solution prior to use in the test.
within the pH range specified by the lysate manufacturer, Confirmation of the lysate sensitivity is carried out when a
usually 6.0 to 8.0. The pH may be adjusted by the use of new lot of lysate is used or when there is any change in the
acid, base or a suitable buffer, as recommended by the lysate test conditions which may affect the outcome of the test.
manufacturer. Acids and bases may be prepared from Prepare standard solutions of at least 4 concentrations
concentrates or solids with water for BET in containers free equivalent to 2A, A, 0.5A and 0.25A by diluting the standard
of detectable endotoxin. Buffers must be validated to be free endotoxin stock solution with water for BET.
of detectable endotoxin and interfering factors. Mix a volume of the lysate solution with an equal volume of
6. DETERMINATION OF THE MAXIMUM VALID 1 of the standard solutions (such as 0.1 mL aliquots) in each
DILUTION tube. When single test vials or ampoules containing
The Maximum Valid Dilution (MVD) is the maximum lyophilised lysate are employed, add solutions of standards
allowable dilution of a sample at which the endotoxin limit directly to the vial or ampoule. Incubate the reaction mixture
can be determined. Determine the MVD using the following for a constant period according to the recommendations of
formulae: the lysate manufacturer (usually at 37 ± 1 °C for
60 ± 2 min), avoiding vibration. Test the integrity of the gel:
cndotoxin limit x concentration of test solution for tubes, take each tube in tum directly from the incubator
MVD = -------,l~------
and invert it through approximately 180° in one smooth
motion. If a firm gel has formed that remains in place upon
inversion, record the result as positive. A result is negative if
an intact gel is not formed.
Endotoxin limit The endotoxin limit for active substances
The test is considered valid when the lowest concentration of
administered parenterally, defined on the basis of dose, is
the standard solutions shows a negative result in all replicate
equal to:
tests.
2023 Appendix XIV C V-A471

Table 2.6.14.-1
Solution Endotoxin concentration/Solution to which Diluent Dilution factor Endotoxin Number of replicates
endotoxin is added concentration
A Noncrrest solution 4

B 2).rrest solution Test solution 4

2 4
4 4
8 4

C 2)JWater for BET Water for BET I 2A 2

2 I)_ 2
4 0.5), 2
8 0.251c 2

D None/Water for BET 2

Solution A = solution of the preparation being examined that is free of detectable endotoxins.
Solution B = test for interference.
Solution C = control of the labelled lysate sensitivity.
Solution D = negative control (water for BET).

The end-point is the lowest concentration in the series of Interference may be overcome by suitable validated
decreasing concentrations of standard endotoxin that clots treatment, such as filtration, neutralisation, dialysis or heat
the lysate. Determine the geometric mean end-point treatment. To establish that the treatment chosen effectively
concentration by calculting the mean of the logarithms of the eliminates interference without loss of endotoxins, repeat the
end-point concentrations of the 4 dilution series, take the test for interfering factors using the preparation being
antilogarithm of this value, as indicated by the following examined to which the standard endotoxin has been added
expression: and which has then been submitted to the chosen treatment.
Geometric mean end-point concentration = antilog ~ e 2. LIMIT TEST (METHOD A)
(i) Procedure
sum of the 1og10 end-point concentrations of the dilution series
Prepare solutions A, B, C and D as shown in
used,
Table 2.6.14.-2, and perform the test on these solutions
f number of replicates. following the procedure described under 1. Preparatory
testing, (i) Confirmation of the labelled lysate sensitivity.
The geometric mean end-point concentration is the
measured sensitivity of the lysate solution (IU/mL). If this is Table 2.6.14.-2
not less than 0.5A. and not more than 21\., the labelled Solution Endotoxin concentration/Solution Number of replicates
sensitivity is confirmed and is used in the tests performed to which endotoxin is added
with this lysate. A None/Diluted test solution 2

(ii) Test for interfering factors B 2AiDiluted test solution 2

Prepare solutions A, B, C and D as shown in C 2AIWater for BET 2

Table 2.6.14.-1, and use the test solutions at a dilution less D None/Water for BET 2

than the MVD, not containing any detectable endotoxins,

operating as described under 1. Preparatory testing, (i)
Confirmation of the labelled lysate sensitivity. Prepare solution A and solution B (positive product control)
The geometric mean end-point concentrations of solutions B using a dilution not greater than the MVD and treatments as
and C are determined using the expression described in described in 1. Preparatory testing, (ii) Test for interfering
1. Preparatory testing, (i) Confirmation of the labelled lysate factors. Solutions B and C (positive controls) contain the
sensitivity. standard endotoxin at a concentration corresponding to twice
the labelled lysate sensitivity. Solution D (negative control)
The test for interfering factors must be repeated when any
consists of water for BET.
changes are made to the experimental conditions that are
likely to influence the result of the test. (ii) Interpretation
The test is considered valid when all replicates of solutions A The test is considered valid when both replicates of
and D show no reaction and the result of solution C solution B and C are positive and those of solution D are
confirms the labelled lysate sensitivity. negative.
If the sensitivity of the lysate determined with solution B is When a negative result is found for both replicates of
not less than 0.5A. and not greater than 2A., the test solution solution A, the preparation being examined complies with the
does not contain interfering factors under the experimental test.
conditions used. Otherwise, the test solution interferes with When a positive result is found for both replicates of
the test. solution A, the preparation being examined does not comply
If the preparation being examined interferes with the test at a with the test.
dilution less than the MVD, repeat the test for interfering When a positive result is found for one replicate of
factors using a greater dilution, not exceeding the MVD. solution A and a negative result is found for the other, repeat
The use of a more sensitive lysate permits a greater dilution the test. In the repeat test, the preparation being examined
of the preparation being examined and this may contribute to complies with the test if a negative result is found for both
the elimination of interference. replicates of solution A. The preparation does not comply
with the test if a positive result is found for one or both
replicates of solution A.
V-A4 72 Appendix XIV C 2023

Table 2.6.14.-3
Solution Endotoxin concentration/Solution to which Diluent Dilution factor Endotoxin Number of replicates
endotoxin is added concentration
A NonefTest solution Water for BET 2
2 2
4 2
8 2
B 2icfTest solution 2

C 2,)Water for BET Water for BET 1 Zic 2

2 lie 2
4 0.51c 2
8 0.25ic 2

D None/Water for BET 2

Solution A = test solution at the dilution, not exceeding the MVD, with which the test for interfering factors was carried out. Subsequent dilution of the test
solution must not exceed the MVD. Use water for BET to make a dilution series of 4 tubes containing the test solution at concentrations of 1, 1/2, 1/4 and 1/8,
relative to the dilution used in the test for interfering factors. Other dilutions up to the MVD may be used as appropriate.
Solution B = solution A containing standard endotoxin at a concentration of 2), (positive product control).
Solution C = a dilution series of 4 tubes of water for BET containing the standard endotoxin at concentrations of 2A, ),, 0.5), and 0.251c.
Solution D = water for BET (negative control).

However, if the preparation does not comply with the test at technique may be classified as being either the end-point-
a dilution less than the MVD, the test may be repeated using turbidimetric test or the kinetic-turbidimetric test.
a greater dilution, not exceeding the MVD. The end-point-turbidimetric test (Method F) is based on the
3. QUANTITATIVE TEST (METHOD BJ quantitative relationship between the endotoxin concentration
(i) Procedure and the turbidity (absorbance or transmission) of the reaction
The test quantifies bacterial endotoxins in the test solution mixture at the end of an incubation period.
by titration to an end-point. Prepare solutions A, B, C and D The kinetic-turbidimetric test (Method C) is a method to
as shown in Table 2.6.14.-3, and test these solutions measure either the time (onset time) needed for the reaction
according to the procedure described under 1. Preparatory mixture to reach a predetermined absorbance or
testing, (i) Confirmation of the labelled lysate sensitivity. transmission, or the rate of turbidity development.
(ii) Calculation and interpretation The test is carried out at the incubation temperature
The test is considered valid when the following 3 conditions recommended by the lysate manufacturer (usually
are met: 37 ± 1 °C).
(a) both replicates of solution D (negative control) are 2. CHROMOGENIC TECHNIQUE (METHODS D AND
negative, E)
(b) both replicates of solution B (positive product control) This technique is used to measure the chromophore released
are positive, from a suitable chromogenic peptide by the reaction of
endotoxins with the lysate. Depending on the test principle
(c) the geometric mean end-point concentration of
employed, this technique may be classified as being either the
solution C is in the range of 0.5A to 2A.
end-point-chromogenic test or the kinetic-chromogenic test.
To determine the endotoxin concentration of solution A,
The end-point-chromogenic test (Method E) is based on the
calculate the end-point concentration for each replicate, by
quantitative relationship between the endotoxin concentration
multiplying each end-point dilution factor by A.
and the quantity of chromophore released at the end of an
The endotoxin concentration in the test solution is the incubation period.
end-point concentration of the replicates. If the test is
The kinetic-chromogenic test (Method D) measures either
conducted with a diluted test solution, calculate the
the time (onset time) needed for the reaction mixture to
concentration of endotoxin in the original solution by
reach a predetermined absorbance, or the rate of colour
multiplying the result by the dilution factor.
development.
If none of the dilutions of the test solution is positive in a
The test is carried out at the incubation temperature
valid test, report the endotoxin concentration as less than A
recommended by the lysate manufacturer (usually
(or, if a diluted sample was tested, report as less than the
37 ± 1 °C).
lowest dilution factor of the sample x A). If all dilutions are
positive, the endotoxin concentration is reported as equal to 3. PREPARATORY TESTING
or greater than the largest dilution factor multiplied by A To assure the precision or validity of the turbidimetric and
(e.g. in Table 2.6.14.-3, the initial dilution factor x 8 x A). chromogenic techniques, preparatory tests are conducted to
show that the criteria for the standard curve are satisfied and
The preparation being examined meets the requirements of
that the test solution does not interfere with the test.
the test if the endotoxin concentration in both replicates is
less than that specified in the monograph. Validation of the test method is required when any changes
are made to the experimental conditions that are likely to
8. PHOTOMETRIC QUANTITATIVE TECHNIQUES influence the result of the test.
(METHODS C, D, E AND F)
(i) Assurance of criteria for the standard curve
1. TURBIDIMETRIC TECHNIQUE (METHODS C
The test must be carried out for each lot of lysate reagent.
ANDF)
This technique is a photometric test to measure the increase Using the standard endotoxin solution, prepare at least
in turbidity. Based on the test principle employed, this 3 endotoxin concentrations within the range indicated by the
lysate manufacturer to generate the standard curve. Perform
2023 Appendix XIV C V-A473

the test using at least 3 replicates of each standard endotoxin the treatment chosen effectively eliminates interference
solution as recommended by the lysate manufacturer (volume without loss of endotoxins, repeat the test for interfering
ratios, incubation time, temperature, pH, etc.). factors using the preparation being examined to which the
If the desired range is greater than 2 log 10 in the kinetic standard endotoxin has been added and which has then been
methods, additional standards must be included to bracket submitted to the chosen treatment.
each log 10 increase in the range of the standard curve. 4. TEST
The absolute value of the correlation coefficient, I r I, must (i) Procedure
be greater than or equal to 0.980, for the range of endotoxin Follow the procedure described in 3. Preparatory testing,
concentrations set up. (ii) Test for interfering factors.
(ii) Test for interfering factors (ii) Calculation
Select an endotoxin concentration at or near the middle of Calculate the endotoxin concentration of each replicate of
the endotoxin standard curve. solution A using the standard curve generated by the positive
Prepare solutions A, B, C and D as shown in control solution C.
Table 2.6.14.-4. Perform the test on at least 2 replicates of The test is considered valid when the following 3
these solutions as recommended by the lysate manufacturer requirements are met:
(volume of test solution and lysate solution, volume ratio of (1) the results obtained with solution C comply with the
test solution to lysate solution, incubation time, etc.). requirements for validation defined under 3. Preparatory
Table 2.6.14.-4 testing, (i) Assurance of criteria for the standard curve,
(2) the endotoxin recovery, calculated from the endotoxin
Solution Endotoxin Solution to which Number of
concentration endotoxin is replicates concentration found in solution B after subtracting the
added endotoxin concentration found in solution A, is within the
A None Test solution Not less than 2 range of 50-200 per cent,
B Middle concentration Test solution Not less than 2 (3) the result obtained with solution D (negative control)
of the standard curve does not exceed the limit of the blank value required in the
description of the lysate employed, or it is less than the
C At least 3 Water for BET Each concentration
concentrations (lowest not less than 2 endotoxin detection limit of the lysate reagent employed.
concentration is (iii) Interpretation
designated Jc)
The preparation being examined complies with the test if the
D None Water for BET Not less than 2 mean endotoxin concentration of the replicates of solution A,
Solution A = test solution, that may be diluted not to exceed the MVD. after correction for dilution and concentration, is less than
Solution B = preparation to be examined at the same dilution as solution A, the endotoxin limit for the product.
containing added endotoxin at a concentration equal to or near the middle of
Guidelines on the test for bacterial endotoxins are given in general
the standard curve.
Solution C = standard endotoxin solution at the concentrations used in the chapter 5.1.10.
validation of the method as described under 3. Preparatory testing,
(i) Assurance of criteria for the standard curve (positive controls). Test for Bacterial Endotoxins using Recombinant
Solution D = water for BET (negative control). Factor C
(Ph. Bur. general text 2. 6. 32)
The test is considered valid when the following conditions The test for bacterial endotoxins using recombinant factor C
are met: (rFC) is carried out to quantify endotoxins from gram-
- the absolute value of the correlation coefficient of the negative bacteria. It is performed using rFC based on the
standard curve generated using solution C is greater than gene sequence of the horseshoe crab (Limulus polyphemus,
or equal to 0.980; Tachypleus tridentatus, Tachypleus gigas or Carcinoscorpius
- the result with solution D does not exceed the limit of the rotundicauda), using a fluorimetric method.
blank value required in the description of the lysate
The test is carried out in a manner that avoids bacterial
reagent employed, or it is less than the endotoxin
endotoxin contamination.
detection limit of the lysate reagent employed.
Calculate the mean recovery of the added endotoxin by 1. EQUIPMENT
subtracting the mean endotoxin concentration in the solution Depyrogenate all glassware and other heat-stable equipment
(if any) (solution A, Table 2.6.14.-4) from that in the in a dry-heat oven using a validated process. A commonly
solution containing the added endotoxin (solution B, used minimum time and temperature is 30 min at 250 °C.
Table 2.6.14.-4). Where plastic equipment (such as microtitre plates and
pipette tips for automatic pipettes) is employed, it must be
The test solution is considered free of interfering factors if
shown to be free of detectable endotoxin and not to interfere
under the conditions of the test, the measured concentration
with the test.
of the endotoxin added to the test solution is within
50-200 per cent of the known added endotoxin 2. REAGENTS
concentration, after subtraction of any endotoxin detected in Reagents
the solution without added endotoxin. Recombinant factor C is based on the gene sequence of the
When the endotoxin recovery is out of the specified range, horseshoe crab (Limulus polyphemus, Tachypleus tridentatus,
the test solution is considered to contain interfering factors. Tachypleus gigas or Carcinoscorpius rotundicauda). All reagents,
Repeat the test using a greater dilution, not exceeding including the fluorogenic substrate and assay buffer, must be
the MVD. Furthermore, interference of the test solution or free of detectable endotoxin.
diluted test solution not to exceed the MVD may be
eliminated by suitable validated treatment, such as filtration,
neutralisation, dialysis or heat treatment. To establish that
V-A4 7 4 Appendix XIV C 2023

Reagent solutions When the product is to be injected at frequent intervals or

If necessary, prepare the reagents according to the test kit infused continuously, M is the maximum total dose
manufacturer's instructions. Store the reagents, refrigerated administered in a single hour period.
or frozen, as indicated by the manufacturer. The endotoxin limit for active substances administered
Water for BET (water for bacterial endotoxins test) parenterally is specified in units such
Water for inJections R or water produced by other procedures as IU/mL, IU/mg, IU/Unit of biological activity, etc., in
that shows no reaction with the reagent employed at the monographs.
detection limit of the reagent. Concentration of test solution:
3. PREPARATION OF THE STANDARD - mgimL if the endotoxin limit is specified by mass
ENDOTOXIN STOCK SOLUTION (IU/mg);
The standard endotoxin stock solution is prepared from an - Units/mL if the endotoxin limit is specified by unit of
endotoxin reference standard that has been calibrated against biological activity (IU/Unit);
the International Standard, for example endotoxin - mUmL if the endotoxin limit is specified by volume
standard BRP. (IU/mL).
Endotoxin is expressed in International Units (IU). ),, = the lowest concentration used in the standard curve.
The equivalence in IU of the International Standard is stated
by the World Health Organization.
7. FLUORIMETRIC QUANTITATIVE TECHNIQUE
NOTE: 1 International Unit (JU) of endotoxin is equal to This technique is used to measure the fluorescence (relative
1 Endotoxin Unit (EU).
fluorescence units; RFU) emitted by a fluorescent substrate
Follow the specifications in the package leaflet and on the (reagent) after cleavage by endotoxin-activated factor C. It is
label for preparation and storage of the standard endotoxin used as an end-point-fluorescent test.
stock solution. The end-point-fluorescent test is based on the quantitative
4. PREPARATION OF THE STANDARD relationship between the endotoxin concentration and the
ENDOTOXIN SOLUTIONS fluorescence of the reagent mixture at the end of the
After vigorously mixing the standard endotoxin stock incubation period, expressed for example as !:i.RFU:
solution, prepare appropriate serial dilutions of this solution
using water for BET. !:i.RFU = RFU,md-pm,, - RFU,o
Use the solutions as soon as possible to avoid loss of activity
by adsorption.
RFUlendfoim fluorescence of the reagent mixture at the end of the
5. PREPARATION OF THE TEST SOLUTIONS incubation period;
Prepare the test solutions by dissolving or diluting active RFUt0 fluorescence of the reagent mixture at the start of the
incubation period.
substances or medicinal products using water for BET. Some
substances or preparations may be more appropriately
The test is carried out at the incubation temperature
dissolved or diluted in other aqueous solutions. If necessary,
recommended by the test kit manufacturer (usually
adjust the pH of the test solution (or dilution thereof) so that
37 ± 1 °C),
the pH of the mixture of the reagent(s) and test solution falls
within the pH range specified by the test kit manufacturer, 8. PREPARATORY TESTING
usually 6.0 to 8.0. The pH may be adjusted by the use of Preparatory tests are conducted to ensure that the
acid, base or a suitable buffer, as recommended by the test fluorometric technique is valid. These tests demonstrate that
kit manufacturer. Acids and bases may be prepared from the criteria for the standard curve are satisfied (8-1) and that
concentrates or solids with water for BET in containers free the test solution does not interfere with the test (8-2).
of detectable endotoxin. Buffers must be validated to be free Validation of the test method is required when any changes
of detectable endotoxin and interfering factors. are made to the experimental conditions that are likely to
6. DETERMINATION OF THE MAXIMUM VALID influence the result of the test.
DILUTION 8-1 ASSURANCE OF CRITERIA FOR THE
The maximum valid dilution (MVD) is the maximum STANDARD CURVE
allowable dilution of a sample at which the endotoxin limit The test must be carried out for each lot of recombinant
can be determined. Determine the MVD using the following factor C reagent.
formula: Instrument sensitivity must be adjusted in accordance with
the recommendations of the test kit manufacturer.
endotoxin limit x concentration of test solution
Using the standard endotoxin solution, prepare at least 3
A endotoxin concentrations within the range indicated by the
Endotoxin limit The endotoxin limit for active substances test kit manufacturer to generate the standard curve. If the
administered parenterally, defined on the basis of dose, is desired range exceeds the range indicated by the
equal to: manufacturer by more than 2 log 10, additional standards
must be included to bracket each log increase in the range.
K Perform the test using at least 3 replicates of each standard
M endotoxin solution as recommended by the manufacturer
(volume ratios, incubation time, temperature, pH, etc.).
K threshold pyrogenic dose of endotoxin per kilogram of body mass;
The absolute value of the correlation coefficient, I r I, must
M maximum recommended bolus dose of product per kilogram of
body mass.
be greater than or equal to 0.980, for the range of endotoxin
concentrations prepared.
2023 Appendix XIV D V-A475

8-2 INTERFERING FACTORS interfering factors using the preparation being examined to
As factor G is absent from the test kit, false-positive results which the standard endotoxin has been added and which has
due to ~-glucan activation are not expected to occur. This then been submitted to the chosen treatment.
must be taken into account when the method is compared to
9. TEST
other bacterial endotoxin quantification methods.
9-1 PROCEDURE
Select an endotoxin concentration at or near the middle of Follow the procedure described in section 8-2.
the endotoxin standard curve.
9-2 CALCULATION
Prepare solutions A, B, C and D as shown in Calculate the endotoxin concentration of each replicate of
Table 2.6.32.-1. Perform the test on at least 2 replicates of solution A using the standard curve generated by the
these solutions as recommended by the test kit manufacturer standard endotoxin solution C.
(volume of test solution and reagent test kit mixture, volume
The test is considered valid when the following 3
ratio of test solution to reagent test kit mixture, incubation
requirements are met:
time, etc.).
(1) the results obtained with solution C comply with the
Table 2.6.32.-1 requirements for validation defined in section 8-1;
Solution Endotoxin Solution to which Number of (2) the endotoxin recovery, calculated from the endotoxin
concentration endotoxin is added replicates concentration found in solution B after subtracting the
A None Test solution Not less than 2 endotoxin concentration found in solution A, is within the
B Middle Test solution Not less than 2 range of 50-200 per cent;
concentration of the (3) the result obtained with solution D (negative control)
standard curve does not exceed the limit of the blank value required in the
C At least 3 Water for BET Each concentration description of the reagent mixture employed, or it is less than
concentrations not less than 2 the endotoxin detection limit of the rFC employed.
(lowest
concentration is 9-3 INTERPRETATION
designated A) The preparation being examined complies with the test if the
D None Water for BET Not less than 2 mean endotoxin concentration of the replicates of solution A,
after correction for dilution and concentration, is less than
Solution A = test solution, which may be diluted but not exceeding the MVD.
the endotoxin limit for the product.
Solution B (positive product control) = preparation to be examined at the
same dilution as solution A, containing added endotoxin at a concentration Guidelines on the test for bacterial endotoxins are given in general
equal to or near the middle of the standard curve chapter 5.1.10.
Solution C = standard endotoxin solution at the concentrations used in the
validation of the method as described in section 8-1.
Solution D (negative control) = water for BET.

The test is considered valid when the following conditions D. Test for Pyrogens
are met: (Ph. Bur. method 2.6.8)
- the absolute value of the correlation coefficient of the
The test consists of measuring the rise in body temperature
standard curve generated using solution C is greater than
evoked in rabbits by the intravenous injection of a sterile
or equal to 0.980;
solution of the substance to be examined.
- the result with solution D does not exceed the limit of the
blank value required in the description of the reagent Selection of animals
mixture employed, or it is less than the endotoxin Use healthy, adult rabbits of either sex weighing not less than
detection limit of the rFC employed. 1.5 kg, fed a complete and balanced diet not containing
Calculate the mean recovery of the added endotoxin by antibiotics, and not showing loss of body mass during
subtracting the mean endotoxin concentration in the solution the week preceding the test. A rabbit is not to be used in a
(if any) (solution A, Table 2.6.32.-1) from that in the pyrogen test if:
solution containing the added endotoxin (solution B, a) it has been used in a negative pyrogen test in the
Table 2.6.32.-1). preceding 3 days; or
The test solution is considered free of interfering factors if, b) it has been used in the preceding 3 weeks in a pyrogen
under the conditions of the test, the measured concentration test in which the substance under examination failed to pass
of the endotoxin added to the test solution is within the test.
50-200 per cent of the known added endotoxin Animals' quarters
concentration, after subtraction of any endotoxin detected in Keep the rabbits individually in a quiet area with a uniform
the solution without added endotoxin. appropriate temperature. Withhold food from the rabbits
When the endotoxin recovery is outside the specified range, overnight and until the test is completed; withhold water
the test solution is considered to contain interfering factors. during the test. Carry out the test in a quiet room where
Repeat the test using a greater dilution, not exceeding the there is no risk of disturbance exciting the animals and in
MVD. Furthermore, interference of the test solution or which the room temperature is within 3 °C of that of the
diluted test solution (not exceeding the MVD) may be rabbits' living quarters, or in which the rabbits have been
eliminated by suitable validated treatment, such as filtration, kept for at least 18 h before the test.
neutralisation, dialysis, heat treatment or endotoxin-specific Materials
binding steps (enrichment of endotoxin from the test solution Thoroughly wash all glassware, syringes and needles with
prior to detection in the absence of the interfering matrix). water for injections and heat in a hot-air oven at 250 °C for
To establish that the treatment chosen effectively eliminates 30 min or at 200 °C for 1 h.
interference without loss of endotoxins, repeat the test for
V-A4 7 6 Appendix XIV F 2023

Retaining boxes 4 groups, depending on the results obtained. If the summed

The retaining boxes for rabbits whose temperature is being response of the 1st group does not exceed the figure given in
measured by an electrical device are made in such a way that the 2nd column of Table 2.6.8.-1, the substance passes the
the animals are retained only by loosely fitting neck-stocks; test. If the summed response exceeds the figure given in the
the rest of the body remains relatively free so that the rabbits 2nd column of the table but does not exceed the figure given
may sit in a normal position. They are not restrained by in the 3rd column of the table, repeat the test as indicated
straps or other similar methods that may harm the animal. above. If the summed response exceeds the figure given in
The animals are put into the boxes not less than 1 h before the 3rd column of the table, the product fails the test.
the 1st recording of the temperature and remain in them
throughout the test. Table 2.6.8.-1
Thermometers Nwnber of rabbits Product passes if Product fails if summed
summed response does response exceeds
Use a thermometer or electrical device that indicates the not exceed
temperature with a resolution of 0.1 °C and insert into the
3 1.15 °C 2.65 "C
rectum of the rabbit to a depth of about 5 cm. The depth of
6 2.80 "C 4.30 "C
insertion is constant for any 1 rabbit in any 1 test. When an
9 4.45 °C 5.95 "C
electrical device is used it may be left in position throughout
12 6.60 "C 6.60 "C
the test.
Preliminary test Rabbits used in a test for pyrogens where the mean rise in
After selection of the animals, 1-3 days before testing the the rabbits' temperature has exceeded 1.2 °C are
product to be examined, treat those animals that have not permanently excluded.
been used during the previous 2 weeks by intravenous
injection of 10 mL per kilogram of body mass of a pyrogen- In accordance with the provisions of the European
free 9 g/L solution of sodium chloride R warmed to about Convention for the Protection of Vertebrate Animals used for
38 °C. Record the temperatures of the animals, beginning at Experimental and Other Scientific Purposes, tests must be
least 90 min before injection and continuing for 3 h after the carried out in such a way as to use the minimum number of
injection of the solution. Any animal showing a temperature animals and to cause the least pain, suffering, distress or
variation greater than 0.6 °C is not used in the main test. lasting harm. Wherever possible and after product-specific
validation, the pyrogen test is replaced by the monocyte-
Main test activation test (2. 6.30).
Carry out the test using a group of 3 rabbits.
Preparation and injection of the product Warm the
liquid to be examined to approximately 38 °C before the
injection. The product to be examined may be dissolved in,
or diluted with, a pyrogen-free 9 g/L solution of sodium
F. Test for Depressor Substances
chloride R or another prescribed liquid. Inject the solution (Depressor Substances, Ph. Eur. method 2. 6.11)
slowly into the marginal vein of the ear of each rabbit over a Carry out the test on a cat weighing not less than 2 kg and
period not exceeding 4 min, unless otherwise prescribed in anaesthetised with chloralose or with a barbiturate that allows
the monograph. The amount of the product to be injected the maintenance of uniform blood pressure. Protect the
varies according to the product to be examined and is animal from loss of body heat and maintain it so that the
prescribed in the monograph. The volume injected is not less rectal temperature remains within physiological limits.
than 0.5 mL per kilogram and not more than 10 mL per Introduce a cannula into the trachea. Insert a cannula filled
kilogram of body mass. with a heparinised 9 g/L solution of sodium chloride into the
Determination of the initial and maximum common carotid artery and connect it to a device capable of
temperatures The 'initial temperature' of each rabbit is giving a continuous record of the blood pressure. Insert into
the mean of 2 temperature readings recorded for that rabbit the femoral vein another cannula, filled with a heparinised
at an interval of 30 min in the 40 min immediately preceding 9 g/L solution of sodium chloride, through which can be
the injection of the product to be examined. The 'maximum injected the solutions of histamine and of the substance to be
temperature' of each rabbit is the highest temperature examined. Determine the sensitivity of the animal to
recorded for that rabbit in the 3 h after the injection. Record histamine by injecting intravenously at regular intervals, doses
the temperature of each rabbit at intervals of not more than of histamine solution R corresponding to 0.1 µg and 0.15 µg of
30 min, beginning at least 90 min before the injection of the histamine base per kilogram of body mass. Repeat the lower
product to be examined and continuing 3 h after the dose at least 3 times. Administer the second and subsequent
injection. The difference between the maximum temperature injections not less than 1 min after the blood pressure has
and the initial temperature of each rabbit is taken to be its returned to the level it was at immediately before the
response. When this difference is negative, the result is previous injection. The animal is used for the test only if a
counted as a zero response. readily discernible decrease in blood pressure that is constant
Rabbits showing a temperature variation greater than 0.2 °C for the lower dose is obtained and if the higher dose causes
between 2 successive readings in the determination of the greater responses. Dissolve the substance to be examined in
initial temperature are withdrawn from the test. In any 1 test, sufficient of a 9 g/L solution of sodium chloride or other
only rabbits having initial temperatures that do not differ prescribed solvent, to give the prescribed concentration.
from one another by more than 1 °C are used. All rabbits Inject intravenously per kilogram of body mass 1.0 mL of
having an initial temperature higher than 39.8 °C or less than histamine solutwn R, followed by 2 successive injections of the
38.0 °C are withdrawn from the test. prescribed amount of the solution to be examined and,
finally, 1.0 mL of histamine solution R. The second, third and
Interpretation of results
fourth injections are given not less than 1 min after the blood
Having carried out the test 1st on a group of 3 rabbits, repeat
pressure has returned to the level it was at immediately
if necessary on further groups of 3 rabbits to a total of
2023 Appendix XIV H V-A477

before the preceding injection. Repeat this series of injections The quantity so determined does not exceed the quantity
twice and conclude the test by giving 1.5 mL of histamine prescribed in the monograph.
solution R per kilogram of body mass. If the solution to be examined does not produce a
If the response to 1.5 mL of histamine solution R per kilogram contraction, prepare a fresh solution adding a quantity of
of body mass is not greater than that to 1.0 mL the test is histamine corresponding to the maximum tolerated in the
invalid. The substance to be examined fails the test if the monograph and note whether the contractions produced by
mean of the series of responses to the substance is greater the preparation with the added histamine correspond to the
than the mean of the responses to 1.0 mL of histamine amount of histamine added. If this is not the case, or if the
solution R per kilogram of body mass or if any one dose of contractions caused by the substance to be examined are not
the substance causes a greater depressor response than the reproducible or if subsequent responses to "high" and "low"
concluding dose of the histamine solution. The test animal doses of histamine are diminished, the results of the tests are
must not be used in another test for depressor substances if invalid and the test for depressor substances (2. 6.11) must be
the second criterion applies or if the response to the high carried out.
dose of histamine given after the administration of the Solution A
substance to be examined is less than the mean response to
the low doses of histamine previously injected. Sodium chloride 160.0 g
Potassium chloride 4.0 g
Calcium chloride, anhydrous 2.0 g
Magnesium chloride, anhydrous 1.0 g
Disodium hydrogen phosphate dodecahydrate 0.10 g
G. Test for Histamine Water for injections R to 1000 mL

(Histamine, Ph. Bur. method 2.6.10)

Solution B
Euthanise a guinea-pig weighing 250 g to 350 g that has
been deprived of food for the preceding 24 h. Remove a Solution A 50.0 mL
portion of the distal small intestine 2 cm in length and empty Atropine sulfate 0.5 mg
the isolated part by rinsing carefully with solution B Sodium hydrogen carbonate 1.0 g
Glucose monohydrate 0.5 g
described below using a syringe. Attach a fine thread to each Water for injecti{JltS R to IOOOmL
end and make a small transverse incision in the middle of the
piece of intestine. Place it in an organ bath with a capacity of Solution B should be freshly prepared and used within 24 h.
10 mL to 20 mL, containing solution B maintained at a
constant temperature (34 °C to 36 °C) and pass through the
solution a current of a mixture of 95 parts of oxygen and
5 parts of carbon dioxide. Attach one of the threads near to
the bottom of the organ bath. Attach the other thread to an H. Monocyte-Activation Test
isotonic myograph and record the contractions of the organ (Ph. Bur. method 2.6.30)
on a kymograph or other suitable means of giving a
1 INTRODUCTION
permanent record. If a lever is used, its length is such that
The monocyte-activation test (l'v1AT) is used to detect or
the movements of the organ are amplified about 20 times.
quantify substances that activate human monocytes or
The tension on the intestine should be about 9.8 mN (1 g)
monocytic cells to release endogenous mediators such as pro-
and it should be adjusted to the sensitivity of the organ.
inflammatory cytokines, for example tumour necrosis factor
Flush out the organ bath with solution B. Allow it to stand
alpha (TNfo), interleukin-I beta (IL-1~) and interleukin-
for 10 min. Flush 2 or 3 times more with solution B.
6 (IL-6). These cytokines have a role in fever pathogenesis.
Stimulate a series of contractions by the addition of
Consequently, the l'v1AT will detect the presence of pyrogens
measured volumes between 0.2 mL and 0.5 mL of a solution
in the test sample. The l'v1AT is suitable, after a product-
of histamine dihydrochloride R having a strength which
specific validation, as a replacement for the rabbit pyrogen
produces reproducible submaximal responses. This dose is
test.
termed the "high dose". Flush the organ bath (preferably by
overflow without emptying the bath) 3 times with solution B Pharmaceutical products that contain non-endotoxin
before each addition of histamine. The successive additions pyrogenic or pro-inflammatory contaminants often show very
should be made at regular intervals allowing a complete steep or non-linear dose-response curves in comparison with
relaxation between additions (about 2 min). Add equal endotoxin dose-response curves. Preparations that contain or
volumes of a weaker dilution of histamine dihydrochloride R may contain non-endotoxin contaminants have to be tested
which produces reproducible responses approximately half as at a range of dilutions that includes minimum dilution.
great as the "high dose". This dose is termed the "low dose". The following 3 methods are described in the present
Continue the regular additions of "high" and "low" doses of chapter.
histamine solution as indicated above, and alternate each Method A. Quantitative test
addition with an equal volume of a dilution of the solution to Method B. Semi-quantitative test
be examined, adjusting the dilution so that the contraction of
Method C. Reference lot comparison test
the intestine, if any, is smaller than that due to the "high
dose" of histamine. Determine whether the contraction, if In addition, further useful information on practical aspects of
any, is reproducible and that the responses to the "high" and the tests can be found in the 'Guidance notes' section at the
"low" doses of histamine are unchanged. Calculate the end of this general chapter.
activity of the substance to be examined in terms of its The test is carried out in a manner that avoids pyrogen
equivalent in micrograms of histamine base from the dilution contamination.
determined as above.
V-A478 Appendix XIV H 2023

2 DEFINITIONS 3 GENERAL PROCEDURE

The maximum valid dilution (MVD) is the maximum A solution of the preparation being examined is incubated
allowable dilution of a sample at which the contaminant limit with a source of human monocytes or human monocytic
can be determined. The calculation of the MVD is based on cells, e.g. from human heparinised peripheral blood that is
the endotoxin reference standard. Determine the MVD using preferably not more than 4 h old, or a monocyte-containing
the following expression: fraction of that blood, such as human peripheral blood
mononuclear cells (PBMC) isolated, e.g. by density-gradient
CLCxC centrifugation, or a human monocytic cell line. Human
LOD heparinised peripheral blood is usually diluted with culture
medium or saline e.g. to 2-50 per cent V/V (final
CLC contaminant limit concentration;
C concentration of test solution;
concentration). PBMC or monocytic cell lines, in culture
LOD limit of detection. medium and with either the donor's own plasma or AB
serum, are typically used at a final cell density of
As the LOD is not always available in advance, an estimated 0.1-1.0 x 106 cells per well, tube or other receptacle.
LOD based on historical data can be used to calculate the For monocytic cell lines, heat-inactivated foetal bovine serum
MVD. may be substituted for AB serum. The cell culture is carried
The acceptance criterion for a pass/fail decision is the out at 37 ± 1 °C, in an atmosphere appropriate for the
contaminant limit concentration (CLC), which is expressed culture medium, e.g. 5 per cent CO 2 in humidified air.
in endotoxin equivalents per milligram or millilitre or The duration of the culture is sufficient to allow
per units of biological activity of the preparation being accumulation of the chosen read-out. The responses of the
examined. chosen read-out, e.g. a pro-inflammatory or pyrogenic
cytokine, to a solution of the preparation being examined are
The CLC is calculated using the following expression:
compared with responses to standard endotoxin or to a
K reference lot of the preparation being examined.
M 4APPARATUS
Depyrogenate all glassware and other heat-stable apparatus in
K threshold pyrogenic dose per kilogram of body mass;
M maximum recommended bolus dose of product per kilogram of
a hot-air oven using a validated process. A commonly used
body mass. minimum time and temperature is 30 min at 250 °C.
If employing plastic apparatus, such as microtitre plates and
When the product is to be injected at frequent intervals or pipette tips for automatic pipetters, use apparatus shown to
infused continuously, M is the maximum total dose be free of detectable pyrogens and which do not interfere
administered in a single hour period. with the test.
Where an endotoxin limit concentration (ELC) has been 5 CELL SOURCES AND QUALIFICATION
specified for a product, the CLC is the same as the ELC, 5-1 WHOLE BLOOD
unless otherwise prescribed. In this case, the concentration of Whole blood is obtained from single donors or from pooled
test solution is expressed in mg/mL if the endotoxin limit is whole blood which are qualified according to the
specified by mass (IU/mg), in Units/mL if the endotoxin requirements described under sections 5-3, 5-4, 5-5 and
limit is specified by unit of biological activity (IU /Unit), where applicable, section 6-3.
in mlJmL if the endotoxin limit is specified by volume 5-2 PERIPHERAL BLOOD MONONUCLEAR CELLS
(IU/mL). (PBMC)
Endotoxin equivalents are values for the contaminant PBMC are isolated from blood obtained from single donors
concentration read off the standard endotoxin dose-response or from pooled whole blood which are qualified according to
curve (Method A) or estimated by comparison with the requirements described under sections 5-3, 5-4, 5-5 and
responses to standard endotoxin solutions (Method B). where applicable, section 6-3.
The standard endotoxin stock solution is prepared from an
5-3 QUALIFICATION OF BLOOD DONORS
endotoxin reference standard that has been calibrated against
Blood donors are to satisfy the following qualification criteria,
the International Standard, for example endotoxin
together with other requirements in force that relate to
standard ERP.
consent, health and safety and ethical considerations. Blood
The LOD is determined using the endotoxin standard curve. donors are to describe themselves as being in good health, as
It is the concentration of endotoxin corresponding to the cut- not to be suffering from any bacterial or viral infections and
off value. For the purpose of the test, the LOD is expressed to have been free from the symptoms of any such infection
as endotoxin equivalents per millilitre. The cut-off value is for a period of at least 1 week prior to the donation of blood.
expressed in units appropriate to the read-out (e.g. for an Blood donors are not to have taken non-steroidal anti-
enzyme-linked immunosorbent assay (ELISA), use the inflammatory drugs during the 48 h prior to donating blood
optical density). and steroidal anti-inflammatory drugs during the 7 days prior
The cut-off value may be calculated using the following to donating blood. Individuals who have been prescribed
expression: immunosuppressant or other drugs known to influence the
production of the chosen readout are not to serve as blood
x+ 3s donors. Blood donations are to be tested for infection
markers according to national requirements for transfusion
x mean of the 4 replicates for the responses to the blank (Ro);
medicine.
standard deviation of the 4 replicates of the responses to the 5-4 QUALIFICATION OF CELLS POOLED FROM A
blank (Ro). NUMBER OF DONORS
Pools (of whole blood or blood fractions, e.g. PBMC), must
consist of donations from a minimum of 4 individual donors
 2023 Appendix XIV H V-A479

but preferably 8 or more donors, where practicable, taking 6-1 ASSURANCE OF CRITERIA FOR THE
from each donation an approximately equal volume of blood, ENDOTOXIN STANDARD CURVE
or cells from an approximately equal volume of blood. Using the standard endotoxin solution, prepare at least
For the qualification of pooled cells proceed as follows: 4 endotoxin concentrations to generate the standard curve.
within 4 h of collection of blood, generate dose-response Perform the test using at least 4 replicates of each
curves from the pool using standard endotoxin with at least concentration of standard endotoxin.
4 geometrically diluted endotoxin concentrations, e.g. in the The basal release of the chosen read-out (blank) in the
range of 0.01 IU/mL to 4 IU/mL. The dose-response curves absence of added standard endotoxin is optimised to be as
are to meet the 2 criteria for the endotoxin standard curve low as possible (e.g. an optical density below 0.1 when using
described under section 6-1. If the pool is to be used for the an ELISA).
detection of non-endotoxin contaminants, pools are to be
There are 2 acceptance criteria for the standard curve:
qualified as described in section 6-3. Where cells are pooled,
- the regression of responses (appropriately transformed if
the averaging effect is to be considered when setting the necessary) on log 10 dose shall be statistically significant
pass/fail specification for a given product.
(p < 0.01);
5-5 QUALIFICATION OF CRYO-PRESERVED CELLS - the regression of responses on log 10 dose must not deviate
The cell source intended for use in a MAT, e.g. human significantly from linearity (p > 0.05) (see chapter 5.3.
whole blood, blood fractions, such as PBMC or monocytic Statistical analysis).
cell lines, may be cryo-preserved. Pools of cryo-preserved
6-2 TEST FOR INTERFERING FACTORS
cells are obtained by pooling before freezing, or by pooling
To assure the validity of the test, preparatory tests are
single cryo-preserved donations immediately after thawing.
conducted to ensure that the preparation being examined
Qualification of cryo-preserved blood or cells is performed
does not interfere with the test. Using an appropriate diluent,
immediately after thawing (and pooling if necessary). Dose-
dilute the preparation being examined in geometric steps,
response curves for the cryo-preserved blood or cells are to
with all dilutions not exceeding the MVD. Make the same
comply with the 2 criteria for the endotoxin standard curve
dilutions of the preparation being examined and add
described under section 6-1. Qualification of the cryo-
endotoxin at a justified concentration. Alternatively, use a
preserved blood or cells is to be performed according to
diluent containing added endotoxin at a justified
section 6-3 if the intended use is for the detection of non-
concentration. In both cases, this concentration is usually
endotoxin contaminants.
equal to or near the estimated middle of the endotoxin
5-6 MONOCYTIC CONTINUOUS CELL LINES standard curve (Method A) or twice the estimated LOD
Monocytic cell lines are appropriate for the detection of (Method B). Test these dilution series in parallel in the same
bacterial endotoxins, but have limited use for the detection of experiment. Use the endotoxin standard curve to calculate
non-endotoxin pyrogens. the concentration of endotoxin-equivalents in each solution.
A human monocytic cell line is cultured in order to ensure a Calculate the mean recovery of the added endotoxin by
sufficient supply for the MAT. To optimise the method, subtracting the mean concentration of endotoxin equivalents
clones derived from the cell line can be used. in the solution (if any) from that in the solution containing
Cells must be maintained under aseptic conditions and the added endotoxin. The test solution is considered free of
regularly tested for the presence of mycoplasma interfering factors if, under the conditions of the test, the
contamination. Additionally, cells must be regularly checked measured endotoxin equivalents in the test solution to which
for identity (e.g. doubling time, morphology, and function) endotoxin is added is within 50-200 per cent of the added
and stability. The functional stability of a cell line is assessed concentration, after subtraction of any endotoxin equivalents
by monitoring its performance in relation to the number of detected in the solution without added endotoxin. When this
passages during routine testing. Criteria for functional criterion is not met, Method C is to be preferred over
stability are to be established and may include growth Methods A and B.
criteria, maximum signal obtained in the test, background In Method C, the dilutions of the test and reference lots
noise and receptor expression. The receptor expression may depend on the type of analysis used to make the comparison
be tested with specific ligands e.g. lipopolysaccharide (LPS) between the two. The type of analysis is to be justified and
for toll-like receptor 4 (TLR4), lipoteichoic acid (LTA) for validated for each product, and is to include assay validity
toll-like receptor 2 (TLR2), synthetic bacterial lipoprotein criteria. In an example, a solution of the preparation being
for TLR2-TLR1, synthetic bacterial lipoprotein for TLR2- examined is tested at 3 dilutions: the highest concentration
TLR6 or flagellin. (lowest dilution) that stimulates the greatest release of the
The dose-response curves are to meet the 2 criteria for the chosen read-out and the 2-fold dilutions immediately below
endotoxin standard curve described under section 6-1. If the and above the chosen dilution. Since the concentration that
cells are to be used for the detection of non-endotoxin stimulates the greatest release of the chosen read-out may be
contaminants, they are to be qualified as described in donor-dependent as well as batch-dependent, the product-
section 6-3. specific validation is to be performed in at least
3 independent tests, each using cells from different donors.
6 PREPARATORY TESTING The highest concentration (lowest dilution) that stimulates
To ensure both the precision and validity of the test, the greatest release of the chosen read-out in the majority of
preparatory tests are conducted, to assure that the criteria for donors, and the 2-fold dilutions immediately below and
the endotoxin standard curve are satisfied, that the solution above that dilution are deemed to be validated for further
does not interfere with the test, that the test detects testing. If undiluted test solution stimulates the greatest
endotoxins and non-endotoxins contaminants and that the release of the chosen read-out, subsequent testing is to be
solution does not interfere in the detection system. performed using undiluted test solution and also test solution
A test for interfering factors is required when any changes are diluted in the ratios 1:2 and 1:4 before its addition to the
made to the experimental conditions that are likely to monocytic cells. The dilution factors for these 3 solutions are
influence the result of the test. designated f 1, f 2 and h-
V-A480 Appendix XIV H 2023

If the pyrogen content of the product is inherently high, it Table 2.6.30.-1

may be more appropriate to carry out, for example, a Added endotoxin Number of
parallel-line analysis on the dose-response curves for the test Solution Solution
(IU/mL) replicates
and reference lots. In this situation, solutions of the
preparations are tested at 3 or more geometric dilutions A Test solutionif None 4
which cover the range of the dose-response curve used for B Test solution/2 x f None 4
the validated analysis (see chapter 5.3. Statistical, analysis).
C Test solution/4 x f None 4
6-3 METHOD VALIDATION FOR NON-ENDOTOXIN
MONOCYTE-ACTIVATING CONTAMINANTS Middle dose from
The preparatory testing is also to show that the chosen test endotoxin
AS Test solution// 4
standard curve
system detects, in addition to bacterial endotoxins, non-
CR,)
endotoxin pro-inflammatory or pyrogenic contaminants.
The suitability of the method for the particular product has Middle dose from
to be verified. This can be achieved using historic batches endotoxin
BS Test solution/2 x f 4
standard curve
found to be contaminated with non-endotoxin contaminants (R,)
that caused positive responses in the rabbit pyrogens test or
adverse drug reactions in man. Where such batches are not Middle dose from
available, the preparatory testing is to include validation of endotoxin
CS Test solution/4 x f standard curve
4
the test system using at least 2 non-endotoxin ligands for toll- (R,)
like receptors, e.g. peptidoglycans, lipoteichoic acids,
synthetic bacterial lipoproteins, flagellin and crude bacterial Pyrogen-free saline None (negative
Ro 4
whole cell extract, at least 1 of which is to be spiked into the or test diluent control)
preparation being examined. The choice of non-endotoxin 4 concentrations
Pyrogen-free saline 4 of each
pyrogens used should reflect the most likely contaminant(s) R,-R, of standard
or test diluent concentration
of the preparation being examined. endotoxin

6-4 INTERFERENCE IN THE DETECTION SYSTEM

Once the optimum dilution of the solution of the preparation Solutions R 1-Ri = solutions of standard endotoxin at the
being examined for further testing has been identified, this concentrations used in the test for interfering factors.
dilution is tested for interference in the detection system 7-1-2 Calculation and interpretation
(e.g. ELlSA) for the chosen read-out. The agreement All data to be included in the data analysis are to relate to
between a dilution series of the standard for the chosen read- cells for which the 2 criteria for the endotoxin standard curve
out, in the presence and absence of the preparation being are satisfied. For each different cell source, e.g. individual
examined, is to be within, for example ± 20 per cent of the donation, donor pool, or cell line, use the endotoxin standard
optical density. curve R1-Ri to calculate the concentration of endotoxin
7 METHODS equivalents in each of the replicates of solutions A, B and C
7-1 METHOD A: QUANTITATIVE TEST and solutions AS, BS and CS. The recovery of endotoxin
Method A involves a comparison of the preparation being equivalents calculated from the endotoxin equivalents
examined with a standard endotoxin dose-response curve. concentration found in solutions AS, BS and CS after
The contaminant concentration of the preparation being subtracting the endotoxin equivalents concentration found in
examined is to be less than the CLC to pass the test. solutions A, B and C is within the range of 50-200 per cent.
Dilutions not fulfilling the spike recovery argument are not
7-1-1 Test procedure
valid and are therefore excluded from further evaluation.
Using the validated test method, prepare the solutions shown
The preparation being examined complies with the
in Table 2.6.30.-1 and culture 4 replicates of each solution
requirements of the test for a given cell source if the mean
with the qualified cells.
concentrations of endotoxin equivalents measured in the
Solution A = solution of the preparation being examined at replicates of solutions A, B and C, after correction for
the dilution, here designated f, at which the test for dilution and concentration, are all less than the CLC
interfering factors was carried out, i.e. the highest specified for the preparation being examined. One valid
concentration (lowest dilution) for which the endotoxin dilution is the minimum required for a valid test.
recovery is within 50-200 per cent.
7-1-3 Pass/fail criteria of the preparation
Solution B = 2-fold dilution of solution A, not exceeding the When cells from individual donors are used, the preparation
MVD. being examined is required to comply with the test with the
Solution C = 2-fold dilution of solution B, not exceeding the cells from each of 4 different donors. If the preparation being
MVD. examined passes the test with cells from 3 of the 4 donors,
Solution AS = solution A spiked with standard endotoxin at the test is continued with cells from a further 4 donors, none
a concentration equal to the middle dose from the endotoxin of whom provided cells for the 1st test, and the preparation
standard curve (R3). being examined is required to pass the test with cells from 7
Solution BS = solution B spiked with standard endotoxin at of the 8 different donors (i.e. a maximum of 1 positive
a concentration equal to the middle dose from the endotoxin reaction in 8 donors is allowed). When the source of
standard curve (R3 ). monocytes consists of cells pooled from a number of
individual donors, the preparation being examined is required
Solution CS = solution C spiked with standard endotoxin at
to pass the test with 1 pool of cells. Where a human
a concentration equal to the middle dose from the endotoxin
standard curve (R3). monocytic cell line is used for the test, the preparation being
examined is required to pass the test with 1 qualified passage
Solution Ro = negative control. of cells.
2023 Appendix XIV H V-A481

7-2 METHOD B. SEMI-QUANTITATIVE TEST Solution C = solution of the preparation being examined at a
Method B involves a comparison of the preparation being dilution, here designated f 2 , not exceeding the MVD, chosen
examined with standard endotoxin. The contaminant after a review of data from the product-specific validation,
concentration of the preparation being examined is to be less e.g. MVD.
than the CLC to pass the test. Solution A must be chosen Solution AS = solution A spiked with standard endotoxin at
for the pass decision, unless otherwise justified and 2 x estimated LOD for the test system (as determined in
authorised. preparatory testing).
7-2-1 Test procedure Solution BS = solution B spiked with standard endotoxin at
Using the validated test method, prepare the solutions shown 2 x estimated LOD for the test system.
in Table 2.6.30.-2 and culture 4 replicates of each solution Solution CS = solution C spiked with standard endotoxin at
with the qualified cells. 2 x estimated LOD for the test system.
Table 2.6.30.-2 Solution Ro = negative control.
Solution R 1 = standard endotoxin at 0.5 x estimated LOD
Added
Nwnber of for the test system.
Solution Solution endotoxin
replicates
(IU/mL) Solution R2 = standard endotoxin at 1 x estimated LOD for
A Test solution/! None 4
the test system.
Solution R 3 = standard endotoxin at 2 x estimated LOD for
B Test solutionlf1 None 4 the test system.
C Test solution/j2 None 4 Solution Ri = standard endotoxin at 4 x estimated LOD for
the test system.
Standard
endotoxin at 7-2-2 Calculation and interpretation
AS Test solution/! 2 x estimated 4 All data to be included in the data analysis are to relate to
LO D for the test
cells for which mean responses to solutions Ro-Ri increase
system
progressively. The mean response to Ro may be equal to the
Standard mean response to R 1 . For each different cell source, the
endotoxin at mean response to solution R2 is to be greater than a positive
BS Test solution/j1 2 x estimated 4
cut-off value. Data below this cut-off value are considered
LOD for the test
system negative. If the mean response to R1 or R2 exceeds the cut-
off value, the response to the solution chosen for the pass/fail
Standard decision must be negative (= pass). For each negative
endotoxin at
CS Test solution/Ji 2 x estimated 4
solution of the preparation being examined (A, B and C), the
LOD for the test mean response to the corresponding spiked solution (AS, BS
system or CS respectively) is compared with the mean response
to R3 to determine the percentage spike recovery.
Pyrogen-free saline None (negative
Ro or test diluent control)
4 The contaminant concentration of the preparation being
examined is less than the CLC for a given cell source if the
Standard solution of the preparation being examined designated for the
endotoxin at pass/fail-decision and the dilutions below all give negative
Pyrogen-free saline
R1 0.5 x estimated 4
or test diluent
LOD for the test results and the endotoxin spike recovery is within the range
system of 50-200 per cent.
Standard
7-2-3 Pass/fail criteria of the preparation
endotoxin at The criteria are the same as for method A (see 7-1-3).
Pyrogen-free saline
R, I x estimated 4
or test diluent 7-3 METHOD C: REFERENCE LOT COMPARISON
LOD for the test
system TEST
Method C involves a comparison of the preparation being
Standard examined with a validated reference lot of that preparation.
endotoxin at
Pyrogen-free saline The type of analysis selected to compare the two is to be
R3 2 x estimated 4
or test diluent justified and validated for each product and is to include
LO D for the test
system assay validity criteria. The reference lot is also selected
according to criteria that have been justified and authorised.
Standard
endotoxin at
The test is intended to be performed in cases where a
Pyrogen-free saline preparation being examined shows marked interference but
R, 4 x estimated 4
or test diluent
LOD for the test cannot be diluted within the MVD to overcome the
system interference or because it contains or is believed to contain
non-endotoxin contaminants. Responses to non-endotoxin
Solution A = solution of the preparation being examined at contaminants may dilute out more rapidly than responses to
the dilution, here designated f, at which the test for endotoxin, which makes it necessary to perform the test at a
interfering factors was completed. range of dilutions that include minimum dilution. The test
procedure is described below and includes an example of a
Solution B = solution of the preparation being examined at a
type of analysis used for the comparison of a test lot and
dilution, here designated f 1, not exceeding the MVD, chosen
reference lot.
after a review of data from the product-specific validation,
e.g. 1:2 x MVD (i.e. 2 times less diluted than the MVD).
V-A482 Appendix XIV H 2023

7-3-1 Test procedure The foUowing section is published for infomiation only.
Using the validated test method, prepare the solutions shown
in Table 2.6.30.-3 and culture 4 replicates of each solution GUIDANCE NOTES
with the qualified cells.
1 INTRODUCTION
Table 2.6.30.-3 The monocyte-activation test (MAT) is primarily intended to
be used as a replacement for the rabbit pyrogen test.
Solution/dilution
Solution
factor
Number of replicates The MAT detects pyrogenic and pro-inflammatory
contaminants, including endotoxins from gram-negative
Solution of reference bacteria and 'non-endotoxin' contaminants, including
A 4
lot//1
pathogen-associated molecular patterns (PAMPs), derived
Solution of reference from gram-positive and gram-negative bacteria, viruses and
B 4
lot//2 fungi, and product-related and process-related biological or
chemical entities.
Solution of reference
C 4
lot/h Since non-endotoxin contaminants are a physico-chemically
diverse class of molecules, and usually the nature of the
Solution of preparation
D
being examined//1
4 contaminant in a preparation being examined is unknown,
the level of contamination is expressed either in endotoxin-
E
Solution of preparation
4
equivalent units, derived by comparison with responses to
being examined//2 standard endotoxin, or by comparison with a reference lot of
Solution of preparation the preparation being examined.
F 4
being examined/ls In the MAT, responses to standard endotoxin usually dilute
Positive control
out over approximatively 1 log 10 and responses to products
G 4 contaminated with non-endotoxin contaminants (alone or in
(standard endotoxin)
combination with endotoxins) often show very steep dose-
Diluent (negative response curves, usually over only 1 or 2 dilution steps when
Ro control)
4
tested for their capability to stimulate monocytes. Frequently,
the largest response to such contaminated products is
Solutions A, B and C are solutions of the reference lot obtained with undiluted solutions of preparations being
diluted by the dilution factors, / 1, / 2 and /J, determined in the examined or small dilutions of the preparations being
test for interfering factors. examined. For this reason test solutions of preparations being
Solutions D, E and F are solutions of the preparation being examined that contain or may contain non-endotoxin
examined diluted by the dilution factors, / 1, fz and /J, contaminants have to be tested at a range of dilutions that
determined for the reference lot in the test for interfering includes minimum dilution.
factors. 2METHODS
Solution G is the positive test control for the viability of the 2-1 INFORMATION REGARDING THE CHOICE OF
cells and is a standard endotoxin concentration that gives a METHODS
clear positive response. Methods A, B and C, are not normally applied where a
Solution Ro is the diluent used to dilute the preparation preparation being examined has the intrinsic activity of
being examined and serves as the test blank. stimulating the release of the chosen read-out or where the
7-3-2 Calculation and interpretation preparation being examined is contaminated with the chosen
All data to be included in the data analysis are to relate to read-out. In both cases, this fact is to be addressed by
cells for which solution G and at least one of solutions A, B modifying and validating the chosen method accordingly.
and C give a response that is greater than the basal release of The product-specific validation of the chosen method would
the read-out (Solution Ro). For each different cell source, be expected to identify the frequency of non-responders to a
e.g. individual donation, donor pool, or cell line, use the particular product/contaminant(s) combination and to
standard curve for the read-out (a calibration curve in identify steps to address this, e.g. screening of donors,
duplicate with a blank and at least 4 geometrically diluted increasing the number of donors per test, and setting pass/fail
concentrations of the standard for the chosen read-out) and criteria of appropriate stringency to maximise the likelihood
calculate the mean responses of the replicates of solutions A- of detecting contaminated batches. Method A is not
F. Sum the mean responses to solutions A, Band C and appropriate if the results of different dilutions (endotoxin
sum the mean responses to solutions D, E and F. Divide the equivalents per millilitre) show that the dose response curve
sum of the mean responses to solutions D, E and F by the is not parallel to the standard endotoxin curve. Method B is
a semi-quantitative test that can be applied when responses
sum of the mean responses to solutions A, B and C.
The preparation being examined complies with the test for a to dilutions of a preparation being examined are not parallel
given cell source if the resulting value complies with a to responses to dilutions of standard endotoxin.
defined acceptance criterion not exceeding a justified value, Method C, the reference lot comparison test, was developed
e.g. 2.5. to address extreme donor variability in responses to certain
product/contaminant(s) combinations. In this regard, it
7-3-3 Pass/fail criteria of the preparation
should be noted that, while monocytes from most donors
The criteria are the same as for method A (see 7-1-3).
respond in a broadly similar marmer to bacterial endotoxin,
To quantify more closely the level of contamination, responses of monocytes from different donors to non-
Methods A, B and C may be performed using other dilutions endotoxin contaminants can differ markedly, so that it is
of the solution of the preparation being examined not possible to identify non-responders along with low and high
exceeding the MVD. responders to certain product/contaminant(s) combinations.
2023 Appendix XIV I V-A483

2-2 CALCULATION OF CONTAMINANT LIMIT together with the validation experiments for the test for
CONCENTRATION bacterial endotoxins (BED using the same 3 batches. Cross-
The acceptance criterion for a pass/fail decision is the validation should be repeated on 3 batches if important
contaminant limit concentration (CLC), which is expressed process parameters have changed, as potential contamination
in endotoxin equivalents per milligram or millilitre or in units by non-endotoxin pyrogens cannot be ruled out.
of biological activity of the preparation being examined. The rabbit pyrogen test (see general chapter 2.6.8. Pyrogens)
Where an endotoxin limit concentration (ELC) has been must only be performed for cross validation if none of the
specified for a product, the CLC is the same as the ELC, MAT methods (A, B or C) can be validated for a certain
unless otherwise prescribed. The CLC is expressed in terms product.
of endotoxin equivalents. The CLC is calculated using the
following expression: 3 REPLACEMENT OF THE RABBIT PYROGEN
TEST BY THE MONOCYTE ACTIVATION TEST
K As noted above, MAT is primarily intended to be used as a
M replacement for the rabbit pyrogen test. Monographs on
pharmaceutical products intended for parenteral
K threshold pyrogenic dose per kilogram of body mass; administration that may contain pyrogenic contaminants
M maximum recommended bolus dose of product per kilogram of
body mass.
require either a test for bacterial endotoxins or a monocyte
activation test.
When the product is to be injected at frequent intervals or As a general policy:
infused continuously, M is the maximum total dose - in any individual monograph, when a test is required,
administered in a single hour period. only 1 test is included, either that for bacterial endotoxins
The CLC depends on the product and its route of or the MAT. Before including the MAT in a monograph,
administration and is stated in some monographs. evidence is required that 1 of the 3 methods described in
the MAT chapter can be applied satisfactorily to the
Values for K are suggested in Table 2.6.30.-4.
product in question;
Table 2.6.30.-4 - the necessary information is sought from manufacturers.
Companies are invited to provide any validation data that
Route of administration K
they have concerning the applicability of the MAT to the
Intravenous 5.0 JU of endotoxin per kilogram of
substances and formulations of interest. Such data include
body mass
details of sample preparation and of any procedures
Intravenous, for radiopharmaceuticals 2.5 IU of endotoxin per kilogram of
body mass necessary to eliminate interfering factors. In addition, any
Intrathecal 0.2 JU of endotoxin per kilogram of available parallel data for rabbit pyrogen testing that
body mass would contribute to an assurance that the replacement of
Parenteral formulations administered 100 IU/m 2 a rabbit pyrogen test by the MAT is appropriate, is to be
per square metre of body surface provided.
4 VALIDATION OF ALTERNATIVE .METHODS
2-3 INFORMATION REGARDING CRYO- Replacement of a rabbit pyrogen test, or replacement of a
PROTECTANTS method for detecting pro-inflammatory/pyrogenic
The influence of cryo-protectants, e.g. dimethyl contaminants by another method, is to be regarded as the
sulfoxide (DMSO), and their residues in thawed cells, is to use of an alternative method in the replacement of a
be considered: DMSO is toxic to cells in culture and, even pharmacopoeia! test, as described in the General Notices.
when cells have been washed thoroughly, cryo-preservation The following procedures are suggested for validating a
may have altered cell properties, e.g. cell membrane
method for the MAT other than the one indicated in the
permeability. monograph:
2-4 INTERFERENCE TESTING - the procedure and the materials and reagents used in the
Where practicable, interference testing is performed on at method should be validated as described for the test
least 3 different lots of the preparation being examined. concerned;
Preparations being examined that show marked batch-to- - the presence of interfering factors (and, if needed, the
batch variation, that effectively renders each batch unique for procedure for removing them) should be tested on
the purposes of interference testing, are to be subjected to samples of at least 3 production batches.
interference testing within each individual test, MAT should be applied to all new products intended for
i.e. concomitant validation. parenteral administration that have to be tested for the
Interference testing is preferably performed on batches of the presence of non-endotoxin pyrogens according to the
preparation being examined that are free of endotoxins and requirements of the European Pharmacopoeia.
other pyrogenic/pro-inflammatory contaminants and, where
this is not practicable, none of the batches are to be heavily
contaminated. If only 1 batch is available the validation has
to be performed on that batch in 3 independent tests.
Precision parameters for reproducibility, e.g. ± 50 per cent,
I. Assay of Pancreatin
are to be fulfilled. The free protease, lipase and amylase activities of pancreatin
are determined by the following methods.
2-5 CROSS-VALIDATION
General chapter 5. 1.10. Guidelines for using the test for bacterial Standard preparation and units
endotoxins, states that an MAT should be performed on The Standard Preparation is the appropriate FIP Standard
products where the presence of non-endotoxin pyrogenic which has been adopted as an official preparation by the
substances cannot be ruled out. It is therefore recommended European Pharmacopoeia Commission and is available as
to perform cross-validation experiments with the MAT
V-A484 Appendix XIV I 2023

pancreas powder (protease) EPBRP or pancreas powder (amylase Measure the absorbances of the filtrates at the maximum at
and lipase) EPBRP as appropriate. 275 nm, Appendix II B, using in the reference cell a mixture
The Unit of protease activity is contained in that amount of of 6.0 mL of borate buffer pH 7.5 and 5.0 mL of the 5% w/v
the Standard Preparation that, under the conditions of the solution of trichwroacetic acid that has been filtered in the
assay, hydrolyses casein at an initial rate such that there is same way. Correct the mean absorbances of the filtrates from
liberated per minute an amount of peptides not precipitated tubes S 1, S2 and S3 by subtracting the mean absorbances of
by trichloroacetic acid that gives the same absorbance at the filtrates from the corresponding control tubes S 1B, S2B
275 nm as one micromole of tyrosine. The Unit of lipase and S3 B.
activity is contained in that amount of the Standard Prepare a reference curve by plotting the mean corrected
Preparation that, under the conditions of the assay, liberates absorbances against the potency of the dilution of the
one micro-equivalent of acid per minute at pH 9.0 and 37°. solution of the Standard Preparation used. Calculate the
The Unit of amylase activity is contained in that amount of corrected mean absorbance of the substance being examined
the standard preparation that, under the conditions of the by subtracting the mean absorbance of the filtrates from
assay, decomposes starch at an initial rate such that one tubes UB from that of the filtrates from tubes U. Using the
micro-equivalent of glycosidic linkage is hydrolysed per corrected mean absorbance, determine the potency of the
minute. solution of the substance being examined from the reference
1. Free protease activity curve and calculate the free protease activity per mg of the
substance being examined by taking into account the dilution
Method factors.
Solution of the standard preparation Triturate for
5 minutes a quantity of the Standard Preparation containing The test is not valid unless the corrected absorbances are
approximately 100 Units of protease activity with 25 mL of between 0.15 and 0.60.
calcium chloride solution cooled to 5°. Dilute to 100 mL with 2. Lipase activity
the cooled calcium chloride solution and then dilute a sufficient Apparatus
quantity of the resulting suspension to 100 mL with borate Use a reaction vessel of about 50 mL capacity fitted with a
buffer pH 7.5, cooled to 5°, so that 1 mL of the final solution device that will maintain a temperature of 36.5° to 37.5°, a
contains 0.065 Units of protease activity. magnetic stirrer and a lid with holes for the insertion of
Solution of the substance being examined Triturate for electrodes, the tip of a burette, a tube for the admission of
5 minutes a quantity of the substance being examined nitrogen and the introduction of reagents. An automatic or
containing approximately 100 Units of protease activity with manual titration apparatus may be used. In the latter case,
25 mL of calcium chloride solution cooled to 5°. Dilute to the burette is graduated in 5-µL divisions and the pH meter
100 mL with the cooled calcium chloride solution and then is provided with a wide reading scale and glass and calomel
dilute a sufficient quantity of the resulting suspension to electrodes. After each test the reaction vessel is evacuated by
100 mL with borate buffer pH 7.5, cooled to 5°, so that the suction and washed several times with water, the washings
estimated free protease activity corresponds approximately to being removed each time by suction.
the activity of the solution of the Standard Preparation. Method
Label 16 test tubes with the following identification in Carry out the assay under nitrogen. In a small mortar cooled
duplicate; S 1, S2 , S3 , S 1B, S2 B, S3 B, U and UB. To tubes S 1 to 0° to 4 ° triturate carefully an amount of the substance
and S 1B add 2.0 mL and to tubes S2 , S 2 B, U and UB, being examined containing approximately 2500 Units of
1.0 mL of borate buffer pH 7.5. Then to tubes S 1 and S 1B lipase activity with 1 mL of cooled lipase solvent until a very
add 1.0 mL, to tubes S2 and S 2B, 2.0 mL and to tubes S3 fine suspension is obtained (about 10 minutes). Dilute with
and S3B, 3.0 mL of the solution of the Standard Preparation. cooled lipase solvent, transfer quantitatively to a graduated
Add 2.0 mL of the solution of the substance being examined flask and dilute to 100.0 mL with the cooled solvent; use
to tubes U and UB. To each of the control tubes (S 1B, S2B, immediately.
S3 B and UB) add 5.0 mL of a 5% w/v solution of Transfer 29.5 mL of olive oil substrate emulsion to the
trichloroacetic acid and mix. Place a stirring rod in each tube assembled reaction vessel equilibrated at 36.5° to 3 7.5° and
and warm to, and maintain at, 35° in a water bath. adjust the pH to 9.2 with O.lM sodium hydroxide. Add about
Add 5.0 mL of concentrated casein substrate to each of the 0.5 mL of the suspension of the preparation being examined
control tubes and mix. and record the time at which the pH reaches 9.0.
At accurately timed intervals add 5.0 mL of concentrated Add continuously from a micrometer syringe sufficient O. lM
casein substrate, previously warmed to 35°, to tubes S 1, S2 , S3 sodium hydroxide VS to maintain the pH at 9.0. Record the
and U and mix immediately. After exactly 30 minutes, in the volume of 0.1 M sodium hydroxide VS consumed at I -minute
same order, stop the reaction in tubes S 1, S2 , S3 and U by intervals for 5 minutes. Discounting the first reading,
adding 5.0 mL of a 5% w/v solution of trichloroacetic acid and calculate the mean rate of alkali consumption U. If necessary,
mix thoroughly. Remove all the test tubes from the water dilute with sufficient lipase solvent to produce an average
bath and allow to stand at room temperature for 20 minutes. alkali consumption of 0.08 to 0.16 mL of O.lM sodium
Filter the contents of the tubes through suitable filter paper 1 , hydroxide VS per minute. Repeat the procedure using the
collect the filtrates and refilter through the same paper. Standard Preparation in place of the substance being
The filtrates must be free from haze. examined and calculate the mean rate of alkali consumption,
S. Calculate the potency (PL) of the substance being
examined in Units per mg from the expression:

1 A suitable filter paper complies with the following test. Filter 5 mL of a u w.

PL =-x_axR
5% wlv solution of trichloroacetic acid through a sample of the paper and
measure the absorbance of the filtrate at 275 nm, Appendix II B, using the
sw
unfiltered 5% wlv solution of trichloroacetic acid in the reference cell.
The absorbance is not more than 0. 04.
2023 Appendix XIV J V-A485

Where u the mean volume in mL of 0. IM sodium hydroxide

VS used per minute in the titration of the J. Blood and Related Products
substance being examined,
s the mean volume in mL of 0. lM sodium hydroxide Coagulants
VS used per minute in the titration of the Standard
Preparation, Al. Assay of Human Coagulation Factor II
w weight in mg of the substance being examined, (Ph. Bur. method 2. 7.18)
w, weight in mg of the Standard Preparation,
potency of the Standard Preparation in Units
Human coagulation factor II is assayed following specific
R
per mg. activation to form factor Ila. Factor Ila is estimated by
comparing its activity in cleaving a specific chromogenic
Calculate the potency of the preparation being examined peptide substrate with the same activity of the International
using the average of three separate titrations for both the Standard or of a reference preparation calibrated in
substance being examined and the Standard Preparation. International Units.
3. Amylase activity The International Unit is the factor II activity of a stated
amount of the International Standard which consists of a
Method
freeze-dried concentrate of human blood coagulation
Triturate an amount of the preparation being examined
factor II. The equivalence in International Units of the
containing approximately 1500 Units of amylase activity with
International Standard is stated by the World Health
60 mL of 0. 2M mixed phosphate buffer pH 6. 8 for 15 minutes
Organization.
and add sufficient 0. 2M mixed phosphate buffer pH 6. 8 to
produce 100 mL. To a stoppered tube (200 mm x 22 mm) The chromogenic assay method consists of 2 steps: snake
add 25.0 mL of starch substrate, 10.0 mL of 0.2M mixed venom-dependent activation of factor II, followed by
phosphate buffer pH 6.8 and 1.0 mL of 0.2M sodium chloride. enzymatic cleavage of a chromogenic factor Ila substrate to
Stopper the tube, mix the contents and place in a water bath form a chromophore that can be quantified
at 24. 9° to 25.1 °. When the temperature of the mixture has spectrophotometrically. Under appropriate assay conditions,
reached 25° add 1.0 mL of the solution of the substance there is a linear relation between factor Ila activity and the
being examined and record the time of addition. cleavage of the chromogenic substrate.
Mix thoroughly and replace in the water bath. After exactly REAGENTS
10 minutes add 2 mL of lM hydrochloric acid to stop the Viper venom specific factor II activator (ecarin).
reaction. Transfer the contents of the tube to a stoppered A protein derived from the venom of the saw-scaled viper
300 mL flask. While shaking continuously add 10.0 mL of (Echis carinatus) which specifically activates factor II.
0.05M iodine VS followed immediately by 45 mL of 0.1M Reconstitute according to the manufacturer's instructions.
sodium hydroxide. Allow to stand in the dark at a temperature Store the reconstituted preparation at 4 °C and use within
of 15° to 25° for 15 minutes. Add 4 mL of a mixture of 1 month.
1 volume of sulfuric acid and 4 volumes of water and titrate Factor Ila chromogenic substrate Specific chromogenic
with 0. JM sodium thiosulfate VS. Repeat the procedure but substrate for factor Ila such as: H-D-phenylalanyl-L-pipecolyl-
add the 2 mL of lM hydrochwric acid before the addition of L-arginine-4-nitroanilide dihydrochloride, 4-toluenesulfonyl-
the solution of the substance being examined. Prepare a glycyl-prolyl-L-arginine-4-nitroanilide, H-D-cyclohexylglycyl-
solution of the Standard Preparation in the same manner as a-aminobutyryl-L-arginine-4-nitroanilide, D-cyclohexylglycyl-
described for the solution of the substance being examined L-alanyl-L-arginine-4-nitroanilide diacetate. Reconstitute
and repeat the procedure beginning at the words 'To a according to the manufacturer's instructions.
stoppered tube ... ' but using 1.0 mL of this solution in Dilution buffer Solution containing 6.06 g/L of m·s
place of the solution of the substance being examined. (hydroxymethyl)aminomethane R, 17.53 g/L of sodium
Calculate the potency (PA) of the preparation being chloride R and 1 g/L of bovine albumin R or human albumin R.
examined in Units per mg from the expression: Adjust to pH 8.4 if necessary, using hydrochloric acid R.
METHOD
Test solution Dilute the preparation to be examined with
dilution buffer to obtain a solution containing 0.015 IU of
factor II per millilitre. Prepare at least 3 further dilutions in
dilution buffer.
Where A volume in mL of 0.1 M sodium thiosulfate VS used Reference solution Dilute the reference preparation to be
in the titration of the substance being examined,
A, volume in mL of 0.1M sodium thiosulfate VS used
examined with dilution buffer to obtain a solution containing
in the titration of the Standard Preparation 0.015 IU of factor II per millilitre. Prepare at least 3 further
B volume in mL of 0. lM sodium thiosulfate VS used dilutions in dilution buffer.
in the titration of the substance being examined
inactivated by the addition of IM hydrochloric
Wann all solutions to 37 °C in a water-bath shortly before
acid, the test.
B, volume in mL of 0. lM sodium thiosuljate VS used The following working conditions apply to microtitre plates.
in the titration of the Standard Preparation
inactivated by the addition of IM hydrochloric
If the assay is carried out in tubes, the volumes are adjusted
acid, while maintaining the proportions in the mixture.
Q potency of the Standard Preparation in Units per Using a microtitre plate maintained at 37 °C, add 25 µL of
mg
w total weight in mg of the substance being
each dilution of the test solution or the reference solution to
examined in the solution prepared for assay, each of a series of wells. To each well add 125 µL of dilution
w, total weight in mg of the Standard Preparation in buffer, then 25 µL of ecarin and incubate for exactly 2 min.
the solution prepared for assay. To each well add 25 µL of factor Ila chromogenic substrate.
Read the rate of change of absorbance (2.2.25) at 405 nm
continuously over a period of 3 min and obtain the mean
V-A486 Appendix XIV J 2023

rate of change of absorbance (M/min). If continuous a final concentration during the first step of the assay of
monitoring is not possible, read the absorbance at 405 nm at 10-350 nmol/L, preferably 14-70 nmol/L. Thromboplastin
suitable consecutive intervals, for instance 40 s, plot the from natural sources (bovine or rabbit brain) or synthetic
absorbances against time on a linear graph and calculate preparations may be used as the tissue factor/phospholipid
M/min as the slope of the line. From the M/min values of component. Thromboplastin suitable for use in prothrombin
each individual dilution of standard and test preparations, time determination is diluted 1:5 to 1:50 in buffer such that
calculate the potency of the preparation to be examined and the final concentration of Ca 2 + is 15-25 mmol/L. The final
check the validity of the assay by the usual statistical methods factor Xa generation is performed in a solution containing
(5.3). human or bovine albumin at a concentration such that
adsorption losses do not occur and which is appropriately
A2. Assay of Human Coagulation Factor VII buffered at pH 7.3-8.0. In the final incubation mixture,
(Ph. Bur. method 2.7.10) factor VII must be the only rate-lin1iting component and
Human coagulation factor VII is assayed by its biological each reagent component must lack the ability to generate
activity as a factor VIIa-tissue factor complex in the factor Xa on its own.
activation of factor X in the presence of calcium ions and The second step comprises the quantification of the formed
phospholipids. The potency of a factor VII preparation is factor Xa employing a chromogenic substrate that is specific
estimated by comparing the quantity necessary to achieve a for factor Xa. Generally this consists of a short peptide of
certain rate of factor Xa formation in a test mixture between three and five amino acids, bound to a chromophore
containing the substances that take part in the activation of group. On cleavage of this group from the peptide substrate,
factor X, and the quantity of the International Standard, or its absorption maximum shifts to a wavelength allowing its
of a reference preparation calibrated in International Units, spectrophotometric quantification. The substrate is usually
required to produce the same rate of factor Xa formation. dissolved in water R and used at a final concentration of
The International Unit is the factor VII activity of a stated 0.2-2 mmol/L. The substrate may also contain appropriate
amount of the International Standard, which consists of inhibitors to stop further factor Xa generation (addition of
freeze-dried plasma. The equivalence in International Units edetate).
of the International Standard is stated by the World Health
ASSAY PROCEDURE
Organization.
Reconstitute the entire contents of one ampoule of the
Human coagulatwn factor VII concentrate ERP is calibrated in reference preparation and the preparation to be examined by
International Units by comparison with the International adding the appropriate quantity of water R; use within 1 h.
Standard. Add sufficient prediluent to the reconstituted preparations to
The chromogenic assay method consists of 2 consecutive produce solutions containing between 0.5 IU and 2.0 IU of
steps: the factor VII-dependent activation of factor X reagent factor VII per millilitre.
mixture containing tissue factor, phospholipids and calcium Prepare further dilutions of reference and test preparations
ions, followed by enzymatic cleavage of a chromogenic using an isotonic non-chelating buffer containing 1 per cent
factor Xa substrate into a chromophore that can be of bovine or human albumin, buffered preferably between
quantified spectrophotometrically. Under appropriate assay pH 7.3 and 8.0. Prepare at least three separate, independent
conditions, there is a linear relation between the rate of factor dilutions for each material, preferably in duplicate. Prepare
Xa formation and the factor VII concentration. The assay is the dilutions such that the final factor VII concentration is
summarised in the following scheme. below 0.005 IU/mL.
Prepare a control solution that includes all components
Step 1
except factor VII.
Prepare all dilutwns in plastic tubes and use within 1 h.
a) factor VI I _ _ _ti_ss_u_e_fa_c_to_r.;..,_c_a_+-----1► factor VIia
2
Step 1
Mix dilutions of the factor VII reference preparation and the
preparation to be examined with an appropriate volume of
b) factor X _ _ _f_a_c_to_r_v_1_1a..;,_c_a_2_+_ _---I► factor Xa
the prewarmed coagulation factor reagent or a combination
tissue factor/phospholipid of its separate constituents, and incubate the mixture in
plastic tubes or microplate wells at 37 °C.
Step 2 The concentrations of the various components during the
chromogenic substrate factor Xa ► peptide + chromophore factor Xa generation must be as specified above under the
description of the reagents.
Allow the activation of factor X to proceed for a suitable
time, usually terminating the reaction before the factor Xa
Both steps employ reagents that may be obtained concentration has reached its maximal level in order to
commercially from a variety of sources. Although the obtain a satisfactory linear dose-response relationship.
composition of individual reagents may be subject to some The activation time is also chosen to achieve linear
variation, their essential features are described in the production of factor Xa in time. Appropriate activation times
following specification. are usually between 2 min and 5 min, but deviations are
REAGENTS permissible if acceptable linearity of the dose-response
The coagulation factor reagent comprises purified proteins relationship is thus obtained.
derived from human or bovine sources. These include Step 2
factor X and thromboplastin tissue factor/phospholipid as Terminate the activation by the addition of a prewarmed
factor VII activator. These proteins are partly purified and do reagent containing a chromogenic substrate. Quantify the rate
not contain impurities that interfere with the activation of of substrate cleavage, which must be linear with the
factor VII or factor X. Factor X is present in amounts giving
2023 Appendix XIV J V-A487

concentration of factor Xa formed, by measuring the Both steps employ reagents that may be obtained
absorbance change at an appropriate wavelength using a commercially from a variety of sources. Although the
spectrophotometer, either monitoring the absorbance composition of individual reagents may be subject to some
continuously, thus allowing the initial rate of substrate variation, their essential features are described in the
cleavage to be calculated, or terminating the hydrolysis following specification. Deviations from this description may
reaction after a suitable interval by lowering the pH by the be permissible provided that it has been shown, using the
addition of a suitable reagent, such as acetic acid (500 g/L appropriate International Standard for blood coagulation
C 2 H 4 O 2 ) or a citrate solution (1 mol/L) at pH 3. Adjust the factor VIII as the standard, that the results obtained do not
hydrolysis time to achieve a linear development of differ significantly.
chromophore with time. Appropriate hydrolysis times are It is important to demonstrate by validation the suitability of
usually between 3 min and 15 min, but deviations are the kit used, notably by checking the time course of
permissible if better linearity of the dose-response relationship factor Xa generation in order to determine the time taken to
is thus obtained. reach 50 per cent of the maximal factor Xa generation.
Check the validity of the assay and calculate the potency of
REAGENTS
the test preparation by the usual statistical methods (for
The coagulation factor reagent comprises purified proteins
example, 5.3).
derived from human or bovine sources. These include
A3. Assay of Factor VIII Fraction (Human factor X, factor IXa, and a factor VIII activator, usually
Coagulation Factor VIII) thrombin. These proteins are partly purified, preferably to at
(Ph. Bur. method 2.7.4) least 50 per cent, and do not contain impurities that interfere
Human coagulation factor VIII is assayed by its biological with the activation of factor VIII or factor X. Thrombin may
activity as a cofactor in the activation of factor X by activated be present in its precursor form prothrombin, provided that
factor IX (factor IXa) in the presence of calcium ions and its activation in the reagent is sufficiently rapid to give almost
phospholipid. Factor VIII activity may be measured in instantaneous activation of factor VIII in the assay.
plasma preparations and therapeutic concentrates (plasma- Phospholipid may be obtained from natural sources or be
derived and recombinant). The potency of a factor VIII synthetically prepared, and must, to a substantial extent,
preparation is estimated by comparing the quantity necessary consist of the species phosphatidylserine. The components of
to achieve a certain rate of factor Xa formation in a test the complete reagent are usually divided into at least
mixture containing the substances that take part in the 2 separate reagents, each lacking the ability to generate
activation of factor X, and the quantity of the International factor Xa on its own. One of the reagents contains calcium
Standard, or of a reference preparation calibrated in ions. After reconstitution, the reagents may be combined
International Units, required to produce the same rate of provided that no substantial amounts of factor Xa are
factor Xa formation. generated in the absence of factor VIII. In the final
incubation mixture, factor VIII must be the only rate-limiting
Quantification of factor VIII activity in plasma preparations is component.
expressed in International Units defined by the International
Standard for blood coagulation factor VIII in plasma, and The 2nd step comprises the quantification of the formed
coagulation factors V, VIII, XI and XIII plasma ERP is suitable
factor Xa, employing a chromogenic substrate that is specific
for use as a reference preparation. Quantification of for factor Xa. Generally this consists of a derivatised short
factor VIII activity in therapeutic concentrates is expressed in peptide of between 3 and 5 amino acids, joined to a
International Units defined by the International Standard for chromophore group. On cleavage of this group from the
blood coagulation factor VIII concentrate, and human peptide substrate, its chromophoric properties shift to a
coagulation factor VIII concentrate ERP is suitable for use as a wavelength allowing its spectrophotometric quantification.
reference preparation. The substrate must also contain appropriate inhibitors to
stop further factor Xa generation, e.g. chelating agents, and
The chromogenic assay method consists of 2 consecutive to suppress thrombin activity.
steps: the factor VIII-dependent activation of factor X in a
coagulation-factor reagent composed of purified components, ASSAY PROCEDURE
and the enzymatic cleavage of a chromogenic factor Xa For the assay of therapeutic concentrates, add sufficient
substrate to yield a chromophore that can be quantified prediluent to the reference and test preparations to produce
spectrophotometrically. Under appropriate assay conditions, solutions containing 0.5-2.0 IU/mL. The prediluent consists
there is a linear relation between the rate of factor Xa of haemophilia A plasma, or of an artificially prepared
formation and the factor VIII concentration. The assay is reagent that contains sufficient von Willebrand factor and
summarised by the following scheme. that gives results that do not differ significantly from those
obtained employing haemophilia plasma. The prediluted
materials must be stable beyond the time required for the
Step 1 assay. Pre-dilution in haemophilia A plasma is not required
for the assay of factor VIII in plasma preparations.
factor X ___(_a_ct_iv_a_te_d_}_f_ac_t_o_rV_lll_----:11► factor Xa Prepare dilutions of the pre-diluted reference and test
factor IXa, phospholipid, Ca 2+ concentrate preparations (or the reference and test plasma
preparations) using a non-chelating, appropriately buffered
Step 2
solution, for example, tris(hydroxymethyl)aminomethane or
imidazole, containing 1 per cent of human or bovine
albumin. Prepare at least 2 dilution series of at least 3 further
chromogenic substrate factor Xa ► peptide + chromophore dilutions for each material. Prepare the dilutions such that
the final factor VIII concentration in the reaction mixture is
preferably below 0.01 IU/mL, during the step of factor Xa
generation.
V-A488 Appendix XN J 2023

Prepare a control solution that includes all components solution pH 7. 3 R) containing 10 g/L of bovine or human
except factor VIII. albumin. Use these dilutions immediately.
Prepare all dilutions in plastic tubes and use immediately. Use an apparatus suitable for measurement of coagulation
Step 1 times or carry out the assay with incubation tubes maintained
Mix prewarmed dilutions of the factor VIII reference in a water-bath at 37 °C. Place in each tube 0.1 mL of
preparation and of the preparation to be examined with an factor IX-deficient plasma (for example plasma substrate R2)
appropriate volume of the prewarmed coagulation factor and O.1 mL of one of the dilutions of the reference
reagent or a combination of its separate constituents, and preparation or of the preparation to be examined. Add to
incubate the mixture in plastic tubes or microplate wells at each tube 0.1 mL of a suitable Activated Partial
37 °C. Allow the activation of factor X to proceed for a Thromboplastin Time (APTT) reagent containing
suitable time, terminating the reaction (step 2) when the phospholipid and contact activator and incubate the mixture
factor Xa concentration has reached approximately for a recommended time at 37 °C. To each tube, add
50 per cent of the maximal (plateau) level. Appropriate 0.1 mL of a 3.7 g/L solution of calcium chloride R previously
activation times are usually between 2 min and 5 min. heated to 37 °C. Using a timer, measure the coagulation
time, i.e. the interval between the moment of the addition of
Step 2 the calcium chloride and the first indication of the formation
Terminate the activation by addition of a prewarmed reagent
of fibrin. The volumes given above may be adapted to the
containing a chromogenic substrate. Quantify the rate of APTT reagent and apparatus used. Calculate the potency
substrate cleavage, which must be linear with the
using the usual statistical methods (for example, 5.3).
concentration of factor Xa formed, by measuring the
absorbance change at an appropriate wavelength using a A5. Assay of Human Coagulation Factor X
spectrophotometer, either monitoring the absorbance (Ph. Bur. method 2.7.19)
continuously, thus allowing the initial rate of substrate Human coagulation factor X is assayed following specific
cleavage to be calculated, or terminating the hydrolysis activation to form factor Xa. Factor Xa is estimated by
reaction after a suitable interval by lowering the pH by comparing its activity in cleaving a specific chromogenic
addition of a suitable reagent, such as a 50 per cent VIV peptide substrate with the same activity of the International
solution of acetic acid, or a 1 M pH 3 citrate buffer solution. Standard or of a reference preparation calibrated in
Adjust the hydrolysis time to achieve a linear development of International Units.
chromophore over time. Appropriate hydrolysis times are
The International Unit is the factor X activity of a stated
usually between 3 min and 15 min, but deviations are
amount of the International Standard which consists of a
permissible if better linearity of the dose-response relationship
freeze-dried concentrate of human coagulation factor X.
is thus obtained.
The equivalence in International Units of the International
Calculate the potency of the test preparation by the usual Standard is stated by the World Health Organization.
statistical methods (for example, 5.3).
The chromogenic assay method consists of 2 steps: snake
A4. Assay of Factor IX Fraction (Human venom-dependent activation of factor X, followed by
Coagulation Factor IX) enzymatic cleavage of a chromogenic factor Xa substrate to
(Ph. Bur. method 2. 7.11) form a chromophore that can be quantified
spectrophotometrically. Under appropriate assay conditions,
The principle of the assay is to measure the ability of a
factor IX preparation to reduce the prolonged coagulation there is a linear relation between factor Xa activity and the
cleavage of the chromogenic substrate.
time of factor IX-deficient plasma. The reaction is
accelerated by addition of a reagent containing phospholipid REAGENTS
and a contact activator, e.g. kaolin, silica or ellagic acid. Russell's viper venom specific factor X activator
The potency is assessed by comparing the dose-response (RVV). A protein derived from the venom of Russell's viper
curve of the preparation to be examined to that of a ( Vipera russellz) which specifically activates factor X.
reference preparation, calibrated in International Units. Reconstitute according to the manufacturer's instructions.
The International Unit is the factor IX activity of a stated Store the reconstituted preparation at 4 °C and use within
amount of the International Standard, which consists of a 1 month.
freeze-dried concentrate of human coagulation factor IX. Factor Xa chromogenic substrate Specific chromogenic
The equivalence in International Units of the International substrate for factor Xa such as: N-cr-benzyloxycarbonyl-D-
Standard is stated by the World Health Organization. arginyl-L-glycyl-L-arginine-4-nitroanilide dihydrochloride,
Human coagulation factor IX concentrate BRP is calibrated in N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-4-
International Units by comparison with the International nitroanilide hydrochloride, methanesulfonyl-o-leucyl-glycyl-L-
Standard. arginine-4-nitroanilide, methoxycarbonyl-o-cyclohexylalanyl-
Reconstitute separately the preparation to be examined and glycyl-L-arginine-4-nitroanilide acetate. Reconstitute
the reference preparation as stated on the label and use according to the manufacturer's instructions.
immediately. Where applicable, determine the amount of Dilution buffer Solution containing 3.7 g/L of tris
heparin present (2. 7.12) and neutralise the heparin, for (hydroxymethyl)aminomethane R, 18.0 g/L of sodium
example by addition of protamine sulfate R ( 10 µg of chloride R, 2.1 g/L of imidazole R, 0.02 g/L of hexadimethrine
protamine sulfate neutralises 1 IU of heparin). Predilute the bromide R and 1 g/L of bovine albumin R or human albumin R.
preparation to be examined and the reference preparation in Adjust to pH 8.4 if necessary using hydrochloric acid R.
factor IX-deficient plasma (for example plasma substrate R2) METHOD
to produce solutions containing 0.5-2.0 IU/mL. Prepare at Test solution Dilute the preparation to be examined with
least 3 dilutions for each material, preferably in duplicate, dilution buffer to obtain a solution containing 0.18 IU of
using a suitable buffer solution (for example imidazole buffer factor X per millilitre. Prepare at least 3 further dilutions in
dilution buffer.
2023 Appendix XIV J V-A489

Reference solution Dilute the reference preparation to be time, i.e. the interval between the moment of the addition of
examined with dilution buffer to obtain a solution containing the calcium chloride and the first indication of the formation
0.18 IU of factor X per millilitre. Prepare at least 3 further of fibrin. The volumes given above may be adapted to the
dilutions in dilution buffer. APTT reagent and apparatus used. Calculate the potency
Warm all solutions to 37 °C in a water-bath shortly before using the usual statistical methods (for example, 5.3).
the test.
A7. Activated Coagulation Factors
The following working conditions apply to microtitre plates. (Ph. Bur. method 2.6.22)
If the assay is carried out in tubes, the volumes are adjusted
Where applicable, determine the amount of heparin present
while maintaining the proportions in the mixture.
(2. 7.12) and neutralise the heparin, for example by addition
Using a microtitre plate maintained at 37 °C, add 12.5 µL of of protamine sulfate R ( 10 µg of protamine sulfate neutralises
each dilution of the test solution or the reference solution to 1 IU of heparin). Prepare 1 to 10 and 1 to 100 dilutions of
each of a series of wells. To each well add 25 µL of RVV the preparation to be examined using tris(hydroxymethyl)
and incubate for exactly 90 s. To each well add 150 µL of aminomethane buffer solution pH 7.5 R. Place a series of
factor Xa chromogenic substrate, diluted 1 in 6 in dilution polystyrene tubes in a water-bath at 37 °C and add to each
buffer. tube 0.1 mL of platelet-poor plasma R and 0.1 mL of a
Read the rate of change of absorbance (2.2.25) (at 405 nm suitable dilution of a phospholipid preparation to act as a
continuously over a period of 3 min and obtain the mean platelet substitute. Allow to stand for 60 s. Add to each tube
rate of change of absorbance (M/min). If continuous either 0 .1 mL of 1 of the dilutions or 0 .1 mL of the buffer
monitoring is not possible, read the absorbance at 405 nm at solution (control tube). To each tube add immediately
suitable consecutive intervals, for instance 40 s, plot the 0.1 mL of a 3.7 g/L solution of calcium chloride R previously
absorbances against time on a linear graph and calculate heated to 37 °C, and measure, within 30 min of preparing
M/min as the slope of the line. From the M/min values of the original dilution, the time that elapses between addition
each individual dilution of standard and test preparations, of the calcium chloride solution and the formation of a clot.
calculate the potency of the preparation to be examined and The test is not valid unless the coagulation time measured
check the validity of the assay by the usual statistical methods for the control tube is 200 s to 350 s.
(5.3).
A8. Assay of Human von Willebrand Factor
A6. Assay of Human Coagulation Factor XI (Ph. Bur. method 2.7.21)
(Ph. Bur. method 2. 7.22) The biological functions of human von Willebrand factor are
The principle of the assay is to measure the ability of a numerous. At present, its ristocetin cofactor activity and its
factor XI preparation to reduce the prolonged coagulation collagen binding activity can be utilised for assays.
time of factor XI-deficient plasma. The reaction is The potency of human von Willebrand factor is determined
accelerated by addition of a reagent containing phospholipid by comparing, in given conditions, its activity with the same
and a contact activator, e.g. kaolin, silica or ellagic acid. activity of a reference preparation calibrated against the
The potency is assessed by comparing the dose-response International Standard, in International Units where
curve of the preparation to be examined to that of a applicable.
reference plasma calibrated against the International The International Unit is the activity of a stated amount of
Standard for blood coagulation factor XI in plasma. the International Standard, which consists of a freeze-dried
Reconstitute separately the preparation to be examined and human von Willebrand factor concentrate. The equivalence
the reference preparation as stated on the label and use in International Units of the International Standard is stated
immediately. Coagulation factors V, VIII, XI and XIII by the World Health Organization (WHO).
plasma ERP is suitable for use as a reference preparation.
RISTOCETIN COFACTOR ASSAY
Where applicable, determine the amount of heparin present
(2. 7.12) and neutralise the heparin, for example by addition The ristocetin cofactor activity of von Willebrand factor is
of pro ta mine sulfate R ( 10 µg of protamine sulfate neutralises determined by measuring agglutination of a suspension of
1 IU of heparin). Predilute the preparation to be examined platelets in the presence of ristocetin A. The assay can be
carried out for quantitative determinations by using
and the reference preparation in factor XI-deficient plasma
automated instruments, or for semi-quantitative
(for example plasma substrate R3) to produce solutions
containing 0.5-2.0 units/mL. Prepare at least 3 appropriate determinations by visually assessing the endpoint of
dilutions for each material, preferably in duplicate, using a agglutination in a dilution series. Quantitative assays are
preferred.
suitable buffer solution (for example imidazole buffer solution
pH 7.3 R) containing 10 g/L of bovine or human albumin. REAGENTS
Use these dilutions immediately. Suspension of platelets Use standardised and, for
Use an apparatus suitable for measurement of coagulation example, formaldehyde- or paraformaldehyde-fixed
times or perform the assay with incubation tubes maintained preparations of freshly isolated and washed human platelets.
in a water bath at 37 °C. Place in each tube 0.1 mL of The suspension may also be freeze-dried. An appropriate
factor XI-deficient plasma (for example plasma substrate R3) amount of ristocetin A is added if necessary. Some platelet
and 0.1 mL of one of the dilutions of the reference reagents may already contain ristocetin A.
preparation or of the preparation to be examined. Add to Reference preparation The reference preparation for von
each tube 0.1 mL of a suitable Activated Partial Willebrand factor is the WHO International Standard for von
Thromboplastin Time (APTT) reagent containing Willebrand factor concentrate.
phospholipid and contact activator and incubate the mixture METHOD
for a recommended time at 37 °C. To each tube, add Semi-quantitative assay Prepare suitable dilutions of the
0.1 mL of a 3.7 g/L solution of calcium chloride R previously preparation to be examined and of the reference preparation,
heated to 37 °C. Using a timer, measure the coagulation using as diluent a solution containing 9 g/L of sodium
V-A490 Appendix XIV J 2023

chloride R and 10-50 g/L of human albumin R. Add to each Microtitre plates Flat-bottomed polystyrene plates with
dilution an appropriate amount of the suspension of platelets surface properties optimised for enzyme immunoassay and
and, if necessary, of ristocetin A. Mix on a glass slide by high protein-binding capacity.
moving it gently in circles for 1 min. Allow to stand for a METHOD
further 1 min and read the result against a dark background Test solutions Reconstitute the preparation to be
with side lighting. The last dilution which clearly shows examined as stated on the label. Dilute with dilution buffer
visible agglutination indicates the ristocetin cofactor titre of to produce a solution containing approximately 1 IU of von
the sample. Use diluent as a negative control. Willebrand factor. Prepare 2 series of at least 3 further
Quantitative Assay Reconstitute the entire contents of dilutions using dilution buffer.
1 ampoule of the reference preparation and the preparation Reference solutions Reconstitute the reference
to be examined by adding the appropriate quantity of the preparation as directed. Dilute with dilution buffer to
recommended diluent (for example water R); use produce a solution containing approximately 1 IU of von
immediately. Add sufficient prediluent to the reconstituted Willebrand factor. Prepare 2 series of at least 3 further
preparations to produce solutions containing 0.5-2.0 IU/mL. dilutions using dilution buffer.
The prediluent consists of an isotonic non-chelating buffer
Allow the solution of collagen to warm to room temperature.
containing, for example, 1-5 per cent of human or bovine
Dilute with collagen diluent to obtain a solution containing
albumin, and tris(hydroxymethyl)aminomethane or
30- 75 µg/mL of collagen, mix gently to produce a uniform
imidazole, appropriately buffered.
suspension of collagen fibrils. Pipette 100 µL into each well
The test is performed in accordance with the manufacturer's of the microtitre plate. Cover the plate with plastic film and
instructions with at least 2 dilution series with as many incubate at 3 7 °C overnight. Empty the wells of the collagen-
dilutions as are needed to obtain a total of at least 3 different coated plate by inverting and draining on a paper towel.
concentrations in the linear range of the assay. Add 250 µL of washing buffer. Empty the wells of the plate
Check the validity of the assay and calculate the potency of by inverting and draining on a paper towel. Repeat this
the test preparation using the usual statistical methods (for operation 3 times. Add 250 µL of blocking reagent to each
example, 5.3). well, cover the plate with plastic film and incubate at 37 °C
COLLAGEN-BINDING ASSAY for 1 h. Empty the wells of the plate by inverting and
Collagen-binding is determined by an enzyme-linked draining on a paper towel. Add 250 µL of washing buffer.
immunosorbent assay on collagen-coated microtitre plates. Empty the wells of the plate by inverting and draining on a
The method is based on the specific binding of von paper towel. Repeat this operation 3 times.
Willebrand factor to collagen fibrils and the subsequent Add 100 µL each of the test solutions or reference solutions
binding of polyclonal anti-von Willebrand factor antibody to the wells. Add 100 µL of dilution buffer to a series of
conjugated to an enzyme, which on addition of a wells to serve as negative control. Cover the plate with plastic
chromogenic substrate yields a product that can be film and incubate at 37 °C for 2 h. Empty the wells of the
quantitated spectrophotometrically. Under appropriate plate by inverting and draining on a paper towel.
conditions, there is a linear relationship between von Add 250 µL of washing buffer. Empty the wells of the plate
Willebrand factor collagen-binding and absorbance. by inverting and draining on a paper towel. Repeat this
operation 3 times.
REAGENTS
Collagen Use native equine or human fibrils of collagen Prepare a suitable dilution of the conjugate (for example, a
type I or III. For ease of handling, collagen solutions may be dilution factor of 1 to 4000) with PBS containing 5 g/L of
used. bovine albumin R and add 100 µL to each well. Cover the
plate with plastic film and incubate at 37 °C for 2 h. Empty
Collagen diluent Dissolve 50 g of glucose R in water R,
the wells of the plate by inverting and draining on a paper
adjust to pH 2.7-2.9 with 1 M hydrochloric acid and dilute to
towel. Add 250 µL of washing buffer. Empty the wells of the
1000 mL with water R.
plate by inverting and draining on a paper towel. Repeat this
Phosphate-buffered saline (PBS) Dissolve 8.0 g of operation 3 times.
sodium chloride R, 1.05 g of disodium hydrogen phosphate
Add I 00 µL of substrate solution to each of the wells and
dihydrate R, 0.2 g of sodium dihydrogen phosphate Rand 0.2 g
incubate at room temperature for 20 min in the dark.
of potassium chloride R in water R. Adjust to pH 7 .2 using
Add I 00 µL of 1 M hydrochloric acid to each of the wells.
1 M sodium hydroxide or 1 M hydrochloric acid and dilute to
1000 mL with water R. Measure the absorbance at 492 nm. Use the absorbance
values to estimate the potency of the preparation to be
Washing buffer PBS containing 1 g/L of polysorbate 20 R.
examined using the usual statistical methods (5.3).
Blocking reagent PBS containing 1 g/L of
The assay is invalid if the absorbances measured for the
polysorbate 20 R and 10 g/L of bovine albumin R.
negative controls are greater than 0.05.
Dilution buffer PBS containing 1 g/L of polysorbate 20 R
and 50 g/L of bovine albumin R. A9. Test for Prekallikrein Activator
Conjugate Rabbit anti-human von Willebrand factor (Ph. Bur. method 2. 6.15)
serum horseradish peroxidase conjugate. Use according to Prekallikrein activator (PKA) activates prekallikrein to
the manufacturer's instructions. kallikrein and may be assayed by its ability to cleave a
Substrate solution Immediately before use, dissolve a chromophore from a synthetic peptide substrate so that the
tablet of o-phenylenediamine dihydrochloride and a tablet of rate of cleavage can be measured spectrophotometrically and
urea hydrogen peroxide in 20 mL of water R or use a suitable the concentration of PKA calculated by comparison with a
volume of hydrogen peroxide. Protect from light. reference preparation calibrated in International Units.
The International Unit is the activity of a stated amount of
the International Standard, which consists of freeze-dried
prekallikrein activator. The equivalence in International Units
2023 Appendix XIV J V-A491

of the International Standard is stated by the World Health change in absorbance per minute for 2-10 min at the
Organization. wavelength specific for the substrate used. Prepare a blank
for each mixture of sample or standard using buffer B instead
REAGENTS
of prekallikrein substrate.
Prekallikrein activator in albumin ERP is calibrated in
International Units by comparison with the International Depending on the method used, M/min has to be corrected
Standard. by subtracting the value obtained for the corresponding blank
without the prekallikrein substrate. The results may be
Buffer A Dissolve 6.055 g of tris(hydroxymethyl)
calculated using a standard curve, a parallel-line or a slope
aminomethane R, 1.17 g of sodium chloride R, 50 mg of
ratio assay or any other suitable statistical method. Plot a
hexadimethrine bromide Rand 0.100 g of sodium azide R in
calibration curve using the values thus obtained for the
water R. Adjust to pH 8.0 with 2 M hydrochloric acid R and
reference preparation and the respective concentrations; use
dilute to 1000 mL with water R.
the curve to determine the PKA activity of the preparation to
Buffer B Dissolve 6.055 g of tris(hydroxymethyl) be examined. During method validation, the spiking
aminomethane Rand 8.77 g of sodium chloride R in water R. experiments must show that the sample matrix has no
Adjust to pH 8.0 with 2 M hydrochloric acid Rand dilute to influence on the results. High blank values may impact assay
1000 mL with water R. validity and should be appropriately investigated.
PREPARATION OF PREKALLIKREIN SUBSTRATE AlO. Assay of Human Plasmin Inhibitor
To avoid coagulation activation, blood or plasma used for the (Ph. Bur. method 2.7.25)
preparation of prekallikrein must come into contact only with
Human plasmin inhibitor, also called human :Xrantiplasmin,
plastics or silicone-treated glass surfaces.
is a plasma protein that inhibits the plasmin (a serine
Draw 9 volumes of human blood into 1 volume of protease) pathway of fibrinolysis by rapidly forming a
anticoagulant solution (ACD, CPD or a 38 g/L solution of complex with free plasmin. Furthermore, upon blood
sodium citrate R) to which 1 mg/mL of hexadimethrine coagulation, human plasmin inhibitor is cross-linked to fibrin
bromide R has been added. Centrifuge the mixture at 3600 g strands by factor XIII, and interferes with binding of the
for 5 min. Separate the plasma and centrifuge again at proenzyme plasminogen to fibrin.
6000 g for 20 min to sediment platelets. Separate the
The potency of human plasmin inhibitor is estimated by
platelet-poor plasma and dialyse against 10 volumes of buffer
comparing the ability of the preparation to be examined to
A for 20 h. Apply the dialysed plasma to a chromatography
inhibit the cleavage of a specific chromogenic substrate by
column containing agarose-DEAE for ion-exchange
plasmin with the same ability of a reference standard of
chromatography R that has been equilibrated in buffer A and
human plasmin inhibitor. Plasmin cleavage of the
is equal to twice the volume of the plasma. Elute from the
chromogenic substrate yields a chromophore that can be
column with buffer A at 20 mUcm 2/h. Collect the eluate in
quantified spectrophotometrically.
fractions and record the absorbance at 280 nm (2.2.25). Pool
the fractions containing the 1st protein peak so that the The individual reagents for the assay may be obtained
volume of the pool is about 1.2 times the volume of the separately or in commercial kits. Both end-point and kinetic
platelet-poor plasma. methods are available. Procedures and reagents may vary
between different kits and the manufacturer's instructions are
Test the substrate pool for absence of kallikrein activity by
followed. The essential features of the procedure are
mixing 1 part with 20 parts of the pre-warmed chromogenic
described in the following example of a microtitre-plate
substrate solution to be used in the assay and incubate at
kinetic method.
37 °C for 2 min. The substrate is suitable if the increase in
absorbance is less than 0.001 per minute. Add to the pooled REAGENTS
solution 7 g/L of sodium chloride R and filter through a Dilution buffer pH 7.5 According to the manufacturer's
membrane filter (nominal pore size 0.45 µm). Freeze the instructions, a suitable buffer is used. Adjust the pH if
filtrate in portions and store at -25 °C; the substrate may be necessary.
freeze-dried before storage. Plasmin A preparation of human plasmin that does not
Carry out all procedures from the beginning of the contain significant amounts of other proteases is preferably
chromatography to freezing in portions during a single used. Reconstitute and store according to the manufacturer's
working day. instructions.
METHOD Plasmin chromogenic substrate A suitable specific
The assay may be carried out using an automated enzyme chromogenic substrate for plasmin is used: H-o-
analyser or a suitable microtitre plate system allowing kinetic cyclohexylalanyl-norvalyl-lysyl-p-nitroaniline hydrochloride
measurements, with appropriate software for calculation of (H-o-CHA-Nva-Lys-pNA.HCl) or L-pyroglutamyl-L-
results. Standards, samples and prekallikrein substrate may phenylalanyl-L-lysyl-p-nitroaniline hydrochloride (Glp-Phe-
be diluted as necessary using buffer B. Lys-pNA.H Cl). Reconstitute in water R to give a suitable
concentration according to the manufacturer's instructions.
Incubate diluted standards or samples with prekallikrein
substrate for 10 min such that the volume of the undiluted METHOD
sample does not exceed 1/10 of the total volume of the Varying quantities of the preparation to be examined are
incubation mixture to avoid errors caused by variation in mixed with a given quantity of plasmin and the remaining
ionic strength and pH in the incubation mixture. Incubate plasmin activity is determined using a suitable chromogenic
the mixture or a part thereof with at least an equal volume of substrate.
a solution of a suitable synthetic chromogenic substrate, Reconstitute or thaw the preparation to be examined
known to be specific for kallikrein (for example, N-benzoyl-L- according to the manufacturer's instructions. Dilute with
prolyl-L-phenylalanyl-L-arginine 4-nitroanilide acetate R or o- dilution buffer pH 7.5 and prepare at least 2 independent
prolyl-L-phenylalanyl-L-arginine 4-nitroanilide series of 3 or 4 dilutions for both the preparation to be
dihydrochloride R), dissolved in buffer B. Record the rate of examined and the reference standard.
V-A492 Appendix XIV J 2023

Mix 0.020 mL of each dilution with 0.020 mL of dilution solution Rand incubate at 37 °C for 30 s. Add 40 µL of a
buffer pH 7.5 and warm to 37 °C. Add 0.040 mL of a 1 mmol/L solution of factor Xa chromogenic substrate and
plasmin solution (test concentration in the range of incubate at 37 °C for 3 min. Terminate the reaction by
0.2 nkat/mL to 1.6 nkat/mL) previously heated to 37 °C and lowering the pH by the addition of a suitable reagent, such as
leave at 37 °C for 1 min. Add 0.020 mL of the chromogenic a 20 per cent V/V solution of glacial, acetic acid R and
substrate solution, previously heated to 37 °C, to each measure the absorbance at 405 nm (2.2.25). Appropriate
mixture. Immediately start measurement of the change in reaction times are usually between 3 min and 15 min, but
absorbance at 405 nm (2.2.25) using a microtitre plate deviations are permissible if better linearity of the dose-
reader. Calculate the rate of change of absorbance (LlA/min). response relationship is thus obtained.
Alternatively, an end-point assay might be used by stopping Kinetic method Transfer 40 µL from each well to a
the reaction with acetic acid and measuring the absorbance at second series of wells, add 20 µL of bovine factor Xa
405 nm. solution Rand incubate at 37 °C for 30 s. Add 40 µL of a
In both cases the duration of the cleavage of the chromogenic 2 mmol/L solution of factor Xa chromogenic substrate,
substrate should be chosen to produce a linear increase in incubate at 37 °C and measure the rate of substrate cleavage
absorbance at 405 nm, before substrate depletion becomes by continuous measurement of the absorbance change at
significant. If the assay is performed in test tubes or cuvettes 405 nm (2.2.25), thus allowing the initial rate of substrate
using a spectrophotometric method, the volumes of reagent cleavage to be calculated. This rate must be linear with the
solutions are changed proportionally. concentration of residual factor Xa.
Substract the optical density of the blank (prepared with Check the validity of the assay and calculate the heparin
dilution buffer pH 7.5) from the optical density of the activity of the test preparation by the usual statistical
preparation to be examined. Check the validity of the assay methods for a slope-ratio assay (for example, 5.3).
and calculate the potency of the preparation to be examined
by the usual statistical methods (5.3). B2. Assay of Heparin
(Ph. Bur. method 2.7.5)
The anticoagulant activity of heparin is determined in vitro by
Anticoagulants its ability to accelerate the inhibition of thrombin, factor Ila
B1. Assay of Heparin in Coagulation Factors (anti-Ila assay), by antithrombin. The International Unit is
the activity contained in a stated amount of the International
(Ph. Bur. method 2. 7.12)
Heparin is assayed as a complex with antithrombin III (AT) Standard for unfractionated heparin. Heparin sodium BRP,
via its inhibition of coagulation factor Xa (anti-Xa activity). calibrated in International Units by comparison with the
International Standard using the 2 assays given below, is
An excess of AT is maintained in the reaction mixture to
ensure a constant concentration of the heparin-AT complex. used as the reference preparation.
Factor Xa is neutralised by the heparin-AT complex and the The assay of anti-factor Xa activity is carried out to
residual factor Xa hydrolyses a specific chromogenic peptide determine the ratio of anti-factor Xa activity to anti-factor Ila
substrate to release a chromophore. The quantity of activity.
chromophore is inversely proportional to the activity of the For anti-Ila and anti-Xa assays, carry out the assay by
heparin. determining the absorbance (end-point method) or the
Factor Xa chromogenic substrate Specific chromogenic change of absorbance per minute (kinetic method).
substrate for factor Xa such as: N-benzoyl-L-isoleucyl-L- ANTI-FACTOR HA ACTIVITY
glutamyl-glycyl-L-arginine-4-nitroanilide hydrochloride. Reference and test solutions
Reconstitute according to the manufacturer's instructions. Prepare 4 independent series of 4 dilutions each of the
Dilution buffer 6.05 g/L solution of tns(hydroxymethyl) substance to be examined and of heparin sodium BRP in tris
aminomethane R. Adjust to pH 8.4 if necessary using (hydroxymethyl)aminomethane-EDTA buffer solution pH 8.4 Rl;
hydrochloric acid R. a concentration range within 0.005 IU and 0.03 IU per
Test solution Dilute the preparation to be examined with millilitre is suitable. The dilutions chosen must give a linear
dilution buffer to obtain a solution expected to contain response when results are plotted as absorbance against log
0.1 IU of heparin per millilitre. concentration.
Reference solution Dilute the heparin reference Procedure
preparation with dilution buffer to obtain a solution Label 16 tubes for the dilutions of the substance to be
containing 0.1 IU of heparin per millilitre. examined and 16 tubes for the dilutions of the reference
The following working conditions apply to microtitre plates. preparation: T 1, T 2 , T 3 , T 4 for each of the 4 series of
If the assay is carried out in tubes, the volumes are adjusted dilutions of the substance to be examined and S 1, S2 , S3, S4
while maintaining the proportions in the mixture. for each of the 4 series of dilutions of the reference
preparation. To each of the 32 tubes add 100 µL of
Warm all solutions to 37 °C in a water-bath shortly before
antithrombin III solution R5 and 50 µL of the appropriate
the test.
dilution of the substance to be examined or the reference
Distribute in a series of wells, 20 µL of normal human preparation. After each addition, mix but do not allow
plasma and 20 µL of antithrombin Ill solution Rl. Add to the bubbles to form. Treating the tubes in 2 subsequent series in
wells a series of volumes (20 µL, 60 µL, 100 µL and 140 µL) the order S 1, S2 , S3, S4, T 1, T 2, T 3 , T 4, T 1, T 2 , T 3 , T 4, S1,
of the test solution or the reference solution and make up the S2 , S3 , S4 , allow to equilibrate at 37 °C (water-bath or
volume in each well to 200 µL using dilution buffer heating block) for at least 1 min and add to each tube 25 µL
(0.02-0.08 IU of heparin per millilitre in the final reaction of human thrombin solution R2. Incubate for exactly 1 min and
mixture). add 50 µL of a chromogenic substrate specific to factor Ila at
End-point method Transfer 40 µL from each well to a a concentration suitable for the assay (for example, D-
second series of wells, add 20 µL of bovine factor Xa phenylalanyl-L-pipecolyl-L-arginine-4-nitroanilide
2023 Appendix XIV J V-A493

dihydrochloride dissolved in water R to give a 1.25 mM Determine the blank amidolytic activity at the beginning and
solution). at the end of the procedure in a similar manner, using tris
For the kinetic method, transfer the mixtures to semi-micro (hydroxymethyl)aminomethane-BDTA buffer solution pH 8.4 Rl
cuvettes and measure the change in absorbance per minute instead of the reference and test solutions; the 2 blank values
(2.2.25) at 405 nm using a suitable reading device. do not differ significantly.
For the end-point method, stop the reaction after exactly Calculate the regression of the absorbance on log
4 min by adding 50 µL of a 20 per cent V/V solution of concentrations of the solutions of the substance to be
glacial acetic acid R. Assess whether exactly 4 min of examined and of heparin sodium ERP, and calculate the
incubation with the chromogenic substrate yields the optimal potency of the substance to be examined in International
absorbance reading and, if necessary, adjust the incubation Units per millilitre using the usual statistical methods for
time to give the best dose-response curve. Then, transfer the parallel-line assays (5.3).
mixtures to semi-micro cuvettes and measure the absorbance B3. Assay of Human Antithrombin III
(2.2.25) at 405 nm using a suitable reading device.
(Ph. Bur. method 2. 7.17)
Determine the blank amidolytic activity at the beginning and
The antithrombin III content of the preparation to be
at the end of the procedure in a similar manner, using tris
examined is determined by comparing its ability to inactivate
(hydroxymethyl)aminomethane-BDTA buffer solution pH 8.4 Rl
thrombin in the presence of an excess of heparin with the
instead of the reference and test solutions; the 2 blank values
same ability of a reference preparation of human
do not differ significantly.
antithrombin III concentrate calibrated in International
Calculate the regression of the absorbance on log Units. Varying quantities of the preparation to be examined
concentrations of the solutions of the substance to be are mixed with a given quantity of thrombin and the
examined and of heparin sodium ERP, and calculate the remaining thrombin activity is determined using a suitable
potency of the substance to be examined in International chromogenic substrate.
Units per millilitre using the usual statistical methods for
The International Unit is the activity of a stated amount of
parallel-line assays (5. 3).
the International Standard for human antithrombin III
ANTI-FACTOR XA ACTIVITY concentrate. The equivalence in International Units of the
Reference and test solutions International Standard is stated by the World Health
Prepare 4 independent series of 4 dilutions each of the Organization.
substance to be examined and of heparin sodium ERP in tris Method Prepare 2 independent series of 3 or 4 dilutions in
(hydroxymethyl)aminomethane-BDTA buffer solution pH 8.4 Rl; the range 1/75 to 1/200 from 1 IU/mL, for both the
a concentration range within 0.03 IU and 0.375 IU per preparation to be examined and the reference preparation,
millilitre is suitable. The dilutions chosen must give a linear using tris-EDTA BSA buffer solution pH 8.4 R containing
response when results are plotted as absorbance against log 15 IU of heparin per millilitre.
concentration. Warm 200 µL of each dilution at 37 cc for 1-2 min. Add to
Procedure each dilution 200 µL of a solution of bovine thrombin R
Label 16 tubes for the dilutions of the substance to be containing 2 IU/mL in m·s-BDTA BSA buffer solution
examined and 16 tubes for the dilutions of the reference pH 8.4 R. Mix and maintain at 37 °C for exactly 1 min.
preparation: T 1, T 2, T 3, T 4 for each of the 4 series of Add 500 µL of a suitable chromogenic substrate (for
dilutions of the substance to be examined and S1, S2, S3, S4 example, D-phenylalanyl-L-pipecolyl-L-arginine-4-nitroanilide,
for each of the 4 series of dilutions of the reference reconstituted in water R to give a solution containing
preparation. To each of the 32 tubes add 50 µL of 4 mmol/L and further diluted to a concentration suitable for
antithrombin III solution R6 and 50 µL of the appropriate the assay using tris-BDTA BSA buffer solution pH 8.4 R
dilution of the substance to be examined or the reference without albumin). Immediately start measurement of the
preparation. After each addition, mix but do not allow change in absorbance at 405 nm (2.2.25), continuing the
bubbles to form. Treating the tubes in 2 subsequent series in measurement for at least 30 s. Calculate the rate of change of
the order S 1, S2, S3, S4, T1, T2, T3, T4, T1, T2, T3, T4, S1, absorbance (AA/min). (Alternatively, an end-point assay may
S2, S3, S4 , allow to equilibrate at 37 °C (water-bath or be used by stopping the reaction with acetic acid and
heating block) for 1 min and add to each tube 100 µL of measuring the absorbance at 405 nm.)
bovine factor Xa solution R2. Incubate for exactly 2 min and The rate of change of absorbance (AA/min) is inversely
add 100 µL of a chromogenic substrate specific to factor Xa proportional to antithrombin III activity.
at a concentration suitable for the assay (for example, N-rx-
Check the validity of the assay and calculate the potency of
benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-4-nitroanilide
the test preparation by the usual statistical methods (5. 3).
dihydrochloride dissolved in water R to give a 1 mM
solution). B4. Assay of Human Protein C
For the kinetic method, transfer the mixtures to semi-micro (Ph. Bur. method 2. 7.30)
cuvettes and measure the change in absorbance per minute
1. CHROMOGENIC ASSAY
(2. 2. 25) at 405 nm using a suitable reading device.
Human protein C is a vitamin K-dependent plasma protein
For the end-point method, stop the reaction after exactly that, upon activation to activated protein C (APC), can
4 min by adding 50 µL of a 20 per cent V/V solution of inhibit blood coagulation through the proteolytic cleavage of
glacial acetic acid R. Assess whether exactly 4 min of factors Va and VIIIa. Human protein C activity is estimated
incubation with the chromogenic substrate yields the optimal using a two-step method: in the 1st step, human protein C in
absorbance reading and, if necessary, adjust the incubation the preparation is activated by a specific activator from snake
time to give the best dose-response curve. Then, transfer the venom; in the 2nd step, APC cleaves a specific chromogenic
mixtures to semi-micro cuvettes and measure the absorbance substrate to form a chromophore that can be quantified
(2.2.25) at 405 nm using a suitable reading device. spectrophotometrically.
V-A494 Appendix XIV J 2023

of human protein C in the preparation. Measure the optical

density at a wavelength of 405 nm. Subtract the optical
Step 1
density of the blank from the optical density of the test
human protein C activator sample. Check the validity of the assay and calculate the
human protein C - - - - - - - - - - ~ APC potency of the preparation to be examined using the usual
statistical methods (5.3).
2. CLOTTING ASSAY
Step 2
Human protein C activity is estimated following cleavage to
APC by a specific activator extracted from the venom of the
APC -pept1·ct e + c hromop hore
chromogenic substrate ----► viper Agkistrodon contortrix contortrix. The resulting APC
inactivates factors Va and VIiia, and thus prolongs the
APTT (Activated Partial Thromboplastin Time) of a system
The potency of human protein C is estimated by comparing in which all the coagulation factors are present, constant and
the ability of the preparation to be examined to cleave a in excess, except for human protein C, which is derived from
chromogenic substrate with the same ability of a reference the preparation being tested. Prolongation of the clotting
standard of human protein C calibrated in International time is proportional to the concentration of human protein C
Units. The International Unit is the activity of a stated in the preparation.
amount of the International Standard for human protein C. The potency of human protein C is estimated by comparing
The equivalence in International Units of the International the ability of the preparation to be examined to prolong the
Standard is stated by the World Health Organization. clotting time with the same ability of a reference standard of
Individual reagents may be obtained separately or in human protein C calibrated in International Units.
commercial kits. Both end-point and kinetic methods are The International Unit is the activity of a stated amount of
available. Procedures and reagents may vary between the International Standard for human protein C.
different kits and the manufacturer's instructions are The equivalence in International Units of the International
followed. The essential features of the procedure are Standard is stated by the World Health Organization.
described in the following example of a microtitre plate Individual reagents may be obtained separately or in
end-point method. commercial kits. Procedures and reagents may vary between
REAGENTS different kits and the manufacturer's instructions are
Dilution buffer pH 8.4 Dissolve 6.055 g of tris followed. The essential features of the procedure are
(hydroxymethy[)aminomethane Rand 16.84 g of caesium described in the following example.
chloride R in water R and adjust the pH if necessary. Dilute to REAGENTS
1000.0 mL with water R. Dilution buffer pH 7.4 Isotonic non-chelating buffer.
Human protein C activator Protein isolated from the Human protein C-deficient plasma Citrated human
venom of the viper Agkistrodon contortrix contortrix that plasma with no measurable human protein C content.
specifically activates human protein C. Reconstitute and store Reconstitute and store according to the manufacturer's
according to the manufacturer's instructions. Dilute to 0.25 instructions.
U/mL with water R before use in the assay. Human protein C activator Protein isolated from the
Activated protein C chromogenic substrate Specific venom of the viper Agkistrodon contortrix contortrix that
chromogenic substrate for APC, for example L-pyroglutamyl- specifically activates human protein C. Reconstitute and store
L-prolyl-L-arginyl-p-nitroaniline hydrochloride (pyroGlu-Pro- according to the manufacturer's instructions.
Arg-pNA.HCI). Reconstitute with water R to give a Coagulation activator A suitable APTT reagent
concentration of 4.5 mmol/L. Further dilute to 1.1 mmol/L containing phospholipids and a contact activator may be
with dilution buffer pH 8.4 before use in the assay. used. It may be combined with the human protein C
METHOD activator.
Reconstitute or thaw the preparation to be examined METHOD
according to the manufacturer's instructions. Dilute with Reconstitute or thaw the preparation to be examined
water R to produce at least 3 separate dilutions for each according to the manufacturer's instructions. Dilute with
preparation in the range 0.050-0.200 IU/mL, preferably in dilution buffer pH 7.4 to produce at least 3 separate dilutions
duplicate. for each preparation in the range 0.010-0.150 IU/mL,
Step 1 preferably in duplicate.
Mix 0.025 mL of each dilution with 0.050 mL of the human Mix 1 volume of each dilution with 1 volume of human
protein C activator, both previously heated to 37 °C, and protein C-deficient plasma and 1 volume of the human
leave at 37 °C for exactly 10 min. For each dilution, prepare protein C activator (combined with the APTT reagent where
a blank in the same manner, using water R instead of the appropriate), all previously heated to 37 °C. Add 1 volume of
human protein C activator. 0.025 M calcium chloride solution R previously heated to
Step 2 37 °C, and record the clotting time.
Add 0.150 mL of diluted chromogenic substrate, previously The clotting time is proportional to the concentration of
heated to 37 °C, to each mixture and leave at 37 °C for human protein C in each dilution. Check the validity of the
exactly 10 min. The incubation time must be adjusted, if assay and calculate the potency of the preparation to be
necessary, to ensure a linear development of chromophore examined using the usual statistical methods (5.3).
with time. Terminate the reaction by adding 0.050 mL of a
50 per cent V/V solution of glacial acetic acid R.
Cleavage of the chromogenic substrate by APC causes release
of the chromophore pNA, in proportion to the concentration
2023 Appendix XIV J V-A495

B5. Assay of Human Protein S Alternative procedures may use a coagulation activator
(Ph. Eur. method 2. 7.31) without calcium chloride, and require a precisely timed
Human protein S is a vitamin K-dependent plasma protein activation period before the addition of calcium chloride and
that acts as a cofactor for the anticoagulant functions of the measurement of clotting time.
activated protein C (APC). Human protein S activity may be The clotting time is proportional to the concentration of
determined by the clotting assay described below, which is human protein S in each dilution. Check the validity of the
sensitive to the ability of human protein S to accelerate the assay and calculate the potency of the preparation to be
inactivation of factor Va by APC. In practice, the assay examined using the usual statistical methods (5.3).
involves the addition of human protein S to a reagent
mixture containing APC, factor Va and human protein S-
deficient plasma. Prolongation of the clotting time is lmmunoglobulins
proportional to the concentration of human protein S in the Cl. Test for Fe Function oflmmunoglobulin
preparation. Methods in which APC is added directly as a (Ph. Eur. method 2.7.9)
reagent are preferred to those in which APC is generated The test for Fe function of immunoglobulin is carried out using
during the assay by the addition of a specific human method A or B. Method B is an adaptation of the procedure of
protein C activator purified from snake venom. Activation of method A for the use of micro titre plates for the measurement of
coagulation is initiated by the addition of an activating complement-mediated haemolysis. Differences in the test procedures
reagent such as thromboplastin or activated factor X, between methods A and B are addressed in the test.
together with phospholipids and calcium chloride. During the
assay, factor Va is generated from factor V in the human REAGENTS
protein S-deficient plasma following the activation of Stabilised human blood Collect group O human blood
coagulation. The assay procedure must ensure that human into ACD anticoagulant solution. Store the stabilised blood
protein S is the only limiting factor. at 4 °C for not more than 3 weeks.
The potency of human protein S is estimated by comparing Phosphate-buffered saline pH 7.2 Dissolve 1.022 g of
the ability of the preparation to be examined to prolong the anhydrous disodium hydrogen phosphate R, 0.336 g of anhydrous
clotting time with the same ability of a reference standard of sodium dihydrogen phosphate Rand 8.766 g of sodium
human protein S calibrated in International Units. chloride R in 800 mL of water R and dilute to 1000 mL with
The International Unit is the activity of a stated amount of the same solvent.
the International Standard for human protein S. Magnesium and calcium stock solution Dissolve
The equivalence in International Units of the International 1.103 g of calcium chloride Rand 5.083 g of magnesium
Standard is stated by the World Health Organization. chloride R in water R and dilute to 25 mL with the same
Individual reagents may be obtained separately or in solvent.
commercial kits. Procedures and reagents may vary between Barbital buffer stock solution Dissolve 207.5 g of sodium
different kits and the manufacturer's instructions are chloride Rand 25.48 g of barbital sodium R in 4000 mL of
followed. The essential features of the procedure are water Rand adjust to pH 7.3 using 1 M hydrochloric acid.
described in the following example. Add 12.5 mL of magnesium and calcium stock solution and
dilute to 5000 mL with water R. Store at 4 °C in transparent
REAGENTS
containers.
Dilution buffer pH 7.4 Isotonic non-chelating buffer
prepared as follows: dissolve 6.08 g of tris(hydroxymethyl) Albumin barbital buffer solution Dissolve 0.150 g of
aminomethane R and 8.77 g of sodium chloride R in water R bovine albumin R in 20 mL of barbital buffer stock solution
and adjust the pH if necessary; add 10 g of bovine albumin R and dilute to 100 mL with water R. Prepare immediately
or human albumin Rand dilute to 1000.0 mL with water R. before use.
Human protein S-deficient plasma Citrated human Tannic acid solution Dissolve 10 mg of tannic add R in
plasma with no measurable human protein S content and, 100 mL of phosphate-buffered saline pH 7.2. Prepare
preferably, also free of C4b-binding protein. immediately before use.
Coagulation activator This reagent is used to initiate Guinea-pig complement Prepare a pool of serum from
coagulation in the human protein S-deficient plasma, and the blood of not fewer than 10 guinea-pigs. Separate the
thereby also provides a source of activated factor V. serum from the clotted blood by centrifugation at about
The activator may consist of tissue factor, activated factor X, 4 °C. Store the serum in small amounts below -70 °C.
or an agent capable of directly activating factor X that may Immediately before starting complement-initiated haemolysis,
be purified from the venom of Russell's viper (Vipera russelh). dilute to 125-200 CH50 per millilitre with albumin barbital
The reagent also contains APC, phospholipids and calcium buffer solution and store in an ice-bath during the test.
chloride R, or, alternatively, calcium chloride may be added Rubella antigen Suitable rubella antigen for
separately after a timed activation period. haemagglutination-inhibition titre (HIT).
Titre > 256 HA units.
METHOD
Reconstitute or thaw the preparation to be examined Preparation of tanned human red blood cells
according to the manufacturer's instructions. Dilute with Separate human red blood cells by centrifuging an
dilution buffer pH 7.4 to produce at least 3 separate dilutions appropriate volume of stabilised human blood, wash the cells
for each preparation in the range 0.020-0.100 IU/mL, at least 3 times with phosphate-buffered saline pH 7.2 and
preferably in duplicate. suspend at 2 per cent V/V in phosphate-buffered saline
pH 7.2. Add 0.2 mL oftannic acid solution to 14.8 mL of
Mix 1 volume of each dilution with 1 volume of human
phosphate-buffered saline pH 7 .2. Mix 1 volume of the
protein S-deficient plasma, both previously heated to 37 °C.
freshly prepared dilution with 1 volume of the human red
Add 2 volumes of the coagulation activator, previously heated
blood cell suspension and incubate at 37 °C for 10 min.
to 37 °C, and record the clotting time.
Collect the cells by centrifugation (800 g for 10 min), discard
V-A496 Appendix XIV J 2023

the supernatant and wash the cells once with phosphate- the thermostatted cuvette holder of a spectrophotometer.
buffered saline pH 7.2. Resuspend the tanned cells at After 2 min, add 200 µL of diluted guinea-pig complement
1 per cent V/V in phosphate-buffered saline pH 7 .2. (125-200 CH 50/mL), mix thoroughly by pipetting twice and
Antigen coating of tanned human red blood cells start immediately after the second pipetting the time-
Take a suitable volume (V,) of tanned cells, add 0.2 mL of dependent recording of absorbance at 541 nm, using
rubella antigen per 1.0 mL of tanned cells and incubate at albumin barbital buffer solution as the compensation liquid.
37 °C for 30 min. Collect the cells by centrifugation (800 g Stop the measurement if absorbance as a function of time
for 10 min) and discard the supernatant. Add a volume of has clearly passed the inflexion point.
albumin barbital buffer solution equivalent to the discarded To measure haemolysis where method Bis performed, add
supernatant, resuspend and collect the cells as described and 900 µL of albumin barbital buffer solution warmed to 37 °C
repeat the washing procedure. Resuspend with albumin to each test-tube and resuspend the cells carefully by
barbital buffer solution using a volume equivalent to 3/4 repeated pipetting (not fewer than 5 times). The microtitre
of Vs, thereby obtaining the initial volume (JI;). Mix 900 µL plate must be prewarmed to 37 °C before starting the test.
of albumin barbital buffer solution with 100 µL of Vi, which Transfer 240 µL of each solution into 4 microtitre plate wells
is thereby reduced to the residual volume (Vr), and then incubate the microplate at 37 °C for 6 min, stirring
determine the initial absorbance at 541 nm (A). Dilute Vr by gently every 10 s. To each microtitre plate well add 60 µL of
a factor equal to A using albumin barbital buffer solution, diluted guinea-pig complement (150 CH50 /mL). Mix for 10 s
thereby obtaining the final adjusted volume Vi = v;. x A of and immediately start recording the absorbance at 541 nm at
sensitised human red blood cells and adjusting A to 37 °C, measuring every 20 s. Stop the measurement if the
1.0 ± 0.1 for a tenfold dilution. absorbance as a function of time has clearly passed the
Antibody binding of antigen-coated tanned human red inflexion point.
blood cells Evaluation
Prepare the following solutions in succession and in For each cuvette/test-tube/well, determine the slope (S) of
duplicate, using for each solution a separate half-micro the haemolysis curve at the approximate inflexion point by
cuvette (for example, disposable type) or test-tube. segmenting the steepest section in suitable time intervals (for
(1) Test solutions. If necessary, adjust the immunoglobulin to example, ~t = 1 min), and calculate S between adjacent
be examined to pH 7. intersection points, expressed as M per minute. The largest
value for S serves as Sexp· In addition, determine the
Where method A is performed, dilute volumes of the
absorbance at the start of measurement (As) by extrapolating
preparation to be examined with albumin barbital buffer to
the curve, which is almost linear and parallel to the time axis
obtain 30 mg and 40 mg of immunoglobulin and adjust the
within the first few minutes. Correct Scxp using the
volume to 900 µL with albumin barbital buffer.
expression:
Where method B is performed, dilute volumes of the
preparation to be examined with albumin barbital buffer to
obtain 15 mg and 30 mg of immunoglobulin and adjust the
volume to 1200 µL with albumin barbital buffer.
(2) Reference solutions. Prepare as for the test solutions using Calculate the arithmetic mean of the values of S' for each
human immunoglobulin for Fe function ERP. preparation (test and reference solution). Calculate the index
(3) Complement control. Albumin barbital buffer solution. of Fe function UFc) from the expression:
Where method A is performed, add to each cuvette/test-tube
100 µL of sensitised human red blood cells and mix well. I = 100 x (s7 - S'c)
Allow to stand for 15 min, add 1000 µL of albumin barbital Fe S's _ S' C
buffer solution, collect the cells by centrifugation (1000 g for
arithmetic mean of the corrected slope for the preparation to be
10 min) of the cuvette/test-tube and remove 1900 µL of the
examined;
supernatant. Replace the 1900 µL with albumin barbital
buffer solution and repeat the whole of the washing s; arithmetic mean of the corrected slope for the reference
procedure, finally leaving a volume of 200 µL. Test samples preparation;
may be stored in sealed cuvettes/test-tubes at 4 °C for not
arithmetic mean of the corrected slope for the complement
longer than 24 h. control.
Where method B is performed, add to each test-tube 300 µL
of sensitised human red blood cells and mix well (the final
immunoglobulin concentration is in the range of Calculate the index of Fe function for the preparation to be
10-20 mg/mL). Allow to stand for 15 min, add 1500 µL of examined: the value is not less than that stated in the leaflet
albumin barbital buffer solution and stir gently until accompanying the reference preparation.
homogeneous. Collect the cells by centrifugation (1000 g for
C2. Test for Anticomplementary Activity of
10 min) of the test-tube, remove the supernatant and add
lmmunoglobulin
approximately 3 mL of albumin barbital buffer solution.
(Ph. Bur. method 2. 6.1 7)
Repeat this operation up to 4 times in total, leaving a final
volume of 300 µL. Test samples may be stored in sealed test- For the measurement of anticomplementary activity (ACA)
tubes at 4 °C for not longer than 24 h. of immunoglobulin, a defined amount of test material
(10 mg of immunoglobulin) is incubated with a defined
Complement-initiated haemolysis amount of guinea-pig complement (20 CH 50 ) and the
To measure haemolysis where method A is performed, add remaining complement is titrated; the anticomplementary
600 µL of albumin barbital buffer solution warmed to 37 °C activity is expressed as the percentage consumption of
to the test sample, resuspend the cells carefully by repeated complement relative to the complement control considered as
pipetting (not fewer than 5 times) and place the cuvette in 100 per cent.
 2023 Appendix XIV J V-A497

The haemolytic unit of complement activity (CH 50 ) is the Table 2.6.17.-1

amount of complement that, in the given reaction conditions, Required Prepared using
will produce the lysis of 2.5 x 10 8 out of a total of 5 x 108 dilution of
optimally sensitised red blood cells. haemolysin

Magnesium and calcium stock solution Dissolve Gelatin barbital

Haemolysin
buffer solution
1.103 g of calcium chloride R and 5 .083 g of magnesium
chloride R in water R and dilute to 25 mL with the same Volume Dilution Volume
(mL) (1/ ... ) (mL)
solvent.
7.5 0.65 undiluted 0.1
Barbital buffer stock solution Dissolve 207. 5 g of sodium
10 0.90 undiluted 0.1
chloride R and 25.48 g of barbital sodium R in 4000 mL of
75 1.80 7.5 0.2
water Rand adjust to pH 7.3 using 1 M hydrochloric acid.
100 1.80 10 0.2
Add 12. 5 mL of magnesium and calcium stock solution and
150 1.00 75 1.0
dilute to 5000 mL with water R. Filter through a membrane
200 1.00 100 1.0
filter (nominal pore size 0.22 µm). Store at 4 °C in glass
300 1.00 150 1.0
containers.
400 1.00 200 1.0
Gelatin solution Dissolve 12.5 g of gelatin R in about 600 1.00 300 1.0
800 mL of water R and heat to boiling in a water-bath. Cool 800 1.00 400 1.0
to 20 °C and dilute to 10 L with water R. Filter through a 1200 1.00 600 1.0
membrane filter (nominal pore size 0.22 µm). Store at 4 °C. 1600 1.00 800 1.0
Use clear solutions only. 2400 1.00 1200 1.0
Citrate solution Dissolve 8.0 g of sodium citrate R, 4.2 g of 3200* 1.00 1600 1.0
sodium chloride Rand 20.5 g of glucose R in 750 mL of 4800* 1.00 2400 1.0
water R. Adjust to pH 6.1 using a 100 g/L solution of citric
* discard 1.0 mL of the mixture.
acid monohydrate R and dilute to 1000 mL with water R.
Gelatin barbital buffer solution Add 4 volumes of Add 1.0 mL of 5 per cent sheep red blood cell suspension to
gelatin solution to 1 volume of barbital buffer stock solution each tube of the haemolysin dilution series, starting at the
and mix. Adjust to pH 7 .3, if necessary, using 1 M sodium 1/75 dilution, and mix. Incubate at 37 °C for 30 min.
hydroxide or 1 M hydrochloric acid. Maintain at 4 °C. Prepare
Transfer 0.2 mL of each of these incubated mixtures to new
fresh solutions daily. tubes and add 1.10 mL of gelatin barbital buffer solution and
Stabilised sheep blood Collect 1 volume of sheep blood 0.2 mL of diluted guinea-pig complement (for
into 1 volume of citrate solution and mix. Store at 4 °C for example, 1/150). Perform this in duplicate.
not less than 7 days and not more than 28 days. (Stabilised As the unhaemolysed cell control, prepare 3 tubes with
sheep blood and sheep red blood cells are available from a 1.4 mL of gelatin barbital buffer solution and 0.1 mL of
number of commercial sources.) 5 per cent sheep red blood cell suspension.
Haemolysin Antiserum against sheep red blood cells As the fully haemolysed control, prepare 3 tubes with 1.4 mL
prepared in rabbits. (Such antisera are available from a of water Rand 0.1 mL of 5 per cent sheep red cell
number of commercial sources.) suspension.
Guinea-pig complement Prepare a pool of serum from Incubate all tubes at 37 °C for 60 min and centrifuge at
the blood of not fewer than 10 guinea-pigs. Separate the 1000 g for 5 min. Measure the absorbance (2.2.25) of the
serum from the cloned blood by centrifugation at about supernatants at 541 nm and calculate the percentage degree
4 °C. Store the serum in small amounts below -70 °C. of haemolysis in each tube using the following expression:
METHOD
Preparation of standardised 5 per cent sheep red blood
cell suspension
Separate sheep red blood cells by centrifuging an appropriate
Aa absorbance of tubes with haemolysin dilution;
volume of stabilised sheep blood and wash the cells at least A, mean absorbance of the 3 tubes with full haemolysis;
3 times with gelatin barbital buffer solution and prepare a A1 mean absorbance of the 3 tubes with no haemolysis.
5 per cent V/V suspension in the same solution. Measure the
cell density of the suspension as follows: add 0.2 mL to Plot the percentage degree of haemolysis as the ordinate
2.8 mL of water R and centrifuge the lysed solution for 5 min against the corresponding reciprocal value of the haemolysin
at 1000 g; the cell density is suitable if the absorbance dilution as the abscissa on linear graph paper. Determine the
(2.2.25) of the supernatant at 541 nm is 0.62 ± 0.01. optimal dilution of the haemolysin from the graph by
Correct the cell density by adding gelatin barbital buffer inspection. Select a dilution such that further increase in the
solution according to the following equation: amount of haemolysin does not cause appreciable change in
the degree of haemolysis. This dilution is defined as
Vi xA 1 minimal haemolytic unit (1 MHU) in 1.0 mL. The optimal
Vr = 0.62 haemolytic haemolysin dilution for preparation of sensitised
sheep red blood cells contains 2 MHU/mL.
v; final adjusted volwne;
Vi the initial volume; The haemolysin titration is not valid unless the maximum
A absorbance of the original suspension at 541 nm. degree of haemolysis is 50 per cent to 70 per cent. If the
maximum degree of haemolysis is not in this range, repeat
The adjusted suspension contains about 1 x 10 9 cells/mL. the titration with more or less diluted complement solution.
Haemolysin titration
Prepare haemolysin dilutions as shown in Table 2.6.17.-1.
V-A498 Appendix XIV J 2023

Preparation of optimised sensitised sheep red blood Test for anticomplementary activity
cells (haemolytic system) Prepare a complement dilution having I 00 CH 5 ofmL by
Prepare an appropriate volume of diluted haemolysin diluting titrated guinea-pig complement with gelatin barbital
containing 2 MHU/mL and an equal volume of standardised buffer solution. Depending on the immunoglobulin to be
5 per cent sheep red blood cell suspension. Add the examined and based on validation data, a pH adjustment to
haemolysin dilution to the standardised cell suspension and 7 may be necessary. Prepare incubation mixtures as follows
mix. Incubate at 37 °C for 15 min, store at 2 °C to 8 °C and for an immunoglobulin containing 50 mglmL:
use within 6 h.
Table 2.6.17.-3
Titration of complement
Prepare an appropriate dilution of complement (for lmmunoglobulin Complement control
to be examined (in duplicate)
example 1/250) with gelatin barbital buffer solution and
lmmunoglobulin (50 mg/mL) 0.2 mL
perform the titration in duplicate as shown in
Table 2.6.17.-2. Gelatin barbital buffer 0.6 mL 0.8 mL
Complement 0.2 mL 0.2 mL
Table 2.6.17.-2
Tube nwnber Volwne of diluted Volwne of gelatin Carry out the test on the immunoglobulin to be examined
complement barbital buffer solution and prepare ACA negative and positive controls using human
(for example 1/250)(mL) (mL) immunoglobulin for anticomplementary activity ERP, as
I 0.1 1.2 indicated in the leaflet accompanying the reference
2 02 1.1 preparation. Higher or lower volumes of sample and of
3 0.3 1.0 gelatin barbital buffer solution are added if the
4 0.4 0.9 immunoglobulin concentration varies from 50 mglmL; for
5 Q5 0.8 example, 0.47 mL of gelatin barbital buffer solution is added
6 Q6 0.7 to 0.33 mL of immunoglobulin containing 30 mglmL to give
7 0.7 0.6 0.8 mL. Close the tubes and incubate at 37 °C for 60 min.
8 0.8 0.5 Add 0.2 mL of each incubation mixture to 9.8 mL of gelatin
9 Q9 0.4 barbital buffer solution to dilute the complement. Perform
10 1.0 0.3 complement titrations on each tube as described above to
11 1.1 0.2 determine the remaining complement activity
12 1.2 0.1 (Table 2.6.17.-2). Calculate the anticomplementary activity
3 tubes as cell control 1.3 of the preparation to be examined relative to the complement
at O per cent control considered as 100 per cent, using the following
haemolysis expression:
3 tubes at 1.3 mL of water
100 per cent a-b
- - xlOO
haemolysis a
a mean complement activity (CH 5 ofmL) of complement control;
Add 0.2 mL of sensitised sheep red blood cells to each tube, b complement activity (CH,./mL) of tested sample.
mix well and incubate at 37 °C for 60 min. Cool the tubes in
an ice-bath and centrifuge at 1000 g for 5 min. Measure the The test is not valid unless:
absorbance of the supernatant at 541 nm and calculate the - the anticomplementary activities found for ACA negative
degree of haemolysis (Y) using the following expression: control and ACA positive control are within the limits
stated in the leaflet accompanying the reference
preparation;
- the mean complement activity of complement control (a)
is in the range 80 CH50/mL to 120 CH5 ofmL.
A, absorbance of tubes 1 to 12;
Ab mean absorbance of tubes with 100 per cent haemolysis; C3. Assay of Human Anti-D Immunoglobulin
A1 mean absorbance of cell controls with O per cent haemolysis.
(Ph. Bur. method 2. 7.13)
Plot Y/(1-Y) as the abscissa against the amount of diluted METHOD A
complement in millilitres as the ordinate on log-log graph The potency of human anti-D immunoglobulin is determined
paper. Fit the best line to the points and determine the by comparing the quantity necessary to produce agglutination
ordinate for the 50 per cent haemolytic complement dose of D-positive red blood cells with the quantity of a reference
where Y/(1-Y) = 1.0. Calculate the activity in preparation, calibrated in International Units, required to
haemolytic units (CH 5 ofmL) using the following expression: produce the same effect.
The International Unit is the activity contained in a stated
amount of the International Reference Preparation.
The equivalence in International Units of the International
Reference Preparation is stated by the World Health
Cd reciprocal value of the complement dilution;
Organization.
Ca volume of diluted complement resulting in 50 per cent
haemolysis, in millilitres; Human anti-D immunoglobulin ERP is calibrated in
5 scaling factor to take account of the number of red blood cells. International Units by comparison with the International
Standard and intended for use in the assay of human anti-D
The test is not valid unless the plot is a straight line between immunoglobulin.
15 per cent and 85 per cent haemolysis and the slope is
Use pooled D-positive red blood cells, collected not more
0.15 to 0.40, and preferably 0.18 to 0.30.
than 7 days earlier and suitably stored, obtained from not
2023 Appendix XIV J V-A499

fewer than 4 group O R 1R 1 donors. To a suitable volume of MATERIALS

the cells, previously washed 3 times with a 9 g/L solution of Reagents not specified are of analytical grade.
sodium chloride R, add an equal volume of bromelains PBS (Phosphate-buffered saline) Dissolve 8.0 g of
solution R, allow to stand at 37 °C for 10 min, centrifuge, sodium chloride R, 0.76 g of anhydrous disodium hydrogen
remove the supernatant and wash 3 times with a 9 g/L phosphate R, 0.2 g of potassium chloride R, 0.2 g of potassium
solution of sodium chloride R. Suspend 20 volumes of the red dihydrogen phosphate Rand 0.2 g of sodium azide R in water R
blood cells in a mixture of 15 volumes of inert serum, and dilute to 1000 mL with the same solvent.
20 volumes of a 300 g/L solution of bovine albumin R and
TBS (Tris-buffered saline) Dissolve 8.0 g of sodium
45 volumes of a 9 g/L solution of sodium chloride R. Stand the
chloride Rand 0.6 g of tris(hydroxymethyl)aminomethane R in
resulting suspension in iced water, stirring continuously.
water R. Adjust to pH 7.2 with JM hydrochloric acid and
Using a calibrated automated dilutor, prepare suitable dilute to 1000 mL with water R.
dilutions of the preparation to be examined and of the
Papain solution Prepare a solution by stirring 1 g of
reference preparation using as diluent a solution containing papain Rat 37 °C for 30 min in 10 mL of 0.067 M phosphate
5 g/L of bovine albumin R and 9 g/L of sodium chloride R. buffer solution pH 5.4 R, centrifuge at 10 000 g for 5 min and
Use a suitable apparatus for automatic continuous analysis. filter through a membrane filter (nominal pore size 0.22 µm).
The following protocol is usually suitable: maintain the To activate, combine 1 mL of the filtrate with 1 mL of a
temperature in the manifold, except for the incubation coils, 48.44 g/L solution of L-cysteine Rand 1 mL of a 3.72 g/L
at 15.0 °C. Pump into the manifold of the apparatus the red solution of sodium edetate R and dilute to 10 mL with
blood cell suspension at a rate of 0.1 mIJmin and a 3 g/L 0.067 M phosphate buffer solution pH 5.4 R. Freeze in aliquots
solution of methylcellulose 450 R at a rate of 0.05 mIJmin. at -20 °C or below.
Introduce the dilutions of the preparation to be examined
Red blood cells Use pooled D-positive red blood cells
and the reference preparation at a rate of 0.1 mIJmin for
obtained from not fewer than 3 group O R 2 R 2 donors. Wash
2 min, followed by the diluent solution at a rate of
the cells 4 times with PBS. Centrifuge the cells at 1800 g for
0.1 mIJmin for 4 min before the next dilution is introduced. 5 min, mix a suitable volume of prewarmed packed cells with
Introduce air at a rate of 0.6 mIJmin. Incubate at 37 °C for a suitable volume of prewarmed papain solution (2 volumes
18 min and then disperse the rouleaux by introducing at a to 1 volume has been found suitable) and incubate at 37 °C
rate of 1.6 mIJmin a 9 g/L solution of sodium chloride R for 10 min. Wash the cells 4 times with PBS. Store at 4 °C
containing a suitable wetting agent (for example, in an appropriate stabiliser for up to 1 week.
polysorbate 20 R at a final concentration of 0.2 g/L) to prevent
Biotinylated Brad-5 Use according to instructions.
disruption of the bubble pattern. Allow the agglutinates to
settle and decant twice, first at 0.4 mIJmin and then at Alkaline phosphatase-conjugated avidinlstreptavidin
0.6 mIJmin. Lyse the unagglutinated red blood cells with a reagent Preferably modified to combine high specific
activity with low non-specific binding. Use according to
solution containing 5 g/L of octoxinol JO R, 0.2 g/L of
potassium ferricyanide R, 1 g/L of sodium hydrogen carbonate R instructions.
and 0.05 g/L of potassium cyanide Rat a rate of 2.5 mIJmin. Substrate solution Use para-nitrophenyl phosphate
A 10-minute delay coil is introduced to allow for conversion according to instructions.
of the haemoglobin. Continuously record the absorbance Cell fixation buffer Dissolve 18.02 g of glucose R, 4.09 g
(2.2.25) of the haemolysate at a wavelength between 540 nm of sodium chloride R, 1.24 g of boric acid R, 10.29 g of sodium
and 550 nm. Determine the range of antibody concentrations citrate Rand 0.74 g of sodium edetate R in water R. Adjust to
over which there is a linear relationship between the pH 7.2-7.3 using 1 M sodium hydroxide or 1 M hydrochloric
concentration and the resultant change in absorbance (M). acid, and dilute to 1000 mL with water R. Use directly from
From the results, prepare a standard curve and use the linear storage at 4 °C.
portion of the curve to determine the activity of the Glutaraldehyde solution Immediately before use, add
preparation to be examined. 750 µL of a 250 g/L solution of glutaraldehyde R to 50 mL of
Calculate the potency of the preparation to be examined cold PBS.
using the usual statistical methods (5.3). Microtitre plates Plates to be coated with red blood cells
METHODB are flat-bottomed polystyrene plates with surface properties
The potency of human anti-D immunoglobulin is determined optimised for enzyme immunoassay and high protein-binding
by competitive enzyme-linked immunoassay on erythrocyte- capacity. Plates used to prepare immunoglobulin dilutions
coated microtitre plates. The method is based on the are U- or V-bottomed polystyrene or poly(vinyl chloride)
competitive binding between a polyclonal anti-D plates.
immunoglobulin preparation and a biotinylated monoclonal METHOD
anti-D antibody directed against a D-antigen-specific epitope. Prepare a 0.1 per cent V/V suspension of papain-treated red
The activity of the preparation to be examined is compared blood cells in cold cell-fixation buffer. Pipette 50 µL into
with a reference preparation calibrated in International Units. each well of the flat-bottomed microtitre plate.
The International Unit is the activity of a stated amount of Centrifuge the plate at 350 g for 3 min, preferably at 4 °C.
International Reference Preparation. The equivalence in Without removing the supernatant, gently add 100 µL of
International Units of the International Reference glutaraldehyde solution to each well and leave for 10 min.
Preparation is stated by the World Health Organization. Drain the wells by quickly inverting the plate and wash
Human anti-D immunoglobulin BRP is calibrated in 3 times with 250-300 µL of PBS. This may be done
International Units by comparison with the International manually or using a suitable automated plate washer. Either
Standard and intended for use in the assay of human anti-D carry out the assay as described below, or store the plate at
immunoglobulin. 4 °C after draining off the PBS and adding 100 µL of cell-
fixation buffer per well and sealing with plastic film. Plates
can be stored at 4 °C for up to 1 month.
V-A500 Appendix XIV J 2023

Test solutions For freeze-dried preparations, reconstitute PBS-BSA solution PBS containing 10.0 g/L of bovine
as stated on the label. Prepare 4 independent replicates of albumin R.
5 serial 2-fold dilutions starting with 30 IU/mL in PBS Red blood cells Use D-positive red blood cells obtained
containing 10 g/L of bovine albumin R. If necessary, adjust from a group O R 1R 1 donor within 2 weeks of collection.
the starting dilution to obtain responses falling in the linear Store if necessary in an appropriate stabiliser at 4 °C. Wash
portion of the dose-response curve. the cells at least twice with PBS-BSA solution and prepare a
Reference solutions Reconstitute the reference suspension containing 1 x 104 cells per microlitre but not
preparation according to instructions. Prepare 4 independent more than 5 x 104 cells per microlitre in PBS-BSA solution.
replicates of 5 serial 2-fold dilutions starting with 30 IU/mL Use D-negative red blood cells obtained from a group Orr
in PBS containing 10 g/L of bovine albumin R. donor and prepared similarly.
Using U- or V-bottomed microtitre plates, add 35 µL of each Secondary antibody Use a suitable fluorescent dye-
of the dilutions of the test solution or reference solution to conjugated anti-lgG antibody fragment specific for human
each of a series of wells. To each well add 35 µL of IgG or parts of it. Store and use according to the
biotinylated Brad-5 at 250 ng/mL. manufacturer's instructions.
Empty the wells of the red cell-coated plate by inverting and Microtitres plates Use flat-bottomed plates without
draining on a paper towel. Add 250 µL of PBS containing surface treatment for enzyme immunoassays.
20 g/L of bovine albumin R and leave at room temperature for
METHOD
30 min.
Test solutions For freeze-dried preparations, reconstitute
Empty the wells of the red cell-coated plate by inverting and as stated on the label. Prepare at least 3 independent
draining on a paper towel and transfer 50 µL from each of replicates of at least 3 serial 1.5- or 2-fold dilutions starting
the dilutions of the test solution or reference solution with a concentration in the range of 1.2-0.15 IU/mL using
containing biotinylated Brad-5 into the wells. Use 50 µL of PBS/BSA solution as diluent. If necessary, adjust the starting
PBS containing 10 g/L of bovine albumin R as negative dilution to obtain responses falling in the linear portion of the
control. Seal the plate with plastic film and incubate at room dose-response curve.
temperature for 1 h.
Reference solutions Reconstitute the reference
Remove the liquid from the wells of the red cell-coated plate preparation according to instructions. Prepare at least
and wash 3 times with 250-300 µL ofTBS. 3 independent replicates of at least 3 serial 1.5- or 2-fold
Dilute the alkaline phosphatase-conjugated avidin/streptavidin dilutions starting with a concentration in the range of
reagent in TBS containing 10 g/L of bovine albumin R and 1.2-0.15 IU/mL using PBS-BSA solution as diluent.
add 50 µL to each well. Incubate for 30 min at room If necessary, adjust the starting dilution to obtain responses
temperature. falling in the linear portion of the dose-response curve.
Remove the liquid from the wells of the red cell-coated plate Distribute 50 µL of the D-positive red blood cells into each
and wash 3 times with 250-300 µL of TBS. well of a microtitre plate. Add 50 µL of each of the dilutions
Add 100 µL of substrate solution to each of the wells and of the test solution or reference solution to each of a series of
incubate at room temperature for 10 min in the dark. wells. Use 50 µL of PBS-BSA solution as negative control.
To stop the reaction, add 50 µL of 3M sodium hydroxide to Distribute 50 µL of the D-negative red blood cells into
each of the wells. 4 wells of the same microtitre plate and add 50 µL of the
Measure the absorbances at 405 nm and substract the lowest dilution of the test preparation. To monitor spurious
negative control reading. Use the absorbance values in the reactions, distribute 50 µL of the D-positive red blood cells
linear range of the titration curve to estimate the potency of into 4 wells of the same microtitre plate and add 50 µL of
the preparation to be examined by the usual statistical PBS-BSA solution. Seal with plastic film and incubate at
methods (5.3). 37 °C for 40 min.
Centrifuge the plates at 50 g for 3 min, discard the
METHODC
supernatant and wash the cells with 200-250 µL of PBS-BSA
The potency of human anti-D immunoglobulin is determined
solution. Repeat this at least once.
by flow cytometry in a microtitre plate format. The method
is based on the specific binding between anti- D Centrifuge the plates at 50 g for 3 min, discard the
immunoglobulin and D-positive red blood cells. The activity supernatant and add 50 µL of the secondary antibody diluted
of the preparation to be examined is compared with a with PBS-BSA solution to a suitable protein concentration.
reference preparation calibrated in International Units. Seal with plastic film and incubate, protected from light, at
room temperature for 20 min.
The International Unit is the activity of a stated amount of
International Reference Preparation. The equivalence in Centrifuge the plates at 50 g for 3 min, discard the
International Units of the International Reference preparation supernatant and wash the cells with 200-250 µL of PBS-BSA
is stated by the World Health Organization. solution. Repeat this at least once.
Human anti-D immunogl.obulin BRP is calibrated in Centrifuge the plates at 50 g for 3 min, resuspend the cells
International Units by comparison with the International into 200-250 µL of PBS. Transfer the cell suspension into a
Standard and intended for use in the assay of human anti-D tube suitable for the flow-cytometry equipment available and
immunoglobulin. further dilute by adding PBS to allow a suitable flow rate.
Proceed immediately with measurement of the median
MATERIALS
Reagents not specified are of analytical grade. fluorescence intensity in a flow cytometer. Record at least
10 000 events without gating but excluding debris.
PBS Dissolve 8.0 g of sodium chloride R, 0.76 g of disodium
Use the median fluorescence intensity in the linear range of
hydrogen phosphate dodecahydrate R, 0.2 g of potassium
the dose-response curve to estimate the potency of the
chloride Rand 0.2 g of potassium dihydrogen phosphate R in
preparation to be examined by the usual statistical methods
water R and dilute to 1000 mL with the same solvent.
(5.3).
2023 Appendix XIV J V-ASOl

C4. Test for Anti-D Antibodies in Human (0.195 g/L IgG). Add 20 µL of each dilution of each
Immunoglobulin preparation in duplicate to the microtitre plate.
(Ph. Bur. method 2.6.26) Test solutions Dilute the preparation to be examined with
MATERIALS PBS-BSA solution to obtain a starting IgG concentration of
Phosphate-buffered saline (PBS) Dissolve 8.0 g of 25 g/L. For 50 g/L preparations, this is a 2-fold dilution;
sodium chloride R, 0.76 g of anhydrous disodium hydrogen adjust the dilution factor accordingly for preparations with an
phosphate R, 0.2 g of potassium chloride R and 0.2 g of IgG concentration other than 50 g/L to obtain a starting
potassium dihydrogen phosphate R in water R and dilute to concentration of 25 g/L for testing. This 25 g/L solution is
1000 mL with the same solvent. If the solution has to be assigned a nominal 2-fold dilution factor for comparison with
kept for several days, 0.2 g of sodium azide R may be added the reference solutions, even if this does not reflect the true
in order to avoid microbial contamination. dilution factor used to achieve 25 g/L. Prepare 7 further
serial 2-fold dilutions of the preparation using PBS-BSA
PBS-BSA solution PBS containing 2 g/L of bovine
solution to extend the nominal dilution range to 1/256
albumin R (Cohn Fraction V, for ELISA). Store the solution
(0.195 g/L IgG) for comparison with the reference
at 2-8 °C but allow it to reach 19-25 °C before use.
preparations over the same IgG concentration range.
Papain solution Use serological-grade papain from a Add 20 µL of each dilution in duplicate to the microtitre
commercial source, the activity of which has been validated. plate.
Red blood cells Use pooled D-positive red blood cells Prepare 3 per cent V/V suspensions of papain-treated
from not fewer than 3 donors, preferably of group OR2 R2 . D-positive (preferably OR2 R2 , but OR 1 R 1 or OR1 R2 may
D-positive red blood cells may also be obtained from OR 1R 1 also be used) and D-negative (Orr) red blood cells in PBS-
or OR1R 2 donors. Mixing phenotypes has not been tested BSA solution. Add 20 µL of D-positive red blood cells to 1
and is therefore not recommended. dilution series of each of the preparation to be examined, the
Use pooled D-negative red blood cells, preferably from positive control and the negative control, and 20 µL of
3 donors of group Orr. When only 1 donor of group Orr is D-negative red blood cells to the other dilution series.
available, D-negative red blood cells from only 1 donor may Mix by shaking the plate on a shaker for 10 s (or until the
be used. cells are resuspended).
Wash the cells 4 times with PBS or until the supernatant is Centrifuge the plate at 80 g at room temperature for 1 min
clear. Each wash consists of suspending the cells in a to pack the cells. Place the plate at an angle of approximately
minimum of 2 volumes of PBS, centrifuging the cells at 70°. Read after 4-5 minor when the negative controls (D-
1800 g for 5 min to pack, and discarding the supernatant. negative red blood cells and negative control solution) have
Treat the packed cells with papain solution according to the streamed. A cell button at the bottom of the well indicates a
manufacturer's instructions and wash the cells 4 times with positive result. A stream of cells represents a negative result.
PBS. Record the endpoint titre as the reciprocal of the highest
Red blood cells may be stored for not more than 1 week in a dilution that gives rise to a positive result.
preservative solution at 2-8 °C. A preparation of the The positive control has a nominal titre of 8 and the negative
following composition is appropriate: controls CD-negative red blood cells and negative control
solution) must not show agglutination at the starting dilution
Trisodium citrate 8 g/L of 1 in 2. Users must validate their own test conditions, and
D-glucose 20 g/L
Citric acid 0.5 g/L
investigate their assay conditions and reagents in the event of
Sodium chloride 4.2 g/L results being significantly different from those expected.
lnosine 0.938 g/L Failure to obtain negative reactions with the negative controls
Adenosine triphosphate (ATP) 0.4 g/L may indicate that, for example, insufficient time has elapsed
Ch!oramphenicol 0.34 g/L
Neomycin sulfate 0.1 g/L
for the cells to stream, or that reagents have been used
directly from cold storage.
If using stored cells, wash the cells at least twice in PBS or The titre of the preparation to be examined must not be
until the supernatant is clear before proceeding. greater than the titre of the positive control when both
Microtitre plates Use V-bottomed rigid microtitre plates. preparations are titrated from 25 g/L.
Reference standards Immunoglobulin (anti-D antibodies C5. Anti-A and Anti-B Haemagglutinins
test) ERP and Immunoglobulin (anti-D antibodies test negative (Ph. Bur. method 2.6.20)
control) ERP are suitable for use as the positive control and
negative control, respectively. METHOD A: INDIRECT METHOD
Prepare in duplicate serial dilutions of the preparation to be
METHOD examined in a 9 g/L solution of sodium chloride R. To each
The test described in this chapter is performed at room dilution of 1 series add an equal volume of a 5 per cent VIV
temperature on the positive control solutions, the negative suspension of group A 1 red blood cells previously washed
control solutions and the test solutions at the same time and 3 times with the sodium chloride solution. To each dilution
under identical conditions. of the other series add an equal volume of a 5 per cent VIV
Reference solutions Reconstitute the positive control and suspension of group B red blood cells previously washed
the negative control according to the instructions. 3 times with the sodium chloride solution. Incubate the
The immunoglobulin G (IgG) concentration is 50 g/L in suspensions at 37 °C for 30 min then wash the cells 3 times
each of the reconstituted preparations. Make a 2-fold dilution with the sodium chloride solution. Leave the cells in contact
of each reconstituted preparation with PBS-BSA solution to with a polyvalent anti-human globulin reagent for 30 min.
obtain solutions containing IgG at 25 g/L. Prepare 7 further Without centrifuging, examine each suspension for
serial 2-fold dilutions of each preparation using PBS-BSA agglutination under a microscope.
solution to extend the dilution range to 1/256
V-A502 Appendix XIV J 2023

METHOD B: DIRECT METHOD of each reconstituted preparation with PBS-BSA solution to

MATERIALS obtain solutions containing IgG at 25 g/L. Prepare 7 further
Phosphate-buffered saline (PBS) Dissolve 8.0 g of serial 2-fold dilutions of each preparation using PBS-BSA
sodium chloride R, 0.76 g of anhydrous disodium hydrogen solution to extend the dilution range to 1/256 (0.195 g/L
phosphate R, 0.2 g of potassium chloride R and 0.2 g of IgG). Add 20 µL of each dilution of each preparation in
potassium dihydrogen phosphate R in water R and dilute to triplicate to the microtitre plate.
1000 mL with the same solvent. If the solution has to be Test solutions Dilute the preparation to be examined with
kept for several days, 0.2 g of sodium azide R may be added PBS-BSA solution to obtain a starting IgG concentration of
in order to avoid microbial contamination. 25 g/L. For 50 g/L preparations, this is a 2-fold dilution;
PBS-BSA solution PBS containing 2 g/L of bovine adjust the dilution factor accordingly for preparations with an
albumin R (Cohn Fraction V, for ELlSA). Store the solution IgG concentration other than 50 g/L to obtain a starting
at 2-8 °C but allow it to reach 19-25 °C before use. concentration of 25 g/L for testing. This 25 g/L solution is
Papain solution Use serological-grade papain from a assigned a nominal 2-fold dilution factor for comparison with
commercial source, the activity of which has been validated. the reference solutions, even if this does not reflect the true
dilution factor used to achieve 25 g/L. Prepare 7 further
Red blood cells Use pooled D-negative A1 (A 1rr),
serial 2-fold dilutions of the preparation using PBS-BSA
D-negative B (Brr) and D-negative O (Orr) red blood cells
solution to extend the nominal dilution range to 1/256
from preferably 3 donors. When Immunoglobulin for anti-A
(0.195 g/L IgG) for comparison with the reference
and anti-B antibodies limit test BRP is used, 3 donors are to be
preparations over the same IgG concentration range.
used. A2 red blood cells are not recommended as they give
Add 20 µL of each dilution in triplicate to the microtitre
weaker reactions.
plate.
Wash the cells 4 times with PBS or until the supernatant is
For preparations with an IgG concentration lower than
clear. Each wash consists of suspending the cells in a
25 g/L, dilute to a starting concentration corresponding to
minimum of 2 volumes of PBS, centrifuging the cells at
the nearest lower concentration of the reference solutions.
1800 g for 5 min to pack, and discarding the supernatant.
This solution is assigned the same nominal dilution factor as
Treat the packed cells with papain solution according to the
the corresponding reference solution having the same lgG
manufacturer's instructions and wash the cells 4 times with
concentration. Proceed with the preparation of the
PBS.
appropriate 2-fold dilution series as described above.
Red blood cells may be stored for not more than 1 week in a
Prepare 3 per cent V/V suspensions of papain-treated
preservative solution at 2-8 °C. A preparation of the
D-negative A 1, B and O red blood cells in PBS/BSA
following composition is appropriate:
solution. Add 20 µL of D-negative A 1, B and O red blood
Trisodium citrate 8 g/L
cells respectively to the 1st, the 2 nd and the 3 rd dilution series
o-glucose 20 g/L of each of the preparation to be examined, the positive
Citric acid 0.5 g/L control and the negative control. Mix by shaking the plate on
Sodium chloride 4.2 g/L a shaker for 10 s (or until the cells are resuspended).
Inosine 0.938 g/L
Adenosine triphosphate (ATP) 0.4 g/L Centrifuge the plate at 80 g at room temperature for 1 min
Chloramphenicol 0.34 g/L to pack the cells. Place the plate at an angle of approximately
Neomycin sulfate 0.1 g/L 70°. Read after 4-5 min or when the negative controls (D-
negative O red blood cells and negative control solution)
If using stored cells, wash the cells at least twice in PBS or have streamed. A cell button at the bottom of the well
until the supernatant is clear before proceeding. indicates a positive result. A stream of cells represents a
Microtitre plates Use V-bottomed rigid microtitre plates. negative result.
Reference standards Immunoglobulin (anti-A, anti-B Record the endpoint titre as the reciprocal of the highest
antibodies test positive control) BRP and lmmunoglobulin (anti- dilution that gives rise to a positive result.
A, anti-B antibodies test negative control) BRP are suitable for The positive control has nominal anti-A and anti-B titres of
use as the positive control and negative control, respectively, 32 (range 32-64 for anti-A; range 16-32 for anti-B) and the
and should be used as guides for operators establishing and negative controls CD-negative O red blood cells and negative
performing the direct method for anti-A and anti-B control solution) must not show agglutination at the starting
haemagglutinins. dilution of 1 in 2. Users must validate their own test
Immunoglobulin for anti-A and anti-B antibodies limit test BRP conditions, and investigate their assay conditions and
defines the recommended maximum limits permissible for reagents in the event of results being significantly different
batches of human immunoglobulin and must be used only from those expected. Failure to obtain negative reactions
for comparison with batches of human immunoglobulin that with the negative controls may indicate that, for example,
have higher titres than the positive control. insufficient time has elapsed for the cells to stream, or that
METHOD reagents have been used directly from cold storage.
The test described in this chapter is performed at room If the nominal anti-A or anti-B titre of the preparation to be
temperature on the positive control solutions, the negative examined is greater than the titre of the positive control, the
control solutions and the test solutions at the same time and test preparation is to be compared with lmmunoglobulin for
under identical conditions. Whenever necessary, a further test anti-A and anti-B antibodies limit test BRP.
is performed with Immunoglobulin for anti-A and anti-B The maximum allowable titre is 64.
antibodies limit test BRP.
Reference solutions Reconstitute the positive control and
the negative control according to the instructions.
The immunoglobulin G (IgG) concentration is 50 g/L in
each of the reconstituted preparations. Make a 2-fold dilution
2023 Appendix XIV K V-A503

D2. Assay of Human Cl-Esterase Inhibitor

Other blood-related components (Ph. Bur. method 2.7.34)
The human C 1-esterase inhibitor content of the preparation
D1. Assay of Human cr-1-proteinase Inhibitor
(Ph. Bur. method 2.7.32) to be examined is determined by comparing its ability to
Human cr-1-proteinase inhibitor (also known as a-1- inhibit Cl-esterase with that of a reference preparation
calibrated in International Units. The International Unit is
antitrypsin or a-1-antiproteinase) content is determined by
comparing the ability of the preparation to be examined to the activity of a stated amount of the International Standard
inactivate the serine protease elastase (porcine pancreatic for human plasma-derived Cl-esterase inhibitor concentrate.
The equivalence in International Units of the International
elastase or human neutrophil elastase) with the same ability
of a reference standard of human cr-1-proteinase inhibitor Standard is stated by the World Health Organization.
calibrated in milligrams of active (functional) a-1-proteinase Varying quantities of the preparation to be examined are
inhibitor. Varying quantities of the preparation to be mixed with an excess of C 1-esterase and the remaining
examined are mixed with a given quantity of elastase and the C 1-esterase activity is determined using a suitable
chromogenic substrate.
remaining elastase activity is determined using a suitable
chromogenic substrate. The method described below is given Individual reagents may be obtained separately or in
as an example. commercial kits. Procedures and reagents may vary between
different kits and the manufacturer's instructions are
REAGENTS followed. The essential features of the procedure are
Tris-albumin buffer solution Dissolve 24.23 g of described in the following example of a microtitre-plate
trometamol R in water R, adjust to pH 8.0 ± 0.3 using kinetic method.
hydrochloric acid Rl and dilute to 1000 mL with water R.
Reconstitute the preparation as stated on the label. Prepare
To 100 mL of this solution add 0.5 mL of a 20 per cent
an appropriate series of at least 3 dilutions, from 1 IU/mL of
solution of human albumin R or bovine albumin R.
Cl-esterase inhibitor, for both the preparation to be
Buffer solution containing human or bovine albumin must be examined and the reference preparation, using a suitable
prepared fresh on the day of its use; otherwise, it can be pH 7.4 buffer solution containing 9 g/L of sodium chloride R
conserved by sterile filtration (0.2 µm) and stored at 2-8 °C and either 10 g/L of human albumin R or 10 g/L of bovine
for up to 2 weeks. albumin R. Warm all solutions to 37 °C. Place a suitable
METHOD quantity of 1 of the dilutions of the reference preparation or
Prepare 2 series of 4 or 5 dilutions in an appropriate human of the preparation to be examined in microtitre plate wells
a-1-proteinase inhibitor concentration range, for both the and incubate at 37 °C. To each well add a suitable quantity
preparation to be examined and the reference standard, using of Cl-esterase solution and incubate at 37 °C for 5 min.
the tris-albumin buffer solution. Add an appropriate quantity of a suitable specific
Transfer 50 µL of the reference solution dilutions into the chromogenic substrate such as methoxycarbonyl-L-lysyl(£-
wells of a microtitre plate and to each well, add 150 µL of a benzyloxycarbonyl)-glycyl-L-arginine-4-nitroanilide. Read the
porcine pancreatic elastase solution diluted to an appropriate rate of increase of absorbance (M/min) at 405 nm.
concentration with the tris-albumin buffer solution. Incubate A positive control, using the pH 7.4 buffer solution instead
for a defined period of time, 3-10 min, at room temperature. of the C 1-esterase inhibitor, is included. For preparations
Since the activity of the solutions of the different porcine that may exhibit proteolytic activity, a test is carried out on a
pancreatic elastases may vary, the concentration of elastase suitable blank composed of the preparation to be examined,
can be adjusted by evaluation of blank values containing the pH 7 .4 buffer solution and the chromogenic substrate.
elastase but no human a-1-proteinase inhibitor, to exhibit a Calculate the Cl-esterase inhibitor content using the usual
suitable change of absorbance at 405 nm under the actual statistical methods, for example slope ratio (5.3).
assay conditions.
Add to each well 100 µL of a solution of chromogenic
substrate N-succinyl-tri-L-alanyl 4-p-nitroanilide (Suc-Ala-
Ala-Ala-pNA), reconstituted in dimethyl sulfoxide R to give a K. Immunological Products
solution containing 4.5 mg/mL, then further diluted with the
tris-albumin buffer solution to a concentration of 1. Assay of Diphtheria Vaccine (Adsorbed)
0.45 mg/mL. Immediately start measurement of the change (Ph. Bur. method 2. 7. 6. An alternative in viva method (Method
in absorbance (2.2.25) at 405 nm using a microtitre plate B) in which the potency is determined by comparing the dose
reader, continuing the measurement for at least 5 min. necessary to protect guinea-pigs against the lethal effect of a
Calculate the rate of change of absorbance (~Nmin). subcutaneous injection of dipththeria toxin with the dose of a
Alternatively, an end-point assay may be used by stopping reference preparation calibrated in International Units necessary to
the reaction with acetic acid and measuring the absorbance at give the same protection is also described in the European
405 nm. If the assay is performed in test tubes using Pharmacopoeia.)
spectrophotometers for monitoring the change in absorbance The potency of diphtheria vaccine is determined by
at 405 nm, the volumes of reagent solutions are changed administration of the vaccine to guinea-pigs followed either
proportionally. by challenge with diphtheria toxin (method A or B) or by
The rate of change of absorbance (~min) is inversely determination of the titre of antibodies against diphtheria
proportional to human cr-1-proteinase inhibitor activity. toxin or toxoid in the serum of guinea-pigs (method C).
Check the validity of the assay and calculate the potency of In both cases, the potency of the vaccine is calculated by
the test preparation by the usual statistical methods (5.3). comparison with a reference preparation, calibrated in
International Units.
The International Unit is the activity contained in a stated
amount of the International Standard, which consists of a
V-A504 Appendix XIV K 2023

quantity of diphtheria toxoid adsorbed on aluminium - the ratio of antitoxin titres or scores for the positive serum
hydroxide. The equivalence in International Units of the control to the serum samples corresponding to the
International Standard is stated by the W odd Health reference vaccine.
Organization (WHO).
METHOD A: INTRADERMAL CHALLENGE TEST
Diphtheria vaccine (adsorbed) ERP is suitable for use as a IN GUINEA-PIGS
reference preparation. SELECTION AND DISTRIBUTION OF THE TEST
The method chosen for the assay of diphtheria vaccine ANIMALS
(adsorbed) depends on the intended purpose. Method A Use in the test healthy, white guinea-pigs from the same
or Bis used: stock and of a size suitable for the prescribed number of
1. during development of a vaccine, to assay batches challenge sites, the difference in body mass between the
produced to validate the production; heaviest and the lightest animal being not greater than 100 g.
2. wherever revalidation is needed following a significant Use guinea-pigs of the same sex or with males and females
change in the manufacturing process. equally distributed between the groups. Distribute the
guinea-pigs in not fewer than 6 equal groups; use groups
Method A or B may also be used for the routine assay of
containing a number of animals sufficient to obtain results
batches of vaccine, but in the interests of animal welfare,
that fulfil the requirements for a valid assay prescribed below.
method C is used wherever possible.
If the challenge toxin to be used has not been shown to be
Method C may be used, except as specified under 1 and 2 stable or has not been adequately standardised, include
above, after verification of the suitability of the method for 5 guinea-pigs as unvaccinated controls.
the product. For this purpose, a suitable number of batches
SELECTION OF THE CHAUENGE TOXIN
(usually 3) are assayed by method C and method A or B.
Select a preparation of diphtheria toxin containing 67 to
Where different vaccines (monovalent or combinations) are
133 lr/100 in 1 Lf and 25 000 to 50 000 minimal reacting
prepared from diphtheria toxoid of the same origin, and with
doses for guinea-pig skin in 1 Lf. If the challenge toxin
comparable levels (expressed in L£'mL) of the same
preparation has been shown to be stable, it is not necessary
diphtheria toxoid, suitability demonstrated for the
to verify the activity for every assay.
combination with the highest number of components can be
assumed to be valid for combinations with fewer components PREPARATION OF THE CHALLENGE TOXIN
and for monovalent vaccines. Any combinations containing a SOLUTION
whole-cell pertussis component or containing haemophilus Immediately before use, dilute the challenge toxin with a
type b conjugate vaccine with diphtheria toxoid or CRM 197 suitable diluent to obtain a challenge toxin solution
diphtheria protein as carrier in the same vial must always be containing about 0.0512 Lfin 0.2 mL. Prepare from this a
assessed separately. further series of 5 four-fold dilutions containing about
For combinations containing diphtheria and tetanus 0.0128, 0.0032, 0.0008, 0.0002 and 0.00005 Lfin 0.2 mL.
components, the serological assay (method C) can be DILUTION OF THE TEST AND REFERENCE
performed with the same group of animals used for the PREPARATIONS
serological assay of the tetanus vaccine (adsorbed) (2. 7.8) Using a 9 g/L solution of sodium chloride R, prepare dilutions
when the common immunisation conditions for the of the vaccine to be examined and of the reference
diphtheria and the tetanus components (for example, doses, preparation, such that for each, the dilutions form a series
duration) have been demonstrated to be valid for the differing by not more than 2.5-fold steps and in which the
combined vaccine. intermediate dilutions, when injected subcutaneously at a
The design of the assays described below uses multiple dose of 1.0 mL per guinea-pig, will result in an intradermal
dilutions for the test and reference preparations. Once the score of approximately 3 when the animals are challenged.
analyst has sufficient experience with this method for a given IMMUNISATION AND CHALLENGE
vaccine, it is possible to apply a simplified model such as a Allocate the dilutions, 1 to each of the groups of guinea-pigs,
single dilution for both test and reference preparations. Such and inject subcutaneously 1.0 mL of each dilution into each
a model enables the analyst to determine whether the guinea-pig in the group to which that dilution is allocated.
potency of the test preparation is significantly higher than the After 28 days, shave both flanks of each guinea-pig and inject
minimum required, but does not give information on 0.2 mL of each of the 6 toxin dilutions intradermally into
linearity, parallelism and the dose-response curve. 6 separate sites on each of the vaccinated guinea-pigs in such
The simplified model allows for a considerable reduction in a way as to minimise interference between adjacent sites.
the number of animals required and must be considered by DETERMINATION OF THE ACTIVI1Y OF THE
each analyst in accordance with the provisions of the CHAUENGE TOXIN
European Convention for the Protection of Vertebrate If necessary, inject the unvaccinated control animals with
Animals Used for Experimental and Other Scientific dilutions containing 80, 40, 20, 10 and 5 x 10-6 Lf of the
Purposes. challenge toxin.
Where a single-dilution assay is used, production and test
READING AND INTERPRETATION OF RESULTS
consistency over time are monitored via suitable indicators Examine all injection sites 48 h after injection of the
and by carrying out a full multiple-dilution assay periodically,
challenge toxin and record the incidence of specific
for example every 2 years. For serological assays, suitable diphtheria erythema. Record also the number of sites free
indicators to monitor test consistency are: from such reactions as the intra-dermal challenge score.
- the mean and standard deviation of relative antitoxin Tabulate the intradermal challenge scores for all the animals
titres or scores of the serum samples obtained after receiving the same dilution of vaccine and use those data
administration of a fixed dose of the vaccine reference
with a suitable transformation, such as (score/ or
preparation; arcsin ((score/6) 2 ), to obtain an estimate of the relative
- the antitoxin titres or scores of run controls (positive and
negative serum samples);
2023 Appendix XIV K V-A505

potency for each of the test preparations by parallel-line PREPARATION OF SERUM SAMPLES
quantitative analysis. Avoid frequent freezing and thawing of serum samples.
REQUIREMENTS FOR A VALID ASSAY To avoid microbial contamination, it is preferable to carry
The test is not valid unless: out manipulations in a laminar-flow cabinet.
- for both the vaccine to be examined and the reference DETERMINATION OF ANTIBODY TITRE
preparation, the mean score obtained at the lowest dose Determine the relative antibody titre or score of each serum
level is less than 3 and the mean score at the highest dose sample by a suitable immunochemical method (2. 7.1).
level is more than 3; The methods shown below (enzyme-linked immunosorbent
- where applicable, the toxin dilution that contains assay (ELISA) and Vero cell assay) have been found to be
40 x 1o- 6 Lf gives a positive erythema in at least suitable.
80 per cent of the control guinea-pigs and the dilution CALCULATION OF POTENCY
containing 20 x 1o- 6 Lf gives a positive erythema in less Calculate the potency of the vaccine to be examined in
than 80 per cent of the guinea-pigs (if these criteria are International Units relative to the reference preparation,
not met a different toxin has to be selected); using the usual statistical methods (for example, 5.3).
- the confidence limits (P = 0.95) are not less than
50 per cent and not more than 200 per cent of the REQUIREMENTS FOR A VALID ASSAY
estimated potency; The test is not valid unless:
- the statistical analysis shows no deviation from linearity - the confidence limits (P = 0.95) are not less than
and parallelism. 50 per cent and not more than 200 per cent of the
estimated potency;
The test may be repeated but when more than 1 test is - the statistical analysis shows a significant slope and no
performed the results of all valid tests must be combined in deviation from linearity and parallelism of the dose-
the estimate of potency. response curves (chapter 5. 3 describes possible
METHOD C. DETERMINATION OF ANTIBODIES alternatives if significant deviations are observed).
IN GUINEA-PIGS The test may be repeated but when more than 1 test is
SELECTION AND DISTRIBUTION OF THE TEST performed the results of all valid tests must be combined in
ANIMALS the estimate of potency.
Use in the test healthy guinea-pigs from the same stock, each The following section is published for information.
weighing 250-350 g. Use guinea-pigs of the same sex or with
males and females equally distributed between the groups.
Distribute the guinea-pigs in not fewer than 6 equal groups;
ASSAY OF DIPHTHERIA VACCINE
use groups containing a number of animals sufficient to (ADSORBED): GUIDELINES
obtain results that fulfil the requirements for a valid assay METHOD C. DETERMINATION OF ANTIBODIES
prescribed below. Use a further group of non-vaccinated IN GUINEA-PIGS
guinea-pigs of the same origin to provide a negative serum PREPARATION OF SERUM SAMPLES
control. If test consistency has been demonstrated, a For the preparation of serum samples, the following
reference negative serum control may be used. technique has been found to be suitable. Invert the tubes
REFERENCE PREPARATION containing blood samples 6 times and allow to stand at
Use a suitable reference preparation such as diphtheria vaccine 37 °C for 2 h, then at 4 °C for 2 h. Centrifuge at room
( adsorbed) ERP or a batch of vaccine shown to be effective in temperature at 800 g for 20 min. Transfer the serum to
clinical studies, or a batch representative thereof, and which sterile tubes and store at a temperature below -20 °C.
has been calibrated in International Units with reference to At least a 40 per cent yield of serum is obtained by this
diphtheria vaccine (adsorbed) ERP or the International procedure.
Standard for diphtheria toxoid (adsorbed). DETERMINATION OF ANTIBODY TITRE
DILUTION OF THE TEST AND REFERENCE The ELISA and Vero cell assays shown below are given as
PREPARATIONS examples of immunochemical methods that have been found
Using a 9 g/L solution of sodium chloride Ras diluent, prepare to be suitable for the determination of antibody titre.
serial dilutions of the vaccine to be examined and the Determination of antibody titre in guinea-pig serum by
reference preparation; series differing by 2.5- to 5-fold steps enzyme-linked immunosorbent assay (ELISA)
have been found to be suitable. Use not fewer than Dilutions of test and reference sera are made on ELISA
3 dilutions within the range of, for example, 0.5-16 IU/mL plates coated with diphtheria toxoid. A positive guinea-pig
for the reference vaccine and within the range of, for serum control and a negative guinea-pig serum control are
example, 1:2 to 1: 125 for the vaccine to be examined. included on each plate to monitor the assay performance.
Use the dilutions for immunisation preferably within 1 h of Peroxidase-conjugated rabbit or goat antibody directed
preparation. Allocate 1 dilution to each group of guinea-pigs. against guinea-pig-lgG is added, followed by a peroxidase
IMMUNISATION substrate. Optical density is measured and the relative
Inject subcutaneously to each guinea-pig 1.0 mL of the antibody titre is calculated using the usual statistical methods
dilution allocated to its group. (for example, 5.3).
BLOOD SAMPUNG Reagents and equipment
35-42 days after immunisation, take a blood sample from - ELISA plates: 96 wells, columns 1-12, rows A-H.
each vaccinated and control guinea-pig using a suitable - Diphtheria guinea-pig antiserum (for vaccines-human use)
method. (positive control serum), obtained by immunisation of
guinea-pigs using diphtheria vaccine ( adsorbed) ERP.
- Peroxidase conjugate. Peroxidase-conjugated rabbit or goat
antibody directed against guinea-pig IgG.
- Diphtheria toxoid.
V-A506 Appendix XIV K 2023

- Carbonate coating buffer pH 9.6. Dissolve 1.59 g of The limit of detection is specific for each antitoxin and is
anhydrous sodium carbonate Rand 2.93 g of sodium usually between 0.015 IU/mL (method 1) and 0.05 IU/mL
hydrogen carbonate R in 1000 mL of water R. Distribute (method 2).
into 150 mL bottles and sterilise by autoclaving at 121 °C The endpoint is taken as the highest serum dilution
for 15 min. protecting cells from the diphtheria toxin effect.
- Phosphate-buffered saline pH 7. 4 (PBS). Dissolve with The antitoxin activity is calculated with respect to guinea-pig
stirring 80.0 g of sodium chloride R, 2.0 g of potassium or WHO reference standard, and expressed in International
dihydrogen phosphate R, 14.3 g of disodium hydrogen Units per millilitre.
phosphate dihydrate Rand 2.0 g of potassium chloride R in
Reagents and equipment
1000 mL of water R. Store at room temperature to - Flat-bottomed tissue culture plates: 96 wells, columns 1-12,
prevent crystallisation. Dilute to 10 times its volume with
rows A-H.
water R before use. - 75 cm 2 tissue culture flasks.
- Citric acid solution. Dissolve 10.51 g of cim·c acid
- Diphtheria toxin.
monohydrate R in 1000 mL of water R and adjust the
- Diphtheria guinea-pig antiserum (for vaccines-human use)
solution to pH 4.0 with a 400 g/L solution of sodium (positive control serum), obtained by immunisation of
hydroxide R.
guinea-pigs with diphtheria vaccine ( adsorbed) ERP.
- Washing buffer. PBS containing 0.5 g/L of - Vero cells (African Green Monkey kidney cells). Cell
polysorbate 20 R.
passages from P2 to P15 are suitable for use.
- Diluent blocking buffer. PBS containing 0.5 g/L of
polysorbate 20 R and 25 g/L of dried skimmed milk. Method 1 The diphtheria toxin causes a cytopathogenic
- Peroxidase substrate. Shortly before use, dissolve 10 mg of
effect on Vero cells leading to cellular lysis. Antibodies
diammonium 2,2 1-azinobis(3-ethylbenzothiazoline-6- directed against diphtheria toxin may inhibit this
sulfonate) R (ABTS) in 20 mL of citric acid solution. cytopathogenic effect. Consequently, the potency of a
Immediately before use add 5 µL of strong hydrogen diphtheria vaccine may be indirectly determined with the
peroxide solution R. help of this cell culture system if different serum dilutions
from immunised animals are cultured with a constant toxin
Method concentration. In the Vero cell assay, yellow colour indicates
The description below is given as an example of a suitable viable cells, red colour dead cells. When only part of the cells
plate layout but others may be used. Wells lA-H are for are dead, the colour may be orange.
negative control serum and wells 2A-H and 12A-H are for Reagents and equipment
positive control serum for assay monitoring. Wells 3-llA-H - Modified MEM. Minimum Essential Medium (MEM)
are for test samples. with Earle's Salts, without L-glutamine and sodium
Coat each well of the ELISA plates with 100 µL of bicarbonate.
diphtheria toxoid solution (0.5 Lfi'mL in carbonate coating - Modified medium 199. Medium 199, with Hanks' Solution
buffer pH 9.6). Allow to stand overnight at 4 °Cina humid and L-glutamine, without sodium bicarbonate.
atmosphere. To avoid temperature gradient effects, do not - Foetal bovine serum.
stack more than 4 plates high. On the following day, wash - Sodium bicarbonate 7. 5 per cent solution.
the plates thoroughly with washing buffer. Block the plates - Trypsin solution: trypsin 2.5 per cent solution.
by addition of 100 µL of diluent block buffer to each well. - EDTA solution: EDTA 0.02 per cent (Versene 1:5000)
Incubate in a humid atmosphere at 37 °C for 1 h. Wash the solution.
plates thoroughly with washing buffer. Place 100 µL of - Modified D-PBS. Dulbecco's phosphate buffered saline
diluent block buffer in each well of the plates, except those of (D-PBS), without calcium, or magnesium.
row A. Prepare suitable dilutions of negative control serum, - L-glutamine 200mM solution.
positive control serum (from about 0.01 IU/mL) and test - Penicillin/streptomycin solution.
sera. Allocate the negative control serum to column 1, - Primary culture medium. To 50 mL of modified MEM add
positive control serum to columns 2 and 12 and test sera to 440 mL of water R, 5 mL of L-glutamine 200 mM
columns 3-11 and add 100 µL of each serum to the first solution, and 10 mL of sodium bicarbonate 7.5 per cent
2 wells of the column to which it is allocated. Using a solution. To 25 mL of this medium add 1.25 mL of
multichannel micropipette, make twofold serial dilutions from foetal bovine serum.
row B, down the plate to row H, by transferring 100 µL - Maintenance culture medium. Similar to the primary culture
from one well to the next well. Discard 100 µL from the last medium except that 0.5 mL instead of 1.25 mL of foetal
row so that all wells contain 100 µL. Incubate at 37 °C for bovine serum is added to 20 mL of the enriched MEM
2 h. Wash thoroughly with washing buffer. Prepare a suitable medium.
dilution (a 2000-fold dilution has been found to be suitable) - Medium A. To 50.0 mL of modified medium 199 add
of peroxidase conjugate in diluent block buffer and add 440.0 mL of water R, 5.0 mL of L-glutamine 200 mM
100 µL to each well. Incubate at 37 °C in a humid solution and 10.0 mL of sodium bicarbonate 7.5 per cent
atmosphere for 1 h. Wash the plates thoroughly with washing solution.
buffer. Add 100 µL of peroxidase substrate to each well. - Medium B. To 150.0 mL of medium A add 3.0 mL of
Allow to stand at room temperature, protected from light, for foetal bovine serum and 0.3 mL of penicillin/streptomycin
30 min. Read the plates at 405 nm in the same order as solution.
addition of substrate was made. - Medium C. To 22.0 mL of medium A add 0.44 mL of
Determination of antibody titre in guinea-pig serum by foetal bovine serum and 0.44 mL of
Vero cell assay penicillin/streptomycin solution.
The method used relies either on metabolic inhibition Vero cells are cultured in tissue culture flasks (for example
(method 1) or on cytotoxicity (method 2) as the end point, 75 cm2 /250 mL) in an incubator at 36 ± 1 °C,
and on either microscopic (cell morphology) or visual 5 per cent CO 2 and 90 per cent relative humidity. Vero cells
(colour) inspection of the cells.
2023 Appendix XIV K V-A507

are first grown in the primary culture medium. After 2-3 days - Antibiotic solutwn (containing 10 000 units of penicillin,
of growth, the primary culture medium is replaced by the 10 mg of streptomycin and 25 µg of amphotericin B per
maintenance culture medium. 'When a confluent monolayer millilitre).
is obtained, the culture supernatant is discarded and the cell - L-glutamine 200mM solutwn.
layer washed gently with modified D-PBS. Add a mixture of - Trypsin-EDTA.
1 volume of trypsin solution and I volume of EDTA solution - Thiazolyl blue MTT [3-( 4,5-dimethylthiazol-2-yl)-2,5-
to the flask. Swirl the flask gently and incubate in the CO 2 diphenyltetrazolium bromide].
incubator for about 3 min until the cells start to break from - 1 M HEPES buffer pH 8.1. Dissolve 18. 75 g of HEPES in
the monolayer. Vigorously tap the side of the flask to make 82.5 mL of water Rand 30.0 mL of 2 M sodium
the cells fall. Resuspend the cells in 5-6 mL of fresh hydroxide R.
medium C to obtain a homogeneous suspension. Prepare a - Glucose solution (10 per cent).
cell suspension in medium C containing approximately - Complete culture medium. Mix 200 mL of MEM with
1 x 10 5 cells/mL. 10 mL of newborn calf serum, 3.0 mL of 1 M HEPES
Place 25 µL of medium B in each well except those of buffer pH 8.1, 2.0 mL of glucose solution (10 per cent),
column 1. Place 25 µL of the diphtheria guinea-pig 2.0 mL of antibiotic solution and 2.0 mL of L-glutamine
antiserum (for vaccines-human use) (positive control serum, 200mM solution.
working dilution in medium B of 0.40 IU/rnL) in wells Al, - Phosphate-buffered saline pH 7.4 (PBS). Dissolve 10.0 g of
A2 and Al 1. Place 25 µL of guinea-pig serum samples in sodium chloride R, 0.75 g of potassium chloride R, 1.44 g of
wells B-G of columns 1, 2 and 11. Place 25 µL of negative disodium hydrogen phosphate dodecahydrate R, and 0.125 g
control serum in row H of columns 1, 2 and 11. Using a of potassium dihydrogen phosphate R in water R, and dilute
multichannel micropipette, make twofold serial dilutions to 1000.0 mL with the same solvent. Adjust the pH if
across the plate (from column 2 up to column 10 for rows A- necessary. Autoclave at 120 °C for 15 min.
G and up to column 8 for row H). Discard 25 µL from the - Thiazolyl blue MTT solution. Dissolve 0.1 g ofthiazolyl
wells in column 10 in rows A-G, and from well H8. blue MTT in 20 mL of PBS. Sterilise by filtration
Reconstitute the diphtheria toxin with saline solution to give (0.2 µm) and store in dark bottle.
- pH adfuster solution. Mix 40 mL of acetic acid R with
a solution of 50 IU/mL. Prepare a SO-fold dilution of this
diphtheria toxin dilution in medium B to obtain a working 1.25 mL of 1 M hydrochloric acid and 8. 75 mL of water R.
- Extractwn buffer pH 4. 7. Dissolve 10 g of sodium
solution of 1.0 IU/mL. Add 25 µL of this working solution to
wells A12 and B12 (toxin control). Make twofold serial laurilsulfate R in water R and add 50 mL of
dilutions by tranferring 25 µL from one well to the next, dimethylfomiamide R, and dilute to 100 mL with water R.
from well B12 down to Hl2. Change the tip between each Adjust the pH with an appropriate volume of pH adjuster
dilution. Discard 25 µL from well H12. Add 25 µL of solution.
medium B to wells B12-Hl2. Then, place 25 µL of the Vero cells are cultured in tissue culture flasks (for example
working dilution of the diphtheria toxin ( 1.0 IU/mL) in each 75 cm2/250 mL) in an incubator at 36 ± 1 °C, 5 per cent
well of rows A-H, from column 1-10, except in wells H9 CO 2 and 90 per cent relative humidity. Vero cells are grown
and HlO (cells only, without serum and without toxin). in the complete culture medium. After 6-7 days of growth, a
Cover the plates with lids or sealer and shake gently. confluent monolayer is obtained, the culture supernatant is
Incubate the plates for at least 2 h in a humid container in a discarded and the cell layer is washed 3 times with trypsin-
CO 2 incubator at 37 °C. Add 200 µL of cell suspension EDTA: gently pipette out the medium, add 0.5-1 mL of
containing 1 x 10 5 cells/mL to all the wells. Cover the plates trypsin-EDTA, swirl the flask and tip the trypsin out. Do this
with sealer. Incubate at 37 °C for 5 days. Check for twice, and the 3rd time, place the flask in the incubator for
microbial contamination by microscopic examination. 5 min until the cells start to break from the monolayer.
Vigorously tap the side of the flask to make the cells fall.
Yellow wells are recorded as negative and red wells indicate Resuspend the cells in 6-25 mL of fresh complete culture
dead cells and are recorded as positive. A colour between medium to obtain a homogeneous suspension. Prepare a cell
yellow and red indicates a mixture of viable and dead cells suspension in complete culture medium containing
and is recorded as positive/negative. The results based on the approximately 4 x 10 5 cells/mL.
change in colour can be confirmed by reading viable and
dead cells under the microscope. Place 50 µL of complete culture medium in each well except
those of column 1. Place 100 µL of diphtheria guinea pig
The potency of the guinea-pig antiserum samples is obtained antiserum (for vaccines-human use) (positive control serum,
by comparing the last well of the standard preparation working dilution in complete culture medium of
showing complete neutralisation of the toxin, with the last 0.12 IU/mL) in well Al and 50 µLin well All. Place
well of the sample demonstrating the same effect. 100 µL of guinea pig test serum samples, diluted if necessary,
For calculations of potency, it must be remembered that the in wells B 1-G 1. Add 50 µL of the same sample to wells B 11-
endpoint may be between a negative well and a G 11 in the corresponding row. Place 100 µL of negative
positive/negative well. control serum in well Hl and 50 µLin well Hll. Using a
Method 2 Thiazolyl blue MTT is reduced to a blue/black multi-channel micropipette, make twofold serial dilutions by
formazan product by the mitochondrial dehydrogenase of transferring 50 µL from one well to the next working across
viable cells, and thus serves as a quantitative measure of the plate (from column 1-10 for rows A-G and from column
living cells present, indicating when the toxin has been 1-8 for row H).
neutralised by the antitoxin. 'White or colourless wells Diphtheria toxin of known activity and Lf content is diluted
indicate absence of viable cells due to insufficient antitoxin to to a suitable working stock containing at least 4 minimum
neutralise the toxin. cytopathic doses in complete culture medium. Add 50 µL of
Reagents and equipment the diluted toxin to each well except H9 and Hl0 (cell
- MEM (Minimal Essential Media). control), Al 1-Hl 1 (serum control) and Al2-H12 (toxin
- Newborn calf serum. control). Add 100 µL of diluted toxin to well A12 and make
V-A508 Appendix XIV K 2023

twofold serial dilutions by transferring 50 µL from one well dilution allocated to its group. After 14 days, count the
to the next working down the plate (from well A12-Hl2). number of mice surviving in each group. From the results,
Discard 50 µL from well H12. Add 50 µL of complete calculate the expected opacity of a suspension containing
medium to wells H9 and Hl0. 100 LD 50 in each challenge dose. For the test of the vaccine
Cover the plates with a lid or sealer and leave for 1 h at to be examined make a fresh subculture from the same strain
room temperature to allow toxin neutralisation to occur. of B. pertussis and prepare a suspension of the harvested
50 µL of cell suspension containing approximately organisms with an opacity corresponding to about 100 LD 50
4 x 10 5 cells/mL is added to each well. The plates are in each challenge dose. Prepare 3 dilutions of the challenge
sealed and incubated at 37 °C for 6 days. Check for suspension.
microbial contamination by microscopic examination. 10 µL Determination of potency
of thyazolyl blue MTT solution is added to each well. Prepare 3 serial dilutions of the vaccine to be examined and
The plates are incubated at 37 °C for a further 2-4 h. Then, 3 similar dilutions of the reference preparation such that in
the medium is removed and 100 µL of extraction buffer each the intermediate dilution may be expected to protect
pH 4.7 is added to each well. The plates are incubated at about 50 per cent of the mice from the lethal effects of the
37 °C and left overnight to aid the extraction process. Once challenge dose of B. pertussis. Suggested doses are 1/8, 1/40
extraction and solubilisation is complete, plates are visually and 1/200 of the human dose of the vaccine to be examined
examined or read at 570 nm. and 0.5 IU, 0.1 IU and 0.02 IU of the reference preparation,
Blue/black wells are recorded as negative (all the cells are each dose being contained in a volume not exceeding
alive, toxin neutralisation by antitoxin) and white or 0.5 mL. Allocate the 6 dilutions, one to each of the groups of
colourless wells indicate dead cells (no toxin neutralisation) not fewer than 16 mice, and inject intraperitoneally into each
and are recorded as positive. mouse one dose of the dilution allocated to its group. After
The potency of the test antitoxin is obtained by comparing 14 - 17 days inject intracerebrally into each animal in the
the last well of the reference antitoxin preparation showing groups of not fewer than 16, one dose of the challenge
neutralisation of the toxin, with the last well of the antitoxin suspension. Allocate the challenge suspension and the
preparation demonstrating the same effect. The neutralising 3 dilutions made from it, one to each of the groups of
antibody titre of the sample being examined can be 10 mice, and inject intracerebrally one dose of each
calculated by multiplication of the dilution factor with total suspension into each mouse in the group to which that
number of International Units per millilitre of the reference suspension is allocated. Exclude from consideration any mice
preparation at the end point. The test is valid if all the cells that die within 48 h of challenge. Count the number of mice
in the toxin control are dead and reference antitoxin gives a surviving in each of the groups after 14 days. Calculate the
neutralisation in at least the first 2 dilutions tested. potency of the vaccine to be examined relative to the potency
of the reference preparation on the basis of the numbers of
2. Assay of Pertussis Vaccine (Whole Cell) animals surviving in each of the groups of not fewer than 16.
(Ph. Bur. method 2. 7. 7) The test is not valid unless:
The potency of pertussis vaccine (whole cell) is determined - for both the vaccine to be examined and the reference
by comparing the dose necessary to protect mice against the preparation, the 50 per cent protective dose lies between
effects of a lethal dose of Bordetella pertussis, administered the largest and the smallest doses given to the mice;
intracerebrally, with the quantity of a reference preparation, - the number of animals that die in the 4 groups of 10
calibrated in International Units, needed to give the same injected with the challenge suspension and its dilutions
protection. indicates that the challenge dose is approximately
The International Unit is the activity contained in a stated 100 LD 50; and
amount of the International Standard which consists of a - the statistical analysis shows no deviation from linearity or
quantity of dried pertussis vaccine. The equivalence in parallelism.
International Units of the International Standard is stated by The test may be repeated but when more than one test is
the World Health Organization. performed the results of all valid tests must be combined.
Selection and distribution of the test animals 3. Assay of Tetanus Vaccine (Adsorbed)
Use in the test healthy mice less than 5 weeks old of a (Ph. Bur. method 2.7.8)
suitable strain from the same stock, the difference in mass
between the heaviest and the lightest being not greater than The potency of tetanus vaccine is determined by
5 g. Distribute the mice in 6 groups of not fewer than 16 and administration of the vaccine to animals (guinea-pigs or
4 groups of 10. The mice must all be of the same sex or the mice) followed either by challenge with tetanus toxin
males and females distributed equally between the groups. (method A or B) or by determination of the titre of
antibodies against tetanus toxoid in the serum of the guinea-
Selection of the challenge strain and preparation of the pigs (method C). In both cases, the potency of the vaccine is
challenge suspension calculated by comparison with a reference vaccine, calibrated
Select a suitable strain of B. pertussis capable of causing the in International Units. For methods A and B, in countries
death of mice within 14 days of intracerebral injection. where the paralysis method is not obligatory, the
If more than 20 per cent of the mice die within 48 h of the LD 50 method may be used. For the LD 50 method, the
injection the strain is not suitable. Make one subculture from number of animals and the procedure are identical to those
the strain and suspend the harvested B. pertussis in a solution described for the paralysis method, but the end-point is the
containing 10 g/L of casein R hydrolysate and 6 g/L of sodium death of the animal rather than paralysis.
chloride Rand having a pH of 7.0 to 7.2 or in another
The International Unit is the activity contained in a stated
suitable solution. Determine the opacity of the suspension.
amount of the International Standard for tetanus toxoid
Prepare a series of dilutions in the same solution and allocate
(adsorbed). The equivalence in International Units of the
each dilution to a group of 10 mice. Inject intracerebrally
International Standard is stated by the World Health
into each mouse a dose (0.02 mL or 0.03 mL) of the
Organization.
2023 Appendix XIV K V-A509

Tetanus vacane (adsorbed) ERP is calibrated in International METHOD A. CHALLENGE TEST IN GUINEA-PIGS
Units with reference to the International Standard. SELECTION AND DISTRIBUTION OF THE TEST
The method chosen for the assay of tetanus vaccine ANIMALS
(adsorbed) depends on the intended purpose. Method A Use in the test healthy guinea-pigs from the same stock, each
or Bis used: weighing 250-350 g. Use guinea-pigs of the same sex or with
1. during development of a vaccine, to assay batches males and females equally distributed between the groups.
produced to validate the production; Distribute the guinea-pigs in not fewer than 6 equal groups;
use groups containing a number of animals sufficient to
2. wherever revalidation is needed following a significant
obtain results that fulfil the requirements for a valid assay
change in the manufacturing process.
prescribed below. If the activity of the challenge toxin has to
Method A or B may also be used for the routine assay of be determined, include 3 further groups of 5 guinea-pigs as
batches of vaccine, but in the interests of animal welfare, unvaccinated controls.
method C is used wherever possible.
SELECTION OF THE CHALLENGE TOXIN
Method C may be used, except as specified under 1 and 2 Select a preparation of tetanus toxin containing not less than
above, after verification of the suitability of the method for 50 times the 50 per cent paralytic dose per millilitre. If the
the product. For this purpose, a suitable number of batches challenge toxin preparation has been shown to be stable, it is
(usually 3) are assayed by method C and method A or B. not necessary to verify the paralytic dose for every assay.
Where different vaccines (monovalent or combinations) are
prepared from tetanus toxoid of the same origin and with PREPARATION OF THE CHALLENGE TOXIN
comparable levels (expressed in L£1mL) of the same tetanus SOLUTION
toxoid, suitability demonstrated for the combination with the Immediately before use, dilute the challenge toxin with a
highest number of components can be assumed to be valid suitable diluent (for example, peptone buffered saline
for combinations with fewer components and for monovalent solution pH 7 .4) to obtain a stable challenge toxin solution
vaccines. Any combinations containing a whole-cell pertussis containing approximately 50 times the 50 per cent paralytic
component or containing haemophilus type b conjugate dose per millilitre. If necessary, use portions of the challenge
vaccine with tetanus toxoid in the same vial must always be toxin solution diluted 1 to 16, 1 to 50 and 1 to 160 with the
assessed separately. same diluent to determine the activity of the toxin.
For combinations containing diphtheria and tetanus DILUTION OF THE TEST AND REFERENCE
components, the serological assay (method C) can be PREPARATIONS
performed with the same group of animals used for the Using a 9 g/L solution of sodium chloride R, prepare dilutions
serological assay of the diphtheria vaccine (adsorbed) (2. 7. 6) of the vaccine to be examined and of the reference
when the common immunisation conditions for the tetanus preparation, such that for each, the dilutions form a series
and the diphtheria components (for example, doses, differing by not more than 2.5-fold steps and in which the
duration) have been demonstrated to be valid for the intermediate dilutions, when injected subcutaneously at a
combined vaccine. dose of 1.0 mL per guinea-pig, protect approximately
50 per cent of the animals from the paralytic effects of the
The design of the assays described below uses multiple
subcutaneous injection of the quantity of tetanus toxin
dilutions for the test and reference preparations. Based on
prescribed for this test.
the potency data obtained in multiple-dilution assays, it may
be possible to reduce the number of animals needed to IMMUNISATION AND CHAUENGE
obtain a statistically significant result by applying a simplified Allocate the dilutions, 1 to each of the groups of guinea-pigs,
model such as a single dilution for both test and reference and inject subcutaneously 1.0 mL of each dilution into each
preparations. Such a model enables the analyst to determine guinea-pig in the group to which that dilution is allocated.
whether the potency of the test preparation is significantly After 28 days, inject subcutaneously into each animal 1.0 mL
higher than the minimum required, but does not give of the challenge toxin solution (containing 50 times the
information on the dose-response curves and their linearity, 50 per cent paralytic dose).
parallelism and significant slope. The simplified model allows DETERMINATION OF THE ACTIV/1Y OF THE
for a considerable reduction in the number of animals CHALLENGE TOXIN
required and must be considered by each analyst in If necessary, allocate the 3 dilutions made from the challenge
accordance with the provisions of the European Convention toxin solution, 1 to each of the 3 groups of 5 guinea-pigs,
for the Protection of Vertebrate Animals Used for and inject subcutaneously 1.0 mL of each solution into each
Experimental and Other Scientific Purposes. guinea-pig in the group to which that solution is allocated.
Where a single-dilution assay is used, production and test The activity and stability of the challenge toxin are
consistency over time are monitored via suitable indicators determined by carrying out a suitable number of
and by carrying out a full multiple-dilution assay periodically, determinations of the 50 per cent paralytic dose. It is then
for example every 2 years. For serological assays, suitable not necessary to repeat the determination for each assay.
indicators to monitor test consistency are: READING AND INTERPRETATION OF RESULTS
- the mean and standard deviation of relative antitoxin Examine the guinea-pigs twice daily. Remove and euthanise
titres or scores of the serum samples obtained after all animals showing definite signs of tetanus paralysis. Count
administration of a fixed dose of the vaccine reference the number of guinea-pigs without paralysis 5 days after
preparation; injection of the challenge toxin. Calculate the potency of the
- the antitoxin titres or scores of run controls (positive and vaccine to be examined relative to the potency of the
negative serum samples); reference preparation on the basis of the proportion of
- the ratio of antitoxin titres or scores for the positive serum challenged animals without paralysis in each group of
control to the serum samples corresponding to the vaccinated guinea-pigs, using the usual statistical methods
reference vaccine. (for example, 5.3).
V-A510 Appendix XIV K 2023

REQUIREMENTS FOR A VALID ASSAY the challenge toxin solution (containing 50 times the
The test is not valid unless: 50 per cent paralytic dose).
- for both the vaccine to be examined and the reference DETERMINATION OF THE ACTIVITY OF THE
preparation, the 50 per cent protective dose lies between CHALLENGE TOXIN
the largest and smallest doses of the preparations given to If necessary, allocate the 3 dilutions made from the challenge
the guinea-pigs; toxin solution, 1 to each of the 3 groups of not fewer than
- where applicable, the number of paralysed animals in the 5 mice, and inject subcutaneously 0.5 rnL of each solution
3 groups of 5 injected with the dilutions of the challenge into each mouse in the group to which that solution is
toxin solution indicates that the challenge was allocated.
approximately 50 times the 50 per cent paralytic dose; READING AND INTERPRETATION OF RESULTS
- the confidence limits (P = 0.95) are not less than Examine the mice twice daily. Remove and euthanise all
50 per cent and not more than 200 per cent of the animals showing definite signs of tetanus paralysis. Count the
estimated potency; number of mice without paralysis 4 days after injection of the
- the statistical analysis shows a significant slope and no challenge toxin. Calculate the potency of the vaccine to be
deviation from linearity and parallelism of the dose- examined relative to the potency of the reference preparation
response curves (chapter 5. 3 describes possible on the basis of the proportion of challenged animals without
alternatives if significant deviations are observed). paralysis in each group of vaccinated mice, using the usual
The test may be repeated but when more than 1 test is statistical methods (for example, 5.3).
performed the results of all valid tests must be combined in REQUIREMENTS FOR A VALID ASSAY
the estimate of potency. The test is not valid unless:
METHOD B. CHALLENGE TEST IN MICE - for both the vaccine to be examined and the reference
SELECTION AND DISTRIBUTION OF THE TEST preparation, the 50 per cent protective dose lies between
ANIMALS the largest and smallest doses of the preparations given to
Use in the test healthy mice from the same stock, about the mice;
5 weeks old and from a strain shown to be suitable. - where applicable, the number of paralysed animals in the
Use mice of the same sex or with males and females equally 3 groups of not fewer than 5 injected with the dilutions of
distributed between the groups. Distribute the mice in not the challenge toxin solution, indicates that the challenge
fewer than 6 equal groups; use groups containing a number dose was approximately 50 times the 50 per cent paralytic
of animals sufficient to obtain results that fulfil the dose;
requirements for a valid assay prescribed below. If the - the confidence limits (P = 0.95) are not less than
challenge toxin to be used has not been shown to be stable 50 per cent and not more than 200 per cent of the
or has not been adequately standardised, include 3 further estimated potency;
groups of not fewer than 5 mice to serve as unvaccinated - the statistical analysis shows a significant slope and no
controls. deviation from linearity and parallelism of the dose-
SELECTION OF THE CHALLENGE TOXIN response curves (chapter 5.3 describes possible
Select a preparation of tetanus toxin containing not less than alternatives if significant deviations are observed).
100 times the 50 per cent paralytic dose per millilitre. If the The test may be repeated but when more than 1 test is
challenge toxin preparation has been shown to be stable, it is performed the results of all valid tests must be combined in
not necessary to verify the paralytic dose for every assay. the estimate of potency.
PREPARATION OF THE CHALLENGE TOXIN METHOD C. DETERMINATION OF ANTIBODIES
SOLUTION IN GUINEA-PIGS
Immediately before use, dilute the challenge toxin with a SELECTION AND DISTRIBUTION OF THE TEST
suitable diluent (for example, peptone buffered saline ANIMALS
solution pH 7.4) to obtain a stable challenge toxin solution Use in the test healthy guinea-pigs from the same stock, each
containing approximately 50 times the 50 per cent paralytic weighing 250-350 g. Use guinea-pigs of the same sex or with
dose in 0 .5 mL. If necessary, use portions of the challenge males and females equally distributed between the groups.
toxin solution diluted 1 to 16, 1 to 50 and 1 to 160 with the Distribute the guinea-pigs in not fewer than 6 equal groups;
same diluent to determine the activity of the toxin. use groups containing a number of animals sufficient to
DILUTION OF THE TEST AND REFERENCE obtain results that fulfil the requirements for a valid assay
PREPARATIONS prescribed below. Use a further group of non-vaccinated
Using a 9 g/L solution of sodium chloride R, prepare dilutions guinea-pigs of the same origin to provide a negative serum
of the vaccine to be examined and of the reference control. If test consistency has been demonstrated, a
preparation, such that for each, the dilutions form a series reference negative serum control may be used.
differing by not more than 2.5-fold steps and in which the REFERENCE PREPARATION
intermediate dilutions, when injected subcutaneously at a Use a suitable reference preparation such as tetanus vaccine
dose of 0.5 mL per mouse, protect approximately (adsorbed) ERP or a batch of vaccine shown to be effective in
50 per cent of the animals from the paralytic effects of the clinical studies, or a batch representative thereof, and which
subcutaneous injection of the quantity of tetanus toxin has been calibrated in International Units with reference to
prescribed for this test. tetanus vaccine ( adsorbed) ERP or the International Standard
IMMUNISATION AND CHALLENGE for tetanus toxoid (adsorbed).
Allocate the dilutions, 1 to each of the groups of mice, and DILUTION OF THE TEST AND REFERENCE
inject subcutaneously 0.5 mL of each dilution into each PREPARATIONS
mouse in the group to which that dilution is allocated. After Using a 9 g/L solution of sodium chloride Ras diluent, prepare
28 days, inject subcutaneously into each animal 0.5 mL of serial dilutions of the vaccine to be examined and the
reference preparation; series differing by 2.5- to 5-fold steps
2023 Appendix XIV K V-AS 11

have been found to be suitable. Use not fewer than - T5: tetanus seizures, continuous tonic spasm of muscles;
3 dilutions within the range of, for example, 0.5-16 IU/mL - D: death.
for each series. Use the dilutions for immunisation preferably
METHOD B. CHALLENGE TEST IN MICE
within 1 h of preparation. Allocate 1 dilution to each group
READING AND INTERPRETATION OF RESULTS
of guinea-pigs.
In order to minimise suffering in the test animals, it is
IMMUNISATION recommended to note the degree of paralysis on a scale such
Inject subcutaneously to each guinea-pig 1.0 mL of the as that shown below. The scale gives typical signs when
dilution allocated to its group. injection of the challenge toxin is made in the dorsal region,
BLOOD SAMPLING close to one of the hind legs. Grade T3 is taken as the
35-42 days after immunisation, take a blood sample from end-point, but with experience grade T2 can be used instead.
each vaccinated and control guinea-pig using a suitable Tetanus toxin produces in the toxin-injected hind leg paresis
method. followed by paralysis that can be recognised at an early stage.
The tetanus grades in mice are characterised by the following
PREPARATION OF SERUM SAMPLES
Avoid frequent freezing and thawing of serum samples. signs:
- Tl: slight stiffness of toxin-injected hind leg, only
To avoid microbial contamination, it is preferable to carry
out manipulations in a laminar-flow cabinet. observed when the mouse is lifted by the tail;
- T2: paresis of the toxin-injected hind leg, which still can
DETERMINATION OF ANTIBODY TITRE function for walking;
Determine the relative antibody titre or score of each serum - T3: paralysis of the toxin-injected hind leg, which does
sample by a suitable immunochemical method (2. 7.1). not function for walking;
The methods shown below (enzyme-linked immunosorbent - T4: the toxin-injected hind leg is completely stiff with
assay (ELISA) and toxin-binding inhibition (ToBI)) have immovable toes;
been found to be suitable. - T5: tetanus seizures, continuous tonic spasm of muscles;
CALCUIATION OF POTENCY - D: death.
Calculate the potency of the vaccine to be examined in
METHOD C. DETERMINATION OF ANTIBODIES
International Units relative to the reference preparation,
IN GUINEA-PIGS
using the usual statistical methods (for example, 5.3).
PREPARATION OF SERUM SAMPLES
REQUIREMENTS FOR A VALID ASSAY For the preparation of serum samples, the following
The test is not valid unless: technique has been found to be suitable. Invert the tubes
- the confidence limits (P = 0.95) are not less than containing blood samples 6 times and allow to stand at
50 per cent and not more than 200 per cent of the 37 °C for 2 h, then at 4 °C for 2 h. Centrifuge at room
estimated potency; temperature at 800 g for 20 min. Transfer the serum to
- the statistical analysis shows a significant slope and no sterile tubes and store at a temperature below -20 °C.
deviation from linearity and parallelism of the dose- At least a 40 per cent yield of serum is obtained by this
response curves (chapter 5. 3 describes possible procedure.
alternatives if significant deviations are observed).
DETERMINATION OF ANTIBODY TITRE
The test may be repeated but when more than 1 test is The ELISA and ToBI tests shown below are given as
performed the results of all valid tests must be combined in examples of immunochemical methods that have been found
the estimate of potency. to be suitable for the determination of antibody titre.
The following section is published for information. Determination of antibody titre in guinea-pig serum by
enzyme-linked immunosorbent assay (ELISA)
ASSAY OF TETANUS VACCINE Dilutions of test and reference sera are made on ELISA
(ADSORBED): GUIDELINES plates coated with tetanus toxoid. A positive guinea-pig
serum control and a negative guinea-pig serum control are
METHOD A. CHALLENGE TEST IN GUINEA-PIGS
included on each plate to monitor the assay performance.
READING AND INTERPRETATION OF RESULTS
Peroxidase-conjugated rabbit or goat antibody directed
In order to minimise suffering in the test animals, it is
against guinea-pig-IgG is added, followed by a peroxidase
recommended to note the degree of paralysis on a scale such
substrate. Optical density is measured and the relative
as that shown below. The scale gives typical signs when
antibody titre is calculated using the usual statistical methods
subcutaneous injection of the challenge toxin is made mid-
(for example, 5.3).
ventrally, directly behind the sternum with the needle
pointing towards the neck of the guinea-pig. Grade T3 is Reagents and equipment
taken as the end-point, but with experience grade T2 can be - ELISA plates: 96 wells, columns 1-12, rows A-H.
used instead. Tetanus toxin produces in at least 1 of the - Clostridium tetani guinea-pig antiserum (for vaccines-human
forelimbs paralysis that can be recognised at an early stage. use) BRP (positive control serum).
The tetanus grades in guinea-pigs are characterised by the - Peroxidase confugate. Peroxidase-conjugated rabbit or goat
following signs: antibody directed against guinea-pig IgG.
- Tl: slight stiffness of 1 forelimb, but difficult to observe; - Tetanus toxoid.
- T2: paresis of 1 forelimb which still can function; - Carbonate coating buffer pH 9. 6. Dissolve 1.59 g of
- T3: paralysis of 1 forelimb. The animal moves reluctantly, anhydrous sodium carbonate Rand 2.93 g of sodium
the body is often slightly banana-shaped owing to hydrogen carbonate R in 1000 mL of water R. Distribute
scoliosis; into 150 mL bottles and sterilise by autoclaving at 121 °C
- T4: the forelimb is completely stiff and the toes are for 15 min.
immovable. The muscular contraction of the forelimb is - Phosphate-buffered saline pH 7. 4 (PBS). Dissolve with
very pronounced and usually scoliosis is observed; stirring 80.0 g of sodium chloride R, 2.0 g of potassium
dihydrogen phosphate R, 14.3 g of disodium hydrogen
V-A512 Appendix XIV K 2023

phosphate dihydrate Rand 2.0 g of potassium chloride R in Reagents and equipment

1000 mL of water R. Store at room temperature to - Round-bottomed, rigid polystyrene microtitre plates.
prevent crystallisation. Dilute to 10 times its volume with - Flat-bottomed ELISA plates.
water R before use. - Tetanus toxin or tetanus toxoid.
- Citric acid solution. Dissolve 10.51 g of citric acid - Clostridium tetani guinea-pig antiserum (for vaccines-human
monohydrate R in 1000 mL of water R and adjust the use) ERP (positive control serum).
solution to pH 4.0 with a 400 g/L solution of sodium - Equine anti-tetanus lgG.
hydroxide R. - Peroxidase-conjugated equine anti-tetanus lgG.
- Washing buffer. PBS containing 0.5 g/L of - Carbonate buffer pH 9.6. Dissolve 1.5 g of anhydrous
polysorbate 20 R. sodium carbonate R, 2.39 g of sodium hydrogen carbonate R
- Diluent block buffer. PBS containing 0.5 g/L of and 0.2 g of sodium azide R in 1000 mL of water R,
polysorbate 20 Rand 25 g/L of dried skimmed milk. adjust to pH 9.6 and autoclave at 121 °C for 20 min.
- Peroxidase substrate. Shortly before use, dissolve 10 mg of - Sodium acetate buffer pH 5.5. Dissolve 90.2 g of anhydrous
diammonium 2,2 '-azinobis (3-ethylbenzothiazoline-6- sodium acetate R in 900 mL of water R, adjust to pH 5.5
sulfonate) R (ABTS) in 20 mL of citric acid solution. using a saturated solution of citric acid monohydrate R and
Immediately before use add 5 µL of strong hydrogen dilute to 1000 mL with water R.
peroxide solution R. - Phosphate-buffered saline pH 7.2 (PBS). Dissolve 135.0 g
Method of sodium chloride R, 20.55 g of disodium hydrogen
The description below is given as an example of a suitable phosphate dihydrate Rand 4.80 g of sodium dihydrogen
phosphate monohydrate R in water R and dilute to 15 L
plate layout but others may be used. Wells lA-H are for
negative control serum and wells 2A-H and 12A-H are for with the same solvent. Autoclave at 100 °C for 60 min.
- Diluent buffer. PBS containing 5 g/L of bovine albumin R
positive control serum for assay monitoring. Wells 3-1 lA-H
are for test samples. and 0.5 g/L of polysorbate 80 R.
- Block buffer. PBS containing 5 g/L of bovine albumin R.
Coat each well of the ELISA plates with 100 µL of tetanus - Tetramethylbenzidine solution. 6 g/L solution of
toxoid solution (0.5 Lfi'mL in carbonate coating buffer tetramethylbenzidine R in ethanol (96 per cent) R.
pH 9.6). Allow to stand overnight at 4 °C in a humid The substance dissolves within 30-40 min at room
atmosphere. To avoid temperature gradient effects, do not temperature.
stack more than 4 plates high. On the following day, wash - Peroxidase substrate. Mix 90 mL of water R, 10 mL of
the plates thoroughly with washing buffer. Block the plates sodium acetate buffer pH 5.5, 1.67 mL of
by addition of 100 µL of diluent block buffer to each well. tetramethylbenzidine solution and 20 µL of strong
Incubate in a humid atmosphere at 37 °C for 1 h. Wash the hydrogen peroxide solution R.
plates thoroughly with washing buffer. Place 100 µL of - Washing solution. Tap water containing 0.5 g/L of
diluent block buffer in each well of the plates, except those of polysorbate 80 R.
row A. Prepare suitable dilutions of negative control serum,
positive control serum (from about 0.01 IU/mL) and test Method
sera. Allocate the negative control serum to column 1, Block the microtitre plates by placing in each well 150 µL of
positive control serum to columns 2 and 12 and test sera to block buffer. Cover the plates with a lid or sealer. Incubate in
columns 3-11 and add 100 µL of each serum to the first a humid atmosphere at 37 °C for 1 h. Wash the plates
2 wells of the column to which it is allocated. Using a thoroughly with washing solution. Place 100 µL of PBS in
multichannel micropipette, make twofold serial dilutions from each well. Place 100 µL of reference guinea-pig tetanus
row B down the plate to row H, by transferring 100 µL from antitoxin in the first well of a row. Place 100 µL of undiluted
one well to the next. Discard 100 µL from the last row so test sera in the first well of the required number of rows.
that all wells contain 100 µL. Incubate at 37 °C for 2 h. Using a multichannel micropipette, make twofold serial
Wash thoroughly with washing buffer. Prepare a suitable dilutions across the plate (up to column 10), by transferring
dilution (a 2000-fold dilution has been found to be suitable) 100 µL from one well to the next. Discard 100 µL from the
of peroxidase conjugate in diluent block buffer and add last column so that all wells contain 100 µL. Prepare a
100 µL to each well. Incubate at 37 °C in a humid 0.1 Lfi'mL solution of tetanus toxin or toxoid using PBS as
atmosphere for 1 h. Wash the plates thoroughly with washing diluent. Add 40 µL of this solution to each well except those
buffer. Add 100 µL of peroxidase substrate to each well. of column 12. The wells of column 11 are a positive control.
Allow to stand at room temperature, protected from light, for Add 40 µL of PBS to the wells of column 12 (negative
30 min. Read the plates at 405 nm in the same order as control). Shake the plates gently and cover them with lids.
addition of substrate was made. Coat the ELISA plates: immediately before use make a
suitable dilution of equine anti-tetanus IgG in carbonate
Determination of antibody titre in guinea-pig serum by
buffer pH 9.6 and add 100 µL to each well. Incubate the
toxin- or toxoid-binding inhibition (ToBI)
2 series of plates overnight in a humid atmosphere at 37 °C.
Tetanus toxin or toxoid is added to serial dilutions of test
To avoid temperature gradient effects, do not stack more
and reference sera; the serum/antigen mixtures are incubated
than 4 plates high. Cover the plates with lids. On the
overnight. To determine unbound toxin or toxoid, the
following day, wash the ELISA plates thoroughly with
mixtures are transferred to an ELISA plate coated with
washing solution. Block the plates by placing in each well
tetanus antitoxin. Peroxidase-conjugated equine anti-tetanus
125 µL of block buffer. Incubate at 37 °C in a humid
IgG is added followed by a peroxidase substrate. Optical
atmosphere for 1 h. Wash the plates thoroughly with washing
density is measured and the antibody titre is calculated using
solution. Transfer 100 µL of the pre-incubation mixture from
the usual statistical methods (for example, 5.3). A positive
the polystyrene plates to the corresponding wells of the
control serum and a negative control serum are included on
ELISA plates, starting with column 12 and then continuing
each plate to monitor assay performance.
from 1 to 11. Cover the plates with a lid. Incubate at 37 °C
in a humid atmosphere for 2 h. Wash the ELISA plates
 2023 Appendix XIV K V-A513

thoroughly with washing solution. Make a suitable dilution Calculations

(a 4000-fold dilution has been found to be suitable) of the Carry out the calculations by the usual statistical methods for
peroxidase-conjugated equine anti-tetanus IgG in diluent an assay with a quanta! response (5.3).
buffer. Add 100 µL of the dilution to each well and cover the From the distribution of reaction levels measured on all the
plates with a lid. Incubate at 37 °C in a humid atmosphere sera in the unvaccinated group, determine the maximum
for 1.5 h. Wash the ELISA plates thoroughly with washing reaction level that can be expected to occur in an
solution. Add 100 µL of peroxidase substrate to each well. unvaccinated animal for that particular assay. Any response
A blue colour develops. Incubate the plates at room in vaccinated animals that exceeds this level is by definition a
temperature. Stop the reaction at a given time (within seroconversion.
10 min) by the addition of 100 µL of a 2 M solution of
Make a suitable transformation of the percentage of animals
sulfuric acid prepared from sulfuric acid R to each well in the showing seroconversion in each group (for example, a probit
same order as the addition of substrate. The colour changes transformation) and analyse the data according to a parallel-
from blue to yellow. Measure the absorbance at 450 nm line log dose-response model. Determine the potency of the
immediately after addition of the sulfuric acid or maintain test preparation relative to the reference preparation.
the plates in the dark until reading.
Validity conditions
4. Assay of Hepatitis A Vaccine The test is not valid unless:
(Ph. Bur. method 2. 7.14) - for both the test and the reference vaccine, the ED 50 lies
The assay of hepatitis A vaccine is carried out either in vitro, between the smallest and the largest doses given to the
by an immunochemical determination of the antigen content animals;
(method A), or in vivo, by comparing under given conditions - the statistical analysis shows no significant deviation from
its capacity to induce specific antibodies in mice with the linearity or parallelism;
same capacity of a reference preparation (method B). - the confidence limits (P = 0.95) are not less than
33 per cent and not more than 300 per cent of the
METHOD A. IN VITRO ASSAY estimated potency.
Carry out an immunochemical determination (2. 7.1) of the
hepatitis A antigen content. Potency requirement
The upper confidence limit (P = 0.95) of the estimated
Enzyme-linked immunosorbent assay (ELISA) using
relative potency is not less than 1. 0.
monoclonal antibodies specific for the detection of a
hepatitis A epitope that induces neutralising antibodies has 5. Assay of Hepatitis B Vaccine (rDNA)
been shown to be suitable. (Ph. Bur. method 2. 7.15)
Suitable numbers of dilutions of the vaccine to be examined The assay of hepatitis B vaccine (rDNA) is carried out either
and the reference preparation are used, and the potency of in vivo, by comparing in given conditions its capacity to
the vaccine to be examined is calculated using the usual induce specific antibodies against hepatitis B surface antigen
statistical methods (5.3). (HBsAg) in mice or guinea-pigs with the same capacity of a
The antigen content is within the limits approved for the reference preparation, or in vitro, by an immunochemical
particular product. determination of the antigen content.
Hepatitis A vaccine (inactivated, non-adsorbed) ERP is IN VIVO ASSAY
calibrated in International Units and may be used as a Selection and distribution of the test animals
reference preparation. Use in the test healthy mice from the same stock, about
METHOD B. IN VIVO ASSAY 5 weeks old. The strain of mice used for this test must give a
The test in mice shown below is given as an example of a significant slope for the dose-response curve to the antigen;
method that has been found suitable for a given vaccine; mice with haplotype H-2q or H-2ct are suitable. Healthy
other validated methods may also be used. guinea-pigs weighing 300 g to 350 g (about 7 weeks old)
from the same stock are also suitable. Use animals of the
Selection and distribution of the test animals same sex. Distribute the animals in at least 7 equal groups of
Use in the test healthy mice from the same stock, about a number appropriate to the requirements of the assay.
5 weeks old and from a strain shown to be suitable.
Use animals of the same sex. Distribute the animals in at Determination of potency of the vaccine to be
least 7 equal groups of a number suitable for the examined
requirements of the assay. Using a 9 g/L solution of sodium chloride R containing the
aluminium adjuvant used for the vaccine or another
Determination of potency of the vaccine to be
appropriate diluent, prepare at least 3 dilutions of the vaccine
examined to be examined and matching dilutions of the reference
Using a 9 g/L solution of sodium chloride R containing the
preparation. Allocate the dilutions, 1 to each of the groups of
aluminium adjuvant used for the vaccine, prepare at least
animals, and inject intraperitoneally not more than 1.0 mL of
3 dilutions of the vaccine to be examined and matching each dilution into each animal in the group to which that
dilutions of the reference preparation. Allocate the dilutions
dilution is allocated. One group of animals remains
one to each of the groups of animals and inject unvaccinated and is injected intraperitoneally with the same
subcutaneously not more than 1.0 mL of each dilution into volume of diluent. After an appropriate time interval (for
each animal in the group to which that dilution is allocated. example, 4-6 weeks), anaesthetise and bleed the animals,
Maintain a group of unvaccinated controls, injected keeping the individual sera separate. Assay the individual sera
subcutaneously with the same volume of diluent. After for specific antibodies against HBsAg by a suitable
28-32 days, anaesthetise and bleed all animals, keeping the immunochemical method (2. 7.1).
individual sera separate. Assay the individual sera for specific
antibodies against hepatitis A virus by a suitable Calculations
immunochemical method (2. 7.1). Calculations are carried out by the usual statistical methods
for an assay with a quanta! response (5.3).
V-AS 14 Appendix XIV K 2023

From the distribution of reaction levels measured on all the The guinea-pig model allows for a further reduction in the
sera in the unvaccinated group, the maximum reaction level number of animals required and must be considered by each
that can be expected to occur in an unvaccinated animal for analyst in accordance with the provisions of the European
that particular assay is determined. Any response in Convention for the Protection of Vertebrate Animals Used
vaccinated animals that exceeds this level is by definition a for Experimental and Other Scientific Purposes.
seroconversion. Methods A and B described below are developed by testing
Make a suitable transformation of the percentage of animals multiple dilutions of the test vaccine and the reference
showing seroconversion in each group (for example, a probit vaccine or internal control (see Glossary), to determine which
transformation) and analyse the data according to a parallel- dilutions are suitable. Once the suitable dilutions have been
line log dose-response model. Determine the potency of the confirmed for a given vaccine, it is recommended, in
test preparation relative to the reference preparation. accordance with 3R principles (Replacement, Reduction,
Validity conditions Refinement), to apply a simplified model such as a single
The test is not valid unless: dilution for both the test vaccine and the reference vaccine or
- for both the test and the reference vaccine, the ED 50 lies internal control. Such a model enables the analyst to
between the smallest and the largest doses given to the determine whether the immunogenicity of the test vaccine is
animals; satisfactory.
- the statistical analysis shows no significant deviation from Suitable indicators to monitor the performance of the
linearity or parallelism; serological assay (single or multiple dilutions) are:
- the confidence limits (P = 0.95) are not less than - the geometric mean and geometric standard deviation of
33 per cent and not more than 300 per cent of the antibody titres in the serum samples obtained after
estimated potency. administration of a fixed dose of the reference vaccine or
Potency requirement internal control;
The upper confidence limit (P = 0.95) of the estimated - the antibody titres of run controls (positive control and
relative potency is not less than 1.0. negative serum samples).
It may be necessary to reconfirm the suitability of the
IN VITRO ASSAY selected dilution with a multiple-dilution assay, e.g. after
Carry out an immunochemical determination (2. 7.1) of major process changes or for investigational purposes.
antigen content with acceptance criteria validated against the
in vivo test. METHOD A. SEROLOGY IN MICE
Enzyme-linked immunosorbent assay (ELISA) and radio- The following test model is given as an example of a multiple-
immunoassay (RIA) using monoclonal antibodies specific for dilutwn assay which may fonn the basis for the establishment of a
protection-inducing epitopes of HBsAg have been shown to single-dilution assay.
be suitable. Suitable numbers of dilutions of the vaccine to Selection and distribution of the test animals
be examined and the reference preparation are used and a Use healthy mice (for example, CDI strain from the same
parallel-line model is used to analyse the data, which may be stock and more than 5 weeks old). Distribute the animals
suitably transformed. Kits for measuring HBsAg in vitro are into not fewer than 6 equal groups; use groups containing a
commercially available and it is possible to adapt their test number of animals sufficient to meet the pre-defined criteria
procedures for use as an in vitro potency assay. for variability of the antibody responses prescribed under
The acceptance criteria are approved for a given reference Calculations and validity criteria. Use serial dilutions of the
preparation by the competent authority in light of the test vaccine and the reference vaccine or internal control and
validation data. allocate each dilution to a group of mice. During validation
studies a further group of mice may be used as a negative
6. Assay of Pertussis Vaccine (Acellular) control by injecting the animals with diluent alone.
(Ph. Bur. method 2. 7.16) Immunisation
The assay of acellular pertussis vaccine measures the capacity Inject intraperitoneally or subcutaneously into each mouse
of the vaccine to induce the formation of specific antibodies 0.5 mL of the dilution allocated to its group.
in mice or guinea-pigs. Antibody titres for each antigen are
Collection of serum samples
determined using a suitable immunochemical method (2. 7.1) 4-5 weeks after vaccination, bleed the mice individually
such as enzyme-linked immunosorbent assay (ELISA). under anaesthesia. Store the sera at -20 °C until used for
The assay results can be expressed: antibody determination. Avoid frequent freezing and thawing
- either as a ratio of the geometric mean titre (GMT) of of serum samples.
antibodies produced following administration of the test
Reference antiserum
vaccine to the GMT of antibodies produced following
A reference antiserum of assigned activity serves as the basis
administration of a reference vaccine examined in parallel
for calculation of the antibody titres in the test sera. Bordetella
(relative potency assay);
pertussis mouse antiserum ERP is suitable for use as a reference
- or directly as a GMT of antibodies induced by the test
antiserum.
vaccine (geometric mean unit assay or GMU assay).
For combinations containing pertussis components together Antibody determination
with diphtheria and tetanus components, the serological assay Assay the individual sera for content of specific antibodies
in guinea-pigs can be performed with the same group of against each acellular pertussis antigen using a validated
animals used for the serological assay of diphtheria vaccine method such as the ELISA test shown below.
(adsorbed) (2. 7. 6) and of tetanus vaccine (adsorbed) (2. 7.8) ELISA test
when the common immunisation conditions for all Microtitre plates (poly(vinyl chloride) or polystyrene as
components (for example, doses, duration) have been appropriate for the specific antigen) are coated with the
demonstrated to be valid for the combined vaccine. purified antigen at a concentration of 100 ng per well. After
washing, unreacted sites are blocked by incubating the plates
2023 Appendix XIV K V-AS 15

with a solution of bovine serum albumin and then washed. plates are washed. A suitable solution of enzyme-conjugated
2-fold dilutions of sera from individual mice immunised with anti-guinea-pig IgG antibody is added to each well and
the test vaccine or either the reference vaccine or the internal incubated at 37 °C for 1 h. After washing, a chromogenic
control are prepared on the plates. Reference antiserum is substrate is added from which the bound enzyme conjugate
included on each plate. After incubation at 22-25 °C for 1 h, liberates a chromophore that can be quantified by
the plates are washed. A suitable solution of enzyme- measurement of absorbance (2.2.25).
conjugated anti-mouse IgG antibody is added to each well CALCULATIONS AND VALIDITY CRITERIA
and incubated at 22-25 °C for 1 h. After washing, a
Relative potency assay
chromogenic substrate is added from which the bound
The antibody titres in the sera of mice or guinea-pigs
enzyme conjugate liberates a chromophore that can be
immunised with the test and reference vaccines are
quantified by measurement of absorbance (2.2.25).
determined for each acellular pertussis antigen using the
METHOD B. SEROLOGY IN GUINEA-PIGS reference antiserum. From the values obtained, the GMT
The following test model is given as an example of a multiple- ratio of the test vaccine in relation to the reference vaccine is
dilution assay which may form the basis for the establishment of a calculated for each antigen.
single-dilution assay. The relative potency assay is not valid unless:
Selection and distribution of the test animals - the number of responder animals for the test and
Use healthy guinea-pigs from the same stock, each weighing reference vaccines meets the pre-defined criteria;
250-350 g. Use guinea-pigs of the same sex or with males - the GMT for the reference vaccine is within the limits of
and females equally distributed between the groups. the control chart;
Distribute the guinea-pigs into not fewer than 6 equal - the variability of the antibody responses meets the pre-
groups; use groups containing a number of animals sufficient defined criteria.
to meet the pre-defined criteria for variability of the antibody GMU assay
responses prescribed under Calculations and validity criteria. The antibody titres in the sera of mice or guinea-pigs
During validation studies a further group of guinea-pigs may immunised with the internal control and test vaccine are
be used as a negative control by injecting the animals with determined for each acellular pertussis antigen using the
diluent alone. reference antiserum, and the GMTs are calculated for each
Dilution of the test and reference preparations antigen.
Using a 9 g/L solution of sodium chloride R as diluent, prepare The GMU assay is not valid unless:
serial dilutions of the test vaccine and the reference vaccine - the number of responder animals for the test vaccine and
or internal control; series differing by 2.5- to 5-fold steps internal control meets the pre-defined criteria;
have been found to be suitable. Use not fewer than 3 - the GMT for the internal control is within the limits of
dilutions within the range found to be suitable for all the the control chart;
components in the test vaccine. Use the dilutions for - the variability of the antibody responses meets the pre-
immunisation preferably within 1 h of preparation. Allocate 1 defined criteria.
dilution to each group of guinea-pigs.
GLOSSARY
Immunisation Internal control for GMU assay
Inject subcutaneously into each guinea-pig 1.0 mL of the A batch of vaccine shown to be representative of the current
dilution allocated to its group. manufacturing process and whose response in mice or guinea
Collection of serum samples pigs has been appropriately measured. The stability of the
5-6 weeks after immunisation, take a blood sample from each internal control shall be monitored and documented.
vaccinated and negative control guinea-pig using a suitable Reference vaccine for relative potency assay
method. Store the sera at -20 °C until used for antibody A batch of vaccine shown to be effective in clinical trials or a
determination. Avoid frequent freezing and thawing of serum batch representative thereof. The stability of the reference
samples. vaccine shall be monitored and documented.
Reference antiserum Responder animals
An in-house guinea-pig reference antiserum of assigned Immunised animals producing antibodies at a titre greater
activity serves as the basis for calculation of the antibody than a threshold defined during the development and
titres in the test sera. validation of the method.
Antibody determination
Assay the individual sera for content of specific antibodies The following section is published for information.
against each acellular pertussis antigen using a validated
method such as the ELISA test shown below. ASSAY OF PERTUSSIS VACCINE
ELISA test (ACELLULAR): GUIDELINES
Suitable 96-well rnicrotitre plates are coated with the purified
antigens (e.g. pertussis toxin (PT), pertactin (PRN), METHOD B. DETERMINATION OF ANTIBODIES
filamentous haemagglutinin (FHA) and/or fimbrial IN GUINEA-PIGS
agglutinogens (Fim 2/3)) representing components in the The ELISA shown below is given as an example of an
combined vaccine at a concentration of 200-400 ng per well. immunochernical method that has been found to be suitable.
After washing, unreacted sites are blocked by incubating the Determination of antibody titre by ELISA method for
plates with a suitable blocking buffer and then washed. 2-fold pertussis toxin (PT), filamentous
dilutions of sera from individual guinea-pigs immunised with haemagglutinin (FHA), fimbrial agglutinogens (Fim
the test vaccine or either the reference vaccine or the internal 2/3) and pertactin (PRN)
control are prepared on the plates. Reference antiserum is 2-fold dilutions of sera from test and reference vaccine or
included on each plate. After incubation at 37 °C for 1 h, the internal control are made on ELISA plates coated with
V-AS 16 Appendix XIV K 2023

acellular pertussis antigens (PRN, PT, FHA or Fim 2/3). Incubate at 37 °C for 2 h. Wash thoroughly with the washing
A guinea-pig reference antiserum and a negative guinea-pig buffer. Prepare a suitable dilution of the peroxidase conjugate
serum are included on each plate. Peroxidase-conjugated in the diluent block buffer and add 100 µL to each well.
rabbit or goat antibody directed against guinea-pig IgG is Incubate at 37 °C in a humid atmosphere for 1 h. Wash the
added, followed by a peroxidase substrate. plates thoroughly with the washing buffer. Add 100 µL of the
Reagents and equipment: peroxidase substrate to each well. Allow to stand at room
- ELISA plates: 96 wells, columns 1-12, rows A-H. temperature, protected from light, for 30 min. Read the
- Reference antiserum (guinea-pig). plates at 405 nm in the same order as the addition of
- Peroxidase conjugate. Peroxidase-conjugated rabbit or goat substrate was made.
antibody directed against guinea-pig IgG. 7. In vivo Assay of Poliomyelitis Vaccine
- Bordetella pertussis antigens (PRN, PT, FHA or Fim 2/3). (Inactivated)
- Carbonate coating buffer pH 9.6. Dissolve 1.59 g of (Ph. Bur. method 2.7.20)
anhydrous sodium carbonate Rand 2.93 g of sodium
The capacity of the vaccine to induce the formation of
hydrogen carbonate R in 1000 mL of water R. Distribute
neutralising antibodies is determined in vivo by one of the
into 150 mL bottles and sterilise by autoclaving at 121 °C
following methods.
for 15 min.
- Phosphate-buffered saline pH 7.4 (PBS). Dissolve with TEST IN CHICKS OR GUINEA-PIGS
stirring 80.0 g of sodium chloride R, 2.0 g of potassium Prepare a suitable series of not fewer than 3 dilutions of the
dihydrogen phosphate R, 14.3 g of disodium hydrogen vaccine to be examined using a suitable buffered saline
phosphate dihydrate R and 2.0 g of potassium chloride R in solution. Distribute either guinea-pigs weighing 250-350 g or
1000 mL of water R. Store at room temperature to 3-week-old chicks into groups of 10, and allocate a group to
prevent crystallisation. Dilute 10-fold with water R before each dilution of the vaccine. Inject intramuscularly into each
use. animal O.5 mL of the dilution intended for its group. Bleed
- Citric acid solution. Dissolve 10.51 g of citric acid the animals after 5-6 days and separate the sera. Examine the
monohydrate R in 1000 mL of water R and adjust to sera for the presence of neutralising antibodies, at a dilution
pH 4.0 with a 400 g/L solution of sodium hydroxide R. of 1 in 4, to each of the human poliovirus types 1, 2 and 3.
- Washing buffer. PBS containing 0.5 g/L of Mix 100 CCID50 of virus with the dilution of serum and
polysorbate 20 R. incubate at 37 °C for 4.5-6 h. Keep at 5 ± 3 °C for 12-18 h
- Diluent block buffer. PBS containing 0.5 g/L of where necessary for consistency of results. Inoculate the
polysorbate 20 R and 25 g/L of dried skimmed milk. mixtures into cell cultures for the detection of unneutralised
- Peroxidase substrate. Shortly before use, dissolve 10 mg of virus and read the results up to 7 days after inoculation.
diammonium 2,2 '-azinobis(3-ethylbenzothiazoline-6- For each group of animals, note the number of sera that have
sulfonate) R (ABTS) in 20 mL of the citric acid solution. neutralising antibodies and calculate the dilution of the
Immediately before use add 5 µL of strong hydrogen vaccine that gives an antibody response in 50 per cent of the
peroxide solution R. animals. Carry out in parallel a control test using a suitable
Method reference preparation. The vaccine complies with the test if a
The description below is given as an example of a suitable dilution of 1 to 100 or more produces an antibody response
plate layout but others may be used. Wells lA-H are used for for each of the 3 types of virus in 50 per cent of the animals.
negative control serum. Wells 2-12 A-Hare used for guinea- TEST IN RATS
pig reference antiserum (usually in 2 positions) and A suitable in vivo assay method consists of intramuscular
individual sera from guinea-pigs immunised with the test injection into the hind limb(s) of not fewer than 3 dilutions
vaccine, or either the reference vaccine or the internal of the vaccine to be examined and a reference vaccine, using
control. for each dilution a group of 10 specific pathogen-free rats of
Coat each well of the ELISA plates with 100 µL of the a suitable strain. Use of 4 dilutions is often necessary to
appropriate antigen solution (PT, FHA and Fim 2/3 at obtain valid results for all 3 serotypes. The number of
2 µg/mL and PRT at 4 µg/mL, in carbonate coating buffer animals per group must be sufficient to obtain results that
pH 9.6). Allow to stand overnight at 4 °C in a humid meet the validity criteria; groups of 10 rats are usually
atmosphere. To avoid temperature gradient effects, do not sufficient, although valid results may be obtained with fewer
stack more than 4 plates high. On the following day, wash animals per group. If animals of different sex are used, males
the plates thoroughly with the washing buffer. Block the and females are evenly distributed between all groups.
plates by addition of 150 µL of the diluent block buffer to A weight range of 175-250 g has been found to be suitable.
each well. Incubate in a humid atmosphere at 37 °C for 1 h. An inoculum of 0.5 mL per rat is used. The dose range is
Wash the plates thoroughly with the washing buffer. Place chosen such that a dose response to all 3 poliovirus types is
100 ~1L of the diluent block buffer in each well of the plates, obtained. Bleed the animals after 20-22 days. Neutralising
except those of row A. Prepare suitable dilutions of titres against all 3 poliovirus types are measured separately
individual test and reference vaccine or internal control using 100 CCID 50 of the Sabin strains as challenge viruses,
serum samples, reference antiserum and negative control Vero or Hep2 as indicator cells, and neutralisation conditions
serum samples. Allocate the negative control serum to of 3 hat 35-37 °C followed by 18 hat 2-8 °C where
column 1, the reference antiserum to at least 2 other necessary for consistency of results. Results are read following
columns and individual test and reference vaccine or internal fixation and staining after 7 days of incubation at 35 °C.
control sera to the remaining columns and add 100 µL of For a valid antibody assay, the titre of each challenge virus
each serum to the first 2 wells of the column to which it is must be shown to be within the range 10 CCID 50 to
allocated. Using a multichannel micropipette, make 2-fold 1000 CCID 50 and the neutralising antibody titre of a control
serial dilutions from row B down the plate to row H, by serum must be within 2 twofold dilutions of the geometric
transferring 100 µL from one well to the next. Discard mean titre of the serum. The potency is calculated by
100 µL from the last row so that all wells contain 100 µL. comparison of the proportion of responders for the vaccine to
2023 Appendix XIV K V-AS 17

be examined and the reference vaccine by the probit method - establishment of acceptance criteria for the D-antigen
or, after validation, using a parallel-line model. For the probit assay based on a suitable number of consecutive final lots;
method it is necessary to establish a cut-off neutralising - establishment of production consistency on recent final
antibody titre for each poliovirus type to define a responder. bulks using the currently approved in vivo assay; the final
Due to interlaboratory variation, it is not possible to define bulks should correspond to the final lots used to establish
cut-off values that could be applied by all laboratories. the acceptance criteria for the D-antigen assay and should
Rather, the cut-off values are determined for each laboratory represent different inactivated harvests of each of the 3
based on a minimum series of 3 tests with the reference types of poliovirus.
vaccine. The mid-point on a log2 scale of the minimum and The validation study should be performed on:
maximum geometric mean titres of the series of 3 or more - a final bulk/lot that is representative of the current
tests is used as the cut-off value. For each of the 3 poliovirus production method;
types, the potency of the vaccine is not significantly less than - 2 sub-potent batches prepared, for example, by heating
that of the reference preparation. The test is not valid unless: normal vaccine or mixing it with heat-treated vaccine; the
- for both the vaccine to be examined and the reference sub-potent batches should have expected titres of about
vaccine, the ED 50 lies between the smallest and the half that of the representative final bulk/lot.
largest doses given to the animals; These batches are assayed using as reference standard a
- the statistical analysis shows no significant deviation from homologous production batch:
linearity or parallelism; - by the currently approved in vivo assay for the vaccine;
- the confidence limits (P = 0.95) are not less than - by the rat assay where this is not the currently approved
25 per cent and not more than 400 per cent of the in vivo assay;
estimated potency. - by the D-antigen assay.
The fallowing section is published for information. Waiving of the in vivo assay is acceptable if the representative
final bulk/lot complies with the in vivo and in vitro assays and
the sub-potent batches fail to comply. If a sub-potent batch
GUIDELINE ON WAIVING OF THE IN fails to comply with the D-antigen assay but complies with
VIVO ASSAY OF POLIOMYELITIS the in i1ivo assay, the latter may be repeated.
VACCINE (INACTIVATED) AND ITS
COMBINATIONS 8. Flocculation Value (Lf) of Diphtheria and
Tetanus Toxins and Toxoids (Ramon Assay)
This guideline applies to vaccines derived from wild strains of (Ph. Eur. method 2.7.27)
poliovirus. The validatwn described should be carried out for each
The content of toxin or toxoid in a sample can be expressed
product before waiving of the in vivo assay, and should be
as a flocculation value (Lt) using the Ramon assay. In this
repeated wherever there is a substantial change to the
assay, antitoxin is added in increasing concentrations to a
manufacturing process that may affect the in vitro or in vivo
series of tubes containing a constant amount of toxin or
assays.
toxoid. At the equivalence point of toxin/toxoid and
The European convention on the protection of vertebrate antitoxin, flocculation occurs in 1 or more tubes. The first
animals used for experimental and other scientific purposes tube in which flocculation occurs is used to determine the Lf
requires that tests in animals shall not be carried out if a value of the sample.
scientifically satisfactory alternative is reasonably and
The Lf value of a toxin or toxoid is determined by the
practically available. The aim of this guideline is therefore to
number of units of antitoxin that, when mixed with the
promote waiving of the in vivo assay wherever it can be
sample, produces an optimally flocculating mixture (Ramon
shown for a given product that the in vitro assay CD-antigen
assay).
determination) gives sufficient assurance of satisfactory
potency for routine batch control. Practical experience has shown that the results of the
calibration of antitoxins in International Units (IU), for
For the in vivo assay, the test in rats is considered to be the
example by comparison to international antitoxin standards,
method of choice. For vaccines that are assayed using chicks
depends on the immunochemical method used. For this
or guinea-pigs and that have an established record of
reason, antitoxins used for the Ramon assay must be directly
production history, the in vivo assay may be waived if the rat
calibrated against the international bwlogical reference reagents
assay is also applied to the batches included in the validation
for diphtheria or tetanus toxoid for fiocculatwn tests, using the
study described below. For vaccines not yet approved, the
principles described below. The concentration thus
results of the rat assay on all final bulks should be included
determined may be indicated in Lf-equivalents per millilitre
in all data generated for demonstration of consistency of
(Lf-eq./mL).
production before waiving of the in vivo assay.
Once the in vivo assay has been waived, batches of vaccine By definition, 1 Lf is the quantity of toxin or toxoid that
will be released on the basis of the in vitro assay, and the in flocculates in the shortest time with 1 Lf-eq. of specific
vivo assay should not be used as an alternative for the release antitoxin.
of a batch that fails the in vitro assay. Repetition of the in A range of volumes of the reference standard of antitoxin
vitro assay may be performed according to an authorised adjusted to a concentration of 100 Lf-eq./mL is dispensed
procedure. into a series of, for example, 7 cm x 1 cm flocculation
tubes. A sufficient quantity of a 9 g/L solution of sodium
PROCEDURE
chloride R is added to each tube to give a constant total
The following conditions should be met before performance volume of, for example, 1 mL. The test sample is diluted to
of the validation study: give an expected concentration of approximately 50 L£'mL,
- appropriate experience of the rat assay; and, for example, 1 mL aliquots of this dilution are
- full validation of the D-antigen assay (linearity, dispensed into each of the tubes containing antitoxin.
repeatability, intermediate precision, accuracy and limits The tubes are properly mixed by shaking, then placed in a
of quantification);
V-A518 Appendix XIV K 2023

water-bath at a constant temperature between 30 °C and comparison of the Ll value of a known toxin or toxoid and
52 °C, and observed at regular intervals for the first that of a mixture of the sample with that toxin or toxoid.
appearance of floccules. This may require the use of a When a toxin or toxoid with a known Lf value and a toxin or
magnifying lens and strong illumination. toxoid with an unknown Lf value are flocculated together,
The first and the second mixtures to flocculate are recorded the mixture will flocculate as the sum of their values if they
as well as the time taken for the first flocculation to appear. are homogeneous. If non-homogenous toxins or toxoids are
2 tubes may flocculate simultaneously. mixed they will produce an aberrant pattern with
The first tube to flocculate is the one that contains the 2 flocculation maxima.
amount of antitoxin closest in equivalence to the amount of
9. Residual Pertussis Toxin
antigen in the sample. The antitoxin content of this tube can
(Ph. Bur. method 2.6.33)
be used to calculate the Lf value of the sample. If 2 tubes
flocculate at the same time, the mean from the tubes are The test for residual pertussis toxin is performed in vitro,
given as the result. using a Chinese Hamster Ovary (CHO) cell-based assay, and
is intended for the assay of non-adsorbed purified pertussis
The time taken for the first tube to flocculate (Kf) is a useful
components.
indicator of the quality of the antigen. If at a given
temperature and concentration of toxoid and antitoxin the Pertussis toxin ERP is suitable as a reference pertussis toxin
Kf value is increased compared with normal, this indicates preparation.
that the antigen has been damaged. The Kf value may also The CHO cell-clustering assay (CHO assay) is based on the
change with the quality of the antitoxin used. induction of clusters in non-confluent CHO cell cultures by
Example active pertussis toxin. The cultures are then examined under
a microscope and any clusters present are counted. The assay
Tube A B C D E F may be used as a quantitative test or as a limit test with the
sensitivity determined within each assay using dilutions of a
Antitoxin added 40 45 50 55 60 65
(Lf-eq.) reference pertussis toxin preparation.
Antitoxin added 0.40 0.45 0.50 0.55 0.60 0.65 The sensitivity of the CHO assay to pertussis toxin is verified
(mL) with pertussis toxin ERP or a suitable reference preparation
Saline added 0.60 0.55 0.50 0.45 0.40 0.35 calibrated in International Units using a suitable CHO assay.
(mL)
The assay sensitivity is defined as the lowest concentration in
Diluted sample 1.0 1.0 1.0 1.0 1.0 1.0
the reference preparation dilution series to produce a positive
added
response, i.e. at least 10 CHO cell clusters. A suitable assay
has a sensitivity of at least 5 mIU/mL.
If in this example the first tube to flocculate is tube C then
The fallowing method is given as an example.
the Lf value of the diluted sample is 50 Lf'mL. However, if
the first tube to flocculate is tube A or tube F this does not Reagents and equipment
indicate equivalence at that level. It would be necessary to - Pertussis toxin ERP.
perform a repeat test using either a different dilution of test - CHO-Kl cell line (ATCC No. CCL-61 or ECACC
sample or selecting a different range of doses of reference No. 85051005).
antitoxin. - Kaighn's modified Ham's F-12K medium.
More precision can be obtained by making allowance for the Media that have a slightly different composition from that in
sequence of flocculation after the first tube. Thus, in the Table 2.6.33.-1 may also be used. Proline is an essential
example quoted, if the second tube to flocculate had been component of the medium. Adjust to pH 7.2 ± 0.2 if
tube D, the final value for the diluted sample would be 52, necessary.
whereas if the second tube to flocculate was tube B, the final
Table 2.6.33.-1. - Kaighn's modified Ham's F-12K medium
value would be 48. The test may be performed in duplicate
with slightly different dilutions of the test sample. Amino acids
If there is no indication of the expected Ll value of the L-Alanine 18.0 mg
sample available, it is advisable to obtain a rough estimate by
use of a wider range of antitoxin content in the tubes before L-Arginine hydrochloride 0.4220 g
proceeding to the final test. L-Asparagine monohydrate 30.0 mg
Example
L-Aspartic acid 26.6 mg

Tube A B C D E F L-Cysteine hydrochloride 70.0 mg

Antitoxin 20 30 45 70 100 150 monohydrate
content (Lf-
eq.) L-Glutamic acid 29.0 mg

L-Glutamine 0.2920 g
The level of toxin or toxoid and antitoxin concentration in
Glycine 15.0 mg
the test may be varied, but this will markedly affect the
flocculation time, so that at very low levels the test will take L-Histidine hydrochloride 45.8 mg
too long, whilst at a high concentration the onset of monohydrate
flocculation may be so rapid as to make it difficult to L-lsoleucine 7.88 mg
distinguish the first and second tubes to flocculate.
L-Leucine 26.2 mg
Assay of low concentrations by blend flocculation
For very low concentrations, it is preferable to measure toxin L-Lysine hydtochloride 73.0 mg
or toxoid by the method of blend flocculation. This involves
2023 Appendix XIV K V-AS 19

Amino acids concentration of 1 per cent V/V. An antibiotic/antimycotic

solution may be added if required. Adjust to
L-Methionine 8.96 mg
pH 7 .2 ± 0.2 if necessary. Store at 5 ± 3 °C for a
L-Phenylalanine 9.92 mg maximum of 3 weeks, protected from light.
- Trypsin-EDTA solution (trypsin 0.25 per cent).
L-Proline 69.0 mg
- Phosphate-buffered saline pH 7.4 (PBS), without calcium or
L-Serine 21.0 mg magnesium. Dissolve 9.0 g of sodium chloride R, 0.144 g
of potassium dihydrogen phosphate Rand 0.795 g of
L-Threoninc 23.0 mg
disodium hydrogen phosphate heptahydrate R in water R, and
L-Tryptophan 4.1 mg dilute to 1000.0 mL with the same solvent. Adjust the
pH if necessary.
L-Tyrosine disodium salt dihydrate 13.5 mg - 24-well fiat-bottomed assay plates with li.d.
L-Valine 23.0 mg - 75 cm 2 tissue culture flasks.
- Low protein binding polypropylene microtubes.
Vitamins
CHO cell culture
Biotin 70 µg The cells are obtained from a cell bank and used between
defined passage levels. CHO cells are cultured in tissue
D-Calcium pantothenate 0.5 mg
culture flasks containing 20 mL of CHO cell culture medium
Choline chloride 14.0mg in a humidified incubator set at 37 °C and 5 per cent CO 2 •
The cells are passaged at a ratio of 1:5 to 1:20 as they
Folic acid 1.3 mg
approach confluence.
myo-Inositol 18.0 mg Before use, allow all reagents to reach room temperature or
warm to 37 °C.
Nicotinamide 37 (Lg
To passage the cells, remove the CHO cell culture medium.
Pyridoxine hydrochloride 60 µg Wash the cell layer gently by rinsing briefly 2-3 times with
Riboflavin 40 µg PBS. Remove the PBS and add 2 mL of trypsin-EDTA
solution to the flask, allowing it to cover the monolayer for
Thiamine hydrochloride 0.3mg 20 s. Remove the trypsin-EDTA solution by aspiration.
Vitamin B12 1.4 mg Immediately observe the culture using phase-contrast
microscopy. When the cells begin to contract, tap the side of
Inorganic salts the culture flask firmly to dislodge the cells. When the
Calcium chloride, anhydrous 0.1020 g
majority of cells are free-floating, add 10 mL of CHO cell
culture medium to stop the trypsin digestion. Trypsinisation
Cupric sulfate pentahydrate 2 µg used for cell detachment must be carefully controlled to
Disodium hydrogen phosphate, 0.1155 g
avoid over-digestion which causes cleavage of either pertussis
anhydrous toxin receptors or cell adherence proteins, or both.
Determine the cell concentration and viability using trypan
Ferric sulfate heptahydrate 0.8 mg
blue exclusion or another appropriate method. The cell
Magnesium chloride, anhydrous 49.7 mg viability must be greater than 95 per cent to continue.
Transfer a sufficient volume of the cell suspension to a fresh
Magnesium sulfate, anhydrous 0.1920 g
flask for a 1:5 to 1:20 passage ratio and add CHO cell
Potassium chloride 0.2850 g culture medium to bring the volume to 20 mL. Incubate the
cells in a humidified incubator set at 37 °C and 5 per cent
Sodium bicarbonate 2.5000 g
CO 2 for at least 48 h prior to use in the CHO assay.
Sodium chloride 7.5300g Seeding density for CHO assay
Zinc sulfate heptahydratc 0.144 mg
Prepare a suspension of CHO cells using the trypsinisation
procedure described above. Count and dilute the cell
Other components suspension to between 4 x 104 and 8 x 104 CHO cells/mL
D-Glucose 1.2600 g
in CHO cell culture medium. The cell concentration selected
must allow clusters to be identified at the end of the
Hypoxanthine sodium salt 4.0 mg stimulation period. Transfer 250 µL of the cell suspension
into each well of the 24-well plates. Store the seeded plates
Lipoic acid 0.21 mg
in a humidified incubator set at 37 °C and 5 per cent CO 2
Phenol red 3.0 mg while the reference preparation and test sample are prepared.
Putrescine dihydrochloride 0.32 mg Preparation of pertussis toxin reference
Reconstitute pertussis toxin ERP as stated in the leaflet
Sodium pyruvate 0.2200 g accompanying the reference preparation. Prepare 7 two-fold
Thymidine 0.7 mg serial dilutions in CHO cell culture medium, in such a way
that the assay end-point occurs in the middle of the dilution
Water to 1000 mL series. The dilutions are prepared in low protein binding
polypropylene microtubes. One dilution series for the
CHO cell culture medium. Supplement Kaighn's modified reference preparation is included in each assay plate.
Ham's F-12K medium with foetal bovine serum to obtain
a final concentration of 10 per cent VIV. Add a sufficient
quantity of 0.2 M L-glutamine solution to obtain a final
V-A520 Appendix XIV L 2023

Preparation of non-adsorbed purified pertussis Table 2.6.33.-2 - Reference preparation dilutions and scores
component samples
Reference preparation Score
Dilute the test sample in CHO cell culture medium to obtain dilution
the highest concentration of the test sample which does not
significantly dilute the nutrients of the medium, in order to Rep. I Rep. 2
maximise the sensitivity of the assay. Prepare a dilution series 1:100 000 + +
as described for the reference preparation.
1:200 000 + +
Stimulation of CHO cells
Transfer 250 µL from each tube of the reference preparation 1:400 000 + +
dilution series into the assigned wells of the assay plates
seeded with CHO cells. Similarly, transfer 250 µL of each
1:800 000 + -
dilution of the test sample into the assigned well(s). Transfer 1:1 600 000 - -
250 µL of CHO cell culture medium into the negative
control wells. Return the assay plates to a humidified 1:3 200 000 - -

incubator set at 37 °C and 5 per cent CO 2 for 48 h. 1:6 400 000 - -

Scoring and interpretation of results End-point dilutions 1:800 000 1:400 000
Observe the cell cultures using phase contrast microscopy at
a magnification of 4 x or 10 x . In all wells the cell cultures Reciprocal of end-point dilutions 800 000 400 000
must not be confluent and must have sufficient growth space
GM(end-point)R 565 685
to allow any clusters present to be counted. Assign a positive
score when 10 or more CHO cell cluster formations are
clearly evident within a single well. Assign a negative score to
Table 2.6.33.-3 - Test sample dilutions and scores
wells containing fewer than 10 clusters. The end-point
concentration is the lowest concentration at which there is a Test sample dilution Score
positive score. Rep. 1 Rep. 2
Assay validity
The test is not valid unless: 1:100 + +
- the negative control wells show no evidence of clustering. 1:200 + +
- wells containing the reference toxin preparation at a
concentration greater than or equal to 5 mill/mL exhibit 1:400 + +
a positive response (i.e. 10 or more CHO cell clusters). 1:800 + +
- a clear end-point dilution is observed for the reference
preparation. 1:1600 + -
- replicate values for the end-point of the reference 1:3200 - -
preparation do not differ by more than one 2-fold dilution
within one assay. 1:6400 - -

Calculation End-point dilutions 1:1600 1:800

Calculate the residual pertussis toxin activity in the test
Reciprocal of end-point dilutions 1600 800
sample in International Units per millilitre, relative to the
reference preparation, using the following expression: GM(end-point) s 1131

GM(end - point)s AR
------.-~ X The residual pertussis toxin activity in the test sample can be
GM(end - pomt)R determined as follows: (1131/565685) x 1000 = 2 IU/mL.
GM (end-point) s geometric mean of the reciprocal of the
end-point dilutions (last dilution at which there
is a positive score) for the test sample;
GM (end-point) R geometric mean of the reciprocal of the
end-point dilutions (last dilution at which there
L. Nucleic Acid Amplification Techniques
is a positive score) for the reference preparation; (Ph. Bur. method 2.6.21)
activity of the reference preparation (stock
solution), in International Units per millilitre. 1. INTRODUCTION
Nucleic acid amplification techniques are based on
Example 2 different approaches:
Series of 7 two-fold dilutions were prepared for the reference 1. amplification of a target nucleic acid sequence using, for
preparation and test sample. In this example, the reference example, polymerase chain reaction (PCR), ligase chain
stock solution has an activity of 1000 IU/mL. The scores reaction (LCR), or isothermal ribonucleic acid (RNA)
observed at each dilution of the reference preparation and amplification;
test sample, the reciprocal end-point dilutions and the 2. amplification of a hybridisation signal using, for example,
geometric means of reciprocal end-point dilutions are shown for deoxyribonucleic acid (DNA), the branched DNA
in Tables 2.6.33.-2 and 2.6.33.-3. (bDNA) method; in this case signal amplification is achieved
without subjecting the nucleic acid to repetitive cycles of
amplification.
In this general chapter, the PCR method is described as the
reference technique. Alternative methods may be used, if
they comply with the quality requirements described below.
2023 Appendix XIV L V-A521

2. SCOPE - post-PCR detection (the only area where the amplified

This section establishes the requirements for sample material is handled in an open system).
preparation, in vitro amplification of DNA sequences and If closed systems are used, the strict segregation of areas is
detection of the specific PCR product. With the aid of PCR, not required.
defined DNA sequences can be detected. RNA sequences
5.2. Sample preparation
can also be detected following reverse transcription of the
When preparing samples, the target sequence to be amplified
RNA to complementary DNA (cDNA) and subsequent
needs to be efficiently extracted or liberated from the test
amplification.
material in a reproducible manner and in such a way that
3. PRINCIPLE OF THE METHOD amplification under the selected reaction conditions is
PCR is a procedure that allows specific in vitro amplification possible. A variety of physico-chemical extraction procedures
of segments of DNA or of RNA after reverse transcription and/or enrichment procedures may be employed.
into cDNA. Additives present in test material may interfere with PCR.
Following denaturation of double-stranded DNA into single- The procedures described under 7.3.2. must be used as a
stranded DNA, 2 synthetic oligonucleotide primers of control for the presence of inhibitors originating from the test
opposite polarity anneal to their respective complementary material.
sequences in the DNA to be amplified. The short double- In the case of RNA-templates, care must be taken to avoid
stranded regions that form as a result of specific base pairing ribonuclease activity.
between the primers and the complementary DNA sequence
5.3. Amplification
border the DNA segment to be amplified, and serve as
PCR amplification of the target sequence is conducted under
starting positions for in vitro DNA synthesis by means of a
defined cycling conditions (temperature profile for
heat-stable DNA polymerase.
denaturation of double-stranded DNA, annealing and
Amplification of the DNA occurs in cycles consisting of: extension of primers; incubation times at selected
- heat denaturation of the nucleic acid (target sequence) temperatures; ramp rates). These depend on various
into 2 single strands; parameters such as:
- specific annealing of the primers to the target sequence - the length and base composition of primer and target
under suitable reaction conditions; sequences;
- extension of the primers, which are bound to both single - the type of DNA polymerase, buffer composition and
strands, by DNA polymerase at a suitable temperature reaction volume used for the amplification;
(DNA synthesis). - the type of thermocycler used and the thermal
Repeated cycles of heat denaturation, primer annealing and conductivity rate between the apparatus, reaction tube
DNA synthesis results in an exponential amplification of the and reaction fluid.
DNA segment limited by the primers. 5.4. Detection
The specific PCR product known as an amplicon can be The amplicon generated by PCR may be identified by size,
detected by a variety of methods of appropriate specificity sequence, chemical modification or a combination of these
and sensitivity. parameters. Detection and characterisation by size may be
Multiplex PCR assays use several primer pairs designed for achieved by gel electrophoresis (using agarose or
simultaneous amplification of different targets in one polyacrylamide slab gels or capillary electrophoresis) or
reaction. column chromatography (for example, liquid
chromatography). Detection and characterisation by sequence
4. TEST MATERIAL
composition may be achieved by the specific hybridisation of
Because of the high sensitivity of PCR, the samples must be
probes having a sequence complementary to the target
protected against external contamination with target
sequence or by cleavage of the amplified material reflecting
sequences. Sampling, storage and transport of the test
target-specific restriction-enzyme sites. Detection and
material are performed under conditions that minimise
characterisation by chemical modification may be achieved
degradation of the target sequence. In the case of RNA target
by, for example, incorporation of a fluorophore into the
sequences, special precautions are necessary since RNA is
amplicons and subsequent detection of fluorescence following
highly sensitive to degradation by ribonucleases. Care must
excitation.
be taken since some added reagents, such as anticoagulants
or preservatives, may interfere with the test procedure. Detection of amplicons may also be achieved by using probes
labelled to permit a subsequent chemiluminescent,
5. TEST METHOD radioisotopic or immuno-enzyme-coupled detection.
5.1. Prevention of contamination
The risk of contamination requires a strict segregation of the 6. EVALUATION AND INTERPRETATION OF
areas depending on the material handled and the technology RESULTS
used. Points to consider include movement of personnel, A valid result is obtained within a test only if the positive
gowning, material flow and air supply and decontamination control(s) is unambiguously positive and the negative
procedures. control(s) is unambiguously negative. Due to the very high
sensitivity of the PCR method and the inherent risk of
The system should be sub-divided into compartments such
contamination, it is necessary to confirm positive results by
as:
repeating the complete test procedure in duplicate, where
- master-mix area (area where exclusively template-free
possible on a new aliquot of the sample. The sample is
material is handled, e.g. primers, buffers, etc.);
considered positive if at least one of the repeat tests gives a
- pre-PCR (area where reagents, samples and controls are
positive result. As soon as a measurable target threshold is
handled);
defined, a quantitative test system is required.
- PCR amplification (amplified material is handled in a
closed system);
V-A522 Appendix XIV L 2023

7. QUALITY ASSURANCE 7.3.3. Threshold control

7.1. Validation of the PCR assay system The threshold control for quantitative assays is a test sample
The validation programme must include validation of with the analyte at a concentration that is defined as the
instrumentation and the PCR method employed. Reference threshold not to be exceeded. It contains the analyte suitably
should be made to the ICH guideline Q2(Rl) Validation of calibrated in International Units and is analysed in parallel in
Analytical Procedures: Text and Methodology. each run of a quantitative assay.
Appropriate official working reference preparations or 7.4. External quality assessment
in-house reference preparations calibrated against Participation in external quality assessment programmes is an
International Standards for the target sequences are used for important PCR quality assurance procedure for each
validation of a PCR test, when available. laboratory and each operator.
7.1.1. Determination of the positive cut-off point The following sections are published for information.
During validation of qualitative tests, the positive cut-off
point must be determined. The positive cut-off point is VALIDATION OF NUCLEIC ACID
defined as the minimum number of target sequences per AMPLIFICATION TECHNIQUES (NAT)
volume sample that can be detected in 95 per cent of test
runs. The positive cut-off point depends on interrelated
FOR THE DETECTION OF HEPATITIS C
factors such as the volume of the sample extracted and the VIRUS (HCV) RNA IN PLASMA POOLS:
efficacy of the extraction methodology, the transcription of GUIDELINES
the target RNA into cDNA, the amplification process and 1. SCOPE
the detection. The majority of nucleic acid amplification analytical
To define the detection limit of the assay system, reference procedures are qualitative (quanta!) tests for the presence of
must be made to the positive cut-off point for each target nucleic acid with some quantitative tests (either in-house or
sequence and the test performance above and below the commercial) being available. For the detection of HCV RNA
positive cut-off point. contamination of plasma pools, qualitative tests are adequate
7.1. 2. Quantitative assay systems and may be considered to be a limit test for the control of
For a quantitative assay, the following parameters are impurities as described in the Pharmeuropa Technical Guide
determined during validation: accuracy, precision, specificity, for the elaboration of monographs, December 1999, Chapter
quantitation limit, linearity, range and robustness. III 'Validation of analytical procedures'. These guidelines
describe methods to validate only qualitative nucleic acid
7.2. Quality control of reagents amplification analytical procedures for assessing HCV RNA
All reagents crucial for the methodology used have to be contamination of plasma pools. Therefore, the
controlled prior to use in routine applications. Their 2 characteristics regarded as the most important for
acceptance/withdrawal is based on pre-defined quality validation of the analytical procedure are the specificity and
criteria. the detection limit. In addition, the robustness of the
Primers are a crucial component of the PCR assay and as analytical procedure should be evaluated.
such their design, their purity and the validation of their use However, this document may also be used as a basis for the
in a PCR assay require careful attention. Primers may be
validation of nucleic acid amplification in general.
modified (for example, by conjugation with a fluorophore or
antigen) in order to permit a specific method of detection of For the purpose of this document, an analytical procedure is
the amplicon, provided such modifications do not inhibit defined as the complete procedure from extraction of nucleic
accurate and efficient amplification of the target sequence. acid to detection of the amplified products.
Where commercial kits are used for part or all of the
7.3. Run controls
analytical procedure, documented validation points already
7. 3. 1. External controls
covered by the kit manufacturer can substitute for the
In order to minimise the risk of contamination and to ensure validation by the user. Nevertheless, the performance of the
adequate sensitivity, the following external controls are kit with respect to its intended use has to be demonstrated by
included in each PCR assay: the user (e.g. detection limit, robustness, cross-
- positive control: this contains a defined number of target- contamination).
sequence copies, the number being close to the positive
cut-off value, and determined individually for each assay 2. SPECIFICITY
system and indicated as a multiple of the positive cut-off Specificity is the ability to assess unequivocally nucleic acid
value of the assay system; in the presence of components that may be expected to be
- negative control: a sample of a suitable matrix already present.
proven to be free of the target sequences. The specificity of nucleic acid amplification analytical
7.3.2. Internal control procedures is dependent on the choice of primers, the choice
Internal controls are defined nucleic acid sequences of probe (for analysis of the final product) and the stringency
containing, unless otherwise prescribed, the primer binding of the test conditions (for both the amplification and the
sites. Internal controls must be amplified with defined detection steps).
efficacy, and the amplicons must be clearly discernible. When designing primers and probes, the specificity of the
Internal controls must be of the same type of nucleic acid primers and probes to detect only HCV RNA should be
(DNNRNA) as the material to be tested. The internal investigated by comparing the chosen sequences with
control is preferably added to the test material before sequences in published data banks. For HCV, primers (and
isolating the nucleic acid and therefore acts as an overall probes) will normally be chosen from areas of the 5' non-
control (extraction, reverse transcription, amplification, coding region of the HCV genome which are highly
detection). conserved for all genotypes.
2023 Appendix XIV L V-A523

The amplified product should be unequivocally identified by 4. ROBUSTNESS

using one of a number of methods such as amplification with The robustness of an analytical procedure is a measure of its
nested primers, restriction enzyme analysis, sequencing, or capacity to remain unaffected by small but deliberate
hybridisation with a specific probe. variations in method parameters and provides an indication
In order to validate the specificity of the analytical procedure, of its reliability during normal usage.
at least 100 HCV RNA-negative plasma pools should be The evaluation of robustness should be considered during
tested and shown to be non-reactive. Suitable samples of the development phase. It should show the reliability of the
non-reactive pools are available from the European analytical procedure with respect to deliberate variations in
Directorate for the Quality of Medicines & method parameters. For NAT, small variations in the
HealthCare (EDQM). method parameters can be crucial. However, the robustness
The ability of the analytical procedure to detect all HCV of the method can be demonstrated during its development
genotypes will again depend on the choice of primers, probes when small variations in the concentrations of reagents
and method parameters. This ability should be demonstrated (e.g. MgC1 2, primers or dNTP) are tested. To demonstrate
using characterised reference panels. However, in view of the robustness, at least 20 HCV RNA negative plasma pools
difficulty in obtaining samples of some genotypes (selected at random) spiked with HCV RNA to a final
(e.g. genotype 6), the most prevalent genotypes concentration of 3 times the previously determined
(e.g. genotypes 1 and 3 in Europe) should be detected at a 95 per cent cut-off value should be tested and found positive.
suitable level. Problems with robustness may also arise with methods that
3. DETECTION LIMIT use an initial ultracentrifugation step prior to extraction of
The detection limit of an individual analytical procedure is the viral RNA. Therefore, to test the robustness of such
the lowest amount of nucleic acid in a sample that can be methods, at least 20 plasma pools containing varying levels of
detected but not necessarily quantitated as an exact value. HCV RNA, but lacking HCV-specific antibodies, should be
tested and found positive.
The nucleic acid amplification analytical procedure used for
the detection of HCV RNA in plasma pools usually yields Cross-contamination prevention should be demonstrated by
qualitative results. The number of possible results is limited the accurate detection of a panel of at least 20 samples
to 2: either positive or negative. Although the determination consisting of alternate samples of negative plasma pools and
of the detection limit is recommended, for practical purposes, negative plasma pools spiked with high concentrations of
a positive cut-off point should be determined for the nucleic HCV (at least 102 times the 95 per cent cut-off value or at
acid amplification analytical procedure. The positive cut-off least 104 IU/mL).
point (as defined in the general chapter 2.6.21) is the 5. QUALITY ASSURANCE
minimum number of target sequences per volume sample For biological tests such as NAT, specific problems may arise
that can be detected in 95 per cent of test runs. This positive that influence both the validation and the interpretation of
cut-off point is influenced by the distribution of viral results. The test procedures must be described precisely in
genomes in the individual samples being tested and by the form of standard operating procedures (SOPs). These
factors such as enzyme efficiency, and can result in different should cover:
95 per cent cut-off values for individual analytical test runs. - the mode of sampling (type of container, etc.);
In order to determine the positive cut-off point, a dilution - the preparation of mini-pools (where appropriate);
series of a working reagent or of the hepatitis C virus BRP, - the conditions of storage before analysis;
which has been calibrated against the WHO HCV - the exact description of the test conditions, including
International Standard, should be tested on different days to precautions taken to prevent cross-contamination or
examine variation between test runs. At least 3 independent destruction of the viral RNA, reagents and reference
dilution series should be tested with a sufficient number of preparations used;
replicates at each dilution to give a total number of 24 test - the exact description of the apparatus used;
results for each dilution, to enable a statistical analysis of the - the detailed formulae for calculation of results, including
results. statistical evaluation.
For example, a laboratory could test 3 dilution series on The use of a suitable run control (for example, an
different days with 8 replicates for each dilution, 4 dilution appropriate dilution of hepatitis C virus BRP or plasma spiked
series on different days with 6 replicates for each dilution, or with an HCV sample calibrated against the WHO HCV
6 dilution series on different days with 4 replicates for each International Standard) can be considered a satisfactory
dilution. In order to keep the number of dilutions at a system-suitability check and ensures that the reliability of the
manageable level, a preliminary test (using, for example, analytical procedure is maintained whenever used.
log 10 dilutions of the plasma pool sample) should be carried Technical qualification An appropriate installation and
out in order to obtain a preliminary value for the positive operation qualification programme should be implemented
cut-off point (i.e. the highest dilution giving a positive signal). for each critical piece of the equipment used.
The range of dilutions can then be chosen around the For confirmation of analytical procedure performance after a
predetermined preliminary cut-off point (using, for example, change of critical equipment (e.g. thermocyclers), the change
a dilution factor of 0.5 log 10 or less and a negative plasma should be documented by conducting a parallel test on
pool for the dilution matrix). The concentration of HCV 8 samples of a plasma pool that is spiked with HCV RNA to
RNA that can be detected in 95 per cent of test runs can a final concentration of 3 times the previously determined
then be calculated using an appropriate statistical evaluation. 95 per cent cut-off value. All results should be positive.
These results may also serve to demonstrate the intra-assay Operator qualification An appropriate qualification
variation and the day-to-day variation of the analytical programme should be implemented for each operator
procedure. involved in the testing.
V-A524 Appendix XIV L 2023

VALIDATION OF NUCLEIC ACID positive sample suitably calibrated in International Units

AMPLIFICATION TECHNIQUES (NAT) and covering the whole quantitative range of the assay;
the coefficient of variation for the individual samples is
FOR THE QUANTIFICATION OF B19
calculated (intra-assay variability);
VIRUS (B19V) DNA IN PLASMA POOLS: - intermediate precision expresses the intra-laboratory
GUIDELINES variations (inter-assay precision); it is established by
1. SCOPE assaying replicates (as routinely used for the assay) of
The European Pharmacopoeia requires that plasma pools appropriate dilutions of a BI 9V DNA-positive sample
used for manufacture of certain products are tested for the suitably calibrated in International Units covering the
presence of B 19 virus (B 19V) DNA with a threshold whole quantitative range of the assay under different
concentration that must not be exceeded. In order to comply circumstances (e.g. different days, different analysts,
with these requirements, quantitative NAT tests are different equipment, different reagents); the coefficient of
preferred. The characteristics regarded as the most important variation for the individual samples is calculated;
for validation of the quantitative NAT procedure are - reproducibility expresses the precision between different
accuracy, precision, specificity, quantitation limit, linearity laboratories (inter-laboratory precision); it is assessed by
and range. In addition, the robustness of the analytical participation in quantitative collaborative studies on B 19V
procedure should be evaluated. DNA-NAT assays, e.g. under the Proficiency Testing
Scheme (PTS), including the comparative analysis of the
This guideline describes methods to validate NAT analytical
obtained quantitative results, where appropriate.
procedures for assessing B 19V DNA contamination of
plasma pools based on the ICH guidelines. However, this 4. SPECIFICITY
document may also be used as a basis for the validation of Specificity expresses the ability to assess unequivocally
quantitative NAT in general. nucleic acid in the presence of components that may be
For the purpose of this document, an analytical procedure is expected to be present. The specificity of NAT analytical
defined as the complete procedure from extraction of nucleic procedures is dependent on the choice of primers, the choice
acid to detection of the amplified products. of probe (for analysis of the final product) and the stringency
Where commercial kits are used for part or all of the of the test conditions (for both the amplification and the
analytical procedure, documented validation points already detection steps).
covered by the kit manufacturer can substitute for the When designing primers and probes, the specificity of the
validation by the user. Nevertheless, the performance of the primers and probes to detect only human BI 9V DNA should
kit with respect to its intended use has to be demonstrated by be investigated by comparing the chosen sequences with
the user (e.g. precision, accuracy, range, robustness). sequences in published data banks. There should be no
major homology found with sequences unrelated to BI 9V.
2. ACCURACY
The amplified product should be unequivocally identified by
Accuracy expresses the closeness of agreement between the
using one of a number of methods such as amplification with
value that is accepted as either a conventional true value or
nested primers, restriction enzyme analysis, sequencing, or
an accepted reference value and the value found.
hybridisation with a specific probe.
The accuracy of an assay is dependent on the calibration of
the assay and on the variance of the different assay steps. In order to examine the specificity of the analytical
Though it is recommended to establish the accuracy across procedure, at least 20 B 19V DNA-negative plasma pools
the specified range of the analytical procedure, the most should be tested and shown to be non-reactive.
important assessment of accuracy is in the range of the Parvovirus B19 genotypes The International Committee
threshold concentration. In the case of B 19V NAT assays for on Taxonomy of Viruses (ICTV) has classified
investigation of plasma pools it is recommended to assess the representatives of the 3 genotypes as strains of human
accuracy of the calibrated assay by assaying at least parvovirus B 19. Genotype 1 represents prototype B 19V,
5 concentrations (dilution factor of 0.5 log 10) of B19 virus genotype 2 represents viral sequences like A6, and genotype
DNA for NAT testing ERP or another material, suitably 3 represents V9-like sequences. By performing sequence
calibrated in International Units against the actual WHO alignment with respective B 19V genotype sequences available
BI 9 DNA International Standard, covering the range of the from nucleic acid sequence databases, primers and probes
currently recommended threshold concentration of should be designed to detect and quantify consistently the
10.0 IU/µL B19V DNA (e.g. 10 5 IU/mL, 1045 IU/mL, different human parvovirus Bl9 genotypes. Reference
104 IU/mL, 10 35 IU/mL and 103 IU/mL), with at least 3 materials should be used to check the approach chosen.
replicates for each dilution. Accuracy should be reported for Since biological reference preparations reflecting some
the different concentrations in terms of percentage genotypes might be difficult to obtain, respective plasmid
determined compared with the known amount of B 19V preparations or synthetic nucleic acids may also serve as a
DNA. It should reflect the level of technology of the characterised target sequence source. However, those cannot
respective assays, which should also be defined, for example be used to validate the extraction procedure.
in collaborative studies. 5. QUANTITATION LIMIT
3. PRECISION The quantitation limit is the lowest amount of nucleic acid in
Precision expresses the closeness of agreement between a a sample that can be determined quantitatively with suitable
series of measurements, obtained from multiple sampling of precision and accuracy. The quantitation limit of the Bl9V
the same homogenous sample. The precision is defined at NAT assay is assessed during the repeatability and
3 levels: intermediate-precision studies by limiting dilution analysis.
- repeatability expresses the precision under the same The lowest concentration of target nucleic acids that is
operating conditions over a short interval of time (intra- quantitated with suitable precision and accuracy is defined.
assay precision); it is assessed by using 1 assay and testing
3 replicates of appropriate dilutions of a Bl9V DNA-
2023 Appendix XIV M V-A525

6. LINEARITY suitability check and ensures that the reliability of the

The linearity of an assay is its ability to obtain test results analytical procedure is maintained whenever used.
that are directly proportional to the concentration of the Technical qualification An appropriate installation and
nucleic acid. The linearity of the B 19V NAT assay is operation qualification programme should be implemented
assessed during the repeatability and intermediate-precision for each critical piece of the equipment used.
studies by testing replicates of diluted samples with the For confirmation of analytical procedure performance after a
concentrations covering the whole quantitative range. change of critical equipment (e.g. thermocyclers), the change
The interval between the upper and the lower concentration should be documented by conducting a parallel test on
of the target nucleic acid where test results are directly 8 samples of a plasma pool that is spiked with a
proportional to the concentrations is defined. concentration of B 19V DNA around the threshold
7. RANGE concentration. All results should be acceptable and reflect the
The range of an assay is the interval between the upper and features of the assay as determined during the validation
the lower concentration of nucleic acid in the sample for phase.
which it has been demonstrated that the procedure has a Operator qualification An appropriate qualification
suitable level of precision, accuracy and linearity. The range programme should be implemented for each operator
of the B 19V NAT assay is assessed during the repeatability involved in the testing.
and intermediate-precision studies by testing replicates of
diluted samples. The interval between the upper and the
lower concentration that can be expressed with an acceptable
degree of accuracy and precision is defined. M. Assay of Interferons
8. ROBUSTNESS (Ph. Bur. general texts 5. 6)
The robustness of an analytical procedure is a measure of its
The fallowing chapter is published for infonnatwn.
capacity to remain unaffected by small but deliberate
variations in method parameters and provides an indication 1. INTRODUCTION
of its reliability during normal usage. The evaluation of Monographs on human interferons generally contain a
robustness should be considered during the development bioassay based on the inhibitory activity of the interferon on
phase. It should show the reliability of the analytical the cytopathic action of a virus on a cell line in culture.
procedure with respect to deliberate variations in method In most cases, however, the virus, cell line and the assay
parameters. For NAT, small variations in the method details are not specified, in order to allow the appropriate
parameters can be crucial. Nonetheless, the robustness of flexibility, where the monograph covers more than one sub-
NAT can be demonstrated during the development of the class of interferon.
method when small variations in the concentrations of The present text is intended to provide outline information
reagents, for example MgC1 2 , primers or dNTP, are tested. for the analyst on how to design, optimise and validate such
To demonstrate robustness, at least 20 BI 9V DNA-negative an assay once an appropriate combination of cell line and
plasma pool samples spiked with B19V DNA at the cytopathic virus has been identified. A detailed procedure for
threshold concentration should be tested and found to have a particular cytopathic antiviral assay is described as an
acceptable quantitative values. example of a suitable method, together with information on
Cross-contamination prevention should be demonstrated by other virus-cell line combinations and guidance on how to
the accurate detection of a panel of at least 20 samples adapt and validate the procedure for these other
consisting of alternate samples of plasma pools without B 19V combinations.
DNA or with levels below the threshold concentration 2. ANTIVIRAL (CYTOPATHIC EFFECT
(10 samples) and plasma pools spiked with high REDUCTION) ASSAYS
concentrations of B 19V DNA (at least 102 times the
The antiviral assay of human interferons is based on the
threshold level, 10 samples).
induction of a cellular response in human cells, which
9. QUALITY ASSURANCE prevents or reduces the cytopathic effect of an infectious
For biological tests such as NAT, specific problems may arise virus. The potency of interferon is estimated by comparing its
that may influence both the validation and the interpretation protective effect against a viral cytopathic effect with the
of results. The test procedures must be described precisely in same effect of the appropriate reference preparation
the form of standard operating procedures (SOPs). These calibrated in International Units.
should cover: 3. INTERFERON ASSAY USING HEP2C CELLS AND
- the mode of sampling (type of container, etc.); INFECTIOUS ENCEPHALOMYOCARDITIS VIRUS
- the preparation of mini-pools by manufacturers (where
The antiviral assay of human interferons described is of the
appropriate);
cytopathic effect reduction type. It uses human Hep2c cells
- the conditions of storage before analysis;
infected by encephalomyocarditis virus (EMCV) to measure
- the exact description of the test conditions including
the potencies of different human interferon test preparations.
precautions taken to prevent cross-contamination or
This assay has been used in three World Health Organization
destruction of the viral nucleic acids, reagents and
(WHO) international collaborative studies of candidate
reference preparations used;
International Standards for human interferon alpha, human
- the exact description of the apparatus used;
interferon beta and human interferon gamma and has
- the detailed formulae for calculation of results, including
repeatedly been demonstrated to be sensitive, reliable and
statistical evaluation.
reproducible for potency estimations of the different types of
The inclusion of an appropriate threshold control (for human interferon.
example, plasma spiked with a BI 9V DNA sample suitably
For the culture of mammalian cells, all procedures are
calibrated in International Units, such as B19 virus DNA far
carried out using standard operating procedures for the
NAT testing ERP) is considered to be a satisfactory system-
V-A526 Appendix XIV M 2023

maintenance of such cell lines in culture. Volumes of bottles, in quantities of 20 mL, 10 mL, 5 mL, 1 mL, 0.5 mL
reagents are indicated for cell cultures carried out in 75 cm2 or 0.2 mL, as appropriate. Store at -70 °C. Larger volumes
flasks. Other types of containers (flasks or plates) may be can be thawed, dispensed into smaller quantities and
used but volumes must be adapted accordingly. re-frozen when required. The EMCV stock will retain its
3.1. MAINTENANCE AND PREPARATION OF HEP2C original titre if stored permanently at approximately -70 °C,
CELLS but repeated freeze-thaw cycles or storage at higher
Hep2c cells are maintained and passaged in culture temperatures, e.g. at approximately -20 °C, results in
medium A. progressive loss of titre.
Cells are stored as frozen stocks using standard operating 3.3. ASSAY PROCEDURE
procedures. Growing cells may be maintained in culture up 3.3.1. Determination of the dose-response range
to a permitted passage number of 30, after which new Preparation of the solutions
cultures are established from frozen stocks. Dilute the appropriate standard for interferon (for example a
At the beginning of the assay procedure, harvest the cells specific WHO sub-type interferon standard) in culture
from the flasks showing 90 per cent confluent monolayers medium A, in 10-fold dose increments, to give doses
using the trypsin-treatment procedure described below. covering the range of 1000 - 0.001 IU/mL. Carry out the
- Remove the culture medium from the flasks. assay procedure in 96-well microtitre plates. To each well
- To each flask, add 5 mL of trypsin solution heated at add 100 µL of culture medium A. Add approximately
37 °C (a trypsin stock solution contains 4 mg/mL of 100 µL of each dilution of the reference preparation to each
trypsin R and 4 mg/mL of sodium edetate R; immediately well except for those intended for virus controls. Using a
before use, dilute 50 times with phosphate buffered multichannel pipette set at 100 µL, mix the contents of the
saline). Swirl the capped flask to wash the cell monolayer. wells.
Remove the excess of trypsin solution. Dispensing of the cell suspension
- Incubate the flasks for 5 min to 10 min at 37 °C.
Pour the cell suspension of Hep2c cells, which has been
Microscopically or visually observe the cells for signs of
adjusted to contain approximately 6 x 10 5 cells/mL of
detachment. When viewed microscopically, the cells
culture medium A, into a plastic Petri-dish. Dispense the cell
appear rounded up or detached and free-floating. Shake
suspension from the Petri-dish into each well of the
the flask vigorously to detach all the cells, add
microtitre plates, using a multichannel pipette set at 100 µL.
approximately 5 mL of culture medium A. Shake
vigorously to yield a suspension of single cells. Incubate the plates for about 24 h in an incubator set at
- To prepare the cell suspensions for the assay procedure, 37 °C and 5 per cent CO 2 •
carefully disperse the cells by pipetting up and down to Viral infection
disrupt cell aggregates, count the cells and resuspend at a At this stage, using an inverted microscope, check that the
concentration of 6 x 105 cells/mL. monolayers of Hep2c cells are confluent, that they show a
3.2. PROPAGATION OF ENCEPHALOMYOCARDITIS relatively even distribution of cells, that they have correct
VIRUS morphology and that they are healthy.
Encephalomyocarditis virus is propagated in mouse Remove most of the culture medium from the wells by
L-929 cells in order to produce a stock of progeny virus. inverting the plate and shaking it and blotting on a paper
L-929 cells are maintained by trypsin treatment and passage towel (proceed in an identical way when discarding fluids
as described for Hep2c cells (NOTE: it may be necessary to from micro-titre plates as described later). Dilute the EMCV
substitute neonatal calf serum with foetal bovine serum if the cells stock with fresh culture medium A to a titre of approximately
show poor growth). 3 x 107 PFU/mL (NOTE: each plate requires approximately
Take several flasks containing confluent cultures of 20 mL of diluted virus, plus 5 per cent to 10 per cent of extra
L-929 cells. Pour off the medium from the flasks. Inoculate volume). Dispense the diluted suspension from a 9 cm sterile
with 2 mL of the EMCV suspension appropriately diluted in Petri-dish using a multichannel pipette set at 200 µL to all
culture medium B so that it contains approximately wells including virus controls, but excluding cell controls.
2.5 x 10 8 plaque forming units (PFU) per millilitre. Each Add approximately 200 µL of culture medium A without
flask will contain 4-6 x 10 7 L-929 cells and therefore the virus to each of the cell control wells.
multiplicity of infection (m.o.i.) will be approximately Return the plates to the incubator set at 37 °C and
10 PFU/cell. Carefully swirl the virus suspension over the 5 per cent CO 2 for approximately 24 h.
entire cell monolayer and return the flasks to the incubator Staining
for approximately 1 h. Maintain the medium at pH 7.4 to Examine the plates microscopically to check that the EMCV
7.8. has caused a cytopathic effect (c.p.e.) in the virus controls.
After adsorption of the EMCV, add approximately 40 mL of The time interval for maximum c.p.e. may vary from one
culture medium B to each flask and return the flasks to the assay to the next because of inherent variation of Hep2c cells
incubator at 37 °C for about 30 h. Maintain the medium at to virus challenge over a given period of continuous
pH 7.4 to 7 .8 to obtain a maximum virus yield. Remove the cultivation.
culture fluid and store at approximately 4 °C. Remove most of the culture medium from the wells by
Place the flasks at -20 °C to freeze the cell monolayer. Then discarding into an appropriate decontaminating solution
thaw to room temperature. Add approximately 5 mL of (sodium hypochlorite is suitable). Dispense phosphate buffered
culture medium and shake the flask to disrupt the cell walls. saline pH 7. 4 R into each well. Discard the phosphate buffered
Transfer the contents of each flask to the container of culture saline pH 7. 4 R into a decontaminating solution. Dispense
fluid. Transfer the culture fluid containing the EMCV to into each well 150 µL of staining solution. Stain the cells for
50 mL plastic centrifuge tubes and centrifuge at approximately 30 min at room temperature. Discard the
approximately 500 g for about 10 min to remove cell debris. staining solution into a decontaminating solution. Dispense
Dispense the clarified culture fluid into glass screw-capped approximately 150 µL of fixing solution. Fix for 10 min at
2023 Appendix XIV NI V-A527

room temperature. Discard the fixing solution into a 4.3. STATISTICAL VALIDATION
decontaminating solution and wash the cell monolayers by As with all parallel line bioassays, the assay must satisfy the
immersing the assay plates in a plastic box containing usual statistical criteria of linearity of response, parallelism
running water. Discard the water and superficially dry the and variance.
plates with paper towels. Dry the assay plates at 20 °C to 4.4. VALIDATION OF ASSAY LAYOUT
37 °C until all moisture has evaporated. As with all microtitre plate assay procedures, attention must
Add 150 µL of 0.1 M sodium hydroxide to each well. Elute be given to validating the assay layout. In particular, bias due
the stain by gentle agitation of the plates or by hitting them to non-random pipetting order or plate edge effects must be
against the palm of the hand. Make sure that the stain is investigated and eliminated, by randomising the assay layout,
evenly distributed in all wells before making or by avoiding the use of edge wells.
spectrophotometric readings.
REAGENTS AND CULTURE MEDIA
Read the absorbance at 610 nm to 620 nm, using a Culture medium A (10 per cent neonatal calf serum)
microtitre plate reader, taking as a blank a well or a column
of wells containing no cells and approximately 150 µL of RPMI 1640 culture medium, supplemented with antibiotics if 450 mL
0.1 M sodium hydroxide. necessary (penicillin 10 000 IU/mL; streptomycin 10 nglmL)
Estimate the concentrations of interferon standard that give L-Glutamine, 200 mM, sterile 5mL
Neonatal calf serum 50 mL
the maximum and minimum reduction of cytopathic effect.
This is the dose response corresponding to the working range
Culture medium B (2 per cent foetal bovine serum)
of the assay.
3.3.2. Assay procedure RPM! 1 640 culture medium, supplemented with antibiotics if 490 mL
Carry out the assay as described above, using: necessary (penicillin 10 000 IU/mL; streptomycin 10 ng/mL)
- as test solutions, the substance to be examined, diluted in L-Glutamine, 200 mM, sterile 5mL
Foetal bovine scrum 10 mL
two-fold increments with culture medium A to give
nominal concentrations covering the working range of the
Staining solution
assay,
- as reference solutions, the appropriate standard for Naphthalene black 0.5 g
interferon (for example, a specific WHO sub-type Acetic acid, glacial 90 mL
interferon) in culture medium A, diluted in two-fold Sodium acetate, anhydrous 8.2 g
increments to give nominal concentrations covering the Water to 1000 mL
working range of the assay.
Fixing solution
3.3.3. Data analysis
Results of the cytopathic effect reduction assay generally fit a Formaldehyde, 40 per cent 100 mL
sigmoidal dose-response curve, when the interferon Acetic acid, glacial 90mL
concentration (the log of the reciprocal of the interferon Sodium acetate, anhydrous 82 g
dilution) is plotted versus stain absorbance. Water to IO00mL

Plot the interferon concentration (log reciprocal of dilution)

versus the stain absorbance for the interferon reference
preparation and for the interferon test solutions. Using the
linear portion of the curve, calculate the concentration of
interferon in the sample by comparing the responses for test N1. Numeration of CD34/CD45+ Cells in
and reference solutions, using the usual statistical methods Haematopoietic Products
for a parallel line assay.
(Ph. Bur. method 2.7.23)
4. VALIDATION OF OTHER PROCEDURES
This chapter describes immunolabelling and analysis by flow
4.1. CHOICE OF CELL LINE AND VIRUS
cytometry (2. 7. 24) to determine the number of
A number of other combinations of cell line and virus have
CD34/CD45+ cells contained in haematopoietic products.
been used in anti-viral assays for interferons. For example,
The determination is carried out by a single platform method
EMCV has been used in combination with the A549
using calibrated fluorospheres, after lysis of the sample red
epithelial lung carcinoma cell line, Semliki Forest virus or
blood cells if necessary.
Sindbis virus have been used with human fibroblasts, and
vesicular stomatitis virus has been used with either human This method applies to all types of preparations and whole
diploid fibroblasts, the human amnion WISH cell line or the blood. However, the characteristics of this method make it
Madin-Darby bovine kidney cell line. In each case the choice particularly suitable for preparations containing very low
of the cell line/virus combination is usually based on that percentages of CD34/CD45+ cells.
which gives the most sensitive response to the interferon Graft quality assessment by CD34/CD45+ cell
preparation to be assayed, and gives parallel responses when enumeration
comparing the test preparation and interferon standard. A variety of studies have established that the 1-3 per cent of
4.2. CHOICE OF RESPONSE cells in the bone marrow that express the CD34 cell surface
The staining procedure described above measures remaining antigen are capable of reconstituting long-term, multilineage
viable cells. A number of other responses have been used, haematopoiesis after myeloablative therapy.
including methyl violet or crystal violet staining, or the CD34/CD45+ cells are also found in the peripheral
thiazolyl blue (MTT) conversion procedure. In each case, the circulation of normal individuals but are extremely rare
method is selected on the basis of producing a suitably linear (0.01-0.1 per cent). However, CD34/CD45+ cells may also
and sensitive relationship between response colour and viable be mobilised from marrow to the peripheral circulation in
cell count. greater numbers by haematopoietic cytokines such as
granulocyte colony-stimulating factor and/or chemotherapy.
V-A528 Appendix XIV Nl 2023

The technique used for enumeration of CD34/CD45+ cells Number of events analysed
must meet the following requirements: A sufficient number of events are analysed to maintain
- high sensitivity, since haematopoietic stem cells are rare acceptable accuracy and precision, for example not fewer
events; than 100 CD34+ events and not fewer than
- accuracy, to provide clinically relevant results; 60 000 CD45+ events; the total number of cells counted
- reproducibility, to provide clinically reliable results; may be greater if the percentage of CD34 is 0.1 per cent or
- speed, to provide real-time analysis. less.
Selection of parameters Specimen collection
The flow cytometry assay uses commercially available, Acid citrate dextrose (ACD) formula A is the anticoagulant
directly conjugated fluorochrome-labelled monoclonal used in apheresis procedures. This anticoagulant allows both
antibodies, routine staining and whole blood lysing an automated leucocyte count and flow cytometry evaluation
procedures, and a gating strategy using light scatter and to be performed on the same specimen. Edetic acid (EDTA)
irnmunofluorescence analysis using a pan-CD45/CD34 is the anticoagulant of choice for peripheral blood sampling.
monoclonal antibody combination. Specimen transport
It is possible to determine CD34/CD45+ cell viability by Transport conditions guarantee the physical and thermal
appropriate nucleic acid staining with a stain that does not safety of samples.
cross the intact cell membrane (for example, with Specimen integrity and storage
7-aminoactinomycin D). Fresh (less than 24 h old) apheresis products, whole blood
Selection of monoclonal antibodies samples, umbilical cord blood specimens or bone marrow
CD34 antibodies Use class III CD34 antibodies that samples can be processed. Old specimens (more than 24 h
detect all glycosylation variants of the molecule (for example, old) and specimens that have been frozen and thawed are
clone 8G12 or 581). To detect rare events, use an antibody stained with a viability dye. On receipt, the temperature
conjugated to the brightest fluorochrome excitable using an within the package is verified.
argon laser-based flow cytometer, for example
TECHNIQUE
phycoerythrin (PE).
Sample preparation
CD45 antibodies Pan-CD45 antibodies that detect all Ensure that the concentration of leucocytes is suitable prior
isoforms and all glycoforms of this structure are required. to staining with monoclonal antibodies. If necessary, dilute
A CD45 antibody conjugated to fluorescein isothiocyanate the sample with medium that is compatible with the product
(FITC) fluorochrome is generally used (for example, J33, to be tested and the lysing system. Record the dilution factor.
HLel, 2Dl). It is recommended to perform the test with a negative
Isotypic or isoclonic controls A negative control is control.
analysed to detect any non-specific signal in the PE
Flow cytometry analysis
fluorescence region. If using an isotypic control (a
Autostandardisation
monoclonal antibody to an irrelevant antigen of the same
isotype as the CD34 antibody employed), the PE-conjugated For analysis of cells labelled with a commercially available
isotype is combined with CD45-FITC (or PerCP). If using kit, manufacturers have developed some quality tools for
an isoclonic control, the unconjugated (in excess) and setting the flow cytometer. These settings are then
PE-conjugated CD34 identical monoclonal antibody is automatically transferred on protocol analysis of samples.
combined with conjugated CD45. Alternative combinations Specific fluorospheres are used to set the photomultiplier
may be used. tube (PMT) on target values, compensation is set and the
system is checked using a control preparation.
Absolute count of CD34/CD45+
System settings
Calibrated .fluorospheres Depending on the technique
- Discriminator/threshold: the forward angle light scatter
used, the internal standard either consists of calibrated beads
threshold is set to exclude debris (low forward scatter)
in suspension or is directly introduced into the associated
but not small lymphocytes from the light-scatter plot.
tubes by the manufacturer.
- PMT high voltage settings: these must be consistent with
The absolute count of the CD34/CD45+ cells per micro litre cell-surface marker analysis and established within each
is calculated using the following expression: laboratory so that negative and positive cell populations of
moderate antigen density can be distinguished; PMT
A
-xC voltages are reviewed and adjusted periodically according
B to standardised laboratory procedures.
A number of CD34/CD45+ cells counted; - Compensation: this must be acceptable for the colour
B number of fluorosphere singlets counted; spectra overlap (for example, FITC/PE) encountered in
C known fluorosphere concentration. cell-surface marker analysis; colour compensation is
analysed and adjusted according to standardised
Gating strategies laboratory procedures.
The purpose of sequential gating is to select the population - Flow rate: this must be consistent with routine cell-surface
of interest and simultaneously minimise interference from marker analysis.
debris and mature cells to which antibodies can bind non- - Gating regions: the gating regions established for the
specifically. If using a commercial kit, apply the gating CD34/CD45 samples are maintained unaltered for the
recommended by the manufacturer. If using an in-house analysis of the negative region.
assay, it is preferable to apply a currently recommended
Calculation of absolute number of CD34/CD45+ cells
strategy. A gating strategy that uses light scattering
The absolute number of CD34/CD45+ cells is calculated
parameters and CD34/CD45 fluorescence will aid in the
using the following expression:
accurate identification and enumeration of
CD34/CD45+ cells.
 2023 Appendix XIV N2 V-A529

nxDx V
number of wells positive for cell proliferation is measured
n total number of CD34/CD45+ cells per microlitre;
with an automated system.
D dilution factor; The other main source of variability stems from the use of
V volume of the product to be tested, in microlitres, undefined materials (for example, foetal bovine serum or
bovine serum albumin) in the CFC assay. These products
Results are reported as both the percentage of derive from pools of source materials and provide a non-
CD34/CD45+ cells and the absolute number per microlitre. specific stimulation of cellular proliferation. However, it is
They may also be reported as the absolute number per not uncommon to have batches with particular characteristics
kilogram of recipient body mass, where this is possible. that selectively stimulate the proliferation of specific
haematopoietic lineages.
Finally, a low level of endotoxins (less than 0.01 IU/mL or
less than 0.01 IU/mg) in all the materials used for the
N2. Colony-forming Cell Assay for clonogenic assay is advisable, as higher levels result first in a
Human Haematopoietic Progenitor Cells progressive skewing of the haematopoietic lineages expression
in the cultures, and afterwards in a more general inhibition of
(Ph. Bur. method 2. 7.28) cell proliferation and clonogenesis.
The haematopoietic system represents a continuum of cells CFC CLONOGENIC ASSAY
whose phenotype and properties change as they progress The CFC assay is based on the capacity of progenitor cells to
from stem cells to differentiated cells. form a colony when plated in a semi-solid medium or in a
Haematopoietic progenitor cells (HPCs) are capable of gel in the presence of specific growth factors. Different types
forming colonies or 'cell clusters' in cultures grown in semi- of semi-solid media may be used (for example,
solid media and are said to be 'clonogenic'. methylcellulose, collagen, agar and plasma-clot) depending
The determination of the number of colony-forming cells on the desired readout. Commercially available media usually
(CFCs) in a cellular product is an indicator of the functional give more reproducible results.
capacity of the progenitor cells and is a predictor of
MATERIALS
haematopoietic reconstitution. The measured number of
A validation is performed at least for the following critical
CFCs correlates with the minimum number of progenitors
materials.
present in the sample.
Growth factors
CELL-SURFACE MARKERS Both multilineage (such as Kit-ligand or stem cell factor
The capacity of colony-forming cells to give rise to (SCF), interleukin-3, granulocyte-macrophage colony-
haematopoietic colonies in vitro and/or to reconstitute the stimulating factor (GM-CSF)) and lineage-specific
haematopoietic system has been correlated with the (erythropoietin, granulocyte colony-stimulating factor (G-
expression of specific cell-surface antigens. The expression of CSF)) growth factors are required to obtain the highest
the membrane antigen CD34 is an accepted marker for most number of colonies from a cell suspension containing a
of the haematopoietic progenitors and stem cells. mixed population of HPCs.
COLONY ASSAY SPECIFICITY Other media components
Colony-forming cells are identified with a nomenclature Media may be supplemented by serum (notably by foetal
based on the lineages of mature cells present in the colony bovine serum) and/or albumin.
(for example, CFU-Mix, CFU-GEMM, CFU-GM, CFU-G, CELL CULTURE
CFU-M, BFU-E, CFU-E, CFU-Meg) and are a population
of progenitors able to give rise to colonies containing one or Cells
more lineages of haematopoietic cells. No or low capacity for The sample placed in culture must be representative of the
self-renewal has been ascribed to this population of human cellular product injected. Cell suspensions are required for
HPCs compared with the most immature stem cells. this assay. In the case of bone marrow aspirates, such
suspensions can be obtained by forcing the bone marrow
The amount and type of growth factors supplied during the
through a sieve or through progressively smaller calibre
culture modulate the type and size of colonies that will be
needles. Repeated passages through a 21-gauge needle are
formed.
usually sufficient to disperse cell clusters into a cell
Greater specificity on the general class of HPCs and on their suspension.
relative proliferative potential is provided by the time
PUT/NG AND SCORING
required to differentiate in vitro into mature cells. The time
The cells diluted in the culture medium are mixed in the
required by post-natal colony-forming cells to give rise to a
semi-solid medium. It is common to plate 1 mL of the
colony formed of mature cells in vitro is 10-14 days.
mixture in an untreated sterile Petri dish (0 35 mm).
QUALITY ASSURANCE FOR A CFC ASSAY Because of the viscosity of the medium, the solution cannot
It is paramount for the overall quality of the colony-forming be plated with air displacement pipettes and the use of
cell assay to apply a strictly standardised approach. It is syringes equipped with large bore (::; 18-gauge) needles is
therefore recommended to carry out intra- and inter- required.
laboratory validations. The source of the materials, including
The number of cells to be plated depends on the HPC
reagents, growth factors and disposables, is identified.
concentration in the sample to be tested. So that no colony is
The main factors affecting variability in the CFC assay are derived from 2 different HPCs, the number of cells plated
the number of cells plated and the identification of colonies. must allow between 40 and 80 colonies per plate (0 35 mm)
Up to 15 per cent intra-laboratory variability may be to be counted. The 'target' number of colonies per plate may
observed for the same test. If it is necessary to evaluate the be obtained either from the percentage of CD34+ (or
number of colony-forming cells in a purified cell population, concentration of CD34+ cells/mL) determined by flow
it is possible to use a limiting dilution approach where the
V-A530 Appendix XIV N3 2023

cytometry (2. 7.24) or from different dilutions of the cell

suspension (usually 2 concentrations are tested).
a number of cells counted;
The plates are incubated in aerobic conditions with a carbon d dilution factor (where applicable);
dioxide concentration of 5 per cent, at 37 °C in a humid n factor varying with the volume of the haemocytometer chamber.
(saturated) atmosphere for 10-14 days, and the number of
colonies is then scored under an inverted microscope. Care It is possible to distinguish between mixed cell populations
must be taken when manipulating the dishes containing the provided they differ in size or pigmentation (for example,
colonies as the methylcellulose-based medium is viscous but leukocytes and erythrocytes).
not jellified. An inclined plate will result in mixed and Preparation of the counting chamber and analysis
'comet' -shaped colonies making the scoring likely to be Mount the coverslip (slightly moistened on the edges) on the
incorrect. slide. Move the coverslip back and forth over the slide,
IDENTIFICATION OF THE COLONIES pressing slightly on the sides. Prepare a suitable dilution of
The size and structure of the colonies depend on the type of the cell suspension in isotonic buffer or in haemolysis buffer.
mature cells that are their constituents. 50 cells per colony is Add an appropriate volume of the dilution to the counting
usually considered a minimum. The presence of chamber. The liquid is added to the border of the coverslip
haemoglobinised cells identifies progenitors of the erythroid and is drained inside the chamber by capillarity. Carefully
lineage. As the amount of mature cells for each lineage place the haemocytometer under the microscope and focus.
largely depends on the growth factors added to the cultures, Count the cells in a zone of the grid. Calculate the cell
performing differentiated counts is not recommended unless concentration in the diluted and original samples.
otherwise prescribed. To increase the accuracy of the measurement, it is important
EXPRESSION OF THE RESULTS to respect the following basic precautions:
The results of CFC culture are usually expressed as the - use only suitably thickened coverslips;
arithmetic mean of the number of colonies counted in at - wherever possible, count more than 100 cells (if
least 3 plates in the test. The mean number of colonies is necessary, count more areas);
then related to 104 or 105 viable nucleated cells placed in - where cell clustering is detected (i.e. the cell suspension is
culture. not monocellular), resuspend the cells before sampling
and count again;
- avoid underfilling or overflowing the chamber, otherwise
the volume will no longer be accurate.
N3. Nucleated Cell Count and Viability AUTOMATED COUNTING METHODS
Particle counters based on conductivity variation
(Ph. Eur. method 2.7.29)
Electronic particle counting devices measure the size and
The determination of the quality of cell suspensions requires number of particles in a solution.
accurate measurements of both cell concentration and Particle counters are calibrated before use with a solution of
percentage of viable cells. These data are essential to the particles of known concentration and size. To allow the
decision-making process for preparing cellular products and counting of larger particles, tubes fitted with differently
for maintaining optimum culture conditions. The cell count calibrated orifices are available. These apparatuses do not
may be expressed as the number of cells per volume of cell allow the discrimination between dead and live cells. As cell
suspension and the cell viability as the number of viable cells debris may also generate pulses that may cause errors,
per volume of cell suspension. The cell-count procedure may counters are also fitted with a threshold control allowing only
be performed manually (haemocytometer) or with an larger particles to be counted.
automated apparatus (for example, particle counter, flow
The apparatus must be qualified for the counting of cellular
cytometer). Other methods than that described below may be products (in terms of linearity, accuracy, etc.).
used.
Particle counters based on flow cytometry (2. 7. 2 4).
CELL NUMBER The flow cytometer is calibrated with reference particles of
MANUAL COUNTING known concentration and size to give an absolute cell
Description of the apparatus and test principle The number per volume. However, a calibrating solution is no
following materials are required: longer necessary in instruments using 2 electrodes inserted in
- a haemocytometer: a specialised microscope counting the sampling chamber where the fixed size of the sampling
chamber available in different designs. It consists of a chamber and distance between the 2 electrodes allow the
thick slide and a coverslip mounted to delimit a chamber measurement of the content of a fixed volume. This type of
with a specific volume for each design. The thick slide of instrument rarely needs to be calibrated after the initial
the various haemocytometers consists of counting setting.
chambers separated by deep grooves to avoid cross-filling.
VIABILITY
The counting chamber is etched in the glass and contains
a grid which is specific for each model; This section applies to cell staining by viability dyes and
- a light microscope - low power 10 x to 40 x manual or automated analysis, under a light microscope or
magnification; by flow cytometry, of a cell suspension in order to determine
- pipettes of a suitable volume range. the percentage of viable cells.
The haemocytometer is used to quantify the number of cells Depending on the type of cells and the method used, the
in a given solution by calculation of the cell concentration results may differ.
per millilitre (C) using the following expression: MANUAL DYE-EXCLUSION METHOD
Test principk This test is based on the exclusion of the
dye from viable cells whereas dead or damaged cells absorb
the dye and are coloured. It provides information on the
2023 Appendix XIV O V-A531

cytoplasmic membrane integrity but its results do not contrast to PI, the 7-AAD/DNA complex shows minimal
necessarily reflect cell functionality. Recently trypsinised or overlap with FITC and PE.
thawed viable cells may have leaky membranes, causing them PI binds to double-stranded DNA by intercalating between
to absorb the dye. bases with little or no sequence preference and with a
Dye Trypan blue is the stain most commonly used to stoichiometry of 1 dye molecule per 4-5 DNA base pairs.
distinguish between viable and non-viable cells, but other Once the dye is bound to nucleic acids, its fluorescence is
suitable dyes such as etythrosin B or nigrosin may also be enhanced 20- to 30-fold, the fluorescence excitation
used. It is an acid dye (Mr 961), an anion with 4 sulfonate maximum is shifted around 30-40 nm towards the red and
groups that can easily bind to proteins; therefore the protein the fluorescence emission maximum (615 nm) is shifted
concentration of the preparation to be tested must be as low around 15 nm towards the blue. Although its absorptivity is
as possible. quite low, PI exhibits a sufficiently large Stokes shift to allow
Test conditions Dye fixation is strongly influenced by pH, simultaneous detection of nucleic acids and fluorescein-
within a range of 6.6 to 7 .6. Fixation is optimal at pH 7.5. labelled antibodies, provided that the suitable optical filters
The other conditions, such as the dye concentration and the are used.
staining time are validated. Storage conditions of nucleic acid dye solution
Storage conditions of the dye Generally a 0.4 or 5 ± 3 "C.
0.5 per cent trypan blue solution in sterile phosphate- Test preparation and analysis In the case of
buffered saline is used. Store protected from light and air. haematopoietic cells, the dye may be added after CD45
Test preparation and analysis Stain the cell suspension labelling to obtain a better separation of cells from debris and
at the required dilution (usually in phosphate-buffered saline) platelets with a side scatter (SS)/CD45+ gating region.
with, for example, a trypan blue solution having a final The incubation conditions of the cell suspension with the dye
concentration of0.l to 0.2 per cent. Mix gently. Incubate for are validated previously.
not more than 2-4 min at room temperature. Mix gently and Incubation is performed at room temperature protected from
place a suitable volume in a counting chamber. Count light. Where necessary, lysis of red blood cells is performed
without delay. using, for example, ammonium chloride. If not, add buffer
Determine the percentage of viable cells from the ratio of the alone.
number of unstained cells to the total number of cells under Percentages of viable cells are directly given by the flow
a light microscope, considering all stained cells as dead cells. cytometer and deduced from the analysis of positive cells
Viability (V) is calculated as a percentage using the following (dead cells) in the SS/7-AAD or SS/PI cytogram (dot plots).
expression: Positive controls may consist of stabilised cells (dead cells)
n mixed with fresh viable cells at a target value.
N x 100 Digital imaging of stained cells
Digital imaging allows the automation of dye-exclusion
n number of unstained (viable) cells;
methods. The cell suspension and viability-dye solution are
N total number of cells (stained and unstained).
directly mixed by a machine. The system, which allows
sample aspiration, reagent handling, and subsequent
It is essential that the incubation time be not more than
4 min as the number of stained cells may increase instrument cleaning is fully automated. Once the cellular
significantly afterwards. For a new determination, it may suspension has been aspirated and mixed with the dye
therefore be necessary to prepare a new test. solution, it is pumped to the flow cell for imaging.
The stained cell suspension is aspirated through a chamber
AUTOMATED METHODS where stroboscopic light allows a camera to photograph the
Flow cytometry flowing cells. The images are digitalised and the number of
Test principle The test is based on the ability of certain dead or live cells counted by the software.
dyes to cross damaged membranes and bind to DNA by
intercalating between bases so that dead cells may fluoresce
and be detected by flow cytometry (2. 7.24). Non-viable cells
are evaluated and discriminated by focusing on positive 0. Host-cell Protein Assays
staining whereas viable cells remain unstained. This analysis
is generally performed with 7-aminoactinomycin D (7-AAD) (Ph. Bur. method 2.6.34)
or propidium iodide (PI) but other suitable dyes may also be This general chapter provides guidance for the development and
used. validation of host-cell protein (HCP) assays used to test products
Dye 7-AAD and PI are given as examples ofmembrane- obtained by recombinant DNA technology. It does not exclude the
impermeants that may be used as viability dyes. use of alternative approaches that are acceptable to the competent
7-AAD is an analogue of actinomycin D that contains a authon·1y.
substituted amino group at position 7 of the chromophore. INTRODUCTION
It intercalates between cytosine and guanine DNA bases. Host-cell proteins (HCPs) are process-related impurities
The spectral properties of 7-AAD make this molecule derived from the host organism used for the production of a
particularly suitable for flow-cytometry analysis. medicinal product by recombinant DNA technology.
The maximum absorption of the 7-AAD/DNA complex is In order to mitigate their potential adverse effects
situated in the green spectral region and is thus suitable for (e.g. immunogenicity), HCP content is expected to be
an argon laser-equipped cytometer (excitation wavelength of reduced to the lowest possible level.
488 nm). The deep red fluorescence emission of the 7-AAD HCP clearance during the purification process must be
viability dye (635 nm to 675 nm) eases the use of the probe assessed and the HCP content determined using an HCP
in combination with fluorescein isothiocyanate (FITC) and
phycoetythrin (PE)-conjugated antibodies, because in
V-A532 Appendix XIV 0 2023

assay that has been evaluated and validated for a given a combination of strains of an expression host species, and
product. the process(es) used may not mimic the process applied for
The HCP acceptance limit, typically expressed in nanograms the product of interest. The suitability of the antiserum
of HCP per milligram of active substance (ppm), must be should be evaluated as described above for process-specific
justified with regard to the HCP clearance capacity of the assays.
purification process and with regard to the potential impact CRITERIA FOR ASSAY SELECTION
of residual HCP on patients, taking into account the worst- In view of the potential safety issues associated with residual
case quantity of HCP that could be administered with the HCPs in the active substance, a risk assessment is performed
product. to support the choice between a generic, a platform or a
HCPs are generally measured using an immuno-based assay process-specific strategy, taking into account the stage of
containing, as reagents, the HCP antigen preparation development of the product.
(hereinafter 'the HCP antigens') or HCP reference standard For early development a generic assay or a platform assay
and the corresponding polyclonal antibodies (antisera). may be used. For later development phases, process-specific
Antisera must cover a broad spectrum of HCPs assays must be considered, as they are generally regarded as
representative of the product concerned. superior, especially when compared to generic assays. This is
Sandwich-type enzyme-linked immunosorbent assays because process-specific assays are more likely to show
(ELISA) are the most commonly employed assays to assess immunoreactivity against representative HCPs.
quantitatively the level of HCPs. It should be noted that Platform or generic assays may be used, provided that the
HCP content measured by ELISA does not represent assay is appropriately characterised and validated against
absolute HCP mass content. The sensitivity is the result of process-specific HCPs.
the observed cumulative responses of many individual HCPs
PRODUCTION AND TESTING OF THE HCP
in comparison to the response of an HCP reference standard.
ANTIGEN PREPARATION
The use of orthogonal analytical methods
The HCP reagents (HCP antigens and anti-HCP antibodies)
(e.g. electrophoresis, HPLC, Western blot, mass
are produced in such a way as to facilitate replication of the
spectrometry) to characterise the various HCPs in the
production when a replenishment for the HCP assay is
product is recommended to support the development and
selection of the assay. needed.
The HCP antigens are used to generate the polyclonal
ASSAY SELECTION antibody reagent for the HCP immunoassay by immunising
Several types of assay are available, with selection taking into one or more suitable animal species. In addition, they serve
account several factors, including the stage of development of as the HCP reference standard in the HCP immunoassay.
the product, the nature of the host cell and the protein
As far as possible, the HCP antigens must cover the relevant
immunogenicity, the expression mode, the manufacturing
HCP population expected to be derived from the
process, and prior knowledge. When selecting and developing
manufacturing process of the protein of interest.
the assay, its life cycle (e.g. reagent supply, consistency, assay
validation, process change) must also be considered. The HCP population must also be broad enough to cover
worst-case purification scenarios and to provide robusmess
IYPES OF ASSAY against potential manufacturing process changes during the
Process-specific assays life cycle of the product.
Process-specific HCP assays (also called product-specific
PROCESS-SPECIFIC ASSAYS
HCP assays) are developed and validated taking into account
the specificity of the production process, and using the same Null cell line
host organism expressing the recombinant product. Development of a process-specific assay involves the selection
of a null cell line that does not contain the expression gene
The HCP antigens are derived from a mock run of the active
for the product of interest and is derived from the same cell
substance manufacturing process (or a process representative
line that has been used to establish the production cell line.
of it) up to a step capable of generating a broad spectrum of
This null cell line may be non-transfected or mock-
HCPs in sufficient quantities.
transfected. A mock-transfected cell line is created by
The antisera raised must cover a broad range of HCPs, in transfecting the parental cell line with a blank plasmid,
order to detect as many different HCPs as possible and also i.e. the plasmid used to create the production cell line, but
to accommodate process variations. missing the gene coding for the protein of interest.
Platform assays Mock production process
Platform assays are developed by individual manufacturers Upstream
and customised for the processes and host organism used by
The antigens produced for process-specific assays are
the manufacturer for production. The same sets of reference
obtained by a mock production process that mimics the
standards and reagents may be used to monitor HCPs in
intended manufacturing process, using the null cell line and,
several products manufactured in the same host organism,
as far as possible, the same operating conditions.
provided that upstream processes (and downstream, if
relevant) are sufficiently similar for these products. As for any mock production, the process used represents an
The suitability of the antiserum should be evaluated as approximation of the intended manufacturing process and
described above for process-specific assays. leads to differences (e.g. different scale, operating parameters,
product interaction). However, the impact of those
Generic assays differences needs to be considered carefully because they may
Commercially available HCP test kits are commonly referred affect the composition of the HCP population.
to as generic HCP assays. They are intended to work broadly
across similar expression hosts. Detailed information on the For example, a mock fermentation of an inclusion body
preparation of the reagents may not be disclosed by the manufacturing process may not deliver the desired inclusion
vendor. For instance, the HCP antigens may be derived from bodies if the product is not present. Therefore, depending on
2023 Appendix XIV O V-A533

the null cell line used (e.g. mock-transfected or not), the Downstream
antigens may need to be isolated differently compared to the As for other assays, the HCP antigens derived from the
intended manufacturing process. upstream process are, in general, only minimally processed
In some situations, operating parameters for the mock (e.g. no or limited number of purification steps) to obtain a
production may be adjusted to cover worst-case scenarios broad spectrum of HCPs, although mixing and pooling
(e.g. to deliver antigens covering a broad spectrum of strategies may also be used to widen the spectrum of HCP
different HCP species). For example, the antigen-containing species.
cell culture supernatant may be harvested beyond the Characterisation and testing
minimum level of cell viability in order to include more As for process-specific assays, both the protein content and
cytosolic proteins, which are released by additional cell lysis. the absence of the protein of interest are tested. Comparison
Downstream of the HCP population with the mock and the intended
The HCP antigens derived from the upstream process are production process is performed.
usually only minimally processed (filtration, concentration), GENERIC ASSAYS
in order to obtain a representative spectrum of HCPs. Generic assays are commercially available and are developed
Further purification is generally not recommended as there by the vendor.
will be a risk of losing HCP species.
Detailed information on the preparation of the reagents may
However, in cases where the antigens are not representative
not be disclosed by the vendor. For instance, the null cell
(e.g. resulting in low coverage), mixing of mock materials
line may be derived from a combination of strains of an
from different processing steps can be considered.
expression host species, and the process(es) used may not
Enrichment may also be achieved by pooling materials from
mimic the process applied for the product of interest.
mock fermentation or purification runs using different
operating conditions, or from selective purification steps Nevertheless, the generic assay must be selected with
(e.g. to reduce large amounts of the few immunodominant consideration given to the intended manufacturing process
HCPs). (e.g. appropriate host cell line), and be appropriately
validated for the product of interest and phase of
Cross-contamination with the protein of interest
development. As a consequence, if generic assays are used in
The HCP antigens must be produced in a manner that
later stages of development or during commercial
avoids contamination with even minute traces of the product
manufacturing, it is recommended to validate the assay and
in order to avoid cross-reactivity with the polyclonal
control lot-to-lot reagent consistency using either appropriate
antibodies.
upstream fractions from the production process or a mock
To achieve this goal, dedicated or single-use equipment is preparation generated using a null cell line.
used as much as possible. Where multi-purpose equipment is
used, it must be cleaned appropriately. In addition, the risk PRODUCTION AND CHARACTERISATION OF
of contamination when filling or handling the antigens in the THE ANTI-HCP ANTIBODY REAGENT
laboratory environment must also be considered. PROCESS-SPECIFIC AND PLATFORM ASSAYS
Characterisation and testing Immunisation
Before using the HCP antigens for immunisation, the protein One of the challenges of the immunisation step is to generate
content is assessed (total protein assay) and the absence of polyclonal antibodies that are highly specific and sensitive for
the protein of interest verified. each of the antigenic proteins in the complex mixture of
Comparison of the HCP population with the mock and the HCPs used as an irnmunogen. An animal's immune response
intended production process is performed, typically by SDS- must be stimulated against both the stronger and the weaker
PAGE and/or two-dimensional (2D) electrophoresis with a antigens.
high sensitivity stain. The aim of this comparison is to show An animal host that yields a sufficient quantity and diversity
that the HCP antigens resulting from the mock production of HCP-specific immunoglobulin G (IgG) is selected.
process contain most of the representative HCP species of Where both the polyclonal capture and the polyclonal
the intended manufacturing process. Where necessary, detection antibodies are from the same source, it can be
complementary information may be gathered by orthogonal assumed that they recognise different epitopes on the same
methods, e.g. mass spectrometry. HCP in the assay. Alternatively, polyclonal anti-HCP
PLATFORM ASSAYS antibodies from different animal species may be used. Using
Null cell line several animals for a given species may reduce the impact of
Development of a platform assay involves a null cell line that individual variations in immune competence and provide
does not contain the expression gene for the product of additional response diversity, resulting in maximised antibody
interest, and uses the same host species. This null cell line coverage against the HCP antigens.
may be non-transfected or mock-transfected, and may be An immune response to a limited number of HCP antigens
used for the production of HCP antigens for products from a may be obtained rapidly, particularly when adjuvants are
given company's manufacturing platform. used to boost the immune response. However, in complex
Mock production process mixtures, differential enhancement of the immune response
Upstream towards weaker antigens or those at lower concentrations
The HCP antigens produced for platform assays are obtained may be necessary.
by a mock production process that mimics the platform It usually takes several immunisations to reach a maximum
upstream process that is used for several products, and immunological response and, depending on the frequency of
typically uses the same media components. As for any mock immunisation, the process can take 3-6 months to complete.
production, the process used represents an approximation of The immune response against the HCPs for a given
the intended manufacturing process, which may impact the immunisation scheme has to be monitored by determining
composition of the HCP population (see process-specific the antibody titre using, for example, an ELISA, and by
assays).
V-A534 Appendix XIV 0 2023

comparing the results of lD or 2D electrophoresis after During the life cycle of the product, a full or partial
protein staining and a Western blot, where the polyclonal revalidation of the assay may be required, for example when
anti-HCP antibodies are used as primary antibody. implementing a manufacturing process change that may
In practice, some minor proteins that elicit a strong immune impact the suitability of the HCP reagent.
response may not be visible in the protein-stained gel, and Accuracy
some poorly antigenic proteins that are detectable by protein Accuracy is demonstrated by spike/recovery analysis of the
staining may not elicit a detectable immune response. HCP reference standard in a relevant background matrix
To achieve sample-dilution linearity in complex multi-analyte (e.g. the active substance or a sample from a relevant
immunoassays, it is essential that the immune reagent purification step).
simultaneously and specifically recognises as many individual
Specificity
analytes as possible in an assay sample and that it is present
Specificity is demonstrated by the absence of interference
in stoichiometric excess. For this purpose, a series of sample
from the matrix background (including the active substance).
dilutions from different process steps may be tested by
For instance, data from the accuracy study can be used to
ELISA using purified anti-HCP antibodies from bleedings
assess specificity.
that have shown suitable coverage by Western blot.
Finally, based on the results of the tests described above, Precision
antisera from different animals are pooled, retested and As for any other quantitative assay, repeatability, intermediate
purified. precision and reproducibility are appropriately demonstrated.
Purification and preparation Quantitation and detection limits
The HCP antibodies must be purified before an assay can be Sensitivity is usually in the ppm range and is normally
developed. described through the quantitation limit (QL). QL is typically
determined by HCP spike recovery studies in the active
Typically, this is achieved by protein A- or protein
substance or an appropriate sample matrix, and is calculated
G-chromatography and/or HCP antigen affinity
from the minimal spike providing a response with predefined
chromatography. In the case of HCP antigen affinity
accuracy and precision from replicate analyses.
chromatography, the antigens used for immunisation are
immobilised on column chromatography media and the Detection limit (DL) is often not determined (optional
specific antibodies are captured by applying the antisera onto validation parameter).
the column. Linearity
Additional purification to remove potential aggregates might The linearity of the HCP assay is demonstrated using
be required by gel permeation chromatography. dilution series of the HCP standard and spike/recovery
experiments (accuracy study).
For the ELISA, a part of the purified anti-HCP antibodies is
conjugated to a detection label (e.g. biotin or horseradish Additionally, due to the nature of HCP assays, the multiple
peroxidase). HCP analytes and polyclonal anti-HCP antibodies, sample-
dilution non-linearity may be observed, i.e. back-calculated
The purified anti-HCP antibodies and the sera must be
results increase with increasing dilutions of samples, which in
stored at a temperature that ensures their stability.
most cases is related to the excess of one or more individual
Characterisation and testing HCPs in the sample when compared to the available
The suitability of the derived HCP assay reagent is assessed antibodies in the HCP immunoassay. As a consequence,
by demonstrating the coverage of the HCPs representative of dilution linearity must be properly assessed for the relevant
the manufacturing process by the anti-HCP antibodies. process steps by comparison of target versus measured HCP
For this purpose, 2D electrophoresis of the HCP antigens is concentrations at varying sample dilutions. Dilution linearity
performed. The protein pattern of the immunostain is is demonstrated if the acceptance criteria for assay variation
compared with the protein pattern of the total stain. are met for different sample dilutions. Studies demonstrating
The anti-HCP antibodies must recognise a broad range of dilution linearity can be carried out either during method
HCPs over the full range of charge and molecular size. Other development or at the latest during method validation.
methods using native conditions may be considered. If a sample shows dilution non-linearity, multiple sample
GENERIC ASSAYS dilutions are prepared beyond the range where non-linear
Immunisation, purification and preparation of the anti-HCP behaviour is observed.
antibody reagent are carried out by the vendor and details The final HCP value is typically reported as the average
may not be available. HCP concentration obtained for a minimum of 2 dilutions
Characterisation and testing of anti-HCP antibodies are within the linear dilution range. If justified, 1 dilution may be
performed as for other assay types. Typically, there is limited sufficient.
control over lot-to-lot reagent consistency. Appropriate Range
comparative lot testing is therefore required. The range of the assay is typically defined by the HCP
VALIDATION OF THE HCP ASSAY concentrations for which a suitable level of precision,
The HCP ELISA is developed to detect and quantify a accuracy and linearity has been demonstrated.
heterogeneous mixture of antigens at varying concentrations, Robustness
and with a reagent containing antibodies that are not The evaluation of robustness is considered during the
represented at a one-to-one ratio. The section below is development phase.
intended to target the specifics in development and validation
CHANGE OF HCP ASSAY AND/OR REAGENT
of this type of ELISA.
The quantities of antigens and antibodies must be large
HCP assays such as ELISA are validated with regard to enough to supply the HCP assay for several years. Therefore,
accuracy, specificity, precision, quantitation and detection the supply, quality and consistency of reagents must be
limits, linearity, range and robustness. appropriately managed throughout the life cycle of the assay.
2023 Appendix XIV O V-A535

Table 2.6.34.-1
Depleted reagents
Process change
HCP reference standard Anti-HCP antibody

Reagent characterisation The protein concentration of the new Total protein concentration of the new The effects of process changes that have
reference standard is determined antibody is determined. The final assay the potential to impact the HCP
preferably using the same method as for concentration must be titrated for the new composition are analysed by suitable
the current reference standard to ensure lot in order to achieve a similar standard methods (e.g. 1D-l2D-PAGE, Western
that the protein concentrations are curve as for the current lot. blot, HCP assay).
comparable. For detection antibodies, the detection If the process change does not lead to a
Using suitable methods (e.g. ID-/2D- label: protein stoichiomerry is controlled relevant change in HCP composition, the
PAGE, 2D-DIGE), the similarity in and ensured to be similar to the current current HCP reagents are also suitable for
protein composition between the new and antibody lot. the new process.
current HCP reference standards is Immunoreactivity of the new antibody is If the process change does lead to a
assessed. compared qualitatively (by visual relevant change in HCP composition, but
comparison) or semi-quantitatively the suitability of the current HCP reagents
(coverage determination) against the was demonstrated, the current HCP
current lot by suitable methods reagents are also suitable for the new
(e.g. ID or 2D Western blot). Due to the process.
variability of the method, it is particularly If the process change does lead to a
advisable to perform this characterisation relevant change in HCP composition, but
side-by-side with the current antibody lot. the current HCP reagents were shown to
be unsuitable for the new process, a new
assay must to be developed including a
mock fermentation according to the new
process and a new immunisation.

Testing of reagents in The new HCP reference standard is Standard curves obtained when using new A mock run harvest from the new process
HCP assays quantitatively tested against the current versus current antibody lots are compared. is tested for spike recovery using the
reference standard for spike recovery at A bridging study is performed with testing current HCP assay.
different concentrations covering the of relevant process samples Relevant process samples (e.g. purification
validated assay range. (e.g. purification steps from harvest to the steps from harvest to the final active
Standard curves obtained with the new final active substance). In a side-by-side substance) from the new and the previous
versus the current reference standard are experiment, new antibodies must detect process are tested side-by-side.
assessed for similarity. HCP levels at different process steps
equally or with an improved quantitation
limit.

Assessment of validation If reagent characterisation and ELlSA If reagent characterisation and ELlSA If, for the new process, the antibody shows
status testing demonstrate suitability of the new testing demonstrate that the new antibody similar or higher immunoreactivity
HCP reference standard, the current is suitable, the current antibody can be compared to the previous process, and the
reference standard can be replaced. replaced. No revalidation of the test HCP assay shows adequate recovery from
No revalidation of the test method is method is required. the mock harvest of the new process and
required. If the new antibody differs significantly in also similar or higher sensitivity for
If the new HCP reference standard differs Western blot immunoreactivity or samples from the relevant process steps,
significantly in protein composition and/or immunoassay sensitivity and/or assay then the current assay and reagents are
assay performance from the current performance compared to the current considered suitable for the new process.
reference standard, revalidation is antibody, revalidation is required. No revalidation of the test method is
required. required.
If reagents appear suitable to detect HCP
from the new process, but the ELlSA
indicates significant differences in spike
recovery of the mock sample or of HCP
levels at relevant process steps, then
revalidation is required. The process
change might also impact dilution linearity
of test samples from certain process steps;
if these steps are essential for the HCP
control strategy_, revalidation or even
generation of new antibody reagents might
be required.
In case of a major change in
HCP composition with the new process
that leads to either a mismatch in protein
composition compared to the current assay
standard, reduced immunoreactivity of the
antibody, or significantly decreased
immunoassay sensitivity, then new assay
reagents are prepared and the HCP assay
is validated with the new reagents.

PAGE: polyacrylamide gel electrophoresis

DIGE: differential gel electrophoresis
V-A536 Appendix XIV 0 2023

For generic HCP assays, in order to ensure the consistency Characteristics qPCR Immunoenzymatic method
and quality of the reagents, recharacterisation or revalidation (Method A) (Method B)
of the assay may be required for each new batch of reagent, Specificity (total
as their quality may change from one batch to another. DNA versus Specific DNA Total DNA
For process-specific and for platform assays, there are specific DNA)
generally 2 situations where new HCP assay reagents may be Interfering
required: Proteins Detergents/proteins/solvents/RNA
substances
- the HCP reference standard and/or antibody are depleted;
Not applicable for DNA-based
antibody may then be purified from a frozen serum stock
products.
or a new immunisation is required;
The DNA content is
a manufacturing process change can impact the HCP
underestimated for fragments below
composition for the purification intermediates or the final 1000 base pairs (bp).
Fragments
product; the assay reagent may not be properly suited to
smaller than the Detectability depends on DNA size.
detect and quantify the modified HCPs; a manufacturing PCR product
Llmitations Undetectable short DNA fragments
process change can therefore render the reagents can not be
(below 80 nucleotides) can interfere
unsuitable for assay use. detected and
with the assay through reagent
quantified.
Newly prepared reagents must be thoroughly characterised consumption.
(e.g. by 2D-SDS-PAGE/Westem blot for coverage, 2D-SDS- Products must be free of bacterial
PAGE/differential gel electrophoresis (DIGE)/identification DNA.
by MS). Afterwards, the validation status of the assay using Narrow quantification range:
5-150 pg/well.
the new reagents must be assessed. It is recommended to
perform these experiments side-by-side with the currently
used reagents. SAMPLE PREPARATION
Table 2.6.34.-1 outlines a recommendation for reagent The concentration of residual host-cell DNA may vary
characterisation and assessment of immunoassay validation, depending on the type of biological product and the DNA
as a consequence of a depletion of assay reagents or a process clearance capacity of the manufacturing process.
change. Depending on the sample matrix, pretreatment of the sample
02. Quantification and Characterisation of Residual may be necessary to ensure appropriate recovery of the
Host-cell DNA residual host-cell DNA.
(Ph. Bur. method 2.6.35) When analysing highly purified protein samples such as
recombinant proteins or monoclonal antibodies, simple
This general chapter describes analytical methods that may be used
digestion with proteinase or affinity chromatography may be
to measure the content and to characterise the size of residual host-
sufficient to recover the residual host-cell DNA. For more
cell DNA in biological products produced in cell substrates. It does
complex matrices such as viral vaccines and viral vectors, an
not exclude the use of alternative approaches that are acceptable to
additional virus lysis step may be required to release the
the competent authority.
residual host-cell DNA from the viral particles.
INTRODUCTION The immunoenzymatic method is especially sensitive to
Several sensitive analytical methods exist for the interference from proteins. This can be avoided by
quantification of residual host-cell DNA, including real-time performing an initial pretreatment step, which may include
quantitative PCR (qPCR) (Method A) and an digestion with proteinase K and sodium dodecyl sulfate
immunoenzymatic method (Method B). (SDS). This step may suffice to recover the residual host-cell
qPCR may also be used to assess the residual host-cell DNA DNA. However, in other cases, the residual host-cell DNA
size distribution, as a characterisation test depending on the may be bound to the sample components, and/or soluble
nature of the cell substrate (e.g. continuous cell lines) and on interfering substances may be present, in which case it may
the amount of residual host-cell DNA. be necessary to extract the residual host-cell DNA from the
A suitable method is selected depending on the nature of the sample.
biological product to be tested and taking into account the The DNA can be extracted using protocols which have
characteristics and limitations of each method as summarised demonstrated a satisfactory recovery rate during spiking
in Table 2.6.35.-1. experiments. Several suitable methods exist, including DNA
precipitation or DNA-specific binding to a matrix
Table 2.6.35.-1 - Comparison of the characteristics of qPCR and (e.g. magnetic beads or silica columns). Commercial kits can
immunoenzymatic methods be used to extract residual host-cell DNA samples. Some of
Characteristics qPCR Immunoenzymatic method these kits use a chaotrope (sodium iodide) and a detergent
(Method A) (Method B) (sodium lauroylsarcosinate) to disrupt the association
between the residual host-cell DNA and the sample
Can be used to
assess DNA size Yes No components. The residual host-cell DNA in the sample is
distribution then recovered by co-precipitation with a carrier molecule
such as glycogen in the presence of ethanol or 2-propanol.
Limit of
Several independent extraction procedures may be required,
quantification
(may vary depending on the reproducibility of the spike recovery.
depending on the 0.01-10 pg/mL 2-10 pg/mL Negative controls must be included in each extraction
matrix, method and procedure. In some cases, dilution of the samples may be
interfering
recommended to reduce the matrix effect. A correction factor
substances)
may also be applied to take into account the recovery rate of
the spike.
2023 Appendix XIV O V-A537

METHOD A-REAL-TIME QUANTITATIVE PCR The coefficient of variation for the different extracts or
(QPCR) replicates is not higher than a predefined criterion.
This method can be used to quantify a cellular DNA target Calculation
sequence from a variety of samples. For the quantification of If several extractions are performed, each extracted sample is
residual host-cell DNA, qPCR targeting either a stable analysed individually. The residual host-cell DNA content is
sequence within a highly conserved host-cell region or calculated from the genomic standard curve by averaging the
targeting repetitive elements to enhance the sensitivity of the values obtained for the different extractions or replicates.
test can be used. When repetitive elements are targeted, it A correction factor may also be applied to take into account
may be difficult to eliminate potential background noise due the recovery rate for total DNA quantification in the samples.
to environmental DNA (e.g. when using Alu human
For the characterisation of residual host-cell DNA size, the
sequences). The specificity of the qPCR method must be
distribution of the overlapping fragments of different sizes is
established during the validation studies by demonstrating
calculated as the ratio of the number of copies for each
the absence of cross-reactivity with unrelated sequences.
amplicon size to the number of copies of the smallest
Alternatively, digital PCR methods may be used. amplicon size.
qPCR amplification
METHOD B - IMMUNOENZYMATIC METHOD
The detection and quantification of residual host-cell DNA
The immunoenzymatic method is a non-specific technique
by qPCR may involve the use of either a non-specific
for the quantification of residual host-cell DNA (regardless of
fluorescent dye that intercalates with any double-stranded
its origin). It is therefore also a total DNA assay, and
DNA, or sequence-specific DNA probes. The qPCR
consequently, it is not only critical to avoid contamination
principle described in general chapter 2.6.21. Nucleic acid
through environmental DNA, but all materials and reagents
amplification techniques applies.
used must be DNA-free. The samples to be tested must be
The number of cycles required for the fluorescent free of microbial contamination and all samples, controls and
measurement to exceed a threshold value (Ct or Cp) standards must be processed under controlled conditions
correlates to the starting amount of residual host-cell DNA in until the denaturation step.
the sample.
By design, the method detects single-stranded DNA.
If several extractions are performed, the resulting samples
must be analysed at an appropriate dilution. Principle
This total DNA assay consists of 4 steps:
PCR negative controls are used. - denaturation and formation of complexes, where the DNA is
A standard curve is plotted using serial dilutions of host-cell denatured into single-stranded DNA by heating the
genomic DNA in order to allow residual host-cell DNA sample. The denatured DNA is mixed with a single
levels in biological products to be determined based on their reagent that contains a single-stranded DNA-binding
Ct or Cp values. The use of carefully characterised protein conjugated to streptavidin and a monoclonal anti-
representative genomic DNA extracted from the cells used DNA antibody conjugated to urease. The DNA-binding
for the production of the biological product is recommended protein and the monoclonal antibody are specific for
for the preparation of the standard. single-stranded DNA but are not sequence-specific.
The same methodology is applied for residual host-cell DNA The liquid phase, in the presence of streptavidin,
size evaluation. At least 2 sets of primers can be designed to facilitates the formation of a complex with the single-
amplify overlapping fragments of different sizes in the target stranded DNA from the sample.
sequence. - filtration, where the complex is filtered through a
Suitability criteria biotinylated nitrocellulose membrane. The biotin in the
Control samples In order to control the risk of membrane captures the complexes by binding to
contamination and to ensure adequate sensitivity, each PCR streptavidin. The membrane is washed to remove any
assay includes the following controls: unbound reagents. Non-specific binding is avoided by the
- a negative control for qPCR and a negative control for use of an albumin-coated nitrocellulose membrane.
extraction, composed of a sample of a suitable matrix - detection, where the membrane is placed in a reader,
already proven to be free of the target sequence(s); which contains a urea solution that reacts with the urease
- a positive control for qPCR, which contains a defined in the DNA complex and produces ammonia.
number of target sequence copies or a defined DNA The associated change in pH is measured by a
concentration which is determined individually for each potentiometric sensor in µV/s and is directly proportional
assay system; to the amount of DNA in the sample.
- a control for extraction, typically an internal control added - analysis, where the raw data from the sample and from
to the test material as a defined concentration or number the standard curve are analysed using appropriate
of target-sequence copies. In this case, the amplicons software to determine the residual host-cell DNA content
must be clearly discernible and may be detected in a in the sample.
separate qPCR. Alternatively, an external control All samples and negative controls are tested spiked and
consisting of test sample spiked with a well-characterised unspiked. The spike solution (1000 pg/mL) is prepared by
level of genomic DNA may be used. dilution of a concentrated standard (calf thymus DNA) at
Extraction recovery must fall within predetermined values 5000 pg/mL.
based on the performance of the assay as demonstrated Suitability criteria
during assay validation. Control samples
Genomic DNA standard curve The standard curve is - the amount of DNA in the positive control falls within
linear over the chosen range. the range indicated on the batch certificate provided by
The coefficient of determination R 2 associated with the the supplier;
standard curve must be greater or equal to 0.98. The PCR
efficiency falls within pre-established limits.
V-A538 Appendix XIV P 2023

- spike recovery in the negative control is between each of the microbial test strains separately as described in
80 per cent and 120 per cent. Table 5.1.11.-1.
Samples
Table 5.1.11.-1. - Test micro-organisms and growth conditions
- when several replicates are analysed, the coefficient of
variation for the different replicates is not higher than a Strains for bactericidal activity testing
predefined criterion;
Staphylococcus aureus such as ATCC 6538, NCIMB
- spike recovery is between 80 per cent and 120 per cent. 9518, CIP 4.83, NBRC 13276
Calculation
Enterococcus h£rae such as ATCC 10541, NCIMB
The content of residual host-cell DNA is calculated in 8192, CIP 58.55, DSM 3320
picograms per millilitre using the following expression:
Escherichia coli such as NCIMB 10083, CIP 54.117,
IDx (C-A) NCTC 10538, DSM 11250

V Pseudomonas aeruginosa such as ATCC 15442, NCIMB

8626, CIP 103467, NBRC 13275
ID ratio for dilution and sampling;
C raw mean value (in picograms per tube) for the test tubes Bacterial growth conditions
containing the diluted sample;
A raw mean value (in picograms per tube) for the test tubes Casein soya bean digest agar or number of CFU in test suspension:
containing the negative control; casein soya bean digest broth 1-5 x 108 CFU/mL
V volume in the test tube, in millilitres (usually 0.5 mL per tube). - for preparation of test strains:
30-35 "C, 18-24 hand subculture at
Where necessary, this result may be corrected by the least twice
- for testing of the product and
extraction recovery (e.g. mean recovery for a given product). validation of the test:
30-35 C, S 3 days

Strain for yeasticidal activity testing

P. Determination of Bactericidal, Candida albicans such as ATCC 10231, NCPF 3179,

CIP 48.72, NBRC 1594
Fungicidal or Yeasticidal Activity of
Yeast growth conditions
Antiseptic Medicinal Products
Sabouraud-dextrose agar or number of CFU in test suspension:
(Ph. Bur. method 5.1.11) Sabouraud-dextrose broth 1-5 x 107 CFU/mL
- for preparation of test strains:
This general chapter describes a test that can be used for the 20-25 "C, 2-3 days and subculture
detennination of antimicrobial activity in antiseptic medicinal at least twice
products that are miscible with water and intended for - for testing of the product and
administration by direct contact with the skin or mucous validation of the test:
20-25 'C, S 5 days
membranes. The extent of testing is dependent on the declared
antimicrobial activity of the product. Strains for fungicidal activity testing
The test detennines whether a product exhibits bactericidal, Candida albicans such as ATCC 10231, NCPF 3179,
fungicidal or yeasticidal activity and compli.es with an established CIP 48.72, NBRC 1594
specificatwn for such activity.
Aspergi.1/us brasiliensis such as ATCC 16404, IM! 149007,
This test cannot replace or confirm the assessment of the clinical CIP 1431.83, NBRC 9455
efficacy of such preparations.
Fungal growth conditions
1 PRINCIPLE
Antimicrobial activity is determined by adding test Sabouraud-dextrose agar or number of CFU in test suspension:
suspensions of micro-organisms (bacteria, fungi or yeasts, Sabouraud-dextrose broth 1-5 x 107 CFU/mL
- for preparation of test suspension
separately) to the sample antiseptic product. The mixture is of C. albicans: 20-25 'C, 2-3 days
maintained at 33 ± 1 °C for contact times of 5 min for - for preparation of test suspension
bactericidal activity and 15 min for fungicidal or yeasticidal of A. brasiliensis spores: 20-25 'C, at
activity. Additional contact times may be chosen, according least 5 days until good sporulation
- for testing of the product and
to the stated use of the antiseptic medicinal product. At the validation of the test with C. albicans
end of the contact time, an aliquot is taken and the and A. brasiliensis:
antimicrobial activity in this aliquot is immediately stopped 20-25 'C, S 5 days
by a validated method. 2 methods are available: dilution-
neutralisation and membrane filtration.
The recommended solutions and media are described in
The procedure is validated to verify its ability to demonstrate general chapter 2.6.13. Purified water is used. All reagents
the required reduction in the count of viable micro-organisms are sterile prior to use.
by the use of appropriate controls.
The test for bactericidal, fungicidal or yeasticidal activity is
2 TEST MICRO-ORGANISMS AND GROWTH performed with the designated strains as described in
CONDITIONS Table 5.1.11.-1. In addition to these micro-organisms, it may
Prepare standardised stable suspensions of test strains as be necessary to add other bacterial or fungal strains that
stated in section 2-1. Seed-lot culture maintenance represent the indications of the antiseptic medicinal product
techniques (seed-lot systems) are used so that the viable tested.
micro-organisms used for inoculation are not more than 5
passages removed from the original master seed-lot. Grow
2023 Appendix XIV P V-A539

Single-strain challenges are used. The counts are performed incubation is not less than 0.5 x (number of CFU in the
in duplicate and the arithmetic mean of the results is validation suspension)/! 0.
calculated and expressed in CFU/mL. 3-1-1-3 Dilution-neutralisation method control
2-1 PREPARATION OF TEST SUSPENSION Transfer 1.0 mL of a 3 g/L solution of bovine albumin R into
For harvesting the micro-organisms use a sufficient volume of a tube, add 1.0 mL of a 9 g/L solution of sodium chloride R
a 9 g/L solution of sodium chloride R (for bacteria and and 8.0 mL of the product test solution and maintain at
C. albicans) or a solution containing 9 g/L of sodium 33 ± 1 °C for the chosen contact time. Transfer 1.0 mL of
chloride Rand 0.5 g/L of polysorbate 80 R (for A. brasiliensis), this mixture into a tube containing 8.0 mL of the neutralising
to obtain a test suspension with the number of CFU agent and maintain at 33 ± 1 "C for the appropriate
described in Table 5.1.11.-1. Use the suspension within 2 h neutralisation time. Then add 1.0 mL of t.l-1e validation
or within 24 h if stored at 2-8 °C. suspension and mix. After 30 min, take a sample of 1.0 mL
2-2 PREPARATION OF ANTISEPTIC PRODUCT of the mixture, in duplicate, and inoculate using the pour-
TEST SOLUTION plate or surface-spread method. For incubation conditions,
The concentration of the antiseptic product test solution shall see Table 5.1.11.-1. After incubation, perform the count.
be, if possible, 1.25 times the in-use test concentration The number of CFU recovered following incubation is not
because it is diluted to 80 per cent during the test and the less than 0.5 x (number of CFU in the validation
method validation. suspension)/! 0.
2-3 NEUTRALISING AGENTS 3-2 MEMBRANE FILTRATION METHOD
Neutralising agents are used to neutralise the antimicrobial Proceed as described in section 3-1, carrying out immediately
activity of the antiseptic product. The common neutralising the filtration step in place of the neutralisation step.
agents are listed in Table 2.6.12.-2 of general chapter 2.6.12. Use membrane filters having a nominal pore size not greater
Microbiological examination of non-sterile products: microbial than 0.45 µm. The type of filter material is chosen such that
enumeration tests. The neutralisation time is not less than 10 s the microbe-retaining efficiency is not affected by the
and not more than 60 s. components of the sample to be investigated. For each of the
3METHODS micro-organisms listed, a single membrane filter is used.
Appropriately dilute 0.1 mL of the test solution and
Prior to testing, equilibrate the temperature of all reagents to
immediately filter the total volume, then rinse the membrane
33 ± 1 °C.
filter with an appropriate volume of the diluent. Perform the
3-1 DILUTION-NEUTRALISATION METHOD test in duplicate. For incubation conditions, see
Transfer 1.0 mL of a 3 g/L solution of bovine albumin R into Table 5.1.11.-1. After incubation, perform the count.
a tube, add 1.0 mL of the test suspension and maintain at
3-2-1 Verification of the selected experimental
33 ± 1 °C for 2 min. Add 8.0 mL of the antiseptic product
conditions and of the membrane filtration method
test solution and maintain at 33 ± 1 °C for the chosen
3-2-1-1 Experimental conditions control
contact time. Then, take a 1.0 mL sample of the test mixture
and transfer into a tube containing 1.0 mL of water R and Proceed as described in section 3-1-1-1, except at the end of
8.0 mL of the neutralising agent and maintain at 33 ± 1 °C the contact time, take the sample in duplicate, and transfer
for the appropriate neutralisation time. Take 1.0 mL of the into a separate membrane filtration apparatus. Filter
neutralised test mixture, in duplicate, and inoculate using the immediately and then transfer each of the membrane filters
pour-plate or surface-spread method. For incubation to the surface of separate plates. For incubation conditions,
conditions, see Table 5.1.11.-1. After incubation, perform see Table 5.1.11.-1. After incubation, perform the count.
the count. The number of CFU recovered following incubation is not
less than 0.5 x (number of CFU in the validation
3-1-1 Suitability of the test/controls
suspension)/! 0.
For all methods, prepare a validation suspension containing
100-1000 CFU of the test micro-organisms per millilitre. 3-2-1-2 Membrane filtration method control
3-1-1-1 Experimental conditions control Proceed as described in section 3-1-1-3, except at the end of
the chosen contact time, take that sample in duplicate, and
Transfer 1.0 mL of a 3 g/L solution of bovine albumin R into
transfer into a separate membrane filtration apparatus. Filter
a tube, add 1.0 mL of the validation suspension and
and rinse as described in section 3-2, then cover the
maintain at 33 ± 1 °C for 2 min. Add 8.0 mL of water R
membranes with rinsing liquid and add a sample of the
and maintain at 33 ± 1 °C for the chosen contact time.
validation suspension. Filter again and transfer each of the
Take 1.0 mL of this mixture, in duplicate, and inoculate
membrane filters to the surface of separate plates.
using the pour-plate or surface-spread method.
For incubation conditions, see Table 5 .1.11.-1. After
For incubation conditions, see Table 5.1.11.-1. After
incubation, perform the count. The number of CFU
incubation, perform the count. The number of CFU
recovered following incubation is not less than
recovered following incubation is not less than
0.5 x (number of CFU in the validation suspension)/10.
0.5 x (number of CFU in the validation suspension)/10.
3-1-1-2 Neutralising agent control 4 ACCEPTANCE CRITERIA
Unless otherwise justified and authorised, the preparation
Transfer 1.0 mL of a 3 g/L solution of bovine albumin R into
has a:
a tube, add 1.0 mL of the validation suspension and 8.0 mL
- bactericidal activity if the defined number of CFU is
of the neutralising agent used in the test and maintain at
reduced by at least 5 log 10;
33 ± 1 °C for the appropriate neutralisation time. Take
- fungicidal activity if the defined number of CFU is
1.0 mL of this mixture, in duplicate, and inoculate using the
reduced by at least 4 log 10;
pour-plate or surface-spread method. For incubation
- yeasticidal activity if the defined number of CFU is
conditions, see Table 5.1.11.-1. After incubation, perform
reduced by at least 4 log 10 .
the count. The number of CFU recovered following
V-A540 Appendix XV 2023

indefinitely in culture and may be obtained from healthy or

Appendix XV tumoral tissue. Some continuous cell lines have oncogenic
potential under certain conditions.
Production and Testing of Vaccines Production cell culture
A culture of cells intended for use in production; it may be
derived from one or more containers of the working cell bank
(working cell seed) or it may be a primary cell culture.
A. Terminology used in Monographs on Control cells
A quantity of cells set aside, at the time of virus inoculation,
Biological Products as uninfected cell cultures. The uninfected cells are incubated
(Ph. Bur. general texts 5. 2.1) under similar conditions to those used for the production cell
Tenninology: Vaccines cultures.
Single harvest
For some items, alternative terms commonly used in
Material derived on one or more occasions from a single
connection with veterinary vaccines are shown in parenthesis.
production cell culture inoculated with the same working
Seed-lot system seed lot or a suspension derived from the working seed lot,
A seed-lot system is a system according to which successive incubated, and harvested in a single production run.
batches of a product are derived from the same master seed
Monovalent pooled harvest
lot. For routine production, a working seed lot may be
Pooled material containing a single strain or type of micro-
prepared from the master seed lot. The origin and the
organism or antigen and derived from a number of eggs, cell
passage history of the master seed lot and the working seed
culture containers etc. that are processed at the same time.
lot are recorded.
Final bulk vaccine
Master seed lot Material that has undergone all the steps of production
A culture of a micro-organism distributed from a single bulk
except for the final filling. It consists of one or more
into containers and processed together in a single operation
monovalent pooled harvests, from cultures of one or more
in such a manner as to ensure uniformity and stability and to
species or types of micro-organism, after clarification, dilution
prevent contamination. A master seed lot in liquid form is
or addition of any adjuvant or other auxiliary substance. It is
usually stored at or below -70 °C. A freeze-dried master seed
treated to ensure its homogeneity and is used for filling the
lot is stored at a temperature known to ensure stability.
containers of one or more final lots (batches).
Working seed lot
Final lot (Batch)
A culture of a micro-organism derived from the master seed
A collection of closed, final containers or other final
lot and intended for use in production. Working seed lots are
dosage units that are expected to be homogeneous and
distributed into containers and stored as described above for
equivalent with respect to risk of contamination during filling
master seed lots.
or preparation of the final product. The dosage units are
Cell-bank system (Cell-seed system) filled, or otherwise prepared, from the same final bulk
A system whereby successive final lots (batches) of a product vaccine, freeze-dried together (if applicable) and closed in
are manufactured by culture in cells derived from the same one continuous working session. They bear a distinctive
master cell bank (master cell seed). A number of containers number or code identifying the final lot (batch). Where a
from the master cell bank (master cell seed) are used to final bulk vaccine is filled and/or freeze-dried on several
prepare a working cell bank (working cell seed). The cell- separate sessions, there results a related set of final lots
bank system (cell-seed system) is validated for the highest (batches) that are usually identified by the use of a common
passage level achieved during routine production. part in the distinctive number or code; these related final lots
Master cell bank (Master cell seed) (batches) are sometimes referred to as sub-batches, sub-lots
A culture of cells distributed into containers in a single or filling lots.
operation, processed together and stored in such a manner as Combined vaccine
to ensure uniformity and stability and to prevent A multicomponent preparation formulated so that different
contamination. A master cell bank (master cell seed) is antigens are administered simultaneously. The different
usually stored at -70 °C or lower. antigenic components are intended to protect against
Working cell bank (Working cell seed) different strains or types of the same organism and/or
A culture of cells derived from the master cell bank (master different organisms. A combined vaccine may be supplied by
cell seed) and intended for use in the preparation of the manufacturer either as a single liquid or freeze-dried
production cell cultures. The working cell bank (working cell preparation or as several constituents with directions for
seed) is distributed into containers, processed and stored as admixture before use.
described for the master cell bank (master cell seed).
Primary cell cultures
Cultures of cells obtained by trypsination of a suitable tissue
or organ. The cells are essentially identical to those of the B. Aluminium in Adsorbed Vaccines
tissue of origin and are no more than 5 in vitro passages from (Ph. Bur. method 2.5.13)
the initial preparation from the animal tissue.
Homogenise the preparation to be examined and transfer a
Cell lines suitable quantity, presumed to contain 5 mg to 6 mg of
Cultures of cells that have a high capacity for multiplication aluminium, to a 50 mL combustion flask. Add 1 mL of
in vitro. In diploid cell lines, the cells have essentially the suljuric acid R, 0.1 mL of nitric acid Rand some glass beads.
same characteristics as those of the tissue of origin. Heat the solution until thick, white fumes are evolved.
In continuous cell lines, the cells are able to multiply
2023 Appendix XV F V-A541

If there is charring at this stage add a few more drops of hydrochloride R. Close the tubes, shake and allow to stand for
nitric acid R and continue boiling until the colour disappears. 60 min. Add 1 mL of ferric chloride-sulfamic acid reagent R
Allow to cool for a few minutes, carefully add 10 mL of and allow to stand for 15 min. Measure the absorbance
water R and boil until a clear solution is obtained. Allow to (2.2.25) of the solutions at 628 nm. Calculate the content of
cool, add 0.05 mL of methyl orange solution Rand neutralise formaldehyde in the vaccine to be examined from the
with strong sodium hydroxide solution R (6.5 mL to 7 mL). If a calibration curve established using the reference solutions.
precipitate forms dissolve it by adding, dropwise, sufficient The test is invalid if the correlation coefficient (r) of the
dilute sulfuric acid R. Transfer the solution to a 250 mL calibration curve is less than 0.97.
conical flask, rinsing the combustion flask with 25 mL of Emulsions If the vaccine to be examined is an emulsion,
water R. Add 25.0 mL of 0.02 M sodium edetate, 10 mL of the aqueous phase is separated using a suitable procedure
acetate buffer solution pH 4. 4 R and a few glass beads and boil and used for preparation of the test solution. The following
gently for 3 min. Add 0.1 mL of pyridylazonaphthol solution R procedures have been found suitable.
and titrate the hot solution with 0. 02 M copper sulfate until (a) Add 1.0 mL of the vaccine to be examined to 1.0 mL of
the colour changes to purplish-brown. Carry out a blank isopropyl myristate R and mix. Add 1.3 mL of 1 M hydrochloric
titration omitting the vaccine. acid, 2.0 mL of chloroform Rand 2.7 mL of a 9 g/L solution
1 mL of 0.02 M sodium edetate is equivalent to 0.5396 mg of of sodium chloride R. Mix thoroughly. Centrifuge at 15 000 g
Al. for 60 min. Transfer the aqueous phase to a 10 mL
volumetric flask and dilute to volume with water R. If this
procedure fails to separate the aqueous phase, add 100 g/L of
polysorbate 20 R to the sodium chloride solution and repeat
C. Calcium in Adsorbed Vaccines the procedure but centrifuge at 22 500 g.
(Ph. Eur. method 2. 5.14) (b) Add 1.0 mL of the vaccine to be examined to 1.0 mL of
a 100 g/L solution of sodium chloride R and mix. Centrifuge
All solutions used for this test must be prepared using water R. at 1000 g for 15 min. Transfer the aqueous phase to a
Determine the calcium by atomic emission spectrometry 10 mL volumetric flask and dilute to volume with water R.
(2.2.22, Method I). Homogenise the preparation to be (c) Add 1.0 mL of the vaccine to be examined to 2.0 mL of
examined. To 1.0 mL add 0.2 mL of dilute hydrochloric a 100 g/L solution of sodium chloride Rand 3.0 mL of
acid Rand dilute to 3.0 mL with water R. Measure the chloroform R and mix. Centrifuge at 1000 g for 5 min.
absorbance at 620 nm. Transfer the aqueous phase to a 10 mL volumetric flask and
dilute to volume with water R.

D. Free Formaldehyde
(Ph. Eur. method 2.4.18) E. Phenol in lmmunosera (Antisera) and
Use method A, unless otherwise prescribed. Method B is Vaccines
suitable for vaccines where sodium metabisulfite has been (Ph. Eur. method 2.5.15)
used to neutralise excess formaldehyde.
Homogenise the preparation to be examined. Dilute an
METHOD A appropriate volume with water R so as to obtain a solution
For vaccines for human use, prepare a 1 in 10 dilution of the presumed to contain 15 µg of phenol per millilitre. Prepare a
vaccine to be examined. For bacterial toxoids for veterinary series of reference solutions with phenol R containing 5 µg,
use, prepare a 1 in 25 dilution of the vaccine to be examined. 10 µg, 15 µg, 20 µg and 30 µg of phenol per millilitre
To 1 mL of the dilution, add 4 mL of water Rand 5 mL of respectively. To 5 mL of the solution to be examined and to
acetylacetone reagent Rl. Place the tube in a water-bath at 5 mL of each of the reference solutions respectively, add
40 cc for 40 min. Examine the tubes down their vertical 5 mL of buffer solution pH 9. 0 R, 5 mL of aminopyrazolone
axes. The solution is not more intensely coloured than a solution R and 5 mL of potassium ferricyanide solution R. Allow
standard, prepared at the same time and in the same to stand for 10 min and measure the intensity of colour at
manner, using 1 mL of a dilution of formaldehyde solution R 546 nm.
containing 20 µg of formaldehyde (CH 2 0) per millilitre, Plot the calibration curve and calculate the phenol content of
instead of the dilution of the vaccine to be examined. the preparation to be examined.
METHODB
Test solution Prepare a 1 in 200 dilution of the vaccine to
be examined with water R. If the vaccine is an emulsion,
prepare an equivalent dilution using the aqueous phase F. Neurovirulence
separated by a suitable procedure (see below). If one of the
methods described below is used for separation of the Test for Neurovirulence of Live Virus Vaccines
aqueous phase, a 1 in 20 dilution of the latter is used. (Ph. Eur. method 2. 6.18)
Reference solutions Prepare solutions containing For each test, use not fewer than ten monkeys that are
0.25 g/L, 0.50 g/L, 1.00 g/L and 2.00 g/L of CH 2 0 by seronegative for the virus to be tested. For each monkey,
dilution of formaldehyde solution R with water R. Prepare a 1 inject not more than 0.5 mL of the material to be examined
in 200 dilution of each solution with water R. into the thalamic region of each hemisphere, unless otherwise
To 0.5 mL of the test solution and of each of the reference prescribed. The total amount of virus inoculated in each
solutions in test-tubes, add 5.0 mL of a freshly prepared monkey must be not less than the amount contained in the
0.5 g/L solution of methylbenzothiazolone hydrazone recommended single human dose of the vaccine. As a check
against the introduction of wild neurovirulent virus, keep a
V-A542 Appendix XV G 2023

group of not fewer than four control monkeys as cage-mates per millilitre of dry polysaccharide. Transfer the contents of a
or in the immediate vicinity of the inoculated monkeys. container quantitatively to the flask and dilute to volume with
Observe the inoculated monkeys for 17 to 21 days for water R.
symptoms of paralysis and other evidence of neurological Dilute the test solution if necessary to obtain an absorbance
involvement; observe the control monkeys for the same value suitable for the instrument used. Measure the
period plus 10 days. Animals that die within 48 h of injection absorbance (2.2.25) at 260 nm using water Ras the
are considered to have died from non-specific causes and compensation liquid.
may be replaced. The test is not valid if: more than The absorbance of a 1 g/L solution of nucleic acid at 260 nm
20 per cent of the inoculated monkeys die from nonspecific is 20.
causes; serum samples taken from the control monkeys at the
time of inoculation of the test animals and 10 days after the Phosphorus
latter are euthanised show evidence of infection by wild virus (Ph. Bur. method 2.5.18)
of the type to be tested or by measles virus. At the end of the Test solution Use a volumetric flask with a suitable
observation period, carry out autopsy and histopathological volume for preparation of a solution containing about 5 mg
examinations of appropriate areas of the brain for evidence of per millilitre of dry polysaccharide. Transfer the contents of a
central nervous system involvement. The material complies container quantitatively to the flask and dilute to volume with
with the test if there is no unexpected clinical or water R. Dilute the solution so that the volume used in the
histopathological evidence of involvement of the central test (1 mL) contains about 6 µg of phosphorus. Transfer
nervous system attributable to the inoculated virus. 1.0 mL of the solution to a 10 mL ignition tube.
Reference solutions Dissolve 0.2194 g of potassium
dihydrogen phosphate R in 500 mL of water R to give a
solution containing the equivalent of 0.1 mg of phosphorus
G. Composition of Polysaccharide per millilitre. Dilute 5.0 mL of the solution to 100.0 mL with
Vaccines water R. Introduce 0.5 mL, 1.0 mL and 2.0 mL of the dilute
solution into 3 ignition tubes.
Protein Prepare a blank solution using 2.0 mL of water R in an
(Ph. Bur. method 2.5.16) ignition tube.
Test solution Use a volumetric flask with a suitable To all the tubes add 0.2 mL of sulfuric acid R and heat in an
volume for preparation of a solution containing about 5 mg oil bath at 120 °C for 1 hand then at 160 °C until white
per millilitre of dry polysaccharide. Transfer the contents of a fumes appear (about 1 h). Add 0.1 mL of perchloric acid R
container quantitatively to the flask and dilute to volume with and heat at 160 °C until the solution is decolorised (about
water R. Place 1 mL of the solution in a glass tube and add 90 min). Cool and add to each tube 4 mL of water Rand
0.15 mL of a 400 g/L solution of trichloroacetic acid R. Shake, 4 mL of ammonium molybdate reagent R. Heat in a water-bath
allow to stand for 15 min, centrifuge for 10 min at at 37 °C for 90 min and cool. Adjust the volume to 10.0 mL
5000 r/min and discard the supernatant. Add 0.4 mL of with water R. The blue colour is stable for several hours.
0.1 M sodium hydroxide to the centrifugation residue. Measure the absorbance (2.2.25) of each solution at 820 nm
Reference solutions Dissolve 0.100 g of bovine albumin R using the blank solution as the compensation liquid. Draw a
in 100 mL of 0.1 M sodium hydroxide (stock solution calibration curve with the absorbances of the 3 reference
containing 1 g of protein per litre). Dilute 1 mL of the stock solutions as a function of the quantity of phosphorus in the
solution to 20 mL with 0.1 M sodium hydroxide (working solutions and read from the curve the quantity of phosphorus
dilution 1: 50 mg of protein per litre). Dilute 1 mL of the in the test solution.
stock solution to 4 mL with 0.1 M sodium hydroxide (working 0-Acetyl Groups
dilution 2: 250 mg of protein per litre). Place in 6 glass tubes
(Ph. Bur. method 2.5.19)
0.10 mL, 0.20 mL and 0.40 mL of working dilution 1 and
0.15 mL, 0.20 mL and 0.25 mL of working dilution 2. Make Test solution Use a volumetric flask with a suitable
up the volume in each tube to 0.40 mL using 0.1 M sodium volume for preparation of a solution containing about 5 mg
hydroxide.
per millilitre of dry polysaccharide. Transfer the contents of a
container quantitatively to the flask and dilute to volume with
Prepare a blank using 0.40 mL of 0.1 M sodium hydroxide.
water R. Dilute the solution so that the volumes used in the
Add 2 mL of cupri-tartaric solution R3 to each tube, shake test contain 30 µg to 600 µg of acetylcholine chloride (0-
and allow to stand for 10 min. Add to each tube 0.2 mL of a acetyl). Introduce 0.3 mL, 0.5 mL and 1.0 mL in duplicate
mixture of equal volumes of phosphomolybdotungstic reagent R into 6 tubes (3 reaction solutions and 3 correction solutions).
and water R, prepared immediately before use. Stopper the
Reference solutions Dissolve O.150 g of acetylcholine
tubes, mix by inverting and allow to stand in the dark for chloride R in 10 mL of water R (stock solution containing
30 min. The blue colour is stable for 60 min. If necessary,
15 g of acetylcholine chloride per litre). Immediately before
centrifuge to obtain clear solutions.
use, dilute 1 mL of the stock solution to 50 mL with water R
Measure the absorbance (2.2.25) of each solution at 760 nm (working dilution 1: 300 µg of acetylcholine chloride per
using the blank as the compensation liquid. Draw a millilitre). Immediately before use, dilute 1 mL of the stock
calibration curve from the absorbances of the 6 reference solution to 25 mL with water R (working dilution 2: 600 µg
solutions and the corresponding protein contents and read of acetylcholine chloride per millilitre). Introduce O.1 mL and
from the curve the content of protein in the test solution. 0.4 mL of working dilution 1 in duplicate (reaction and
Nucleic Acids correction solutions) into 4 tubes and 0.6 mL and 1.0 mL of
working dilution 2 in duplicate (reaction and correction
(Ph. Bur. method 2.5.17)
solutions) into another 4 tubes.
Test solution Use a volumetric flask with a suitable
Prepare a blank using 1 mL of water R.
volume for preparation of a solution containing about 5 mg
2023 Appendix XV G V-A543

Make up the volume in each tube to 1 mL with water R. hexosamine and read from the curve the quantity of
Add 1.0 mL of a 4 M solution of hydrochloric acid prepared hexosamine in the test solution.
from hydrochloric acid R to each of the correction tubes and
to the blank. Add 2.0 mL of alkaline hydroxylamine solution R Methylpentoses
to each tube. Allow the reaction to proceed for exactly 2 min (Ph. Bur. method 2.5.21)
and add 1.0 mL of the 4 M solution of hydrochloric acid to Test solution Use a volumetric flask with a suitable
each of the reaction tubes. To each tube, add 1.0 mL of a volume for preparation of a solution containing about 5 mg
100 g/L solution of ferric chloride Rina 0.1 M solution of per millilitre of dry polysaccharide. Transfer the contents of a
hydrochloric acid prepared from hydrochloric acid R, stopper container quantitatively to the flask and dilute to volume with
the tubes and shake vigorously to remove bubbles. water R. Dilute the solution so that the volumes used in the
Measure the absorbance (2.2.25) of each solution at 540 nm test contain 2 µg to 20 µg of rhamnose (methylpentoses).
using the blank as the compensation liquid. For each reaction Introduce 0.25 mL, 0.50 mL and 1.0 mL of the diluted
solution, subtract the absorbance of the corresponding solution into 3 tubes.
correction solution. Draw a calibration curve from the Reference solutions Dissolve 0.100 g of rhamnose R in
corrected absorbances for the 4 reference solutions and the 100 mL of water R (stock solution containing 1 g of
corresponding content of acetylcholine chloride and read methylpentose per litre). Immediately before use, dilute 1 mL
from the curve the content of acetylcholine chloride in the of the stock solution to 50 mL with water R (working
test solution for each volume tested. Calculate the mean of dilution: 20 mg of methylpentose per litre). Introduce
the 3 values. 0.10 mL, 0.25 mL, 0.50 mL, 0.75 mL and 1.0 mL of the
1 mole of acetylcholine chloride (181. 7 g) is equivalent to working dilution into 5 tubes.
1 mole of O-acetyl (43.05 g). Prepare a blank using 1 mL of water R.
Make up the volume in each tube to 1 mL with water R.
Hexosainines
Place the tubes in iced water and add dropwise and with
(Ph. Bur. method 2.5.20)
continuous stirring to each tube 4.5 mL of a cooled mixture
Test solution Use a volumetric flask with a suitable of 1 volume of water R and 6 volumes of sulfuric acid R.
volume for preparation of a solution containing about 5 mg Warm the tubes to room temperature and place in a water-
per millilitre of dry polysaccharide. Transfer the contents of a bath for a few minutes. Cool to room temperature. Add to
container quantitatively to the flask and dilute to volume with each tube 0.10 mL of a 30 g/L solution of cysteine
water R. Dilute the solution so that the volumes used in the hydrochloride R, prepared immediately before use. Shake and
test contain 125 µg to 500 µg of glucosamine (hexosamine). allow to stand for 2 h.
Introduce 1.0 mL of the diluted solution into a graduated
Measure the absorbance (2.2.25) of each solution at 396 nm
tube. and at 430 nm using the blank as compensation liquid.
Reference solutions Dissolve 60 mg of glucosamine For each solution, calculate the difference between the
hydrochloride R in 100 mL of water R (stock solution absorbance measured at 396 nm and that measured at
containing 0.500 g of glucosamine per litre). Introduce 430 nm. Draw a calibration curve from the absorbance
0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working differences for the 5 reference solutions and the
dilution into 4 graduated tubes. corresponding content of methylpentose and read from the
Prepare a blank using 1 mL of water R. curve the quantity of methylpentose in the test solution for
Make up the volume in each tube to 1 mL with water R. each volume tested. Calculate the mean of the 3 values.
Add 1 mL of a solution of hydrochloric acid R (292 g/L) to
Uronic Acids
each tube. Stopper the tubes and place in a water-bath for
(Ph. Bur. method 2.5.22)
1 h. Cool to room temperature. Add to each tube 0.05 mL
of a 5 g/L solution of thymolphthalein R in alcohol R; add a Test solution Use a volumetric flask with a suitable
solution of sodium hydroxide R (200 g/L) until a blue colour is volume for preparation of a solution containing about 5 mg
obtained and then 1 M hydrochloric acid until the solution is per millilitre of dry polysaccharide. Transfer the contents of a
colourless. Dilute the volume in each tube to 10 mL with container quantitatively to the flask and dilute to volume with
water R (neutralised hydrolysates). water R. Dilute the solution so that the volumes used in the
test contain 4 µg to 40 µg of glucuronic acid (uronic acids).
In a second series of 10 mL graduated tubes, place 1 mL of
Introduce 0.25 mL, 0.50 mL and 1.0 mL of the diluted
each neutralised hydrolysate. Add 1 mL of acetylacetone
solution into 3 tubes.
reagent (a mixture, prepared immediately before use, of
1 volume of acetylacetone R and 50 volumes of a 53 g/L Reference solutions Dissolve 50 mg of sodium
solution of anhydrous sodium carbonate R) to each tube. glucuronate R in 100 mL of water R (stock solution containing
Stopper the tubes and place in a water-bath at 90 °C for 0.4 g of glucuronic acid per litre). Immediately before use,
45 min. Cool to room temperature. Add to each tube dilute 5 mL of the stock solution to 50 mL with water R
2.5 mL of alcohol R and 1.0 mL of (working dilution: 40 mg of glucuronic acid per litre).
dimethylaminobenzaldehyde solution (immediately before use Introduce 0.10 mL, 0.25 mL, 0.50 mL, 0.75 mL, and
dissolve 0.8 g of dimethylaminobenzaldehyde R in 15 mL of 1.0 mL of the working dilution into 5 tubes.
alcohol R and add 15 mL of hydrochloric acid R) and dilute Prepare a blank using 1 mL of water R.
the volume in each tube to 10 mL with alcohol R. Stopper Make up the volume in each tube to 1 mL with water R.
the tubes, mix by inverting and allow to stand in the dark for Place the tubes in iced water and add dropwise and with
90 min. Measure the absorbance (2.2.25) of each solution at continuous stirring to each tube 5.0 mL of borate solution R.
530 nm using the blank as the compensation liquid. Stopper the tubes and place in a water-bath for 15 min. Cool
Draw a calibration curve from the absorbances for the 4 to room temperature. Add 0.20 mL of a 1.25 g/L solution of
reference solutions and the corresponding content of carbazole R in ethanol R to each tube. Stopper the tubes and
place in a water-bath for 15 min. Cool to room temperature.
V-A544 Appendix XV H 2023

Measure the absorbance (2.2.25) of each solution at 530 nm Reference solutions Dissolve 25 mg of ribose R in water R
using the blank as the compensation liquid. and dilute to 100.0 mL with the same solvent (stock solution
Draw a calibration curve from the absorbances for the 5 containing 0.25 g/L of ribose). Immediately before use, dilute
reference solutions and the corresponding content of 1 mL of the stock solution to 10.0 mL with water R (working
glucuronic acid and read from the curve the quantity of dilution: 25 mg/L ofribose). Introduce 0.10 mL, 0.20 mL,
glucuronic acid in the test solution for each volume tested. 0.40 mL, 0.60 mL, 0.80 mL and 1.0 mL of the working
Calculate the mean of the 3 values. dilution into 6 tubes.
Prepare a blank using 2 mL of water R.
Sialic Acid
(Ph. Bur. method 2.5.23) Make up the volume in each tube to 2 mL with water R.
Shake. Add 2 mL of a 0.5 g/L solution of ferric chloride R in
Test solution Transfer quantitatively the contents of one
hydrochloric acid R to each tube. Shake. Add 0.2 mL of a
or several containers to a volumetric flask of a suitable
100 g/L solution of orcinol R in ethanol R. Place the tubes in
volume that will give a solution with a known concentration
a water-bath for 20 min. Cool in iced water. Measure the
of about 250 µg per millilitre of polysaccharide and dilute to
absorbance (2.2.25) of each solution at 670 nm using the
volume with water R. Using a syringe, transfer 4.0 mL of this
blank as the compensation liquid. Draw a calibration curve
solution to a 10 mL ultrafiltration cell suitable for the
from the absorbance readings for the 6 reference solutions
passage of molecules of relative molecular mass less than
and the corresponding content of ribose and read from the
50 000. Rinse the syringe twice with water R and transfer the
curve the quantity of ribose in the test solution for each
rinsings to the ultrafiltration cell. Carry out the ultrafiltration,
volume tested. Calculate the mean of the 3 values.
with constant stirring, under nitrogen R at a pressure of about
150 kPa. Refill the cell with water R each time the volume of
liquid in it has decreased to 1 mL and continue until
200 mL has been filtered and the remaining volume in the
cell is about 2 mL. Using a syringe, transfer this residual H. Chicken Flocks Free from Specified
liquid to a 10 mL volumetric flask. Wash the cell with Pathogens for the Production and
3 quantities, each of 2 mL, of water R, transfer the washings
to the flask and dilute to 10.0 mL with water R (test Quality Control of Vaccines
solution). In each of 2 test-tubes place 2.0 mL of the test (Ph. Bur. method 5.2.2)
solution.
Where specified, chickens, embryos or cell cultures used for
Reference solutions Use the reference solutions
the production or quality control of vaccines are derived from
prescribed in the monograph.
eggs produced by chicken flocks free from specified
Prepare 2 series of 3 test-tubes, place in the tubes of each pathogens (SPF). The SPF status of a flock is ensured by
series 0.5 mL, 1.0 mL and 1.5 mL respectively, of the means of the system described below. The list of micro-
reference solution corresponding to the type of vaccine to be organisms given is based on current knowledge and will be
examined and adjust the volume in each tube to 2.0 mL with updated as necessary.
water R.
A flock is defined as a group of birds sharing a common
Prepare blank solutions using 2.0 mL of water R in each of environment and having their own caretakers who have no
2 test-tubes. contact with non-SPF flocks. Once a flock is defined, no
To all the tubes add 5.0 mL of resorcinol reagent R. Heat at non-SPF birds are added to it.
105 °C for 15 min, cool in cold water and transfer the tubes Each flock is housed so as to minimise the risk of
to a bath of iced water. To each tube add 5 mL of isoamyl contamination. The facility in which the flock is housed must
alcohol R and mix thoroughly. Place in the bath of iced water not be sited near to any non-SPF flocks of birds with the
for 15 min. Centrifuge the tubes and keep them in the bath exception of flocks that are in the process of being
of iced water until the examination by absorption established as SPF flocks and that are housed in facilities and
spectrophotometry. Measure the absorbance (2.2.25) of each conditions appropriate to SPF flocks. The SPF flock is
supernatant solution at 580 nm and 450 nm using isoamyl housed within an isolator or in a building with filtered air
alcohol R as the compensation liquid. For each wavelength, under positive pressure. Appropriate measures are taken to
calculate the absorbance as the mean of the values obtained prevent entry of rodents, wild birds, insects and unauthorised
with 2 identical solutions. Subtract the mean value for the personnel.
blank solution from the mean values obtained for the other
Personnel authorised to enter the facility must have no
solutions.
contact with other birds or with agents potentially capable of
Draw a graph showing the difference between the infecting the flock. It is advisable for personnel to shower and
absorbances at 580 nm and 450 nm of the reference change clothing or to wear protective clothing before entering
solutions as a function of the content of N-acetylneuraminic the controlled facility.
acid and read from the graph the quantity of
Wherever possible, items taken into the facility are sterilised.
N-acetylneuraminic acid (sialic acid) in the test solution.
In particular it is recommended that the feed is suitably
Ribose treated to avoid introduction of undesirable micro-organisms
(Ph. Bur. method 2.5.31) and that water is at least of potable quality, for example from
Test solution Use a volumetric flask with a suitable a chlorinated supply. No medication is administered to birds
volume for preparation of a solution containing about 5 mg within the flock that might interfere with detection of any
per millilitre of dry polysaccharide. Transfer the contents of a disease.
container quantitatively to the flask and dilute to volume with A permanent record is kept of the general health of the flock
water R. Dilute the solution so that the volumes used in the and any abnormality is investigated. Factors to be monitored
test contain 2.5 µg to 25 µg of ribose. Introduce 0.20 mL include morbidity, mortality, general physical condition, feed
and 0.40 mL of the diluted solution into tubes in triplicate. consumption, daily egg production and egg quality, fertility
2023 Appendix XV H V-A545

and hatchability. Records are maintained for a period of at are as described under Routine testing of designated SPF
least 5 years. Details of any deviation from normal in these flocks. Only when all tests have confirmed the absence of
performance parameters or detection of any infection are infection may the new generation be designated as SPF.
notified to the users of the eggs as soon as practicable. ROUTINE TESTING OF DESIGNATED SPF
The tests or combination of tests described below must have FLOCKS
suitable specificity and sensitivity with respect to relevant General examination and necropsy Clinical
serotypes of the viruses. Samples for testing are taken at examination is carried out at least once per week throughout
random. the life of the flock in order to verify that the birds are free
A positive result for chicken anaemia virus (CAV) does not from fowl-pox virus and signs of any other infection. In the
necessarily exclude use of material derived from the flock, event of mortality exceeding 0.2 per cent per week, necropsy
but live vaccines for use in birds less than 7 days old shall be is performed on all available carcasses to verify that there is
produced using material from CAV-negative flocks. no sign of infection. Where appropriate, histopathological
Inactivated vaccines for use in birds less than 7 days old may and/or microbiological/virological studies are performed to
be produced using material from flocks that have not been confirm diagnosis. Specific examination for tuberculosis
shown to be free from CAV, provided it has been lesions is carried out and histological samples from any
demonstrated that the inactivation process inactivates CAV. suspected lesions are specifically stained to verify freedom
ESTABLISHMENT OF AN SPF FLOCK from Mycobacterium avium. Caecal contents of all available
A designated SPF flock is derived from chickens shown to be carcasses are examined microbiologically for the presence of
Salmonella spp. using the techniques described below. Where
free from vertically-transmissible agents listed in
Table 5.2.2-1. This is achieved by testing of 2 generations appropriate, caecal samples from up to 5 birds may be
prior to the designated SPF flock. A general scheme for the pooled.
procedure to be followed in establishing and maintaining an Cultural testing for Salmonella spp Cultural testing for
SPF flock is shown diagrammatically in Table 5.2.2.-2. Salmonella spp. is performed either by testing samples of
In order to establish a new SPF flock, a series of tests must droppings or cloaca! swabs or by testing of drag swabs.
be conducted on 3 generations of birds. All birds in the 1st Where droppings or cloaca! swabs are tested, a total of
generation must be tested at least once before the age of 60 samples within each 4-week period is tested throughout
20 weeks for freedom from avian leucosis group-antigen and the entire life of the flock. Tests may be performed on pools
tested by an enzyme immunoassay (EIA) or by virus of up to 10 samples. Where drag swabs are tested, a
neutralisation (VN) for freedom of antibodies to avian minimum of 2 drag swabs are tested during each 4-week
leucosis virus subtypes A, B and J. All birds must also be period throughout the entire life of the flock. Detection of
tested for freedom from antibodies to the vertically- Salmonella spp. in these samples is performed by pre-
transmissible agents listed in Table 5.2.2-1. From the age of enrichment of the samples followed by culture using
8 weeks the flock is tested for freedom from Salmonella. Salmonella-selective media.
Clinical examination is carried out on the flock from 8 weeks Tests for avian leucosis antigen Prior to the
of age and the birds must not exhibit any signs of infectious commencement of laying, cloaca! swabs or blood samples
disease. The test methods to be used for these tests are given (using buffy coat cultivation) are tested for the presence of
in the table and further guidance is also given in the section group-specific leucosis antigen. A total of 5 per cent
below on routine testing of designated SPF flocks. From (minimum 10, maximum 200) of the flock is sampled during
20 weeks of age, the flock is tested as described under each 4-week period. During lay, albumen samples from
Routine testing of designated SPF flocks. All stages of this 5 per cent (minimum 10, maximum 200) of the eggs are
testing regime are also applied to the subsequent tested in each 4-week period. Tests are performed by EIA for
2 generations, except the testing of every bird before lay for group-specific antigen using methods that are capable of
vertically-transmissible agents. All test results must indicate detecting antigen from subgroups A, B and J.
freedom from pathogens in all 3 generations for the flock Test for antibodies to other agents Tests for antibodies
consisting of the 3rd generation to be designated as SPF. to all agents listed in Table 5.2.2.-1 are performed
SPF embryos derived from another designated SPF flock throughout the laying period of the flock. In each 4-week
contained within a separate facility on the same site may be period, samples are taken from 5 per cent (minimum 10,
introduced. From 8 weeks of age, these replacement birds are maximum 200) of the flock. It is recommended that
regarded as a flock and are tested in accordance with test 1.25 per cent of the flock is sampled each week since some
procedures described above. test methods for some agents must be conducted on a weekly
INITIAL TESTING REQUIREMENTS FOR basis. Table 5.2.2.-1 classifies the agents into those that
SUBSEQUENT GENERATIONS DERIVED FROM A spread rapidly through the flock and those that spread slowly
DESIGNATED SPF FLOCK or may not infect the entire flock. For those agents listed as
slowly spreading, each sample is tested individually.
Where a replacement flock is derived exclusively from a fully
For those agents listed as rapidly spreading, at least
established SPF flock the new generation is tested prior to
20 per cent of the samples collected in each 4-week period
being designated as SPF. In addition to the tests for
are tested individually or, where serum neutralisation or
Salmonella and monitoring of the general health and
ELISA tests are employed, all of the samples may be tested
performance of the flock, further specific testing from the age
individually or by preparing pools of 5 samples, collected at
of 8 weeks is required. Tests are performed on two
the same time.
5 per cent samples of the flock (minimum 10, maximum 200
birds) taken with an interval of at least 4 weeks between the Suitable methods to be used for detection of the agents are
ages of 12-16 weeks and 16-20 weeks. shown in Table 5.2.2.-1. Subject to agreement by the
competent authority, other test methods may be used
All samples are collected and tested individually. Blood
provided they are shown to be at least as sensitive as those
samples for antibody tests and suitable samples for testing for
indicated and of appropriate specificity.
leucosis antigen are collected. The test methods to be used
V-A546 Appendix XV H 2023

Table 5.2.2.-1
Agent Test Vertical Rapid/slow
to be usedtt transmission spread
Avian adenoviruses, group l AGP, EIA yes slow
Avian encephalomyeliris virus AGP, EIA yes rapid
Avian infectious bronchitis virus HI, EIA no rapid
Avian infectious laryngotracheitis virus VN, EIA no slow
Avian leucosis viruses EIA for virus, yes slow
VN, EIA for antibody
Avian nephritis virus IS no slow
Avian orthoreoviruses IS, EIA yes slow
Avian reticuloendotheliosis virus AGP, IS, EIA yes slow
Chicken anaemia virus IS, EIA, VN yes slow
Egg drop syndrome virus HI, EIA yes slow
Infectious bursal disease virus Serotype I: AGP, EIA, VN no rapid
Serotype 2: VN
Influenza A virus AGP, EIA, HI no rapid
Marek's disease virus AGP no rapid
Newcastle disease virus HI, EIA no rapid
Turkey rhinotracheitis virus EIA no slow
Mycoplasma gallisepticum Agg and HI to confirm a positive yes slow
test,
EIA, HI
Mycoplasma synoiiae Agg and HI to confirm a positive yes rapid
test,
EIA, HI
Salmonella pullorum Agg yes slow

Agg: agglutination HI: haemagglutination inhibition

AGP: agar gel precipitation; the technique is suitable where testing is carried out IS: immunostaining
weekly VN: virus neutralisation
EIA: enzyme immunoassay
**Subject to agreement by the competent authority, other types of test may be used provided they are at least as sensitive as those indicated and of appropriate
specificity.

Table 5.2.2-2. - Schematu description of the establishment and maintenance of SPF flocks
NEW STOCK Establish freedom from venically-transmissible agents

Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age

Test for Salmonella spp. and perform general clinical observation from 8 weeks of age

Carry out routine testing for specified agents from 20 weeks of age

2 nd GENERATION Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age

Test for Salmonella spp. and perform general clinical observation from 8 weeks of age

Carry out routine testing for specified agents from 20 weeks of age

3'd GENERATION Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age

Test for Salmonella spp. and perform general clinical observation from 8 weeks of age

DESIGNATE FLOCK AS SPF IF ALL TESTS ARE SATISFACTORY

3'" GENERATION Carry out routine testing for specified agents from 20 weeks of age

Carry out post-lay testing for venically-transmissible agents

SUBSEQUENT GENERATIONS Test two 5 per cent samples for avian leucosis antigen and for antibodies against specified agents between 12 and
20 weeks of age

Test for Salmonella spp. and perform general clinical observation from 8 weeks of age

Carry out routine testing for specified agents from 20 weeks of age

Carry out post-lay testing for venically-transmissible agents

TESTS TO BE CONDUCTED AT THE END OF THE 200) is retained for at least 4 weeks. Blood samples are
LAYING PERIOD collected from every bird in the group during the 4-week
Following the last egg collection, final testing to confirm the period with at least 1.25 per cent of the birds (25 per cent of
absence of vertically-transmissible agents indicated in the sample) being bled not earlier than 4 weeks after the final
Table 5.2.2.-1 is performed. After the last egg collection, a egg collection. Serum samples are tested for vertically-
minimum of 5 per cent of the flock (minimum 10, maximum transmissible agents (as defined by Table 5.2.2.-1) using the
2023 Appendix XV J V-A54 7

methods indicated. Where sampling is performed on a weekly There are perceived theoretical risks associated with the use
basis, at least 1.25 per cent of the birds (25 per cent of the of continuous cell lines, especially if their tumorigenic
sample) are tested each week during this period. potential has been demonstrated experimentally. These risks
Alternatively, within 4 weeks of the final egg collection blood are linked to the potential biological activity of the residual
and/or other suitable sample materials are collected from at host-cell DNA present in the vaccine. The residual host-cell
least 5 per cent of the flock and tested for the presence of DNA may be associated with an infectivity risk if the genome
vertically-transmissible agents using validated nucleic acid of a DNA virus or a provirus is present in the cellular DNA
amplification techniques (2. 6.21). (either integrated or extra chromosomal). In addition, there is
ACTION TO BE TAKEN IN THE EVENT OF a potential risk of oncogenicity if the cell substrate is
DETECTION OF A SPECIFIED AGENT tumorigenic.
If evidence is found of contamination of the flock by an For vaccines produced in continuous cell lines, whether
agent listed as slowly spreading in Table 5.2.2.-1, all tumorigenic or not, risk assessment and risk mitigation must
materials derived from the flock during the 4-week period be performed to evaluate the suitability of the cell substrate,
immediately preceding the date on which the positive sample to define the acceptable criteria for residual host-cell DNA in
was collected are considered unsatisfactory. Similarly, if the final product and to evaluate the consistency of host-cell
evidence is found of contamination of the flock by an agent proteins.
listed as rapidly spreading in Table 5.2.2.-1, all materials Cell-bank system
derived from the flock during the 2-week period immediately Production of vaccines in diploid or continuous cell lines is
preceding the date on which the positive sample was based on a cell-bank system. The in vitro age of the cells is
collected are considered unsatisfactory. Any product counted from the MCB. Each WCB is prepared from one or
manufactured with such materials, and for which the use of more containers of the MCB. The use, identity and
SPF materials is required, is considered unsatisfactory and inventory control of the containers is carefully documented.
must be discarded; any quality control tests conducted using Media and substances of human or animal origin
the materials are invalid. The composition of media used for isolation and all
Producers must notify users of all eggs of the evidence of subsequent culture is recorded in detail, and if substances of
contamination as soon as possible following the outbreak. human or animal origin are used they must be free from
Any flock in which an outbreak of any specified agent is extraneous agents (2. 6. 16) and must comply with general
confirmed may not be redesignated as an SPF flock. chapter 5.1. 7. Viral safety.
Any progeny derived from that flock during or after the If human albumin is used, it complies with the monograph
4-week period prior to the last negative sample being Human albumin solution (0255).
collected may not be designated as SPF. If bovine serum is used, it complies with the monograph
Bovine serum (2262).
Unless of recombinant origin, trypsin used for the
preparation of cell cultures is tested by suitable methods and
J. Cell Substrates for the Production of shown to be sterile and free from mycoplasmas and viruses.
Vaccines for Human Use Cell seed
(Ph. Bur. general texts 5.2.3) The data used to assess the suitability of the cell seed
comprises information, where available, on source, history
This general chapter deals with diploid cell lines and and characterisation.
continuous cell lines used as cell substrates for the Source of the cell seed For human cell lines, the
production of vaccines for human use; additional issues following information concerning the donor is recorded:
specifically related to vaccines prepared by recombinant ethnic and geographical origin; age; sex; general physiological
DNA technology are covered by the monograph Products of condition; tissue or organ used; results of any tests for
recombinant DNA technology (0784). The testing to be carried pathogens.
out at the various stages (cell seed, master cell bank (MCB),
working cell bank (WCB), end of productions cells (EOPC) For animal cell lines, the following information concerning
or extended cell bank (ECB) corresponding to cells at or the source of the cells is recorded: species; strain; breeding
beyond the maximum population doubling level used for conditions; geographical origin; age; sex; general physiological
production) is indicated in Table 5.2.3.-1. General provisions condition; tissue or organ used; results of any tests for
for the use of cell lines and test methods are given below. pathogens.
Where primary cells or cells that have undergone a few Cells of neural origin, such as neuroblastoma and Pl2 cell
passages without constitution of a cell bank are used for lines are not used for vaccine production since they may
vaccine production, requirements are given in the individual contain substances that concentrate agents of spongiform
monograph for the vaccine concerned. encephalopathies.
Diploid cell lines History of the cell seed The following information is
A diploid cell line has a high but finite capacity for recorded: the method used to isolate the cell seed; culture
multiplication in vitro. methods; any other procedures used to establish the MCB,
notably any that might expose the cells to extraneous agents.
Continuous cell lines
A continuous cell line has the capacity to multiply Full information may not be available on the media
indefinitely in vitro; the cells often have differences in ingredients used in the past for cultivation of cells, for
karyotype compared to the original cells; they may be example on the source of substances of animal origin; where
obtained from healthy or tumour tissue either from mammals justified and authorised, cell banks already established using
or from insects. such media may be used for vaccine production.
Characterisation of the cell seed The following
properties are investigated:
V-A548 Appendix XV J 2023

(1) the identity of the cells, using methods such as isoenzyme The MRC-5, WI-38 and FRhL-2 cell lines are recognised as
analysis, in vitro immunochemical assays, nucleic acid being non-tumorigenic and further testing is not necessary.
fingerprinting and nucleic acid amplification techniques Known tumorigenic cell lines (e.g. CHO) do not need to be
(NAT); documented further.
(2) the growth characteristics of the cells and their When a previously uncharacterised cell line is tumorigenic,
morphological properties (optical and electron microscopy); an oncogenicity study must be performed using purified
(3) for diploid cell lines, karyotype; DNA from the cell line and/or cell line lysate to demonstrate
(4) for diploid cell lines, the in vitro life span in terms of the absence of oncogenic components. The results are used
population doubling level. as part of the risk analysis performed to support the use of
the cell line for vaccine production. The determination of the
Cell substrate stability TPD 50 (tumour-producing dose in 50 per cent of animals)
Suitable viability of the cell line in the intended storage and the capacity to form metastases are characteristic
conditions must be demonstrated. For a given product to be properties that must be determined as part of the risk
prepared in the cell line, it is necessary to demonstrate that analysis.
consistent production can be obtained with cells at passage
Despite the difficulty in demonstrating a perfect and
and/or population doubling levels at the beginning and end
conclusive correlation with a tumorigenic phenotype,
of the intended period of use.
additional in vitro characterisation tests may be performed to
Infectious extraneous agents document other cell substrate properties, such as the ability
For cell lines for vaccine production, the testing for infectious to grow in soft agar gels, the ability to induce invasive cell
extraneous agents must be carried out based on a risk growth in muscle and/or the ability of the cell substrate to
assessment. The origin of the cell substrate as well as the induce transformation of 3T3 cells.
potential extraneous agents that may be inadvertently
Residual host-cell DNA
introduced during production processes or through the use of
For each particular vaccine produced on continuous cell
animal or plant derived raw materials must be taken into
lines, residual host-cell DNA content must be tested and an
account in the choice of suitable permissive cells. One such
acceptable upper limit, based on a risk assessment, must be
strategy is given in Table 5.2.3.-1, but alternative strategies
established in the final product taking into consideration:
could focus on more extensive testing at the MCB or WCB
level. In any case, any strategy must be justified and lead to (1) the nature of the cell substrate (non-tumorigenic, level of
the same level of safety as outlined in Table 5.2.3-1. New, tumorigenicity) and its origin (human/non-human);
sensitive molecular techniques with broad detection (2) the presence in the production process of any steps to
capabilities are available, including massive parallel inactivate the potential biological activity (oncogenicity,
sequencing (MPS) methods, degenerate polymerase chain infectivity) of the residual host-cell DNA (e.g. chemical
reaction (PCR) for whole virus families or random-priming agents such as betapropiolactone and/or DNase treatment);
methods (associated or not with sequencing), hybridisation to (3) the capacity of the process to reduce the amount and size
oligonucleotide arrays and mass spectrometry. These of the contaminating residual host-cell DNA;
methods may be used either as an alternative to in viva or (4) the intended use of the vaccine (e.g. route of
specific NAT tests or as a supplement/alternative to in vitro administration);
culture tests, in agreement with the competent authority.
(5) the method used to measure the residual host-cell DNA.
The capacity of the process to remove/inactivate specific
viruses must take into account the origin and culture history In general, a purification process for parenteral vaccines is
of the cell line and adventitious viruses that are known to able to reduce residual host-cell DNA in final products to
persistently infect the species of origin, for example, simian less than 10 ng per dose, but the acceptance limits must be
virus 40 in rhesus monkeys, Flock house virus in insect cells approved by the competent authority.
or viruses that may inadvertently be introduced during Once validation studies (e.g. spiking studies using an
production processes or through the use of raw materials of adequate size distribution of DNA) have been performed and
animal or plant origin. For cell lines of insect origin, tests for the reproducibility of the production process in reducing
specific viruses relevant to the species of origin of the insect residual host-cell DNA to the level expected has been
cells and for arboviruses (arthropod-borne viruses) are carried demonstrated, residual host-cell DNA testing may be omitted
out. The panel of viruses tested is chosen according to the after agreement from the competent authority.
current state of scientific knowledge. For cell lines shown to Chromosomal characterisation
express endogenous retroviral particles (e.g. rodent cells), the Diploid cell lines shall be shown to be diploid. More
test for reverse transcriptase is not needed because it is extensive characterisation of a diploid cell line by karyotype
expected to be positive, and thus infectivity tests must be analysis is required if the removal of intact cells during post-
performed to determine whether these endogenous retroviral harvest processing has not been validated. Samples from 4
particles are infectious or not. passage levels evenly spaced over the life span of the cell line
Cell lines that show the presence of infectious retroviruses are are examined. A minimum of 200 cells in metaphase are
not acceptable for production of vaccines, unless otherwise examined for exact chromosome count and for the frequency
justified and authorised. of hyperploidy, hypoploidy, polyploidy, breaks and structural
Tumorigenicity abnormalities.
Tumorigenicity is defined as the potential of a given cell line The MRC-5, WI-38 and FRhL-2 cell lines are recognised as
to induce a tumour after injection of intact live cells into being diploid and well characterised; where they are not
immunodeficient/immunosuppressed animals (usually genetically modified, further characterisation is not necessary.
rodents). The tumorigenicity test is carried out using cells at
or beyond the maximum population doubling level that will
be used for vaccine production.
 2023 Appendix XV J V-A549

Table 5.2.3.-1. - Testing of cell lines

EOPC/ECB (Cells at or
Master cell bank Working cell bank beyond the maximum
Test Cell seed
(MCB) (WCB) population doubling level
used for production)
I. IDENTITY AND PURITY
Morphology + + + +
Identification + + + +
Karyotype (diploid cell lines) + + +(l) +Ol

Life span (diploid cell lines) + +

2. EXTRANEOUS AGENTS
Bacterial and fungal contamination + +
M ycobacteria +C2) +C2)

Mycoplasmas + +
Spiroplasmasl 3l + +
Electron microscopy +l4l +(4)

Tests for extraneous agents in cell cultures (with viable + + +

cells or equivalent cell lysate)
Tests in suckling mice and eggs +C5l +(5)

Test for specific viruses by NAT +(6) +(6) +(6)

Test for viruses using broad molecular methods +<7) +C7J +C7l +<7)

Retroviruses +OJ +(4)

3. TUMORIGENICITY
+(8, 9) +C8J
Tumorigenicity

(I) The diploid character is established for each WCB but using cells at or beyond the maximum population doubling level used for production.
(2) If the cells are susceptible to infection with Mycobacteriurn tuberculosis or other species.
(3) If insect cells or raw materials of plant origin are used.
(4) Testing is carried out for the MCB, but using cells at or beyond the maximum population doubling level used for production.
(5) Testing is carried out for each WCB, but using cells at or beyond the maximum population doubling level used for production.
(6) Specific tests for possible contaminants (e.g. viruses) defined according to a risk assessment based on the origin of the cells and on the potential extraneous
agents inadvertently introduced during production processes or through the use of animal or plant derived raw materials. The appropriate testing stages should be
selected based on the risk assessment.
(7) These methods may be used either as alternative to in vivo tests and specific NAT or as supplement or alternative to in vitro culture tests based on the risk
assessment and in agreement with the competent authority. The appropriate testing stages should be selected based on the risk assessment,
(8) The MRC-5, Wl-38 and FRhL-2 cell lines are recognised as being non-tumorigenic and they do not need to be tested. Tests are not carried out on cell lines
that are known or assumed to be tumorigenic, for example CHO and BHK-21.
(9) Testing is carried out on the cell seed, but using cells at or beyond the maximum population doubling level used for production.

TEST METHODS FOR CELL CULTURES an assay is validated and shown to be comparable to the
Morphology culture method.
The morphology of the cells is adequately described and Mycoplasmas (2. 6. 7)
documented. The MCB and each WCB comply with the test for
Identification mycoplasmas. Use one or more containers for the test.
Nucleic acid fingerprint analysis and a relevant selection of Spiroplasmas
the following are used to establish the identity of the cells: Spiroplasmas may be introduced into cell substrates through
(1) biochemical characteristics (isoenzyme analysis); contamination of raw materials of plant origin or when insect
(2) immunological characteristics (histocompatibility cell lines are used. When appropriate, the MCB and each
antigens, in vitro immunochemical assays); WCB are demonstrated to be free of spiroplasmas using a
(3) cytogenetic markers; validated method approved by the competent authority.
NAT methods for detection of mycoplasmas (2. 6. 7) may be
(4) NAT. used to detect spiroplasmas after validation and agreement
Contaminating cells from the competent authority. Use one or more containers
The nucleic acid fingerprint analysis carried out for for the test.
identification also serves to demonstrate freedom from Electron microscopy
contaminating cells. The MCB is examined by electron microscopy for the
Bacterial and fungal contamination presence of extraneous agents. Cell lines are maintained at
The MCB and each WCB comply with the test for sterility the temperature routinely used for production and taken at
(2. 6.1), carried out using, for each medium, 10 mL of or beyond the maximum population doubling level used for
supernatant from cell cultures. Carry out the test on production. In addition, insect cell lines are maintained at
I per cent of the containers, with a minimum of 2 temperatures above and below those routinely used for
containers. production and may also be subjected to other treatments
Mycobacteria such as exposure to chemical stressors. For insect cell lines
If the cells are susceptible to infection with Mycobacterium the maintenance temperatures and treatments used are
tuberculosis or other species, the MCB and each WCB comply agreed with the competent authority, along with the number
with the test for mycobacteria (2.6.2). NAT (2.6.21) may be of sectioned cells to be examined.
used as an alternative to this culture method provided such
V-A550 Appendix XV J 2023

Test for extraneous agents in cell cultures decision on the acceptability of a cell substrate is based on all
For mammalian cells, viable cells (at least 10 7 cells) or the available data.
equivalent cell lysate, in their culture supernatant, are either Tests in suckling mice
co-cultivated (for viable cells) or inoculated (for cell lysate) The test is carried out if a risk assessment indicates that it
onto monolayer cultures of: provides a risk mitigation taking into account the overall
(1) human diploid cells; testing package applied to a given cell substrate.
(2) continuous simian kidney cells; and Inject 107 viable cells or the equivalent cell lysate, in their
(3) for cell substrates other than human or simian, cells of culture supernatant into 2 litters of suckling mice less than
that species, from a separate batch. 24 h old, comprising not fewer than 10 animals;
For insect cell lines, cell lysates are inoculated onto Inject at least 0.1 mL intraperitoneally and 0.01 mL
monolayer cultures of other cell systems, including human, intracerebrally.
simian and, in addition, at least 1 cell line that is different Observe the suckling mice for at least 4 weeks. Investigate
from that used in production, is permissible to insect viruses suckling mice that become sick or show any abnormality to
and allows detection of human arboviruses (e.g. BHK-21). establish the cause of illness. The cell substrate complies with
The resulting co-cultivated cell culture (for viable cells) or the test if no evidence of any extraneous agent is found.
inoculated cell cultures (for cell lysate) are observed for The test is invalid if fewer than 80 per cent of the suckling
evidence of viruses by cytopathic effect for at least 2 weeks. mice in each group remain healthy and survive to the end of
If the cell line is known to be capable of supporting the the observation period.
growth of human or simian cytomegalovirus, the human Tests in eggs (only required for avian cell substrates)
diploid cultures are observed for at least 4 weeks. The test is carried out if a risk assessment indicates that it
The extended 4-week cell culture of human diploid cells, for provides a risk mitigation taking into account the overall
the purpose of detecting human or simian cytomegalovirus, testing package applied to a given cell substrate. Inject an
can be replaced by the use of NAT (2.6.21). In cases where inoculum of 106 viable cells or the equivalent cell lysate, in
it is difficult to keep the cell cultures healthy for the their culture supernatant, into the allantoic cavity of ten 9- to
additional 2 weeks, it may be necessary to introduce fresh 11-day-old SPF embryonated hens' eggs (5.2.2) and into the
medium or to subculture after 2 weeks onto fresh cultures in yolk sac of ten 5- to 7-day-old SPF embryonated hens' eggs.
order to be able to detect viral agents. At the end of the Incubate for not less than 5 days. Test the allantoic fluids for
observation period, carry out tests on the cell culture the presence of haemagglutinins using mammalian and avian
supernatants for haemagglutinating viruses, or on the viable red blood cells; carry out the test at 5 ± 3 °C and 20-25 °C
cells for haemadsorbing viruses using guinea-pig red blood and read the results after 30-60 min. The cell substrate
cells. If the guinea-pig red blood cells have been stored, they complies with the test if no evidence of any extraneous agent
shall have been stored at 5 ± 3 °C for not more than 7 days. is found. The test is invalid if fewer than 80 per cent of the
Analyse half of the cultures after incubation at 5 ± 3 °C for embryos remain healthy and survive to the end of the
30 min and the other half after incubation at 20-25 °C for observation period.
30 min. The test for haemagglutinating viruses is not valid
Tests for specific viruses
for arboviruses.
The list of specific viruses to be tested is defined based on a
The test is not valid unless at least 80 per cent of the cell viral contamination risk assessment in accordance with the
cultures remain viable. The cell substrate complies with the principles detailed in general chapter 5.1. 7. Viral Safety, and
test if no evidence of any extraneous agent is found. takes into account (but is not limited to) the origin of the
Retroviruses cells and the potential sources of viral contamination
If the cell line is not known to produce retroviral particles, (e.g. raw material of animal or plant origin). NAT tests
examine for the presence of retroviruses using: (2.6.21) are carried out with or without prior amplification in
(1) product-enhanced reverse transcriptase (PERT) assay cells. For cell lines ofrodent origin, NAT (2.6.21) or
(2.6.21) carried out for cell bank supernatants using cells at antibody production tests in mice, rats or hamsters are used
or beyond the maximum population doubling level that will to detect species-specific viruses.
be used for production; Tests for viruses using broad molecular methods
(2) transmission electron microscopy. In agreement with the competent authority, broad molecular
If tests (1) and/or (2) give a positive result, infectivity assays methods (e.g. High Throughput Sequencing) may be used
are carried out on permissible human cells with a PERT either as an alternative to in vivo tests and specific NAT or as
assay end-point on the supernatant. a supplement or alternative to in vitro culture tests based on
the risk assessment.
If the cell line is shown to produce retroviral particles
(e.g. rodent cell lines), examine for the presence of For both NAT (2.6.21) and broad molecular methods, the
retroviruses using: stage at which testing is to be conducted (e.g. MCB, WCB,
- transmission electron microscopy; EOPC/ECB) is also based on the risk assessment and
- infectivity assays carried out on permissible human cells depends on the steps where viral contaminants may be
and on relevant additional cells (e.g. Mus dunni cells or introduced. In case of positive results with either broad
SC-I cells for CHO cell substrate) with a PERT assay molecular methods or NAT tests, a follow-up investigation
end-point on the supernatant, except when the must be conducted to determine whether detected nucleic
amplification cells are positive for reverse transcriptase, in acids are due to the presence of infectious extraneous agents
which case the readout is performed using plaque assay or and/or are known to constitute a risk to human health.
a fluorescent focus assay. Tests for tumorigenicity in vivo
Since the sensitivity of PERT assays is very high, The test establishes a comparison between the continuous
interpretation of a positive signal may be equivocal and a cell line and a suitable positive control cell line as reference
(for example, HeLa or Hep2 cells).
2023 Appendix XV K V-A551

Animal systems that have been shown to be suitable for this

test include:
K. Carrier Proteins for the Production of
(1) athymic mice (Nu/Nu genotype); Conjugated Polysaccharide Vaccines for
(2) newborn mice, rats or hamsters that have been treated Human Use
with antithymocyte serum or globulin;
(Ph. Bur. general text 5.2.11)
(3) thymectomised and irradiated mice that have been
reconstituted (Y-, B+) with bone marrow from healthy mice. The use of alternative carrier proteins, production methods and
Whichever animal system is selected, the cell line and the tests are acceptable provided they have been authorised by the
positive control cells are injected into separate groups of competent authority.
10 animals each. In both cases, the inoculum for each animal Bacterial polysaccharides are not able to induce a T-cell-
is 107 cells suspended in a volume of 0.2 mL, and the dependent B-cell immune response, which is needed to
injection may be given by the intramuscular or the obtain an immunological memory response, and are generally
subcutaneous route. Newborn animals are treated with poorly immunogenic in children under 2 years of age.
0.1 mL of antithymocyte serum or globulin on days 0, 2, 7 The limitations are overcome by conjugating polysaccharides
and 14 after birth. A potent serum or globulin is one that to carrier proteins. Carrier proteins are highly immunogenic
suppresses the immune mechanisms of growing animals to and, when conjugated to bacterial polysaccharides, increase
the extent that the subsequent inoculum of 10 7 positive the capacity of polysaccharides to induce a protective
control cells regularly produces tumours and metastases. response in infants.
Severely affected animals showing evident, progressively Carrier proteins currently used in polysaccharide vaccines for
growing tumours are euthanised before the end of the test to human use are toxoids, non-toxic mutated toxins, surface or
avoid unnecessary suffering. outer membrane proteins extracted from micro-organisms.
At the end of the observation period all animals, including Micro-organisms used for the production of the protein may
the positive control group, are euthanised and examined for be of genetically modified origin.
gross and microscopic evidence of the proliferation of The production method used for a carrier protein shall have
inoculated cells at the site of injection and in other organs been shown to yield consistently batches suitable for
(for example, lymph nodes, lungs, kidneys and liver). conjugation of the carrier protein to a polysaccharide antigen.
In all test systems, the animals are observed and palpated at Appropriate acceptance criteria for low bioburden before
regular intervals for the formation of nodules at the sites of conjugation with the polysaccharide may be established. It is
injection. Any nodules formed are measured in a prerequisite that the carrier protein is filtered through a
2 perpendicular directions, the measurements being recorded bacteria-retentive filter prior to storage and that adequate
regularly to determine whether there is progressive growth of measures are in place to avoid contamination and growth of
the nodule. Animals showing nodules that begin to regress micro-organisms during storage.
during the period of observation are euthanised before the The production of carrier proteins is based on a seed-lot
nodules are no longer palpable, and processed for histological system. The seed lots are shown to be free from
examination. Animals with progressively growing nodules are contamination using suitable methods of appropriate
observed for 1-2 weeks. Among those without nodule sensitivity. The culture may be inactivated and the carrier
formation, half are observed for 3 weeks and half for protein is purified by a suitable method.
12 weeks before they are euthanised and processed for
The protein is characterised by one or more suitable
histological examination. A necropsy is performed on each method(s) (such as SDS-PAGE, isoelectric focusing, HPLC,
animal and includes examination for gross evidence of size-exclusion chromatography with multiple-angle laser light
tumour formation at the site of injection and in other organs
scattering detection (MAUS), amino-acid analysis, amino-
such as lymph nodes, lungs, brain, spleen, kidneys and liver. acid sequencing, circular dichroism, fluorescence
All tumour-like lesions and the site of injection are examined
spectroscopy, peptide mapping and mass spectrometry) and
histologically. In addition, since some cell lines may give rise
its purity is verified by a suitable method. Suitable tests are
to metastases without evidence of local tumour growth, any
carried out, for validation or routinely, to demonstrate that
detectable regional lymph nodes and the lungs of all animals
where applicable, the product is free from specific toxins.
are examined histologically.
If purification steps are present, the reduction of selected
The test is invalid if fewer than 9 of the 10 animals injected process-related impurities and residuals is monitored to
with the reference positive-control cells show progressively establish consistency of the purification process. In the case
growing tumours. of recombinant carrier proteins, tests for at least the following
For a new tumorigenic cell line, in order to document the impurities are also carried out:
level of tumorigenicity, a dose range of cell substrate - residual host-cell proteins, including proteins derived from
(e.g. dose of cells in the range of 105, 10 6 and 107 ) is the expression vector;
injected in different groups of 10 animals. The number of - residual cellular DNA.
animals showing progressively growing nodules within the Only a carrier protein that complies with the following tests
animal groups is monitored to calculate the TPD 50 . may be used in the preparation of the conjugate.
Identification
The carrier protein is identified using a suitable method.
pH (2.2.3)
Where applicable, the pH of the carrier protein prior to
conjugation is monitored and is within the limits approved
for the particular product.
V-A552 Appendix XV L 2023

Protein content (2.5.16) to immune complexes formed by the antigen-antibody

The content of the carrier protein is determined using a reaction. Although immunonephelometry is applicable for the
suitable method and is within the limits approved by the quantification of both antibodies and antigens, this general
competent authority. chapter describes the quantification of antigens as vaccine
Bacterial endotoxins (2. 6.14) components.
The content is within the limits approved for the particular APPARATUS
product. The nephelometer used is described in general chapter 2. 2.1.
In addition, the following requirements apply for the carrier Clarity and degree of opalescence of liquids. However the
proteins listed below. measurement may be made at a non-zero angle different
Diphtheria toxoid from 90°.
It is produced as described in the monograph Diphtheria METHODS
vaccine (adsorbed) (0443) and complies with the requirements In general, the progress of the reaction measured by light
prescribed therein for bulk purified toxoid, except that the scattering can be described in 3 steps.
test for sterility (2. 6.1) is not required. Initially, a baseline level of light scattering due to the reaction
Tetanus toxoid medium is detected. After the 1st reagent (antigen) is added,
It is produced as described in the monograph Tetanus vaccine an increase in the signal is observed followed by a plateau.
(adsorbed) (0452) and complies with the requirements When the 2nd reagent (antibody) is added, a 2nd increase of
prescribed therein for bulk purified toxoid, except that the the signal is observed with a 2nd plateau followed by a final
antigenic purity is not less than 1500 Ll per milligram of increase in the intensity of the signal, which continues until a
protein nitrogen and that the test for sterility (2. 6.1) is not 3rd plateau is reached. The measurement zone starts from the
required. addition of the 2nd reagent until the higher intensity of light
CRM 197 diphtheria protein scattering (the 3rd plateau) is reached depending on the
It may be prepared from the growth of genetically modified concentrations of the component to be assayed.
(C7 /~197) or non-genetically modified (mCRM) The following methods can be used to quantify the immune
Corynebacterium diphthetiae or prepared by recombinant DNA complexes formed during this type of reaction.
technology in organisms such as Escherichia coli. The culture END-POINT NEPHELOMETRY
supernatant may be concentrated by ultrafiltration and The light scattering is measured after the immune complex
purified by successive precipitation, filtration, and has formed, i.e. after about 60 min. The value of the
chromatography steps. When it is produced on the same end-point is in this case directly proportional to the content
premises as diphtheria toxin, CRM 197 diphtheria protein of the component to be assayed when an excess of antibodies
should be distinguished from the active toxin by a suitable is used. A blank is necessary in order to subtract the value of
method. The purity is not less than 90 per cent of diphtheria nonspecific scattering due to the reaction mixture and to the
protein. reaction/measurement cell.
OMP (Neisseria rneningitidis group B outer RATE NEPHELOMETRY
membrane protein complex) Rate nephelometry is based on the rate of immune complex
Neisseria rneningitidis group B outer membrane protein formation (rate of increase in light scattering), which is
complex is extracted from bacterial cell cultures with a buffer proportional to the concentration of the component to be
containing a detergent. Cell debris is first removed. assayed.
The membrane protein complex may be concentrated and
There are 2 types of rate nephelometry.
purified by successive filtration and additional suitable
purification steps. The lipopolysaccharide content does not Fixed-time rate nephelometry
exceed 8 per cent. The relative quantity of the major OMP is A 1st measurement of light scattering is carried out a few
set and approved by the competent authority. The OMP seconds after the last reagent has been added. A 2nd
complex complies with the test for pyrogens (2. 6.8): inject measurement is carried out after a fixed time interval
into each rabbit 0.25 µg of OMP per kilogram of body mass. established during development of the method.
The difference between the 2 values is proportional to the
Recombinant protein D
quantity of the component to be assayed.
Protein D is a surface protein of nontypable Haemophilus
infiuenzae. It is produced using a specific strain of E. coli Rate nephelometry using the peak of the first derivative
carrying a plasmid containing the coding sequence for The formation of the immune complex is described by the
protein D. In order to express protein D, the modified strain curve of the first derivative of the variation in light scattering
is grown in a suitable liquid medium. At the end of with time (d0n/dt). Two successive peaks are observed due
cultivation, a purification step is carried out. The purity of to the addition of the 1st and then the 2nd reagent.
the product is not less than 95 per cent of protein D. The height of the 3rd peak of the first derivative is
proportional to the quantity of component to be assayed in
the reaction medium. Successive measurements are carried
out a few seconds after the 2nd reagent has been added.
L. lmmunonephelometry for Vaccine In this case, the light scattered by the reaction medium, the
component and the reagent does not affect the initial value of
Component Assay the derived signal.
(Ph. Bur. method 2.7.35) OPERATING CONDITIONS
GENERAL PRINCIPLE REAGENTS AND REFERENCE STANDARDS
The general principle of nephelometry is described in general The reagents, diluents and samples to be examined must be
chapter 2.2.1. Clarity and degree of opalescence of liquids. clear and free from any suspended particles. To this end, a
Immunonephelometry measures turbidity that is mainly due
 2023 Appendix XV M V-A553

preliminary filtration or centrifugation step may be

considered.
M. Substitution of in Vivo Method(s) by
The antisera or antibodies are selected based on their in Vitro Method(s) for the Quality
specificity, their precipitating capacity and their avidity for Control of Vaccines
the antigen. Indeed, high avidity results in clearly detectable
signals being generated rapidly, which thus improves the (Ph. Bur. general texts 5. 2.14)
quality of the results. PURPOSE
In addition, it is important to define the range of antigen The purpose of this general chapter is to provide guidance to
concentrations to be assayed with an excess of antibodies and facilitate the implementation of in vitro methods as
to verify the absence of a zone effect in this range. substitutes for existing in vivo methods, in cases where a
It is recommended to ensure that the profiles of response will typical one-to-one assay comparison is not appropriate for
be similar to that of an appropriate reference standard. reasons unrelated to the suitability of one or more in vitro
PRE-TREATMENT OF THE SAMPLES TO BE methods. This general chapter will not discuss the details of
EXAMINED assay validation as such, since those principles are described
An appropriate pre-treatment could be envisaged to eliminate elsewhere.
an adjuvant or another interfering substance. The general chapter applies primarily to vaccines for human
or veterinary use, however the principles described may also
ASSAY
apply to other biologicals such as sera.
Ensure that all the reagents are at a suitable temperature to
optimise the reaction. CONTEXT
To the samples diluted with a suitable medium in the The test methods used for routine quality control of vaccines
reaction measurement cell, add a fixed quantity of antibodies. are intended to monitor production consistency and to
Carry out several determinations of the dilution being ensure comparability of the quality attributes between
examined. commercial batches and those batches originally found to be
In some cases, the progress of the reaction may be improved safe and efficacious in clinical studies or, for veterinary
by introducing additional substances into the reaction vaccines, in the target species.
medium (e.g. polyethylene glycol). While the in viva potency and safety assays described within
The reference standards and samples must be treated in the Ph. Eur. vaccine monographs have historically played a
same manner. central role in safeguarding the quality of vaccines, the
inherent variability of in vivo assays can make them less
PERFORMANCE OF A BUNK TEST suitable than appropriately designed in vitro assays for
To ensure that the assays are specific, only the scattering due monitoring consistency of production and for assessing the
to immune complexes must be measured. Other particles, the potential impact of manufacturing changes. As a result, it is
matrix of the sample, the reagents or the reaction essential continually to challenge the scientific value and
measurement cell may interfere. In this case and if the relevance of these in viva test methods. When in vivo tests
end-point method is used, it is necessary to carry out a blank are found to be of limited or no value, it is imperative to
test to avoid taking non-specific scattering into account. eliminate them, given the ethical considerations and the
CALIBRATION obligations under the relevant conventions. In addition, there
The calibration curve is obtained by preparing a set of is a substantial effort to develop in vitro methods (including
dilutions of a reference standard. The concentrations are immunological, molecular and physico-chemical tests) to
chosen in order to encompass the working range. replace the animal tests. In several cases this has led to the
METHODS OF CALCUUTION successful introduction of new in vitro methods in vaccine
The parallel line method (see general chapter 5.3) or a monographs. The use of appropriate in vitro methods not
calibration curve is used for the calculations. only reduces the use of animals while maintaining or
improving the scientific relevance of the assays involved, but
VALIDITY OF THE TESTS also substantially reduces assay variability and the time and
VALIDI1Y OF THE CALIBRATION CURVES resources required, and enhances the predictability of the
Only calibration curves that comply with the defined validity release of safe and effective vaccine lots for use.
criteria may be used.
In addition to the benefits resulting from the substitution of
The following parameters are examples to be used to define appropriate in vitro methods for existing in viva methods,
validity criteria: under the European Convention for the Protection of
- minimum of light scattering units (LSU) obtained for the Vertebrate Animals Used for Experimental and Other
lowest concentration point of the calibration curve; Scientific Purposes, the Ph. Eur. Commission has committed
- coefficient of variation for the LSU measured for each to the reduction of animal usage wherever possible in
point on the curve; pharmacopoeia! testing. Under the convention, those
- coefficients of regression or of correlation of the associated with the work of the European Pharmacopoeia are
calibration curve; encouraged to develop and/or implement in vitro procedures,
- slope and intercept of the calibration curve. and the General Notices support the introduction of
VALIDI1Y OF THE RESULTS alternatives to in vivo methods described in Ph. Eur.
The following criteria are examples of validity criteria of the monographs.
samples to be assayed: GENERAL CONSIDERATIONS
- coefficient of variation or the range of values for the
One of the consequences associated with the inherent
determinations carried out on the sample;
variability of in vivo assays is the problem this poses with
- assay of an in-house reference standard with trend
their replacement by the more-consistent in vitro methods,
monitoring or a control chart.
which typically requires one-to-one assay comparison. This
V-A554 Appendix XV M 2023

can be a challenge in some cases as repeated efforts through In the Ph. Eur., in viva assays for vaccines are typically
multicentre international collaborative studies can fail due to replaced by in vitro assays following multicentre collaborative
the variability inherent in the in viva methods. Another studies, but this should not be a prerequisite for in viva assay
consideration is that many of the legacy in viva safety and replacement initiatives for individual products. Additionally,
potency assays for vaccines were generally shown to be fit for while it may be desirable to have assays that are widely
purpose and have historically proven their value in ensuring applicable to a class of products, this should not be a
the efficacy and safety of vaccines. However, this was in an requirement.
era when validation requirements, such as ICH Q2 (Rl) or As explained in the guidance below, in some cases an
VICH Gl2 guideline, were not in place, making a formal existing method may need to be substituted by more than
one-to-one comparison challenging or even impossible in 1 in vitro test, in order to characterise the key qualitative and
some cases. Since precision, reproducibility, limits of quantitative attributes measured by the existing test.
detection and quantification were not established for the in
POTENCY TESTS
viva method, the comparability of one method to another
When it is not possible to show agreement between the
becomes difficult to evaluate. It should also be noted that,
in vitro and in viva methods due to low discriminating power
because Ph. Eur. methods are considered validated under the
and/or high variability of the in viva assay, the following
General Notices, it is not only impractical and excessively
approach can be used. It is assumed that the product under
costly now to undertake a retrospective ICHMCH validation
consideration has a well-established safety and efficacy
of these methods, but it would also be unethical given the
profile, with consistent manufacturing.
above-mentioned convention on animal use in
pharmacopoeia) testing. The in vitro test(s) should be able to detect differences that
are relevant to the control of the production process as
When considering the transition from an in viva-based to an
justified scientifically. This should be supported by data
in vitro-based quality control assay system, it is important to
demonstrating the capability of the proposed assay(s) to
understand what in viva assays can and cannot offer.
control key quality attributes of the vaccine and maintain the
Although properly established in viva potency assays in
link between the quality of the batches to be released and
laboratory animals have the potential to measure complex
those batches found to be safe and efficacious through
functional responses for demonstrating proof of concept,
clinical studies or routine use. With the setting of appropriate
these do not necessarily predict the actual responses in the
specifications, the consistency of manufacturing with the
target population. In addition, in vitro bioassays have the
in vitro method(s) will be maintained.
potential to mimic specific elements of complex in viva
responses with generally lower variability and higher The design of an assay/assay system for vaccine quality
sensitivity. control needs to reflect both antigen content and
functionality. If a single method is used, it should preferably
Another key consideration is that when an in viva test for a
measure the content and integrity of the antigen by targeting
given product is to be replaced with an in vitro test, the
epitope(s) relevant to the protection offered by the vaccine.
quality attribute(s) of the product will likely be assessed
An example of this would be a monoclonal antibody or
differently. Examples include: the determination of antigen
monoclonal antibodies against an epitope or epitopes as the
content or a functional response (e.g. virus or toxin
main target for generating neutralising antibodies.
neutralisation) in an in vitro bioassay instead of in viva
The epitope or epitopes should preferably be conformational
potency; molecular consistency instead of in viva
in order to have a stability-indicating assay (as is the case for
neurovirulence or attenuated phenotype; absence of the
rabies vaccine). In some cases, a single in vitro method may
extraneous agent genomes using molecular methods instead
not adequately reflect the content and functionality. This can
of absence of micro-organisms through in viva testing; and
be remedied through the use of multiple assays, as is the case
demonstration of toxin binding and enzyme activity instead
with conjugate polysaccharide vaccines, where molecular size,
of in viva specific toxicity. As a consequence, a
conjugate integrity, and total and free polysaccharides are
demonstration of agreement between the 2 methods is
examples of relevant measures.
generally not scientifically justified and should not always be
expected. Even where pass/fail results from the 2 test To establish quantitative measurements with an in vitro
procedures are in agreement, the correlation between method, samples that differ in the magnitude of the response
2 quantitative methods across the assay range may still be will be needed. In most cases, samples that are below the
low. Regardless, the in vitro method(s) or testing strategy minimum approved potency specification with the in viva
must provide at least the same confidence that the key method will not be available because production consistency
quality attributes, which are necessary to ensure the is generally well maintained, and potency between batches
consistency of a product's safety and effectiveness, are does not differ significantly and/or the precision of the in viva
adequately controlled. assay is such that it cannot discriminate between batches
unless the difference is very large. Therefore, initial assay
While the focus of this general chapter is on the replacement
evaluation should be performed with samples at different
of existing methods for approved products, it is important to
concentrations, which could be followed by testing of
consider the use of in vitro methods for quality control during
samples subjected to different types of stress conditions to
product development and to understand that the use of in
assess further the stability-indicating potential of the new
viva assays is not mandatory.
method. The inability to demonstrate agreement between an
ALTERNATIVE APPROACHES FOR THE in vitro and an in viva method does not necessarily mean that
SUBSTITUTION OF IN VIVO METHODS the in vitro method is not suitable/relevant. In many cases, an
The primary focus for the implementation of any proposed in in vitro method will detect changes in the product profile that
vitro methods within a quality control system should be the would not be detected by the in viva method. In such cases,
scientific relevance of in vitro assays for control of the the in vitro method may be considered superior for
relevant quality attributes. Additionally, any in vitro methods monitoring the consistency of production and may be more
will have to meet the current validation requirements. relevant to assess the impact of manufacturing changes.
 2023 Appendix XV M V-A555

SAFEIY TESTS limited validation data exist for the in vivo methods.
Specific toxicity It should also be emphasised that the outcome of the new
An in vitro method for detection of residual toxic components molecular methods is not the final result since the detection
should be specific and at least as sensitive as the existing in of a genome or fragments of a genome does not necessarily
vivo method. Where possible, a fully functional in vitro indicate the presence of an infectious virus.
system should be used (e.g. toxin-sensitive cell line). Where
no functional in vitro system is available, an in vitro testing
strategy could be based on the detection/measurement of
more than 1 parameter, sequentially where relevant, that
together reflect the mode of action for the toxic components
in question. Examples include the use of assays with
immunological and biochemical steps to detect receptor
binding and enzyme activity. In most cases, where an in vivo
assay is to be replaced there will be data available on the
sensitivity of that model for detection of the toxin in
question. Therefore, new in vitro methods can be
characterised to demonstrate that they are sufficiently
sensitive using spiking experiments and referring to historic
data for the in vivo assay. Such assays, in conjunction with
the appropriate time and temperature conditions, could also
be used to demonstrate the absence of reversion of a specific
toxoid.
Molecular consistency by deep sequencing versus the
neurovirulence test
An in vitro genotypic method to assess the molecular
consistency of a viral vaccine has the potential to replace an
existing in vivo neurovirulence test. A prerequisite for any in
vitro genotypic method is an in-depth knowledge of the
molecular markers responsible for the attenuation of the live
viral vaccine (as is the case for oral poliovirus vaccine, for
example). In such a case, monitoring the consistency of the
vaccine lots would be achieved by confirming the presence of
the required molecular attenuation markers and percentage of
mutants with methods such as deep sequencing.
Detection of viral extraneous agents by novel molecular
methods
Detection of viral extraneous agents in cell banks, seed lots
and cell culture harvests is currently conducted using a panel
of in vivo and in vitro methods at different stages of the
manufacturing process. Novel, sensitive molecular techniques
with broad detection capabilities are available, including deep
sequencing or high-throughput sequencing methods,
degenerate polymerase chain reaction (PCR) for whole virus
families or random-priming methods (associated or not with
sequencing), hybridisation to oligonucleotide arrays and mass
spectrometry. The use of these new molecular methods has
highlighted gaps in the existing testing strategy by identifying
previously undetected viral contaminants in final product, the
cell banks from which it was produced and intermediate
manufacturing stages. These new molecular methods
(e.g. deep sequencing or high-throughput sequencing) detect
genomes while the existing in vivo methods are based on
observations of the effects viruses have on experimental
animals. The implementation of such new molecular
methods as substitutes for in vivo methods requires a
comparison of the specificity (breadth of detection) and the
sensitivity of the new and existing methods. For this purpose,
an appropriate panel of representative, well-characterised
model viruses should be used to assess the ability of the new
method to detect viruses that are (or are not) detected by the
in vivo methods, and to determine if the sensitivity is at least
equivalent to the sensitivity of the in vivo methods. This last
element is particularly complex since these new molecular
methods do not detect the same characteristic of the viral
contaminant (genome for molecular methods versus
infectious virus for in vivo methods) and also since no or
V-A556 Appendix XVI A 2023

container. If more than the upper one-third of the medium

Appendix XVI has acquired a pink colour, the medium may be restored
once by heating the containers in a water-bath or in free-
A. Test for Sterility flowing steam until the pink colour disappears and cooling
quickly, taking care to prevent the introduction of non-sterile
Sterility 1 air into the container. Do not use the medium for a longer
(Ph. Bur. method 2.6.1) storage period than has been validated.
The test is applied to substances, preparations or articles which, Fluid thioglycollate medium is to be incubated at 30-35 °C.
according to the Pharmacopoeia, are required to be sterile. For products containing a mercurial preservative that cannot
However, a satisfactory result only indicates that no contaminating be tested by the membrane-filtration method, fluid
micro-organism has been found in the sample examined in the thioglycollate medium incubated at 20-25 °C may be used
conditions of the test. instead of soya-bean casein digest medium provided that it
has been validated as described in growth promotion test.
PRECAUTIONS AGAINST MICROBIAL
CONTAMINATION Where prescribed or justified and authorised, the following
alternative thioglycollate medium may be used. Prepare a
The test for sterility is carried out under aseptic conditions.
mixture having the same composition as that of the fluid
In order to achieve such conditions, the test environment has
thioglycollate medium, but omitting the agar and the
to be adapted to the way in which the sterility test is
resazurin sodium solution, sterilise as directed above.
performed. The precautions taken to avoid contamination are
The pH after sterilisation is 7 .1 ± 0.2. Heat in a water-bath
such that they do not affect any micro-organisms which are
prior to use and incubate at 30-35 °C under anaerobic
to be revealed in the test. The working conditions in which
conditions.
the tests are performed are monitored regularly by
appropriate sampling of the working area and by carrying out Soya-bean casein digest medium
appropriate controls.
Pancreatic digest of casein 17.0 g
CULTURE MEDIA AND INCUBATION Papaic digest of soya-bean meal 3.0 g
TEMPERATURES Sodium chloride 5.0 g
Media for the test may be prepared as described below, or Dipotassium hydrogen phosphate 2.5 g
Glucose monohydrate/Glucose 2.5 g/2.3 g
equivalent commercial media may be used provided that they WaterR IO00mL
comply with the growth promotion test. pH after sterilisation 7 .3 ± 0.2
The following culture media have been found to be suitable
for the test for sterility. Fluid thioglycollate medium is Dissolve the solids in water R, warming slightly to effect
primarily intended for the culture of anaerobic bacteria; solution. Cool the solution to room temperature. Add 1 M
however, it will also detect aerobic bacteria. Soya-bean casein sodium hydroxide, if necessary, so that after sterilisation the
digest medium is suitable for the culture of both fungi and solution will have a pH of 7 .3 ± 0.2. Filter, if necessary, to
aerobic bacteria. clarify, distribute into suitable vessels and sterilise using a
validated process. Store at a temperature between 2 °C and
Fluid thioglycollate medium
25 °C in a sterile well-closed container, unless it is intended
L-Cystine 0.5 g
for immediate use. Do not use the medium for a longer
Agar 0.75 g storage period than has been validated.
Sodium chloride 2.5 g Soya-bean casein digest medium is to be incubated at
Glucose monohydrate/Glucose 5.5 g/5.0 g
Yeast extract (water-soluble) 5.0 g
20-25 °C.
Pancreatic digest of casein 15.0 g The media used comply with the following tests, carried out
Sodium thioglycollate or 0.5 g before or in parallel with the test on the product to be
Thioglycollic acid 0.3mL
examined.
Resazurin sodium solution (I g/L of resazurin l.0mL
sodium), freshly prepared Sterility
WaterR IO00mL Incubate portions of the media for 14 days. No growth of
pH after sterilisation 7. I ± 0.2
micro-organisms occurs.
Mix the L-cystine, agar, sodium chloride, glucose, water- Growth promotion test of aerobes, anaerobes and fungi
soluble yeast extract and pancreatic digest of casein with the Test each batch ofready-prepared medium and each batch
water R and heat until solution is effected. Dissolve the of medium prepared either from dehydrated medium or from
sodium thioglycollate or thioglycollic acid in the solution and, ingredients. Suitable strains of micro-organisms are indicated
if necessary, add 1 M sodium hydroxide so that, after in Table 2.6.1.-1.
sterilisation, the solution will have a pH of7.l ± 0.2. Inoculate portions of fluid thioglycollate medium with a small
If filtration is necessary, heat the solution again without number (not more than 100 CFU) of the following micro-
boiling and filter while hot through moistened filter paper. organisms, using a separate portion of medium for each of
Add the resazurin sodium solution, mix and place the the following species of micro-organism: Clostridium
medium in suitable vessels which provide a ratio of surface to sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus.
depth of medium such that not more than the upper half of Inoculate portions of soya-bean casein digest medium with a
the medium has undergone a colour change indicative of small number (not more than 100 CFU) of the following
oxygen uptake at the end of the incubation period. Sterilise micro-organisms, using a separate portion of medium for
using a validated process. If the medium is stored, store at a each of the following species of micro-organism: Aspergillus
temperature between 2 °C and 25 °C in a sterile, airtight brasiliensis, Bacillus subtilis, Candida albicans. Incubate for not
more than 3 days in the case of bacteria and not more than
1 This chapter has undergone pharmacopoeia/ harmonisati<m. See chapter 5 days in the case of fungi.
5. 8 Phamzacopoeial harmonisation.
2023 Appendix XVI A V-A557

Table 2.6.1.-1. - Strains of the test muro-organisms suitable for use in the growth promotion test and the method suitabiliry test
Aerobic bacteria

Staphylococcus aureus ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276
Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134
Pseudomonas aernginosa ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275

Anaerobic bacterium

Clostridium sporogenes ATCC 19404, CIP 79.3, NCTC 532, ATCC 11437, NBRC 14293

Fungi

Canduia a/means ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594

Aspergil/us brasiliensis ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455

Seed lot culture maintenance techniques (seed-lot systems) TEST FOR STERILITY OF THE PRODUCT TO BE
are used so that the viable micro-organisms used for EXAMINED
inoculation are not more than 5 passages removed from the The test may be carried out using the technique of
original master seed-lot. membrane filtration or by direct inoculation of the culture
The media are suitable if a clearly visible growth of the media with the product to be examined. Appropriate negative
micro-organisms occurs. controls are included. The technique of membrane filtration
is used whenever the nature of the product permits, that is,
METHOD SUITABILITY TEST
for filterable aqueous preparations, for alcoholic or oily
Carry out a test as described below under Test for sterility of preparations and for preparations miscible with or soluble in
the product to be examined using exactly the same methods aqueous or oily solvents provided these solvents do not have
except for the following modifications. an antimicrobial effect in the conditions of the test.
Membrane filtration Membrane filtration
After transferring the contents of the container or containers Use membrane filters having a nominal pore size not greater
to be tested to the membrane add an inoculum of a small than 0.45 µm whose effectiveness to retain micro-organisms
number of viable micro-organisms (not more than 100 CFU) has been established. Cellulose nitrate filters, for example, are
to the final portion of sterile diluent used to rinse the filter.
used for aqueous, oily and weakly alcoholic solutions and
Direct inoculation cellulose acetate filters, for example, for strongly alcoholic
After transferring the content of the container or containers solutions. Specially adapted filters may be needed for certain
to be tested (for catgut and other surgical sutures for products, e.g. for antibiotics.
veterinary use: strands) to the culture medium add an The technique described below assumes that membranes
inoculum of a small number of viable micro-organisms (not about 50 mm in diameter will be used. If filters of a different
more than 100 CFU) to the medium. diameter are used the volumes of the dilutions and the
In both cases use the same micro-organisms as those washings should be adjusted accordingly. The filtration
described above under Growth promotion test of aerobes, apparatus and membrane are sterilised by appropriate means.
anaerobes and fungi. Perform a growth promotion test as a The apparatus is designed so that the solution to be
positive control. Incubate all the containers containing examined can be introduced and filtered under aseptic
medium for not more than 5 days. conditions; it permits the aseptic removal of the membrane
If clearly visible growth of micro-organisms is obtained after for transfer to the medium or it is suitable for carrying out
the incubation, visually comparable to that in the control the incubation after adding the medium to the apparatus
vessel without product, either the product possesses no itself.
antimicrobial activity under the conditions of the test or such Aqueous solutions If appropriate, transfer a small quantity
activity has been satisfactorily eliminated. The test for sterility of a suitable, sterile diluent such as a 1 g/L neutral solution
may then be carried out without further modification. of meat or casein peptone pH 7 .1 ± 0.2 onto the membrane
If clearly visible growth is not obtained in the presence of the in the apparatus and filter. The diluent may contain suitable
product to be tested, visually comparable to that in the neutralising substances and/or appropriate inactivating
control vessels without product, the product possesses substances for example in the case of antibiotics.
antimicrobial activity that has not been satisfactorily Transfer the contents of the container or containers to be
eliminated under the conditions of the test. Modify the tested to the membrane or membranes, if necessary after
conditions in order to eliminate the antimicrobial activity and diluting to the volume used in the method suitability test
repeat the method suitability test. with the chosen sterile diluent but in any case using not less
This method suitability test is performed: than the quantities of the product to be examined prescribed
a) when the test for sterility has to be carried out on a new in Table 2.6.1.-2. Filter immediately. If the product has
product; antimicrobial properties, wash the membrane not less than
3 times by filtering through it each time the volume of the
b) whenever there is a change in the experimental conditions
chosen sterile diluent used in the method suitability test.
of the test.
Do not exceed a washing cycle of 5 times 100 mL per filter,
The method suitability test may be performed simultaneously even if during the method suitability test it has been
with the test for sterility of the product to be examined. demonstrated that such a cycle does not fully eliminate the
antimicrobial activity. Transfer the whole membrane to the
culture medium or cut it aseptically into 2 equal parts and
V-A558 Appendix XVI A 2023

Table 2.6.1.-2. - Minimum quantity to be used for each medium

Minhnwn quantity to be used for each medium unless otherwise
Quantity per container
justified and authorised

Liquids

- less than I mL The whole contents of each container

- 1-40 mL Half the contents of each container but not less than 1 mL
- greater than 40 mL and not greater than 100 mL 20 mL
- greater than 100 mL 10 per cent of the contents of the container but not less than 20 mL

Antibiotic liquids 1 mL

Insoluble preparations, creams and ointments to be suspended or emulsified Use the contents of each container to provide not less than 200 mg

Solids

- less than 50 mg The whole contents of each container

- 50 mg or more but less than 300 mg Half the contents of each container but not less than 50 mg
- 300 mg to 5 g 150 mg
- greater than 5 g 500 mg

Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30 cm long)

transfer one half to each of 2 suitable media. Use the same so that the volume of the product is not more than
volume of each medium as in the method suitability test. 10 per cent of the volume of the medium, unless otherwise
Alternatively, transfer the medium onto the membrane in the prescribed.
apparatus. Incubate the media for not less than 14 days. If the product to be examined has antimicrobial activity,
Soluble solids Use for each medium not less than the carry out the test after neutralising this with a suitable
quantity prescribed in Table 2.6.1.-2 of the product dissolved neutralising substance or by dilution in a sufficient quantity
in a suitable solvent such as the solvent provided with the of culture medium. When it is necessary to use a large
preparation, water for injections, saline or a 1 g/L neutral volume of the product it may be preferable to use a
solution of meat or casein peptone and proceed with the test concentrated culture medium prepared in such a way that it
as described above for aqueous solutions using a membrane takes account of the subsequent dilution. Where appropriate,
appropriate to the chosen solvent. the concentrated medium may be added directly to the
Oils and oily solutions Use for each medium not less product in its container.
than the quantity of the product prescribed in Table 2.6.1.-2. Oily liquids Use media to which have been added a
Oils and oily solutions of sufficiently low viscosity may be suitable emulsifying agent at a concentration shown to be
filtered without dilution through a dry membrane. Viscous appropriate in the method suitability test, for example
oils may be diluted as necessary with a suitable sterile diluent polysorbate 80 at a concentration of 10 g/L.
such as isopropyl myristate shown not to have antimicrobial Ointments and creams Prepare by diluting to about 1 in
activity in the conditions of the test. Allow the oil to 10 by emulsifying with the chosen emulsifying agent in a
penetrate the membrane by its own weight then filter, suitable sterile diluent such as a 1 g/L neutral solution of
applying the pressure or suction gradually. Wash the meat or casein peptone. Transfer the diluted product to a
membrane at least 3 times by filtering through it each time medium not containing an emulsifying agent.
about 100 mL of a suitable sterile solution such as 1 g/L
Incubate the inoculated media for not less than 14 days.
neutral meat or casein peptone containing a suitable
Observe the cultures several times during the incubation
emulsifying agent at a concentration shown to be appropriate
period. Shake cultures containing oily products gently each
in the method suitability test, for example polysorbate 80 at a
day. However when fluid thioglycollate medium is used for
concentration of 10 g/L. Transfer the membrane or
the detection of anaerobic micro-organisms keep shaking or
membranes to the culture medium or media or vice versa as
mixing to a minimum in order to maintain anaerobic
described above for aqueous solutions, and incubate at the
conditions.
same temperatures and for the same times.
Catgut and other surgical sutures for veterinary use
Ointments and creams Use for each medium not less Use for each medium not less than the quantities of the
than the quantities of the product prescribed in
product prescribed in Table 2.6.1.-2. Open the sealed
Table 2.6.1.-2. Ointments in a fatty base and emulsions of
package using aseptic precautions and remove 3 sections of
the water-in-oil type may be diluted to 1 per cent in
the strand for each culture medium. Carry out the test on
isopropyl myristate as described above, by heating, if
3 sections, each 30 cm long, cut off from the beginning, the
necessary, to not more than 40 °C. In exceptional cases it
centre and the end of the strand. Use whole strands from
may be necessary to heat to not more than 44 °C. Filter as
freshly opened cassette packs. Transfer each section of the
rapidly as possible and proceed as described above for oils
strand to the selected medium. Use sufficient medium to
and oily solutions.
cover adequately the material to be tested (20 mL to
Direct inoculation of the culture medium 150 mL).
Transfer the quantity of the preparation to be examined
prescribed in Table 2.6.1.-2 directly into the culture medium
2023 Appendix XVI A V-A559

Table 2.6.1.-3. -Minimum number of items to be tested

Minimum number of items to be tested for each medium, unless
Number of items in the batch*
otherwise justified and authorised**

Paremeral preparations

- Not more than I 00 containers 10 per cent or 4 containers, whichever is the greater

- More than 100 but not more than 500 containers I 0 containers

2 per cent or 20 containers (10 containers for large-volume parenterals)

- More than 500 containers
whichever is less

Ophthalmic and other non-injectable preparations

- Not more than 200 containers 5 per cent or 2 containers, whichever is the greater

- More than 200 containers 10 containers

- If the product is presented in the form of single-dose containers, apply the

scheme shown above for preparations for parenteral administration

2 per cent or 5 packages whichever is the greater, up to a maximum total of 20

Catgut and other surgical sutures for veterinary use
packages

Bulk solid products

- Up to 4 containers Each container

- More than 4 containers but not more than 50 containers 20 per cent or 4 containers, whichever is the greater
- More than 50 containers 2 per cent or 10 containers, whichever is the greater

* If the batch size is not known, use the maximum number of items prescribed.
**If the contents of one container are enough to inoculate the 2 media, this column gives the number of containers needed for both the media together.

OBSERVATION AND INTERPRETATION OF APPLICATION OF THE TEST TO PARENTERAL

RESULTS PREPARATIONS, OPHTHALMIC AND OTHER
At intervals during the incubation period and at its NON-INJECTABLE PREPARATIONS REQUIRED
conclusion, examine the media for macroscopic evidence of TO COMPLY WITH THE TEST FOR STERILITY
microbial growth. If the material being tested renders the When using the technique of membrane filtration, use,
medium turbid so that the presence or absence of microbial whenever possible, the whole contents of the container, but
growth cannot be readily determined by visual examination, not less than the quantities indicated in Table 2.6.1.-2,
14 days after the beginning of incubation transfer portions diluting where necessary to about 100 mL with a suitable
(each not less than 1 mL) of the medium to fresh vessels of sterile solution, such as 1 g/L neutral meat or casein peptone.
the same medium and then incubate the original and transfer When using the technique of direct inoculation of media, use
vessels for not less than 4 days. the quantities shown in Table 2.6.1.-2, unless otherwise
If no evidence of microbial growth is found, the product to justified and authorised. The tests for bacterial and fungal
be examined complies with the test for sterility. If evidence of sterility are carried out on the same sample of the product to
microbial growth is found the product to be examined does be examined. When the volume or the quantity in a single
not comply with the test for sterility, unless it can be clearly container is insufficient to carry out the tests, the contents
demonstrated that the test was invalid for causes unrelated to of 2 or more containers are used to inoculate the different
the product to be examined. The test may be considered media.
invalid only if one or more of the following conditions are MINIMUM NUMBER OF ITEMS TO BE TESTED
fulfilled:
The minimum number of items to be tested in relation to the
a) the data of the microbiological monitoring of the sterility size of the batch is given in Table 2.6.1.-3.
testing facility show a fault;
Guidelines on the test for sterility are given in general
b) a review of the testing procedure used during the test in chapter 5.1.9.
question reveals a fault;
c) microbial growth is found in the negative controls;
d) after determination of the identity of the micro-organisms
isolated from the test, the growth of this species or these
species may be ascribed unequivocally to faults with respect
to the material and/or the technique used in conducting the
sterility test procedure.
If the test is declared to be invalid it is repeated with the
same number of units as in the original test.
If no evidence of microbial growth is found in the repeat test
the product examined complies with the test for sterility.
If microbial growth is found in the repeat test the product
examined does not comply with the test for sterility.
V-A560 Appendix XVI B 2023

Use buffered sodium chloride-peptone solution pH 7.0 or

B. Microbiological Examination of Non- phosphate buffer solution pH 7.2 to make test suspensions.
sterile Products Use the suspensions within 2 h or within 24 h if stored at
2-8 °C.
1. Test for Specified Micro-organisms 1 3-1-2 Clostridia
(Ph. Bur. method 2.6.13) Use Clostridium sporogenes such as ATC C 1143 7 (NBRC
1 INTRODUCTION 14293, NCIMB 12343, CIP 100651) or ATCC 19404
The tests described hereafter will allow determination of the (NCTC 532 or CIP 79.03). Grow the clostridial test strain
absence or limited occurrence of specified micro-organisms under anaerobic conditions in reinforced medium for
that may be detected under the conditions described. clostridia at 30-35 °C for 24-48 h. As an alternative to
The tests are designed primarily to determine whether a preparing and then diluting down a fresh suspension of
substance or preparation complies with an established vegetative cells of C. sporogenes, a stable spore suspension is
specification for microbiological quality. When used for such used for test inoculation. The stable spore suspension may be
purposes, follow the instructions given below, including the maintained at 2-8 °C for a validated period.
number of samples to be taken, and interpret the results as 3-2 NEGATIVE CONTROL
stated below. To verify testing conditions, a negative control is performed
Alternative microbiological procedures, including automated using the chosen diluent in place of the test preparation.
methods, may be used, provided that their equivalence to the There must be no growth of micro-organisms. A negative
Pharmacopoeia method has been demonstrated. control is also performed when testing the products as
described in section 4. A failed negative control requires an
2 GENERAL PROCEDURES investigation.
The preparation of samples is carried out as described in
3-3 GROWTH PROMOTION AND INHIBITORY
general chapter 2. 6.12.
PROPERTIES OF THE MEDIA
If the product to be examined has antimicrobial activity, this Test each batch of ready-prepared medium and each batch
is insofar as possible removed or neutralised as described in of medium prepared either from dehydrated medium or from
general chapter 2. 6. 12. ingredients.
If surface-active substances are used for sample preparation, Verify suitable properties of relevant media as described in
their absence of toxicity for micro-organisms and their Table 2.6.13.-1.
compatibility with inactivators used must be demonstrated as
Test for growth promoting properties, liquid media
described in general chapter 2. 6.12.
Inoculate a portion of the appropriate medium with a small
3 GROWTH-PROMOTING AND INHIBITORY number (not more than 100 CFU) of the appropriate micro-
PROPERTIES OF THE MEDIA, SUITABILITY OF organism. Incubate at the specified temperature for not more
THE TEST AND NEGATIVE CONTROLS than the shortest period of time specified in the test. Clearly
The ability of the test to detect micro-organisms in the visible growth of the micro-organism comparable to that
presence of the product to be tested must be established. previously obtained with a previously tested and approved
Suitability must be confirmed if a change in testing batch of medium occurs.
performance, or the product, which may affect the outcome Test for growth promoting properties, solid media
of the test is introduced. Perform the surface-spread method, inoculating each plate
3-1 PREPARATION OF TEST STRAINS with a small number (not more than 100 CFU) of the
Use standardised stable suspensions of test strains or prepare appropriate micro-organism. Incubate at the specified
them as stated below. Seed lot culture maintenance temperature for not more than the shortest period of time
techniques (seed-lot systems) are used so that the viable specified in the test. Growth of the micro-organism
micro-organisms used for inoculation are not more than comparable to that previously obtained with a previously
5 passages removed from the original master seed-lot. tested and approved batch of medium occurs.
3-1-1 Aerobic micro-organisms Test for inhibitory properties, liquid or solid media
Grow each of the bacterial test strains separately in casein Inoculate the appropriate medium with at least 100 CFU of
soya bean digest broth or on casein soya bean digest agar at the appropriate micro-organism. Incubate at the specified
30-35 °C for 18-24 h. Grow the test strain for Candida temperature for not less than the longest period of time
albicans separately on Sabouraud-dextrose agar or in specified in the test. No growth of the test micro-organism
Sabouraud-dextrose broth at 20-25 °C for 2-3 days. occurs.
- Staphylococcus aureus such as ATCC 6538, NCIMB 9518, Test for indicative properties Perform the surface-spread
CIP 4.83 or NBRC 13276; method, inoculating each plate with a small number (not
- Pseudomonas aeruginosa such as ATCC 9027, more than 100 CFU) of the appropriate micro-organism.
NCIMB 8626, CIP 82.118 or NBRC 13275; Incubate at the specified temperature for a period of time
- Escherichia coli such as ATCC 8739, NCIMB 8545, within the range specified in the test. Colonies are
CIP 53.126 or NBRC 3972; comparable in appearance and indication reactions to those
- Salmonella enterica subsp. enterica serovar Typhimurium, previously obtained with a previously tested and approved
such as ATCC 14028 or, as an alternative, Salmonella batch of medium.
enterica subsp. enterica serovar Abony such as 3-4 SUITABILITY OF THE TEST METHOD
NBRC 100797, NCTC 6017 or CIP 80.39; For each product to be tested, perform the sample
- Candida albicans such as ATCC 10231, NCPF 3179, preparation as described in the relevant paragraph in section
IP 48. 72 or NBRC 1594. 4. Add each test strain at the time of mixing, in the
prescribed growth medium. Inoculate the test strains
1 This chapter has undergone phannacopoeial hannonisation. See chapter

5. 8 Pharmacopoeia/ hannonisation.
2023 Appendix XVI B V-A561

Table 2.6.13.-1 - Growth promoting, inhibitory and indicative properties of media

Medium Property Test strains

Test for bile-tolerant gram-negative Enterobactcria enrichment broth- Growth promoting E. coli
bacteria Mossel P. aeruginosa

Inhibitory S. aureus

Violet red bile glucose agar Growth promoting + indicative E.coli

P. aeruginosa

Test for Escherichia coli MacConkey broth Growth promoting E.coli

Inhibitory S. aureus

MacConkey agar Growth promoting + indicative E.coli

Test for Salmonella Rappaport Vassiliadis Salmonella Growth promoting Salmonella enterica subsp. enterica
enrichment broth serovar Typhimurium or Salmonella
enter£ca subsp. enterica serovar Abony

Inhibitory S. aureus

Xylose, lysine, deoxycholate agar Gro"th promoting + indicative Salmonella enteri.ca subsp. enterica
serovar Typhimurium or Salmonella
enrerica subsp. enrerica serovar Abony

Test for Pseudomonas aeruginosa Cetrimide agar Growth promoting P. aeruginosa

Inhibitory E. coli

Test for Staphylococcus aureus Mannitol salt agar Growth promoting + indicative S. aureus

Inhibitory E.coli

Test for clostridia Reinforced medium for clostridia Growth promoting C. sporogenes

Columbia agar Growth promoting C. sporogenes

Test for Candida albicans Sabouraud dextrose broth Growth promoting C. albicans

Sabouraud dextrose agar Growth promoting + indicative C. albicans

individually. Use a number of micro-organisms equivalent to The product complies with the test if there is no growth of
not more than 100 CFU in the inoculated test preparation. colonies.
Perform the test as described in the relevant paragraph in 4-1-3 Quantitative test
section 4 using the shortest incubation period prescribed. 4-1-3-1 Selection and subculture. Inoculate suitable
The specified micro-organisms must be detected with the quantities of enterobacteria enrichment broth-Mossel with
indication reactions as described in section 4. the preparation as described under 4-1-1 and/or dilutions of
Any antimicrobial activity of the product necessitates a it containing respectively 0.1 g, 0.01 g and 0.001 g (or
modification of the test procedure (see 4-5-3 of general 0.1 ml., 0.01 mL and 0.001 mL) of the product to be
chapter 2.6.12). examined. Incubate at 30-35 °C for 24-48 h. Subculture
each of the cultures on a plate of violet red bile glucose agar.
If for a given product the antimicrobial activity with respect
Incubate at 30-35 °C for 18-24 h.
to a micro-organism for which testing is prescribed cannot be
neutralised, then it is to be assumed that the inhibited micro- 4-1-3-2 Interpretation. Growth of colonies constitutes a
organism will not be present in the product. positive result. Note the smallest quantity of the product that
gives a positive result and the largest quantity that gives a
4 TESTING OF PRODUCTS negative result. Determine from Table 2.6.13.-2 the probable
4-1 BILE-TOLERANT GRAM-NEGATIVE BACTERIA number of bacteria.
4-1-1 Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than 1 g Table 2.6.13.-2 - Interpretation of results
of the product to be examined as described in general Results for each quantity of product Probable
chapter 2.6.12, but using casein soya bean digest broth as the number of
0.1 g or 0.01 g or 0.001 g or bacteria per
chosen diluent, mix and incubate at 20-25 °C for a time
0.1 mL 0.01 mL 0.001 mL gram or
sufficient to resuscitate the bacteria but not sufficient to millilitre of
encourage multiplication of the organisms (usually 2 h but product
not more than 5 h).
+ + + > 103
4-1-2 Test for absence
Unless otherwise prescribed, use the volume corresponding + + -
< 10 1 and
> 102
to 1 g of the product, as prepared in 4-1-1, to inoculate
enterobacteria enrichment broth-Mossel. Incubate at + - - < 102 and> 10
30-35 °C for 24-48 h. Subculture on plates of violet red bile - - -
< 10
glucose agar. Incubate at 30-35 °C for 18-24 h.
V-A562 Appendix XVI B 2023

4-2 ESCHERICHIA COLi The product complies with the test if colonies are not present
4-2-1 Sample preparation and pre-incubation or if the confirmatory identification tests are negative.
Prepare a sample using a 1 in 10 dilution of not less than 1 g 4-5 STAPHYLOCOCCUS AUREUS
of the product to be examined as described in general 4-5-1 Sample preparation and pre-incubation
chapter 2. 6.12, and use 10 mL or the quantity corresponding Prepare a sample using a 1 in 10 dilution of not less than 1 g
to 1 g or 1 mL to inoculate a suitable amount (determined as of the product to be examined as described in general
described under 3-4) of casein soya bean digest broth, mix chapter 2. 6.12, and use 10 mL or the quantity corresponding
and incubate at 30-35 °C for 18-24 h. to 1 g or 1 mL to inoculate a suitable amount (determined as
◊When testing orodispersible films, filter the volume of described under 3-4) of casein soya bean digest broth and
sample corresponding to 1 film of the preparation described mix. When testing transdermal patches ◊or orodispersible
under 4-5-1 in general chapter 2. 6.12 through a sterile filter films◊, filter the volume of sample corresponding to 1 patch
membrane and place in 100 mL of casein soya bean digest ◊or 1 film◊ of the preparation described under 4-5-1 in
broth. Incubate at 30-35 °C for 18-24 h.◊ general chapter 2. 6.12 through a sterile filter membrane and
4-2-2 Selection and subculture place in 100 mL of casein soya bean digest broth. Incubate
Shake the container, transfer 1 mL of casein soya bean digest at 30-35 °C for 18-24 h.
broth to 100 mL of MacConkey broth and incubate at 4-5-2 Selection and subculture
42-44 °C for 24-48 h. Subculture on a plate of MacConkey Subculture on a plate of mannitol salt agar and incubate at
agar at 30-35 °C for 18-72 h. 30-35 °C for 18-72 h.
4-2-3 Interpretation 4-5-3 Interpretation
Growth of colonies indicates the possible presence of E. coli. The possible presence of S. aureus is indicated by the growth
This is confirmed by identification tests. of yellow/white colonies surrounded by a yellow zone. This is
The product complies with the test if no colonies are present confirmed by identification tests.
or if the identification tests are negative. The product complies with the test if colonies of the types
4-3 SALMONELLA described are not present or if the confirmatory identification
tests are negative.
4-3-1 Sample preparation and pre-incubation
Prepare the product to be examined as described in general 4-6 CLOSTRIDIA
chapter 2. 6.12, and use the quantity corresponding to not 4-6-1 Sample preparation and heat treatment
less than 10 g or 10 mL to inoculate a suitable amount Prepare a sample using a 1 in 10 dilution (with a minimum
(determined as described under 3-4) of casein soya bean total volume of 20 mL) of not less than 2 g or 2 mL of the
digest broth, mix and incubate at 30-35 °C for 18-24 h. product to be examined as described in general chapter
4-3-2 Selection and subculture 2.6.12. Divide the sample into 2 portions ofat least 10 mL.
Transfer 0.1 mL of casein soya bean digest broth to 10 mL Heat 1 portion at 80 °C for 10 min and cool rapidly. Do not
of Rappaport Vassiliadis Salmonella enrichment broth and heat the other portion.
incubate at 30-35 °C for 18-24 h. Subculture on plates of 4-6-2 Selection and subculture
xylose, lysine, deoxycholate agar. Incubate at 30-35 °C for Use 10 mL or the quantity corresponding to 1 g or 1 mL of
18-48 h. the product to be examined of both portions to inoculate
4-3-3 Interpretation suitable amounts (determined as described under 3-4) of
The possible presence of Salmonella is indicated by the reinforced medium for clostridia. Incubate under anaerobic
growth of well-developed, red colonies, with or without black conditions at 30-35 °C for 48 h. After incubation, make
centres. This is confirmed by identification tests. subcultures from each container on Columbia agar and
The product complies with the test if colonies of the types incubate under anaerobic conditions at 30-35 °C for 48-72 h.
described are not present or if the confirmatory identification 4-6-3 Interpretation
tests are negative. The occurrence of anaerobic growth of rods (with or without
4-4 PSEUDOMONAS AERUGINOSA endospores) giving a negative catalase reaction indicates the
presence of clostridia. This is confirmed by identification
4-4-1 Sample preparation and pre-incubation tests.
Prepare a sample using a 1 in 10 dilution of not less than 1 g
The product complies with the test if colonies of the types
of the product to be examined as described in general
described are not present or if the confirmatory identification
chapter 2. 6.12, and use 10 mL or the quantity corresponding
tests are negative.
to 1 g or 1 mL to inoculate a suitable amount (determined as
described under 3-4) of casein soya bean digest broth and 4-7 CANDIDA ALBICANS
mix. When testing transdermal patches ◊or orodispersible 4-7-1 Sample preparation and pre-incubation
films◊, filter the volume of sample corresponding to 1 patch Prepare the product to be examined as described in general
◊or 1 film◊ of the preparation described under 4-5-1 in chapter 2. 6.12, and use 10 mL or the quantity corresponding
general chapter 2. 6.12 through a sterile filter membrane and to not less than 1 g or 1 mL to inoculate 100 mL of
place in 100 mL of casein soya bean digest broth. Incubate Sabouraud-dextrose broth and mix. Incubate at 30-35 °C for
at 30-35 °C for 18-24 h. 3-5 days.
4-4-2 Selection and subculture 4-7-2 Selection and subculture
Subculture on a plate of cetrimide agar and incubate at Subculture on a plate of Sabouraud-dextrose agar and
30-35 °C for 18-72 h. incubate at 30-35 °C for 24-48 h.
4-4-3 Interpretation 4-7-3 Interpretation
Growth of colonies indicates the possible presence of P. Growth of white colonies may indicate the presence of C.
aeruginosa. This is confirmed by identification tests. albicans. This is confirmed by identification tests.
 2023 Appendix XVI B V-A563

The product complies with the test if such colonies are not Sabouraud-dextrose broth
present or if the confirmatory identification tests are negative.
Dextrose 20.0 g
The following section is given for information. Mixture of peptic digest of animal tissue and pancreatic 10.0 g
digest of casein (I: 1)
5 RECOMMENDED SOLUTIONS AND CULTURE
Purified water IOOOmL
MEDIA
The following solutions and culture media have been found Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at
to be satisfactory for the purposes for which they are 25 °C. Sterilise in an autoclave using a validated cycle.
prescribed in the test for microbial contamination in the
Pharmacopoeia. Other media may be used provided that Enterobacteria enrichment broth-Mossel
their suitability can be demonstrated. Pancreatic digest of gelatin 10.0 g
Stock buffer solution Place 34 g of potassium dihydrogen Glucose monohydrate 5.0 g
Dehydrated ox bile 20.0 g
phosphate in a 1000 mL volumetric flask, dissolve in 500 mL
Potassium dihydrogen phosphate 2.0 g
of purified water, adjust to pH 7 .2 ± 0.2 with sodium Disodium hydrogen phosphate dihydrate 8.0 g
hydroxide, dilute to 1000.0 mL with purified water and mix. Brilliant green 15 mg
Dispense into containers and sterilise. Store at 2-8 °C. Purified water IOOOmL

Phosphate buffer solution pH 7.2 Prepare a mixture of Adjust the pH so that after heating it is 7.2 ± 0.2 at 25 °C.
stock buffer solution and purified water (1:800 V/V) and Heat at 100 °C for 30 min and cool immediately.
sterilise.
Violet red bile glucose agar
Buffered sodium chloride-peptone solution pH 7 .0
Yeast extract 3.0 g
Potassium dihydrogen phosphate 3.6 g Pancreatic digest of gelatin 7.0 g
Disodium hydrogen phosphate 7.2 g, equivalent to 0.067 M Bile salts 1.5 g
dihydrate phosphate Sodium chloride 5.0 g
Sodium chloride 4.3 g Glucose monohydrate 10.0 g
Peptone (meat or casein) 1.0 g Agar 15.0 g
Purified water IOOOmL Neutral red 30 mg
Crystal violet 2 mg
Sterilise in an autoclave using a validated cycle. Purified water IOOOmL

Casein soya bean digest broth Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C.
Pancreatic digest of casein 17.0 g
Heat to boiling; do not heat in an autoclave.
Papaic digest of soya bean 3.0 g
Sodium chloride 5.0 g
MacConkey broth
Dipotassium hydrogen phosphate 2.5 g
Pancreatic digest of gelatin 20.0 g
Glucose monohydrate 2.5 g
Lactose monohydrate 10.0 g
Purified water 1000 mL
Dehydrated ox bile 5.0 g
Bromocresol purple 10mg
Adjust the pH so that after sterilisation it is 7 .3 ± 0.2 at Purified water IOOOmL
25 °C. Sterilise in an autoclave using a validated cycle.
Adjust the pH so that after sterilisation it is 7 .3 ± 0.2 at
Casein soya bean digest agar
25 °C. Sterilise in an autoclave using a validated cycle.
Pancreatic digest of casein 15.0 g
Papaic digest of soya bean 5.0 g
MacConkey agar
Sodium chloride 5.0 g
Pancreatic digest of gelatin 17.0 g
Agar 15.0 g
Peprones (meat and casein) 3.0 g
Purified water IOOOmL
Lactose monohydrate 10.0 g
Sodium chloride 5.0 g
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at Bile salts 1.5 g
25 °C. Sterilise in an autoclave using a validated cycle. Agar 13.5 g
Neutral red 30.0 mg
Sabouraud-dextrose agar Crystal violet 1 mg
Purified water IOOOmL
Dextrose 40.0 g
Mixture of peptic digest of animal tissue and pancreatic 10.0 g Adjust the pH so that after sterilisation it is 7 .1 ± 0.2 at
digest of casein (I: 1)
Agar 15.0 g
25 °C. Boil for 1 min with constant shaking then sterilise in
Purified water IOOOmL an autoclave using a validated cycle.
Rappaport Vassiliadis Salmonella enrichment broth
Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at
25 °C. Sterilise in an autoclave using a validated cycle. Soya peptone 4.5 g
Magnesium chloride hexahydrate 29.0 g
Potato dextrose agar Sodium chloride 8.0 g
Dipotassium phosphate 0.4 g
Infusion from potatoes 200 g Potassium dihydrogen phosphate 0.6 g
Dextrose 20.0 g Malachite green 0.036 g
Agar 15.0 g Purified water 1000 mL
Purified water 1000 mL

Dissolve, warming gently. Sterilise in an autoclave using a

Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at validated cycle, at a temperature not exceeding 115 °C.
25 °C. Sterilise in an autoclave using a validated cycle. The pH is to be 5.2 ± 0.2 at 25 °C after heating and
autoclaving.
V-A564 Appendix XVI B 2023

Xylose, lysine, deoxycholate agar Hydrate the agar, dissolve by heating to boiling with
continuous stirring. If necessary, adjust the pH so that after
Xylose 3.5 g
L-Lysine 5.0 g
sterilisation it is 7 .3 ± 0.2 at 25 °C. Sterilise in an autoclave
Lactose monohydrate 7.5 g using a validated cycle. Allow to cool to 45-50 °C; add,
Sucrose 7.5 g where necessary, gentamicin sulfate corresponding to 20 mg
Sodium chloride 5.0 g of gentamicin base and pour into Petri dishes.
Yeast extract 3.0 g
Phenol red 80 mg 2. Microbial Enumeration Tests2
Agar 13.5 g
Sodium deoxycholate 2.5 g
(Ph. Bur. method 2. 6.12)
Sodium thiosulfate 6.8 g 1 INTRODUCTION
Ferric ammonium citrate 0.8 g
Purified water IO00mL
The tests described hereafter will allow quantitative
enumeration of mesophilic bacteria and fungi that may grow
Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. under aerobic conditions.
Heat to boiling, cool to 50 °C and pour into Petri dishes. The tests are designed primarily to determine whether a
Do not heat in an autoclave. substance or preparation complies with an established
specification for microbiological quality. When used for such
Cetrimide agar purposes follow the instructions given below, including the
Pancreatic digest of gelatin 20.0 g number of samples to be taken, and interpret the results as
Magnesium chloride 1.4 g stated below.
Dipotassium sulfate 10.0 g
Cetrimide 0.3 g
The methods are not applicable to products containing viable
Agar 13.6 g micro-organisms as active ingredients.
Purified water IO00mL Alternative microbiological procedures, including automated
Glycerol 10.0 mL
methods, may be used, provided that their equivalence to the
Pharmacopoeia method has been demonstrated.
Heat to boiling for l min with shaking. Adjust the pH so that
after sterilisation it is 7 .2 ± 0.2 at 25 °C. Sterilise in an 2 GENERAL PROCEDURES
autoclave using a validated cycle. Carry out the determination under conditions designed to
avoid extrinsic microbial contamination of the product to be
Mannitol salt agar examined. The precautions taken to avoid contamination
Pancreatic digest of casein 5.0 g must be such that they do not affect any micro-organisms
Peptic digest of animal tissue 5.0 g that are to be revealed in the test.
Beef extract 1.0 g
If the product to be examined has antimicrobial activity, this
D-Mannitol 10.0 g
Sodium chloride 75.0 g is insofar as possible removed or neutralised. If inactivators
Agar 15.0 g are used for this purpose, their efficacy and their absence of
Phenol red 0.025 g toxicity for micro-organisms must be demonstrated.
Purified water 1000 mL
If surface-active substances are used for sample preparation,
Heat to boiling for 1 min with shaking. Adjust the pH so that their absence of toxicity for micro-organisms and their
after sterilisation it is 7 .4 ± 0.2 at 25 °C. Sterilise in an compatibility with inactivators used must be demonstrated.
autoclave using a validated cycle. 3 ENUMERATION METHODS
Use the membrane filtration method or the plate-count
Reinforced medium for clostridia
methods, as prescribed. The most-probable-number (MPN)
Beef extract 10.0 g method is generally the least accurate method for microbial
Peptone 10.0 g counts, however, for certain product groups with a very low
Yeast extract 3.0 g bioburden, it may be the most appropriate method.
Soluble starch 1.0 g
Glucose monohydrate 5.0 g The choice of method is based on factors such as the nature
Cysteine hydrochloride 0.5 g of the product and the required limit of micro-organisms.
Sodium chloride 5.0 g The chosen method must allow testing of a sufficient sample
Sodium acetate 3.0 g
Agar 0.5 g
size to judge compliance with the specification.
Purified water 1000 mL The suitability of the method chosen must be established.
4 GROWTH PROMOTION TEST, SUITABILITY OF
Hydrate the agar, dissolve by heating to boiling with THE COUNTING METHOD AND NEGATIVE
continuous stirring. If necessary, adjust the pH so that after CONTROLS
sterilisation it is 6.8 ± 0.2 at 25 °C. Sterilise in an autoclave 4-1 GENERAL CONSIDERATIONS
using a validated cycle. The ability of the test to detect micro-organisms in the
Columbia agar presence of product to be tested must be established.
Suitability must be confirmed if a change in testing
Pancreatic digest of casein 10.0 g
Meat peptic digest 5.0 g
performance, or the product, which may affect the outcome
Heart pancreatic digest 3.0 g of the test is introduced.
Yeast extract 5.0 g 4-2 PREPARATION OF TEST STRAINS
Maize starch 1.0 g
Sodium chloride 5.0 g
Use standardised stable suspensions of test strains or prepare
Agar, according to gelling power 10.0-15.0 g them as stated below. Seed lot culture maintenance
Purified water 1000 mL

2 This chapter has undergone pharmacopoeia/ harmonisation. See chapter

5. 8 Pharmacopoeia/ harmonisation.
2023 Appendix XVI B V-A565

Table 2.6.12.-1. - Preparation and use of test micro-organisms

Micro-organism Preparation of test Growth promotion Suitability of counting method in the presence
strain of the product

Total aerobic Total yeasts and Total aerobic Total yeasts and
microbial count moulds count microbial count moulds count

Staphylococcus aureus Casein soya bean digest Casein soya bean digest Casein soya bean digest
such as: agar or casein soya bean agar and casein soya agar/MPN casein soya
ATCC 6538 digest broth bean digest broth beao digest broth
30-35 'C
- S 100 CFU
-
NCIMB 9518 S 100 CFU
CIP 4.83 18-24 h 30-35 'C 30-3, 'C
NBRC 13276 S 3 days S 3 days

Pseudomonas aernginosa Casein soya bean digest Casein soya beao digest Casein soya bean digest
such as: agar or casein soya bean agar and casein soya agar/MPN casein soya
ATCC 9027 digest broth bean digest broth beao digest broth
- -
NCIMB 8626 30-35 ·c S 100 CFU S 100 CFU
CIP 82.118 18-24 h 30-35 'C 30-35 "C
NBRC 13275 S 3 days S 3 days

Bacillus subtilis such as: Casein soya bean digest Casein soya beao digest Casein soya bean digest
ATCC 6633 agar or casein soya bean agar and casein soya agar/MPN casein soya
NCIMB 8054 digest broth bean digest broth beao digest broth
CIP 52.62 30-35 ·c S 100 CFU
-
S 100 CFU
-
NBRC 3134 18-24 h 30-35 ·c 30-35 °C
S 3 days S 3 days

Candida albicans Sabouraud-dextrose Casein soya bean digest Sabouraud-dextrose Casein soya bean digest Sabouraud-dextrose agar
such as: agar or Sabouraud- agar agar agar S JOO CFU
ATCC 10231 dextrose broth 20-25 -c S 100 CFU S 100 CFU S 100 CFU 20-25 ·c
NCPF 3179 2-3 days 30-35 'C 20-25 ·c 30-35 ·c S 5 days
IP 48.72 S 5 days S 5 days S 5 days
NBRC 1594 MPN: not applicable

Aspergillus brasiliensis Sabouraud-dextrose Casein soya beao digest Sabouraud-dextrose Casein soya bean digest Sabouraud-dextrose agar
such as: agar or potato-dextrose agar agar agar S 100 CFU
ATCC 16404 agar S 100 CFU S 100 CFU S 100 CFU 20-25 ·c
IMI 149007 20-25 'C 30-35 "C 20-25 ·c 30-35 "C S 5 days
IP 1431.83 5-7 days, or until good S 5 days S 5 days S 5 days
NBRC 9455 sporulation is achieved MPN: not applicable

techniques (seed-lot systems) are used so that the viable Table 2.6.12.-1, using a separate portion/plate of medium for
micro-organisms used for inoculation are not more than each. Inoculate plates of Sabouraud-dextrose agar with a
5 passages removed from the original master seed-lot. Grow small number (not more than 100 CFU) of the micro-
each of the bacterial and fungal test strains separately as organisms indicated in Table 2.6.12.-1, using a separate plate
described in Table 2.6.12.-1. of medium for each. Incubate in the conditions described in
Use buffered sodium chloride-peptone solution pH 7 .0 or Table 2.6.12.-1.
phosphate buffer solution pH 7.2 to make test suspensions; For solid media, growth obtained must not differ by a factor
to suspend A. brasiliensis spores, 0.05 per cent of greater than 2 from the calculated value for a standardised
polysorbate 80 may be added to the buffer. Use the inoculum. For a freshly prepared inoculum, growth of the
suspensions within 2 h or within 24 h if stored at 2-8 °C. micro-organisms comparable to that previously obtained with
As an alternative to preparing and then diluting a fresh a previously tested and approved batch of medium occurs.
suspension of vegetative cells of A. brasiliensis or B. subtilis, a Liquid media are suitable if clearly visible growth of the
stable spore suspension is prepared and then an appropriate micro-organisms comparable to that previously obtained with
volume of the spore suspension is used for test inoculation. a previously tested and approved batch of medium occurs.
The stable spore suspension may be maintained at 2-8 cc for 4-5 SUITABILI1Y OF THE COUNTING METHOD IN
a validated period of time. THE PRESENCE OF PRODUCT
4-3 NEGATIVE CONTROL 4-5-1 Preparation of the sample
To verify testing conditions, a negative control is performed The method for sample preparation depends upon the
using the chosen diluent in place of the test preparation. physical characteristics of the product to be tested. If none of
There must be no growth of micro-organisms. A negative the procedures described below can be demonstrated to be
control is also performed when testing the products as satisfactory, an alternative procedure must be developed.
described in section 5. A failed negative control requires an
Water-soluble products Dissolve or dilute (usually a I in
investigation.
10 dilution is prepared) the product to be examined in
4-4 GROWTH PROMOTION OF THE MEDIA buffered sodium chloride-peptone solution pH 7 .0,
Test each batch of ready-prepared medium and each batch phosphate buffer solution pH 7.2 or casein soya bean digest
of medium, prepared either from dehydrated medium or broth. If necessary, adjust to pH 6-8. Further dilutions,
from the ingredients described. where necessary, are prepared with the same diluent.
Inoculate portions/plates of casein soya bean digest broth and Non-fatty products insoluble in water Suspend the
casein soya bean digest agar with a small number (not more product to be examined (usually a 1 in 10 dilution is
than 100 CFU) of the micro-organisms indicated in prepared) in buffered sodium chloride-peptone solution
V-A566 Appendix XVI B 2023

pH 7 .0, phosphate buffer solution pH 7 .2 or casein soya procedure may include, for example, ( 1) an increase in the
bean digest broth. A surface-active agent such as 1 g/L of volume of the diluent or culture medium, (2) incorporation
polysorbate 80 may be added to assist the suspension of of specific or general neutralising agents into the diluent, (3)
poorly wenable substances. If necessary, adjust to pH 6-8. membrane filtration, or (4) a combination of the above
Further dilutions, where necessary, are prepared with the measures.
same diluent. Neutralising agents Neutralising agents may be used to
Fatty products Dissolve in isopropyl myristate, sterilised neutralise the activity of antimicrobial agents
by filtration or mix the product to be examined with the (Table 2.6.12.-2). They may be added to the chosen diluent
minimum necessary quantity of sterile polysorbate 80 or or the medium preferably before sterilisation. If used, their
another non-inhibitory sterile surface-active agent, heated if efficacy and their absence of toxicity for micro-organisms
necessary to not more than 40 °C, or in exceptional cases to must be demonstrated by carrying out a blank with
not more than 45 °C. Mix carefully and if necessary maintain neutraliser and without product.
the temperature in a water-bath. Add sufficient of the pre- If no suitable neutralising method can be found, it can be
warmed chosen diluent to make a 1 in 10 dilution of the assumed that the failure to isolate the inoculated organism is
original product. Mix carefully whilst maintaining the attributable to the microbicidal activity of the product. This
temperature for the shortest time necessary for the formation information serves to indicate that the product is not likely to
of an emulsion. Further serial tenfold dilutions may be be contaminated with the given species of the micro-
prepared using the chosen diluent containing a suitable organism. However, it is possible that the product only
concentration of sterile polysorbate 80 or another non- inhibits some of the micro-organisms specified herein, but
inhibitory sterile surface-active agent. does not inhibit others not included amongst the test strains
Fluids or solids in aerosol form Aseptically transfer the or for which the latter are not representative. Then, perform
product into a membrane filter apparatus or a sterile the test with the highest dilution factor compatible with
container for further sampling. Use either the total contents microbial growth and the specific acceptance criterion.
or a defined number of metered doses from each of the
containers tested. Table 2.6.12.-2. - Common neutralising agents for inteifering
Transdermal patches Remove the protective cover sheets substances
('release liners') of the transdermal patches and place them, Interfering substance Potential neutralising
adhesive side upwards, on sterile glass or plastic trays. Cover method
the adhesive surface with a sterile porous material, for Glutaraldehyde, mercurials Sodium hydrogensulfite
example sterile gauze, to prevent the patches from sticking (sodium bisulfite)
together, and transfer the patches to a suitable volume of the
Phenolics, alcohol, aldehydes, sorbate Dilution
chosen diluent containing inactivators such as polysorbate 80
and/or lecithin. Shake the preparation vigorously for at least Aldehydes Glycine
30 min.
Quaternary Anunonium Compounds Lecithin
◊Orodispersible films. Dissolve 10 films to be examined in
(QACs), parahydroxybenzoates (parabens),
buffered sodium chloride-peptone solution pH 7.0, bis-biguanides
phosphate buffer solution pH 7 .2 or casein soya bean digest
QACs, iodine, parabens Polysorbate
broth. It may be necessary to heat the preparation to not
more than 40 °C, or in exceptional cases to not more than Mercurials Thioglycollate
45 °C, with or without shaking, to achieve dissolution.
If necessary, adjust to pH 6-8. Further dilutions, where Mercurials, halogens, aldehydes Thiosulfate

necessary, are prepared with the same diluent.◊ EDTA (edetate) Mg2 + or Ca 2 1- ions
4-5-2 Inoculation and dilution
Add to the sample prepared as described above (4-5-1) and
4-5-4 Recovery of micro-organism in the presence of
to a control (with no test material included) a sufficient
product
volume of the microbial suspension to obtain an inoculum of
For each of the micro-organisms listed, separate tests are
not more than 100 CFU. The volume of the suspension of
performed. Only micro-organisms of the added test strain are
the inoculum should not exceed 1 per cent of the volume of
counted.
diluted product.
4-5-4-1 Membrane filtration. Use membrane filters having a
To demonstrate acceptable microbial recovery from the
nominal pore size not greater than 0.45 µm. The type of
product, the lowest possible dilution factor of the prepared
filter material is chosen such that the bacteria-retaining
sample must be used for the test. Where this is not possible
efficiency is not affected by the components of the sample to
due to antimicrobial activity or poor solubility, further
be investigated. For each of the micro-organisms listed, one
appropriate protocols must be developed. If inhibition of
membrane filter is used.
growth by the sample cannot otherwise be avoided, the
aliquot of the microbial suspension may be added after Transfer a suitable amount of the sample prepared as
neutralisation, dilution or filtration. described under 4-5-1 to 4-5-3 (preferably representing 1 g
of the product, or less if large numbers of CFU are expected)
4-5-3 Neutralisation/removal of antimicrobial activity to the membrane filter, filter immediately and rinse the
The number of micro-organisms recovered from the prepared membrane filter with an appropriate volume of diluent.
sample diluted as described in 4-5-2 and incubated following
For the determination of total aerobic microbial count
the procedure described in 4-5-4, is compared to the number
(TAMC), transfer the membrane filter to the surface of
of micro-organisms recovered from the control preparation.
casein soya bean digest agar. For the determination of total
If growth is inhibited (reduction by a factor greater than 2), combined yeasts/moulds count (TYMC), transfer the
then modify the procedure for the particular enumeration test membrane to the surface of Sabouraud-dextrose agar.
to ensure the validity of the results. Modification of the
2023 Appendix XVI B V-A567

Incubate the plates as indicated in Table 2.6.12.-1. Perform Table 2.6.12.-3. - Most-probable-number values of micro-
the counting. organisms
4-5-4-2 Plate-count methods. Perform plate-count methods Observed combinations of numbers of tubes
at least in duplicate for each medium and use the mean showing growth in each set
MPNper
95 per cent
count of the result. Number of grams or millilitres of product per
gram or per
confidence
millilitre of
4-5-4-2-1 Pour-plate method tube limits
product
For Petri dishes 9 cm in diameter, add to the dish 1 mL of 0.1 0.01 0.001
the sample prepared as described under 4-5-1 to 4-5-3 and 0-9.4
0 0 0 <3
15-20 mL of casein soya bean digest agar or Sabouraud-
dextrose agar, both media being at not more than 45 °C. 0 0 1 3 0.1-9.5

If larger Petri dishes are used, the amount of agar medium is 0 1 0 3 0.1-10
increased accordingly. For each of the micro-organisms listed
0 1 1 6.1 1.2-17
in Table 2.6.12.-1, at least 2 Petri dishes are used. Incubate
the plates as indicated in Table 2.6.12.-1. Take the 0 2 0 6.2 1.2-17
arithmetic mean of the counts per medium and calculate the
0 3 0 9.4 3.5-35
number of CFU in the original inoculum.
4-5-4-2-2 Surface-spread method 1 0 0 3.6 0.2-17

For Petri dishes 9 cm in diameter, add 15-20 mL of casein 1 0 1 7.2 1.2-17

soya bean digest agar or Sabouraud-dextrose agar at about
1 0 2 11 4-35
45 °C to each Petri dish and allow to solidify. If larger Petri
dishes are used, the volume of the agar is increased 1 1 0 7.4 1.3-20

accordingly. Dry the plates, for example in a laminar-air-flow 1 1 1 11 4-35

cabinet or an incubator. For each of the micro-organisms
1 2 0 11 4-35
listed in Table 2.6.12.-1, at least 2 Petri dishes are used.
Spread a measured volume of not less than 0.1 mL of the l 2 1 15 5-38
sample prepared as described under 4-5-1 to 4-5-3 over the
1 3 0 16 5-38
surface of the medium. Incubate and count as prescribed
under 4-5-4-2-1 2 0 0 9.2 1.5-35

4-5-4-3 Most-probable-number (MPN) method. 2 0 1 14 4-35

The precision and accuracy of the MPN method is less than
2 0 2 20 5-38
that of the membrane filtration method or the plate-count
method. Unreliable results are obtained particularly for the 2 1 0 15 4-38
enumeration of moulds. For these reasons the MPN method
2 1 1 20 5-38
is reserved for the enumeration ofTAMC in situations where
no other method is available. If the use of the method is 2 1 2 27 9-94
justified, proceed as follows. 2 2 0 21 5-40
Prepare a series of at least 3 serial tenfold dilutions of the
2 2 l 28 9-94
product as described under 4-5-1 to 4-5-3. From each level
of dilution, 3 aliquots of 1 g or 1 mL are used to inoculate 3 2 2 2 35 9-94
tubes with 9-10 mL of casein soya bean digest broth.
2 3 0 29 9-94
If necessary, a surface-active agent such as polysorbate 80 or
an inactivator of antimicrobial agents may be added to the 2 3 l 36 9-94

medium. Thus, if 3 levels of dilution are prepared, 9 tubes 3 0 0 23 5-94

are inoculated.
3 0 1 38 9-104
Incubate all tubes at 30-35 °C for not more than 3 days.
If reading of the results is difficult or uncertain owing to the 3 0 2 64 16-181
nature of the product to be examined, subculture in the same 1 0 43 9-181
3
broth, or in casein soya bean digest agar, for 1-2 days at the
same temperature and use these results. Determine the most 3 1 1 75 17-199

probable number of micro-organisms per gram or millilitre of 3 1 2 120 30-360

the product to be examined from Table 2.6.12.-3.
3 1 3 160 30-380
4-6 RESULTS AND INTERPRETATION
When verifying the suitability of the membrane filtration 3 2 0 93 18-360

method or the plate-count method, a mean count of any of 3 2 1 150 30-380

the test organisms not differing by a factor greater than 2
3 2 2 210 30-400
from the value of the control defined in 4-5-2 in the absence
of the product must be obtained. When verifying the 3 2 3 290 90-990
suitability of the MPN method the calculated value from the
3 3 0 240 40-990
inoculum must be within 95 per cent confidence limits of the
results obtained with the control. 3 3 1 460 90-1980

If the above criteria cannot be met for one or more of the 3 3 2 1100 200-4000
organisms tested with any of the described methods, the
3 3 3 > 1100
method and test conditions that come closest to the criteria
are used to test the product.
V-A568 Appendix XVI B 2023

5 TESTING OF PRODUCTS Prepare the sample using a method that has been shown to
5-1 AMOUNT USED FOR THE TEST be suitable as described in section 4. Prepare at least 2 Petri
Unless otherwise prescribed, use 10 g or 10 mL of the dishes for each medium and each level of dilution.
product to be examined taken with the precautions referred For incubation and calculation of the number of CFU
to above. For fluids or solids in aerosol form, sample 10 proceed as described for the pour-plate method.
containers. For transdermal patches, sample 10 patches. ◊For 5-2-3 Most-probable-number method
orodispersible films, sample 10 films.◊ Prepare and dilute the sample using a method that has been
The amount to be tested may be reduced for active shown to be suitable as described in section 4. Incubate all
substances that will be formulated in the following tubes at 30-35 °C for 3-5 days. Subculture if necessary, using
conditions: the amount per dosage unit (e.g. tablet, capsule, the procedure shown to be suitable. Record for each level of
injection) is less than or equal to 1 mg or the amount per dilution the number of tubes showing microbial growth.
gram or millilitre (for preparations not presented in Determine the most probable number of micro-organisms
dose units) is less than 1 mg. In these cases, the amount to per gram or millilitre of the product to be examined from
be tested is not less than the amount present in 10 Table 2.6.12.-3.
dosage units or 10 g or 10 mL of the product. 5-3 INTERPRETATION OF THE RESULTS
For materials used as active substances where sample The total aerobic microbial count (TAMC) is considered to
quantity is limited or batch size is extremely small (i.e. less be equal to the number of CFU found using casein soya
than 1000 mL or 1000 g), the amount tested shall be bean digest agar; if colonies of fungi are detected on this
1 per cent of the batch unless a lesser amount is prescribed medium, they are counted as part of the TAMC. The total
or justified and authorised. combined yeasts/mould count (TYMC) is considered to be
For products where the total number of entities in a batch is equal to the number of CFU found using Sabouraud-
less than 200 (e.g. samples used in clinical trials), the sample dextrose agar; if colonies of bacteria are detected on this
size may be reduced to 2 units, or 1 unit if the size is less medium, they are counted as part of the TYMC. When the
than 100. TYMC is expected to exceed the acceptance criterion due to
Select the sample(s) at random from the bulk material or the bacterial growth, Sabouraud-dextrose agar containing
from the available containers of the preparation. To obtain antibiotics may be used. If the count is carried out by the
the required quantity, mix the contents of a sufficient MPN method the calculated value is the T AMC.
number of containers to provide the sample. When an acceptance criterion for microbiological quality is
prescribed it is interpreted as follows:
5-2 EXAMINATION OF THE PRODUCT
10 1 CFU: maximum acceptable count= 20;
5-2-1 Membrane filtration - 102 CFU: maximum acceptable count = 200;
Use a filtration apparatus designed to allow the transfer of - 10 3 CFU: maximum acceptable count = 2000, and so
the filter to the medium. Prepare the sample using a method forth.
that has been shown suitable as described in section 4 and
The recommended solutions and media are described in
transfer the appropriate amount to each of 2 membrane
general chapter 2.6.13.
filters and filter immediately. Wash each filter following the
procedure shown to be suitable. 3. Test for Absence of Mycoplasmas
For the determination ofTAMC, transfer one of the (Ph. Bur. metlwd 2. 6. 7 as applied to vaccines for human use)
membrane filters to the surface of casein soya bean digest Where the test for mycoplasmas is prescribed for a master
agar. For the determination of TYMC, transfer the other cell bank, for a working cell bank, for a virus seed lot or for
membrane to the surface of Sabouraud-dextrose agar. control cells, both the culture method and the indicator cell
Incubate the plate of casein soya bean digest agar at culture method are used. Where the test for mycoplasmas is
30-35 °C for 3-5 days and the plate of Sabouraud-dextrose prescribed for a virus harvest, for a bulk vaccine or for the
agar at 20-25 °C for 5-7 days. Calculate the number of CFU final lot (batch), the culture method is used. The indicator
per gram or per millilitre of product. cell culture method may also be used, where necessary, for
When examining transdermal patches ◊or orodispersible screening of media.
films◊, filter 10 per cent of the volume of the preparation Nucleic acid amplification techniques (NAT) may be used as
described under 4-5-1 separately through each of 2 sterile an alternative to one or both of the other methods after
filter membranes. Transfer one membrane to casein soya suitable validation.
bean digest agar for T AMC and the other membrane to
Sabouraud-dextrose agar for TYMC.
CULTURE METHOD
CHOICE OF CULTURE MEDIA
5-2-2 Plate-count methods The test is carried out using a sufficient number of both solid
5-2-2-1 Pour-plate method and liquid media to ensure growth in the chosen incubation
Prepare the sample using a method that has been shown to conditions of small numbers of mycoplasmas that may be
be suitable as described in section 4. Prepare for each present in the product to be examined. Liquid media must
medium at least 2 Petri dishes for each level of dilution. contain phenol red. The range of media chosen is shown to
Incubate the plates of casein soya bean digest agar at have satisfactory nutritive properties for at least the micro-
30-35 °C for 3-5 days and the plates of Sabouraud-dextrose organisms shown below. The nutritive properties of each new
agar at 20-25 °C for 5-7 days. Select the plates batch of medium are verified for the appropriate micro-
corresponding to a given dilution and showing the highest organisms in the list. When testing for mycoplasmas in the
number of colonies less than 250 for T AMC and 50 for product to be examined, at least 1 of the following species
TYMC. Take the arithmetic mean per culture medium of will be included as a positive control:
the counts and calculate the number of CFU per gram or per - Acholeplasma laidlawii (vaccines for human and veterinary
millilitre of product. use where an antibiotic has been used during production);
5-2-2-2 Surface-spread method
2023 Appendix XVI B V-A569

- Mycoplasma gallisepticum (where avian material has been neutralised or their effect otherwise countered, for example
used during production or where the vaccine is intended by passage in substrates not containing inhibitors or dilution
for use in poultry); in a larger volume of medium before the test. If dilution is
- Mycoplasma hyorhinis (non-avian veterinary vaccines); used, larger medium volumes may be used or the inoculum
- Mycoplasma orale (vaccines for human and veterinary use); volume may be divided among several 100 mL flasks.
- Mycoplasma pneumoniae (vaccines for human use) or other The effectiveness of the neutralisation or other process is
suitable species of D-glucose fermenter such as checked by repeating the test for inhibitory substances after
Mycoplasma fermentans; neutralisation.
- 1\1.ycoplasma synoviae (where avian material has been used TEST FOR MYCOPIASMAS IN THE PRODUCT TO
during production or where the vaccine is intended for BE EXAMINED
use in poultry). Inoculate 10 mL of the product to be examined per 100 mL
The test strains are field isolates having undergone a limited of each liquid medium. If it has been found that a significant
number of subcultures (not more than 15), and are stored pH change occurs upon the addition of the product to be
frozen or freeze-dried. After cloning, the strains are identified examined, the liquid medium is restored to its original pH
as being of the required species by comparison with type value by the addition of a solution of either sodium
cultures, for example: hydroxide or hydrochloric acid. Inoculate 0.2 mL of the
product to be examined on each plate of each solid medium.
A. laidlawii NCTC 10116 CIP 75.27 ATCC 23206 Incubate liquid media for 20-21 days. Incubate solid media
M. gal/isepticum NCTC 10115 CIP 104967 ATCC 19610
for not less than 14 days, except those corresponding to the
M. fermentans NCTC10117 CIP 105680 ATCC 19989
M. hyorhinis NCTC10130 CIP 104968 ATCC 17981 20-21 day subculture, which are incubated for 7 days. At the
M. orale NCTC 10112 CIP 104969 ATCC 23714 same time incubate an uninoculated 100 mL portion of each
M. pneumoniae NCTC 10119 CIP 103766 ATCC 15531 liquid medium and agar plates, as a negative control.
M. synoviae NCTC 10124 CIP 104970 ATCC 25204 On days 2-4 after inoculation, subculture each liquid
medium by inoculating 0.2 mL on at least 1 plate of each
Acholeplasma laidlawii ERP, Mycoplasma fermentans ERP, solid medium. Repeat the procedure between the 6th and
Mycoplasma hyorhinis ERP, Mycoplasma orate ERP and 8th days, again between the 13th and 15th days and again
Mycoplasma synoviae ERP are suitable for use as low-passage between the 19th and 21 s t days of the test. Observe the liquid
reference strains. media every 2 or 3 days and if a colour change occurs,
INCUBATION CONDITIONS subculture. If a liquid medium shows bacterial or fungal
Incubate liquid media in tightly stoppered containers at contamination, the test is invalid. The test is valid if at least
35-38 "C. Incubate solid media in microaerophilic conditions 1 plate per medium and per inoculation day can be read.
(nitrogen containing 5-10 per cent of carbon dioxide and Include in the test positive controls prepared by inoculation
sufficient humidity to prevent desiccation of the agar surface) of not more than 100 CFU of at least 1 test micro-organism
at 35-38 °C. on agar medium or into broth medium. Where the test for
NUTRITIVE PROPERTIES mycoplasmas is carried out regularly and where possible, it is
Carry out the test for nutritive properties for each new recommended to use the test micro-organisms in regular
batch of medium Inoculate the chosen media with the rotation. The test micro-organisms used are those listed
appropriate test micro-organisms; use not more than under Choice of culture media.
100 CFU per 60 mm diameter plate containing 9 mL of INTERPRETATION OF RESULTS
solid medium and per 100 mL container of liquid medium; At the end of the prescribed incubation period, examine all
use a separate plate and container for each species of micro- inoculated solid media microscopically for the presence of
organism. Incubate the media and make subcultures from mycoplasma colonies. The product complies with the test if
0.2 mL of liquid medium to solid medium at the specified growth of typical mycoplasma colonies has not occurred.
intervals (see below under Test for mycoplasmas in the The product does not comply with the test if growth of
product to be examined). The solid medium complies with typical mycoplasma colonies has occurred on any of the solid
the test if adequate growth is found for each test micro- media. The test is invalid if 1 or more of the positive controls
organism (growth obtained does not differ by a factor greater do not show growth of mycoplasmas on at least 1 subculture
than 5 from the value calculated with respect to the plate. The test is invalid if 1 or more of the negative controls
inoculum). The liquid medium complies with the test if show growth of mycoplasmas. If suspect colonies are
growth on agar plates subcultured from the broth is found observed, a suitable validated method may be used to
for at least 1 subculture for each test micro-organism. determine whether they are due to mycoplasmas.
INHIBITORY SUBSTANCES The following section is published for information.
The test for inhibitory substances is carried out once for a
RECOMMENDED MEDIA FOR THE CULTURE
given product and is repeated whenever there is a change in METHOD
production method that may affect the detection of
The following media are recommended. Other media may be
mycoplasmas.
used, provided that their ability to sustain the growth of
To demonstrate absence of inhibitory substances, carry out mycoplasmas has been demonstrated on each batch in the
the test for nutritive properties in the presence and absence presence and absence of the product to be examined.
of the product to be examined. If growth of a test micro-
organism occurs more than 1 subculture sooner in the
absence of the product to be examined than in its presence,
or if plates directly inoculated with the product to be
examined have fewer than 1/5 of the number of colonies of
those inoculated without the product to be examined,
inhibitory substances are present and they must be
V-A570 Appendix XVI B 2023

HAYFLICK MEDIA (RECOMMENDED FOR THE (1) Beef heart infusion broth
GENERAL DETECTION OF MYCOPIASMAS)
Beef heart (for preparation of the infusion) 500 g
Liquid medium
Peptone 10 g
Sodium chloride 5g
Beef heart infusion broth (1) 90.0 mL Distilled water to 1000 mL
Horse serum (unheated) 20.0 mL
Yeast extract (250 gJL) 10.0mL
Phenol red (0.6 gJL solution) 5.0mL
Sterilise by autoclaving.
Penicillin (20 000 IU/mL) 0.25 mL (2) Essential vitamins
Deoxyribonucleic acid (2 giL solution) 1.2 mL
Biotin 100 mg
Adjust to pH 7.8. Calcium pantothenate 100 mg
Choline chloride 100 mg
Solid medium Folic acid 100 mg
Prepare as described above replacing beef heart infusion ,~Inositol 200 mg
broth by beef heart infusion agar containing 15 g/L of agar. Nicotinamide 100 mg
Pyridoxal hydrochloride 100 mg
FREY MEDIA (RECOMMENDED FOR THE Riboflavine 10mg
DETECTION OF M. SYNOVIAE) Thiamine hydrochloride 100 mg
Distilled water to 1000 mL
Liquid medium

90.0mL
(3) Agar, purified
Beef heart infusion broth (1)
Essential vitamins (2) 0.025 mL A highly refined agar for use in microbiology and
Glucose monohydrate (500 gJL solution) 2.0mL immunology, prepared by an ion-exchange procedure that
Swine serum (inactivated at 56 °C for 30 min) 12.0mL
~-Nicotinamide adenine dinucleotide (10 gJL solution) l.OmL
results in a product having superior purity, clarity and gel
Cysteine hydrochloride (10 gJL solution) 1.0mL strength. It contains about:
Phenol red (0.6 gJL solution) 5.0mL
Penicillin (20 000 IU/mL) 0.25 mL Water 12.2 per cent
Ash 1.5 per cent
Mix the solutions of ~-nicotinamide adenine dinucleotide and Acid-insoluble ash 0.2 per cent
Chlorine 0
cysteine hydrochloride and after 10 min add to the other
Phosphate (calculated as P 2 0 5 ) 0.3 per cent
ingredients. Adjust to pH 7 .8. Total nitrogen 0.3 per cent
Solid medium Copper 8ppm
Iron 170 ppm
Calcium 0.28 per cent
Beef heart infusion broth (1) 90.0 mL Magnesium 0.32 per cent
Agar, purified (3) 1.4 g

(4) Hanks' balanced salt solution (modified)

Adjust to pH 7 .8, sterilise by autoclaving then add:
Sodium chloride 6.4 g
Essential vitamins (2) 0.025 mL Potassium chloride 0.32 g
Glucose monohydrate (500 gJL solution) 2.0mL Magnesium sulfate heptahydrate 0.08 g
Swine serum (unheated) 12.0mL Magnesium chloride hexahydrate 0.08 g
~-Nicotinamide adenine dinucleotide (10 gJL solution) l.OmL Calcium chloride, anhydrous 0.112 g
Cysteine hydrochloride (10 gJL solution) 1.0 mL Disodium hydrogen phosphate dihydrate 0.0596 g
Phenol red (0.6 gJL solution) 5.0mL Potassium dihydrogen phosphate, anhydrous 0.048 g
Penicillin (20 000 IU/mL) 0.25 mL Distilled water to 800 mL

FRIIS MEDIA (RECOMMENDED FOR THE (5) Brain heart infusion

DETECTION OF NON-AVIAN MYCOPIASMAS)
Liquid medium Calf-brain infusion 200 g
Beef-heart infusion 250 g
Protease peptone 10 g
Hanks' balanced salt solution (modified) (4) 800 mL Glucose monohydrate 2g
Distilled water 67 mL Sodium chloride 5g
Brain heart infusion (5) 135 mL Disodium hydrogen phosphate, anhydrous 2.5 g
PPLO Broth ( 6) 248 mL to 1000 mL
Distilled water
Yeast extract (170 gJL) 60 mL
Bacitracin 250 mg
Meticillin 250 mg (6) PPLO broth
Phenol red (5 gJL) 4.5 mL
Horse serum 165 mL Beef-heart infusion 50 g
Swine serum 165 mL Peptone 10 g
Sodium chloride 5g
Adjust to pH 7.40-7.45. Distilled water to 1000 mL

Solid medium
INDICATOR CELL CULTURE METHOD
Hanks' balanced salt solution (modified) (4) 200 mL Cell cultures are stained with a fluorescent dye that binds to
DEAE-dextran 200mg DNA. Mycoplasmas are detected by their characteristic
Agar, purified (3) 15.65 g particulate or filamentous pattern of fluorescence on the cell
surface and, if contamination is heavy, in surrounding areas.
Mix well and sterilise by autoclaving. Cool to 100 °C. Add to Mitochondria in the cytoplasm may be stained but are readily
1740 mL of liquid medium as described above. distinguished from mycoplasmas.
2023 Appendix XVI B V-A571

If for viral suspensions the interpretation of results is affected INTERPRETATION OF RESULTS

by marked cytopathic effects, the virus may be neutralised The product to be examined complies with the test if
using a specific antiserum that has no inhibitory effects on fluorescence typical of mycoplasmas is not present. The test
mycoplasmas or a cell culture substrate that does not allow is invalid if the positive controls do not show fluorescence
growth of the virus may be used. To demonstrate the typical of mycoplasmas. The test is invalid if the negative
absence of inhibitory effects of serum, carry out the positive controls show fluorescence typical of mycoplasmas.
control tests in the presence and absence of the antiserum.
NUCLEIC ACID AMPLIFICATION TECHNIQUES
VERIFICATION OF THE SUBSTRATE (NAT)
Use Vero cells or another cell culture (for example, the NAT (2.6.21) may be used for detection of mycoplasmas by
production cell line) that is equivalent in effectiveness for amplification of nucleic acids extracted from a test sample
detecting mycoplasmas. Test the effectiveness of the cells to with specific primers that reveal the presence of the target
be used by applying the procedure shown below and nucleic acid. NAT indicate the presence of a particular
inoculating not more than 100 CFU or CFU-like micro- nucleic acid sequence and not necessarily the presence of
organisms of suitable reference strains of M. hyorhinis and M. viable mycoplasmas. A number of different techniques are
orate. The following strains have been found to be suitable: available. This general chapter does not prescribe a particular
method for the test. The procedure applied must be
M. hyorhinis ATCC 29052 validated as described, taking account of the guidelines
M. orale NCTC 10112 CIP 104969 ATCC 23714
presented at the end of this section. Where a commercial kit
is used, certain elements of the validation may be carried out
The cells are suitable if both reference strains are detected.
by the manufacturer and information provided to the user
The indicator cells must be subcultured without an antibiotic but it must be remembered that full information on the
before use in the test. primers may not be available and that production of the kit
TEST METHOD may be modified or discontinued.
1. Seed the indicator cell culture at a suitable density (for NAT are applied where prescribed in a monograph. They
example, 2 x 104 to 2 x 10 5 cells/mL, 4 x 10 3 to may also be used instead of the culture method and the
2.5 x 104 cells/cm2 ) that will yield confluence after 3 days of indicator cell culture method after suitable validation.
growth. Inoculate 1 mL of the product to be examined into
Direct NAT Can be applied in the presence of cytotoxic
the cell culture vessel and incubate at 35-38 °C.
material and where a rapid method is needed.
2. After at least 3 days of incubation, when the cells have
Cell-culture enrichment followed by NAT The test
grown to confluence, make a subculture on cover slips in
sample and a suitable cell substrate (as described under the
suitable containers or on some other surface (for example,
indicator cell-culture method) are cultured together for a
chambered slides) suitable for the test procedure. Seed the
suitable period; the nucleic acids are then extracted from
cells at low density so that they reach 50 per cent confluence
cells and supernatant and used for detection by NAT.
after 3-5 days of incubation. Complete confluence impairs
visualisation of mycoplasmas after staining and must be VALIDATION
avoided. Reference standards are required at various stages during
validation and for use as controls during routine application
3. Remove the medium and rinse the indicator cells with
of the test. The reference standards may be mycoplasmas or
phosphate buffered saline pH 7. 4 R, then add a suitable fixing
nucleic acids.
solution (a freshly prepared mixture of 1 volume of glacial
acetic acid R and 3 volumes of methanol R is suitable when For validation of the limit of detection, the following species
bisbenzimide R is used for staining). represent an optimal selection in terms of the frequency of
occurrence as contaminants and phylogenetic relationships:
4. Remove the fixing solution and wash the cells with sterile
- A. laidlawii;
water R. Dry the slides completely if they are to be stained
- M. fermentans;
more than I h later (particular care is needed for staining of
- M. hyorhinis (where cell-culture enrichment is used, a
slides after drying owing to artefacts that may be produced).
fastidious strain such as ATCC 29052 is included);
5. Add a suitable DNA stain and allow to stand for a suitable - M. orate;
time (bisbenzimide working solution R and a standing time of - M. pneumoniae or M. gallisepticum;
10 min are suitable). - M. synoviae (where there is use of or exposure to avian
6. Remove the stain and rinse the monolayer with water R. material during production);
7. Mount each coverslip, where applicable (a mixture of - Mycoplasma arginini;
equal volumes of glycerol R and phosphate-citrate buffer solution - Spiroplasma citri (where there is use of or exposure to
pH 5.5 R is suitable for mounting). Examine by fluorescence insect or plant material during production).
(for bisbenzimide stain a 330 nm/380 nm excitation filter and Demonstration of specificity requires the use of a suitable
an LP 440 nm barrier filter are suitable) at range of bacterial species other than mycoplasmas. Bacterial
400 x magnification or greater. genera with close phylogenetic relation to mycoplasmas are
8. Compare the microscopic appearance of the test cultures most appropriate for this validation; these include Clostridium,
with that of the negative and positive controls, examining for Lactobacillus and Streptococcus.
extranuclear fluorescence. Mycoplasmas produce pinpoints or Comparability studies for use of NAT as an alternative
filaments over the indicator cell cytoplasm. They may also method For each mycoplasma test species:
produce pinpoints and filaments in the intercellular spaces. - as an alternative to the culture method: the NAT test
Multiple microscopic fields are examined according to the system must be shown to detect 10 CFU/mL;
protocol established during validation. - as an alternative to the indicator cell culture method: the
NAT test system must be shown to detect 100 CFU/mL;
V-A572 Appendix XVI B 2023

or an equivalent limit of detection in terms of the number of - an alternative method to replace an official method
copies of mycoplasma nucleic acid in the test sample (using (indicator cell culture method or culture method).
suitable reference standards of mycoplasma nucleic acid). These guidelines will thus separate these 2 objectives by
CONTROLS presenting first a guideline for the validation of the NAT
Internal controls themselves, and second, a guideline for a comparability study
Internal controls are necessary for routine verification of between NAT and official methods.
absence of inhibition. The internal control may contain the 2 GUIDEUNE FOR MYCOPIASMA NAT
primer binding-site, or some other suitable sequence may be VALIDATION
used. It is preferably added to the test material before 3 parameters should be evaluated: specificity, detection limit
isolating the nucleic acid and therefore acts as an overall and robustness.
control (extraction, reverse transcription, amplification, 2-1 Specificity
detection). Specificity is the ability to unequivocally assess target nucleic
External controls acid in the presence of components that may be expected to
The external positive control contains a defined number of be present.
target-sequence copies or CFUs from 1 or more suitable The specificity of NAT is dependent on the choice of
species of mycoplasma chosen from those used during primers, the choice of probe (for analysis of the final
validation of the test conditions. 1 of the positive controls is product) and the stringency of the test conditions (for both
set close to the positive cut-off point to demonstrate that the the amplification and detection steps).
expected sensitivity is achieved. The external negative control The ability of the NAT to detect a large panel of
contains no target sequence but does not necessarily mycoplasma species will depend on the choice of primers,
represent the same matrix as the test article. probes and method parameters. This ability should be
INTERPRETATION OF RESULTS demonstrated using characterised reference panels
The primers used may also amplify non-mycoplasmal (e.g. reference strains provided by the EDQM). Since NAT
bacterial nucleic acid, leading to false positive results. systems are usually based on a mix of primers, the theoretical
Procedures are established at the time of validation for analysis of primers and probes by comparison with databases
dealing with confirmation of positive results, where necessary. is not recommended, because interpretation of the results
may be quite complex and may not reflect the experimental
The foll,owing section is published for information. results.
Moreover, as it is likely that the primers will detect other
VALIDATION OF NUCLEIC ACID bacterial species, the potential cross-detection should be
AMPLIFICATION TECHNIQUES (NAT) documented in the validation study. Bacterial genera such as
gram-positive bacteria with close phylogenetic relation to
FOR THE DETECTION OF
mycoplasmas are most appropriate for this validation; these
MYCOPLASMAS: GUIDELINES include Clostridium, Lacwbacillus and Streptococcus. However,
1 SCOPE this is not an exhaustive list and species to be tested will
n
Nucleic acid amplification techniques (NA are either depend on the theoretical ability (based on primers/probes
qualitative or quantitative tests for the presence of nucleic sequences) of the NAT system to detect such other species.
acid. For the detection of mycoplasma contamination of Based on the results from this validation of the specificity, if
various samples such as vaccines and cell substrates, a gap in the specificity of the method is identified (such as
qualitative tests are adequate and may be considered to be detection of non-mycoplasmal bacterial nucleic acid), an
limit tests. appropriate strategy must be proposed in the validation study
These guidelines describe methods to validate qualitative to allow interpretation of positive results on a routine basis.
nucleic acid amplification analytical procedures for assessing For example, a second test may be performed using an
mycoplasma contamination. They may also be applicable for alternative method without this specificity gap or using an
real-time NAT used as limit tests for the control of official method.
contaminants. 2-2 Detection limit
The 2 characteristics regarded as the most important for The detection limit of an individual analytical procedure is
validation of the analytical procedure are the specificity and the lowest amount of target nucleic acid in a sample that can
the detection limit. In addition, the robustness of the be detected but not necessarily quantitated as an exact value.
analytical procedure should be evaluated. For establishment of the detection limit, a positive cut-off
For the purpose of this document, an analytical procedure is point should be determined for the nucleic acid amplification
defined as the complete procedure from extraction of nucleic analytical procedure. The positive cut-off point (as defined in
acid to detection of the amplified products. general chapter 2. 6. 21) is the minimum number of target
Where commercial kits are used for part or all of the sequence copies per volume of sample that can be detected
analytical procedure, documented validation points already in 95 per cent of test runs. This positive cut-off point is
covered by the kit manufacturer can replace validation by the influenced by the distribution of mycoplasmal genomes in the
user. Nevertheless, the performance of the kit with respect to individual samples being tested and by factors such as
its intended use has to be demonstrated by the user enzyme efficiency, and can result in different 95 per cent cut-
(e.g. detection limit, robustness, cross-detection of other off values for individual analytical test runs.
classes of bacteria). To determine the positive cut-off point, a dilution series of
NAT may be used as: characterised and calibrated (either in CFUs or nucleic acid
- a complementary test (for example, for cytotoxic viral copies) in-house working strains or EDQM standards should
suspensions) or for in-process control purposes; be tested on different days to examine variation between test
runs.
2023 Appendix XVI B V-A573

For validation of the limit of detection, the following species shown to detect 100 CFU/mL for each mycoplasma test
represent an optimal selection in terms of the frequency of species described in paragraph 2-2
occurrence as contaminants and phylogenetic relationships: For both cases, suitable standards calibrated for the number
- A. laidlawii; of nucleic acid copies and the number of CFUs may be used
- A1. f ermentans; for establishing that these acceptability criteria are reached.
- M. hyorhinis; The relation between CFUs and nucleic acid copies for the
- M. orate; reference preparations should be previously established to
- M. pneumoniae or M. gallisepticum; compare the performance of the alternative NAT method
- i\1. synoviae (where there is use of or exposure to avian with the performance of the official methods.
material during production); 1 of the following 2 strategies can be used to perform this
- M. arginini; comparability study:
- S. citri (where there is use of or exposure to insect or - perform the NAT alternative method in parallel with the
plant material during production). official method(s) to evaluate simultaneously the detection
For each strain, at least 3 independent 10-fold dilution series limit of both methods using the same samples of
should be tested, with a sufficient number of replicates at calibrated strains;
each dilution to give a total number of 24 test results for - compare the performance of the NAT alternative method
each dilution, to enable a statistical analysis of the results. using previously obtained data from official method
For example, a laboratory may test 3 dilution series on validation. In this case, calibration of standards used for
different days with 8 replicates for each dilution, 4 dilution both validations as well as their stabilities should be
series on different days with 6 replicates for each dilution, or documented carefully.
6 dilution series on different days with 4 replicates for each Comparability study reports should describe all the validation
dilution. In order to keep the number of dilutions at a elements described in section 2 (specificity, limit of detection
manageable level, a preliminary test should be performed to and variability, as well as robustness) in order to assess all
obtain a preliminary value for the positive cut-off point the advantages and/or disadvantages of the alternative NAT
(i.e. the highest dilution giving a positive signal). The range method compared to official methods.
of dilutions can then be chosen around the predetermined
preliminary cut-off point. The concentration of mycoplasmas 4. Mycobacteria
(CFUs or copies) that can be detected in 95 per cent of test (Ph. Eur. method 2.6.2)
runs can then be calculated using an appropriate statistical If the sample to be examined may be contaminated by
evaluation. micro-organisms other than mycobacteria, treat it with a
These results may also serve to evaluate the variability of the suitable decontamination solution, such as acetylcysteine-
analytical procedure. sodium hydroxide solution or sodium laurilsulfate solution.
2-3 Robustness Inoculate 0.2 mL of the sample in triplicate onto each of
The robustness of an analytical procedure is a measure of its 2 suitable solid media (Lowenstein-Jensen medium and
capacity to remain unaffected by small but deliberate Middlebrook 7H10 medium are considered suitable).
variations in method parameters, and provides an indication Inoculate 0.5 mL in triplicate into a suitable liquid medium.
of its reliability during normal usage. Incubate all media at 37 °C for 56 days.
The evaluation of robustness should be considered during Establish the fertility of the media in the presence of the
the development phase. It should show the reliability of the preparation to be examined by inoculation of a suitable strain
analytical procedure with respect to deliberate variations in of a Mycobacterium sp. such as BCG and if necessary use a
method parameters. For NAT, small variations in the suitable neutralising substance.
method parameters can be crucial. However, the robustness If contaminating micro-organisms develop during the first
of the method can be demonstrated during its development 8 days of incubation, repeat the test and carry out at the
when small variations in the concentrations of reagents same time a bacteriological sterility test.
(e.g. MgC1 2, primers or deoxyribonucleotides) are tested. If at the end of the incubation time no growth of
Modifications of extraction kits or extraction procedures as mycobacteria occurs in any of the test media, the preparation
well as different thermal cycler types may also be evaluated. complies with the test.
Finally, robustness of the method can be evaluated through
collaborative studies. 5. Extraneous Agents in Viral Vaccines
(Ph. Eur. method 2. 6.16)
3 GUIDELINE FOR COMPARABILIIY STUDY
NAT may be used instead of official methods (indicator cell INTRODUCTION
culture method and/or culture method). In this case a A strategy for testing extraneous agents in viral vaccines must
comparability study should be carried out. This comparability be developed based on a risk assessment following the
study should include mainly a comparison of the respective principles of viral contamination risk detailed in general
detection limits of the alternative method and official chapter 5.1. 7. Viral safety. This strategy includes a full
methods. However, specificity (mycoplasma panel detected, package of suitable tests that are able to detect different
putative false positive results) should also be considered. families of extraneous agents that may infect the source of
For the detection limit, acceptability criteria are defined as virus strains including cell substrates and raw material of
follows: animal or plant origin. It also takes into account the capacity
- if the alternative method is proposed to replace the of the manufacturing process to remove or inactivate viruses.
culture method, the NAT system must be shown to The list oftests summarised in Table 2.6.16.-1 must be
detect 10 CFU/mL for each mycoplasma test species adapted depending on the extraneous agents that have the
described in paragraph 2-2; potential to contaminate the product: for in vitro tests, the
- if the alternative method is proposed to replace the risk assessment may allow, with the agreement of the
indicator cell culture method, the NAT system must be competent authority, the use of other permissive cell lines or
V-A574 Appendix XVI B 2023

molecular biology methods depending on the manufacturing Table 2.6.16.-1. - Relevant tests for extraneous agents at various
process and the incubation temperature for the growth of production stages
particular viruses. If in viva tests are more relevant than in Production of culture
vitro tests for the detection of some adventitious viruses substrates
Virus Virus
(e.g. suckling mice for the vesicular stomatitis virus and seed lots harvests
fertilised SPF eggs for the influenza virus) the decision to Control Control
cells eggs
maintain or to introduce such in viva assays in a testing
strategy must be justified by the risk assessment. Bacterial and fungal
- -
contamination + +
New, sensitive molecular methods with broad detection
capabilities are available. These new approaches include Mycoplasmas + + - -
high-throughput sequencing (HTS) methods, nucleic acid
amplification techniques (NAT) (e.g. polymerase chain SpiroplasmasC 1J + - - -

reaction (PCR), reverse transcriptase PCR (RT-PCR), Mycobacteria + + - -

product-enhanced reverse transcriptase (PERT) assays) for
whole virus families or random-priming methods (associated Test in suckling miceC 2J + - - -
or not with sequencing), hybridisation to oligonucleotide Avian virusesC 3) + + - -
arrays, and mass spectrometry with broad-spectrum PCR.
These methods may be used either as an alternative to in Test for extraneous
agents in cell culturesc•J + + + +
viva tests and specific NAT or as a supplement/alternative to
in vitro culture tests based on the risk assessment and with Insect viruses( 5) + + - -
the agreement of the competent authority.
Test on control cells
In tests that require prior neutralisation of the virus, use (microscopic - - + -
specific antibodies of non-human, non-simian origin; if the examination)
virus has been propagated in avian tissues, the antibodies
must also be of non-avian origin. To prepare antiserum, use Haemadsorbing viruses - - + -

an immunising antigen produced in cell cultures from a Test on control eggs

species different from that used for the production of the (haemagglutinating - - - +
vaccine and free from extraneous agents. Where the use of agents)

SPF eggs is prescribed, the eggs are obtained from a flock Avian 1eucosis viruses< 6) - - + +
free from specified pathogens (5.2.2).
Test for specific viruses
TEST METHODS byNATC 7l + + - -
Relevant tests for extraneous agents to be carried out at
Test for viruses using
various production stages are indicated in Table 2.6.16.-1 - -
broad molecular + +
using the methods described below, based on the risk methodscsJ
assessment.
(1) If insect cells or raw materials of plant origin are used.
Take samples at the time of harvesting, and if not tested (2) If the risk assessment indicates that this test provides a risk mitigation
immediately, keep at a temperature below -40 °C. taking into account the overall testing package.
Bacterial and fungal contamination (3) If the virus is propagated in avian or primary avian tissues. If the risk
assessment indicates that this test provides a risk mitigation taking into
Each virus seed lot and virus harvest complies with the test account the overall testing package.
for sterility (2. 6.1). (4) Test performed in suitable permissive cell cultures. Based on a risk
assessment.
Mycoplasmas (2. 6. 7)
(5) If the virus is propagated in insect cells.
Each virus seed lot and virus harvest complies with the test (6) If the virus is propagated in primary avian tissues or in eggs.
for mycoplasmas. (7) Based on a risk assessment.
(8) These methods may be used either as an alternative to in vivo tests and
Spiroplasmas specific NAT or as a supplement/alternative to in vitro culture tests based on
Spiroplasmas may be introduced into virus seed lots through the risk assessment and in agreement with the competent authority.
contamination of raw materials of plant origin or when insect
cell lines are used for virus propagation. When appropriate,
virus seed lots are demonstrated to be free of spiroplasmas intracerebrally with 0.01 mL and intraperitoneally with at
using a validated method approved by the competent least 0.1 mL of the virus seed lot. Observe the suckling mice
authority. NAT methods for detection of mycoplasmas daily for at least 4 weeks. Carry out an autopsy of all suckling
(2. 6. 7) may be used to detect spiroplasmas after validation mice that die after the first 24 h of the test or that show signs
and agreement from the competent authority. of illness, and examine for evidence of viral infection by
direct macroscopical observation. The virus seed lot passes
Mycobacteria (2. 6. 2) the test if no suckling mice show evidence of infection
A 2. 7 mL sample of each virus seed lot and each virus attributable to the seed lot. The test is not valid unless at
harvest is tested for the presence of Mycobacterium spp. least 80 per cent of the original inoculated suckling mice
by culture methods known to be sensitive for the detection of survive the observation period.
these organisms. NAT (2.6.21) may be used as an
alternative, provided such an assay is validated and shown to Avian viruses
be comparable to the culture method. Each virus seed lot propagated in avian tissues and each virus
harvest propagated in primary avian tissues is tested for avian
Test in suckling mice viruses if the risk assessment indicates that this test provides
Each virus seed lot is tested in suckling mice if the risk a risk mitigation taking into account the overall testing
assessment indicates that this test provides a risk mitigation, package. Neutralise a sample equivalent to 100 human doses
taking into account the overall testing package. Inoculate no of vaccine or 10 mL, whichever is the greater. Using 0.5 mL
fewer than 20 suckling mice, each less than 24 h old, per egg, inoculate a group of fertilised SPF eggs, 9-11 days
2023 Appendix XVI B V-A575

old, by the allantoic route and a second group, 5-7 days old, than 14 days beyond the time of inoculation of the
into the yolk sac. Incubate for 7 days. The virus seed lot or production vessels, whichever is the longer. The test is not
harvest complies with the test if the allantoic and yolk sac valid unless at least 80 per cent of the control cell cultures
fluids show no sign of the presence of any haemagglutinating survive to the end of the observation period.
agent and if all embryos and chorio-allantoic membranes At 14 days or at the time of the last virus harvest, whichever
examined for gross pathology, are normal. The test is not is the longer, pool the supernatant fluids from the control
valid unless at least 80 per cent of the inoculated eggs survive cells and examine for the presence of extraneous agents over
for 7 days. a period of 14 days as described above for the virus seed lot
Test for extraneous agents in cell cultures and the virus harvest by inoculation of relevant cell cultures
For each virus seed lot, each virus harvest and each depending on the type of cells used for virus growth.
production cell culture (control cells or control eggs), tests Haemadsorbing viruses
for other extraneous agents must be carried out based on a Where cell cultures are used for virus production, a
risk assessment. The origin of the cell substrate and virus microscopic examination of the control cells is carried out as
strain, as well as the potential extraneous agents that may be described above for the test for extraneous agents in cell
inadvertently introduced during production processes or cultures. At 14 days or at the time of the last virus harvest,
through the use of animal- or plant-derived raw materials, whichever is the longer, examine no fewer than 25 per cent
must be taken into account when choosing suitable of the control cultures for the presence of haemadsorbing
permissive cells. viruses by the addition of guinea-pig red blood cells. If the
For each virus seed lot and virus harvest, neutralised test for haemadsorbing viruses is not feasible, carry out a test
samples, equivalent (unless otherwise prescribed) to 500 for haemagglutinating viruses. If the guinea-pig red blood
human doses of vaccine or 50 mL, whichever is the greater, cells have been stored, they shall have been stored at
are tested for the presence of extraneous agents by 5 ± 3 °C for not more than 7 days. Read half of the cultures
inoculation into continuous simian and human cell cultures. after incubation at 5 ± 3 cc for 30 min and the other half
If the virus is grown in simian or human cells, the neutralised after incubation at 20-25 °C for 30 min. No evidence of
virus harvest is tested on a separate culture of these cells. haemadsorbing agents is found.
If the virus is grown in a mammalian cell system other than Tests on control eggs
simian or human, or in avian cells, cells of that species, but Where eggs are used for virus production, examine 0.25 mL
from a separate batch, are also inoculated. The cells are of the allantoic fluid from each control egg for
incubated at 36 ± 1 °C and observed for a period of haemagglutinating agents by mixing directly with chicken red
14 days. If the production cell culture is maintained at a blood cells and after a passage in SPF eggs carried out as
temperature other than 36 ± 1 °C, a supplementary test for follows: inoculate a 5 mL sample of the pooled amniotic
extraneous agents is carried out at the production fluids from the control eggs in 0.5 mL volumes into the
temperature using the same type of cells used for growth of allantoic cavity and into the amniotic cavity of SPF eggs.
the virus. A subculture of 14 days is carried out followed by The control eggs comply with the test if no evidence of the
a haemadsorbing test. The virus seed lot or harvest passes presence of haemagglutinating agents is found in either test.
the tests if none of the cell cultures show evidence of the
In addition, inoculate 5 mL samples of the pooled amniotic
presence of any extraneous agents after 14 and 28 days of
fluids from the control eggs into suitable permissive cells
incubation, and no evidence of any haemadsorbing viruses
including human, simian and avian cells. Observe the cell
after 28 days. The test is not valid unless at least 80 per cent
cultures for 14 days at a suitable incubation temperature.
of the cell cultures remain viable.
The control eggs comply with the test if no evidence of the
Insect viruses presence of extraneous agents is found. The test is not valid
Each virus seed lot and virus harvest propagated in insect unless 80 per cent of the inoculated cultures survive to the
cells is tested for insect viruses. Neutralised samples, end of the observation period.
equivalent (unless otherwise prescribed) to 500 human doses
Avian leucosis viruses
of vaccine or 50 mL, whichever is the greater, are tested for
For each virus propagated in primary avian cell tissues or in
the presence of extraneous agents by inoculation into at least
eggs, the production cell culture (control cells or control
1 cell culture different from that used in production and
eggs) is tested for avian leucosis viruses. When cell cultures
permissible to insect viruses, and that also allows detection of
are used for virus production, a microscopic examination of
human arboviruses (e.g. BHK-21). The choice of cells is
the control cells is carried out as described above for the test
approved by the competent authority and takes into account
for extraneous agents prior to the test for avian leucosis
the origin of the production cells and the likely contaminants
viruses. At 14 days or at the time of the last virus harvest,
that may be detected by the chosen cells. The cells are
whichever is the longer, carry out the test for avian leucosis
incubated at an appropriate temperature and observed for a
viruses on DF-1 cells or leucosis-free chick-embryo cell
period of 14 days. A subculture of 14 days is carried out
cultures with amplification through 5 passages using at least
followed by a haemadsorbing test. The virus seed lot or
5 mL of the supernatant fluid from the control cells or at
harvest passes the tests if none of the cell cultures show
least 10 mL of a sample of the pooled amniotic fluids from
evidence of the presence of any extraneous agents after 14
the control eggs. PERT assay end-point can be used after
and 28 days of incubation, and no evidence of any
D F-1 amplification for detection of exogenous avian
haemadsorbing virus after 28 days. The test is not valid
retroviruses (including avian leucosis virus). For specific
unless at least 80 per cent of the cell cultures remain viable.
detection of avian leucosis virus, several end-points can be
Tests on control cells used such as immunostaining, enzyme-linked immunosorbent
Where cell cultures are used for virus production, examine assay (ELISA) or complement fixation for avian leucosis
the control cells microscopically for the absence of any virus (COFAL). Control cells or control eggs comply with the test
causing cytopathic degeneration throughout the incubation if there is no evidence of the presence of any avian leucosis
time of the inoculated production cell cultures or for no less vrrus.
V-A576 Appendix XVI C 2023

Tests for specific viruses by NAT decrease in the number of micro-organisms with time, vaty
Based on a risk assessment related to the manufacturing for different types of preparations according to the degree of
process, each virus seed lot and each virus harvest may be protection intended (see Tables 5.1.3.-1/2/3).
tested by NAT (2.6.21) for specific viruses that are not Test micro-organisms
detected by conventional in vivo or cell culture assays. Pseudomonas aeruginosa ATCC 9027; NCIMB 8626; CIP 82.118.
Test for viruses using broad molecular methods Staphylococcus aureus ATCC 6538; NCTC 10788;
With the agreement of the competent authority, broad NCIMB 9518; CIP 4.83.
Candida allncans ATCC 10231; NCPF 3179; IP 48.72.
molecular methods (e.g. HTS) may be used either as an Aspergillus brasiliensis ATCC 16404; IMI 149007; IP 1431.83.
alternative to in vivo tests and specific NAT, or as a
supplement/alternative to in vitro culture tests based on the Single-strain challenges are used and the designated micro-
risk assessment. organisms are supplemented, where appropriate, by other
Both NAT (2.6.21) and broad molecular methods are carried strains or species that may represent likely contaminants to
out with or without prior amplification in suitable permissive the preparation. It is recommended, for example, that
cells. In cases of positive results with either broad molecular Escherichia coli (ATCC 8739; NCIMB 8545; CIP 53.126) is
methods or NAT, a follow-up investigation must be used for all oral preparations and Zygosaccharomyces rouxii
conducted to determine whether detected nucleic acids are (NCYC 381; IP 2021.92) for oral preparations containing a
due to the presence of infectious extraneous agents and/or high concentration of sugar.
are known to constitute a risk to human health. Preparation of inoculum
Preparatory to the test, inoculate the surface of casein soya
bean digest agar (2. 6.12) for bacteria or Sabouraud-dextrose
agar without the addition of antibiotics (2. 6.12) for fungi,
C. Efficacy of Antimicrobial Preservation with the recently grown stock culture of each of the specified
(Ph. Bur. general texts 5.1.3) micro-organisms. Incubate the bacterial cultures at 30-35 °C
for 18-24 h, the culture of C. albicans at 20-25 °C for 48 h,
If a pharmaceutical preparation does not itself have adequate and the culture of A. brasiliensis at 20-25 °C for 1 week or
antimicrobial activity, antimicrobial preservatives may be until good sporulation is obtained. Subcultures may be
added, particularly to aqueous preparations, to prevent needed after revival before the micro-organism is in its
proliferation or to limit microbial contamination which, optimal state, but it is recommended that their number be
during normal conditions of storage and use, particularly for kept to a minimum.
multidose containers, could occur in a product and present a
To harvest the bacterial and C. albicans cultures, use a sterile
hazard to the patient from infection and spoilage of the suspending fluid, containing 9 g/L of sodium chloride R, for
preparation. Antimicrobial preservatives must not be used as dispersal and transfer of the surface growth into a suitable
a substitute for good manufacturing practice.
vessel. Add sufficient suspending fluid to reduce the
The efficacy of an antimicrobial preservative may be microbial count to about 108 micro-organisms per millilitre.
enhanced or diminished by the active constituent of the To harvest the A. brasiliensis culture, use a sterile suspending
preparation or by the formulation in which it is incorporated fluid containing 9 g/L of sodium chloride Rand 0.5 g/L of
or by the container and closure used. The antimicrobial polysorbate 80 R and adjust the spore count to about 108 per
activity of the preparation in its final container is investigated millilitre by adding the same solution.
over the shelf life to ensure that such activity has not been
Remove immediately a suitable sample from each suspension
impaired by storage. Such investigations may be carried out
and determine the number of colony-forming units per
on samples removed from the final container immediately millilitre in each suspension by plate count or membrane
prior to testing. filtration (2.6.12). This value serves to determine the
During development of a pharmaceutical preparation, it shall inoculum and the baseline to use in the test. The suspensions
be demonstrated that the antimicrobial activity of the shall be used immediately.
preparation as such or, if necessary, with the addition of a
suitable preservative or preservatives provides adequate METHOD
protection from adverse effects that may arise from microbial To count the viable micro-organisms in the inoculated
contamination or proliferation during storage and use of the products, use the agar medium used for the initial cultivation
preparation. of the respective micro-organisms.
The efficacy of the antimicrobial activity may be Inoculate a series of containers of the product to be
demonstrated by the test described below. The test is not examined, each with a suspension of one of the test
intended to be used for routine control purposes. organisms to give an inoculum of 10 5 to 106 micro-organisms
per millilitre or per gram of the preparation. The volume of
TEST FOR EFFICACY OF ANTIMICROBIAL the suspension of inoculum does not exceed 1 per cent of the
PRESERVATION volume of the product. Mix thoroughly to ensure
The test consists of challenging the preparation, wherever homogeneous distribution.
possible in its final container, with a prescribed inoculum of Maintain the inoculated product at 20-25 °C, protec..1:ed from
suitable micro-organisms, storing the inoculated preparation
light. Remove a suitable sample from each container,
at a prescribed temperature, withdrawing samples from the
typically 1 mL or 1 g, at zero hour and at appropriate
container at specified intervals of time and counting the
intervals according to the type of the product and determine
organisms in the samples so removed. the number of viable micro-organisms by plate count or
The preservative properties of the preparation are adequate membrane filtration (2. 6.12). Ensure that any residual
if, in the conditions of the test, there is a significant fall or no antimicrobial activity of the product is eliminated by dilution,
increase, as appropriate, in the number of micro-organisms in by filtration or by the use of a specific inactivator. When
the inoculated preparation after the times and at the dilution procedures are used, due allowance is made for the
temperatures prescribed. The acceptance criteria, in terms of
2023 Appendix XVI D V-A577

reduced sensitivity in the recovery of small numbers of viable

micro-organisms. When a specific inactivator is used, the
D. Microbiological Quality of Non-sterile
ability of the system to support the growth of the test Pharmaceutical Preparations and
organisms is confirmed by the use of appropriate controls.
The procedure is validated to verify its ability to demonstrate
Substances for Pharmaceutical Use1
the required reduction in count of viable micro-organisms. (Ph. Bur. general texts 5.1.4)
ACCEPTANCE CRITERIA ◊This chapter does not apply to products containing viable micro-
The criteria for evaluation of antimicrobial activity are given organisms as active ingredients.◊
in Tables 5.1.3.-1/2/3 in terms of the log 10 reduction in the The presence of certain micro-organisms in non-sterile
number of viable micro-organisms against the value obtained preparations may have the potential to reduce or even
for the inoculum. inactivate the therapeutic activity of the product and has a
potential to adversely affect the health of the patient.
Table 5.1.3.-1. - Parenteral preparatwns, eye preparations, Manufacturers therefore have to ensure a low bioburden of
intrauterine preparations and intramammary preparations finished dosage forms by implementing current guidelines on
Log 10 reduction Good Manufacturing Practice during the manufacture,
6h 24 h 7d 14 d 28 d storage and distribution of pharmaceutical preparations.
Bacteria A 2 3 NR Microbial examination of non-sterile products is performed
B 3 NI according to the methods given in general chapters 2. 6.12
and 2.6.13. Acceptance criteria for non-sterile pharmaceutical
Fungi A 2 NI
products based upon the total aerobic microbial count
B NI
(TAMC) and the total combined yeasts/moulds count
NR: no recovery. (TYMC) are given in Tables 5.1.4.-1 and 5.1.4.-2.
NI: no increase in number of viable micro-organisms compared to the previous
reading.
Acceptance criteria are based on individual results or on the
average of replicate counts when replicate counts are
The A criteria express the recommended efficacy to be performed (e.g. direct plating methods).
achieved. In justified cases where the A criteria cannot be When an acceptance criterion for microbiological quality is
attained, for example for reasons of an increased risk of prescribed it is interpreted as follows:
adverse reactions, the B criteria must be satisfied. 10 1 CFU: maximum acceptable count = 20;
- 10 2 CFU: maximum acceptable count = 200;
Table 5.1.3.-2. - Ear preparations, nasal preparations, - 103 CFU: maximum acceptable count = 2000, and so
preparations for cutaneous application and preparations for forth.
inhalation Table 5 .1.4.-1 includes a list of specified micro-organisms for
Log 10 reduction which acceptance criteria are set. The list is not necessarily
2d 7d 14 d 28 d exhaustive and for a given preparation it may be necessary to
Bacteria A 2 NI test for other micro-organisms depending on the nature of
B 3 NI the starting materials and the manufacturing process.
Fungi A 2 NI
If it has been shown that none of the prescribed tests will
B NI
allow valid enumeration of micro-organisms at the level
prescribed, a validated method with a limit of detection as
NI: no increase in number of viable micro-organisms compared to the previous
close as possible to the indicated acceptance criterion is used.
reading.
In addition to the micro-organisms listed in Table 5.1.4.-1,
The A criteria express the recommended efficacy to be the significance of other micro-organisms recovered is
achieved. In justified cases where the A criteria cannot be evaluated in terms of:
attained, for example for reasons of an increased risk of - use of the product: hazard varies according to the route of
adverse reactions, the B criteria must be satisfied. administration (eye, nose, respiratory tract);
- nature of the product: its ability to support growth, the
Table 5.1.3.-3. - Oral preparations, oromucosal preparations and presence of adequate antimicrobial preservation;
rectal preparations - method of application;
Log 10 reduction - intended recipient: risk may differ for neonates, infants,
14 d 28 d
the debilitated;
- use of immunosuppressive agents, corticosteroids;
Bacteria 3 NI
- presence of disease, wounds, organ damage.
Fungi NI
Where warranted, a risk-based assessment of the relevant
NI: no increase in number of viable micro-organisms compared to the previous factors is conducted by personnel with specialised training in
reading. microbiology and the interpretation of microbiological data.
For raw materials, the assessment takes account of processing
The above criteria express the recommended efficacy to be to which the product is subjected, the current technology of
achieved. testing and the availability of materials of the desired quality.

1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter

5. 8 Pharmacopoeia/ harmonisation.
V-A578 Appendix XVI E 2023

Table 5.1.4.-1. - Acceptance crit,eria for microbiologi,cal quality of non-sterile dosage forms
TAMC TYMC
Route of administration Specified micro-organisms
(CFU/g or CFU/mL) (CFU/g or CFU/mL)

Non-aqueous preparations for oral use 10' 102 Absence of Escherichia coli ( 1 g or l mL)

Aqueous preparations for oral use 102 101 Absence of Escherichia coli (1 g or 1 mL)

Rectal use 103 102 -

Oromucosal use
Gingival use
Absence of Staphylococcus aureus ( 1 g or 1 mL)
Cutaneous use 102 10 1
Absence of Pseudmnonas aeruginosa (1 g or 1 mL)
Nasal use
Auricular use

Absence of Pseudmnonas aeruginosa (1 g or 1 mL)

Vaginal use 102 101 Absence of Staphylococcus aureus (1 g or 1 mL)
Absence of Candida albicans (1 g or 1 mL)

Transdermal patches (limits for 1 patch including 102 101 Absence of Staphylococcus aureus (1 patch)
adhesive layer and backing) Absence of Pseudomonas aeruginosa (1 patch)

Absence of Escherichia coh (1 film)

◊Orodispersible films (limits for 1 film) 102 10 1 Absence of Staphylococcus aureus (1 film)
Absence of Pseudomonas aeruginosa (1 film)◊

Absence of Staphylococcus aureus (1 g or 1 mL)

Inhalation use (special requirements apply to 102 101 Absence of Pseudomonas aeruginosa ( 1 g or 1 mL)
liquid preparations for nebulisation) Absence of bile-tolerant gram-negative bacteria (1 g or
lmL)

♦Special Ph. Eur. provision for oral dosage forms

Not more than 10 2 CFU of bile-tolerant gram-negative
containing raw materials of natural (animal,
bacteria (1 g or 1 mL)
vegetal or mineral) origin for which antimicrobial
10• 102 Absence of Salmonella (10 g or 10 mL)
pretreatment is not feasible and for which the
Absence of Escherichia coli (1 g or 1 mL)
competent authority accepts T AMC of the raw
Absence of Staphylococcus aureus (1 g or 1 mL) ♦
material exceeding 103 CFU/g or CFU/mL.

♦ Special Ph. Eur. provision for premixes for Not more than 104 CFU of bile-tolerant gram-negative
medicated feeding stuffs for veterinary use using bacteria (I g or 1 mL)
10' 10•
excipients of plant origin for which antimicrobial Absence of Escherichia coli (1 g or 1 mL)
treatment is not feasible. Absence of Salmonella (25 g or 25 mL) ♦

Table 5.1.4.-2. -Acceptance criteria for microbiologi,cal quality of examination tests before administration to the patient, as well
non-sterik substances for pharmaceutical use as the amounts available for testing and sampling-related
TAMC TYMC issues. These approaches may be applied when the test for
(CFU/g or CFU/mL) (CFU/g or CFU/mL) sterility, described in general chapter 2. 6.1. Sterility, is
Substances for required but cannot be performed for technical reasons or
pharmaceutical use due to the characteristics of the specific cell-based
preparation.
♦Recommended acceptance criteria for microbiologi,cal quality of 1-1 SHELF-LIFE
herbal medicinal products for oral use and extracts used in their The shelf-life of cell-based preparations is dependent on the
preparation are given in general chapter 5.1. 8. ♦ cell characteristics and on the preservation conditions.
For non-cryopreserved cell-based preparations, the shelf-life
usually does not exceed 3-4 days and sometimes not more
than a few hours. In such cases the microbiological status of
E. Microbiological Examination of Cell- the final preparation cannot be determined, according to
general chapter 2. 6. 1, before the time of administration.
based Preparations 1-2 SAMPLE COMPOSITION
(Ph. Bur. method 2. 6. 2 7) Microbial contaminants may be found either inside or on the
This chapter does not concern the examination of human blood or surface of cells or other components of the cell-based
blood components, which is covered by Directive 2002/98/EC of preparation and may not be detected if only supernatants,
the European Parliament and of the Council, of 2 7 January 2003 such as culture or transport media, are analysed. The sample
and Commission Directive 2004133/EC of 22 March 2004 tested must be representative of all of the components of the
implementing Directive 2002/98/EC. cell-based preparation, unless otherwise justified.
1 INTRODUCTION 1-3 SAMPLE SIZE
Due to constraints surrounding the use of a single donor or
The approaches to microbiological examination of cell-based
manufacturing-related capacities, the sample volume available
preparations outlined in this general chapter take into
for testing at the end of the production process may be
account the characteristics and limitations of these
limited. Nevertheless, with regard to the sampling error,
preparations, in particular their shelf-life, which may not
which may lead to microbial contamination not being
always allow for completion of conventional microbiological
2023 Appendix XVI E V-A579

detected, the sample size must be sufficient to ensure suitable At least 2 suitable culture media intended for detection of
sensitivity and specificity of the chosen test method. fungi and aerobic and anaerobic bacteria are used. Each
1-4 RATIONALE FOR METHOD SELECTION batch of sterile medium is tested for its growth-promoting
Method selection must be based on the characteristics of the capacities by inoculating duplicate test containers of each
final preparation and the manufacturing process. To ensure medium with not more than 100 CFU of each of the strains
safety for the intended use, the choice of method or listed in Table 2.6.27.-1, and incubating for a maximum of
combination of methods could be supported by a risk 7 days for detection of microbial growth at the temperature
analysis of the potential exposure to microbiological defined for testing (see Table 2.6.27.-3). The test media are
contaminants, and the characteristics and intended use of the satisfactory if there is clear evidence of growth in all
cell-based preparation. The media and incubation times used inoculated media containers within this incubation period.
in these methods must be chosen taking into account the
Table 2.6.27.-1. - Micro-organisms used for growth promotion
properties of the source material and the conditions during
test
the manufacturing process that may support growth of
specific micro-organisms (e.g. psychrophilic, thermophilic or Aerobic medium
fastidious bacteria or fungi). The composition of the cell- Staphy/,ococcus aureus for example, ATCC 6538, CIP 4.83,
NCTC 10788, NCIMB 9518
based preparation may impede certain test methods for
Bacillus subtilis for example, ATCC 6633, CIP 52.62,
physical reasons such as initial turbidity of the culture media
NCIMB 8054
after addition of the test sample.
Pseudomonas aeruginosa for example, ATCC 9027, NCIMB 8626,
The following approaches to microbiological examination CIP 82.118
may be applied: Candida albicans for example, ATCC 10231, IP 48.72,
- automated growth-based methods; NCPF 3179
- a combination of preculturing and detection by alternative Aspergillus brasiliensis for example, ATCC 16404, IP 1431.83, IMI
methods (5.1.6); 149007
- direct detection by alternative methods (5.1.6); Anaerobic medium
- methods based on the sterility test prescribed in general Cwstridium sporoge:nes for example, ATCC 19404, CIP 79.3,
chapter 2. 6.1. NCTC 532 or ATCC 11437
2 GENERAL CONSIDERATIONS Bacteroides fragilis for example, ATCC 25285, CIP 77.16,
NCTC 9343
2-1 GENERAL PRECAUTIONS
The test is carried out under aseptic conditions according to
current regulations for potentially infective material. 3-1-2 Method suitability
The precautions taken to avoid contamination are such that For a validated automated growth-based method only a
they do not affect any micro-organisms that are to be confirmation of the suitability of the method for the given
revealed in the test. The test is performed under working cell-based preparation must be performed with respect to
conditions that are monitored regularly by appropriate specificity (absence of false positive results), sensitivity,
sampling of the working area and by carrying out appropriate reproducibility and robustness. Regardless of the type of cell-
controls. Testing shall take account of the potential for the based preparation, the manufacturing process, the sample
presence of inhibitory substances in the sample that may volume analysed or the type of test system, the suitability of
affect the outcome of the test. the method is to be confirmed in the presence of the test
sample. During the confirmation of the suitability of the
2-2 HANDLING CONSTRAINTS
method, particularly to determine sensitivity, the test is
2-2-1 Shelf-life carried out using the micro-organisms listed in
'Negative-to-date' is understood as an intermediate reading Table 2.6.27.-2. Sensitivity is meant as the capacity to detect
of a test method (2. 6.1 or an automated growth-based 100 CFU or less. Not more than 100 CFU of the chosen
method) that has not yet been completed. Where cell-based micro-organisms are inoculated into the medium in the
preparations have limiting shelf-lives, 'negative-to-date' presence of the test sample, using at least 3 replicates.
results may be used as the readout, where justified. Based on The microbial count in the micro-organism suspensions used
the risk analysis of the characteristics and intended use of the for inoculation is determined by streaking an appropriate
cell-based preparation, results from additional microbiological sample on agar plates. If between 1 and 100 CFU are
in-process control may be needed at the time of use. detected for each strain within the duration of the assay, the
2-2-2 Sampling method is suitable for the intended test sample.
The test sample must be representative of all of the It may be necessary to modify the list of micro-organisms in
components of the cell-based preparation and be taken from Table 2.6.27.-2 depending on the origin of the cells and any
the final preparation. Where this is not possible, surrogate micro-organisms previously found or associated with the
testing may be performed, for example on the liquids last in particular type of cells.
contact with the cells being processed. In some cases, the cell-based preparation itself can inactivate
3 METHODS FOR MICROBIOLOGICAL contaminating micro-organisms. Appropriate measures must
EXAMINATION OF CELL-BASED PREPARATIONS be taken to ensure the suitability of any additional microbial
3-1 AUTOMATED GROWTH-BASED METHOD strains used for confirmation of method suitability.
3-1-1 Growth promotion test
This section outlines the conditions for confirming the
suitability of the culture media used for microbiological
examination.
V-A580 Appendix XVI E 2023

Table 2.6.27.-2. - Micro-organisms used for method suitability Table 2.6.27.-3. - Possible temperature settings in automated
Aerobic medium culturing systems used al,one or in combination with manual
Aspergillus brasiliensis for example, ATCC 16404, IP 1431.83, IMI
testing
149007 Aerobic incubation Anaerobic incubation
Bacillus subtilis for example, ATCC 6633, CIP 52.62, Option I 20-25 "C (automated system), if 30-35 'C (automated system)
NCIMB 8054 necessary 30-35 "C (automated
Candida al/Jicans for example, ATCC 10231, IP 48.72, system)
NCPF 3179 Option 2 35-37 "C (automated system); 35-37 'C (automated system)
Pseudomonas aerugi.nosa for example, ATCC 9027, NCIMB 8626, where relevant, additional
CIP 82.118 incubation at a lower
Staphylococcus aureus for example, ATCC 6538, CIP 4.83, temperature (manual method)*
NCTC 10788, NCIMB 9518 Option 3 30-32 "C (automated system) 30-32 'C (automated system)
Strepwcoccus pyogenes for example, ATCC 19615, CIP 1042.26, Option 4 30-32 ''C (automated system) 35-37 ''C (automated system)
NCIMB 13285
* Where relevant, incubate in addition at a temperature between 20 ''C and
Micrococcus sp. for example, ATCC 700405
30 'C. Incubation can be performed using commercially available
Anaerobic medium microbiological media, either aerobic bottles intended for automated systems
or casein soya bean digest broth.
Clostridium sporogenes for example, ATCC 19404, CIP 79.3,
NCTC 532 or ATCC 11437 3-1-4 Observation and interpretation of results
Cutibacterium acnes for example, ATCC I 1827 Media are examined, visually or with automated systems, at
least daily and at the end of the observation period for
3-1-3 Testing of the preparation to be examined evidence of microbial growth. If no growth is observed
Sample A sample that is representative of the during or at the end of the observation period, the product is
characteristics of the cell-based preparation is tested. 'culture negative'. If growth is observed in a valid test, the
The sample is added to the culture medium as soon as product is 'culture positive'.
possible. If it is necessary to store samples, the impact of the If the inoculated bottles are stored for more than 12 h before
storage on potential contaminants is evaluated. being placed into the automated culturing system, a
For cell-based preparations where the total volume (V) of the subculture of each incubated bottle is performed to check for
batch is between 1 mL and 1 L in a single container, the false negatives. This addresses cases in which, where a micro-
following table indicates the inoculation volume to be used. organism is fast-growing and the conditions are optimal,
micro-organisms may start to proliferate during storage. As a
Total cell-based preparation Total inoculum volume (divided
consequence, there may not be a significant increase in the
volume (mL) between aerobic and anaerobic relevant parameters at the time of testing and microbial
bottles) contaminants may not be recognised by the system (false
10 S: V S: 1000 1 per cent of total volume of negative result).
preparation to be tested 3-2 ALTERNATIVE METHODS
I S: V < JO JOO µL
3-2-1 Combination ofpreculturing and detection by
V<l Not applicable
alternative methods
The samples to be tested are incubated in both aerobic and
For other volumes or multiple containers, alternative anaerobic liquid cultivation media or equivalent solid media
approaches should be used and have to be justified (see for a short period of time (e.g. 12-24 h depending on the
section 2-2-2). Enlargement of the total volume by means of sensitivity of the alternative approach used). An alternative
dilution may be envisaged to assure complete inoculation of method suitable for rapid detection of micro-organisms is
sample volumes of 100 µL. For preparation volumes less then performed (e.g. nucleic acid amplification techniques
than 1 mL, where final sampling is not possible, surrogate (2.6.21), flow cytometry (2.7.24), bioluminescence (5.1.6')).
testing, in-process testing or other appropriate testing should 3-2-2 Direct detection by alternative methods
be used and has to be justified. (5.1. 6')
Analysis Samples are inoculated into containers of culture Where a cell-based preparation has a very short shelf-life
medium as soon as possible and incubated for not less than (e.g. a few hours) or where standard methods do not provide
7 days. Depending on the results obtained during method satisfactory detection of micro-organisms, non-growth-based,
suitability testing and considering relevant micro-organisms, direct detection methods may be carried out for
the incubation period may be extended up to 14 days. microbiological examination (e.g. nucleic acid amplification
Selection of incubation temperatures should enable detection techniques (2.6.21), flow cytometry (2.7.24),
of a broad range of micro-organisms. This is typically in the bioluminescence ( 5. J. 6')).
range of 30-37 °C; however, for cell-based preparations with
This approach enables a result to be obtained within a very
a very short shelf-life, a growth-accelerating temperature of
short time, although at the expense of lower sensitivity, in
not less than 35 °C may be more appropriate to obtain a
comparison to growth-based methods. Depending on the
relevant 'negative-to-date' readout of the test. In addition, for
approach used, both viable and non-viable micro-organisms
preparations where there is a significant risk of contamination
may be detected.
from the environment, 2 temperature ranges of, for example,
20-25 °C (for aerobic) and 30-37 °C (for anaerobic) are 3-2-3 Method validation
used, in order to cover both environmental and clinical Validation is carried out according to the general
micro-organisms. recommendations of general chapter 5. 1. 6 and according to
the recommendations specific to cell-based preparations in
Table 2.6.27 .-3 lists possible alternative approaches for the
section 3-1-2 for automated growth-based methods.
choice of incubation temperatures. The temperature and time
The sensitivity of these approaches must be validated
for incubation are based on the results of the suitability study
considering the doubling times of potentially contaminating
for the specific cell-based preparation.
micro-organisms during pre-incubation.
2023 Appendix XVI F V-A581

- Salmonella enterica subsp. enterica serovar Typhimurium

F. Microbiological Examination of Herbal such as ATCC 14028 or, as an alternative, S. enterica
Medicinal Products for Oral Use and subsp. enterica serovar Abony such as NBRC 100797,
NCTC 6017 or CIP 80.39;
Extracts used in their Preparation - Bacillus subtilis such as ATCC 6633, NCIMB 8054,
(Ph. Bur. method 2.6.31) CIP 52.62 or NBRC 3134.
1 MICROBIAL ENUMERATION TESTS Use buffered sodium chloride-peptone solution pH 7.0 or
Total aerobic microbial count (TAMC) phosphate buffer solution pH 7 .2 to make test suspensions.
Perform as described in general chapter 2. 6.12. Use the suspensions within 2 h, or within 24 h if stored at
2-8 °C. As an alternative to preparing and then diluting a
Total combined yeasts/moulds count (ITMC) fresh suspension of vegetative cells of B. subtilis, a stable
Perform as described in general chapter 2.6. 12. Due to the spore suspension is prepared and then an appropriate volume
natural high bioburden in the products covered by general is used for test inoculation. The stable spore suspension may
chapter 5.1.8, use of Sabouraud-dextrose agar containing be maintained at 2-8 °C for a validated period of time.
antibiotics is suitable.
2-3-2 NEGATIVE CONTROL
2 TEST FOR SPECIFIED MICRO-ORGANISMS To verify testing conditions, a negative control is performed
2-1 INTRODUCTION using the chosen diluent in place of the test preparation.
The tests described hereafter will allow determination of the There must be no growth of micro-organisms. A negative
absence or limited occurrence of specified micro-organisms control is also performed when testing the products as
that may be detected under the conditions described. described in section 2-4. A failed negative control requires an
The tests are designed primarily to determine whether a investigation.
product, substance or preparation (hereinafter referred to as 2-3-3 GROWTH-PROMOTING AND INHIBITORY PROPERTIES
'the product') complies with an established specification for OF THE MEDIA
microbiological quality. When used for such purposes, follow
Test each batch ofready-prepared medium and each batch
the instructions given below, including the number of
of medium prepared either from dehydrated medium or from
samples to be taken, and interpret the results as stated below.
the ingredients described.
Alternative microbiological procedures, including automated
Verify suitable properties of relevant media as described in
methods, may be used, provided that their equivalence to the
Table 2.6.31.-1.
Pharmacopoeia method has been demonstrated.
Test for growth-promoting properties, liquid media
2-2 GENERAL PROCEDURES
Inoculate a portion of the appropriate medium with a small
The preparation of samples is carried out as described in
number (not more than 100 CFU) of the appropriate micro-
general chapter 2. 6.12.
organism. Incubate at the specified temperature for not more
If the product to be examined has antimicrobial activity, this than the shortest period of time specified in the test. Incubate
is as far as possible removed or neutralised as described in the casein soya bean digest broth at 30-35 °C for not more
general chapter 2. 6.12. than 3 days. Clearly visible growth of the micro-organism
If surface-active substances are used for sample preparation, comparable to that obtained with a previously tested and
their absence of toxicity for micro-organisms and their approved batch of medium occurs.
compatibility with inactivators used must be demonstrated as
Test for growth-promoting properties, solid media
described in general chapter 2.6.12.
Perform the surface-spread method, inoculating each plate
2-3 GROWTH-PROMOTING AND INHIBITORY with a small number (not more than 100 CFU) of the
PROPERTIES OF THE MEDIA, SUITABIUTY OF appropriate micro-organism. Incubate at the specified
THE TEST AND NEGATIVE CONTROLS temperature for not more than the shortest period of time
The ability of the test to detect micro-organisms in the specified in the test. Growth of the micro-organism
presence of the product to be examined must be established. comparable to that obtained with a previously tested and
Suitability must be confirmed if a change in testing approved batch of medium occurs.
performance, or the product, which may affect the outcome Test for inhibitory properties, liquid or solid media
of the test is introduced. Inoculate the appropriate medium with at least 100 CFU of
2-3-1 PREPARATION OF TEST STRAINS the appropriate micro-organism. Incubate at the specified
Use standardised stable suspensions of test strains or prepare temperature for not less than the longest period of time
them as stated below. Seed lot culture maintenance specified in the test. No growth of the test micro-organism
techniques (seed-lot systems) are used so that the viable occurs.
micro-organisms used for inoculation are not more than Test for indicative properties
5 passages removed from the original master seed lot. Perform the surface-spread method, inoculating each plate
2-3-1-1 Aerobic micro-organisms with a small number (not more than 100 CFU) of the
Grow each of the bacterial test strains separately in casein appropriate micro-organism. Incubate at the specified
soya bean digest broth or on casein soya bean digest agar at temperature for a period of time within the range specified in
30-35 °C for 18-24 h. the test. Colonies are comparable in appearance and
- Staphylococcus aureus such as ATCC 6538, NCIMB 9518, indication reactions to those obtained with a previously tested
CIP 4.83 or NBRC 13276; and approved batch of medium.
- Pseudomonas aeruginosa such as ATCC 9027, 2-3-4 SUITABILITY OF THE TEST METHOD
NCIMB 8626, CIP 82.118 orNBRC 13275;
For each product to be examined, perform the sample
- Escherichia coli such as ATCC 8739, NCIMB 8545,
preparation as described in the relevant paragraph in
CIP 53.126 or NBRC 3972;
section 2-4. Add each test strain at the time of mixing, in the
prescribed growth medium (casein soya bean digest broth or
V-A582 Appendix XVI F 2023

Table 2.6.31.-1. - Growth-promoting, inhibitory and indicative Use a number of micro-organisms equivalent to not more
properties of media than 100 CFU per gram or millilitre of product. Perform the
test as described in the relevant paragraph in section 2-4
Medium Property Test strains
using the shortest incubation period prescribed. The dilution
Casein soya bean Growth
S. aureus corresponding to 0.1 g or 0.1 mL of product must be
P. aerngi.nosa positive.
digest broth promoting
B. subtilis
2-4 TESTING OF PRODUCTS
Growth E. coli 2-4-1 BILE-TOLERANT GRAM-NEGATIVE BACTERIA
Test for bile- Enterobacteria
promoting P. aerugirwsa
tolerant gram- enrichment 2-4-1-1 Enumeration test
negative bacteria broth-Mossel
Inhibitory S. aureus Semi-quantitative test (probable-number method).
Growth 2-4-1-1-1 Sample preparation and pre-incubation. Prepare a
Violet red bile E.coli
glucose agar
promoting
P. aerugirwsa
sample using a 10-fold dilution of not less than 1 g of the
+ indicative product to be examined as described in general
S. aureus
chapter 2. 6.12, but using casein soya bean digest broth as the
Casein soya bean Growth chosen diluent, mix and incubate at 20-25 °C for a time
P. aeruginosa
digest broth promoting
B. subtilis sufficient to resuscitate the bacteria but not sufficient to
encourage multiplication of the organisms (2-3 h).
Growth
E. coli 2-4-1-1-2 Selection and subculture. Inoculate suitable
Test for MacConkey promoting
Escherichia coli broth quantities of enterobacteria enrichment broth-Mossel with
Inhibitory S. aureus
the preparation as described above and/or, depending on the
Growth limit applied for the particular product, with 3 of the 4
MacConkey agar promoting E.coli dilutions of the preparation, which contain respectively 0 .1 g,
+ indicative 0.01 g, 0.001 g and 0.0001 g (or 0.1 mL, 0.01 mL,
S. enterica subsp. 0.001 mL and 0.0001 mL) of the product to be examined.
enterica serovar Incubate at 30-35 °C for 24-48 h. Subculture each of the
Buffered peptone Growth Typhimurium or cultures on a plate of violet red bile glucose agar. Incubate at
medium promoting S. enterica subsp.
30-35 °C for 18-24 h.
enterica serovar
Abony 2-4-1-1-3 Interpretation. Growth of colonies constitutes a
positive result. Note the smallest quantity of the product that
S. enterica subsp.
gives a positive result and the largest quantity that gives a
enterica serovar
Rappaport Growth Typhimurium or negative result.
Test for Vassiliadis promoting S. enterica Determine from Table 2.6.31.-2 the probable number of
Salmonella Salmonella subsp. enterica
bacteria.
enrichment broth serovar Abony

Inhibitory S. aureus Table 2.6.31.-2. - Interpretation of results

Results for each quantity of product Probable
S. enterica subsp.
number of
enterica serovar
Xylose, lysine, Growth bacteria
Typhimurium or
deoxycholate promoting 0.1 g or 0.01 g or 0.001 g or 0.0001 g or per gram
S. enterica
agar + indicative 0.1 mL 0.01 mL 0.001 mL 0.0001 mL or millilitre
subsp. enterica
of product
serovar Abony

+ + + + > 104

buffered peptone medium). For the enumeration method for < 104 and>
bile-tolerant gram-negative bacteria, inoculate E. coli and + + + - 10'
P. aeruginosa individually. For the tests for E. coli and
<10 3 and>
Salmonella, inoculate the specified micro-organism + + - - 102
individually.
Any antimicrobial activity of the product necessitates a <10 2 and>
+ - - - 10
modification of the test procedure (see section 4-5-3 of
general chapter 2. 6.12). - - - - < 10
If for a given product the antimicrobial activity with respect
to a micro-organism for which testing is prescribed cannot be 2-4-2 ESCHERICHIA COLi
neutralised, then it is to be assumed that the inhibited micro-
organism will not be present in the product. 2-4-2-1 Test for absence
2-4-2-1-1 Sample preparation and pre-incubation. Prepare a
2-3-4-1 Test for absence sample using a 10-fold dilution of not less than I g of the
Use a number of micro-organisms equivalent to not more product to be examined as described in general
than I 00 CFU in the inoculated test preparation. Perform chapter 2. 6.12, and use 10 mL or the quantity corresponding
the test as described in the relevant paragraph in section 2-4 to 1 g or 1 mL to inoculate a suitable amount (determined as
using the shortest incubation period prescribed. The specified described in section 2-3-4) of casein soya bean digest broth,
micro-organisms must be detected with the indication mix and incubate at 30-35 °C for 18-24 h.
reactions as described in section 2-4.
2-4-2-1-2 Selection and subculture. Shake the container,
2-3-4-2 Enumeration test transfer 1 mL of casein soya bean digest broth to 100 mL of
Semi-quantitative test (probable-number method). MacConkey broth and incubate at 42-44 °C for 24-48 h.
2023 Appendix XVI G V-A583

Subculture on a plate of MacConkey agar at 30-35 °C for The following section is given for information.
18-72 h. RECOMMENDED SOLUTIONS AND CULTURE
2-4-2-1-3 Interpretation. Growth of colonies indicates the MEDIA
possible presence of E. coli. This is confirmed by The solutions and culture media mentioned in this chapter
identification tests. and described in general chapter 2. 6.13 and the following
The product complies with the test if no colonies are present buffered peptone medium have been found to be satisfactory
or if the identification tests are negative. for the purposes for which they are prescribed in this chapter.
2-4-2-2 Enumeration test Other media may be used provided that their suitability can
Semi-quantitative test (probable-number method). be demonstrated.
2-4-2-2-1 Sample preparation and pre-incubation. Prepare a Buffered peptone medium
sample using a 10-fold dilution of not less than 1 g of the
Potassium dihydrogen phosphate I .5 g
product to be examined as described in general Disodium hydrogen phosphate dodecahydrate 9.0 g
chapter 2. 6.12, and use the quantities corresponding Sodium chloride 5.0 g
respectively to 0.1 g, 0.01 g and 0.001 g (or 0.1 mL, Peptone 10.0 g
0.01 mL and 0.001 mL) to inoculate a suitable amount Purified water 1000 mL
(determined as described in section 2-3-4) of casein soya
bean digest broth, mix and incubate at 30-35 °C for 18-24 h. Adjust the pH so that after sterilisation it is 7.0 ± 0.2 at
25 °C. Sterilise in an autoclave using a validated cycle.
2-4-2-2-2 Selection and subculture. Shake the container,
transfer 1 mL of casein soya bean digest broth to 100 mL of
MacConkey broth and incubate at 42-44 °C for 24-48 h.
Subculture on a plate of MacConkey agar at 30-35 °C for
18-72 h. G. Microbiological Quality of Herbal
2-4-2-2-3 Interpretation. Growth of colonies indicates the Medicinal Products for Oral Use and
possible presence of E. coli. This is confirmed by
identification tests.
Extracts Used in their Preparation
Note the smallest quantity of the product that gives a positive (Ph. Bur. general texts 5.1.8)
result and the largest quantity that gives a negative result. This general chapter presents recommended acceptance criteria for
Determine from Table 2.6.31.-3 the probable number of the microbiological quality of both herbal medicinal products for
bacteria. oral use and the extracts that are used in their preparation.
Microbial examination of non-sterile products is performed
Table 2.6.31.-3. - Interpretation of results
according to the methods given in general chapters 2. 6.12,
Results for each quantity of product Probable 2.6.13 and 2.6.31. Acceptance criteria based upon the total
number of aerobic microbial count (TAMC) and the total combined
bacteria per
0.01 g or 0.001 g or gram or
yeasts/moulds count (TYMC) are given below.
0.1 g or 0.1 mL
0.01 mL 0.001 mL millilitre of Acceptance criteria are based on individual results or on the
product average of replicate counts when replicate counts are
> 103 performed (e.g. direct plating methods).
+ + +
A list of specified micro-organisms for which acceptance
< 103 and> criteria are set can be found below. The list is not necessarily
+ + - 102
exhaustive and for a given preparation it may be necessary to
+ - - < 10 2 and> 10 test for other micro-organisms depending on the nature of
the starting materials, the manufacturing process and the
- - - < 10
intended use.
Medicinal products containing live yeasts (live biotherapeutic
2-4-3 SAIMONELLA products) are not within the scope of this general chapter.
2-4-3-1 Test for absence HERBAL MEDICINAL PRODUCTS
2-4-3-1-1 Sample preparation and pre-incubation. Use 25 g
A. Herbal medicinal products containing herbal
or 25 mL of the product to be examined to inoculate
drugs, with or without excipients, intended for the
225 mL of buffered peptone medium and mix
preparation of infusions and decoctions using boiling
(e.g. homogenise in a filter bag by using a blender). Incubate
water (for example herbal teas, with or without added
at 30-35 °C for 18-24 h.
flavourings)
2-4-3-1-2 Selection and subculture. Transfer 0.1 mL of
buffered peptone medium to 10 mL of Rappaport Vassiliadis Acceptance criterion: 10 7 CFU/g
TAMC (2.6.12)
Salmonella enrichment broth and incubate at 30-35 °C for Maximum acceptable count: 50 000 000 CFU/g
18-24 h. Subculture on plates of xylose, lysine and
deoxycholate agar. Incubate at 30-35 °C for 18-48 h. TYMC (2.6.12) Acceptance criterion: I 0 5 CFU/g
Maximum acceptable count: 500 000 CFU/g
2-4-3-1-3 Interpretation. The possible presence of
Salmonella is indicated by the growth of well-developed, red Escherichia coli
Acceptance criterion: 10 3 CFU/g
colonies, with or without black centres. This is confirmed by (2.6.31)

identification tests. Salmonella (2.6.31) Absence (25 g)

The product complies with the test if colonies of the types
described are not present or if the identification tests are
negative.
V-A584 Appendix XVI H 2023

B. Herbal medicinal products containing, for characterisation of the microbial contamination and the intended
example, extracts and/or herbal drugs, with or without use of the herbal medicinal product or extract.
excipients, where the method of processing (for If it has been shown that none of the prescribed tests for a herbal
example, extraction) or, where appropriate, in the case medicinal product or extract will allow valid enumeration of micro-
of herbal drugs, of pre-treatment reduces the levels of organisms at the level prescribed, a validated method with a limit
organisms to below those stated for this category of detection as close as possible to the indicated acceptance criterion
is used.
TAMC (2.6.12) Acceptance criterion: 104 CFU/g or CFU/mL
Maximum acceptable count: 50 000 CFU/g
orCFU/mL

TYMC (2.6.12) Acceptance criterion: 102 CFU/g or CFU/mL H. Microbiological Examination of Live
Maximum acceptable count: 500 CFU/g
or CFU/mL Biotherapeutic Products
Bile-tolerant gram-
negative bacteria Acceptance criterion: 102 CFU/g or CFU/mL
Microbiological Examination of Live Biotherapeutic
(2.6.31) Products: Tests for Enumeration of Microbial
Contaminants
Escherichia coli
Absence (I g or I mL) (Ph. Bur. method 2. 6.36)
(2.6.31)
1. INTRODUCTION
Salmonella (2.6.31) Absence (25 g or 25 mL)
The tests described hereafter will allow enumeration of live
biotherapeutic product (LBP) contaminants: mesophilic
C. Herbal medicinal products containing, for bacteria and yeasts/moulds that may grow under aerobic
example, extracts and/or herbal drugs, with or without conditions.
excipients, where it can be demonstrated that the The tests are designed primarily to determine whether an
method of processing (for example, extraction with LBP substance or preparation complies with the established
low-strength ethanol or water that is not boiling, or specifications for microbial contamination.
low-temperature concentration) or, in the case of Alternative microbiological procedures (5.1. 6), including
herbal drugs, of pre-treatment, would not reduce the
automated methods, may be used, provided that their
level of organisms sufficiently to reach the criteria equivalence to the Pharmacopoeia method has been
required under B
demonstrated.

TAMC (2.6.12) Acceptance criterion: 10 5 CFU/g or CFU/mL

2. GENERAL PROCEDURES
Maximum acceptable count: 500 000 CFU/g Carry out the determination under conditions designed to
or CFU/mL avoid extrinsic microbial contamination of the LBP to be
TYMC (2. 6.12) Acceptance criterion: 104 CFU/g or CFU/mL
tested. The precautions taken to avoid contamination must
Maximum acceptable count: 50 000 CFU/g be such that they do not affect any contaminant micro-
or CFU/mL organisms that are to be revealed in the test.
Bile-tolerant gram-
If the LBP to be tested prevents the enumeration of
negative bacteria Acceptance criterion: 104 CFU/g or CFU/mL contaminant micro-organisms, apply the decision tree shown
(2.6.31) in Figure 2.6.36.-1.
Escherichia coli
If components other than the active substance
Absence (I g or I mL) (i.e. the micro-organism) of the LBP to be tested have
(2. 6.31)
inhibitory activity, this is insofar as possible removed or
Salmonella (2.6.31) Absence (25 g or 25 mL)
neutralised as described in general chapter 2. 6.12.
If inactivators are used for this purpose, their efficacy and
EXTRACTS their absence of toxicity for the target contaminant micro-
Extracts should fulfil! the acceptance criteria for category B organisms must be demonstrated.
herbal medicinal products. However, where it can be If surface-active substances are used for sample preparation,
demonstrated that the method of processing would not their absence of toxicity for the target contaminant micro-
reduce the level of micro-organisms sufficiently to reach the organisms and their compatibility with inactivators used must
category B criteria, the extracts shall meet the requirements be demonstrated.
for category C herbal medicinal products. 3. ENUMERATION METHODS
The recommended acceptance criteria apply to extracts that Use the plate-count or the most-probable-number (MPN)
are to be incorporated into herbal medicinal products for oral method. The MPN method is generally the least accurate
use. More-stringent acceptance criteria may be required for method for microbial counts.
extracts that are to be incorporated into pharmaceutical The choice of method is based on factors such as the nature
preparations to be administered by other routes in order to of the LBP and the required limit of microbial
satisfy the acceptance criteria for the intended route of contamination. The chosen method must allow testing of a
administration (5.1.4'). sufficient sample size to judge compliance with the
It is recognised that for some herbal medicinal products and specification. The suitability of the method chosen must be
extracts used in their preparation the criteria given above for established.
TAMC, TYMC and bile-tolerant gram-negative bacteria cannot
be met because of the typical level of microbial contamination.
Less-stringent acceptance criteria may be applied on the basis of a
risk assessment that takes account of qualitative and quantitative
2023 Appendix XVI H V-A585

No Yes

Modification of the method may include:

- increase in the volume of diluent or culture medium;
- incorporate a neutralising agent, surface-active agent or
solubilising agent into the diluent or medium;
- combination of the above measures.

Verify the suitability of the method in presence of the LBP.

Yes No

Modification of the procedure may include:

- modify the growth conditions (temperature, time,
pH, etc.);
- use micro-organism growth inhibitors;
- use other suitable media; given in
section 6.
- combination of the above measures.

Validate the test procedure by verifying the appropriate parameters

depending on the extent of the modifications while ensuring that
the modifications do not reduce the AMCC or the YMCC.

Yes Perform the test with the modified method

and proceed as described in section 5.

Perform the test using the validated method. Follow the procedure
described in sections 3, 4
and 5.

• This section should be understood as informative suggestion. Other procedures are possible as far as they are justified.

Figure 2.6.36.-1. - Decisi.on tree for the microbial contamination enumerati.on methods
4. GROWTH PROMOTION TEST, SUITABILITY OF 4-2. PREPARATION OF TEST STRAINS
THE COUNTING METHOD AND NEGATIVE Use standardised stable suspensions of test strains or prepare
CONTROLS them as stated below. Seed lot culture maintenance
4-1. GENERAL CONSIDERATIONS techniques (seed-lot systems) are used so that the viable
The ability of the test to detect microbial contamination in micro-organisms used for inoculation are not more than
the presence of the LBP to be tested must be established. 5 passages removed from the original master seed-lot. Grow
Suitability must be confirmed if a change in testing each of the bacterial and fungal test strains separately as
performance, or in the LBP, which may affect the outcome described in Table 2.6.36.-1.
of the test is introduced.
V-A586 Appendix XVI H 2023

Use buffered sodium chloride-peptone solution pH 7.0 or is not possible due to inhibitory activity of the LBP, further
phosphate buffer solution pH 7 .2 to make test suspensions; appropriate protocols must be developed (see decision tree
to suspend A. brasz7iensis spores, 0.05 per cent of shown in Figure 2.6.36.-1). If inhibition of growth by the
polysorbate 80 may be added to the buffer. Use the sample cannot otherwise be avoided, the aliquot of the test
suspensions within 2 h or within 24 h if stored at 2-8 °C. micro-organisms may be added after neutralisation or
As an alternative to preparing and then diluting a fresh dilution.
suspension of vegetative cells of A. brasiliensis or B. subtilis, a Inhibitory activity The number of micro-organisms
stable spore suspension is prepared and then an appropriate recovered from the prepared sample diluted as described in
volume of the spore suspension is used for test inoculation. 4-5-2 and incubated following the procedure described in
The stable spore suspension may be maintained at 2-8 °C for 4-5-3, is compared to the number of micro-organisms
a validated period of time. recovered from the control preparation.
4-3. NEGATIVE CONTROL If growth is inhibited (reduction by a factor greater than 2),
To verify testing conditions, a negative control is performed follow the decision tree shown in Figure 2.6.36.-1 and
using the chosen diluent in place of the test preparation. modify the procedure for the particular enumeration test to
There must be no growth of micro-organisms. A negative ensure the validity of the results. Modification of the
control is also performed when testing the LBP as described procedure may include, for example, (1) an increase in the
in section 5. A failed negative control requires an volume of the diluent or culture medium, (2) incorporation
investigation. of specific or general neutralising agents into the diluent, (3)
4-4. GROWTH PROMOTION OF THE MEDIA membrane filtration, or (4) a combination of the above
Test each batch of ready-prepared medium and each batch measures.
of medium, prepared either from dehydrated medium or If growth is still inhibited (reduction by a factor greater than
from the ingredients described. 2) continue to follow the decision tree shown in
Inoculate portions/plates of casein soya bean digest broth and Figure 2.6.36.-1.
casein soya bean digest agar with a small number (not more A new approach needs to be validated using the appropriate
than 100 CFU) of the micro-organisms indicated in parameters depending on the extent of modification, while
Table 2.6.36.-1, using a separate portion/plate of medium for ensuring that the modifications do not reduce the AMCC or
each. Inoculate plates of Sabouraud-dextrose agar with a the YMCC.
small number (not more than 100 CFU) of the micro- 4-5-3. Recovery of the test micro-organisms in the
organisms indicated in Table 2.6.36.-1, using a separate plate presence of an LBP
of medium for each. Incubate in the conditions described in For each of the test micro-organisms listed, separate tests are
Table 2.6.36.-1. performed. Only micro-organisms of the added test strain are
For solid media, growth obtained must not differ by a factor counted.
greater than 2 from the calculated value for a standardised 4-5-3-1. Plate-count methods Perform plate-count
inoculum. For a freshly prepared inoculum, growth of the methods at least in duplicate for each medium and use the
micro-organisms comparable to that previously obtained with mean count of the result.
a previously tested and approved batch of medium occurs.
4-5-3-1-1. Pour-plate method
Liquid media are suitable if clearly visible growth of the
micro-organisms comparable to that previously obtained with For Petri dishes 9 cm in diameter, add to the dish 1 mL of
a previously tested and approved batch of medium occurs. the sample prepared as described under 4-5-1 and 4-5-2, and
15-20 mL of casein soya bean digest agar or Sabouraud-
4-5. SUITABILIIY OF THE COUNTING METHOD IN
dextrose agar, both media being at not more than 45 °C.
THE PRESENCE OF THE LBP TO BE TESTED
If larger Petri dishes are used, the amount of agar medium is
4-5-1. Preparation of the sample increased accordingly. For each of the micro-organisms listed
The method for sample preparation depends upon the in Table 2.6.36.-1, at least 2 Petri dishes are used. Incubate
physical characteristics of the LBP to be tested. If none of the plates as indicated in Table 2.6.36.-1. Take the
the procedures described below can be demonstrated to be arithmetic mean of the counts per medium and calculate the
satisfactory, an alternative procedure must be developed. number of CFU in the original inoculum.
Prepare a homogenous suspension of the LBP to be tested 4-5-3-1-2. Surface-spread method
(usually a 1 in 10 dilution is prepared) in buffered sodium For Petri dishes 9 cm in diameter, add 15-20 mL of casein
chloride-peptone solution pH 7.0, phosphate buffer solution soya bean digest agar or Sabouraud-dextrose agar at about
pH 7.2 or casein soya bean digest broth. A surface-active 45 °C to each Petri dish and allow to solidify. If larger Petri
agent such as a 1 g/L solution of polysorbate 80 may be dishes are used, the volume of the agar is increased
added to assist the suspension of poorly wettable substances. accordingly. Dry the plates, for example in a laminar-air-flow
If necessary, adjust to pH 6-8. Further dilutions, where cabinet or an incubator. For each of the micro-organisms
necessary, are prepared with the same diluent. listed in Table 2.6.36.-1, at least 2 Petri dishes are used.
4-5-2. Inoculation and dilution Spread a measured volume of not less than 0.1 mL of the
Add to the sample prepared as described above (4-5-1) and sample prepared as described under 4-5-1 and 4-5-2 over the
to a control (with no test material included) a sufficient surface of the medium. Incubate and count as prescribed
volume of the microbial suspension to obtain an inoculum of under 4-5-3-1-1.
not more than 100 CFU. The volume of the suspension of 4-5-3-2. Most-probable-number (MPN) method The
the inoculum should not exceed 1 per cent of the volume of precision and accuracy of the MPN method is less than the
diluted LBP. plate-count method. Unreliable results are obtained
To demonstrate acceptable recovery of the test micro- particularly for the enumeration of moulds. For these reasons
organisms from the LBP, the lowest possible dilution factor the MPN method is reserved for the enumeration of AMCC
of the prepared sample must be used for the test. Where this
2023 Appendix XVI H V-A587

Table 2.6.36.-1. - Preparation and use of test micro-organisms

Micro-organism Preparation of test Growth promotion Suitability of counting method in the presence
strain oftheLBP

Aerobic microbial Yeasts and moulds Aerobic microbial Yeasts and moulds
contamination count contamination count contamination count contamination count
(AMCC) (YMCC) (AMCC) (YMCC)

Staphylococcus aureus such Casein soya bean digest Casein soya bean digest
Casein soya bean digest
as: agar and casein soya agarlMPN: casein soya
agar or casein soya bean
ATCC 6538 bean digest broth bean digest broth
digest broth - -
NCIMB 9518 -S 100 CFU -S 100 CFU
30-35 C
CIP 4.83 30-35 "C 30-35 'C
18-24 h
NBRC 13276 -S 3 days -S 3 days

Pseudomonas aeruginosa Casein soya bean digest Casein soya bean digest
Casein soya bean digest
such as: agar and casein soya agar/MPN: casein soya
agar or casein soya bean
ATCC 9027 bean digest broth bean digest broth
NCIMB 8626
digest broth
-S 100 CFU
- -S 100 CFU
-
30-35 "C
CIP 82.118 30-35 °C 30-35 'C
18-24 h
NBRC 13275 -S 3 days -S 3 days

Casein soya bean digest Casein soya bean digest

Bacillus subtilis such as: Casein soya bean digest
agar and casein soya agar/MPN: casein soya
ATCC 6633 agar or casein soya bean
bean digest broth bean digest broth
NCIMB 8054 digest broth - -
CIP 52.62 30-35 cc
-S 100 CFU -S 100 CFU
30-35 "C 30-35 'C
NBRC 3134 18-24 h
-S 3 days -S 3 days

Casein soya bean digest

Candida albicans such as: Sabouraud- dextrose Casein soya bean digest Sabouraud- dextrose Sabouraud- dextrose
agar
ATCC 10231 agar or Sabouraud- agar agar agar
-S 100 CFU
NCPF 3179 dextrose broth -S 100 CFU -S 100 CFU -S 100 CFU
30-35 'C
IP 48.72 20-25 "C 30-35 'C 20-25 'C 20-25 "C
-S 5 days
NBRC 1594 2-3 days -S 5 days -S 5 days -S 5 days
MPN: not applicable

Aspergillus brasiliensis such Sabouraud- dextrose Casein soya bean digest

Casein soya bean digest Sabouraud- dextrose Sabouraud- dextrose
as: agar or potato-dextrose agar
agar agar agar
ATCC 16404 agar -S 100 CFU
20-25 'C
-S 100 CFU -S 100 CFU -S 100 CFU
IMl 149007 30-35 C
30-35 'C 20-25 'C 20-25 C
IP 1431.83 5-7 days, or until good -S 5 days
-S 5 days -S 5 days -S 5 days
NBRC 9455 sporulation is achieved MPN: not applicable

in situations where no other method is available. If the use of 5. TESTING OF LIVE BIOTHERAPEUTIC
the method is justified, proceed as follows. PRODUCTS
Prepare a series of at least 3 serial tenfold dilutions of the 5-1. AMOUNT USED FOR THE TEST
LBP as described under 4-5-1 and 4-5-2. From each level of Unless otherwise prescribed, use 10 g or 10 mL of the LBP
dilution, 3 aliquots of 1 g or 1 mL are used to inoculate to be tested taken with the precautions referred to above.
3 tubes with 9-10 mL of casein soya bean digest broth. For LBP where the total number of entities in a batch is less
If necessary, a surface-active agent such as polysorbate 80 than 200 (e.g. samples used in clinical trials), the sample size
may be added to the medium. Thus, if 3 levels of dilution may be reduced to 2 units, or 1 unit if the size is less than
are prepared, 9 tubes are inoculated. 100.
Incubate all tubes at 30-35 °C for not more than 3 days. Select the sample(s) at random from the bulk material or
If reading of the results is difficult or uncertain owing to the from the available containers of the preparation. To obtain
nature of the LBP to be tested, subculture in the same broth, the required quantity, mix the contents of a sufficient
or in casein soya bean digest agar, for 1-2 days at the same number of containers to provide the sample.
temperature and use these results. Determine the most
probable number of micro-organisms per gram or millilitre of Table 2.6.36.-2. - Most-probable-number ·values of micro-
the LBP to be tested from Table 2.6.36.-2. orgamsms
4-6. RESULTS AND INTERPRETATION Observed combinations of numbers of MPNper 95 per cent
When verifying the suitability of the plate-count method, a tubes showing growth in each set gram or confidence
per limits
mean count of any of the test organisms not differing by a Number of grams or millilitres of LBP millilitre of
factor greater than 2 from the value of the control defined in per tube LBP
4-5-2 in the absence of the LBP must be obtained. When
0.1 0.01 0.001
verifying the suitability of the MPN method the calculated
value from the inoculum must be within 95 per cent 0 0 0 <3 0 - 9.4
confidence limits of the results obtained with the control.
0 0 1 3 0.1 - 9.5
If the above criteria cannot be met, follow the decision tree
shown in Figure 2.6.36.-1 and modify the procedure for the 0 1 0 3 0.1 - 10
particular enumeration test to ensure the validity of the
0 l l 6.1 1.2 - 17
results.
0 2 0 6.2 1.2 - 17
V-A588 Appendix XVI H 2023

Observed combinations of numbers of MPNper 95 per cent digest agar at 30-35 °C for 3-5 days and the plates of
tubes showing growth in each set gram or confidence Sabouraud-dextrose agar at 20-25 °C for 5-7 days. Select the
per limits plates corresponding to a given dilution and showing the
Number of grams or millilitres of LBP millilitre of
per tube LBP highest number of contaminating colonies less than 250 for
AMCC and 50 for YMCC. Take the arithmetic mean per
0.1 0.01 0.001 culture medium of the counts and calculate the number
0 3 0 9.4 3.5 - 35 of CFU per gram or per millilitre of LBP.
5-2-1-2. Surface-spread method Prepare the sample
I 0 0 3.6 0.2 - 17
using a method that has been shown to be suitable as
I 0 I 7.2 1.2 - 17 described in section 4. Prepare at least 2 Petri dishes for each
medium and each level of dilution. For incubation and
I 0 2 II 4 - 35
calculation of the number of contaminant CFU proceed as
I 1 0 7.4 1.3 - 20 described for the pour-plate method.
I 1 I II 4 - 35
5-2-2. Most-probable-number method
Prepare and dilute the sample using a method that has been
I 2 0 II 4 - 35 shown to be suitable as described in section 4. Incubate all
5 - 38
tubes at 30-35 °C for 3-5 days. Subculture if necessary, using
I 2 I 15
the procedure shown to be suitable. Record for each level of
1 3 0 16 5 - 38 dilution the number of tubes showing microbial growth.
Determine the most probable number of contaminant micro-
2 0 0 9.2 1.5 - 35
organisms per gram or millilitre of the LBP to be tested from
2 0 I 14 4 - 35 Table 2.6.36.-2.
2 0 2 20 5 - 38 5-3. INTERPRETATION OF THE RESULTS
The aerobic microbial contamination count (AMCC) is
2 I 0 15 4 - 38 considered to be equal to the number of contaminating CFU
2 I I 20 5 - 38
found using casein soya bean digest agar; if colonies of
contaminating yeasts/moulds are detected on this medium,
2 1 2 27 9 - 94 they are counted as part of the AMCC. The combined
2 2 0 21 5 - 40
yeasts/moulds contaminants count (YMCC) is considered to
be equal to the number of CFU found using Sabouraud-
2 2 I 28 9 - 94 dextrose agar; if colonies of contaminating bacteria are
2 2 2 35 9 - 94
detected on this medium, they may be excluded from the
YMCC. When the YMCC is expected to exceed the
2 3 0 29 9 - 94 acceptance criterion due to bacterial growth, Sabouraud-
I
dextrose agar containing antibiotics can be used (see decision
2 3 36 9 - 94
tree shown in Figure 2.6.36.-1). If the count is carried out by
3 0 0 23 5 - 94 the MPN method the calculated value is the AMCC.
3 0 I 38 9 - 104 When an acceptance criterion for microbial contamination is
prescribed it is interpreted as follows:
3 0 2 64 16 - 181 10 1 CFU: maximum acceptable count = 20;
3 I 0 43 9 - 181
- 102 CFU: maximum acceptable count = 200;
- 103 CFU: maximum acceptable count = 2000.
3 I 1 75 17 - 199
The recommended solutions and media are described in
3 I 2 120 30 - 360 general chapter 2.6.13.

3 I 3 160 30 - 380 6. APPROACHES FOR ENUMERATION OF

MICROBIAL CONTAMINANTS IN THE PRESENCE
3 2 0 93 18 - 360 OF INHIBITION DUE TO THE LBP
3 2 1 150 30 - 380 This section should be understood as informative suggestion.
Other procedures are possible as far as they are justified.
3 2 2 210 30 - 400 Examples of approaches for enumeration of microbial
3 2 3 290 90 - 990
contaminants for LBP inhibiting the microbial contamination
enumeration are indicated below.
3 3 0 240 40 - 990
Each approach needs to be validated using the appropriate
3 3 1 460 90-1980 parameters depending on the extent of modification, while
ensuring that the modifications do not reduce the AMCC or
3 3 2 1100 200 - 4000
the YMCC.
3 3 3 > 1100 The test strains indicated in Table 2.6.36.-1 are used.
Preparation of test strains is performed as described in 4-2.
Negative control is performed as described in 4-3. Growth
5-2. EXAMINATION OF THE LBP
promotion and suitability of new culture media chosen is
5-2-1. Plate-count methods carried out by inoculating each batch of the medium with a
5-2-1-1. Pour-plate method Prepare the sample using a small number (not more than 100 CFU) of the micro-
method that has been shown to be suitable as described in organisms. Growth obtained does not differ by a factor
section 4. Prepare for each medium at least 2 Petri dishes for greater than 2 from the calculated value for a standardised
each level of dilution. Incubate the plates of casein soya bean
2023 Appendix XVI H V-A589

inoculum counted on casein soya bean digest agar (AMCC) If components other than the active substance
or Sabouraud-dextrose agar (YMCC). (i.e. the micro-organism) of the LBP to be tested have
6-1. ENUMERATION OF AEROBIC MICROBIAL inhibitory activity against the target contaminant micro-
CONTAMINANTS organisms, this is insofar as possible removed or neutralised
LBP containing lactic acid bacteria may be tested for aerobic as described in general chapter 2. 6.36.
microbial contamination on sugar-free agar plates incubated If surface-active agents are used for sample preparation, their
aerobically at 30-35 °C for 72 h. Lactic acid bacteria grow absence of toxicity for the target contaminant micro-
slowly as pinpoint colonies, while contaminants can easily be organisms and their compatibility with inactivators used must
detected as larger colonies and are fast growing. be demonstrated as described in general chapter 2.6.36.
Alternatively, the aerobic microbial contamination may be 3. GROWTH-PROMOTING AND INHIBITORY
tested on casein soya bean digest agar plates supplemented PROPERTIES OF THE MEDIA, SUITABILITY OF
with 5 per cent of sheep blood at 30-35 °C for 44-48 h. THE TEST AND NEGATIVE CONTROLS
The addition of blood enhances growth of contaminants and The ability of the test to detect the target micro-organisms in
formation of distinctive colony morphology that are better the presence of the LBP to be tested must be established.
discriminated in the presence of lactic acid bacteria. Suitability must be confirmed if a change is introduced in the
For LBP containing Bacillus clausii spores, the aerobic testing procedure or the LBP, which may affect the outcome
microbial contamination may be enumerated on sporulating of the test.
agar. The medium, by promoting the sporulation of Bacillus
clausii, inhibits the vegetative growth of the LBP micro-
3-1. PREPARATION OF TEST STRAINS
Use standardised stable suspensions oftest strains or prepare
organism, making the detection of contaminants possible.
them as stated below. Seed lot culture maintenance
Plates are incubated at 33-37 °C for 48 h.
techniques (seed-lot systems) are used so that the viable
LBP containing Saccharomyces cerevisiae, var. boulardii may be
micro-organisms used for inoculation are not more than
tested for aerobic microbial contamination on casein soya
5 passages removed from the original master seed-lot.
bean digest agar containing cycloheximide, a suitable
inhibitor of Saccharomyces. Plates are incubated at 30-35 °C 3-1-1. Aerobic micro-organisms
for 3-5 days. Grow each of the bacterial test strains separately in casein
For LBP for which suitable media and growth conditions to soya bean digest broth or on casein soya bean digest agar at
enumerate aerobic microbial contamination are not available, 30-35 °C for 18-24 h. Grow the test strain for Candida
not only the absence of specific contaminants (Escherichia albicans separately on Sabouraud-dextrose agar or in
coli, Staphylococcus aureus, Pseudomonas aemgi,nosa, Salmonella Sabouraud-dextrose broth at 20-25 °C for 2-3 days.
spp. and bile-tolerant Gram-negative bacteria) is verified - Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
using the method described in general chapter 2.6.38, and CIP 4.83 or NBRC 13276;
tests for other contaminating micro-organisms are carried out - Pseudomonas aemgi,nosa such as ATCC 9027,
based on a risk assessment (e.g. specific environmental NCIMB 8626, CIP 82.118 or NBRC 13275;
contaminants). - Escherichia coli such as ATCC 8739, NCIMB 8545,
CIP 53.126 or NBRC 3972;
6-2. ENUMERATION OF YEAST AND MOULD
- Salmonella enterica subsp. enterica serovar Typhimurium,
CONTAMINANTS
such as ATCC 14028 or, as an alternative, Salmonella
For LBP containing bacteria, the test may be carried out
enterica subsp. enterica serovar Abony such as
with Sabouraud-dextrose agar containing antimicrobials
NBRC 100797, NCTC 6017 or CIP 80.39;
(e.g. chloramphenicol) incubated at 20-25 °C for 5-7 days.
- Candida albicans such as ATCC 10231, NCPF 3179,
For LBP containing Saccharomyces cerevisae, var. boulardii, IP 48. 72 or NBRC 1594.
yeasts and moulds may be determined using several media
Use buffered sodium chloride-peptone solution pH 7.0 or
(Sabouraud-dextrose agar supplemented with
phosphate buffer solution pH 7.2 to make test suspensions.
chloramphenicol and cycloheximide, Czapek-Dox agar,
Use the suspensions within 2 h or within 24 h if stored at
potato dextrose agar).
2-8 °C.
Microbiological Examination of Live Biotherapeutic 3-1-2. Anaerobic micro-organisms
Products: Tests for Specified Micro-organisms Clostridia. Use a Clostridium sporogenes strain such as
(Ph. Eur. method 2.6.38) ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651)
1. INTRODUCTION or ATCC 19404 (NCTC 532 or CIP 79.03) or
The tests described hereafter are intended for determination NBRC 14293. Grow the clostridial test strain under
of the absence or limited occurrence of specified micro- anaerobic conditions in reinforced medium for Clostridia at
organisms that may be detected under the conditions 30-35 °C for 24-48 h. As an alternative to preparing and
described. The tests are designed primarily to determine then diluting down a fresh suspension of vegetative cells of C.
whether a live biotherapeutic product (LBP) complies with sporogenes, a stable spore suspension is used for test
the established specification for microbiological quality. inoculation. The stable spore suspension may be maintained
at 2-8 °C for a validated period.
Alternative microbiological procedures, including automated
methods, may be used, provided that their equivalence to the 3-2. NEGATIVE CONTROL
Pharmacopoeia method has been demonstrated. To verify testing conditions, a negative control is performed
using the chosen diluent in place of the test preparation.
2. GENERAL PROCEDURES
There must be no growth of micro-organisms. A negative
The preparation of samples is carried out as described in control is also performed when testing the LBP as described
general chapter 2.6.36. in section 4. A failed negative control requires an
If the LBP to be tested prevents the detection of specified investigation.
micro-organisms apply the decision tree shown in Figure
2.6.38.-1.
V-A590 Appendix XVI H 2023

Is the method
No detection of a Yes
·sm described i
itable for the L

Modification of the method may include:

- increase in the volume of diluent or culture medium;
- incorporate a neutralising agent, surface-active agent or
solubilising agent into the diluent or medium;
- membrane filtration;
- combination of the above measures.

Verify the suitability of the method in presence of the LBP.

Yes No

Modification of the procedure may include:

- modify the growth conditions (temperature, time,
pH, etc.);
- use micro-organism growth inhibitors;
given in
- use other suitable media; section 5.
- combination of the above measures.

Validate the test procedure by verifying the appropriate parameters depending

on the extent of the modifications while ensuring that the same result
(presence/absence) is achieved for the specified micro-organism.

Yes Perform the test for absence with the modified

method and proceed as described in the chapter.

Perform the test using the validated method. Perform the test for
absence as described
in this chapter.

* This section should be understood as informative suggestion. Other procedures are possible as far as they are justified.

Figure 2.6.38.-1. - Deciswn tree for the test for specified micro-organisms

3-3. GROWTH PROMOTION AND INHIBITORY 3-3-1. Test for growth promoting properties, liquid
PROPERTIES OF THE MEDIA media
Test each batch of ready-prepared medium and each batch Inoculate a portion of the appropriate medium of the same
of medium prepared either from dehydrated medium or from volume to be used in the test (see sections 3-4 and 4) with a
ingredients. small number (not more than 100 CFU) of the appropriate
Verify suitable properties of relevant media as described in micro-organism. Incubate at the specified temperature for not
Table 2.6.38.-1. more than the shortest period of time specified in the test.
Clearly visible growth of the micro-organism comparable to
2023 Appendix XVI H V-A591

that previously obtained with a previously tested and Table 2.6.38.-1. - Growth promoting, inhibitory and indicative
approved batch of medium occurs. properties of media
3-3-2. Test for growth promoting properties, solid Medium Property Test strains
media
Perform the surface-spread method, inoculating each plate Growth E. co/i
Enterobacteria
promoting P. aerugi"nosa
with a small number (not more than 100 CFU) of the enrichment
Test for bile- broth-Mossel
appropriate micro-organism. Incubate at the specified Inhibitory S. aureus
tolerant Gram-
temperature for not more than the shortest period of time negative bacteria
Growth
specified in the test. Growth of the micro-organism is Violet red bile
promoting
E. coli
comparable to that previously obtained with a previously glucose agar P. aerugi1Wsa
+ indicative
tested and approved batch of medium and does not differ by
a factor greater than 2. Growth
Mac Conkey
E.coli
promoting
3-3-3. Test for inhibitory properties, liquid or solid broth
media Test for Inhibitory S. aureus
Escherichia coli
Inoculate the appropriate medium with at least 100 CFU of Growth
the appropriate micro-organism. Incubate at the specified MacConkey agar promoting E.coli
temperature for not less than the longest period of time + indicative
specified in the test. No growth of the test micro-organism
Salmonella
occurs. enterica subsp.
3-3-4. Test for indicative properties enten·ca serovar
Rappaport
Growth Typhimurium or
Perform the surface-spread method, inoculating each plate Vassiliadis
promoting Salmonella
with a small number (not more than 100 CFU) of the Salmonella
emerica subsp.
enrichment
appropriate micro-organism. Incubate at the specified broth
enterica serovar
temperature for not more than the shortest validated period Abony
of time specified in the test. Colonies are comparable in Test for
Inhibitory S. aureus
appearance and indication reactions to those previously Salmonella
obtained with a previously tested and approved batch of Salmonella
enterica subsp.
medium.
Growth enterica serovar
Xylose, lysine,
3-4. SUITABILITY OF THE TEST METHOD deoxycholate
promoting Typhimurium or
For each LBP to be tested, perform the sample preparation + Salmonella
agar
indicative enterica subsp.
as described in the relevant paragraph in section 4. Inoculate enten·ca serovar
the test strains individually. Add each test strain at the time Abony
of mixing, in the prescribed growth medium. Use a number
of micro-organisms equivalent to not more than 100 CFU in Growth
Test for P. aerugi.nosa
promoting
the inoculated test preparation. The inoculum should not Pseudomonas Cetrimide agar
aeruginosa
exceed 1 per cent of the volume of the growth medium. Inhibitory E. coli
Perform the test as described in the relevant paragraph in
Growth
section 4 using for each step of the test the shortest promoting
incubation time to be used in the test. Test for S. aureus
Mannitol salt +
Staphylococcus
The specified micro-organisms must be detected with the agar indicative
aureus
appearance and indication reactions described in section 4. Inhibitory E. coli
If non-characteristic colonies and reactions are obtained, the
method may still be suitable provided that all colony types Reinforced
Growth
medium for C. sporogenes
are identified when performing the test. promoting
Test for Clostridia
Any inhibitory activity of the LBP against the target micro- Clostridia
organisms necessitates a modification of the test procedure Growth
Columbia agar C. sporogenes
promoting
(see decision tree shown in Figure 2.6.38.-1) and then the
suitability for the LBP must be confirmed. Sabouraud- Growth
C. albicans
dextrose broth promoting
4. TESTING OF LIVE BIOTHERAPEUTIC
Test for Candida
PRODUCTS Growth
albicans
4-1. BILE-TOLERANT GRAM-NEGATIVE BACTERIA Sabouraud- promoting
C. albicans
dextrose agar +
4-1- 1. Sample preparation and pre-incubation indicative
Prepare a sample using a 1 in 10 dilution of not less than 1 g
or 1 mL of the LBP to be tested as described in general
30-35 °C for 24-48 h. Subculture on violet red bile glucose
chapter 2.6.36, but using casein soya bean digest broth as the
agar. Incubate at 30-35 °C for 18-24 h.
chosen diluent, mix and incubate at 20-25 °C for a time
sufficient to resuscitate the bacteria but not sufficient to The LBP complies with the test if there is no growth of
encourage multiplication of the micro-organisms (usually 2 h colonies.
but not more than 5 h). 4-2. ESCHER/CHIA COLi
4-1-2. Test for absence 4-2-1. Sample preparation and pre-enrichment
Unless otherwise prescribed, use the volume corresponding Prepare a sample using a 1 in 10 dilution of not less than 1 g
to 1 g of the LBP, as prepared in 4-1-1, to inoculate or 1 mL of the LBP to be tested as described in general
enterobacteria enrichment broth-Mossel. Incubate at chapter 2.6.36 and use 10 mL or the quantity corresponding
to 1 g or 1 mL to inoculate a suitable amount (determined as
V-A592 Appendix XVI H 2023

described under 3-4) of casein soya bean digest broth, mix The LBP complies with the test if colonies of the types
and incubate at 30-35 °C for 18-24 h. described are not present or if the confirmatory identification
4-2-2. Selection and subculture tests are negative.
Shake the container, transfer 1 mL of the casein soya bean 4-6. CLOSTRIDIA
digest broth to 100 mL of MacConkey broth and incubate at 4-6-1. Sample preparation and heat treatment
42-44 °C for 24-48 h. Subculture on MacConkey agar at Prepare a sample using a 1 in 10 dilution (with a minimum
30-35 °c for 18-72 h. total volume of 20 mL) of not less than 2 g or 2 mL of the
4-2-3. Interpretation LBP to be tested as described in general chapter 2. 6.36.
Growth of colonies indicates the possible presence of E. coli. Divide the sample into 2 portions of at least 10 mL. Heat 1
This is confirmed by identification tests. portion at 80 °C for 10 min and cool rapidly. Do not heat
The LBP complies with the test if colonies are not present or the other portion.
if the confirmatory identification tests are negative. 4-6-2. Selection and subculture
4-3. SALMONELI.A Use 10 mL or the quantity corresponding to 1 g or 1 mL of
the LBP to be tested of both portions to inoculate suitable
4-3-1. Sample preparation and pre-enrichment
amounts (determined as described under 3-4) of reinforced
Prepare the LBP to be tested as described in general
medium for Clostridia. Incubate under anaerobic conditions
chapter 2.6.36 and use the quantity corresponding to not less
at 30-35 °C for 48 h. After incubation, make subcultures
than 10 g or 10 mL to inoculate a suitable amount
from each container on Columbia agar and incubate under
(determined as described under 3-4) of casein soya bean
anaerobic conditions at 30-35 °C for 48-72 h.
digest broth, mix and incubate at 30-35 °C for 18-24 h.
4-6-3. Interpretation
4-3-2. Selection and subculture
The occurrence of anaerobic growth of rods (with or without
Transfer 0.1 mL of the casein soya bean digest broth to
endospores) giving a negative catalase reaction may indicate
10 mL of Rappaport Vassiliadis Salmonella enrichment broth
the presence of Clostridia. This is confirmed by identification
and incubate at 30-35 °C for 18-24 h. Subculture on xylose,
tests.
lysine, deoxycholate agar. Incubate at 30-35 °C for 18-48 h.
The LBP complies with the test if colonies of the types
4-3-3. Interpretation
described are not present or if the confirmatory identification
Growth of well-developed, red colonies, with or without tests are negative.
black centres indicates the possible presence of Salmonella.
This is confirmed by identification tests. 4-7. CANDIDA ALBICANS
The LBP complies with the test if colonies of the types 4-7-1. Sample preparation and pre-enrichment
described are not present or if the confirmatory identification Prepare the LBP to be tested as described in general
tests are negative. chapter 2. 6. 36 and use 10 mL or the quantity corresponding
to not less than 1 g or 1 mL to inoculate 100 mL of
4-4. PSEVDOMONAS AERVGINOSA
Sabouraud-dextrose broth and mix. Incubate at 30-35 °C for
4-4-1. Sample preparation and pre-enrichment 3-5 days.
Prepare a sample using a 1 in 10 dilution of not less than 1 g
4-7-2. Selection and subculture
or 1 mL of the LBP to be tested as described in general
Subculture on Sabouraud-dextrose agar and incubate at
chapter 2. 6. 36 and use 10 mL or the quantity corresponding
30-35 °C for 24-48 h.
to 1 g or 1 mL to inoculate a suitable amount (determined as
described under 3-4) of casein soya bean digest broth and 4-7-3. Interpretation
mix. Incubate at 30-35 °C for 18-24 h. Growth of white colonies may indicate the presence of C.
albicans. This is confirmed by identification tests.
4-4-2. Selection and subculture
Subculture on cetrimide agar and incubate at 30-35 °C for The LBP complies with the test if colonies of the types
18-72 h. described are not present or if the confirmatory identification
tests are negative.
4-4-3. Interpretation
Growth of colonies indicates the possible presence of P. 5. APPROACHES FOR TESTING SPECIFIED
aeruginosa. This is confirmed by identification tests. MICRO-ORGANISMS AND ADDITIONAL TESTING
The LBP complies with the test if colonies are not present or This section should be understood as informative suggestion.
if the confirmatory identification tests are negative. Other procedures are possible as far as they are justified.
4-5. STAPHYLOCOCCUS AVREUS If the detection of the specified micro-organism is inhibited
by the LBP, its detection is carried out under conditions that
4-5-1. Sample preparation and pre-enrichment neutralise the inhibition or limit the growth of the LBP
Prepare a sample using a 1 in 10 dilution of not less than 1 g micro-organisms. The modified test for specified micro-
or 1 mL of the LBP to be tested as described in general organisms is validated using the appropriate validation
chapter 2.6.36 and use 10 mL or the quantity corresponding parameters depending on the extent of the modifications
to 1 g or 1 mL to inoculate a suitable amount (determined as while ensuring that the same result (presence/absence) is
described under 3-4) of casein soya bean digest broth and achieved for the specified micro-organism.
mix. Incubate at 30-35 °C for 18-24 h.
A suitable combination of antibiotics may be used to inhibit
4-5-2. Selection and subculture the LBP and detect the specified micro-organism.
Subculture on mannitol salt agar and incubate at 30-35 °C For example, for LBP samples containing S. cerevisiae, var.
for 18-72 h. boulardii, pre-enrichment and selective media (Sabouraud-
4-5-3. Interpretation dextrose broth and agar) can be supplemented with
Growth of yellow/white colonies surrounded by a yellow zone chloramphenicol and cycloheximide to detect contaminant
indicates the possible presence of S. aureus. This is confirmed C. albicans.
by identification tests.
2023 Appendix XVII A V-A593

Alternative pre-enrichment media (e.g. buffered peptone

medium for Salmonella - see general chapter 2.6.31), media
and test/growth conditions may be used to support growth of
Appendix XVII
the target micro-organisms while limiting growth of the A. Particle Size of Powders
micro-organisms of the LBP.
LBP containing Bacillus clausii spores are tested for 1. Particle Size Classification of Powders (Sieve
contaminating Bacillus cereus on chromogenic selective agar Test)
media plates. Incubation time and temperature depends on (Ph. Bur. method 2. 9.12)
the specific medium. Where the degree of fineness of powders is determined by
A proposed approach to detect Bacillus cereus is described sieving, it may be expressed with reference to sieves that
below. comply with the specifications for sieves in general chapters
Prepare a sample using a 1 in 10 dilution of not less than 1 g 2.1.4. Sieves and 2. 9. 38. Particle-size distribution estimation by
or 1 mL of the LBP to be tested and use 10 mL or the analytical sieving.
quantity corresponding to 1 g or 1 mL to inoculate a suitable The terms used in general chapter 2.9.35. Powder fineness to
amount of casein soya bean digest broth, mix and incubate at describe the degree of fineness apply.
35-37 °C for 18-24 h. Subculture on selective media (e.g. B. Assemble the sieves and operate according to general
cereus Mossel agar). For each LBP to be tested, validate
chapter 2. 9. 38. Weigh the separated fractions of the powder.
incubation time and temperature. The appearance of
B. cereus colonies depends on the medium used. The LBP The symbol Q3 (x) as defined in general chapter 2.9.35 is
complies with the test if no B. cereus colonies are present. generally used to denote the cumulative distribution by mass
or volume of particles with a dimension less than or equal to
For LBP containing E. coli, use Bnterococcus faecalis and X.
Bnterococcus faecium as microbial indicators of faecal
contamination to replace the test for absence of B. coli. When a single sieve is used, the fineness of the powder can
Use selective or differential chromogenic media and, for each also be expressed as a sieve number; this number
LBP to be tested, validate incubation time and temperature. corresponds to the particular sieve through which not less
The LBP complies with the test when there is no growth of than 97 per cent m/m of the powder passes, unless otherwise
Bnterococcus spp. prescribed.
A search for pathogenic B. coli strains may be carried out by Additional points for monographs other than those
suitable methods (e.g. molecular methods for the detection of of the European Pharmacopoeia
gene stx or eae) based on risk-assessment. Within the monographs of the British Pharmacopoeia, the
6. RECOMMENDED SOLUTIONS AND CULTURE above terms may be used to specify the degree of coarseness
MEDIA or fineness of a medicinal or pharmaceutical substance in
The solutions and culture media mentioned in this chapter powder form that is to be incorporated into a formulated
and described in chapter 2.6.13 have been found satisfactory preparation. The following terms may also be used for such
for bile-tolerant Gram-negative bacteria, B. coli, P. aeruginosa, purposes.
S. aureus, Clostridia, Salmonella and C. albicans. Other media When the use of sieves is inappropriate, the definition is
may be used provided that their suitability can be expressed in terms of the particle size as determined by
demonstrated and the analytical method where they are used suitable microscopical examination.
is validated with the appropriate validation parameters. Moderately coarse powder Not less than 95% by weight
passes through a number 710 sieve and not more than 40%
by weight passes through a number 250 sieve.
Micro.fine powder Not less than 90% by weight passes
through a number 45 sieve.
Superfine powder Not less than 90% by number of the
particles are less than 10 ~1m in size.
2. Powder Fineness 1
(Ph. Bur. method 2.9.35)
Particle-size distribution is estimated by analytical
sieving (2.9.38) or by application of other suitable methods
where appropriate. A simple descriptive classification of
powder fineness is provided in this chapter. For practical
reasons, sieves are commonly used to measure powder
fineness. Sieving is most suitable where a majority of the
particles are larger than about 75 µm, although it can be
used for some powders having smaller particle sizes where
the method can be validated. Light diffraction is also a
widely used technique for measuring the size of a wide range
of particles.

1 This chapter has undergone phamzacopoeial harmonisation. See chapter

5. 8. Phamiacopoeial ham10nziation.
V-A594 Appendix XVII B 2023

Where the cumulative distribution has been determined by

analytical sieving or by application of other methods, particle
B. Sieves and Filters
size may be characterised in the following manner: 1. Sieves
(Ph. Eur. method 2.1.4)
x90 particle size corresponding to 90 per cent of the cumulative
undersize distribution; Sieves are constructed of suitable materials with square
x50 median particle size (i.e. 50 per cent of the particles are smaller meshes. For purposes other than analytical procedures, sieves
and 50 per cent of the particles are larger);
x 10 particle size corresponding to JO per cent of the cumulative with circular meshes may be used, the iriternal diameters of
undersize distribution. which are 1.2 5 times the aperture of the square mesh of the
correspondirig sieve size. There must be no reaction between
It is recognised that the symbol d is also widely used to the material of the sieve and the substance being sifted.
designate these values. Therefore, the symbols dgo, dso, d10 Degree of comminution is prescribed in the monograph using
may be used. the sieve number, which is the size of the mesh in
Toe following parameters may be defined based on the micrometres, given in parenthesis after the name of the
cumulative distribution. substance (Table 2.1.4.-1).
Qh) = cumulative distribution of particles with a dimension Maximum tolerance 1 for an aperture (+ X): no aperture size
less than or equal to x where the subscript r reflects the shall exceed the nominal size by more than X, where:
distribution type.

r Distribution type
w = width of aperture.
0 Number
Tolerance for mean aperture (± Y): the average aperture
1 Length size shall not depart from the nominal size by more
than ± Y, where:
2 Area

3 Volume

Therefore, by definition: Intermediary tolerance (+ Z): not more than 6 per cent of
Qr(x) = 0.90 when x = X90 the total number of apertures shall have sizes between
"nominal + X'' and "nominal + Z", where:
QrCx) = 0.50 when x = x 50
QrCx) =
0.10 when x X10 =
An alternative but less informative method of classifying
powder fineness is by use of the descriptive terms in Wire diameter d: the wire diameters given in Table 2.1.4.-1
Table 2.9.35.-1. apply to woven metal wire cloth mounted in a frame.
The nominal sizes of the wire diameters may depart from
Table 2.9.35.-1.
these values within the limits dmax and dmm- Toe limits define
Classification of powders by fineness a permissible range of choice ± 15 per cent of the
recommended nominal dimensions. The wires in a test sieve
Cumulative
Descriptive term. Xso (µm) distribution by shall be of a similar diameter in warp and weft directions.
volume basis, Q,(x)

Coarse > 355 Q,(355) < 0.50

Q,(180) < 0.50 and

Moderately fine 180 - 355
Q,(355) 2: 0.50

Q,(125) < 0.50 and

Fine 125 - 180
Qs(l 80) :> 0.50

Very fine $ 125 Q 3 (125) :> 0.50

1 See the International Standard ISO 3310/1 (1975).

 2023 Appendix XVII B V-A595

Table 2.1.4.-1 (val,ues in micrometers)

Sieve Tolerances for apertures Wire diameters
numbers
(Nominal Maximum Tolerance for Intermediary Recommended Admissible limits
dimensions of tolerance for mean aperture tolerance nominal
apertures) an aperture dimensions

+X ± y +z d ~~ dmin

11 200 770 350 560 2500 2900 2100

8000 600 250 430 2000 2300 1700

5600 470 180 320 1600 1900 1300

4000 370 130 250 1400 1700 1200

2800 290 90 190 1120 1300 950

2000 230 70 150 900 1040 770

1400 180 50 110 710 820 600

1000 140 30 90 560 640 480

710 112 25 69 450 520 380

500 89 18 54 315 360 270

355 72 13 43 224 260 190

250 58 9.9 34 160 190 130

180 47 7.6 27 125 150 106

125 38 5.8 22 90 104 77

90 32 4.6 18 63 72 54

63 26 3.7 15 45 52 38

45 22 3.1 13 32 37 27

38 - - - 30 35 24

2. Filters2 Special, Uses

(Ph. Bur. method 2.1.2) Diameters in micrometres
< 2.5 Bacteriological filtration
Table 2.1.2.-1 4 - 10 Ultra-fine filtration, separation of micro-organisms of large
diameter
Porosity Maximum Germany France United
10 - 40 Analytical filtration, very fine filtration of mercury, very fine
number diameter of Kingdom
dispersion of gases
(Ph. Eur.) 3 pores in
40 - 100 Fine filtration, filtration of mercury, fine dispersion of gases
micrometres
I 00 - 160 Filtration of coarse materials, dispersion and washing of gases,
1.6 less than 1.6 Sf support for other filter materials
I - 2.5 5 5 160 - 500 Filtration of very coarse materials, dispersion and washing of
gases.
4 1.6 - 4
4-6 5
10 4 - 10 4f 4 3. Particle-size Distribution Estimation by
16 10 - 16 4 4 Analytical Sieving4
40 16 - 40 3 3 3 (Ph. Bur. method 2.9.38)
40 - 50 2 Sieving is one of the oldest methods of classifying powders
100 40 - 100 2 2 and granules by particle-size distribution. When using a
100 - 120 woven sieve cloth, the sieving will essentially sort the particles
160 100 - 160 1 by their intermediate size dimension (i.e. breadth or width).
150-200 0 0 Mechanical sieving is most suitable where the majority of the
250 160 - 250 particles are larger than about 75 µm. For smaller particles,
200 - 500 00 their light weight provides insufficient force during sieving to
overcome the surface forces of cohesion and adhesion that
cause the particles to stick to each other and to the sieve, and
thus cause particles that would be expected to pass through
the sieve to be retained. For such materials other means of
agitation such as air-jet sieving or sonic-sifter sieving may be
more appropriate. Nevertheless, sieving can sometimes be
2 The given limits are only approximate.
3 The European Pharmacopoeia has adopted the system proposed by the 4 This chapter has undergone pharmacopoeia/ harmonisation. See chapter

International Organization for Standardization (ISO). 5. 8 Pharmacopoeia/ harmonisation.

V-A596 Appendix XVII B 2023

used for some powders or granules having median particle progression of the area of the sieve openings is
sizes smaller than 75 µm where the method can be validated. recommended. The nest of sieves is assembled with the
In pharmaceutical terms, sieving is usually the method of coarsest screen at the top and the finest at the bottom.
choice for classification of the coarser grades of single Use micrometres or millimetres in denoting test sieve
powders or granules. It is a particularly attractive method in openings.
that powders and granules are classified only on the basis of Test sieves are made from stainless steel or, less preferably,
particle size, and in most cases the analysis can be carried from brass or another suitable non-reactive wire.
out in the dry state.
Calibration and recalibration of test sieves is in accordance
Among the limitations of the sieving method are the need for with the current edition ofISO 3310-1. Sieves are carefully
an appreciable amount of sample (normally at least 25 g, examined for gross distortions and fractures, especially at
depending on the density of the powder or granule, and the their screen frame joints, before use. Sieves may be calibrated
diameter of the test sieves) and the difficulty in sieving oily or optically to estimate the average opening size, and opening
other cohesive powders or granules that tend to clog the sieve variability, of the sieve mesh. Alternatively, for the evaluation
openings. The method is essentially a two-dimensional of the effective opening of test sieves in the size range of
estimate of size because passage through the sieve aperture is 212-850 µm, standard glass spheres are available. Unless
frequently more dependent on maximum width and thickness otherwise specified in the individual monograph, perform the
than on length. sieve analysis at controlled room temperature and at ambient
This method is intended for estimation of the total particle- relative humidity.
size distribution of a single material. It is not intended for Cleaning test sieves Ideally, test sieves are cleaned using
determination of the proportion of particles passing or only a low-pressure air jet or a liquid stream. If some
retained on 1 or 2 sieves. apertures remain blocked by test particles, careful gentle
Estimate the particle-size distribution as described under Dry brushing may be used as a last resort.
sieving method, unless otherwise specified in the individual Test sample
monograph. Where difficulty is experienced in reaching the If the test sample mass is not given in the monograph for a
endpoint (i.e. material does not readily pass through the particular material, use a test sample having a mass of
sieves) or when it is necessary to use the finer end of the 25-100 g, depending on the bulk density of the material, for
sieving range (below 75 µm), serious consideration must be test sieves having a 200 mm diameter. For 76 mm sieves, the
given to the use of an alternative particle-sizing method. amount of material that can be accommodated is
Sieving is carried out under conditions that do not cause the approximately 1/7 that which can be accommodated by a
test sample to gain or lose moisture. The relative humidity of 200 mm sieve. Determine the most appropriate mass for a
the environment in which the sieving is carried out must be given material by test sieving accurately weighed samples of
controlled to prevent moisture uptake or loss by the sample. different masses, such as 25 g, 50 g and 100 g, for the same
In the absence of evidence to the contrary, analytical test time period on a mechanical shaker (note: if the test results
sieving is normally carried out at ambient humidity. are similar for the 25 g and 50 g samples, but the 100 g
Any special conditions that apply to a particular material sample shows a lower percentage through the finest sieve, the
must be detailed in the individual monograph. 100 g sample size is too large). Where only a sample of
Principles of analytical sieving 10-25 g is available, smaller diameter test sieves conforming
Analytical test sieves are constructed from a woven-wire to the same mesh specifications may be substituted, but the
mesh, which is of simple weave that is assumed to give nearly endpoint must be redetermined. The use of test samples
square apertures and is joined to the base of an open having a smaller mass (e.g. down to 5 g) may be needed.
cylindrical container. The basic analytical method involves For materials with low apparent particle density, or for
stacking the sieves on top of one another in ascending materials mainly comprising particles with a highly iso-
degrees of coarseness, and then placing the test powder on diametrical shape, sample masses below 5 g for a 200 mm
the top sieve. The nest of sieves is subjected to a screen may be necessary to avoid excessive blocking of the
standardised period of agitation, and then the mass of sieve. During validation of a particular sieve-analysis method,
material retained on each sieve is accurately determined. it is expected that the problem of sieve blocking will have
The test gives the mass percentage of powder in each sieve been addressed.
size range. If the test material is prone to absorbing or losing significant
This sieving process for estimating the particle-size amounts of water with varying humidity, the test must be
distribution of a single pharmaceutical powder is generally carried out in an appropriately controlled environment.
intended for use where at least 80 per cent of the particles Similarly, if the test material is known to develop an
are larger than 75 µm. The size parameter involved in electrostatic charge, careful observation must be made to
determining particle-size distribution by analytical sieving is ensure that such charging does not influence the analysis.
the length of the side of the minimum square aperture An antistatic agent, such as colloidal silicon dioxide and/or
through which the particle will pass. aluminum oxide, may be added at a 0.5 per cent (mlm) level
to minimise this effect. If both of the above effects cannot be
TEST SIEVES
eliminated, an alternative particle-sizing technique must be
Test sieves suitable for pharmacopoeia) tests conform to the
selected.
current edition of ISO 3310-1: Test sieves - Technical
requirements and testing - Part 1: Test sieves of metal wire cloth Agitation methods
(see Table 2.9.38.-1). Unless otherwise specified in the Several different sieve and powder-agitation devices are
monograph, use those ISO sieves listed as principal sizes in commercially available, all of which may be used to perform
Table 2.9.38.-1 that are recommended in the particular sieve analyses. However, the different methods of agitation
region. may give different results for sieve analyses and endpoint
determinations because of the different types and magnitudes
Sieves are selected to cover the entire range of particle sizes
of the forces acting on the individual particles under test.
present in the test sample. A nest of sieves having a ,/2
2023 Appendix XVII B V-A597

Table 2.9.38.-1. ISO Nominal Aperture us Recom- Euro- Japan-

Sieve mended pean ese
Principal Supplementary
ISO Nominal Aperture us Recom- Euro- Japan-
sizes sizes
No. USP Sieve Sieve
Sieve mended pean ese Sieves No. No.
Principal Supplementary
No. USP Sieve Sieve (µm)
sizes sizes
Sieves No. No.
(µm) R20/3 R20 R40/3

R 20/3 R 20 R40/3 140 flm

125 µm 125 µm 125 µm 120 125 125 119
11.20mm 11.20 mm 11.20mm II 200
112 µm
10.00 mm
106 µm 140 140
9.50 mm

100 µm
9.00 mm
90 µm 90 µm 90 µm 170 90 90 166
8.00 mm 8.00 mm 8.00 mm
80 µm
7.10mm
75 µm 200 200
6.70 mm

71µm
6.30 mm
63 µm 63 µm 63 µm 230 63 63 235
5.60 mm 5.60 mm 5.60 mm 5600 3.5
56 µm
5.00 mm
53 µm 270 282
4.75 mm 4

50 µm
4.50 mm
45 µm 45 µm 45 µm 325 45 45 330
4.00 mm 4.00 mm 4.00 mm 5 4000 4000 4.7
40 µm
3.55 mm
38 flffi 38 391
3.35 mm 6 5.5

3.15 mm
2.80 mm 2.80 mm 2.80mm 7 2800 2800 6.5
Methods using mechanical agitation or electromagnetic
agitation, and that can induce either a vertical oscillation or a
2.50 mm
horizontal circular motion, or tapping or a combination of
2.36 mm 8 7.5
both tapping and horizontal circular motion are available.
2.24 mm Entrainment of the particles in an air stream may also be
2.00 mm 2.00 mm 2.00 mm 10 2000 2000 8.6 used. The results must indicate which agitation method was
1.80mm used and the agitation parameters used (if they can be
1.70 mm 12 10
varied), since changes in the agitation conditions will give
different results for the sieve analysis and endpoint
1.60 mm determination, and may be sufficiently different to give a
1.40mm 1.40mm 1.40mm 14 1400 1400 12 failing result under some circumstances.
1.25 mm
Endpoint determination
1.18mm 16 14 The test sieving analysis is complete when the mass on any
1.12mm of the test sieves does not change by more than 5 per cent or
1.00 mm I.OD mm 1.00 mm 18 1000 1000 16
0 .1 g ( 10 per cent in the case of 76 mm sieves) of the
previous mass on that sieve. If less than 5 per cent of the
900 µm
total sample mass is present on a given sieve, the endpoint
850 µm 20 18
for that sieve is increased to a mass change of not more than
800 µm 20 per cent of the previous mass on that sieve.
710 µm 710 µm 710 µrn 25 710 710 22 If more than 50 per cent of the total sample mass is found
630 µm on any one sieve, unless this is indicated in the monograph,
600 µm 30 26 the test is repeated, but with the addition to the sieve nest of
a more coarse sieve intermediate between that carrying the
560 µrn
excessive mass and the next coarsest sieve in the original
500 µrn 500 µm 500 µm 35 500 500 30
nest, i.e. addition of the ISO series sieve omitted from the
450 µm nest of sieves.
425 µm 40 36
SIEVING METHODS
400 µm Mechanical agitation (Dry sieving method)
355 pm 355 µm 355 µm 45 355 355 42 Tare each test sieve to the nearest 0.1 g. Place an accurately
315 µm weighed quantity of test sample on the top (coarsest) sieve,
300 µm 50 50 and replace the lid. Agitate the nest of sieves for 5 min, then
carefully remove each sieve from the nest without loss of
280 µm
material. Reweigh each sieve, and determine the mass of
250 flm 250 µm 250 µrn 60 250 250 60
material on each one. Determine the mass of material in the
224 ~tm collecting pan in a similar manner. Re-assemble the nest of
212 ~LID 70 70 sieves, and agitate for 5 min. Remove and weigh each sieve
200 µm
as previously described. Repeat these steps until the endpoint
180 µm
criteria are met (see Endpoint determination under Test
180 ~lffi 180 µm 80 180 180 83
sieves). Upon completion of the analysis, reconcile the
160 µrn
masses of material. Total loss must not exceed 5 per cent of
150 µm 100 100
the mass of the original test sample.
V-A598 Appendix XVII C 2023

Repeat the analysis with a fresh sample, but using a single consisting of particles less than a few micrometres is not
sieving time equal to that of the combined times used above. taken into account in the equation used to calculate the
Confirm that this sieving time conforms to the requirements specific surface area.
for endpoint determination. When this endpoint has been
APPARATUS
validated for a specific material, then a single fixed time of
sieving may be used for future analyses, providing the
particle-size distribution falls within normal variation. 016
If there is evidence that the particles retained on any sieve are
aggregates rather than single particles, the use of mechanical
dry sieving is unlikely to give good reproducibility, and a
different particle-size analysis method must be used.
Air-entrainment methods (Air-jet and sonic-sifter
sieving)
Different types of commercial equipment that use a moving
air current are available for sieving. A system that uses a
single sieve at a time is referred to as air-jet sieving. It uses
the same general sieving methodology as that described - - 3 - - - (C)
under Dry sieving method, but with a standardised air jet
replacing the normal agitation mechanism. It requires
sequential analyses on individual sieves starting with the
finest sieve to obtain a particle-size distribution. Air-jet
sieving often includes the use of finer test sieves than used in
ordinary dry sieving. This technique is more suitable where
0 12.6 ± 0.1
only oversize or undersize fractions are needed.
In the sonic-sifter method, a nest of sieves is used, and the
test sample is carried in a vertically oscillating column of air
that lifts the sample and then carries it back against the mesh
openings at a given number of pulses per minute. It may be
necessary to lower the sample amount to 5 g when sonic
sifting is employed.
The air-jet sieving and sonic-sifter sieving methods may be
useful for powders or granules when the mechanical sieving
techniques are incapable of giving a meaningful analysis. lO
(")
These methods are highly dependent upon proper dispersion
of the powder in the air current. This requirement may be
hard to achieve if the method is used at the lower end of the
sieving range (i.e. below 75 µm), when the particles tend to
be more cohesive, and especially if there is any tendency for
the material to develop an electrostatic charge. For the above
reasons endpoint determination is particularly critical, and it
j
is very important to confirm that the oversize material
comprises single particles and is not composed of aggregates.
INTERPRETATION
The raw data must include the mass of the test sample, the
total sieving time, the precise sieving methodology, and the
set values for any variable parameters, in addition to the
masses retained on the individual sieves and in the pan.
It may be convenient to convert the raw data into a
cumulative mass distribution, and if it is desired to express
the distribution in terms of a cumulative mass undersize, the
range of sieves used must include a sieve through which all
the material passes. If there is evidence on any of the test
sieves that the material remaining on it is composed of Figure 2.9.14.-1. - Permeability cell
aggregates formed during the sieving process, the analysis is Dimensions in millimetres
invalid.
The apparatus consists of the following parts:
(a) a penneabiliry cell (see Figure 2.9.14.-1), which consists of
a cylinder with an inner diameter of 12.6 ± 0.1 mm (A),
C. Specific Surface Area by Air constructed of glass or non-corroding metal. The bottom of
Permeability the cell forms an airtight connection (for example, via an
adapter) with the manometer (Figure 2.9.14.-2). A ledge
(Ph. Bur. method 2. 9.14)
0.5-1 mm in width is located 50 ± 15 mm from the top of
The test is intended for the determination of the specific the cell. It is an integral part of the cell or firmly fixed so as
surface area of dry powders expressed in square metres per to be airtight. It supports a perforated metal disk (B),
gram in the sub-sieve region. The effect of molecular flow constructed of non-corroding metal. The disk has a thickness
("slip flow") which may be important when testing powders
 2023 Appendix XVII C V-A599

of 0.9 ± 0.1 mm and is perforated with 30 to 40 holes (F)

1 mm in diameter evenly distributed over this area.
The plunger (C) is made of non-corroding metal and fits into
the cell with a clearance of not more than 0.1 mm. (E)
The bottom of the plunger has sharp square edges at right
angles to the principal axis. There is an air vent 3 mm long

I
and 0.3 mm deep on one side of the plunger. The top of the
+--09
plunger has a collar such that when the plunger is placed in
the cell and the collar is brought into contact with the top of
the cell, the distance between the bottom of the plunger and
the top of the perforated disk (B) is 15 ± 1 mm. 0
s:t
The filter paper disks (D) have smooth edges and the same I/')
diameter as the inside of the cell. C\I

(b) a U-tube manometer (E) (Figure 2.9.14.-2) is made of (G)

nominal 9 mm outer diameter and 7 mm inner diameter I/')
I/')
glass tubing with standard walls. The top of one arm of the
manometer forms an airtight connection with the
permeability cell (F). The manometer arm connected to the
permeability cell has a line etched around the tube at I/')

125-145 mm below the top of the side outlet and three other
lines at distances of 15 mm, 70 mm and 110 mm above that
line (G). The side outlet, 250-305 mm above the bottom of r
the manometer, is used to evacuate the manometer arm
connected to the permeability cell. A tap is provided on the
side outlet not more than 50 mm from the manometer arm. 0
CD
The manometer is mounted firmly in such a manner that the
arms are vertical. It is filled to the lowest mark with the I/')
C\I
liquid recommended by the manufacturer of the equipment
or water.
METHOD
If prescribed, dry the powder to be examined and sift
through a suitable sieve (for example no. 125) to disperse
agglomerates. Calculate the mass (M) of the powder to be
used from the following expression:
Vxpx(l-z)
Figure 2.9.14.-2. - Manometer
Dimensions in millimetres
V bulk volume of the compacted bed of powder,
p density of the substance to be examined in grams per millilitre, Using the measured flow time, calculate the specific surface
porosity of the compacted bed of powder.
area (S), expressed in square metres per gram, from the
following expression:
Assume first a porosity of 0.5 and introduce this value in
expression (1) to calculate the mass (M) of the powder to be KxRx/i
examined. (2)
px(l -r)x,/ii
Place a filter paper disk on top of the perforated metal
disk (B). Weigh the calculated mass (M) of the powder to be flow time in seconds,
examined to the nearest 1 mg. Carefully transfer the powder dynamic viscosity of air in millipascal seconds (see
into the cleaned, tared permeability cell and carefully tap the Table 2.9.14.-1),
K apparatus constant determined according to expression ( 4),
cell so that the surface of the powder bed is level and cover it
p density of the substance to be examined in grams per millilitre,
with a second filter paper disk. Slowly compact the powder porosity of the compacted bed of powder.
by means of the plunger, avoiding rotary movement.
Maintain the pressure until the plunger is completely inserted CALIBRATION OF THE APPARATUS
into the permeability cell. If this is not possible, decrease the
Bulk volume of the compacted powder bed
quantity of the powder used. If, on the contrary, there is not
It is determined by the mercury displacement method as
enough resistance, increase the quantity of the powder. follows:
In this case calculate the porosity again. After at least 10 s,
remove the plunger. Place two filter paper disks in the permeability cell, pressing
down the edges with a rod slightly smaller than the cell
Attach the permeability cell to the tube of the manometer by
diameter until the filter disks lie flat on the perforated metal
means of an airtight connection. Evacuate the air from the disk; fill the cell with mercury, removing any air bubbles
manometer by means of a rubber bulb until the level of the adhering to the wall of the cell and wipe away the excess to
coloured liquid is at the highest mark. Close the tap and
create a plane surface of mercury at the top of the cell. If the
check that the apparatus is airtight by closing the upper end
cell is made of material that will amalgamate, grease the cell
of the cell, for example with a rubber stopper. Remove the
and the metal disk first with a thin layer of liquid paraffin.
stopper and, using a timer, measure the time taken for the Pour out the mercury into a tared beaker and determine the
liquid to fall from the second to the third mark.
mass (MA) and the temperature of the mercury.
V-A600 Appendix XVII E 2023

0 110
Make a compacted bed using the reference powder and again
fill the cell with mercury with a planar surface at the top of
the cell. Pour out the mercury in a tared beaker and again
40° ± 5'
determine the mass of the mercury (MB). Calculate the bulk
volume (V) of the compacted bed of powder from the ~
following expression: I
(3)
I

difference between the determined masses of

mercury in grams,

PHg density of mercury at the determined temperarure in

grams per millilitre.

Repeat the procedure twice, changing the powder each time;

the range of values for the calculated volume (V) is not
greater than 0.01 mL. Use the mean value of the three
determined volumes for the calculations.
Apparatus constant (K) 0 30.2_g,
It is determined using a reference powder with known
specific surface area and density as follows: Nozzle 1, 2 or 3
Calculate the required quantity of the reference powder to be enlarged
used (expression (1)) using the stated density and the
determined volume of the compacted powder bed
(expression (3)).
Homogenise and loosen up the powder by shaking it for
2 min in a 100 mL bottle. Prepare a compacted powder bed
and measure the flow time of air as previously described.
Calculate the apparatus constant (K') from the following
expression: Diameter (d) of the outflow
Nozzle
opening (millimetres)
S,pxpx(l-,)xfo
(4)
J7i X ✓i I JO ± 0.01
2 15 ± 0.01
Ssp stated specific surface area of the reference powder,
p density of the substance to be examined in grams per millilitre, 3 25 ± 0.01
porosity of the compacted bed of powder,
flow time in seconds,
dynamic viscosity of air in millipascal seconds (see Figure 2.9.16.-1. - Flow funnel and nozzle. Nozzle is made of
Table 2.9.14.-1). stainless, acid-resistant steel (V4A,CrNi)
Dimensions in millimetres
The density of mercury and the viscosity of air over a range
of temperatures are shown in Table 2.9.14.-1.
APPARATUS
Table 2.9.14.-1. According to the flow properties of the material to be tested,
Temperature Density of mercury Viscosity of air (T))
funnels with or without stem, with different angles and orifice
/ii diameters are used. Typical apparatuses are shown in
CC) (g/mL) (mPa-s)
16 13.56 0.01800 0.1342
Figures 2.9.16.-1 and 2.9.16.-2. The funnel is maintained
17 13.56 0.01805 0.1344
upright by a suitable device. The assembly must be protected
18 13.55 0.01810 0.1345
from vibrations.
19 13.55 0.01815 0.1347 METHOD
20 13.55 0.01819 0.1349 Into a dry funnel, whose bottom opening has been blocked
21 13.54 0.01824 0.1351 by suitable means, introduce without compacting a test
22 13.54 0.01829 0.1353 sample weighed with 0.5 per cent accuracy. The amount of
23 13.54 0.01834 0.1354 the sample depends on the apparent volume and the
24 13.54 0.01839 0.1356 apparatus used. Unblock the bottom opening of the funnel
and measure the time needed for the entire sample to flow
out of the funnel. Carry out three determinations.
EXPRESSION OF RESULTS
The flowability is expressed in seconds and tenths of
E. Flowability seconds, related to 100 g of sample.
(Ph. Bur. Method 2. 9.16) The results depend on the storage conditions of the material
The test for flowability is intended to determine the ability of to be tested.
divided solids (for example, powders and granules) to flow
vertically under defined conditions.
2023 Appendix XVII F V-A601

12
a
.,.__ B
+I
K1
.,.__ C

D
45°

Figure 2.9.16.-2
Dimensions in millimetres

The results can be expressed as the following:

a) the mean of the determinations, if none of the individual
values deviates from the mean value by more than
10 per cent;
b) as a range, if the individual values deviate from the mean
value by more than 10 per cent;
c) as a plot of the mass against the flow time;
d) as an infinite time, if the entire sample fails to flow
through.
Figure 2.9.9.-1. - Penetrometer

The stand is made up of:

F. Measurement of Consistency and - a vertical shaft to maintain and guide the penetrating
object;
Texture Analysis - a horizontal base;
1. Measurement of Consistency by Penetrometry - a device to ensure that the penetrating object is vertical;
- a device to check that the base is horizontal;
(Ph. Bur. method 2. 9. 9)
- a device to retain and release the penetrating object;
This test is intended to measure, under determined and - a scale showing the depth of penetration, graduated in
validated conditions, the penetration of an object into the tenths of a millimetre.
product to be examined in a container with a specified shape
The penetrating object, made of a suitable material, has a
and size.
smooth surface, and is characterised by its shape, size and
APPARATUS mass (m).
The apparatus consists of a penetrometer made up of a stand Suitable penetrating objects are shown in Figures 2.9.9.-2
and a penetrating object. A suitable apparatus is shown in and 2.9.9.-3.
Figure 2.9.9.-1.
PROCEDURE
A. Scale showing the depth of penetration, graduated in
Prepare the test samples according to one of the following
tenths of millimetres.
procedures.
B. Vertical shaft to maintain and guide the penetrating
A. Carefully and completely fill 3 containers, without
object.
forming air bubbles. Level if necessary to obtain a flat
C. Device to retain and to release the penetrating object surface. Store the samples at 25 ± 0.5 °C for 24 h, unless
automatically and for a constant time. otherwise prescribed.
D. Device to ensure that the penetrating object is vertical B. Store 3 samples at 25 ± 0.5 °C for 24 h, unless
and that the base is horizontal. otherwise prescribed. Apply a suitable shear to the samples
E. Penetrating object (see Figures 2.9.9.-2 and 3). for 5 min. Carefully and completely fill 3 containers, without
F. Container. forming air bubbles, and level if necessary to obtain a flat
G. Horizontal base. surface.
H. Control for the horizontal base.
V-A602 Appendix XVII F 2023

65 ± 0.25 0

0d

N h
oi
+I "'
N
0
a,
.,; +I

ai
~ -+--------~......- - N

+I
a,
,
,._:-+--------~---------_.,_
0
+I
a,
.;

0.38 ± 0.02 0

8.38 ± 0.02 0

Figure 2.9.9.-2. - Cone (m = 102.5 ± 0.05 g), suitable container (d = 102 mm or 75 mm; h 2: 62 mm)
and shaft (l = 162 mm; m = 47.5 ± 0.05 g).
Dimensions in millimetres

0 9.5

0 3.2

0 8.2 ~

76°
.--
..-
r--.
LO
I
r--.

11
LO
M
(!)

Figure 2.9.9.-3 - Micro-cone (m = 7.0 g), suitable container and shaft (l = 116 mm; m = 16.8 g)
Dimensions in millimetres
 2023 Appendix XVII G V-A603

Travelling arm moves probe up or down

◄

Probe

Sample stage
◄

Fig. 17F-1 Texture Analyser

C. Melt 3 samples and carefully and completely fill - a probe attachment, which may be of various shapes such
3 containers, without forming air bubbles. Store the samples as a platen, a cylinder, a cone, a needle, a sphere or a
at 25 ± 0.5 °C for 24 h, unless otherwise prescribed. wire,
Determination of penetration - a load cell capable of measuring the load or tensile forces
Place the test sample on the base of the penetrometer. Verify experienced by the probe as the mobile arm moves up or
that its surface is perpendicular to the vertical axis of the down.
penetrating object. Bring the temperature of the penetrating Calibration The apparatus is calibrated using a suitable
object to 25 ± 0.5 °C and then adjust its position such that certified weight.
its tip just touches the surface of the sample. Release the Method Check that the apparatus is vertical.
penetrating object and hold it free for 5 s. Clamp the
Place the sample being examined in a suitable holder as
penetrating object and measure the depth of penetration. specified in the monograph. Programme the apparatus in
Repeat the test with the 2 remaining containers.
accordance with the manufacturer's specifications to move
EXPRESSION OF THE RESULTS the probe up or down at a defined speed or force as specified
The penetration is expressed in tenths of a millimetre as the in the monograph. Measure the forces experienced by the
arithmetic mean of the 3 measurements. If any of the probe through the sample.
individual results differ from the mean by more than The maximum peak force (in g) required to penetrate the
3 per cent, repeat the test and express the results of the 6 sample is measured by the load cell.
measurements as the mean and the relative standard
Carry out each measurement six times.
deviation.
2. Texture Analysis of Semi-solids or Gels
(No Ph. Bur. method)
This test determines, under defined conditions, the force G. Friability
required to penetrate a semi-solid or gel sample.
This test applies to samples consisting of a semi solid or gel- 1. Uncoated Tablets 1
like mass which retains its form. It is not applicable to (Ph. Bur. method 2. 9. 7)
suspensions consisting of fine solid particles in a liquid. This chapter provides guidelines for the friability
Apparatus A suitable texture analyser, (see Fig. 17F-1) determination of compressed, uncoated tablets. The test
consisting of: procedure presented in this chapter is generally applicable to
- a suitable platform or device to hold the sample being most compressed tablets. Measurement of tablet friability
examined,
- a mobile arm that can be moved in a vertical direction ' This chapter has undergone phannacopoeial harmonisation. See chapter
towards or away from the sample, 5. 8 Phannacopoeial hannonisation.
V-A604 Appendix XVII G 2023

0 10.0 ± 0.1 mm

156.0 ± 2.0 mm

Drop 0 287.0 ± 4.0 mm

height (int.)

0 25.0 ± 0.5 mm

' 0 302.5 ± 4.0 mm

_J
38.0 ± 2.0 mm
Figure 2.9.7.-1. - Tabletfriability apparatus

supplements other physical strength measurements, such as lying next to each other, which prevents them from falling
tablet breaking force. freely.
Use a drum, with an internal diameter between 283-291 mm Effervescent tablets and chewable tablets may have different
and a depth between 36-40 mm, of transparent synthetic specifications as far as friability is concerned. In the case of
polymer with polished internal surfaces, and subject to hygroscopic tablets, a humidity-controlled environment is
minimum static build-up (see Figure 2.9.7.-1.). One side of required for testing.
the drum is removable. The tablets are tumbled at each tum A drum with dual scooping projections, or apparatus with
of the drum by a curved projection with an inside radius more than one drum, for the running of multiple samples at
between 75.5-85.5 mm that extends from the middle of the one time, are also permitted.
drum to the outer wall. The outer diameter of the central
ring is between 24.5-25.5 mm. The drum is attached to the 2. Granules and Spheroids
horizontal axis of a device that rotates at 25 ± 1 r/min. (Ph. Bur. method 2.9.41)
Thus, at each tum the tablets roll or slide and fall onto the This chapter describes 2 methods for determination of the friability
drum wall or onto each other. of granules and spheroids, which may be used during development
For tablets with a unit mass equal to or less than 650 mg, studies. It is recognised, however, that many methods with equal
take a sample of whole tablets corresponding as near as suitability may be used.
possible to 6.5 g. For tablets with a unit mass of more than This test is intended to determine, under defined conditions,
650 mg, take a sample of 10 whole tablets. The tablets are the friability of granules and spheroids. Friability is defined as
carefully dedusted prior to testing. Accurately weigh the a reduction in the mass of the granules or spheroids or in the
tablet sample, and place the tablets in the drum. Rotate the formation of fragments of granules or spheroids, occurring
drum 100 times, and remove the tablets. Remove any loose when the granules or spheroids are subjected to mechanical
dust from the tablets as before, and accurately weigh. strain during handling (tumbling, vibration, fluidisation, etc.).
Generally, the test is run once. If obviously cracked, cleaved, Examples of changes are abrasion, breakage or deformation
or broken tablets are present in the tablet sample after of granules or spheroids.
tumbling, the sample fails the test. If the results are difficult METHOD A
to interpret or if the weight loss is greater than the targeted Apparatus (fluidised-bed apparatus)
value, the test is repeated twice and the mean of the 3 tests The apparatus (see Figure 2.9.41.-1) consists of a glass
determined. A maximum loss of mass (obtained from a single cylinder (A) with a conical lower part. The cylinder is
test or from the mean of 3 tests) not greater than provided with a sieve lid (B) having an aperture size of
1.0 per cent is considered acceptable for most products. 500 µm or any other suitable sieve. The conical end is
If tablet size or shape causes irregular tumbling, adjust the connected to a U-shaped glass tube (C) that can be
drum base so that the base forms an angle of about 10° with disconnected from the cylinder for removal of the granules or
the horizontal and the tablets no longer bind together when spheroids. The U-tube is attached to a T-coupling (D).
2023 Appendix XVII G V-A605

E
(.)
I{)

A 'SI'

4.5cm 5cm E
(.)
0
1 cm
NS 145/2

0
E
(.)
I{)

Figure 2.9.41.-1. - Fluidised-bed apparatus

D
Glass
container
N
+I

l•••I D 0
r--
C')

Time Frequency
107 ± 3 ml glass container,
42.0 ± 0.5 mm in internal diameter
and 85.0 ± 1 mm in height,
with a twist-off cap.

Figure 2.9.41.-2. - Osci7lating apparatus

One inlet of the T-coupling is joined by a silicone tube to a Procedure

manometer for regulating the compressed-air flow (use The following procedure is usually suitable Remove
compressed air complying with the test for water in the the fine particles by sieving (sieve having an aperture size of
monograph Medicinal air (1238)), the other one is connected 710 µm or any other suitable sieve). Introduce about
via a silicone tube to a by-pass flowmeter (E) 8.0 g (m 1) of granules or spheroids into the cylinder (A).
(0.10-1.00 m 3 ·h- 1). Close the apparatus with the sieve lid (B). Adjust the flow
rate of the compressed air to 0.45 m 3 ·h- 1 • After 15 min,
remove the granules or spheroids from the apparatus by
V-A606 Appendix XVII H 2023

disconnecting the U-tube and weigh again (m 2). Test are perpendicular to the direction of movement.
3 samples and calculate the mean value. It is recommended The crushing surfaces of the jaws are flat and larger than the
to spray the inside of the apparatus with an antistatic agent zone of contact with the tablet. The apparatus is calibrated
every 3 determinations in order to prevent electrostatic using a system with a precision of 1 newton.
charging.
OPERATING PROCEDURE
Loss on drying Dry in an oven at 105 °C, unless Place the tablet between the jaws, taking into account, where
otherwise prescribed. Alternatively, other drying conditions as applicable, the shape, the break-mark and the inscription; for
described in general chapter 2.2.32 may be used. each measurement orient the tablet in the same way with
Calculation respect to the direction of application of the force. Carry out
the measurement on 10 tablets, taking care that all fragments
m1 (100 - T1) - m2(IO0 - T2) of tablets have been removed before each determination.
F = - - - - - - - - - - x 100
mi This procedure does not apply when ftdly automated equipment is
F friability; used.
T1 percentage loss on drying before the test (mean of
2 determinations); EXPRESSION OF RESULTS
T2 percentage loss on drying after the test (mean of Express the results as the mean, minimum and maximum
2 determinations); values of the forces measured, all expressed in newtons.
m1 mass of the granules or spheroids before the test, in grams;
m2 mass of the granules or spheroids after the test, in grams. Indicate the type of apparatus and, where applicable, the
orientation of the tablets.
METHODB
Apparatus (oscillating apparatus)
The apparatus (see Figure 2.9.41.-2) consists of a glass
container, containing the granules or spheroids to be J. Softening Time Determination of
examined, which is subjected to horizontal oscillations. Lipophilic Suppositories
The frequency and duration of the oscillations can be varied
continuously. The frequency can be adjusted, using a scale, (Ph. Eur. method 2.9.22)
to a value in the range 0-400 oscillations/min. The duration The test is intended to determine, under defined conditions,
can be set to a value in the range 0-9999 s. the time which elapses until a suppository maintained in
Procedure water softens to the extent that it no longer offers resistance
The following procedure is usually suitable Remove when a defined weight is applied.
the fine particles by sieving (sieve having an aperture size of APPARATUS A
355 µm or any other suitable sieve). In the glass container, The apparatus (see Figure 2.9.22.-1) consists of a glass tube
weigh about 10.00 g (m 1) of the granules or spheroids. Install 15.5 mm in internal diameter with a flat bottom and a length
the container in the apparatus. Shake for 240 s at the highest of about 140 mm. The tube is closed by a removable plastic
frequency for hard granules or spheroids, or for 120 s at a cover having an opening 5.2 mm in diameter. The apparatus
lower frequency (e.g. 140 oscillations/min) for soft granules comprises a rod 5.0 mm in diameter which becomes wider
or spheroids. Sieve (355 µm, or the same sieve as used towards the lower end, reaching a diameter of 12 mm.
previously) and weigh the granules or spheroids again (m 2). A metal needle 2 mm in length and 1 mm in diameter is
Test 3 samples and calculate the mean value. fixed on the flat underside.
Loss on drying Dry in an oven at 105 °C, unless The rod consists of 2 parts, a lower part made of plastic
otherwise prescribed. Alternatively, other drying conditions as material and an upper part made of plastic material or metal
described in general chapter 2.2.32 may be used. with a weight disk. The upper and lower parts are either
Calculation fitted together (manual version) or separate (automated
version). The weight of the entire rod is 30 ± 0.4 g.
The upper part of the rod carries a sliding mark ring. When
the rod is introduced into the glass tube so that it touches
F friability; the bottom, the mark ring is adjusted to coincide with the
T1 percentage loss on drying before the test (mean of upper level of the plastic cover.
2 determinations);
T2 percentage loss on drying after the test (mean of Method
2 detenninations); Place the glass tube containing 10 mL of water in a water-
m1 mass of the granules or spheroids before the test, in grams; bath and equilibrate at 36.5 ± 0.5 °C. Fix the glass tube
m2 mass of the granules or spheroids after the test, in grams. vertically and immerse to a depth of at least 7 cm below the
surface but without touching the bottom of the water-bath.
Introduce a suppository, tip first, into the tube followed by
the rod with the free gliding plastic cover into the glass tube
until the metal needle touches the flat end of the suppository.
H. Resistance to Crushing of Tablets Put the cover on the tube (beginning of time measurement).
(Ph. Eur. method 2.9.8) Note the time which elapses until the rod sinks down to the
bottom of the glass tube and the mark ring reaches the upper
This test is intended to determine, under defined conditions,
level of the plastic cover.
the resistance to crushing of tablets, measured by the force
needed to disrupt them by crushing.
APPARATUS
The apparatus consists of 2 jaws facing each other, one of
which moves towards the other. The flat surfaces of the jaws
2023 Appendix XVII K V-A607

lO
C')
P·I
II

0
~I
N
"'""
\
\
\

0 A 0 C1 C2
0 0 -
"'
0
a, B
C')
r-
l ~1
0
"J @'
~
~~-
'q"
H,0
I 14

-·~
u 05.0 37°C
~

J
50 ~E-t©
Figure 2.9.22.-2. -Apparatus B for measuring the softening time
of lipophilic suppositories
Dimensions in millimetres

K. Pycnometric Density of Solids1

(Ph. Bur. method 2.9.23)
I 01a
I~ 5 Gas pycnometric density is determined by measuring the
volume occupied by a known mass of powder, which is
equivalent to the volume of gas displaced by the powder
Figure 2.9.22.-1. - Apparatus A for measuring the softening time using a gas displacement pycnometer. In gas pycnometric
of lipophilic suppositories density measurements, the volume determined excludes the
Dimensions in millimetres volume occupied by open pores; however, it includes the
volume occupied by sealed pores or pores inaccessible to the
APPARATUSB gas.
The apparatus (see Figure 2.9.22.-2) consists of a water- Usually, helium is used as a test gas due to its high diffusivity
bath (B) into which an inner tube (A) is inserted and fixed into small open pores. If gases other than helium are used,
with a stopper. The inner tube is closed by a stopper at the different values would be obtained, since the penetration of
bottom. The apparatus is fitted with a thermometer. 2 insets the gas is dependent on the size of the pore as well as the
are available: cross-sectional area of the gas molecules.
- a glass rod (Cl) in the form of a tube sealed at both The measured density is a volume-weighted average of the
ends, carrying a rim at its lower end weighed with lead densities of individual powder particles. It is called the
shot, which has a weight of 30 ± 0.4 g, particle density, distinct from the true density of a solid or
- a penetration inset (C2) consisting of a rod (7.5 ± 0.1 g) the bulk density of a powder. The density of solids is
in a tube which has an enlargement for the suppository, expressed in grams per cubic centimetre (g/cm3 ), although
both made of stainless steel. the International Unit is the kilogram per cubic metre
Method (1 g/cm 3 = 1000 kg/m 3).
Pour 5 mL of water at 36.5 ± 0.5 °C into the inner APPARATUS
tube (A), introduce a suppository with the tip downwards The apparatus (see Figure 2.9.23.-1) consists of the
and onto that, place the inset (Cl or C2). Note the time following:
which elapses between this moment and the moment when - a sealed test cell, with empty cell volume Ve, connected
the lower, rimmed end of the glass rod (Cl) or the steel through a valve to an expansion cell, with volume V,.;
rod (C2) reaches the narrowed part of the inner glass tube. - a system capable of pressurising the test cell with the
Melting or dissolution is then considered as complete. measurement gas until a defined pressure (P) indicated by
a manometer;

1 This chapter has undergone pharmacopoeia! harnwnisation. See chapter

5. 8. Phamiacopoeial harmonisation.
V-A608 Appendix XVII M 2023

- the system is connected to a source of measurement gas, shown in Figure 2.9.23.-1, follow the instructions of the
preferably helium, unless another gas is specified. manufacturer of the pycnometer.
The gas pycnometric density measurement is performed at a EXPRESSION OF THE RESULTS
temperature between 15 °C and 30 °C that does not vary by The sample volume (V,) is given by the equation:
more than 2 °C during the course of measurement.
The apparatus is calibrated, which means that the Vr
volumes Ve and Vr are determined using a suitable calibration V, = Ve - p. -P
_,__r_l
standard whose volume is known to the nearest 0.001 cm3 • P1-Pr
The procedure described below is followed in 2 runs, firstly
with an empty test cell, and secondly with the calibration The density (p) is given by the equation:
standard placed in the test cell. The volumes Ve and Vr are
calculated using the equation for the sample volume (V,), m
p=-
taking into account that V, is zero in the first run. v,
The sample conditioning is indicated with the results.
For example, indicate whether the sample was tested as is or
dried under specific conditions such as those described for
loss on drying.

M. Specific Surface Area by Gas

Adsorption 1
M
(Ph. Bur. method 2.9.26)
INTRODUCTION
The specific surface area of a powder is determined by
physical adsorption of a gas on the surface of the solid and
by calculating the amount of adsorbate gas corresponding to
a monomolecular layer on the surface. Physical adsorption
results from relatively weak forces (van der Waals forces)
between the adsorbate gas molecules and the adsorbent
surface of the test powder. The determination is usually
carried out at the temperature of liquid nitrogen.
The amount of gas adsorbed can be measured by a
A valve; volumetric or continuous flow procedure.
V, expansion volume, in cubic centimetres; BRUNAUER, EMMETT AND TELLER (BET)
V, cell volume, in cubic centimetres;
V, sample volume, in cubic centimetres;
THEORY AND SPECIFIC SURFACE AREA
M manometer. DETERMINATION
MULTI-POINT MEASUREMENT
Figure 2.9.23.-1. - Schematic diagram of a gas pycnometer The data are treated according to the Brunauer, Emmett and
Teller (BET) adsorption isotherm equation:
METHOD
Volatile contaminants in the powder are removed by
degassing the powder under a constant purge of helium prior
to the measurement. Occasionally, powders may have to be
degassed under vacuum. Because volatiles may be evolved
during the measurement, weighing of the sample is carried
out after the pycnometric measurement of volume. P partial vapour pressure of adsorbate gas in equilibrium with the
surface at 77 .4 K (b.p. of liquid nitrogen), in pascals,
Weigh the test cell of the pycnometer and record the mass. P0 saturated pressure of adsorbate gas, in pascals,
Fill the test cell with a given mass of powder of the substance Va volume of gas adsorbed at standard temperature and pressure
to be examined. Seal the test cell in the pycnometer. Record (STP) [273.15 Kand atmospheric pressure (1.013 x 105 Pa)],
in millilitres,
the system reference pressure (Pr) as indicated by the Vm volume of gas adsorbed at STP to produce an apparent
manometer while the valve that connects the expansion cell monolayer on the sample surface, in millilitres,
with the test cell is open. Close the valve to separate the C dimensionless constant that is related to the enthalpy of
expansion cell from the test cell. Pressurise the test cell with adsorption of the adsorbate gas on the powder sample.
the gas to an initial pressure (P;) and record the value
obtained. Open the valve to connect the expansion cell with A value of Va is measured at each of not less than 3 values of
the test cell. Record the final pressure (P1). Repeat the PIP0 •
measurement sequence for the same powder sample until
consecutive measurements of the sample volume (V,) agree
to within 0.2 per cent. Unload the test cell and measure the
final powder mass (m), expressed in grams. If the
pycnometer differs in operation or construction from the one 1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter

5. 8 Phannacopoeial harmonisation.
2023 Appendix XVII M V-A609

Then the BET value error associated with the single-point method can be reduced
or eliminated by using the multiple-point method to evaluate
C for one of the samples of the series from the BET plot,
from which C is calculated as (1 + slope/intercept). Then Vm is
calculated from the single value of Va measured at a single
value of PIP0 by the equation:
is plotted against PIP0 according to equation (1). This plot
should yield a straight line usually in the approximate relative
(4)
pressure range 0.05 to 0.3. The data are considered
acceptable if the correlation coefficient, r, of the linear
regression is not less than 0. 9975; that is, I is not less than
The specific surface area is calculated from Vm by
0.995. From the resulting linear plot, the slope, which is
equation (2) given above.
equal to (C -1)/V,,,C, and the intercept, which is equal to
1/VmC, are evaluated by linear regression analysis. From EXPERIMENTAL TECHNIQUES
these values, Vm is calculated as 1/(slope + intercept), while C This section describes the methods to be used for the sample
is calculated as (slope/intercept) + 1. From the value of Vm so preparation, the dynamic flow gas adsorption technique
determined, the specific surface area, S, in m 2 ·g-1, is (Method I) and the volumetric gas adsorption technique
calculated by the equation: (Method II).
SAMPLE PREPARATION
S = VmNa (2)
m X 22400 Outgassing
Before the specific surface area of the sample can be
determined, it is necessary to remove gases and vapours that
N Avogadro constant (6.022 x 10 23 mol- 1), may have become physically adsorbed onto the surface after
a effective cross-sectional area of one adsorbate molecule., in manufacture and during treatment, handling and storage.
square metres (0.162 nm 2 for nitrogen and 0.195 nm 2 for If outgassing is not achieved, the specific surface area may be
krypton),
m mass of test powder, in grams,
reduced or may be variable because an intermediate area of
22400 volume occupied by I mole of the adsorbate gas at STP the surface is covered with molecules of the previously
allowing for minor depanures from the ideal, in millilitres. adsorbed gases or vapours. The outgassing conditions are
critical for obtaining the required precision and accuracy of
A minimum of 3 data points is required. Additional specific surface area measurements on pharmaceuticals
measurements may be carried out, especially when non- because of the sensitivity of the surface of the materials.
linearity is obtained at a PIP0 value close to 0.3. Because Conditions The outgassing conditions must be
non-linearity is often obtained at a PIP0 value below 0.05, demonstrated to yield reproducible BET plots, a constant
values in this region are not recommended. The test for weight of test powder, and no detectable physical or chemical
linearity, the treatment of the data, and the calculation of the changes in the test powder.
specific surface area of the sample are described above.
The outgassing conditions defined by the temperature,
SINGLE-POINT MEASUREMENT pressure and time should be chosen so that the original
Normally, at least 3 measurements of Va each at different surface of the solid is reproduced as closely as possible.
values of P/P0 are required for the determination of specific Outgassing of many substances is often achieved by applying
surface area by the dynamic flow gas adsorption technique a vacuum, by purging the sample in a flowing stream of a
(Method I) or by volumetric gas adsorption (Method II). non-reactive, dry gas, or by applying a desorption-adsorption
However, under certain circumstances described below, it cycling method. In either case, elevated temperatures are
may be acceptable to determine the specific surface area of a sometimes applied to increase the rate at which the
powder from a single value of Va measured at a single value contaminants leave the surface. Caution should be exercised
of PIP0 such as 0.300 (corresponding to 0.300 mole of when outgassing powder samples using elevated temperatures
nitrogen or 0.001038 mole fraction of krypton), using the to avoid affecting the nature of the surface and the integrity
following equation for calculating Vm: of the sample.
If heating is employed, the recommended temperature and
(3)
time of outgassing are as low as possible to achieve
reproducible measurement of specific surface area in an
acceptable time. For outgassing sensitive samples, other
The specific surface area is then calculated from the value of outgassing methods such as the desorption-adsorption cycling
Vm by equation (2) given above. method may be employed.
The single-point method may be employed directly for a Adsorbate
series of powder samples of a given material for which the The standard technique is the adsorption of nitrogen of
material constant C is much greater than unity. These analytical quality at liquid nitrogen temperature.
circumstances may be verified by comparing values of
For powders of low specific surface area ( < 0.2 m 2 ·g- 1) the
specific surface area determined by the single-point method
proportion adsorbed is low. In such cases the use of krypton
with that determined by the multiple-point method for the
at liquid nitrogen temperature is preferred because the low
series of powder samples. Close similarity between the single-
vapour pressure exerted by this gas greatly reduces error.
point values and multiple-point values suggests that 1/C
The use of larger sample quantities where feasible (equivalent
approaches zero.
to 1 m 2 or greater total surface area using nitrogen) may
The single-point method may be employed indirectly for a compensate for the errors in determining low surface areas.
series of very similar powder samples of a given material for
All gases used must be free from moisture.
which the material constant C is not infinite but may be
assumed to be invariant. Under these circumstances, the
V-A61 O Appendix XVII M 2023

Self seals quick connection ~

0 ring seals Flow
meter
Path Diffusion

Flow
l selection
valve
baffle

control -I
::::r
valve CD
3
9d.
CD
..Q
Outgassing
Cold ~ Sample
trap o' cell station
~
o·
;;:,
Differential
C:
flow O" Long
CD Short
controller path
path
ballast ballast

On-off
valve
Digital
A display B
Vent
Gas inlet
Detector Detector

Figure 2. 9 .26.-1. - Schematic diagram of the dynamic flow method apparatus

Quantity of sample Remove from the coolant; this gives a desorption peak equal
Accurately weigh a quantity of the test powder such that the in area and in the opposite direction to the adsorption peak.
total surface of the sample is at least 1 m2 when the Since this is better defined than the adsorption peak, it is the
adsorbate is nitrogen and 0.5 m2 when the adsorbate is one used for the determination.
krypton. To effect the calibration, inject a known quantity of
Lower quantities of sample may be used after appropriate adsorbate into the system, sufficient to give a peak of similar
validation. magnitude to the desorption peak and obtain the proportion
MEASUREMENTS of gas volume per unit peak area.
Because the amount of gas adsorbed under a given pressure Use a nitrogen/helium mixture for a single-point
tends to increase on decreasing the temperature, adsorption determination and several such mixtures or premixing
measurements are usually made at a low temperature. 2 streams of gas for a multiple-point determination.
Measurement is performed at 77.4 K, the boiling point of Calculation is essentially the same as for the volumetric
liquid nitrogen. method.
Method I: the dynamic flow method Method II: the volumetric method
Principle Principle
In the dynamic flow method (see Figure 2.9.26.-1), the In the volumetric method (see Figure 2.9.26.-2), the
recommended adsorbate gas is dry nitrogen or krypton, while recommended adsorbate gas is nitrogen which is admitted
helium is employed as a diluent gas, which is not adsorbed into the evacuated space above the previously outgassed
under the recommended conditions. powder sample to give a defined equilibrium pressure, P, of
A minimum of 3 mixtures of the appropriate adsorbate gas the gas. The use of a diluent gas, such as helium, is therefore
with helium are required within the PIP range 0.05 to 0.30.
0 unnecessary, although helium may be employed for other
The gas detector-integrator should provide a signal that is purposes, such as to measure the dead volume.
approximately proportional to the volume of the gas passing Since only pure adsorbate gas, instead of a gas mixture, is
through it under defined conditions of temperature and employed, interfering effects of thermal diffusion are avoided
pressure. For this purpose, a thermal conductivity detector in this method.
with an electronic integrator is one among various suitable Procedure
types. A minimum of 3 data points within the recommended Admit a small amount of dry nitrogen into the sample tube
range of 0.05 to 0.30 for PIP0 is to be determined. to prevent contamination of the clean surface, remove the
Procedure sample tube, insert the stopper, and weigh it. Calculate the
A known mixture of the gases, usually nitrogen and helium, weight of the sample. Attach the sample tube to the
is passed through a thermal conductivity cell, through the volumetric apparatus. Cautiously evacuate the sample down
sample, again through the thermal conductivity cell and then to the specified pressure (e.g. between 2 Pa and 10 Pa).
to a recording potentiometer. Alternatively, some instruments operate by evacuating to a
Immerse the sample cell in liquid nitrogen, then the sample defined rate of pressure change (e.g. less than 13 Pa/30 s)
adsorbs nitrogen from the mobile phase. This unbalances the and holding for a defined period of time before commencing
thermal conductivity cell, and a pulse is generated on a the next step.
recorder chart.
2023 Appendix XVII N V-A611

9
Vacuum
To cold traps and
gauge
vacuum pumps

7 10 11

Helium
reservoir

Vacuum
Vapour
Air
pressure
manometer

Figure 2.9.26.-2. - Schematic diagram of the volumetric method apparatus

If the principle of operation of the instrument requires the The purpose of this chapter is to review the methods for
determination of the dead volume in the sample tube, for characterising powder flow that have appeared most
example, by the admission of a non-adsorbed gas, such as frequently in the pharmaceutical literature. In addition, while
helium, this procedure is carried out at this point, followed it is clear that no single and simple test method can
by evacuation of the sample. The determination of dead adequately characterise the flow properties of pharmaceutical
volume may be avoided using difference measurements, that powders, this chapter proposes the standardisation of test
is, by means of reference and sample tubes connected by a methods that may be valuable during pharmaceutical
differential transducer. The adsorption of nitrogen gas is then development.
measured as described below. 4 commonly reported methods for testing powder flow are:
Raise a Dewar vessel containing liquid nitrogen at 77.4 K up - angle of repose,
to a defined point on the sample cell. Admit a sufficient - compressibility index or Hausner ratio,
volume of adsorbate gas to give the lowest desired relative - flow rate through an orifice,
pressure. Measure the volume adsorbed, Va. For multipoint - shear cell.
measurements, repeat the measurement of Va at successively In addition, numerous variations of each of these basic
higher PIP values. When nitrogen is used as the adsorbate
0 methods are available. Given the number of test methods
gas, PIP values of 0.10, 0.20, and 0.30 are often suitable.
0 and variations, standardising the test methodology, where
REFERENCE MATERIALS possible, would be advantageous.
Periodically verify the functioning of the apparatus using With this goal in mind, the most frequently used methods
appropriate reference materials of known surface area, such are discussed below. Important experimental considerations
as l'.l-alumina, which should have a specific surface area are identified and recommendations are made regarding
similar to that of the sample to be examined. standardisation of the methods. In general, any method of
measuring powder flow must be practical, useful,
reproducible and sensitive, and must yield meaningful results.
It bears repeating that no simple powder flow method will
N1. Powder Flow1 adequately or completely characterise the wide range of flow
properties experienced in the pharmaceutical industry.
(Ph. Bur. method 2.9.36) An appropriate strategy may well be the use of multiple
The widespread use of powders in the pharmaceutical standardised test methods to characterise the various aspects
industry has generated a variety of methods for characterising of powder flow as needed by the pharmaceutical scientist.
powder flow. Not surprisingly, scores of references appear in ANGLE OF REPOSE
the pharmaceutical literature, attempting to correlate the The angle of repose has been used in several branches of
various measures of powder flow to manufacturing science to characterise the flow properties of solids. Angle of
properties. The development of such a variety of test repose is a characteristic related to interparticulate friction, or
methods was inevitable; powder behavior is multifaceted and resistance to movement between particles. Angle of repose
thus complicates the effort to characterise powder flow. test results are reported to be very dependent upon the
method used. Experimental difficulties arise due to
segregation of material and consolidation or aeration of the
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter
powder as the cone is formed. Despite its difficulties, the
5. 8 Phamzacopoeial harmonisation.
V-A612 Appendix XVII N 2023

method continues to be used in the pharmaceutical industry, used to form the cone of powder. On this subject, the
and a number of examples demonstrating its value in existing literature raises these important considerations:
predicting manufacturing problems appear in the literature. - the peak of the cone of powder can be distorted by the
The angle of repose is the constant, three-dimensional angle impact of powder from above. By carefully building the
(relative to the horizontal base) assumed by a cone-like pile powder cone, the distortion caused by impact can be
of material formed by any of several different methods, minimised;
described briefly below. - the nature of the base upon which the powder cone is
formed influences the angle of repose. It is recommended
Basic methods for angle of repose
that the powder cone be formed on a 'common base',
A variety of angle of repose test methods are described in the
which can be achieved by forming the cone of powder on
literature. The most common methods for determining the
a layer of powder. This can be done by using a base of
static angle of repose can be classified based on 2 important
fixed diameter with a protruding outer edge to retain a
experimental variables:
layer of powder upon which the cone is formed.
- the height of the 'funnel' through which the powder
passes may be fixed relative to the base, or the height may Recommended procedure for angle of repose
be varied as the pile forms; Form the angle of repose on a fixed base with a retaining lip
- the base upon which the pile forms may be of fixed to retain a layer of powder on the base. The base must be
diameter or the diameter of the powder cone may be free of vibration. Vary the height of the funnel to carefully
allowed to vary as the pile forms. build up a symmetrical cone of powder. Care must be taken
to prevent vibration as the funnel is moved. The funnel
Variations in angle of repose methods
height is maintained at approximately 2-4 cm from the top of
Variations of the above methods have also been used to some
the powder pile as it is being formed in order to minimise the
extent in the pharmaceutical literature:
impact of falling powder on the tip of the cone. If a
- drained angle of repose: this is determined by allowing an
symmetrical cone of powder cannot be successfully or
excess quantity of material positioned above a fixed
reproducibly prepared, this method is not appropriate.
diameter base to 'drain' from the container. Formation of
Determine the angle of repose by measuring the height of the
a cone of powder on the fixed diameter base allows
cone of powder and calculating the angle of repose, rx, from
determination of the drained angle of repose;
the following equation:
- dynamic angle of repose: this is determined by filling a
cylinder (with a clear, flat cover on one end) and rotating height
it at a specified speed. The dynamic angle of repose is the tan (oc ) = - - - -
0.5 x base
angle (relative to the horizontal) formed by the flowing
powder. The internal angle of kinetic friction is defined COMPRESSIBILITY INDEX AND HAUSNER RATIO
by the plane separating those particles sliding down the In recent years the compressibility index and the closely
top layer of the powder and those particles that are related Hausner ratio have become the simple, fast, and
rotating with the drum (with roughened surface). popular methods of predicting powder flow characteristics.
The compressibility index has been proposed as an indirect
General scale of flowability for angle of repose
measure of bulk density, size and shape, surface area,
While there is some variation in the qualitative description of
moisture content, and cohesiveness of materials, because all
powder flow using the angle of repose, much of the
of these can influence the observed compressibility index.
pharmaceutical literature appears to be consistent with the
The compressibility index and the Hausner ratio are
classification by Carr2 , which is shown in Table 2.9.36.-1.
determined by measuring both the bulk volume and tapped
There are examples in the literature of formulations with an
volume of a powder.
angle of repose in the range of 40-50 degrees that
manufactured satisfactorily. When the angle of repose Basic methods for compressibility index and Hausner
exceeds 50 degrees, the flow is rarely acceptable for ratio
manufacturing purposes. While there are some variations in the method of determining
the compressibility index and Hausner ratio, the basic
Table 2.9.36.-1. - Flow properties and corresponding angles of procedure is to measure the unsettled apparent volume, (Va),
repose2 and the final tapped volume, ( Vj-), of the powder after
Flow property Angle of repose (degrees) tapping the material until no further volume changes occur.
Excellent 25-30 The compressibility index and the Hausner ratio are
Good 31-35 calculated as follows:
Fair (aid not needed) 36-40
Passable (may hang up) 41-45 Compressibility Index = 100 x Vo - V1
Vo
Poor (must agitate, vibrate) 46-55
Very poor 56-65
.
Very, very poor > 66 Hausner Ratw = -Vo
V1
Experimental considerations for angle of repose Alternatively, the compressibility index and Hausner ratio
Angle of repose is not an intrinsic property of the powder, may be calculated using measured values of bulk density
that is to say, it is very much dependent upon the method (Pbutk) and tapped density (P,apped) as follows:

Compressibility Index = 100 x P,apped - Pbuik

P,apped

2 Carr RL. Evaluating flow properties of solids. Chem. Eng 1965; Hausner Ratio= Piapped
72: 163-168. Pbulk
2023 Appendix XVII N V-A613

In a variation of these methods, the rate of consolidation is - the size and shape of the orifice used. The orifice
sometimes measured rather than, or in addition to, the diameter and shape are critical factors in determining
change in volume that occurs on tapping. For the powder flow rate;
compressibility index and the Hausner ratio, the generally - the method of measuring powder flow rate. Flow rate can
accepted scale of flowability is given in Table 2.9.36.-2. be measured continuously using an electronic balance
with some sort of recording device (strip chart recorder,
Table 2.9.36.-2. - Scale of fiowability 3 computer). It can also be measured in discrete samples
Compressibility index Flow character Hausner ratio (for example, the time it takes for 100 g of powder to
(per cent) pass through the orifice to the nearest tenth of a second
1-10 Excellent 1.00-1.ll or the amount of powder passing through the orifice in
11-15 Good 1.12-1.18 10 s to the nearest tenth of a gram).
16-20 Fair 1.19-1.25
Variations in methods for flow through an orifice
21-25 Passable 1.26-1.34
Either mass flow rate or volume flow rate can be determined.
26-31 Poor 1.35-1.45
Mass flow rate is the easier of the methods, but it biases the
32-37 Very poor 1.46-1.59
results in favour of high-density materials. Since die fill is
> 38 Very, very poor > 1.60 volumetric, determining volume flow rate may be preferable.
A vibrator is occasionally attached to facilitate flow from the
Experimental considerations for the compressibility container, however, this appears to complicate interpretation
index and Hausner ratio of results. A moving orifice device has been proposed to
Compressibility index and Hausner ratio are not intrinsic more closely simulate rotary press conditions. The minimum
properties of the powder, that is to say, they are dependent diameter orifice through which powder flows can also be
upon the methodology used. The existing literature points identified.
out several important considerations affecting the General scale of flowability for flow through an orifice
determination of the unsettled apparent volume, V0, of the No general scale is available because flow rate is critically
final tapped volume, Vi, of the bulk density, Pbuik, and of the dependent on the method used to measure it. Comparison
tapped density, Ptappel between published results is difficult.
- the diameter of the cylinder used,
- the number of times the powder is tapped to achieve the Experimental considerations for flow through an
tapped density, orifice
Flow rate through an orifice is not an intrinsic property of
- the mass of material used in the test,
the powder. It is very much dependent upon the
- rotation of the sample during tapping.
methodology used. The existing literature points out several
Recommended procedure for compressibility index and important considerations affecting these methods:
Hausner ratio - the diameter and shape of the orifice,
Use a 250 mL volumetric cylinder with a test sample mass of - the type of container material (metal, glass, plastic),
100 g. Smaller amounts and volumes may be used, but - the diameter and height of the powder bed.
variations in the method must be described with the results.
Recommended procedure for flow through an orifice
An average of 3 determinations is recommended.
Flow rate through an orifice can be used only for materials
FLOW THROUGH AN ORIFICE that have some capacity to flow. It is not useful for cohesive
The flow rate of a material depends upon many factors, some materials. Provided that the height of the powder bed (the
of which are particle-related and some related to the process. 'head' of powder) is much greater than the diameter of the
Monitoring the rate of flow of material through an orifice has orifice, the flow rate is virtually independent of the powder
been proposed as a better measure of powder flowability. head. It is advisable to use a cylinder as the container,
Of particular significance is the utility of monitoring flow because the walls of the container must have little effect on
continuously, since pulsating flow patterns have been flow. This configuration results in flow rate being determined
observed even for free-flowing materials. Changes in flow rate by the movement of powder over powder, rather than
as the container empties can also be observed. Empirical powder along the wall of the container. Powder flow rate
equations relating flow rate to the diameter of the opening, often increases when the height of the powder column is less
particle size, and particle density have been determined. than twice the diameter of the column. The orifice must be
However, determining the flow rate through an orifice is circular and the cylinder must be free of vibration. General
useful only with free-flowing materials. guidelines for dimensions of the cylinder are as follows:
The flow rate through an orifice is generally measured as the - diameter of the opening greater than 6 times the diameter
mass per time flowing from any of a number of types of of the particles,
containers (cylinders, funnels, hoppers). Measurement of the - diameter of the cylinder greater than twice the diameter
flow rate can be in discrete increments or continuous. of the opening.
Basic methods for flow through an orifice Use of a hopper as the container may be appropriate and
There are a variety of methods described in the literature. representative of flow in a production situation. It is not
The most common for determining the flow rate through an advisable to use a funnel, particularly one with a stem,
orifice can be classified based on 3 important experimental because flow rate will be determined by the size and length
variables: of the stem as well as the friction between the stem and the
- the type of container used to contain the powder. powder. A truncated cone may be appropriate, but flow will
Common containers are cylinders, funnels, and hoppers be influenced by the powder-wall friction coefficient, thus,
from production equipment; selection of an appropriate construction material is
important.
For the opening in the cylinder, use a flat-faced bottom plate
' Can- RL. Evaluating flow properties of solids. Chem. Eng 1965;
72: 163-168.
with the option to vary orifice diameter to provide maximum
V-A614 Appendix XVII N 2023

flexibility and better ensure a powder-over-powder flow equipment, and an approach commonly used to characterise
pattern. Rate measurement can be either discrete or these properties is to submit the powder to shear tests, which
continuous. Continuous measurement using an electronic are experiments designed to determine the flow properties of
balance can more effectively detect momentary flow rate the powder by applying different states of stress and strain to
variations. it. Using these tests, a wide variety of parameters describing
SHEAR CELL METHODS the flow properties of a powder can be studied, including the
In an effort to put powder flow studies and hopper design on yield locus, the angle of internal friction, the compressive
a more fundamental basis, a variety of powder shear testers strength, the tensile strength, and a variety of derived
and methods that permit more thorough and precisely parameters such as the flowability ratio. Because of the ability
defined assessment of powder flow properties have been to control experimental parameters precisely, flow properties
developed. Shear cell methodology has been used extensively can also be determined as a function of consolidation load,
in the study of pharmaceutical materials. From these time, and environmental conditions such as temperature and
methods, a wide variety of parameters can be obtained, humidity.
including the yield loci representing the shear stress-shear Many suitable shear cell configurations and test methods are
strain relationship, the angle of internal friction, the available; the most widely used are those based on the
unconfined yield strength, the tensile strength, and a variety Jenike-type shear cell or on ring shear cells such as the
of derived parameters such as the flow factor and other Schulze-type ring shear tester.
flowability indices. Because of the ability to control PRINCIPLE
experimental parameters more precisely, flow properties can UN/AXIAL COMPRESSION TEST
also be determined as a function of consolidation load, time, The uniaxial compression test illustrates the concept of
and other environmental conditions. These methods have flowability. As shown in Figure 2.9.49.-1, a hollow cylinder
been successfully used to determine critical hopper and bin (cross-sectional area A, internal wall assumed as frictionless)
parameters. is filled with a powder. The powder is loaded by the
Basic methods for shear cell consolidation stress (cr 1 ) in the vertical direction. The more
One type of shear cell is the cylindrical shear cell which is the volume of the powder can be reduced, the more
split horizontally, forming a shear plane between the lower compressible the powder is. In addition to the increase in
stationary base and the upper moveable portion of the shear bulk density (pb) from the consolidation stress, an increase in
cell ring. After powder bed consolidation in the shear cell the strength of the powder is also observed. Hence, the
(using a well-defined procedure), the force necessary to shear powder is both consolidated and compressed through the
the powder bed by moving the upper ring is determined. effect of the consolidation stress.
Annular shear cell designs offer some advantages over the
cylindrical shear cell design, including the need for less cr C ·A
material. A disadvantage, however, is that because of its
design, the powder bed is not sheared as uniformly because area A
material on the outside of the annulus is sheared more than
material in the inner region. A third type of shear cell (plate-
type) consists of a thin sandwich of powder between a lower
stationary rough surface and an upper rough surface that is
moveable.
All of the shear cell methods have their advantages and /
disadvantages, but a detailed review is beyond the scope of
this chapter. As with the other methods for characterising Figure 2.9.49.-1. - Uniaxial compression test
powder flow, many variations are described in the literature.
A significant advantage of shear cell methodology in general After consolidation, the powder is relieved of the
is a greater degree of experimental control. The methodology consolidation stress (cr 1) and the hollow cylinder is removed.
generally is rather time-consuming and requires significant The consolidated cylindrical powder sample is subsequently
amounts of material and a well-trained operator. loaded with an increasing vertical compressive stress and, at a
Recommendations for shear cell certain stress, the sample fails along the shear plane and the
The many existing shear cell configurations and test methods powder starts to flow. The compressive strength (crc), or
provide a wealth of data and can be used very effectively to unconfined yield strength, is defined as the stress causing
characterise powder flow. They are also helpful in the design failure. Since the powder fails only at a sufficiently large
of equipment such as hoppers and bins. Because of the vertical stress, there is a specific yield limit for the powder.
diversity of available equipment and experimental procedures, The yield limit of a powder is dependent on its stress history,
no specific recommendations regarding methodology are i.e. its previous consolidation. The greater the consolidation
presented in this chapter. It is recommended that the results stress (cr 1) the greater the bulk density (Pb) and compressive
of powder flow characterisation using shear cell methodology strength (cr c). Thus, uniaxial compression tests conducted at
include a complete description of equipment and different consolidation stresses (cr 1) yield different pairs of
methodology used. values (cr1, crc) and (cr1, Pb)- Plotting these pairs of values as
points in a (cri, crc) diagram and a (cr 1, Pb) diagram,
N2. Powder Flow Properties by Shear Cell Methods
respectively, and drawing in each diagram a curve through
(Ph. Bur. method 2. 9. 49) these points, usually results in curves similar to those shown
The methodology in this general chapter is based on for product A in Figure 2.9.49.-2, where bulk density (pb)
standard test methods ASTM D6773-08 and and compressive strength (crc) typically increase with
ASTM D6128-14. consolidation stress (cr 1). Very rarely a progressive slope
Powder flow properties play an important role in the design similar to that of the left part of curve B is observed.
of formulations, processes and pharmaceutical production The graph of crc versus cr 1 is called the flow function.
2023 Appendix XVII N V-A615

I
Furthermore, the flow function may change depending on
the consolidation time, for example, at the same CJ 1 level,
higher CJc values may be obtained with longer consolidation
times; this is known as the time consolidation effect. Possible
mechanisms are solid or liquid bridges, solid crystallisation,
viscoelastic or viscoplastic deformation, or chemical reactions
at the particle contacts.

-
YIELD LIMIT AND MOHR STRESS CIRCLE
a, Assuming that the force of gravity and wall friction effects
can be disregarded, the uniaxial compression test can be

I
.,,,,. represented as shown in Figure 2.9.49.-4, in a (CJ, ,) diagram
/ where CJ is the normal stress and t is the shear stress. In such
~B a diagram, all pairs of values (CJ, ,) form a circle representing
/ the stresses in the powder; this is called a 'Mohr stress circle'.
/ The Mohr stress circle is centred on the sigma axis. The two
intersect points with the sigma axis are called the minor and
major principal stresses. Figure 2.9.49.-4 shows the Mohr
stress circles corresponding to the uniaxial compression test
and a possible yield limit of the sample (the real course of
the yield limit cannot be determined with only the uniaxial
compression test).

a,------
Figure 2.9.49.-2. - Bulk density (pi,) and compressive strength
Mohr stress circle A describes the stresses in the powder
sample at consolidation. Since no shear stress is applied, CJ 1
corresponds to the normal stress or vertical stress ( CJv) and CJ2
corresponds to the horizontal stress (CJh).
( CJ J as a function of consolidation stress (CJ J

NUMERICAL CHARACTERISATION OF T
FLOWABILI1Y
The flow function can be used to characterise the flow
behaviour of a powder in terms of the flowability ratio (ffc),
which is given by: 0
a 1 =aV
a-

The larger the ffc, the better a powder flows. The ff;, value
can be used to classify the flow behaviour as follows: a=
2
canst
ffc < 1 not flowing

l<ffc<2 very cohesive B3: incipient flow C: incipient flow A: consolidation

2<ff,<4 cohesive a 2 =0 a2 >0
4 < ffc < 10 easy-flowing
Figure 2.9.49.-4. - Measurement of compressive strength and
10 < ff, free-flowing
corresponding (CJ, r) diagram

Figure 2.9.49.-3 shows the flow function A taken from the In the second part of the test shown in Figure 2.9.49.-1, the
(CJ 1, CJc) diagram in Figure 2.9.49.-2. Additionally, the sample is loaded with increasing vertical stress after it has
boundaries of the ranges of the classifications listed above are been relieved of the consolidation stress and the hollow
shown as straight lines, each representing a constant value of cylinder has been removed. As the vertical load increases, the
the flowability ratio (ffc)- This diagram clearly shows that the stress states at different load steps are represented by stress
flowability ratio (ffc) of a specific powder is dependent on the circles with increasing diameter (stress circles B 1, B2 , B3 in
consolidation stress (CJ 1) applied. Figure 2.9.49.-4). The minor principal stress, i.e. the
horizontal stress, is equal to zero for all stress circles since the
ffc = 1 lateral surface of the sample is uncovered and not loaded.
not flowing
very cohesive Mohr stress circle B3 represents the stresses in the powder
sample at failure of the sample. Since the load corresponding
to this Mohr stress circle causes incipient flow of the sample,
ffC = 4 Mohr stress circle B3 must be tangential to the yield limit in
the (CJ, t) diagram.
ffC = 10
If, during the second part of the experiment shown in
Figure 2.9.49.-1 (i.e. the measurement of compressive
free-flowing
0 strength), a constant horizontal stress CJh > 0 were also
0 a,- applied to the sample in addition to the vertical stress (CJv),
stress circles that indicate failure of the sample and are
Figure 2.9.49.-3. - Flow function and lines of constant tangential to the yield limit (e.g. stress circle C in Figure
fiowability ratio 2.9.49.-4) would likewise be found. Thus the yield limit is
V-A616 Appendix XVII N 2023

powder sample lid of shear cell shearing plane. The lid of the shear cell is allowed to move
vertically in order to adjust to changes in the sample's bulk
density.
When a point of a yield locus is measured, as in the uniaxial
V= constant compression test, 2 steps are necessary: first the powder
sample is consolidated in a preshear step, and then a point of
i.;:..;~......,;,.:,,:~..::.,;.:,..;.;:s;s.;-+';J-- bottom of the yield locus is measured in a shear or shear-to-failure step.
shear cell PRESHEAR STEP
For preshear, the powder sample is loaded in the vertical
Figure 2.9.49.-5. - Principle of shear defmnation in a shear cell direction under a well-defined normal stress (er= erpre), and
is then sheared. Preshear is stopped when steady-state flow,
the envelope of all stress circles that indicate failure of a
characterised by constant shear stress (tpre), is achieved.
powder sample.
The pair of values of normal stress and shear stress at steady-
MEASUREMENT WITII A SHEAR TESTER state flow (erpre, tprc) is plotted in a normal stress - shear
PRINCIPLE stress diagram ((er, t) diagram, Figure 2.9.49.-6, right). Point
The test sample must be representative of the powder with (erpre, 'tpre) is called the preshear point.
respect to particle-size distribution, moisture, temperature
and other properties that may have an influence on the flow
SHEAR-TO-FAILURE STEP
After the powder has been consolidated by the preshear
behaviour. The test sample is filled into a shear cell of
procedure, the shear stress (t) is reduced to zero by reversing
circular or annular cross-section, depending on the type of
the relative motion of the lid with respect to the bottom of
tester used. In order to achieve a homogenous and
the shear cell.
representative powder bed, filling should be carried out
uniformly in small horizontal layers using a spoon or spatula, For the next step of the test procedure, the so-called shear or
without applying force to the surface of the material, until the shear-to-failure step, the normal stress acting on the sample
cell is slightly overfilled. Excess powder is then removed by is decreased to a value er,h, which is less than the normal
scraping off with a blade until the powder is flush with the stress at preshear (erpre).
top of the shear cell. The shear cell is then completed with a If the consolidated sample is sheared under the normal stress
lid placed on top of the sample. The latter step may vary ersh < erpre, it will start to flow when a sufficiently large shear
with the type of apparatus and is therefore carried out stress (t,0 is attained. At that point particles start to move
according to the manufacturer's instructions to achieve a against each other. The material will start to dilate,
homogenous and representative powder bed. i.e. decrease in bulk density and shear resistance, and thus
In general, a shear test measures the yield limit of a shear stress will decrease. The maximum shear stress (t,0
consolidated powder bed. This yield limit is also called the characterises incipient flow. The corresponding pair of values
yield locus. It depends to a certain extent on the apparatus (er,h, tm} is a point of the yield locus in the (er, t) diagram
and experimental conditions. Typically the yield locus is (Figure 2.9.49.-6, right). Such a point is called a shear point
measured immediately after consolidation of the powder bed. or a point of incipient flow.
If however the yield locus is measured after a certain In order to measure the course of the yield locus, several of
consolidation time, then it is called the time yield locus. the tests described above must be performed, where the
When running a shear test, a normal stress (er) is applied samples must first be consolidated at the same normal stress
vertically to the powder in the shear cell. A shear (erpre, preshear). Then the samples are sheared to failure
deformation of the sample is then induced by moving the lid under different normal stresses er,h < erpre• The yield locus
of the shear cell relative to the bottom of the shear cell in a follows a curve plotted through all measured shear points
horizontal direction with constant velocity (V) (see Figure (Figure 2.9.49.-6, right). In general, at least 3 shear points
2.9.49.-5). This results in a horizontal shear stress (t), which should be measured.
develops due to the friction between the particles in the

yield locus

incipient flow
,t shear points
(incipient flow)
preshear point
(steady flow)

I
-- - - 'tpre -- --
7

steady-state
flow
I 'sh

1
I
I
I I
o,___ __,1-----1------+-----
-If-
Preshear shear time
... 0
cr

(crp,e) (cr,h < crP,.) next test (J'pre

Figure 2.9.49.-6. - Plot of shear stress versus time (left) and corresponding yiel,d locus (right)
2023 Appendix XVII O V-A61 7

linearised yield locus effective yield locus

With the Jenike-type shear cell a new sample has to be (slope cp 110 )
prepared for each shear point (indicated as 'next test' in
Figure 2.9.49.-6). However, with ring shear testers a yield locus
complete yield locus is usually measured with 1 sample.
In addition, with the Schulze-type ring shear tester the bulk
density is measured during the test, depending on the
consolidation stress applied.
DATA EVALUATION
Parameters that describe the flow properties can be
determined from the yield locus as shown in cr-
Figure 2.9.49.-7.

steady-state flow Figure 2.9.49.-8. - Yield locus and characteristic values for flow
incipient flow properties

The cohesion (le) is the value of the shear stress where the
yield locus intersects with the ,-axis, i.e. at normal stress
cr = 0. The uniaxial tensile strength (crJ is the normal stress
end point of the yield locus at the left end of the yield locus at shear stress ,: = 0. As it is
difficult to measure the yield locus at small and negative
stress levels, cohesion may be obtained by extrapolating the
yield locus to the intersection with the ,-axis. Due to the
0 ,.__ _....u.,._ _ _ _ _ _....__ _ _ _ _ _ _ _ _ __ _ . _ pronounced non-linearity of the yield locus at low stresses,
the cohesion obtained in this way is less accurate than the
0 0'2 /(Jc c r - / c r1 compressive strength, and it is hardly possible to determine
crC ·A cr 1 ·A
tensile strength by extrapolation.
If several yield loci are measured at different stress levels,
i.e. with different normal stresses at preshear (crp,e), each
yield locus represents another state of consolidation and
another bulk density. The above-mentioned flow properties,
namely compressive strength, effective angle of internal
/ friction or slope angle of the linearised yield locus, can be
indicated as functions of the consolidation stress (cr 1), similar
Figure 2.9.49.-7. - Yield locus, Mohr stress circles and analogy to Figure 2.9.49.-2 where bulk density and compressive
with uniaxial compression test strength are plotted versus consolidation stress.

The consolidation stress (cr 1) is equal to the major principal

stress of the Mohr stress circle, which is tangential to the
yield locus, and intersects at the point of steady-state flow
( crp,e, 'pre). This stress circle represents the stress distribution
01. Optical Microscopy1
in the sample at the end of the consolidation procedure (Ph. Bur. method 2.9.37)
(stresses at steady-state flow). It corresponds to the stress Optical microscopy for particle characterisation can generally
circle at the end of consolidation in the uniaxial compression be applied to particles of 1 µm and greater. The lower limit
test. is imposed by the resolving power of the microscope.
The compressive strength (crc) is determined from the stress The upper limit is less definite and is determined by the
circle that is tangential to the yield locus and runs through increased difficulty associated with the characterisation of
the origin (minor principal stress cr 2 = 0). This stress circle larger particles. Various alternative techniques are available
represents a similar stress state to the one that prevails in the for particle characterisation outside the applicable range of
second step of the uniaxial compression test. optical microscopy. Optical microscopy is particularly useful
A straight line through the origin of the (cr, ,) diagram, for characterising particles that are not spherical. This
tangent to the greater Mohr circle, is the effective yield locus, method may also serve as a base for the calibration of faster
shown as a broken line in Figure 2.9.49.-7. It encloses the and more routine methods that may be developed.
cr-axis with the effective angle of internal friction (cpe)- Apparatus
Because the largest Mohr stress circle indicates a state of Use a microscope that is stable and protected from vibration.
steady-state flow, the angle cpe can be regarded as a measure The microscope magnification (product of the objective
of the internal friction at steady-state flow. magnification, ocular magnification, and additional
Further flow properties can be determined from the yield magnifying components) must be sufficient to allow adequate
locus as illustrated in Figure 2.9.49.-8. characterisation of the smallest particles to be classified in the
For many applications the yield locus is linearised as the test sample. The greatest numerical aperture of the objective
tangent to both Mohr circles. The linearised yield locus is is sought for each magnification range. Polarising filters may
characterised by its slope angle (jl1in· be used in conjunction with suitable analysers and
retardation plates. Colour filters of relatively narrow spectral
transmission are used with achromatic objectives, and are

1 This chapter has undergone phamzacopoeial hamzonisation. See chapter

5. 8 Phamzacopoeial hamzonisation.
V-A618 Appendix XVII 0 2023

preferable with apochromats; they are required for oil on a clean glass slide. Examine the mixture using a
appropriate colour rendition in photomicrography. polarising microscope: the particles show birefringence
Condensers, corrected at least for spherical aberration are (interference colors) and extinction positions when the
used in the microscope substage and with the lamp. microscope stage is revolved.
The numerical aperture of the substage condenser matches Limit test of particle size by microscopy
that of the objective under the conditions of use; this is Weigh a suitable quantity of the powder to be examined (for
affected by the actual aperture of the condenser diaphragm example, 10-100 mg), and suspend it in 10 mL of a suitable
and the presence of immersion oils. medium in which the powder does not dissolve, adding, if
Adjustment necessary, a wetting agent. A homogeneous suspension of
The precise alignment of all elements of the optical system particles can be maintained by suspending the particles in a
and proper focusing are essential. The focusing of the medium of similar or matching density and by providing
elements is done in accordance with the recommendations of adequate agitation. Introduce a portion of the homogeneous
the microscope manufacturer. Critical axial alignment is suspension into a suitable counting cell, and scan under a
recommended. microscope an area corresponding to not less than 10 µg of
IDumination the powder to be examined. Count all the particles having a
A requirement for good illumination is a uniform and maximum dimension greater than the prescribed size limit.
adjustable intensity of light over the entire field of view; The size limit and the permitted number of particles
Kohler illumination is preferred. With coloured particles, exceeding the limit are defined for each substance.
choose the colour of the filters so as to control the contrast Particle size characterisation
and detail of the image. The measurement of particle size varies in complexity
Visual characterisation depending on the shape of the particle, and the number of
The magnification and numerical aperture must be particles characterised must be sufficient to ensure an
sufficiently high to allow adequate resolution of the images of acceptable level of uncertainty in the measured parameters.
the particles to be characterised. Determine the actual Additional information on particle size measurement, sample
magnification using a calibrated stage micrometer to calibrate size, and data analysis is available, for example, in ISO 9276.
an ocular micrometer. Errors can be minimised if the For spherical particles, size is defined by the diameter.
magnification is sufficient that the image of the particle is at For irregular particles, a variety of definitions of particle size
least 10 ocular divisions. Each objective must be calibrated exist. In general, for irregularly shaped particles,
separately. To calibrate the ocular scale, the stage micrometer characterisation of particle size must also include information
scale and the ocular scale must be aligned. In this way, a on the type of diameter measured as well as information on
precise determination of the distance between ocular stage particle shape. Several commonly used measurements of
divisions can be made. Several different magnifications may particle size are defined in Figure 2.9.37.-1.
be necessary to characterise materials having a wide particle
size distribution.
Photographic characterisation
If particle size is to be determined by photographic methods,
take care to ensure that the object is sharply focused at the
plane of the photographic emulsion. Determine the actual
magnification by photographing a calibrated stage
micrometer, using photographic film of sufficient speed,
resolving power, and contrast. Exposure and processing must
be identical for photographs of both the test sample and the
determination of magnification. The apparent size of a Martin's diameter
photographic image is influenced by the exposure,
development, and printing processes as well as by the
resolving power of the microscope.
Preparation of the mount
The mounting medium will vary according to the physical
properties of the test sample. Sufficient, but not excessive,
contrast between the sample and the mounting medium is
required to ensure adequate detail of the sample edge.
The particles must rest in one plane and be adequately
dispersed to distinguish individual particles of interest. Figure 2.9.37.-1. - Commonly used measurements of particle size
Furthermore, the particles must be representative of the
- Feret's diameter. the distance between imaginary parallel
distribution of sizes in the material and must not be altered
lines tangent to a randomly oriented particle and
during preparation of the mount. Care must be taken to
perpendicular to the ocular scale,
ensure that this important requirement is met. Selection of
- Martin's diameter. the diameter of the particle at the point
the mounting medium must include a consideration of the
that divides a randomly oriented particle into 2 equal
analyte solubility.
projected areas,
Crystallinity characterisation - projected area diameter. the diameter of a circle that has the
The crystallinity of a material may be characterised to same projected area as the particle,
determine compliance with the crystallinity requirement - length: the longest dimension from edge to edge of a
where stated in the individual monograph of a drug particle oriented parallel to the ocular scale,
substance. Unless otherwise specified in the individual - width: the longest dimension of the particle measured at
monograph, mount a few particles of the sample in mineral right angles to the length.
2023 Appendix XVII O V-A619

dIJEquant
Flake

Plate

Columnar

Figure 2.9.37.-2. - Commonly used descriptions of partide shape

Particle shape characterisation microscopy in terms of resolution, magnification and depth

For irregularly shaped particles, characterisation of particle of field.
size must also include information on particle shape. In addition, by exploiting the different interactions between
The homogeneity of the powder must be checked using the electrons and the specimen, it provides topographical and
appropriate magnification. The following defines some compositional information and can thus also be used as an
commonly used descriptors of particle shape (see analytical tool. When it is combined with elemental X-ray
Figure 2.9.37.-2). microanalysis, in particular, chemical analysis can be
- acicular: slender, needle-like particle of similar width and performed (see general chapter 2.2.37. X-ray fluorescence
thickness, spectrometry).
- columnar: long, thin particle with a width and thickness
SEM is a well-established technique and, increasingly, it is
that are greater than those of an acicular particle,
being applied to examine and characterise a wide range of
- flake: thin, flat particle of similar length and width,
pharmaceutical materials in solid form but also, with the use
- plate: flat particle of similar length and width but with
of specialised specimen preparation methods, in semi-solid
greater thickness than a flake particle,
and liquid form.
- lath: long, thin, blade-like particle,
- equant. particle of similar length, width, and thickness; PRINCIPLE
both cubical and spherical particles are included. To produce magnified images, a scanning electron
General observations microscope uses a finely-focused beam of accelerated
A particle is generally considered to be the smallest electrons instead of light; the specimen is scanned (rastered)
discrete unit. A particle may be a liquid or semi-solid droplet; in a rectangular pattern with the electron beam. This
a single crystal or polycrystalline; amorphous or an technique exploits the interactions between the electron beam
agglomerate. Particles may be associated. This degree of and the specimen using different types of detectors. Due to
association may be described by the following terms: the nature of these interactions, a variety of signals are
- lamellar: stacked plates, produced and these can be used to provide characteristic
- aggregate: mass of adhered particles, information about the specimen at and from just below its
- agglomerate: fused or cemented particles, surface.
- conglomerate: mixture of 2 or more types of particles, INTERACTION WITH THE SPECIMEN
- spherulite: radial cluster, As the surface of the specimen is scanned by the electron
- drusy: particle covered with tiny particles. beam, these electrons (also known as primary electrons)
Particle condition may be described by the following terms: interact with the specimen and can penetrate to a depth of
- edges: angular, rounded, smooth, sharp, fractured, up to a few tens of micrometres. Electrons are scattered and
- optical: color (using proper color balancing filters), absorbed within a teardrop-shaped volume just below the
transparent, translucent, opaque, surface of the specimen, known as the interaction volume
- defects: occlusions, inclusions. (Figure 2.9.52-1). The depth to which the electron beam
Surface characteristics may be described as: penetrates is directly proportional to the beam voltage (a
- cracked: partial split, break, or fissure, high-energy beam will penetrate deeper than a low-energy
- smooth: free of irregularities, roughness, or projections, beam) and inversely proportional to the average atomic
- porous: having openings or passageways, number of the constituent elements of the specimen (the
- rough: bumpy, uneven, not smooth, beam will penetrate much deeper into a specimen rich in
- pitted: small indentations. light elements than into one rich in heavy elements).
Types of emitted signals
02. Scanning Electron Microscopy Figure 2. 9 .52-1 illustrates the sources of the principal
(Ph. Bur. method 2. 9. 52) emitted signals, which are described as follows:
This general chapter focuses on scanning electron microscopy - secondary electrons are low-energy electrons that are ejected
applied to pharmaceutical materials, from research to quality from just below the specimen surface as a result of the
control. inelastic scattering of the incident electrons;
Scanning electron microscopy (SEM) is a powerful imaging - backscattered electrons are high-energy electrons that result
technique that is superior to and complements light from the elastic scattering of incident electrons deeper in
V-A620 Appendix XVII 0 2023

the interaction volume; as a consequence, backscattered texture, porosity and shapes of the crystals. This information
electron images typically have a lower spatial resolution can be correlated with dissolution behaviour, bioavailability
than secondary electron images and show less surface and the crystallinity of the components in solid dosage forms.
detail; SEM has particular value in supporting the development and
- characteristic X-rays are produced when the incident optimisation of manufacturing processes for most solid
electron beam interacts with the elements in the dosage forms, such as tablets, powder mixtures for oral
specimen; suspensions, granules, powders for inhalation, spray- or
- cathodoluminescence is the emission of photons in the freeze-dried powders, and powders for injections.
visible spectrum when atoms and molecules deep within SPECIFICITIES OF THE TECHNIQUE
the interaction volume return to their ground state after SEM offers many distinct advantages over conventional
being excited by the electron beam. optical light microscopy because specimens can be examined
at higher magnifications (250 000 x compared to 1000 x,
for example) with greater lateral resolution (3 nm or better
compared to about 200 nm) and superior depths of field.
However, the interaction between the electrons and the
specimen could induce a strong charging effect due to the
accumulation of electrical charge on its surface. Specific
preparation procedures may thus be required for non-
conducting specimens so that the accumulated charges can
be dissipated.
Therefore, the ultimate performance of an electron
microscope depends upon a number of factors, with the
nature of the specimen having a major influence on the
quality of the final image.
EQUIPMENT
Figure 2.9.52-2 is a schematic diagram of an electron
microscope. It typically consists of:

electron
gun

0
d,

condenser
lens

beam scan
coils
Figure 2.9.52-1. - Schematic diagram showing the signals specimen
generated from a teardrop-shaped interaction volume image
X-ray emission
APPLICATIONS spectrum

[AJ
SEM is used to support many pharmaceutical processes
during the formulation and analytical development,
manufacturing and quality control of medicinal products.
Its applications include:
- physical and chemical analysis of samples;
- comparison of raw materials from different suppliers;
- support for the development of particle sizing methods;
- particle size analysis (using image analysis); ~cioc
- characterisation of the surface roughness of tablet film
specimen
coatings;
- investigation of complaints;
- analysis and identification of particulate matter Figure 2.9.52-2. - Schematic diagram of a scanning electron
contamination; microscope
- trouble-shooting of manufacturing problems; - an electron gun that is housed at the top of an electron-
- examination of falsified products. optical column (electron column) and emits a beam of
SEM is used to examine a wide range of materials, such as electrons; the electrons are accelerated down the column
active pharmaceutical ingredients (APis), excipients, powder under the influence of a high voltage that is typically
blends, solid dosage forms, medical devices, primary and variable from 100 V to 30 kV;
secondary packaging materials and contaminants. - an electron column, which contains various components
The examination of SEM images permits the qualitative and used to control and focus the electron beam:
quantitative assessment of the homogeneity and consistency - a condenser lens system that controls the diameter
of powders as raw materials or after processing and energy of the electron beam;
(e.g. compressed into tablets), with respect to the shapes, - scan coils that are used to move the beam in a raster-
sizes and size distributions of particles in powders and of the fashion across a rectangular area on the specimen;
2023 Appendix XVII O V-A621

- an objective lens that focuses the beam to a fine point specimen that happen to be in the direct line of sight of the
and directs it towards the specimen; detector. As a consequence, secondary electron images
- a specimen chamber positioned at the bottom of the include contributions from both secondary electrons and
electron column; it contains the specimen and an array of backscattered electrons and it is this combination that
detectors that collect the emitted signals; produces the three-dimensional effect that is a distinctive
- a system of pumps that maintains the electron gun characteristic of secondary electron images.
chamber and electron column under high vacuum to An E-T detector cannot be used in ESEM or VPSEM
preserve the reliability and stability of the electron source. because secondary electrons emitted from a specimen are
VACUUM IN THE SPECIMEN CHAMBER scattered and absorbed by the gas molecules in the chamber.
Conventional SEM is performed with the electron column To observe secondary electron images in ESEM or VPSEM,
and specimen chamber maintained at a high vacuum so that a detector that is sensitive to the small amount of light that is
conductive specimens can be examined. However, recent emitted from the ionised gas molecules surrounding the
developments have enabled the examination of non- specimen (a process called gas luminescence) in the specimen
conductive specimens and hydrated materials either in a chamber is used. It is this cloud of positive ions around the
humid or wet atmosphere using environmental SEM specimen that also neutralises any negative charge
(ESEM), or in a dry partial vacuum using variable pressure accumulation.
SEM (VPSEM) at pressures ranging from 10 Pa to 103 Pa. Backscattered electron detectors
A differential vacuum pumping system keeps the gun Backscattered electrons have higher energies than secondary
chamber at a high vacuum relative to the low vacuum electrons and are emitted over a broad angular range.
(sometimes close to atmospheric pressure) in the specimen Consequently, the detector is positioned close to and above
chamber. An advantage of using ESEM or VPSEM is that the specimen. Most high-vacuum and low-vacuum electron
the microscope can also be operated in high vacuum mode. microscopes are fitted with a dedicated backscattered electron
ELECTRON EMIITERS detector that uses either a scintillator or solid-state diodes.
There are 2 main types of electron sources: Scintillator detectors work in the same way as E-T secondary
- thermwnic emission. A widely-used thermionic source electron detectors by detecting electrons as flashes of light,
consists of a filament, usually a tungsten wire, heated by but have no biased grid to attract electrons towards them.
passing an electrical current directly through it. Solid-state backscattered electron detectors consist of an
An alternative is a lanthanum hexaboride (LaB 6 ) emitter, annulus having 4 or 5 separate photodiodes arranged as an
which consists of an indirectly heated single crystal; it has array of segments. The diodes can be turned on or off in
a much greater electron yield and is much brighter than a different combinations to allow topographic imaging,
tungsten emitter. compositional imaging or both together.
- field emission (FE). Electrons are emitted from the ultra- The emission of backscattered electrons increases with the
sharp tip of a fine-pointed tungsten wire immersed within average atomic number of the various components (pure or
an intense electrostatic field. Used in high-resolution composite) of the specimen. This behaviour can be exploited
electron microscopes, FE emitters give an intensely to examine the spatial distribution and homogeneity of
bright, small-diameter and low-current electron beam. components containing light and heavy atoms in mixtures
They are very stable and have an operating life of many such as powder blends, compressed tablets and specimens
thousands of hours. FE-SEM offers significant contaminated with foreign matter. Backscattered electron
advantages, because it produces high-resolution images at compositional images do not reveal which elements are
low accelerating voltages (down to a few hundreds of present in a specimen but they do show where high atomic
volts), with increased signal-to-noise ratios (giving low- number materials are relative to low atomic number
noise, high-quality images) and with a superior depth of materials. Materials consisting mainly of heavy atoms
field. (e.g. iron, bromine) appear brighter than those having lighter
DETECTORS atoms (e.g. carbon, nitrogen, oxygen, aluminium).
A cluster of different detectors (see Figure 2.9.52-2) are To determine which elements are actually present, elemental
positioned at optimised distances close to the specimen and X-ray microanalysis is used.
collect the variety of signals emitted during interaction with Unlike secondary electron detectors, backscattered electron
the electron beam. detectors are not greatly affected by electrical charging at the
Secondary electron detectors specimen surface. Therefore, non-conducting specimens can
Most high-vacuum electron microscopes are fitted with an be imaged in high-vacuum mode and in low-vacuum mode
Everhart-Thornley (E-T) detector positioned to one side of using a backscattered electron detector.
the specimen. E-T detectors are sensitive to the low-energy Both scintillator and solid-state backscattered electron
secondary electrons that originate from a shallow depth detectors are also very sensitive to visible light and can
(typically less than 50 nm) below the surface of a specimen. therefore also function as cathodoluminescence detectors.
The E-T detector has a positively biased wire grid in front of Cathodoluminescence detectors
it to attract electrons towards a scintillator which converts the The weak-intensity light emitted from cathodoluminescent
secondary electrons into flashes of light that are directed into materials is collected by a retractable paraboloidal mirror and
a photomultiplier. Secondary electrons can follow curved directed into a light-sensitive scintillator detector (not shown
paths from areas on specimens that may not be in the direct in Figure 2.9.52-2). The mirror is inserted just above the
line of sight of the detector. As a consequence, secondary specimen and is retracted when not in use. The electron
electron images tend to have high contrast and show beam passes through a small hole in the mirror so that it
considerable surface details that emphasise topographical strikes the specimen to induce the emission of visible light.
features and variations in surface roughness across specimens.
In its simplest form, the detector does not discriminate
In addition to secondary electrons, E-T detectors are also between different wavelengths of light and greyscale images
sensitive to backscattered electrons emitted from areas on the are produced with cathodoluminescent areas on a specimen
V-A622 Appendix XVII 0 2023

being shown as bright. More complex detectors have the since contaminant particles could potentially be released from
capability to select different wavelengths of light being metal tools.
emitted to produce monochromatic images tuned to a single SPECIMEN COATING
wavelength that represents a specific material. Most pharmaceutical materials are readily examined without
Cathodoluminescence spectral analysis is achieved by passing the need for prolonged and complicated preparation
polychromatic light emitted from the specimen into a techniques. However, such materials tend to be electrically
spectrometer where it is dispersed to produce a visible light non-conductive and so specimens that are examined using
spectrum. high-vacuum SEM need to be coated with an ultra-thin layer
Spectral analysis of the emitted light is not widely used but of conductive material. Without a conductive coating,
has applications in the examination and characterisation of specimens will acquire a net negative charge as the electron
some organic and inorganic materials. It can be used to beam scans across them; this causes the specimen to glow
acquire diagnostic information about the chemistry and brightly and, when it discharges, disturbing flashes and bright
structure of single materials and spectral imaging can be used streaks will be seen in the image. Suitable conductive
for analysing mixtures of materials. materials for coating include metals, such as platinum and
Elemental X-ray microanalysis detectors gold, and these can be applied to rough and smooth
An X-ray analyser fitted to the specimen chamber of an specimen surfaces using a plasma sputter coater. If carbon is
electron microscope enables the rapid and non-destructive to be used, it needs to be evaporated in a very high vacuum
elemental analysis of materials being examined. 2 main because it cannot be sputtered. In addition to reducing
detector types are available and these detect either the charging effects, a conductive coating will increase the
dispersed energies (energy-dispersive X-ray microanalysis, emission of secondary electrons to give brighter images and
EDX) or the dispersed wavelengths (wavelength-dispersive will also transfer localised electron beam-induced heat away
X-ray microanalysis, WDX) of the characteristic X-rays from the specimen. Some powders consisting of very small
emitted from specimens (for more details, see general particles (such as colloidal silica) may need to be sputter
chapter 2.2.37). coated 3 or 4 times to prevent charging because the coating
material initially fails to form a continuous conductive layer.
PROCEDURE
Some beam-sensitive specimens, such as semi-solids or wet
SPECIMEN PREPARATION I PRESENTATION
materials, may have to be cooled or frozen using a cooling
Prior to preparing a specimen for examination by SEM, the
stage to minimise degradation or evaporation. With respect
purpose of the examination must be considered because this
to this, FE-SEM allows the surfaces of labile specimens to be
can affect the way the sample is treated.
examined with an electron beam that has a very low energy
In addition, the electrical conductivity of the specimen must (e.g. 500 volts or less), and this can help to minimise or even
be considered to minimise charge accumulation as the eliminate beam-induced degradation.
electron beam scans across the specimen. Numerous
Specimens to be analysed by elemental X-ray microanalysis
methods have been developed to prepare specimens to
are typically not coated with metal because the coat would
maintain integrity whilst minimising artefacts.
add extra peaks to the X-ray spectrum and this could
The preparation of most pharmaceutical samples is simple
interfere with the analysis or even obscure minor amounts of
and quick and does not require specialised equipment or
some elements, thereby precluding the possibility of
complex processes.
quantitative determinations of unknowns. Most elemental
Specimen holders, called stubs, are used to support powders, analysers are capable of detecting light elements, so even a
single particles, tablets, freeze-dried cakes, capsules and thin carbon coat could hinder the interpretation of the
beads. Rapid-curing, vacuum compatible glue or electrically spectrum. To minimise or exclude potential problems and
conductive silver or carbon paints are ideal for holding large artefacts associated with specimen coating, non-conductive
objects that are up to a few millimetres in diameter. Fine materials can be examined uncoated using ESEM or
powders and small objects can be attached to stubs on VPSEM. Software correction for any coating may need to be
double-sided adhesive tape or on sticky tabs. Large samples applied.
may have to be reduced in size to fit onto a stub or into the
specimen chamber and tablets can be broken open to expose OPERATION
their cores before being attached securely to a stub. The electron microscope is operated with a beam-
accelerating voltage that is appropriate to produce images
Powders can be simply sprinkled or poured onto a stub that that reveal the information of interest about a specimen.
has had a thin layer of adhesive applied to it and excess loose For example, a low voltage (e.g. less than 5 kV) can be used
powder is then tapped off or blown off with a gentle stream to image surface details and a higher voltage (e.g. greater
of inert compressed gas. A rapid and simple method for than 10 kV) increases the resolving power and can be used
powders is to attach double-sided adhesive tape onto a stub for the analysis of a wide range of elements. The most useful
and dip it gently into the powder so that a thin layer sticks to accelerating voltage range is about 2 kV to 20 kV because
the tape, and excess powder is then blown off (taking care most elements of interest can be ionized by electrons with
not to inhale the airborne powder). energies in this range.
When preparing powder samples, cross-contamination must Adjustment of the objective lens focuses the beam on the
be avoided because the presence in the specimen of just a specimen over a wide range of working distances to
single particle from another material can lead to an incorrect accommodate large and small specimens. The depth of field
interpretation of an image or chemical analysis. For this can also be increased by selecting smaller beam apertures to
reason, it is not good practice to disperse powder particles enable the full depth of thick specimens to be in acceptable
onto a specimen stub using a brush, because the brush must focus simultaneously.
be decontaminated or discarded after a single use.
For microanalysis, the use of inert/plastic tools (needles, When SEM is used to examine specimens in a high vacuum,
spatulas, tweezers, etc.) is preferred for sample manipulation very high magnifications in excess of 10 000 x can be
achieved to resolve fine detail down to about 3 nm,
2023 Appendix XVII P V-A623

depending upon the specimen. This is possible because the instrument variations using, for example, a bracketing
electron beam can be focused without being scattered by gas concept. Accuracy of scale should be checked in orthogonal
molecules. For ESEM or VPSEM, a consequence of gas or directions and the typical accuracy of the displayed scale
water vapour being present in the specimen chamber is that should be 10 per cent or better. If justified, other acceptance
the electron beam suffers some lateral scattering (called criteria, such as image resolution, might be applied.
skirting). This scattering can inhibit high-resolution imaging For elemental analysis by EDX, the accuracy and
because the beam cannot be focused as finely as it would be reproducibility of the energy scale over a specific energy
in a high vacuum. By selecting a shorter working distance range (e.g. 0-5 keV or 0-20 keV) represents a parameter that
and optimising the gas pressure, this adverse effect can be should be checked and calibrated according to the
minimised. manufacturer's instrument operating instructions using
Lateral spatial resolutions of 1 nm or better become possible suitable reference materials (see general chapter 2.2.37).
(depending upon the specimen being examined) when using These single element materials are used because they have
FE-SEM. To achieve this high resolving power, a beam- multiple X-ray emission peaks at well-defined energies and
accelerating voltage in excess of about 15 kV may be intensities across the spectral range being calibrated.
required, but a high beam voltage could damage beam- The intensities of the emitted X-rays can be influenced by
sensitive materials. For most organic solids, the electron other instrumental parameters, such as accelerating voltage or
beam will penetrate many micrometres into the specimen and the working distance, and should be checked for relevant
surface detail will be lost due to the scattering and absorption instrumental parameters using, for example, a bracketing
of the emitted electrons. In order to reveal the surface details concept. If justified, other acceptance criteria (such as
of specimens, the electron microscope must be operated at a detector resolution) might be used.
much lower voltage, and this is at the expense of resolution.
Most pharmaceutical specimens studied by SEM are likely to
be examined at a magnification less than 10 000 x and so a
high resolving power is typically not needed. P. Particle Size Analysis by Laser Light
COMBINATION OF SEM AND X-RAY
MICROANALYSIS
Diffraction 1
This combination enables the identification of the elemental (Ph. Bur. method 2.9.31)
compositions of specimens and elemental mapping to The method is based on the ISO standards 13320-1(1999)
visualise the distributions of elements, for example in tablets and 9276-1(1998).
and blends.
INTRODUCTION
Element maps are easily and quickly generated to visualise
the spatial distribution of the elements present in a specimen The laser light diffraction technique used for the
at low and high magnifications. Cross-sections cut or determination of particle-size distribution is based on the
fractured through tablet cores and powders that have been analysis of the diffraction pattern produced when particles are
lightly pressed flat can be mapped to reveal the relationships exposed to a beam of monochromatic light. Historically, the
between their components. This technique is useful to early laser diffraction instruments only used scattering at
compare the differences between samples to indicate why small angles. However, the technique has since been
some perform better than others. It is important to have broadened to include laser light scattering in a wider angular
surfaces that are not rough because emitted X-rays follow range and application of the Mie theory, in addition to the
straight paths and so may not be detected in areas (effectively Fraunhofer approximation and anomalous diffraction.
in shadows) that are not in direct line of sight of the X-ray The technique cannot distinguish between scattering by
detector. This artefact can be reduced by tilting the specimen single particles and scattering by clusters of primary particles,
towards the detector. i.e. by agglomerates or aggregates. As most particulate
X-ray microanalysis is also used for the investigation of samples contain agglomerates or aggregates and as the focus
particulate matter. Black spots in tablets, for example, are of interest is generally on the size distribution of primary
often caused by particles of metal or rubber debris, caused by particles, the clusters are usually dispersed into primary
u'tc wcc:11.i..11b uf Lablc:L _lJlC~~c~, u'1aL hc.1.vc ~ub~cyucuJ.y bccu
particles before measurement.
incorporated into the tablet as a visible defect. SEM-EDX is For non-spherical particles, an equivalent sphere-size
an ideal technique to identify them because it is non- distribution is obtained because the technique assumes
destructive and can often be used to distinguish between spherical particles in its optical model. The resulting particle-
different metals so that the source and root cause can be size distribution may differ from those obtained by methods
identified. Also, particles in vials and syringes of solutions for based on other physical principles (e.g. sedimentation,
injection can be examined and analysed after they have been sieving).
isolated by filtration or removed using a micropipette. This chapter provides guidance for the measurement of size
EQUIPMENT PERFORMANCE distributions of particles in different dispersed systems, for
example, powders, sprays, aerosols, suspensions, emulsions,
SEM user requirements, in addition to manufacturer's
and gas bubbles in liquids, through analysis of their angular
recommendations, provide a basis for the system
light-scattering patterns. It does not address specific
performance evaluation. For size measurements, accuracy of
requirements of particle size measurement of specific
the displayed scale bar represents a critical parameter that
products.
should be checked at both low and high magnifications using
certified calibration test specimens (available from SEM
accessory suppliers). As accuracy of scale can be influenced
by instrumental parameters, such as accelerating voltage and
working distance, or by the specimen itself, it should be
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter
checked under different operating conditions to allow for
5. 8. Pharmacopoeia! harmonisation.
V-A624 Appendix XVII P 2023

9 11

7
r

(/)

1. Obscuration detector 5. Scattered light not collected by lens (4) 9. Working distance of lens (4)
2. Scattered beam 6. Particle ensemble 10. Multi-element detector
3. Direct beam 7. Light source laser 11. Focal distance of lens (4)
4. Fourier lens 8. Beam processing unit

Figure 2.9.31.-1. - Example of a set-up of a laser light diffraction instrument

PRINCIPLE It is assumed that the measured scattering pattern of the

A representative sample, dispersed at an adequate particle ensemble is identical to the sum of the patterns from
concentration in a suitable liquid or gas, is passed through a all individual single scattering particles presented in random
beam of monochromatic light, usually a laser. The light relative positions. Note that only a limited angular range of
scattered by the particles at various angles is measured by a scattered light is collected by the lens(es) and, therefore, by
multi-element detector. Numerical values representing the the detector.
scattering pattern are then recorded for subsequent analysis. DEVELOPMENT OF THE METHOD
These scattering pattern values are then transformed, using The measurement of particle size by laser diffraction can give
an appropriate optical model and mathematical procedure, to reproducible data, even in the sub-micron region, provided
yield the proportion of total volume to a discrete number of the instrument used and the sample tested are carefully
size classes, forming a volumetric particle-size distribution. controlled to limit variability of the test conditions
INSTRUMENT (e.g. dispersion medium, method of preparation of the
The instrument is located in an environment where it is not sample dispersion).
affected by electrical noise, mechanical vibrations, Traditionally, the measurement of particle size using laser
temperature fluctuations, humidity or direct bright light. diffraction has been limited to particles in the range of
An example of a set-up of a laser light diffraction instrument approximately O.1 µm to 3 mm. Because of recent advances
is given in Figure 2.9.31.-1. Other equipment may be used. in lens and equipment design, newer instruments are capable
The instrument comprises a laser light source, beam of exceeding this range routinely. With the validation report
processing optics, a sample measurement region (or cell), a the user demonstrates the applicability of the method for its
Fourier lens, and a multi-element detector for measuring the intended use.
scattered light pattern. A data system is also required for Sampling
deconvolution of the scattering data into a volumetric size The sampling technique must be adequate to obtain a
distribution and associated data analysis and reporting. representative sample of a suitable volume for the particle-
The particles can enter the laser beam in 2 positions. In the size measurement. Sample splitting techniques such as
conventional case the particles enter the parallel beam before rotating riffler or the cone and quartering method may be
the collecting lens and within its working distance. applied.
In so-called reversed Fourier optics the particles enter behind Evaluation of the dispersion procedure
the collecting lens and thus, in a converging beam. Inspect the sample to be analysed, visually or with the aid of
The advantage of the conventional set-up is that a reasonable a microscope, to estimate its size range and particle shape.
path length for the sample is allowed within the working The dispersion procedure must be adjusted to the purpose of
distance of the lens. The second set-up allows only small the measurement. The purpose may be such that it is
path lengths but enables measurement of scattered light at preferable to deagglomerate clusters into primary particles as
larger angles, which is useful when submicron particles are far as possible, or it may be desirable to retain clusters as
present. intact as possible. In this sense, the particles of interest may
The interaction of the incident light beam and the ensemble be either primacy particles or clusters.
of dispersed particles results in a scattering pattern with For the development of a method it is highly advisable to
different light intensities at various angles. The total angular check that comminution of the particles does not occur, and
intensity distribution, consisting of both direct and scattered conversely, that dispersion of particles or clusters is
light, is then focused onto a multi-element detector by a lens satisfactory. This can usually be done by changing the
or a series of lenses. These lenses create a scattering pattern dispersing energy and monitoring the change of the particle-
that, within limits, does not depend on the location of the size distribution. The measured size distribution must not
particles in the light beam. Hence, the continuous angular change significantly when the sample is well dispersed and
intensity distribution is converted into a discrete spatial the particles are neither fragile nor soluble. Moreover, if the
intensity distribution on a set of detector elements. manufacturing process (e.g. crystallisation, milling) of the
2023 Appendix XVII P V-A625

material has changed, the applicability of the method must Optimisation of the gas dispersion
be verified (e.g. by microscopic comparison). For sprays and dry powder dispersions, a compressed gas
Sprays, aerosols and gas bubbles in a liquid should be free from oil, water and particles may be used. To remove
measured directly, provided that their concentration is such materials from the compressed gas, a dryer with a filter
adequate, because sampling or dilution generally alters the can be used. Any vacuum unit should be located away from
particle-size distribution. the measurement zone, so that its output does not disturb
In other cases (such as emulsions, pastes and powders), the measurement.
representative samples may be dispersed in suitable liquids. Detennination of the concentration range
Dispersing aids (wetting agents, stabilisers) and/or In order to produce an acceptable signal-to-noise ratio in the
mechanical forces (e.g. agitation, sonication) are often detector, the particle concentration in the dispersion must
applied for deagglomeration or deaggregation of clusters and exceed a minimum level. Likewise, it must be below a
stabilisation of the dispersion. For these liquid dispersions, a maximum level in order to avoid multiple scattering.
recirculating system is most commonly used, consisting of an The concentration range is influenced by the width of the
optical measuring cell, a dispersion bath usually equipped laser beam, the path length of the measurement zone, the
with stirrer and ultrasonic elements, a pump, and tubing. optical properties of the particles, and the sensitivity of the
Non-recirculating, stirred cells are useful when only small detector elements.
amounts of a sample are available or when special dispersion In view of the above, measurements must be performed at
liquids are used. different particle concentrations to determine the appropriate
Dry powders can also be converted into aerosols through the concentration range for any typical sample of material. (Note:
use of suitable dry powder dispersers, which apply in different instruments, particle concentrations are usually
mechanical force for deagglomeration or deaggregation. represented by differently scaled and differently named
Generally, the dispersers use the energy of compressed gas or numbers, e.g. obscuration, optical concentration,
the differential pressure of a vacuum to disperse the particles proportional number of total mass).
to an aerosol, which is blown through the measuring zone, Detennination of the measuring time
usually into the inlet of a vacuum unit that collects the The time of measurement, the reading time of the detector
particles. However, for free flowing, coarser particles or and the acquisition frequency are determined experimentally
granules the effect of gravity may be sufficient to disperse the in accordance with the required precision. Generally, the
particles adequately. time for measurement permits a large number of detector
If the maximum particle size of the sample exceeds the scans or sweeps at short time intervals.
measuring range of the instrument, the material that is too Selection of an appropriate optical model
coarse can be removed by sieving and the mass and Most instruments use either the Fraunhofer or the Mie
percentage of removed material are reported. However, after theory, though other approximation theories are sometimes
pre-sieving, note that the sample is no longer representative, applied for calculation of the scattering matrix. The choice of
unless otherwise proven. the theoretical model depends on the intended application
Optimisation of the liquid dispersion and the different assumptions (size, absorbance, refractive
Liquids, surfactants, and dispersing aids used to disperse index, roughness, crystal orientation, mixture, etc.) made for
powders must: the test material. If the refractive index values (real and
- be transparent at the laser wavelength and practically free imaginary parts for the used wavelength) are not exactly
from air bubbles or particles; known, then the Fraunhofer approximation or the Mie theory
- have a refractive index that differs from that of the test with a realistic estimate of the refractive index can be used.
material; The former has the advantages that it is simple and it does
- be non-solvent of the test material (pure liquid or pre- not need refractive index values; the latter usually provides
filtered, saturated solution); less-biased particle-size distributions for small particles.
- not alter the size of the test materials (e.g. by solubility, For instance, if the Fraunhofer model is used for samples
solubility enhancement, or recrystallisation effects); containing an appreciable amount of small, transparent
- favour easy formation and stability of the dispersion; particles, a significantly larger amount of small particles may
- be compatible with the materials used in the instrument be calculated. In order to obtain traceable results, it is
(such as O-rings, gaskets, tubing, etc.); essential to document the refractive index values used, since
- possess a suitable viscosity to facilitate recirculation, small differences in the values assumed for the real and
stirring and filtration. imaginary part of the complex refractive index may cause
Surfactants and/or dispersing aids are often used to wet the significant differences in the resulting particle-size
particles and to stabilise the dispersion. For weak acids and distributions. Small values of the imaginary part of the
weak bases, buffering of the dispersing medium at low or refractive index (about 0.01-0.1 i) are often applied to allow
high pH respectively can assist in identifying a suitable the correction of the absorbance for the surface roughness of
dispersant. the particles. It should be noted, in general, that the optical
A preliminary check of the dispersion quality can be properties of the substance to be tested, as well as the
performed by visual or microscopic inspection. It is also structure (e.g. shape, surface roughness and porosity), bear
possible to take fractional samples out of a well-mixed stock upon the final result.
dispersion. Such stock dispersions are formed by adding a Validation
liquid to the sample while mixing it with, for example, a glass Typically, the validity of a procedure may be assessed by the
rod, a spatula or a vortex mixer. Care must be taken to evaluation of its specificity, linearity, range, accuracy,
ensure the transfer of a representative sample and that precision and robustness. In particle-size analysis by laser
settling of larger particles does not occur. Therefore a sample light diffraction, specificity as defined by ICH is not
paste is prepared or sampling is carried out quickly from a applicable as it is not possible to discriminate between
suspension maintained under agitation. different components in a sample, nor is it possible to
V-A626 Appendix XVII P 2023

discriminate agglomerates from dispersed particles unless lens determine the range of scattering angles for each
properly complemented by microscopic techniques. Exploring element. Most instruments also measure the intensity of the
a linear relationship between concentration and response, or central (unscattered) laser beam. The ratio of the intensity of
a mathematical model for interpolation, is not applicable to a dispersed sample to that in its absence (a blank
this procedure. Rather than evaluating linearity, this method measurement) indicates the proportion of scattered light and
requires the definition of a concentration range within which hence the particle concentration.
the result of the measurements does not vary significantly. Conversion of scattering pattern into particle-size
Concentrations below that range produce an error due to a distribution
poor signal-to-noise ratio, while concentrations above that This deconvolution step is the inverse of the calculation of a
range produce an error due to multiple scattering. The range scattering pattern for a given particle-size distribution.
depends mostly on the instrument hardware. Accuracy The assumption of spherical particle shape is particularly
should be confirmed through an appropriate instrument important as most algorithms use the mathematical solution
qualification and comparison with microscopy, while for scattering from spherical particles. Furthermore, the
precision may be assessed by means of a repeatability measured data always contain some random and systematic
determination. errors, which may vitiate the size distributions. Several
The attainable repeatability of the method mainly depends mathematical procedures have been developed for use in the
on the characteristics of the material (milled/not milled, available instruments. They contain some weighting of
robust/fragile, width of its size distribution, etc.), whereas the deviations between measured and calculated scattering
required repeatability depends on the purpose of the patterns (e.g. least squares), some constraints (e.g. non-
measurement. Mandatory limits cannot be specified in this negativity for amounts of particles), and/or some smoothing
chapter, as repeatabilities (different sample preparations) may of the size distribution curve.
vary appreciably from one substance to another. However, it The algorithms used are specific to each make and model of
is good practice to aim at acceptance criteria for repeatability equipment, and are proprietary. The differences in the
such as sre1 ~ 10 per cent [n = 6] for any central value of the algorithms between different instruments may give rise to
distribution (e.g. for x50). Values at the sides of the differences in the calculated particle-size distributions.
distribution (e.g. x 10 and x90) are oriented towards less
Replicates
stringent acceptance criteria such as SreI ~ 15 per cent
[n = 6]. Below 10 µm, these values must be doubled. The number of replicate measurements (with individual
Robustness may be tested during the selection and sample preparations) to be performed depends on the
optimisation of the dispersion media and forces. The change required measurement precision. It is recommended to set
of the dispersing energy may be monitored by the change in this number in a substance-specific method.
the particle-size distribution. REPORTING OF RESULTS
MEASUREMENT The particle-size distribution data are usually reported as
Precautions cumulative undersize distribution and/or as density
The instructions given in the instrument manual are distribution by volume. The symbol x is used to denote the
followed: particle size, which in turn is defined as the diameter of a
- never look into the direct path of the laser beam or its volume-equivalent sphere. Q3(x) denotes the volume fraction
reflections; undersize at the particle size x. In a graphical representation,
x is plotted on the abscissa and the dependent variable Q3
- earth all instrument components to prevent ignition of
solvents or dust explosions; on the ordinate. Most common characteristic values are
- check the instrument set-up (e.g. warm-up, required calculated from the particle-size distribution by interpolation.
measuring range and lens, appropriate working distance, The particle sizes at the undersize values of 10 per cent,
position of the detector, no direct bright daylight); 50 per cent, and 90 per cent (denoted as x 10, x50, and x 90
- in the case of wet dispersions, avoid air bubbles, respectively) are frequently used. x50 is also known as the
evaporation of liquid, schlieren or other inhomogeneities median particle size. It is recognised that the symbol d is also
in the dispersion; similarly, avoid improper mass-flow widely used to designate the particle size, thus the symbol x
from the disperser or turbulent air-flow in the case of dry may be replaced by d.
dispersions; such effects can cause erroneous particle-size Moreover, sufficient information must be documented about
distributions. the sample, the sample preparation, the dispersion
conditions, and the cell type. As the results depend on the
Measurement of the light scattering of dispersed
particular instrument, data analysis program, and optical
sample(s)
model used, these details must also be documented.
After proper alignment of the optical part of the instrument,
a blank measurement of the particle-free dispersion medium CONTROL OF THE INSTRUMENT PERFORMANCE
must be performed using the same method as that used for Use the instrument according to the manufacturer's
the measurement of the sample. The background signal must instructions and carry out the prescribed qualifications at an
be below an appropriate threshold. The detector data are appropriate frequency, according to the use of the instrument
saved in order to substract them later from the data obtained and substances to be tested.
with the sample. The sample dispersion is measured Calibration
according to the developed method. Laser diffraction systems, although assuming idealised
For each detector element, an average signal is calculated, properties of the particles, are based on first principles of
sometimes together with its standard deviation. laser light scattering. Thus, calibration in the strict sense is
The magnitude of the signal from each detector element not required. However, it is still necessary to confirm that the
depends upon the detection area, the light intensity and the instrument is operating correctly. This can be undertaken
quantum efficiency. The co-ordinates (size and position) of using any certified reference material that is acceptable in
the detector elements together with the focal distance of the industrial practice. The entire measurement procedure is
2023 Appendix XVII Q V-A627

examined, including sample collection, sample dispersion,

sample transport through the measuring zone, measurement,
Q. Characterisation of Crystalline and
and the deconvolution procedure. It is essential that the total Partially Crystalline Solids by X-ray
operational procedure is fully described.
The preferred certified reference materials consist of spherical
Powder Diffraction (XRPD) 1
particles of a known distribution. They must be certified as (Ph. Bur. method 2.9.33)
to the mass-percentage size distribution by an absolute Every crystalline phase of a given substance produces a
technique, if available, and used in conjunction with an characteristic X-ray diffraction pattern.
agreed, detailed operation procedure. It is essential that the
Diffraction patterns can be obtained from a randomly oriented
real and imaginary parts of the complex refractive index of
crystalline powder composed of crystallites (crystalline region
the material are indicated if the Mie theory is applied in data
within a particle) or crystal fragments of finite size. Essentially
analysis. The representation of the particle-size distribution
3 types of information can be derived from a powder
by volume will equal that of the distribution by mass,
diffraction pattern: angular position of diffraction lines
provided that the density of the particles is the same for all
(depending on geometry and size of the unit cell); intensities
size fractions.
of diffraction lines (depending mainly on atom type and
The response of a laser diffraction instrument is considered arrangement, and preferred orientation within the sample);
to meet the requirements if the mean value of x 50 from at and diffraction line profiles (depending on instrumental
least 3 independent measurements does not deviate by more resolution, crystallite size, strain and specimen thickness).
than 3 per cent from the certified range of values of the
Experiments giving angular positions and intensities of lines
certified reference material. The mean values for x10 and x 90
can be used for applications such as qualitative phase analysis
must not deviate by more than 5 per cent from the certified
(for example, identification of crystalline phases) and
range of values. Below 10 µm, these values must be doubled.
quantitative phase analysis of crystalline materials.
Although the use of materials consisting of spherical particles An estimate of the amorphous and crystalline fractions 2 can
is preferable, non-spherical particles may also be employed. also be made.
Preferably, these particles have certified or typical values from
The X-ray powder diffraction (XRPD) method provides an
laser diffraction analyses performed according to an agreed,
advantage over other means of analysis in that it is usually
detailed operating procedure. The use of reference values
non-destructive in nature (specimen preparation is usually
from methods other than laser diffraction may cause a
limited to grinding to ensure a randomly oriented sample).
significant bias. The reason for this bias is that the different
XRPD investigations can also be carried out under in situ
principles inherent in the various methods may lead to
conditions on specimens exposed to non-ambient conditions,
different sphere-equivalent diameters for the same non-
such as low or high temperature and humidity.
spherical particle.
Although the use of certified reference materials is preferred, PRINCIPLE
other well-defined reference materials may also be employed. X-ray diffraction results from the interaction between X-rays
They consist of substances of typical composition and and electron clouds of atoms. Depending on the atomic
particle-size distribution for a specified class of substances. arrangement, interferences arise from the elastically scattered
Their particle-size distribution has proven to be stable over X-rays. These interferences are constructive when the path
time. The results must comply with previously determined difference between 2 diffracted X-ray waves differs by an
data, with the same precision and bias as for the certified integral number of wavelengths. This selective condition is
reference material. described by the Bragg equation, also called Bragg's law (see
Figure 2.9.33.-1):
Qualification of the system
In addition to the calibration, the performance of the 2dhkl sin 0hkl = Ilic
instrument must be qualified at regular time intervals or as
frequently as appropriate. This can be undertaken using any The wavelength 'A of the X-rays is of the same order of
suitable reference material as mentioned in the previous magnitude as the distance between successive crystal lattice
paragraph. planes, or dhkl (also called 'd-spacings'). 0hkl is the angle
The qualification of the system is based on the concept that between the incident ray and the family of lattice planes, and
the equipment, electronics, software and analytical operations sin0hk1 is inversely proportional to the distance between
constitute an integral system, which can be evaluated as an successive crystal planes or d-spacings.
entity. Thus the entire measurement procedure is examined, The direction and spacing of the planes with reference to
including sample collection, sample dispersion, sample the unit cell axes are defined by the Miller indices {hkl} .
transport through the measuring zone, and the measurement These indices are the reciprocals, reduced to the next-lower
and deconvolution procedure. It is essential that the total integer, of the intercepts that a plane makes with the unit cell
operational procedure is fully described. axes. The unit cell dimensions are given by the spacings a, b
In general, unless otherwise specified in the individual and c and the angles between them, a., ~, and y.
monograph, the response of a laser diffraction instrument is The interplanar spacing for a specified set of parallel hkl
considered to meet the requirements if the x 50 value does not planes is denoted by dhkl· Each such family of planes may
deviate by more than 10 per cent from the range of values of
the reference material. If optionally the values at the sides of 1 This chapter has undergone pharmacopoeia! harmonisation. See chapter
the distribution are evaluated (e.g. x 10 and x 90 ), then these 5. 8. Pharmacopoeia/ harmonisation.
2 There are many other applications of the X-ray powder diffraction
values must not deviate by more than 15 per cent from the
certified range of values. Below 10 µm, these values must be technique that can be applied to crystalline phamzaceutical substances such
doubled. as: determination of crystal structures, refinement of crystal structures,
determination of crystallographic purity of crystalline phases, characterisation
NOTE: for calibration of the instrument, stricter requirements are of crystallographic texture, etc. These applications are not described in this
laid down in the paragraph Calibration. chaprer.
V-A628 Appendix XVII Q 2023

Figure 2.9.33.-1. - Diffraction of X-rays by a crystal according w Bragg's law

show higher orders of diffraction where the d values for the background, upon which the peaks are superimposed.
related families of planes nh, nk, nl are diminished by the Besides specimen preparation, other factors contribute to the
factor 1/n (n being an integer: 2, 3, 4, etc.). background, for instance the sample holder, diffuse scattering
Every set of planes throughout a crystal has a corresponding from air, the sample and/or the equipment, other
Bragg diffraction angle, 0hk1' associated with it (for a specific instrumental parameters such as detector noise, general
wavelength A). radiation from the X-ray tube, etc. The peak-to-background
For polycrystalline powder specimens, at any angle 0hkl there ratio can be increased by minimising background and by
are always crystallites in an orientation allowing diffraction choosing prolonged exposure times.
according to Bragg's law3 • For a given X-ray wavelength, the INSTRUMENT
positions of the diffraction peaks (also referred to as 'lines', Instrument set-up
'reflections' or 'Bragg reflections') are characteristic of the X-ray diffraction experiments are usually performed using
crystal lattice (d-spacings), their theoretical intensities depend powder diffractometers or powder cameras.
on the crystallographic unit cell content (nature and positions A powder diffractometer generally comprises 5 main parts: an
of atoms), and the line profiles on the perfection and extent X-ray source; incident beam optics, which may perform
of the crystal lattice. Under these conditions the diffraction monochromatisation, filtering, collimation and/or focusing of
peak has a finite intensity arising from atomic arrangement, the beam; a goniometer; the diffracted beam optics, which
type of atoms, thermal motion and structural imperfections, may perform monochromatisation, filtration, collimation
as well as from instrument characteristics. and/or focusing of the beam; and a detector. Data-collection
The intensity is also dependent upon many factors such as and data-processing systems are also required and are
crystallinity and the structure, temperature, polarisation, generally included with current diffraction measurement
multiplicity, Lorentz and microabsorption factors. equipment.
The main characteristics of diffraction line profiles Depending on the type of analysis to be performed (phase
are 20 position, peak height, peak area and shape identification, quantitative analysis, lattice parameter
(characterised by, for example, peak width or asymmetry, determination, etc.), different XRPD instrument
analytical function, empirical representation). An example of configurations and performance levels are required.
powder patterns obtained for 5 different solid phases of a The simplest instruments used to measure XRPD patterns
substance is shown in Figure 2.9.33.-2. are powder cameras. The replacement of photographic film
In addition to the diffraction peaks, an X-ray diffraction as the detection method by photon detectors has led to the
design of diffractometers in which the geometric arrangement
experiment also generates a more-or-less uniform
of the optics is not truly focusing but parafocusing, such as in
3
the Bragg-Brentano geometry. The Bragg-Brentano
An 'ideal' powder for diffraction experiments consists of a large number of
parafocusing configuration is currently the most widely used
small, randomly oriented spherical particles (coherendy diffracting crystalline
domains). If this number is sufficiently large, there are always enough and is therefore briefly described here.
crystallites in any diffracting orientation w give reproducible diffraction
patterns.
2023 Appendix XVII Q V-A629

Form D

Form C

Form B

Form A

amorphous

i-l ' I,-~

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 28 29 30 31

2 0(A Cu) - Scale

Figure 2.9.33.-2. - X-ray powder diffraction patterns collected for 5 different solid phases of a substance
(the intensities of crystalline fonns A-D are normalised)

A given instrument may provide a horizontal or vertical detector, usually a scintillation counter, or a sealed-gas
0/20 geometry or a vertical 0/0 geometry. For both proportional counter. However, nowadays a position-sensitive
geometries, the incident X-ray beam forms an angle 0 with solid-state detector or hybrid photon counting detectors are
the specimen surface plane and the diffracted X-ray beam more common. The receiving slit assembly and the detector
forms an angle 20 with the direction of the incident X-ray are coupled together and move tangentially to the focusing
beam (an angle 0 with the specimen surface plane). circle. For 0/20 scans the goniometer rotates the specimen
One example of a basic geometric arrangement is represented about the same axis as that of the detector, but at half the
in Figure 2.9.33.-3. The divergent beam of radiation from rotational speed, in a 0/20 motion. The surface of the
the X-ray tube (the so-called 'primary beam') passes through specimen thus remains tangential to the focusing circle.
the Soller slits and a divergence slit assembly and illuminates The Soller slit limits the axial divergence of the beam and
the fiat surface of the specimen. All the rays diffracted by hence partially controls the shape of the diffracted line
suitably oriented crystallites in the specimen at an angle 20 profile.
converge to a line at the receiving slit. A second set of Soller A diffractometer may also be used in transmission mode.
slits and a scatter slit may be placed either behind or before The advantage with this technology is to lessen the effects
the receiving slit; the second receiving slit is normally only due to preferred orientation. A capillary of about 0.5-2 mm
used when a OD detector is present. The axes of the line thickness can also be used for small sample amounts.
focus and of the receiving slit are at equal distances from the
axis of the goniometer. The X-rays are counted by a
V-A630 Appendix XVII Q 2023

\
/

/ 0 )
- ~

/ \
'\

~
/ ,__
D
EI

G
H

\ I

\ /
/ F

'\

~ /
/
K ' -------- - ---------

A. X-ray tube C. sample E. receiving slit G. detector receiving slit J.diffractometer circle
B. divergence slit D. anti-diffusion slit F. monochroniator H. detector K. focusing circle

Figure 2.9.33.-3. - Geometric arrangement of the Bragg-Brentano parafocusing geometry

X-ray radiation monochromator crystal (usually referred to as a

In the laboratory, X-rays are obtained by bombarding a 'monochromator'). This crystal is placed before or behind
metal anode with electrons emitted by the thermionic effect the specimen and diffracts the different characteristic peaks
and accelerated in a strong electric field (using a high-voltage of the X-ray beam (i.e. Kc, and Kp) at different angles, so
generator). Most of the kinetic energy of the electrons is that only one of them may be selected to enter into the
converted to heat, which limits the power of the tubes and detector. It is even possible to separate l<c, 1 and l<c,2
requires efficient anode cooling. A 20- to 30-fold increase in radiations by using a specialised monochromator.
brilliance can be obtained using rotating anodes and by using Unfortunately, the gain in getting a monochromatic beam by
X-ray optics. Alternatively, X-ray photons may be produced using a filter or a monochromator is counteracted by a loss in
in a large-scale facility (synchrotron). intensity. Another way of separating Kc, and Kp wavelengths
The spectrum emitted by an X-ray tube operating at is by using curved X-ray mirrors that can simultaneously
sufficient voltage consists of a continuous background of monochromate and focus or parallelise the X-ray beam.
polychromatic radiation (bremsstrahlung or white radiation) RADIATION PROTECTION. Exposure of any part of the
and additional characteristic radiation that depends on the human body to X-rays can be injurious to health. It is therefore
type of anode. Normally, only this characteristic radiation is essential that whenever X-ray equipment is used, adequate
used in X-ray diffraction experiments. The principal radiation precautions are taken to protect the operator and any other person
sources utilised for X-ray diffraction are vacuum tubes in the vicinity. Recommended practice for radiation protection as
utilising copper, molybdenum, iron, cobalt, silver or well as limits for the levels of X-radiation exposure are those
chromium as anodes; copper and molybdenum X-rays are established by national legislation in each country. If there are no
employed most commonly for organic substances. The choice official regulations or recommendations in a country, the latest
of radiation to be used depends on the absorption recommendations of the International Commission on Radiological
characteristics of the specimen and possible fluorescence by Protection should be applied.
atoms present in the specimen. The wavelengths used in
SPECIMEN PREPARATION AND MOUNTING
powder diffraction generally correspond to the Kc, radiation
The preparation of the powdered material and mounting of
from the anode. Consequently, it is advantageous to make
the specimen in a suitable holder are critical steps in many
the X-ray beam 'monochromatic' by eliminating all the other
analytical methods, and are particularly so for XRPD
components of the emission spectrum. This can be partly
analysis, since they can greatly affect the quality of the data
obtained using Kp filters, i.e. metal filters selected as having
to be collected4. The main sources of error due to specimen
an absorption edge between the Kc, and Kp wavelengths
preparation and mounting are briefly discussed here for
emitted by the tube.
instruments in Bragg-Brentano parafocusing geometry.
Such a filter is usually inserted between the X-ray tube and
the specimen. Another, more-and-more-commonly used way 4 Similarly, changes in the specimen can occur during data collection in the
to obtain a monochromatic X-ray beam is via a large case of a non-equilibrium specimen (temperature, humidity).
 2023 Appendix XVII Q V-A631

SPECIMEN PREPARATION transmission mode is that problems with sample thickness

In general, the morphology of many crystalline particles tends and specimen transparency are less important. The use of an
to give a specimen that exhibits some degree of preferred appropriate internal standard allows the detection and
orientation in the specimen holder. This is particularly correction of this effect simultaneously with that arising from
evident for needle-like or plate-like crystals when size specimen displacement.
reduction yields finer needles or platelets. Preferred CONTROL OF THE INSTRUMENT PERFORMANCE
orientation in the specimen influences the intensities of
Goniometers and the corresponding incident and diffracted
various reflections, so that some are more intense and others
X-ray beam optics have many mechanical parts that need
are less intense, compared to what would be expected from a
adjustment. The degree of alignment or misalignment
completely random specimen. Several techniques can be
directly influences the quality of the results of an XRPD
employed to improve randomness in the orientation of
investigation. Therefore, the different components of the
crystallites (and therefore to minimise preferred orientation),
diffractometer must be carefully adjusted (optical and
but further reduction of particle size is often the best and
mechanical systems, etc.) to minimise adequately systematic
simplest approach. The optimum number of crystallites
errors, while optimising the intensities received by the
depends on the diffractometer geometry, the required
detector. The search for maximum intensity and maximum
resolution and the specimen attenuation of the X-ray beam.
resolution is always antagonistic when aligning a
In some cases, particle sizes as large as 50 µm will provide
diffractometer. Hence, the best compromise must be sought
satisfactory results in phase identification. However, excessive
whilst performing the alignment procedure. There are many
milling (particle sizes less than approximately 0.5 µm) may
different configurations and each supplier's equipment
cause line broadening and significant changes to the sample
requires specific alignment procedures.
itself such as:
- specimen contamination by particles abraded from the The overall diffractometer performance must be tested and
milling instruments (mortar, pestle, balls, etc.); monitored periodically using suitable certified reference
- reduced degree of crystallinity; materials, e.g. silicon powder or IX-alumina (corundum).
- solid-state transition to another polymorph; Depending on the type of analysis, other well-defined
- chemical decomposition; reference materials may also be employed, although the use
- introduction of internal stress; of certified reference materials is preferred.
- solid-state reactions. QUALITATIVE PHASE ANALYSIS
Therefore, it is advisable to compare the diffraction pattern of (IDENTIFICATION OF PHASES)
the non-ground specimen with that corresponding to a The identification of the phase composition of an unknown
specimen of smaller particle size (e.g. a milled specimen). If the sample by XRPD is usually based on the visual or computer-
XRPD pattern obtained is of adequate quality considering its assisted comparison of a portion of its XRPD pattern to the
intended use, then grinding may not be required. experimental or calculated pattern of a reference material.
It should be noted that if a sample contains more than one Ideally, these reference patterns are collected on well-
phase and if sieving is used to isolate particles to a specific characterised single-phase specimens. This approach makes it
size, the initial composition may be altered. possible in most cases to identify a crystalline substance by
its 20 diffraction angles or d-spacings and by its relative
SPECIMEN MOUNTING
intensities. The computer-aided comparison of the diffraction
Effect of spechnen displacement pattern of the unknown sample to the reference data can be
A specimen surface that is offset by D with reference to the based either on a more-or-less extended 20-range of the
diffractometer rotation axis causes systematic errors that are whole diffraction pattern or on a set of reduced data derived
very difficult to avoid entirely; for the reflection mode, this from the pattern. For example, the list of d-spacings and
results in absolute D-cos0 shifts 5 in 20 positions (typically of normalised intensities UnorrrJ, a so-called (d, Inorm)-list
the order of 0.01 ° in 20 at low angles (cos0 ::,,: 1) for a extracted from the pattern, is the crystallographic fingerprint
displacement D = 15 µm) and asymmetric broadening of the of the material, and can be compared to (d, Inorm)-lists of
profile towards low 20 values. Use of an appropriate internal single-phase samples compiled in databases.
standard allows the detection and correction of this effect For most organic crystals, when using Cu Ka radiation, it is
simultaneously with that arising from specimen transparency. appropriate to record the diffraction pattern in a 20-range
This is by far the largest source of error in data collected on from as near 0° as possible to at least 30°. The agreement in
well-aligned diffractometers. the 20-diffraction angles between specimen and reference is
Effect of spechnen thickness and transparency expected to be within O.2° for the same crystal form, while
When the XRPD method in reflection mode is applied, it is relative intensities between specimen and reference may vary
often preferable to work with specimens of 'infinite considerably due to preferred orientation effects. By their
thickness'. To minimise the transparency effect, it is very nature, variable hydrates and solvates are recognised to
advisable to use a non-diffracting substrate (zero background have varying unit cell dimensions and as such shifting occurs
holder), for example a plate of single crystalline silicon cut in peak positions of the measured XRPD patterns for these
parallel to the 510 lattice planes6. One advantage of the materials. In these unique materials, variance in 20-positions
of greater than 0.2° is not unexpected. As such, peak position
5 Note that a goniometer zero alignment shift would result in constant shift variances such as 0.2° are not applicable to these materials.
on all observed 20-line positions, in other words, the whole diffraction pattern For other types of samples (e.g. inorganic salts), it may be
is in this case translated by an offset of Z in 20. necessary to extend the 20-region scanned to well beyond
6 In rhe case of a thin specimen with low attenuation, accurate 30°. It is generally sufficient to scan past the 10 strongest
measurements of line positions can be made with focusing diffractometer reflections identified in single phase XRPD database files.
configurations in either transmission or reflection geometry. Accurate
measurements of line positions on specimens with low attenuation are
preferably made using diffractometers with parallel beam optics. This helps to
reduce the effects of specimen thickness.
V-A632 Appendix XVII Q 2023

It is sometimes difficult or even impossible to identify phases with a diffraction pattern that does not overlap at all with
in the following cases: that of the sample to be analysed, can be used. A known
- non-crystallised or amorphous substances; quantity of this reference material is added to the sample to
- the components to be identified are present in low mass be analysed and to each of the reference mixtures. Under
fractions of the analyte amounts (generally less than these conditions, a linear relationship between line intensity
10 per cent m/m); and concentration exists. This application, called the 'internal
- pronounced preferred orientation effects; standard method', requires a precise measurement of
- the phase has not been filed in the database used; diffraction intensities.
- formation of solid solutions; In the 'spiking method' (or 'standard addition method'),
- presence of disordered structures that alter the unit cell; some of the pure phase a is added to the mixture containing
- the specimen comprises too many phases; the unknown concentration of phase a. Multiple additions
- presence of lattice deformations; are made to prepare an intensity-versus-concentration plot in
- structural similarity of different phases. which the negative x intercept is the concentration of the
QUANTITATIVE PHASE ANALYSIS phase a in the original sample.
If the sample under investigation is a mixture of 2 or more ESTIMATE OF THE AMORPHOUS AND
known phases, of which not more than 1 is amorphous, the CRYSTALLINE FRACTIONS
percentage (by volume or by mass) of each crystalline phase In a mixture of crystalline and amorphous phases, the
and of the amorphous phase can, in many cases, be crystalline and amorphous fractions can be estimated in
determined. Quantitative phase analysis can be based on the several ways. The choice of the method used depends on the
integrated intensities, on the peak heights of several nature of the sample:
individual diffraction Jines 7, or on the full pattern. These - if the sample consists of crystalline fractions and an
integrated intensities, peak heights or full-pattern data points amorphous fraction of different chemical compositions,
are compared to the corresponding values of reference the amounts of each of the individual crystalline phases
materials. These reference materials shall be single-phase or a may be estimated using appropriate standard substances
mixture of known phases. The difficulties encountered during as described above; the amorphous fraction is then
quantitative analysis are due to specimen preparation (the deduced indirectly by subtraction;
accuracy and precision of the results require in particular - if the sample consists of one amorphous and one
homogeneity of all phases and a suitable particle size crystalline fraction, either as a !-phase or a 2-phase
distribution in each phase) and to matrix effects. Amounts of mixture, with the same elemental composition, the
crystalline phases as small as 10 per cent may usually be amount of the crystalline phase ('the degree of
determined in solid matrices, and in favourable cases crystallinity') can be estimated by measuring 3 areas of
amounts of crystalline phases less than 10 per cent may be the diffractogram:
determined.
POLYMORPHIC SAMPLES A sum of the area of all the peaks arising from diffraction of the
For a sample composed of 2 polymorphic phases a and b, the crystalline fraction of the sample;
B area under the curve in the diffractogram generated by the
following expression may be used to quantify the fraction Fa sample (excluding area A);
of phase a: C area of the background signal (due to scattering from air,
fluorescence, equipment, etc).
1
F------
a - 1 +K(Ib/Ia) When these areas have been measured, the degree of
crystallinity can be roughly estimated using the following
The fraction is derived by measuring the intensity ratio formula:
between the 2 phases, knowing the value of the constant K.
K is the ratio of the absolute intensities of the 2 pure % crystallinity = l00A/(A + B - C)
polymorphic phases I 0 jl06 • Its value can be determined by
measuring standard samples. It is noteworthy that this method does not yield absolute
METHODS USING A STANDARD degree-of-crystallinity values and hence is generally used for
The most commonly used methods for quantitative analysis comparative purposes only.
are: More sophisticated methods are also available, such as the
- the 'external standard method'; Ruland method.
- the 'internal standard method'; SINGLE CRYSTAL STRUCTURE
- the 'spiking method' (often also called the 'standard In general, the determination of crystal structures is
addition method'). performed from X-ray diffraction data obtained using single
The 'external standard method' is the most general method crystals. However, crystal structure analysis of organic crystals
and consists of comparing the X-ray diffraction pattern of the is a challenging task, since the lattice parameters are
mixture, or the respective line intensities, with those comparatively large, the symmetry is low and the scattering
measured in a reference mixture or with the theoretical properties are normally very low.
intensities of a structural model, if it is fully known. For any given crystalline form of a substance, knowledge of
To limit errors due to matrix effects, an internal reference the crystal structure allows the calculation of the
material with crystallite size and X-ray absorption coefficient corresponding XRPD pattern, thereby providing a 'preferred-
comparable to those of the components of the sample, and orientation-free' reference XRPD pattern, which may be used
for phase identification.
7 If the crystal structures of all components are known, the Rierveld method
can be used to quantify them with good accuracy. If the crystal structures of
the components are not known, the Pawley or least squares methods can be
used.
2023 Appendix XVII R V-A633

Mercury porosimetry is considered to be comparative, as for

R. Porosity and Pore-size Distribution of most porous media a theory is not available to allow an
Solids by Mercury Porosimetry absolute calculation of results of pore-size distribution.
Therefore this technique is mainly recommended for
(Ph. Bur. method 2.9.32)
development studies.
INTRODUCTION Mercury is toxic. Appropriate precautions must be observed
In general, different types of pores may be pictured as to safeguard the health of the operator and others working in
apertures, channels or cavities within a solid body, or as the area. Waste material must also be disposed of in a
space (i.e. interstices or voids) between solid particles in a suitable manner, according to local regulations.
bed, compact or aggregate. Porosity is a term that is often
used to indicate the porous nature of solid material, and is PRINCIPLE
more precisely defined as the ratio of the volume of The technique is based on the measurement of the mercury
accessible pores and voids to the total volume occupied by a volume intruded into a porous solid as a function of the
given amount of the solid. In addition to the accessible pores, applied pressure. The measurement includes only those pores
a solid may contain closed pores, which are isolated from the into which mercury can penetrate at the pressure applied.
external surface and into which fluids are not able to A non-wetting liquid penetrates into a porous system only
penetrate. The characterisation of closed pores, i.e. cavities under pressure. The pressure to be applied is in inverse
with no access to an external surface, is not covered in this proportion to the inner diameter of the pore aperture. In the
chapter. case of cylindrical pores, the correlation between pore
Porous materials may take the form of fine or coarse diameter and pressure is given by the Washburn equation:
powders, compacts, extrudates, sheets or monoliths. Their
characterisation usually involves the determination of the
total pore volume or porosity as well as the pore-size
distribution. dp pore diameter, in metres;
It is well established that the performance of a porous solid cr surface tension, in newtons per metre;
(e.g. its strength, reactivity, permeability or adsorbent power) 0 contact angle of mercury on the sample, in degrees;
p applied pressure, in pascals.
is dependent upon its pore structure. Many different
methods have been developed for the characterisation of pore
structure. In view of the complexity of most porous solids, it APPARATUS
is not surprising to find that the results obtained are not The sample holder, referred to as penetrometer or
always in agreement and that no single technique can be dilatometer, has a calibrated capillary tube, through which
relied upon to provide a complete picture of the pore the sample can be evacuated and through which mercury can
structure. The choice of the most appropriate method enter. The capillary tube is attached to a wider tube in which
depends on the application of the porous solid, its chemical the test sample is placed. The change in the volume of
and physical nature and the range of pore-size. mercury intruded is usually measured by the change in
This chapter provides guidance for measurement of porosity capacitance between the mercury column in the capillary
and pore-size distribution by mercury porosimetry. It is a tube and a metal sleeve around the outside of the capillary
comparative test, usually destructive, in which the volume of tube. If precise measurements are required the expected total
mercury penetrating a pore or void is determined as a void and pore volume of the sample should be between
function of an applied hydrostatic pressure, which can be 20 per cent and 90 per cent of the internal volume of the
related to a pore diameter. Other information such as pore capillary tube. Since different materials exhibit a wide range
shape and inter-connectivity, the internal and external surface of open porosities, a number of penetrometers with different
area, powder granulometry, bulk and tapped density could capillary tube diameters and sample volumes may be
also be inferred from volume-pressure curves; however, these required. A typical set-up for a mercury porosimeter
aspects of the technique do not fall under the scope of this instrument is given in Figure 2.9.32.-1. The porosimeter may
chapter. have separate ports for high- and low-pressure operation, or
the low-pressure measurement may be carried out on a
Practical considerations presently limit the maximum applied separate unit.
absolute pressure reached by some equipment to about
400 MPa, corresponding to a minimum equivalent pore The pressure range is typically 4-300 kPa for low-pressure
diameter of approximately 0.003 µm. The maximum operation and above 300 kPa for high-pressure operation,
diameter will be limited for samples having a significant depending on the design of the particular apparatus and on
depth due to the difference in hydrostatic head of mercury the intended use.
from the top to the bottom of the sample. For most purposes METHOD
this limit may be regarded as 400 µm. Sample preparation
Inter-particle and intra-particle porosity can be determined, The sample is pre-treated to remove adsorbed material that
but the method does not distinguish between these porosities can obscure its accessible porosity, for example by heating
where they co-exist. and/or evacuation, or by flowing inert gas. It may be possible
The method is suitable for the study of most porous to passivate the surface of wettable or amalgam-forming
materials. Samples that amalgamate with mercury, such as solids, for example by producing a thin layer of oxide, or by
certain metals, may be unsuitable for this technique or may coating with stearate.
require a preliminary passivation. Other materials may The sample of the pre-treated solid is weighed and
deform or compact under the applied pressure. In some cases transferred to the penetrometer. The pore system of the
it may be possible to apply sample-compressibility corrections sample is then degassed in a vacuum to a maximum residual
and useful comparative data may still be obtained. pressure of 7 Pa.
V-A634 Appendix XVII R 2023

~ I
I
I
I
I
I
I
I
I
I
I

-8
A. Low-pressure hydraulic fluid E. High-pressure hydraulic fluid J. Penetration volume N. Sample
reservoir reservoir indicator
B. Hydraulic pump F. Vacuum pump with gauge K. Capillary tube
C. Pressure multiplier G. Mercury reservoir L. High-pressure chamber
D. Pressure transducer H.Oil M. Mercury

Figure 2.9.32.-1. - Example of the set-up of a mercury porosimeter instrument

Filling the penetrometer with mercury pressure. The extent of the corrections may be determined
The mercury used is of analytical quality. Overlay the sample by means of blank measurements under the same conditions.
with mercury under vacuum. The vacuum is required to An experimentally determined volume-pressure curve is
ensure the transfer of mercury from the reservoir to the shown in Figure 2.9.32.-2.
penetrometer. In a filled penetrometer the filling pressure
comprises the applied pressure plus the pressure contribution
created by the head of mercury contacting the sample.
~
~
300------------------
.S 240
E
A typical filling pressure would be about 4 kPa.
Q)
The hydrostatic pressure of the mercury over the sample may E
::,
be minimised by filling the penetrometer in the horizontal §! 180
position. (.)
-=
Low-pressure measurement
Admit air or nitrogen in a controlled manner to increase the
'i 120
If)

pressure either in stages corresponding to the particular pore -~1o 60

sizes of interest, or continuously at a slow rate. 'S
The concomitant change in the length of the mercury E
column in the capillary tube is recorded. When the 60 1L0•,;;;;;;=;;;;;;==;10;:::~----1-0_
0 1.Jo'
1_ _ _ _ _ _

maximum required pressure has been reached, return to

Pressure (MPa)
atmospheric pressure.
High-pressure measurement Figure 2.9.32.-2. - Volume-pressure curve as semilogarithmic pwt
After measurement at low pressure, the penetrometer filled
with mercury is transferred to the high-pressure port or unit REPORTING OF RESULTS
of the instrument and overlaid with hydraulic fluid. Mercury The pressure readings can be converted to pore diameters by
is intruded into the pore system via the hydraulic fluid. means of the Washburn equation or by another model.
Increase the pressure in the system to the maximum pressure The surface tension of mercury (cr) depends not only on the
reached in the low-pressure measurement and record the temperature, but also, in the case of markedly curved
intrusion volume at this pressure, since subsequent intrusion surfaces areas, on the radius of curvature. In general, values
volumes are calculated from this initial volume. Increase the between 0.41 N-m- 1 and 0.52 N-m- 1 are measured at room
pressure either in stages corresponding to the particular pore temperature. If the value is not known, cr = 0.48 N-m- 1 can
sizes of interest, or continuously at a slow rate. The fall in be used.
the mercury column is measured up to the maximum
required pressure. If required the pressure may be decreased The contact angle of mercury (0) in most cases is more
than 90°. It may be determined using a contact angle
either in stages or continuously at a slow rate to determine
instrument. If the value is not known, 0 = 130° can be used.
the mercury extrusion curve.
The values of contact angle and surface tension and the
Corrections are made to take account of changes in the model used in the calculation are reported.
volume of the mercury, the penetrometer and other
components of the volume detector system under elevated Visualisation of the data can be done with several types of
graphs. Frequently, in a graphical representation the pore
diameter is plotted on the abscissa and the intruded volume
2023 Appendix XVII S V-A635

~
C
(])
::::J
er
~
LL

10 1 1O' 10'
Pore diameter (nm)

Figure 2.9.32.-3. - Pore-size distribution as semilogarithmic plots of the cumulative and the normalised density distribution

per sample mass on the ordinate to give the pore-size The bulking properties of a powder are dependent upon the
distribution. It is appropriate here to choose a logarithmic preparation, treatment and storage of the sample, i.e. how it
scale for the abscissa (see Figure 2.9.32.-3). The spaces has been handled. The particles can be packed to have a
between the particles of the solid sample are included as range of bulk densities and, moreover, the slightest
pores in the calculation. If the pores differ in size from the disturbance of the powder bed may result in a changed bulk
voids, the latter can be separated by choosing the appropriate density. Thus, the bulk density of a powder is often very
pore-size range. difficult to measure with good reproducibiliry and, in
Extrusion curves may not be used for calculating the pore- reporting the results, it is essential to specify how the
size distribution (for hysteresis, see Figure 2.9.32.-2), because determination was made.
an intruded part of the mercury always remains in the pore The bulk density of a powder is determined either by
system. The retention ratio may however be useful for the measuring the volume of a known mass of powder sample,
qualitative characterisation of pores that are only accessible which may have been passed through a sieve, in a graduated
via narrow openings ('ink-bottle pores'). cylinder (Method 1), or by measuring the mass of a known
Most common characteristic values, such as the total volume of powder that has been passed through a volumeter
intruded specific volume and the mean and median pore into a cup (Method 2) or has been introduced into a
diameters, are calculated from the pore-size distribution. measuring vessel (Method 3).
Moreover, sufficient information must be documented about Methods I and 3 are favoured.
the sample, the sample preparation, the evacuation METHOD 1: MEASUREMENT IN A GRADUATED
conditions and the instrument used.
CYLINDER
CONTROL OF INSTRUMENT PERFORMANCE Procedure Pass a quantity of powder sufficient to
As mercury porosimetry is considered to be used as a complete the test through a sieve with apertures greater than
comparative test, no details are given in this chapter. or equal to 1.0 mm, if necessary, to break up agglomerates
However, it is recommended that a stable comparison that may have formed during storage; this must be done
material is tested on a regular basis to monitor instrument gently to avoid changing the nature of the material. Into a
calibration and performance. dry, graduated, 250 mL cylinder (readable to 2 mL), gently
introduce, without compacting, approximately 100 g (m) of
the test sample weighed with 0.1 per cent accuracy.
If necessary, carefully level the powder without compacting,
S. Bulk Density and Tapped Density of and read the unsettled apparent volume ( Vo) to the nearest
graduated unit. Calculate the bulk density in grams per
Powders 1 millilitre using the formula m/V0 • Generally, replicate
(Ph. Bur. method 2.9.34) determinations are desirable for the determination of this
property.
BULK DENSITY If the powder density is too low or too high, such that the
The bulk density of a powder is the ratio of the mass of an test sample has an untapped apparent volume of more than
untapped powder sample to its volume, including the 250 mL or less than 150 mL, it is not possible to use 100 g
contribution of the interparticulate void volume. Hence, the of powder sample. In this case, a different amount of powder
bulk density depends on both the density of powder particles is selected as the test sample, such that its untapped apparent
and the spatial arrangement of particles in the powder bed. volume is between 150 mL and 250 mL (apparent volume
The bulk density is expressed in grams per millilitre despite greater than or equal to 60 per cent of the total volume of
the International Unit being kilogram per cubic metre the cylinder); the mass of the test sample is specified in the
(1 g/mL = 1000 kg/m 3), because the measurements are made expression of results.
using cylinders. It may also be expressed in grams per cubic For test samples having an apparent volume between 50 mL
centimetre. and 100 mL, a 100 mL cylinder readable to 1 mL can be
used; the volume of the cylinder is specified in the expression
of results.
1 This chapter has undergone pharmacopoeia/ harmonisation. See chapter

5. 8. Pharmacopoeia/ harmonisation.
V-A636 Appendix XVII S 2023

METHOD 2: MEASUREMENT IN A VOLUMETER METHOD 3: MEASUREMENT IN A VESSEL

Apparatus The apparatus (Figure 2.9.34.-1) consists of a Apparatus The apparatus consists of a 100 mL cylindrical
top funnel fitted with a 1.0 mm sieve, mounted over a baffle vessel of stainless steel with dimensions as specified in
box containing 4 glass baffles over which the powder slides Figure 2.9.34.-2.
and bounces as it passes. At the bottom of the baffle box is a
funnel that collects the powder and allows it to pour into a - - 54.0 ---+- -- 57.0-----+-

□l [
cup mounted directly below it. The cup may be cylindrical
(25.00 ± 0.05 mL volume with an internal diameter of
30.00 ± 2.00 mm) or cubical (16.39 ± 0.20 mL volume 52.0
5 5
o. ] l
50.0
with internal dimensions of 25.400 ± 0.076 mm).
❖ ! 10;0 54.0 !
A

Figure 2.9.34.-2. - Measuring vessel (left) and cap (right)

Dimensions in millimetres
B
Procedure Pass a quantity of powder sufficient to
complete the test through a 1.0 mm sieve, if necessary, to
break up agglomerates that may have formed during storage,
and allow the obtained sample to flow freely into the
C measuring vessel until it overflows. Carefully scrape the
excess powder from the top of the vessel as described under
Method 2. Determine the mass (M0) of the powder to the
nearest 0.1 per cent by subtracting the previously determined
mass of the empty measuring vessel. Calculate the bulk
density in grams per millilitre using the formula M 0 /100 and
record the average of 3 determinations using 3 different
powder samples.
D
TAPPED DENSITY
The tapped density is an increased bulk density attained after
mechanically tapping a receptacle containing the powder
sample.
E
G The tapped density is obtained by mechanically tapping a
graduated measuring cylinder or vessel containing the powder
sample. After observing the initial powder volume or mass,
the measuring cylinder or vessel is mechanically tapped, and
volume or mass readings are taken until little further volume
or mass change is observed. The mechanical tapping is
F achieved by raising the cylinder or vessel and allowing it to
drop, under its own mass, a specified distance by one of
3 methods as described below. Devices that rotate the
cylinder or vessel during tapping may be preferred to
minimise any possible separation of the mass during tapping
down.
A. 1.0 mm sieve E. glass baffle
B. powder funnel F. cup METHOD 1
C. loading funnel G. stand Apparatus The apparatus (Figure 2.9.34.-3) consists of
D. baffle box the following:
- a 250 mL graduated cylinder (readable to 2 mL) with a
Figure 2.9.34.-1. - Volumeter mass of 220 ± 44 g;
Procedure Allow an excess of powder to flow through the - a settling apparatus capable of producing, per minute,
apparatus into the sample receiving cup until it overflows, either nominally 250 ± 15 taps from a height of
using a minimum of 25 cm3 of powder with the cubical cup 3 ± 0.2 mm, or nominally 300 ± 15 taps from a height
and 35 cm 3 of powder with the cylindrical cup. Carefully, of 14 ± 2 mm. The support for the graduated cylinder,
scrape excess powder from the top of the cup by smoothly with its holder, has a mass of 450 ± 10 g.
moving the edge of the blade of a spatula perpendicular to Procedure Proceed as described above for the
and in contact with the top surface of the cup, taking care to determination of the bulk volume (Vo). Secure the cylinder
keep the spatula perpendicular to prevent packing or removal in the support. Carry out 10, 500 and 1250 taps on the same
of powder from the cup. Remove any material from the side powder sample and read the corresponding volumes Vio,
of the cup and determine the mass (M) of the powder to the Vsoo and Vi 250 to the nearest graduated unit. If the
nearest 0.1 per cent. Calculate the bulk density in grams per difference between Vsoo and Vi 250 is less than or equal to
millilitre using the formula MIV0 (where V 0 is the volume of 2 mL, Vi 250 is the tapped volume. If the difference between
the cup) and record the average of 3 determinations using Vsoo and Vi 250 exceeds 2 mL, repeat in increments of, for
3 different powder samples. example, 1250 taps, until the difference between successive
measurements is less than or equal to 2 mL. Fewer taps may
be appropriate for some powders, when validated. Calculate
2023 Appendix XVII S V-A637

I
E
.±.
..... ..J
E
-::I:"
-
I.!)
E E M
0
I.!)
E ..c:: M
0) C
0
N 'ii> ro
co -
+
.....,...... t::'.
0
N ..c ..c
Graduated cylinder -:i=- - ro
0.. C
ro ~

..... - -
..c:: 0
t2 'O
.±.
- E
Cl)
ro en
::::, en 0

- -
Cl)
-::I:" "O C
ro
,_
Cylinder -=I=- (.9
0
C
support

+I E
E E
E "!
MO

Anvil
This dimension is such
that the drop G) meets
_...........,.___ _ _ _ _ _.......,specifications and that, at
t the lowest point of the cam,

(
the cylinder support is sitting
freely on the upper part of
the anvil.
Cam

Figure 2.9.34.-3. - Settling device for pcrwder samples

Dimensions in millimetres

the tapped density in grams per millilitre using the METHOD 3

formula m!V; (where J7;- is the final tapped volume). Procedure Proceed as described under Method 3 for
Generally, replicate determinations are desirable for the measuring the bulk density, using the measuring vessel
determination of this property. Specify the drop height with equipped with the cap shown in Figure 2.9.34.-2.
the results. The measuring vessel with the cap is lifted 50-60 times per
If it is not possible to use a 100 g test sample, use a reduced minute by the use of a suitable tapped density tester. Carry
amount and a suitable 100 mL graduated cylinder (readable out 200 taps, remove the cap and carefully scrape excess
to 1 mL) weighing 130 ± 16 g and mounted on a support powder from the top of the measuring vessel as described
weighing 240 ± 12 g. If the difference between V500 and under Method 3 for measuring the bulk density. Repeat the
Vi 250 is less than or equal to 1 mL, Vi 250 is the tapped procedure using 400 taps. If the difference between the
volume. If the difference between Vioo and Vi 250 exceeds 2 masses obtained after 200 and 400 taps exceeds 2 per cent,
1 mL, repeat in increments of, for example, 1250 taps, until repeat the test using 200 additional taps until the difference
the difference between successive measurements is less than between successive measurements is less than 2 per cent.
or equal to 1 mL. The modified test conditions are specified Calculate the tapped density in grams per millilitre using the
in the expression of the results. formula M/100 (where M1 is the mass of powder in the
measuring vessel). Record the average of 3 determinations
METHOD2
using 3 different powder samples. The test conditions,
Procedure Proceed as directed under Method 1 except including tapping height, are specified in the expression of
that the mechanical tester provides a fixed drop of the results.
3 ± 0.2 mm at a nominal rate of 250 taps per minute.
V-A638 Appendix XVII T 2023

MEASURES OF POWDER express the wettability by a contact angle measurement

COMPRESSIBILITY between the porous solid and a given liquid.
The sessile drop method is based on direct measurement of a
Because the interparticulate interactions influencing the
contact angle of a sessile drop on a compacted powder disc.
bulking properties of a powder are also the interactions that
interfere with powder flow, a comparison of the bulk and With the Washburn method the contact angle is indirectly
tapped densities can give a measure of the relative measured. The method is based on the capillary effect of the
importance of these interactions in a given powder. Such a powder pores. The effect (mass gain) is recorded by special
comparison is often used as an index of the ability of the electronic balances starting the moment when the powder
powder to flow, for example the compressibility index or the sample touches the surface of a liquid, preferably not
Hausner ratio. dissolving or poorly dissolving the sample. The measurement
has very little or no effect on the state of the powder.
The compressibility index and Hausner ratio are measures of
the propensity of a powder to be compressed as described Any pre-treatment of the sample to be examined is
above. As such, they are measures of the powder's ability to disadvantageous, since the properties may be significantly
settle, and they permit an assessment of the relative altered. For example, the compaction of a powder as a disc
importance of interparticulate interactions. In a free-flowing may decrease the surface free energy when the crystalline
powder, such interactions are less significant, and the bulk state of the powder is changed (e.g. metastable forms), or
and tapped densities will be closer in value. For more-poorly may increase surface free energy by creating crystal defects
flowing materials, there are frequently greater interparticulate (disadvantage of the sessile drop method since compacted
interactions, and a greater difference between the bulk and powder discs are tested).
tapped densities will be observed. These differences are The methods are usually applied to examine the following
reflected in the compressibility index and the Hausner ratio. parameters:
Compressibility index: - batch-to-batch consistency of samples in terms of
wettability;
lOO(Va - VJ) - effect of liquid viscosity on wettability;
Va - effect of surface tension of a liquid on wettability;
- alteration of surface properties of samples.
Vi unsettled apparent volume;
]7t final tapped volume. SESSILE DROP METHOD
This method may be used to characterise directly the
Hausner Ratio: wettability of coatings and compacted formulations such as
tablets. Moreover, it is sometimes possible to use the sessile
Vo drop instrument in a dynamic measurement (dynamic
Vr contact angle measurement, Figure 2.9.45.-2) of porous
solid/liquid systems where the contact angle decreases.
Depending on the material, the compressibility index can be
By taking several contact angle measurements as a function
determined using Via instead of V0 • If Via is used, it is
of time, the rate of spreading accompanied by penetration of
clearly stated with the results.
a liquid droplet into a slightly porous solid may be studied.

T. Wettability of Porous Solids Including

Powders
(Ph. Bur. method 2.9.45)
INTRODUCTION
The wettability of solid surfaces is commonly characterised
by direct or indirect contact angle measurements.
The contact angle (0) between a liquid and a solid is the
angle naturally formed when a drop of a liquid is placed on a
solid surface. This is depicted in Figure 2.9.45.-1. For a
given liquid, wettable solids show a low contact angle and
non-wettable solids show a contact angle of 90° or more.

Figure 2.9.45.-2. - Sessile drop determination with visual

inspection of the droplet

Under equilibrium conditions the contact angle of a sessile

drop depends on 3 interrelated surface tensions and is
determined using Young's equation (see Figure 2.9.45.-2,
1st part):
Figure 2.9.45.-1. - Contact angle (0) of a sessik drop observed
on a non-porous surface

Ys surface tension of the solid with air;

2 methods for the determination of wettability are described
YsL interfacial tension of the solid with the liquid;
below. The methods are capable of measuring the wettability surface tension of the liquid with air.
YL
of porous solids like powders or granules. Both methods
 2023 Appendix XVII T V-A639

rr2 x r 5 x 1V 1
PROCEDURE (4)
2
Since powders are unable to form a completely flat surface,
the powder is usually compacted as a disc in an attempt to
make the surface smoother. A liquid drop with a given
r average capillary radius within the porous solid;
volume is placed on the disc (see Figure 2.9.45.-2) allowing N number of capillaries per volumetric unit.
direct measurement of the contact angle using a goniometer
fitted with an eyepiece protractor, or by geometric If a Washburn determination is performed with a liquid
construction on a photomicrograph. Other physical and considered to have a contact angle of 0° (cos 0° = 1) on the
mathematical procedures of data analysis may also be solid, then the solid material constant (c) is the only
appropriate. The drop volume may influence the result. remaining unknown in equation (3) and can thus be
Several determinations of the contact angle (0) (n = 6) are determined. n-Heptane is the liquid of choice for determining
usually carried out and the average is calculated. material constants because of its low surface tension
WASHBURN METHOD (20.14 mN•m- 1 at 25 °C). n-Hexane may also be used
The Washburn method is able to measure the contact angle (18.43 mN-m- 1 at 25 °C) but is more volatile. If the powder
of porous solids with a contact angle in the range of 0-90°. dissolves too quickly in these liquids, hexamethyldisiloxane
The tested material is the combination of the sample, the may be used instead (15.9 mN·m- 1 at 25 °C). Replicate
holder and the filter system. Therefore, an estimation or determinations are performed (n = 6) and the average value
determination of the true value is not possible and only calculated.
apparent values of the contact angle can be determined. Once the material constant (c) has been determined for the
However, the contact angle of the sample is the functional solid to be examined, a sample of the solid can be tested for
property on which the result is significantly dependent. wettability by another liquid. The material constant
The outcome of the test is a ranking order listing the determined by the n-heptane test is used in the Washburn
wettability of different substances or formulations equation, in combination with the capillary penetration
characterised by an apparent contact angle. rate ("f-) data obtained while testing the substance to be
examined in the prescribed liquid. This allows calculation of
PRINCIPLE
the contact angle.
If a porous solid is brought into contact with a liquid, such
that the solid is not submerged in the liquid, but rather is NOTE: if a series of liquids (at least 2 liquids in addition to
just touching the liquid surface, then the rise of liquid into the liquid used to determine the material constant) is tested
the pores of the solid due to capillary action will be governed against a given solid then the resultant contact angle data can
by the following equations: be used to calculate the surface energy of the porous solid.
APPARATUS
2 t
m =-
A
(I) Figure 2.9.45.-3 shows the principal components of the
apparatus. The main device is an electronic balance with a
suitable processor ensuring a suitable resolution in force
measurement and a suitable resolution in lifting up the
m mass of liquid sucked into the solid;
time elapsed since the solid and the liquid were brought into immersion liquid towards the sample.
contact;
A constant, dependent on the properties of the liquid and the
A B

~ ---CJ
solid to be examined, calculated using the following equation:

A= 'I (2)
cxp 2 xyxcos 8

11 viscosity of the liquid;

p density of the liquid;
y surface tension of the liquid;
e contact angle between the solid and the liquid;
material constant, dependent on the porous texture of the solid.

Equations (1) and (2) lead to equation (3):

m2 rJ
COS O= f X CX p?. X/ (3)

In setting up a Washburn determination, a liquid with known

density (p), viscosity (17), and surface tension (y) is used.
Under these conditions, when the mass of liquid rising into
the porous solid is monitored as a function of time (such that
capillary penetration rate ("f-) is the experimental data),
2 unknowns remain according to equation (3): the contact
angle (0) of the liquid on the solid, and the solid material
constant (c). A. electronic balance C. sample holder E. immersion liquid
B. computer D. filter F. lift
Determination of the material constant (c)
The material constant for a porous solid is determined by the Figure 2.9.45.-3. - Apparatus for contact angle measurement by
following equation, considering cylindrical pores: the Washburn method
V-A640 Appendix XVII T 2023

Table 2.9.45.-1 indicates parameters of the electronic balance osmosis) is recommended because of its high porosity and
that are generally considered suitable. minimum flow resistance.
Place a known amount of powder into the cell.
Table 2.9.45.-1. - Technical, parameters of the electronic balance
The reproducibility of material constants and contact angles
Lift Mass measurement will depend on the ability to weigh out the same amount of
Range > 110 mm 0 - 210 g powder for each test when a sufficient and adjusted amount
Resolution 0.1 µm 10 µg of powder is compacted in a uniform way
Speed 0.099 - 500 mm/min (i.e. tapping/compaction of the powder).
For most powders, a correct amount is in the range of a few
Sample holders grams, typically filling about 2/3 of the capacity of the holder.
The sample holder may be a small glass cylinder with a Place a second piece of filter paper on top of the powder in
sintered-glass filter at one end. the cell. This will prevent powder from rising through the
Powder material holders (see Figure 2.9.45-4) may also be holes in the piston during the compaction process and/or
made of aluminium; they are less fragile than those made of during the determination.
glass and have small holes in the bottom that render them Tapping/compaction of the powder
easier to clean than a sintered-glass filter. The cover for the A bulk powder bed is very porous and thus very sensitive to
cell is equipped with 2 screw threads. One connects it with small influences that can easily alter the porosity and
the sample chamber while the other allows the user to guide consequently the c-constant. Therefore a tapped powder may
a piston down onto the sample itself and compact it. be advantageous and will show more reproducible results.
The apparatus is similar to an automatic tensiometer, except The appropriate number of taps must first be evaluated:
for the sample holder. 50-100 taps are usually appropriate.
If the aluminium sample holder is used then it may be
mounted in the cylinder of a stamp volumeter, which can run
the evaluated number of taps.
If tapping is not appropriate, the powder bed is compacted
by screwing the piston of the aluminium sample holder
applying a specified pressure.
A further possibility is centrifugation under defined
conditions. Where applicable, a compacted disc of the
powder sample may also be mounted on the electronic
balance. A sample holder is omitted in this case.
After connecting to the balance, the sample holder is
positioned with the porous solid just above the surface of the
liquid (see Figure 2.9.45.-3), using the lift.
The liquid is raised further until it just touches the bottom of
the porous sample. Mass-versus-time data is then collected as
liquid penetrates into the solid. Data can be presented in
either graphical or tabular format. The apparatus may
perform the whole determination automatically.
CRITICAL PARAMETERS
The following points must be considered.
Sample properties:
- water content of the sample;
- crystalline or solid-state properties of the sample
(polymorphic form, type of solvate).
Sample preparation:
- homogeneity of any powder blend to be examined;
- particle-size distribution; before testing it is sometimes
View of plunger bottom View of apparatus bottom
advisable to sieve the sample (e.g. using a 250 µm sieve);
- the optimal compaction parameters (amount of sample,
A. fixing C. thread E. capillary holes
number of taps or piston mass) must be determined;
B. cover D. plunger F. capillary holes - the compaction state of the different powder samples
must be uniform;
Figure 2.9.45.-4. - Example of sample holder with plunger for - the sample holder or, if used, the glass frit must be
compaction of a powder carefully cleaned;
- uniformity of the results is improved by using a sample
PROCEDURE holder made of aluminium.
Filling of the sample holder Immersion liquid:
Place a disc of filter paper in the bottom of the aluminium or - specifications of the immersion liquid must be indicated.
glass sample holder. This prevents powder from leaking out
of the bottom of the cell. The filter does not have to be made
of paper, but it must be a material that is easily wetted by the
liquid to be tested. A black-band filter (used for reverse
2023 Appendix XVII U V-A641

these 2 extreme crystallinities. Such a powder is obtained

U. Crystallinity w~en pure cry_stalline and amorphous phases are physically
(Ph. Bur. general texts 5.16) mixed. In reality, a powder probably contains particles with
This chapter provides general information on crystallinity and different degrees of crystallinity, just as it may contain
refers to the various techniques described in the European particles with different sizes and shapes.
Phannacopoeia that are used for its detennination. The extent of disorder in a crystalline solid can affect many
physico-chemical properties of substances for pharmaceutical
INTRODUCTION - THE CONCEPT OF
~se. Because of the great relevance of these properties, it is
CRYSTALLINITY important to be able to assess the extent of disorder or the
Most organic and inorganic compounds of pharmaceutical crystallinity of a solid by a suitable quantitative method.
relevance exist as a solid material, which can be characterised
by a structure located between a perfectly ordered crystal and METHODS FOR MONITORING AND
an amorphous material. DETERMINING CRYSTALLINITY
Real crystals lie somewhere between an ideal crystal state and Various methods are available for determining the
the amorphous state. The position of a crystal on a scale crystallinity of a solid. Many techniques cannot detect or
bounded by these 2 extremes is termed its crystallinity. quantify these properties independently; for this reason, it is
useful to combine several of the methods described below.
A perfectly ordered crystal is an ideal state that is seldom if
Such methods often do not give accurate results and limits of
ever, achieved. The structural units of a crystal, termed ~it quantitation are usually much greater than those for chemical
cells, are repeated regularly and indefinitely in 3 dimensions impurities. In addition, certain assumptions have to be made
in space. The unit cell has a definite orientation and shape about the relationship between standards used for calibration
defined by the translational vectors a, b and c, and the angles which are typically mixtures of crystalline and amorphous '
a., Pand y, and hence has a definite volume, V, that contains particles (2-state model), and the samples to be analysed that
the atoms and molecules necessary for forming the crystal. are likely to have at least a small component of material
A crystalline system is defined by 3 long-range order exhibiting I-state model behaviour. Finally, the lack of well-
symmetry operators (translational, orientational and defined standards for 100 per cent crystalline or 100 per cent
conformational); the various mesophases (liquid crystals,
amorphous material complicates the validation of such
crystals and plastic crystals) have 1 or 2 of the long-range
methods. As explained above, it is obvious that different
symmetry operators and the ideal amorphous state is defined
amorphous or non-crystalline phases exist and even co-exist
by the absence of all 3 operators. in a solid powder. These different non-crystalline forms of a
Each crystal can be classified as a member of one of solid can give different responses depending on the
7 possible crystal systems that are defined by the techniques used for determining the degree of crystallinity.
relationships between the individual dimensions a, b and c
X-ray powder diffraction (2.9.33)
and between the individual angles a., p and y of the unit cell.
XRPD is still the most commonly used method for
The structure of a given crystal may be classified according
determining the degree of crystallinity, although this method
to one of the 7 crystal systems, to one of the 14 Bravais
suffers from some limitations due to peak broadening,
lattices and to one of the 230 space groups. All the 230
amorphous halo and preferred orientation, which make
possible space groups, their symmetries and the symmetries
interpretation and quantitation difficult.
of their diffraction patterns are compiled in the International
Tables for Crystallography. XRPD alone is often insufficient to distinguish between the
different non-crystalline phases. The X-ray diffraction pattern
Many substances are capable of crystallising in more than
of a purely amorphous and nanocrystalline phase is
one type of crystal lattice, which is known as polymorphism.
characteristic of a broad diffuse halo. In-depth analysis of the
The occurrence of polymorphism is a common phenomenon
X-ray diffraction patterns will show that the diffuse halo in
among organic molecules, giving rise to different physico-
the pattern of nanocrystalline material shows some
chemical properties. Crystalline polymorphs have the same
correlation to the pattern of the parent crystalline phase
chemical composition but different internal crystal structures
while in the case of a pure amorphous phase such a '
and, therefore, possess different physico-chemical properties.
correlation does not exist. Additional techniques may be
The different crystal structures in polymorphs are due to
required to establish the true nature of X-ray amorphous
different atomic packing arrangements and/or different
materials.
conformations of the molecules (see chapter 5. 9.
Polymorphism). Thermal analysis
The other extreme of a crystal state is the ideal or true Thermal analysis (2.2.34) of crystalline materials exhibits a
amorphous state, where all long-range order is lost. For most melting transition that is often accompanied by
organic systems certain short-range order remains, but this is decomposition or evaporation of solvents. In the case of true
not expected to extend over distances much larger than amorphous materials, thermal analysis reveals a glass
nearest neighbour (NN) or next nearest neighbour (NNN) transition, whereas only a melt would be expected for a
interactions, which are typically less than 2-2.5 nm for small nanocrystalline material.
organic molecules. Microcalorimetry (2.2.61)
Amorphous material is characterised by the absence of It is a highly sensitive technique which allows the
distinct reflections in the X-ray powder diffraction (XRPD) determination of the rate and extent of chemical reactions,
pattern (2.9.33). changes of phase or changes of structure. Amorphous parts
o~ a substance can recrystallise by subjecting the sample to
The crystallinity of a real powder can be considered by
higher relative humidity or an atmosphere containing organic
2 models of crystallinity. In the I-state model all particles will
vapour. The measurement of the heat of recrystallisation
be of the same crystallinity whereas in the 2-state model each
enables the amorphous content to be determined from the
particle can be either crystalline or amorphous, such that the
enthalpy of recrystallisation. By relating the output from the
actual crystallinity of the powder is the weighted average of
microcalorimeter for a sample to that obtained for an
V-A642 Appendix XVII V 2023

amorphous standard, it is possible to quantify the amorphous

content of the sample. Toe range of amorphous content
v. Characterisation of Crystalline Solids
covered by this method depends on the individual substance by Microcalorimetry and Solution
to be tested; in favourable cases limits of detection below
1 per cent can be reached. Calorimetry
Solution calorimetry (2.2.61) (Ph. Bur. method 2.2.61)
Solution calorimetry provides a means of determining For the purpose of this chapter, crystalline material, partially
enthalpy of solution for a solid substance. The crystallinity of crystalline material and amorphous material are considered as
the solid sample to be examined is given by the enthalpy of solids.
solution of the solid sample (/iH~) minus the enthalpy of
solution of the chosen reference standard of the same
INTRODUCTION - THE CONCEPT OF
substance (/iH:) when determined under the same CRYSTALLINITY
conditions. Because the reference standard is usually chosen The perfectly ordered crystal lattice with every molecule in its
for its perceived high crystallinity, its enthalpy of solution is expected lattice position is an ideal that is seldom, if ever,
usually algebraically greater (more endothermic or less achieved. The other extreme is the amorphous state, in
exothermic) than that of the solid sample to be examined in which a solid contains the maximum possible density of
the same solvent. Consequently, the crystallinity determined imperfections (defects of various dimensionalities), such that
is a negative quantity with the SI units kJ/mol or Jig G/kg is all long-range order is lost while only the short-range order,
avoided because of its unwieldiness and potential for error). imposed by its nearest neighbours, remains. Real crystals lie
The preference for a negative value with respect to a highly somewhere between these 2 extremes. A crystal's position on
crystalline reference standard recognises the fact that most a scale bounded by these 2 extremes is termed crystallinity.
samples have a lower crystallinity than this reference All real crystals, even in the pure state, possess some lattice
standard. imperfections or defects, which increase both the energy
(enthalpy under conditions of constant atmospheric pressure)
Near-infrared (NIR) spectroscopy
and the disorder (expressed as the entropy) of the crystal
Near-infrared (NIR) spectroscopy (2.2.40) is another
lattice. A crystal with a relatively low density of imperfections
technique used to measure the degree of crystallinity, and has
is said to be highly crystalline and to possess a high
also been proven to be useful in studies of polymorphism.
crystallinity. By contrast, a particle with a relatively high
The NIR spectrum of a sample contains both physical and
density of imperfections is said to be partially amorphous and
chemical information. Being non-invasive, non-destructive
to possess a low crystallinity. In ideal terms, a totally
and operable at room temperature, the method is a valuable
amorphous particle corresponds to zero crystallinity.
tool to assess changes in the amorphous and crystalline state.
Amorphous particles may contain somewhat ordered
Infrared absorption spectrophotometry and Raman domains that can act as nuclei for crystallisation; such
spectroscopy so-called amorphous particles are said to possess a low-level
Infrared absorption spectrophotometry (2.2.24) and Raman but finite crystallinity.
spectroscopy (2.2.48) are other techniques used to measure
The ability to detect and to quantify the amount of
the degree of crystallinity, and have also been proven to be
amorphous material within a highly crystalline substance is of
useful in studies of polymorphism. The IR spectrum and
great importance during the development and subsequent
Raman spectrum of a sample contain both physical and
manufacture of a pharmaceutical preparation.
chemical information.
In reality, a powder probably contains particles with different
Solid-state NMR degrees of crystallinity, just as it may contain particles with
Solid-state nuclear magnetic resonance spectrometry (ss varying sizes and shapes. The lower the crystallinity of a
NMR) (2.2.33) can be used to provide information about
solid, the greater its enthalpy and entropy. Toe increase in
polymorphism and related relative molecular conformations. enthalpy is never totally compensated for by the increase in
However, some caution has to be exercised in the entropy; therefore, the Gibbs free energy, which reflects the
interpretation of results, since it is not always simple to balance between them, actually increases. Hence, the lower
distinguish between samples that comprise a mixture of the crystallinity of a material (powder), and consequently the
different physical forms (2-state model) and those that greater its amorphous character, the greater its apparent
comprise crystals having disorder with exchange that is slow intrinsic solubility and dissolution rate, but the lower its
on the NMR timescale. Similarly, samples that contain thermodynamic stability. Because of the great relevance of
defects arising from different molecular conformations or these properties, crystallinity is also an important property
slightly different packing arrangements (I-state model) may and requires measurement by a suitable method.
show additional signals in the spectra. Solid-state NMR may
In the following chapter, the crystallinity or the content of
be quite sensitive to this, even if lattice parameters are hardly
affected and, consequently, little or no change is observed by amorphous parts of a powder are measured by calorimetric
methods such as microcalorimetry or solution calorimetry,
XRPD. It is evident that the crystallinity of substances for
pharmaceutical use can be complex, and both crystalline although other methods could be used (e.g. see general
chapter 2. 9. 33. Characterisation of crystalline and partially
defects and amorphous material may co-exist.
crystalline solids by X-ray powder diffraction (XRPD)).
Optical microscopy
Many substances are capable of crystallising in more than
A method to detect whether or not particles are crystalline is
one type of crystal lattice, which is known as polymorphism.
to use a polarising microscope (2.9.37), where particles show
If water or a solvent is incorporated in the crystal lattice the
birefringence and extinction positions when the microscope
crystals are termed hydrates or solvates. Because of the
stage is revolved.
different crystal packing, and/or molecular conformation and
lattice energy, they usually exhibit different physical
properties. For simplicity, calorimetry measurements for
degree of crystallinity determination discussed here assume
2023 Appendix XVII V V-A643

only one solid crystalline form present in the material of 2.5

interest. The theory and experimental technique can be easily
II
expanded to polymorphic systems with proper consideration
of the enthalpy differences among the polymorphs. 2
METHOD 1 - MICROCALORIMETRY
(DETERMINATION OF AMORPHOUS CONTENT)
Most chemical, physical and biological processes are
associated with the exchange of heat. Microcalorimetry is a
highly sensitive technique to monitor and quantify both
exothermic (heat producing) and endothermic (heat
absorbing) changes associated with those processes.
The technique allows the determination of the rate and
extent of chemical reactions, changes of phase or changes of
structure. 0.5

Thermal events producing only a fraction of a microwatt can

be observed using microcalorimetry. This means that
temperature differences less than 1o- 6 K must be detectable.
Microcalorimetry typically uses the heat flow (heat leakage) 0 2 3
principle, where the heat produced (or absorbed) in a
thermally defined vessel flows away (or into) in an effort to Time (Hours)
re-establish thermal equilibrium with its surroundings.
Exceptional thermal stability with its surrounding has to be Figure 2.2.61.-1. - Typical microcawrimetric output of power
achieved either by a heat sink or an electronically regulated (in µ W'.) as a function of time (in hours): amorphous collapse
surrounding. peak (1) and crystallisation peak (11) for mainly amorphous
Heat energy from an active sample in the reaction vessel is lactose at 25 °C and 75 per cent relative humidity
channelled typically through Peltier elements; they act as
microcalorimeter for a sample to that obtained from an
thermoelectric generators using the Seebeck effect. The heat
amorphous standard, it is possible to quantify the amorphous
energy is converted into a voltage signal proportional to the
content of the sample. The range of amorphous content
heat flow.
covered by this method depends on the individual substance
Results are typically presented as a measure of the thermal to be tested; in favourable cases limits of detection below
energy produced per unit of time (Watt) as a function of 1 per cent can be reached.
time.
Recrystallisation can be initiated by subjecting the sample to
APPARATUS higher relative humidity or an atmosphere containing organic
Microcalorimeters are typically designed as twin systems with vapour. The sample is typically placed in an ampoule which
a measuring vessel and a reference vessel. Vessels are also contains a small test-tube containing an aqueous
typically made of glass or stainless steel. For certain saturated salt solution, an organic solvent, or a solvent
applications specially designed vessels which allow the mixture.
addition of a gas, a liquid or a solid material may be used.
The heat of recrystallisation is typically measured using a
CALIBRATION fixed sample mass placed in a glass or steel vessel. The test-
The microcalorimeter is calibrated for heat flow (energy per tube containing a saturated salt solution or an organic solvent
time unit) using either calibrated external or internal is chosen to be large enough to allow a full saturation of the
electrical heat sources or a suitable standard reaction. atmosphere above the sample. The mass of the sample and
SENSITIVIIY the nature of the vapour atmosphere above the sample are
The sensitivity of the microcalorimetric method can be chosen so that recrystallisation occurs in such a way that a
assessed based on an appropriate standard sample analysed distinct peak is observed, clearly separated from initial
according to the corresponding method in conjunction with thermal events caused by introduction of the sample.
the determination of the instrument baseline noise. The conditions under which the transition of the amorphous
PROCEDURE phase to a thermodynamically more stable crystalline state
Weigh in a suitable vessel an appropriate quantity of the occurs will have a significant impact on the time of
substance to be examined. Close the vessel carefully to avoid recrystallisation. In particular, physical mixtures of purely
any evaporation of solvents and place the vessel in the sample amorphous and crystalline material will behave differently
holder. If appropriate, allow the vessel to equilibrate at the from a partially crystalline material. These effects should be
temperature of the measurement before placing it in the considered when developing a method.
measuring position. A typical response for the recrystallisation of a mainly
Begin the analysis and record the heat flow, with the time on amorphous material is shown in Figure 2.2.61.-1. The first
the abscissa and the heat flow on the ordinate (specify the part of the curve represents several concurrent processes
direction of exothermic or endothermic heat flow). taking place simultaneously, such as the absorption of water
vapour into the amorphous parts of the powder and the
DETECTION AND QUANTIFICATION OF
generation of water vapour from the test-tube. After this
AMORPHOUS CONTENT IN POWDERS
initial response there is a large exothermic response caused
The amorphous state is metastable with respect to the
by the recrystallisation of the amorphous material. Also
crystalline state; recrystallisation may therefore occur.
included, but not seen, are the expulsion of excess water
The measurement of the heat of recrystallisation enables the
from the recrystallised parts and its condensation. Thus, the
amorphous content to be determined by the area of the
recrystallisation peak. By relating the output from the
V-A644 Appendix XVII V 2023

area under this exothermic recrystallisation response is calorimeters are more advantageous than isothermal solution
proportional to the heat of recrystallisation. calorimeters when the solution process is relatively fast.
METHOD 2 - SOLUTION CALORIMETRY For all measurements of enthalpy of solution using isoperibol
(DETERMINATION OF CRYSTALLINITY) solution calorimeters, the choice of solvent is critical.
The nature and mass of the solvent and the mass of sample
Solution calorimetry provides a means of determining
allow the total heat change, corresponding to total dissolution
enthalpy of solution (i.e. heat of solution under constant
of the solid, to proceed to completion within 5 min under
atmospheric pressure) of a substance. Enthalpy of solution is
vigorous stirring at a constant rotational speed within the
defined as the enthalpy of the substance dissolved in the
range of 400-600 r/min.
solution to a defined concentration minus the enthalpy of the
original substance. The solvent for the dissolution process The effective heat capacity of the calorimeter cell and its
must be such that the mass of solid dissolves within a time contents is determined for every calorimeter run. This
frame that matches the response time of the calorimeter, as determination is accomplished by electrical heating of the
discussed below. The enthalpy of solution is proportional to contents of the calorimeter cell. The effective heat capacity is
the amount of solid being dissolved. This amount may be determined according to 1 of 2 protocols: either by making 1
defined as 1 mol for molar enthalpy or as 1 g for specific determination after ampoule breakage or by making
enthalpy. If the substance possesses adequate purity (as 1 determination before and a 2nd determination after
determined by the degree of accuracy required) and if its ampoule breakage and then averaging the 2 results.
molecular mass is known, the molar enthalpy is preferred, The accuracy and reliability of the electrical heating are
otherwise the specific enthalpy must be used. The enthalpy established by the accuracy and reliability of the
of solution is weakly dependent on both the temperature, aforementioned chemical calibrations.
which is usually 25.0 °C, and the final concentration of the ISOTHERMAL SOLUTION CALORIMETRY
dissolved solute. In the isothermal (constant temperature) solution
It is usually preferred to express the crystallinity, Pc, of a calorimeter, the heat change during the solution process is
substance on a percentage scale. This procedure requires compensated for by an equal but opposite energy change,
2 reference standards, namely a highly crystalline sample such that the temperature of the solvent-solute system
assuming 100 per cent crystallinity and having a measured (i.e. solution) remains essentially constant. This equal but
enthalpy of solution of /'J.H;, and an amorphous sample opposite energy change is measured and, when its sign is
assuming 0 per cent crystallinity and having a measured reversed, provides the enthalpy of solution. For isothermal
enthalpy of solution of /'J.H~. From these values and from the calorimeters, response is relatively slow, but the
measured enthalpy of solution, /'J.H;, of the solid under study, compensation process eliminates the effects of heat losses to
the percentage crystallinity of the solid, Pc, may be calculated or heat gains from the bath. Therefore, isothermal solution
as follows: calorimeters are more advantageous than isoperibol solution
calorimeters when the solution process is relatively slow.
SOLUTION CALORIMETER CALIBRATION
To ensure the accuracy of the calorimeter, chemical
Clearly, crystallinity expressed on a percentage scale depends
calibrations must be performed on a regular basis. For an
on 3 measured values and the enthalpies of solution may be
endothermic solution process, the calibration of the
replaced by other corresponding physical quantities that
calorimeter is checked by measuring the heat absorbed
depend on crystallinity. The value of the percentage
during the dissolution of potassium chloride in distilled water
crystallinity of a sample, however, depends not only on the
at 298.15 K (25.0 °C).The established enthalpy change in
nature and method of preparation of the 2 reference
this endothermic process is 235.5 Jig (17.56 kJ/mol). For an
standards, but also on the choice of the physical quantity that
exothermic solution process, the calorimeter is checked by
is measured.
measuring the heat evolved during the dissolution of 5 g per
The enthalpy of solution is measured either by an isoperibol litre of tromethamine [tris(hydroxymethyl)aminomethane,
(constant perimeter, i.e. jacket) solution calorimeter or by an THAM] in a 0.1 mol/L aqueous hydrochloric acid solution
isothermal (constant temperature) solution calorimeter. at 298.15 K (25.0 °C). The established heat for the
Typically, at least 3 measurements are made with each aforementioned process is -246.0 J/g (-29.80 kJ/mol).
sample. The mean of these values is then calculated.
The exact requirements will depend upon the equipment SAMPLE HANDLING
capability and degree of accuracy needed. The chemical and physical stability of solids may decrease
with decreasing crystallinity. In particular, solids of low
ISOPERIBOL SOLUTION CALORIMETRY crystallinity, especially amorphous solids, tend to sorb water
In the isoperibol solution calorimeter, the heat change during vapour from the atmosphere, leading to crystallisation and a
the solution process causes a corresponding change in corresponding gain in crystallinity. For these reasons,
temperature of the solvent-solute system (i.e. solution). This anhydrous samples whose crystallinity is to be determined
temperature change is measured by a temperature sensor, must be stored at zero humidity or below critical humidity
which is wired to an electrical circuit that records an levels in sealed chambers containing a desiccant, preferably
electrical signal corresponding to the temperature change. containing an indicator of effectiveness. If crystallinity-
Typically, this temperature change in an electronic form is humidity studies are to be carried out, the sample is stored in
measured at precisely defined time intervals to produce a sealed chamber containing a saturated salt solution to
temperature-time data that are collected, analysed by a provide a defined relative humidity.
computer, and then plotted. A blank run without addition of
the solid solute to the solvent normally shows no discernible
change in the slope of the temperature-time plot.
For isoperibol solution calorimeters, response is fairly rapid,
but corrections must be made for any heat losses to or heat
gains from the bath. Therefore, isoperibol solution
2023 Appendix XVIII V-A645

The efficacy of a sterilisation process is dependent on its

Appendix XVIII nature, the processing conditions (e.g. time, temperature,
moisture), the pre-sterilisation microbial contamination and
Methods of Sterilisation the formulation of the product. The inactivation of micro-
organisms by physical or chemical means follows an
Methods of Sterilisation exponential law and hence there is always a non-zero
(Methods of Preparation of Sterile Products, Ph. Bur. general probability that a micro-organism may survive the
texts 5. 1. 1) sterilisation process.
GENERAL INTRODUCTION Sterility assurance level (SAL)
Sterility is the absence of viable micro-organisms, as defined In the methods described, reference is made to a sterility
by a sterility assurance level equal to or less than 10~ 6 • assurance level (SAL) where appropriate. The SAL for a
Sterility is a critical quality attribute for a wide variety of given sterilisation process is expressed as the probability of
human and veterinary preparations, including but not micro-organisms surviving in a product item after exposure to
restricted to: the process. An SAL of 10- 6 , for example, denotes a
- preparations required to be sterile due to their route of probability of not more than 1 non-sterile item in 1 x 10 6
administration, such as parenteral, ophthalmic and sterilised items of the final product. The SAL of a process for
intramammary preparations, and some inhalation, a given product is established by appropriate validation
irrigation and intrauterine preparations; studies. Microbial contamination may be described by the
- preparations applied to severely injured skin, such as number, type and resistance of any micro-organisms present.
semi-solid preparations for cutaneous application. Microbiological monitoring and setting of suitable limits is
therefore essential for all components of sterile preparations.
The achievement of sterility for any one item in a population
Steps designed to reduce microbial contamination, such as
of items submitted to a sterilisation process can neither be filtration prior to sterilisation, will contribute significantly to
guaranteed nor demonstrated. It is essential to study the
sterility assurance. The composition of a product can affect
effect of the chosen sterilisation procedure on the product the behaviour of micro-organisms present in the product,
(including its final container) to ensure its effectiveness and
which in turn can affect the efficacy of the sterilisation
the integrity of the product, and to validate the procedure
process. The water activity (Aw), the pH and the presence of
before it is applied in practice. Failure to follow meticulously compounds with antimicrobial activity are examples of factors
a validated process introduces the risk of a non-sterile and/or that can influence the resistance of any micro-organisms
deteriorated product. present. The water activity or the product formulation
Sterile products are prepared under appropriate conditions (including the presence of nutrients) can affect the number of
and are packed in suitable containers. It is recommended micro-organisms, which in turn can affect the efficacy of the
that the choice of container permits application of the membrane-filtration process.
optimum sterilisation process for the product. The container
and closure system are required to maintain the sterility of METHODS AND CONDITIONS OF STERILISATION
the product throughout its shelf life. Sterilisation may be carried out by one of the methods
described hereafter. Modifications to, or combinations of,
Sterilisation process conditions are chosen to achieve the
these methods may be used, provided that the chosen
highest level of sterility assurance compatible with the drug
procedure is validated with respect both to its effectiveness
product and, wherever possible, a process in which the
and to the integrity of the product including its container.
product is sterilised in its final container (terminal
For all sterilisation methods, the critical parameters of the
sterilisation) is chosen. When a fully validated terminal
procedure are monitored in order to confirm that any
sterilisation method by steam (moist heat), dry heat or
previously determined requirements or conditions are
ionising sterilisation is used, parametric release
respected throughout the batch during the entire sterilisation
(i.e. the release of a batch of sterilised items based on process
process. This applies in all cases, including those where the
data rather than submission of a sample of the items to
reference conditions are used. Guidance concerning
sterility testing) may be carried out, subject to the approval of
validation of a steam sterilisation process using the F0
the competent authority. If terminal sterilisation is not
concept is described in general chapter 5.1. 5. Biological
possible, aseptic assembly or filtration through a bacterial
indicators of sterilisation are used to develop and validate
retentive filter is used. Wherever possible, an appropriate
sterilisation processes and also to monitor gas sterilisation
additional treatment (e.g. heating) of the product in its final
processes. Guidance on the use of biological indicators is
container is applied to further ensure the sterility assurance
provided in general chapter 5.1.2.
level.
Precautions shall be taken to prevent contamination of the
Requirements for the use of biological indicators for
sterilised articles after the sterilisation phase.
validation of sterilisation processes are given in general
chapter 5. 1.2. STEAM STERIUSATION
The present general chapter provides guidance on conditions Principle
validation and control of sterilisation processes. The method; Steam sterilisation is achieved by heat transfer during
described here apply mainly to the inactivation or removal of condensation of water from a saturated vapour phase on the
bacteria, yeasts and moulds. For biological products of surfaces of the sterilised items. Where items (open or
animal or human origin, or in cases where such material has wrapped) are sterilised in direct contact with steam, the
been used in the production process, it is necessary to hydrating effect of the condensate adds to the sterilising
demonstrate during validation that the process is capable of effect. For direct steam exposure, it is essential that the items
the removal or inactivation of any relevant viral are fully penetrated by saturated steam, i.e. free of air and
contamination. Further guidance is provided in general other non-condensable gases. Where items are sterilised in
chapter 5. 1. 7. Viral safery. closed-containers, the chamber of the steriliser serves as a
steam jacket. Condensation on the surface of the containers
V-A646 Appendix XVIII 2023

still serves as a highly effective mechanism for energy 8 min. The calculation of sterilisation effectiveness by the P0
transfer, but has no additional sterilising effect on its own. concept is performed according to general chapter 5.1.5.
In closed-container sterilisation, the sterilising effect is Calculated effectiveness from physical parameters (Pphys) is
determined by the conditions reached within the closed correlated with biological effectiveness (Pbio). Pbio expresses
containers, where sterilisation must be achieved in the the lethality, in minutes, provided by the process in terms of
product itself and in the head-space. destruction of the biological indicators used. Pbio is calculated
Equipment by the following equation:
Steam sterilisation is performed in autoclaves, i.e. pressure
vessels designed to admit or generate steam continuously and Pbio = D121 (log10 No ~ log10 N)
to remove condensate from the chamber to maintain the
D 121 is the D-value of the biological indicator at an exposure
pressure and temperature at controlled levels.
temperature of 121 °C, N 0 is the number of viable micro-
For equipment used to perform direct steam exposure cycles, organisms in the biological indicator before exposure, and N
the supply of saturated steam, free of non-condensable gases, is the number of viable micro-organisms in the biological
is assured. In autoclaves intended for the sterilisation of indicator after exposure.
closed containers, steam-air mixtures or a superheated water
In cycle validation, the relevant positions in the load that are
spray can be used to achieve heat transfer. Suitable
the most difficult to sterilise are determined and adequate
autoclaves are qualified to achieve homogeneous conditions
biological effectiveness is verified by exposure of biological
within the chamber and the load. The principles of operation
indicators (5.1. 2) in these positions or products, whichever is
are appropriate for the items to be sterilised and the loading
relevant. Protection of spores from the sterilising effect
configuration. The suitability of the equipment for the items
(e.g. by physical occlusion of steam or by the protective
to be sterilised and its performance in the chosen cycle is
properties of the product) are suitably addressed. The Pbio
demonstrated in autoclave performance qualification studies.
determined for the most-difficult-to-sterilise position is used
Temperature profiles in the slowest-to-heat items are
to define the parameters necessary to achieve reliably the
recorded.
required SAL equal to or less than 10- 6 for the chosen
Suitable autoclaves are equipped with temperature and cycle.
pressure sensors of appropriate sensitivity that are placed in
relevant positions to ensure effective process control.
Routine control
Autoclave cycles are monitored by physical determination of
Chamber temperature and pressure profiles are recorded for
chamber pressure and temperature profiles, at a minimum, in
each cycle. There is at least 1 independent thermal probe
the coldest position of the chamber. For each cycle, pressure,
that controls the load temperature at the slowest-to-heat
time and temperature are recorded and, if possible, P0 is
position or in the slowest-to-heat closed container of the
calculated and recorded.
load.
Cooling water sprayed into the chamber at the end of a DRY HEAT STERIUSATION
sterilisation process for closed containers is of sufficient Principle
quality not to impact negatively the sterility of the sterilised Dry heat sterilisation is a terminal sterilisation method based
items. on the transfer of heat to the articles to be sterilised. Heat
Sterilisation cycle may be transferred by means of convection, radiation or
Suitable sterilisation cycles are chosen to be compatible with direct transfer.
the items to be sterilised and the loading configuration. Equipment
Where air is displaced from the chamber by gravity, the items Dry heat sterilisation is carried out in an oven with forced air
to be autoclaved are designed to allow the removal of air and circulation or using other equipment specifically designed for
are arranged within the autoclave to prevent the formation of this purpose, e.g. a tunnel.
inaccessible air pockets. Where air is removed by vacuum Sterilisation cycle
cycles followed by steam pulses, it is assured that the items The steriliser is loaded in such a way that the specified or
are not affected by the evacuation process. For pressure- required temperature is achieved throughout the load.
sensitive products in closed containers, saturated steam Knowledge of the temperature within the steriliser during the
sterilisation may not be possible. Steam-air mixtures may be sterilisation cycle is obtained by means of temperature-
applied to the chamber in order to balance pressure sensing elements suitably placed in or on representative items
conditions inside the closed containers. Steam penetration is situated in the coolest part (as previously established) of the
assured by choosing suitable cycles to remove air from loaded steriliser. The time and temperature throughout each
porous loads or hollow bodies. Steam penetration is verified cycle is suitably recorded.
during cycle development by, for example, the use of
Cycle effectiveness
physical/chemical indicators, while the biological effectiveness
The reference conditions for this method of sterilisation are a
of the cycle is verified by the use of biological indicators
minimum of 160 °C for at least 2 h. Other combinations of
(5.1.2). Appropriate loading patterns are specified.
time and temperature may be used if it has been satisfactorily
Cycle effectiveness demonstrated that the process chosen delivers an adequate
The reference cycle for steam sterilisation is 15 min at and reproducible level of lethality when operated within the
121 °C in saturated steam determined in the coldest position established tolerances. The procedures and precautions
of the chamber. Product- and load-specific cycles, employed are such as to achieve an SAL equal to or less than
e.g. applying another combination of time and temperature, 10- 6 • Dry heat sterilisation processes are validated using a
may be adopted based on cycle development and validation. combination of temperature mapping and biological indicator
The minimum temperature acceptable for a steam studies (5.1.2).
sterilisation process is 110 °C. The minimum P0 , calculated
Dry heat at temperatures greater than 220 °C, for a validated
in the slowest-to-heat position of the load is not less than
time, is frequently used for depyrogenation of glassware.
In this case, demonstration of a 3 log 10 reduction in heat-
2023 Appendix XVIII V-A64 7

resistant endotoxin can be used as validation criteria and sterilising agents include hydrogen peroxide and peracetic
biological indicators will not be needed. acid.
Routine control Development and validation of sterilisation processes
Dry heat sterilisation cycles are monitored by determination Gas sterilisation is performed by exposure of the product to
of temperature profiles, at a minimum, in the coldest position the sterilising agent in a leak-proof chamber under specified
of the chamber. Time and temperature are recorded for each conditions.
cycle. A typical gas sterilisation process consists of 3 phases: (pre)
IONISING RADIATION STERILISATION conditioning, sterilisation and aeration. The parameters
Principle necessary for these phases to produce the required SAL are
Sterilisation by irradiation is achieved by exposure of the established during process development. A combination of
product to ionising radiation in the form of either gamma physical and biological methods is used to determine the
rays from a suitable isotopic source (such as cobalt 60), a optimum sterilisation conditions. The cycle shall not
beam of electrons energised by a suitable electron accelerator, compromise the functionality of either product or the
or X-rays resulting from bombarding a suitable target with container.
energised electrons. Ionising radiation may be used for the Sterilisation cycle
terminal sterilisation of finished dosage forms, the microbial Specialised equipment may be required for the monitoring of
inactivation of tissues and cells, or the sterilisation of temperature, humidity and gas concentration during both
materials or containers to be employed in aseptic processing. validation and routine operation.
Low-energy electrons may be used for the surface sterilisation Cycle effectiveness
of materials upon entry to isolators used in the preparation of Validation of microbiological performance shall confirm the
sterile products. effectiveness of the defined process for the product/load
Cycle effectiveness combination in the steriliser. The lethality of the cycle may
For this method of sterilisation, the reference absorbed dose be determined by using an appropriate approach: after time-
is 25 kGy. Other doses may be used if, during validation of graded exposures, the rate of inactivation CD-value) of the
the sterilising dose, it has been satisfactorily demonstrated test organisms can be established by construction of a
that the dose chosen delivers an adequate and reproducible survivor curve or by using a fraction-negative method.
level of lethality when the process is operated routinely within Biological indicators shall be shown to be, at a minimum, as
the established tolerances. The procedures and precautions resistant to the sterilising agent as the microbiological
employed are such as to achieve an SAL equal to or less than contaminants of the product to be sterilised. They shall be
10- 6 • Biological indicators may be required for the placed within the product at locations where sterilising
development and validation of the sterilisation of tissues and conditions are most difficult to achieve.
cell products. They may also be required for products with a The effectiveness of the process is dependent on a number of
potential to prevent spore inactivation. parameters, including gas concentration, temperature,
Routine control humidity, exposure time, load configuration and
During the sterilisation process, the sterilisation dose characteristics of the product and its packaging materials.
delivered is monitored using a dosimetry system, The effect on the process effectiveness of any change in one
measurements from which are traceable to national or more of these parameters shall be investigated.
standards. Routine control
GAS STERILISATION (VAPOR PHASE The relevant cycle process parameters (including the results
STERILISATION) of the biological indicator test) are recorded.
Principle MEMBRANE FILTRATION
Gas sterilisation of surfaces may be used for the sterilisation Principle
of primary packaging materials, equipment and some Membrane filtration is used for reduction of viable and non-
pharmaceuticals. viable particles in gases and fluid products that are not
It is essential that penetration by gas and moisture into the amenable to sterilisation by heat or irradiation. In contrast to
material to be sterilised is ensured, and that it is followed by other sterilisation methods, the principle of membrane
a process whereby the gas is eliminated under conditions that filtration is not inactivation but removal of microorganisms
have been previously established as sufficient to ensure that from the product. Removal is achieved by a combination of
any residues of gas or related transformation by-products are sieving and surface interaction.
below concentrations that could give rise to toxic effects Equipment
during product use. Membrane filters are available as flat stock (discs) in
Sterilising agents appropriate holders or as cartridges. Pore size ratings are
There are 2 main categories of gaseous sterilising agents as based on the correlation between microbial retention and
distinguished by their antimicrobial action: alkylating agents diffusion characteristics or bubble-point measurement. Many
and oxidising agents. factors contribute to the effectiveness of the filtration process,
Alkylating agents Alkylating agents are highly reactive e.g. shape, pore size, structure, surface properties, the
compounds and interact with many components, such as structure and arrangement of the filter unit, interaction of the
amino, sulfhydryl and hydroxyl groups in proteins and purine filter matrix with the product, applied pressure, flow and
bases in nucleic acids. duration of the process. Filter characteristics have to be
Ethylene oxide is an alkylating agent that is associated with determined in a product-specific validation. Suitable integrity
cytotoxic, carcinogenic and mutagenic effects. test procedures (e.g. diffusive flow measurement, bubble-
point determination or water-intrusion testing) are employed,
Oxidising agents Oxidising agents are highly reactive,
as recommended by filter manufacturers. Chemical and
toxic compounds. Such compounds currently used as
physical compatibility of the membranes with the product to
V-A648 Appendix XVIII 2023

be filtered and the conditions of the filtration process are Development and validation of aseptic assembly
demonstrated in development studies. The filter size is In order to maintain the sterility of the components and the
suitable for the volume of the product to be filtered and the product during assembly, careful attention needs to be given
bioburden. to the following:
For sterilisation of process gases, an appropriate frequency - environment;
for physical integrity testing is established. - personnel;
- critical surfaces;
Filtration effectiveness
- container/closure sterilisation and transfer procedures;
Microbial challenge tests with a suitable model system shall
- the maximum holding period of the product before filling
demonstrate the effectiveness of the filtration process. Where
into the final container.
testing with the product is not possible (e.g. due to the
antimicrobial properties of the product), a fluid that is Process validation includes appropriate checks on all of the
representative of the product shall be used, or the test above and also regular checks on the process, which are
conditions are modified. carried out by means of process simulation tests using
microbial growth media that are then incubated and
It is recommended that the filtration process is carried out as
examined for microbial contamination (media fill tests).
close as possible to the filling point.
In addition, a suitable sample of each batch of any product
Sterilisation of membrane filters that is aseptically processed is tested for sterility (2. 6.1).
Membrane filters may be sterilised off-line or in-line.
If sterilisation is off-line, steam penetration is verified and the Biological Indicators of Sterilisation
filter is suitably protected against contamination. (Biological Indicators and Related Microbial Preparations used in
The sterilised filter is aseptically assembled in the production the Manufacture of Sterile Products, Ph. Bur. general texts 5.1. 2)
line by means of a validated procedure. For in-line 1 INTRODUCTION
sterilisation, steam penetration throughout the filtration The use of biological indicators in this general chapter is
equipment is assured and the pressure difference across the intended to cover the sterilisation of finished products and
membrane is controlled to prevent damage to the membrane relevant related sterilisation processes i.e. sterilisation
itself. processes for items coming into direct contact with the final
Filtration process sterilised product. Other uses of biological indicators to
Sterilisation by membrane filtration is performed by passage validate the sterilisation of other non-terminal units is outside
of the product through a microporous membrane with a the scope of this general chapter.
nominal pore size not greater than 0.22 µm. Biological indicators are test systems containing viable micro-
The pre-sterilisation microbial contamination is determined organisms (usually spores of bacteria) that provide a defined
for each batch of product and process parameters are applied challenge to verify the required effectiveness of a specified
as established and validated in the development of the sterilisation process.
filtration process. Biological indicators are intended for the development and
Where multiple bioburden-reduction filters are used to validation of the sterilisation processes and not for routine
increase the efficacy of the filtration process, the filter closest monitoring unless otherwise stated in this general chapter.
to the filling point in the final container is characterised as The validity of the sterilisation process and the validity of the
the sterilising filter. biological indicators can be assured by the use of reduced
The sterility and integrity of the equipment downstream from sterilisation process conditions, whereby a small proportion of
the point of filtration, the qualified environmental conditions the micro-organisms within the biological indicator will be
and the validated aseptic procedures applied in the handling shown to survive. However, when the validated sterilisation
of the filtered product all contribute to preventing process is used, there will be no surviving viable micro-
recontamination of the product. This is addressed in the organisms (see section 3-1-2).
section on aseptic assembly. Bacterial spores are resistant forms of life, they can be
Routine control produced and standardised, and may be stored for long
Filtration processes are monitored by physical and periods of time under appropriate conditions.
microbiological determination of parameters established Commercially available biological indicators typically contain
during validation studies. These parameters include the a standardised population of spores of a suitable bacterium.
following: pre-sterilisation microbial contamination, pre- In cases where no suitable commercial biological indicators
filtration integrity test results, duration of filtration, volume are available to characterise the sterilising effect in the
filtered, differential pressure and post-filtration integrity test product or at a position difficult to penetrate by the sterilant,
results. custom-made biological indicators may be used. Such
ASEPTIC ASSEMBLY biological indicators can be prepared by inoculating a
standardised spore suspension onto or into the item or
Principle
product to be sterilised, such action may change the
The objective of aseptic assembly is to maintain the sterility
characteristics of the biological indicator.
of a product that is assembled from components, each of
which has been sterilised by one of the above methods. This A suspension of vegetative bacterial cells is used to validate
is achieved by using conditions and facilities designed to the bacterial retention capability of sterilising grade filters
prevent microbial contamination. when applied as a sterilisation step in an aseptic production
process.
Aseptic processing may include aseptic filling of products into
container/closure systems, freeze-drying under aseptic 2 BIOLOGICAL INDICATORS FOR STERILISATION
conditions, aseptic blending of formulations followed by PROCESSES
aseptic filling, and aseptic packaging. In addition to the physical sterilisation parameters, the
effectiveness of a sterilisation process as described in general
chapter 5.1.1 is dependent on a large number of variables,
2023 Appendix XVIII V-A649

which may include, but is not necessarily restricted to, the bacterial spores and must be compatible with the chosen
number and resistance of contaminating micro-organisms, sterilisation process (e.g. strips of filter paper in glassine
penetration of the sterilant, time, temperature, concentration, envelopes are frequently used for steam and ethylene oxide,
pH, moisture content, and the chemical composition of the while metal discs packaged in non-woven fibre envelopes are
product or item being sterilised. used for hydrogen peroxide vapour). After exposure to the
To validate a sterilisation process, physical conditions are sterilisation process, the carrier is aseptically handled
chosen that are expected to sterilise the items in the load to according to the manufacturer's instructions, transferred to a
achieve a sterility assurance level (SAL) equal to or less than suitable culture medium and incubated for a sufficient period
1o- 6 as described in general chapter 5.1.1. In a physical of time at the appropriate temperature.
validation process it is demonstrated that these conditions are 2-1-2 Self-contained biological indicators
delivered homogeneously to all parts and positions of the A self-contained biological indicator may be, for example:
load. It is the aim of biological validation to demonstrate the - a system consisting of an inoculated carrier and a
correlation between the predicted effect of the physical container (e.g. ampoule) with a nutrient medium suitable
conditions applied during the process and the observed for the test micro-organism used; the system is designed
biological effect on biological indicators. Using process in such a way that the sterilising agent comes into contact
parameters that have been demonstrated to deliver the with the inoculated carrier (e.g. through a tortuous path
required biological effect will ensure sterility of the resulting or a filter) while the growth-promoting properties of the
product in routine processing. nutrient medium are not adversely affected by the
The selection of the type of biological indicator used will sterilisation process. After sterilisation, the carrier is
depend on: brought into contact with the nutrient medium by simple
- the nature of the sterilising agent (e.g. heat, gas or manipulation. This type of biological indicator system
radiation); may be used to characterise moist heat sterilisation
- the expected effectiveness of the treatment (e.g. the Fphys processes including assurance of the penetration of steam
calculated from the process parameters); into the system;
- the process conditions (e.g. temperature, time, relative - a container (e.g. ampoule) of a population of the test
humidity, gas concentration, radiation dose); micro-organism in an appropriate nutrient medium. After
- the characteristics of the pharmaceutical product or item sterilisation, the container is incubated without further
(e.g. product in final container, packaging material, manipulation. This type of biological indicator is sensitive
utensils such as tubes or pumps) to be sterilised. only to an exposure time and temperature and may be
In the development of a sterilisation process, the load and the used primarily to monitor sterilisation of aqueous fluids.
product should be assessed to determine the most difficult In order to facilitate detection of growth, the medium
position to sterilise (e.g. cold spots, vial-stopper interface, may contain an indicator (e.g. a pH indicator).
difficult to penetrate areas). When choosing the optimum Self-contained biological indicators might not be suitable for
biological challenge to a sterilisation process, the conditions the validation of certain sterilisation processes.
in the most difficult position to sterilise in the load and the 2-1-3 Characterised spore suspensions
product should be simulated as closely as possible. Characterised spore suspensions consist of a defined
Spores inoculated into a product or onto surfaces are known population of bacterial spores, prepared from a clearly
to react differently to sterilising conditions as compared to characterised and suitably maintained strain of a spore-
biological indicator units. In these cases, commercially forming bacterial species (e.g. of the genera Bacillus or
available biological indicator units may not be suitable to test Clostridium) in a stable suspension.
sterilisation effectiveness and an inoculated test product/item 2-1-4 Custom-made biological indicators
prepared from a well-characterised spore suspension may be Custom-made biological indicators are test items (e.g. rubber
a better model to evaluate the effectiveness of the sterilisation stoppers), or products, inoculated with a suitable test micro-
cycle. organism, usually from a characterised spore suspension but
2-1 DESCRIPTION OF BIOLOGICAL INDICATORS also from spore suspensions prepared from isolates from
FOR STERILISATION PROCESSES environmental monitoring or other microbiological testing
Depending on the process to be characterised, a suitable using a well-defined procedure designed to give satisfactory
biological challenge may consist of biological indicators sporulation. The D-value (time for 90 per cent reduction of
presented as test micro-organism suspensions, inoculated micro-organisms under the stated conditions) and, when
carriers, or self-contained biological indicators. The user appropriate, the z-value (see section 3-1-1) of the spore
must establish a high level of confidence in the suspension must be determined. Also the D-value and
manufacturer's compliance to quality standards for the z-value (if appropriate) of the spores of the inoculated test
biological indicator (e.g. by means of auditing) in order to items/ products must be determined as this may be different
rely on the characteristics stated by the manufacturer (see from the spores in suspension.
section 2-2). Alternatively, the labelled characteristics of After exposure to the sterilisation cycle, the custom-made
biological indicators shall be verified by the user or by an biological indicator is enumerated or tested for the
independent, contract laboratory that is formally approved by presence/absence of surviving test micro-organisms using a
the user. For custom-made biological indicators (see validated, appropriate microbiological technique.
section 2-1-4), the characteristics shall be verified by the user
2-2 QUALITY REQUIREMENTS FOR BIOLOGICAL
or by a contract laboratory.
INDICATORS
2-1-1 Inoculated carriers The following are required to be known by the user per
Inoculated carriers consist of a defined population of delivery of each batch:
bacterial spores inoculated into or onto a suitable carrier, and - genus and species of the micro-organism (including the
in most cases, in a protective envelope. The type of carrier type culture collection number where applicable);
(and the envelope if used) may influence the resistance of the - unique reference (e.g. batch number);
V-A650 Appendix XVIII 2023

- logarithm of the viable spore count expressed to 3 BIOLOGICAL INDICATORS FOR HEAT
1 decimal place in scientific notation; STERILISATION
- recovery method used; 3-1 PARAMETERS OF BIOLOGICAL INDICATORS
- type of carrier; FOR HEAT STERILISATION
- type of packaging (e.g. envelope); 3-1-1 z-Value
- composition of the recovery medium, if needed Sterilisation processes can be operated at temperatures lower
(e.g. in case of self-contained biological indicators); than the standard 121 °C (for longer exposure times) or at
- type of indicator (e.g. pH indicator) for growth, if higher temperatures (for shorter exposure times). The z-value
relevant; (the temperature difference that leads to a 10-fold change in
- type of sterilisation process(es) and the conditions for the D-value of the biological indicator) is used to compare
which the biological indicator has been characterised; the efficacy of 2 cycles operated at different temperatures.
- resistance (D-value) per batch of finished biological For a z-value determination, the D-value must be determined
indicator against the specified sterilisation processes at 3 or more temperatures. The intended process
throughout the labelled shelf-life; the D-value should be temperature should be within the range of the
stated in applicable units (e.g. time or dose) and 3 temperatures. The log 10 of the D-value is plotted against
expressed to 1 decimal place, together with a 95 per cent the temperature in degrees Celsius. The z-value is equal to
confidence interval if feasible; the negative reciprocal of the slope of the best-fit linear curve
- method (inactivation kinetics or fraction negative method) as determined by log 10-linear regression analysis.
used to determine the resistance (D-value); parameters to
verify e.g. exposure conditions, number of replicates 3-1-2 Establishment of validation cycle
tested, medium and incubation conditions used for The characteristics of the sterilisation process (e.g. time-
recovery after exposure, etc.; temperature combination, level of sterility assurance or F0
- the z-value (where relevant) for the biological indicator required) are the basis for the choice of the biological
stated in temperature units expressed to 1 decimal place indicator (type of biological indicator, test micro-organism,
in scientific notation, including the range of temperatures and initial viable count).
used to determine the z-value; Inactivation of micro-organisms under sterilising conditions
- the storage conditions and the expiry date. can be described by lethality kinetics and statistical
probabilities. For a number of biological indicator units with
2-2-1 User requirement specification (URS)
an initial population of N 0 micro-organisms per unit and a
The particular sterilisation process (moist heat, dry heat, gas,
given D-value, the exposure time in minutes where all units
or ionising radiation) is considered as the basis for the choice
are expected to carry survivors (average of 100 surviving
of the biological indicator. This choice includes the selection
spores per unit) is calculated by equation (1).
of the test micro-organism, the type of biological indicator
(inoculated carrier, self-contained, or custom-made), the
t, = D x (log1oN0 - 2) (I)
D-value, and the initial spore count. Moreover, the resistance
of the test strain is suitable for the particular sterilisation
method and is great compared to the resistance of micro- t, survival time
organisms potentially contaminating the product.
The exposure time in minutes where all units are expected to
2-2-2 Quality control
be inactivated (average of 10-4 surviving spores per unit) is
Quality control for biological indicators consists of testing for
calculated by equation (2).
purity, identity and estimation of the number of viable cells.
The biological indicator should be compliant with the URS.
(2)
Users employing biological indicators outside of the
manufacturer's labelled recommendations should thoroughly
characterise the biological indicators for the particular kill time
sterilisation process.
The objective of a validation study is to demonstrate that the
Purity
sterilisation effectiveness anticipated from the physical
Examination of the micro-organisms on a suitable culture process parameters is equivalent to the biological sterilisation
medium incubated under appropriate conditions shall not effectiveness. As part of that objective, the exposure time
show any evidence of contamination. during validation tvb shall not exceed tk. If a too high tv1 is
Identification chosen, even a relatively large increase in the D-value would
Colony morphology and homogeneity of the population are still result in biological indicator units with no surviving
verified, as appropriate. micro-organisms. In this case, the suboptimal sterilising
Viable count conditions would not be detected. It is considered reasonable
to choose a fvt not higher than required to expect 1 in
The viable count is performed according to the
1000 biological indicator units having surviving micro-
manufacturer's instructions or by any other validated
organisms. However, too short a tvt shall not be chosen. If a
method.
tv1 is chosen such that 50 per cent of the biological
2-2-3 Suitability for purpose indicator units have surviving micro-organisms, changes in
The user shall ensure that the biological indicator is the sterilising conditions (e.g. time, temperature) could still
inactivated to the expected survival rate by the particular result in 100 per cent of the biological indicator units having
range of sterilisation conditions used. surviving micro-organisms, and the test would be
meaningless. For these reasons, a tv1 is chosen such that a
theoretical survival rate between 10-1 and 10-3 is expected,
thus:
2023 Appendix XVIII V-A651

D x (logwNo + I) S tv1 S Dx (logwNo + 3) (3) inactivated by 48 log 10 scales. For dry-heat sterilisation
processes, z-values of about 20 °C are typically assumed in
calculations of equivalence of cycle effectiveness (Fw
In general, biological indicators are subjected to the intended
calculations). FH is the equivalent time in minutes at a
sterilisation process. However, for highly effective sterilisation
temperature of 160 °C delivered by the sterilisation process
processes, the calculated effectiveness of the cycle may be
to the product in its final container. For a biological indicator
such that the tk is exceeded by a wide margin. In such
with a D 1 wc-value of 5 min, the D 150 c-value would be
instances, biological validation is carried out with reduced
about 16 min, and inactivation in the reference cycle would
sterilisation cycles. Such reduced cycles may be shorter in
be 7 .5 log 10 scales. The use of a sterilisation process at a
time (e.g. half cycle) or be performed at a lower temperature.
temperature reduced from the target temperature by 10 °C
In the latter case the z-value for the test micro-organism
would give an expected 1 in 30 biological indicator units
under the actual sterilising conditions shall be known.
having surviving micro-organisms.
A reduced cycle is chosen such that the temperature is not
more than 1 z-value below the reference sterilisation process 3-3-1 Test micro-organisms
temperature. Biological indicators of an appropriate resistance Spores of Bacillus atrophaeus (e.g. ATCC 9372,
for that cycle show an expected micro-organism survival rate NCIMB 8058, NRRLB-4418, or CIP 77.18) have been
within a window between the lower tv 1 and the tk (see found to be suitable for use as biological indicators for dry
equation (3)). A decision not to perform this test must be heat sterilisation processes performed at temperatures
justified. between 160 °C and 180 °C. Where a test micro-organism
other than Bacillus atrophaeus is used, to ensure its suitability,
Depending on the D-value of the test micro-organism and
the resistance of the test micro-organism for the sterilisation
the tvt chosen, biological indicators having surviving micro-
process is evaluated as described in section 3-1-2
organisms can be expected with a low frequency (not more
than 1 in 10). If it can be demonstrated that the frequency of 4 BIOLOGICAL INDICATORS FOR GAS
biological indicators having surviving micro-organisms is STERILISATION
within the expected range and is not due to inappropriate The use of biological indicators is necessary for the
sterilising conditions, the process can be accepted. development, validation and monitoring of all gaseous
Following a full sterilisation cycle, all biological indicators in sterilisation processes. Gas sterilisation is a multi-factorial
a validation study must be inactivated, thereby proving at process: gas concentration, humidity, temperature, time,
least a 106 reduction in micro-organisms. It can then be surface characteristics interact in a complex manner.
concluded, from the resistance of the spore preparation used, A number of gas sterilisation processes are currently used,
that the process has delivered sufficient lethality to achieve including ethylene oxide, hydrogen peroxide and peracetic
the required sterility assurance level. acid or combinations of the latter.
3-2 BIOLOGICAL INDICATORS FOR MOIST HEAT Gas surface disinfection is widely used for medical devices,
STERIUSATION isolators, chambers, etc. Use for such purposes is outside the
scope of the European Pharmacopoeia but the use of
3-2-1 Test micro-organisms
biological indicators as described in this general chapter may
Geobacillus stearothennophilus is the most widely accepted
assist in the validation of such disinfection processes.
biological indicator micro-organism for moist heat
sterilisation processes. Reported D 121 c-values for its spores 4-1 TEST MICRO-ORGANISMS
are in the range of 1.5 min to about 4.5 min, depending on 4-1-1 Ethylene oxide sterilisation
sporulation conditions, the carrier material on which the The use of spores of Bacillus atrophaeus (e.g. ATCC 9372,
spores are inoculated, the primary package surrounding the NCIMB 8058, NRRL B-4418, or CIP 77.18), or other
inoculated carrier, and the environment during sterilisation. strains of micro-organism having demonstrated equivalent
Strains ATCC 7953, NCTC 10007, CIP 52.81, performance, is recommended for ethylene oxide sterilisation.
NCIMB 8157 and ATCC 12980 (equivalent to NRRL B- The number of viable spores is greater than or equal to 10 6
4419) have been found to be suitable. Other strains may be per carrier. Test micro-organisms shall have D-values
used, provided equivalent performance has been relevant to the process to be validated. These biological
demonstrated. It is recognised that a 105 or 106 population indicators are used routinely during each sterilisation cycle
of Geobacillus stearothennophilus may not be suitable for thus allowing the effectiveness of the process to be checked.
sterilisation processes delivering an F0 between 8 and 15, 4-1-2 Other processes
therefore a lower spore number (i.e. 10 3 or 104) or a It is the responsibility of the user to define the sterilisation
different test micro-organism may be used. Where a test cycle and the suitability of any biological indicator used.
micro-organism other than Geobacillus stearothennophilus Geobacillus stearothennophilus has been found suitable for
(e.g. Bacillus subtilis ATCC 35021) is used, the resistance of vaporised hydrogen peroxide processes.
the test micro-organism is evaluated to ensure its suitability
for the process. 5 BIOLOGICAL INDICATORS FOR IONISING
RADIATION STERILISATION
3-3 BIOLOGICAL INDICATORS FOR DRY HEAT
Unless otherwise indicated, biological indicators are not
STERIUSATION
generally considered necessary for validation of the sterilising
The reference conditions are stated in general chapter 5.1.1.
dose for radiation sterilisation. The use of biological
Heat transfer is less effective with dry heat than with steam,
indicators may however be required for the development and
and temperature distribution in dry heat sterilisers is less
validation of ionising radiation sterilisation e.g. of tissues, cell
homogeneous compared to steam sterilisers.
preparations or other specific cases (e.g. products with a
For example, biological indicators available for dry heat potential for spore protection).
sterilisation have D 160 c-values within a range of 1 to 5 min.
When exposed to the reference cycle of 2 hat 160 °C, a
5-1 TEST MICRO-ORGANISMS
Spores of Bacz7lus pumilus (e.g. ATCC 27142, NCTC 10327,
biological indicator with a D 160 c-value of 2.5 min would be
NCIMB 10692 or CIP 77.25) or other strains of micro-
V-A652 Appendix XVIII 2023

organisms having demonstrated equivalent or better The F-value of a heat sterilisation process (F0 for moist heat
performance are recommended. sterilisation or FH for dry heat sterilisation) is the lethality
expressed in terms of the equivalent time in minutes at the
6 MICROBIAL PREPARATIONS FOR
reference temperature delivered by the process to the
STERILISATION GRADE FILTRATION
sterilisation load, with reference to micro-organisms
As stated in general chapter 5.1.1, certain products that
possessing the relevant theoretical z-value in Table 5.1.5-1.
cannot be sterilised in their final container may be sterilised
by a filtration process. In contrast to the biological indicators The total F of a process takes into account the heating and
discussed in the previous sections, which assess kill-based cooling phases of a cycle and can be calculated by integration
sterilisation, the biological challenge assesses the retention of of lethal rates with respect to time at discrete temperature
micro-organisms by the filters. intervals above the minimum temperature specified in
Table 5.1.5-1.
To validate the sterilisation process, it must be demonstrated
that the filtration process (usually in a scaled-down model) is The following mathematical relationships apply:
capable of completely retaining a microbial challenge of at Fo = Dl21 (log 10 No - log 10 N)
least 107 CPU per square centimetre of effective filter surface
using a suitable test micro-organism. This test should mimic
the actual filtration process as closely as possible. Where
feasible, the test is carried out in the product using the
specified filtration conditions. If this is not possible, D 121 D-value of the reference spores (5.1.2) at 121 "C;
e.g. due to the antimicrobial properties of the product, a D 160 D-value of the reference spores (5.1.2) at 160 ''C;
medium as similar as possible to the product must be used in N0 initial number of viable micro-organisms;
N final nwnber of viable micro-organisms.
the test.
6-1 TEST MICRO-ORGANISMS
For processes using a filtration system with a nominal pore Table 5.1.5.-1 - Parameters for moist heat and dry heat
size not greater than 0.22 µm, a suspension of Brevundimonas sterilisation
diminuta (ATCC 19146, NCIMB 11091 or CIP 103020) is Reference Minimum
Theoretical z-
recommended. The Brevundimonas diminuta suspension must Sterilisation F temperature temperature
value CC)
be prepared in order to achieve predominantly single cells of (°C) CC)
the smallest possible size. Other micro-organisms, for
Moist heat Fo 10 121 110
example natural flora isolated from the product or process in
question, may be used if presenting a stronger challenge to Dry heat FH 20 160 140
the sterile filtration system than Brevundimonas diminuta.
For filtration systems with a nominal pore size of 0.1 µm or For both dry and moist heat sterilisation cycles, the relevant
less, a suspension of Acholeplasma laidlawii (ATCC 23206) F-value is used to demonstrate that the required sterility
may be used. assurance level of equal to or less than 10-6 is consistently
Methods of Sterilisation achieved.
(Application of the F Concepts to Heat Sterilisation Processes,
Ph. Bur. general texts 5.1.5)
The following chapter is published for information.
INTRODUCTION
Heat sterilisation can be differentiated into 2 types: moist
heat sterilisation using saturated steam or water heated to the
sterilisation temperature; and dry heat sterilisation using hot
air with a moisture content so low as to have an insignificant
biological activity.
DEFINITIONS
The D-value (or decimal reduction value) is the time in
minutes required at a defined temperature to reduce the
number of viable test micro-organisms by 90 per cent. It is
only of significance under precisely defined experimental
conditions.
The z-value is the change in temperature in degrees Celsius
required to alter the D-value by a factor of 10 (the z-value
relates the resistance of a micro-organism to changes in
temperature). The z-value is calculated using the following
equation:

T2 - T1
z=-------
log10D1 - log,0D2

D1 D-value of the micro-organism at temperature T1 ;

D2 D-value of the micro-organism at temperature T2 ;
T temperature.
2023 Appendix XIX B V-A653

Coloured glass Is obtained by the addition of small

Appendix XIX amounts of metal oxides, chosen according to the desired
spectral absorbance.
A. Containers Introduction Neutral glass Is a borosilicate glass containing significant
amounts of boric oxide, aluminium oxide, alkali metal oxides
(Ph. Bur. general texts 3.2)
and/or alkaline earth oxides in the glass network. Due to its
This Appendix provides requirements, guidance and informatwn composition, neutral glass has a high hydrolytic resistance
on containers for pharmaceutical use. Additional guidance is and a high thermal shock resistance.
proiided in a number of British Standards. Attention is drawn in Soda-lime-silica glass Is a silica glass containing alkali
particular to British Standards 1679-5: 1973, 1679-6: 1994, metal oxides, mainly sodium oxide, and alkaline earth oxides,
1679-7: 1968 and 1679-8: 1992. The expresswn 'tamper-evident mainly calcium oxide, in the glass network. Due to its
container' means a closed container fitted with a device that composition, soda-lime-silica glass has only a moderate
reveals irreversibly whether the container has been opened, hydrolytic resistance.
whereas, the expression 'tamper-proof container' means a closed
The hydrolytic stability of glass containers for pharmaceutical
container in which access to the contents is prevented under normal
use is expressed by the resistance to the release of soluble
conditions of use. The two terms are considered to be synonymous
mineral substances into water under the prescribed
by the European Phamzacopoeia Cornmisswn.
conditions of contact between the inner surface of the
A container for pharmaceutical use is an article that contains container or glass grains and water. The hydrolytic resistance
or is intended to contain a product and is, or may be, in is evaluated by titrating released alkali reacting ions.
direct contact with it. The closure is a part of the container. According to their hydrolytic resistance, glass containers are
The container (see General Notices section 1.3) is so classified as follows:
designed that the contents may be removed in a manner - type I glass containers: neutral glass, with a high
appropriate to the intended use of the preparation. hydrolytic resistance due to the chemical composition of
It provides a varying degree of protection depending on the the glass itself;
nature of the product and the hazards of the environment, - type II glass containers: usually of soda-lime-silica glass
and minimises the loss of constituents. The container does with a high hydrolytic resistance resulting from suitable
not interact physically or chemically with the contents in a treatment of the inner surface;
way that alters their quality beyond the limits tolerated by - type III glass containers: usually of soda-lime-silica glass
official requirements. with only moderate hydrolytic resistance.
Single-dose container A single-dose container holds a The following italicised statements constitute general
quantity of the preparation intended for total or partial use recommendations concerning the type of glass container that
on 1 occasion only. may be used for different types of pharmaceutical
Multidose container A multidose container holds a preparations. The manufacturer of a pharmaceutical product
quantity of the preparation suitable for 2 or more doses. is responsible for ensuring the suitability of the chosen
Well-closed container A well-closed container protects container.
the contents from contamination with extraneous solids and Type I glass containers are suitable for most preparations whether
liquids and from loss of contents under ordinary conditions or not for parenteral administration.
of handling, storage and transport. Type II glass containers are suitable for most acidic and neutral,
Airtight container An airtight container is impermeable to aqueous preparations whether or not for parenteral administratwn.
solids, liquids and gases under ordinary conditions of Type III glass containers are in general suitable for non-aqueous
handling, storage and transport. If the container is intended preparations for parenteral administration, for powders for
to be opened on more than 1 occasion, it must be so parenteral administration (except for freeze-dried preparations)
designed that it remains airtight after re-closure. and for preparations not for parenteral administration.
Sealed container A sealed container is a container closed Glass containers with a hydrolytic resistance higher than that
by fusion of the material of the container. recommended above for a particular type of preparation may
Tamper-evident container A tamper-evident container is generally also be used.
a closed container fitted with a device that reveals irreversibly The container chosen for a given preparation shall be such
whether the container has been opened. that the glass material does not release substances in
Child-proof container A container that is fitted with a quantities sufficient to affect the stability of the preparation
closure that prevents opening by children. or to present a risk of toxicity. In justified cases, further
detailed information may be necessary to assess the impact
on chronic use and for vulnerable patient groups.
Preparations for parenteral administration are normally
B. Glass Containers for Pharmaceutical presented in colourless glass, but coloured glass may be used
for substances known to be light-sensitive. Colourless or
Use coloured glass is used for the other pharmaceutical
(Ph. Bur. method 3.2.1) preparations. It is recommended that all glass containers for
liquid preparations and for powders for parenteral
Glass containers for pharmaceutical use are glass articles
administration permit the visual inspection of the contents.
intended to come into direct contact with pharmaceutical
preparations. The inner surface of glass containers may be specially treated
to improve hydrolytic resistance, to confer water-repellancy,
Colourless glass Is highly transparent in the visible
etc. The outer surface may also be treated, for example to
spectrum.
reduce friction and to improve resistance to abrasion.
V-A654 Appendix XIX B 2023

The outer treatment is such that it does not contaminate the HYDROLYTIC RESISTANCE
inner surface of the container.
Table 3.2.1.-1. - Types of glass
Except for type I glass containers, glass containers for
pharmaceutical preparations are not to be re-used. Type of container Test to be performed
Containers for human blood and blood components must Type I and type II glass containers (to Test A (surface test)
not be re-used. distinguish from type III glass
containers)
PRODUCTION Type I glass containers (to distinguish Test B (glass grains test) or test C
When glass containers for pharmaceutical use are from type II and type III glass (etching test)
manufactured under stressed conditions (e.g. temperature- containers)
time profile) and/or are placed in contact with particularly Type I and type II glass containers (if Tests A and B, or tests A and C
there are doubts whether the high
aggressive pharmaceutical preparations, they may undergo hydrolytic resistance is due to the
delamination, i.e the separation of the inner glass surface into chemical composition or to the surface
thin layers called lamellae or flakes. Glass delamination may treatment)
be the result of a chemical attack that occurs according to
well-known glass corrosion mechanisms, such as dissolution The test is carried out by titration of the extraction solutions
by hydrolysis and ion exchange (leaching) as a function of obtained under the conditions described for tests A, B
the pH. The process of interaction between the glass surface and C. Test C is performed if there are uncertainties whether
and the pharmaceutical preparation requires incubation time, the container is type I or type II.
and flaking may only become visible a number of months
EQUIPMENT
after filling.
- An autoclave or steam steriliser capable of withstanding a
Several risk factors are known to increase the propensity of a pressure of 2.5 x 105 N/m2 (equivalent to
glass to delaminate. The chemical composition of the 0.25 MPa = 2.5 bar) or more and capable of carrying out
pharmaceutical preparation, the presence of buffers like the heating cycle described under Autoclaving process.
citrate or phosphate, which are known to corrode glass, and Preferably it is equipped with a constant-pressure
the ionic strength of the liquid medium may all strongly regulator or other suitable means in order to maintain the
favour delamination. The manufacturing process of the temperature at 121 ± 1 °C. The autoclave vessel is
container, chemical treatments of the inner surface, and equipped with a heating device, a thermometer integrated
terminal sterilisation and processing at the pharmaceutical in the autoclave, a pressure gauge, a vent cock (for
filling lines are other important risk factors to be considered. manually operated autoclaves only) and a tray of
It is recommended that the user of the container assesses the sufficient capacity to accommodate, above the water level,
compatibility of the glass container and the pharmaceutical the number of containers needed to carry ou