Quality Control

And
Quality Assurance

Course code :512

Theoretical and practical Book

For
Pharmacy Students

By
Prof. Dr. Khadiga M. Kelani

1
Analytical Chemistry
Department

2
 Contents

Aim of course
Introduction
Definition
Types of quality standards for laboratories
Drug standards
Sampling
Glossary of terms
Analysis of pharmaceutical products
Documentation
Total analytical validation
Method validation
Data elements required for assay validation
Reference standards
Drug stability
Degradation and indicating assay
methods(SIAMs)
Practical part
Contents
references

3
 AIM OF THE COURSE

The aim of this course is to provide pharmacy students with

the basic, principles, theoretical aspects and. practical
experience of different aspects (of quality control and

quality assurance and their applications in the field of drug

analysis.

This course handles chemical aspects of total QC / QA it is

run p.arallel with a G.M.P. course that is thought by the

Dept. Of Pharmceutical Technology. Biological aspects

(pharmacological and micropiological) _are prerequisites.

Chemical· aspects of QC/QA of raw materials; single
components and multicomponent. dosage forms are discussed.
Stability and expiration dating are considered.

· The .approach is a hybrid functional group methodology

one·. Further the organizational aspects, introduction of new
drugs and their selection as well as documentation are
presented and stressed.

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 Introduction

The word Quality has several dictionary definitions According

to Webster's.

· Quality is:

1- Something inherent and distinctive.

2- A usually high level of merit or superiority.

3- Degree of excellence.

Technically, Quality of product may be defined in several

ways:

*"Quality is conformance to requirements to goodness"

* "Conformance to use"

ISO Definition:

"The totally of features and characteristics of a product that

bear on its ability to satisfy stated or implied needs"

For a pharmaceutical productquality means that this product

should be safe and effective.

The following characteristics and features of drug that

determine its quality:

1- Identity : The correct active ingredient with potentially

harmful substances.
2- Purity : The drug is not contaminated with potentially harmful
substances.

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3- Potency: The correct amount of active ingredient is present,
usually between 90 and 110 percent of the labeled amount.
4- Uniformity: Consistency, color, shape and size of the dosage
form do not vary.
5- Bioavailability: Bioavailability refers to the speed and
completeness with which an administered drug enters the
blood stream. This must be consistent to provide a predictable
therapeutic result. Drug bioavailability differences exist
between manufactures of the same product. Therefore, careful
evaluation of generic drugs may be necessary before purchase
and use.
6- Stability: The activity of the drug is ensured for the period of
time stated on the product label, that is, until the expiration
date.
7- Pharmacopoeial standard: A drug is of good quality if its
characteristics meet the standards described in a widely
accepted pharmacopoeia such as the British Pharmacopoeia
(IP), or United States Pharmacopeia (USP).

Definition:

Quality Assurance (QA): is the sum total of all organized

arrangements made with the objective of ensuring that
medicines are of the quality required for their intended use.
As its name implies, QA assures that quality will be achieved.
It is about putting things in place in advance so that
everything goes according to a plan and there are no problems

6
with the quality of the final product. It is about preventing
errors.
Good Manufacturing Practice (GMP): IS that part of
Quality Assurance which ensures that products are
consistently produced and controlled to the quality standards
appropriate for their intended use and the legal requirements.
GMP is thus concerned with both production and quality
control matters.
Quality Control (QC): Is that part of GMP which is
concerned with sampling, specifications and testing, and with
the organization, documentation and release procedures which
ensure that the necessary and relevant test are actually carried
out and that materials are not released for use, or products
released for sale or supply, until their quality has been
judged to be satisfactory.

The Pharmaceutical Industry has traditionally focused upon

the application of Good Manufacturing Practice (GMP) to
achieve quality. Only relatively recently has the
pharmaceutical industry begun to implement quality
management systems (QMS). Therefore GMP compliance is
not sufficient and should be incorporated into an overall
Quality Management Systems. GMP ensures that regulatory
requirements are met while QMS ensures efficiency in
achieving quality.

Traditionally in the pharmaceutical industry, the quality

7
function is divided into two parts, quality control arid quality
assurance. Quality Control centers on testing products to
assure their compliance to specification. It is in general
concerned with evaluating events from the past. FDA has
recognized this deficiency with the often-quoted philosophy
that "you cannot test quality into a product" Quality assurance
· is focused on building quality into a product through
activities like validation, process and environmental control,
and documentation. Quality assurance is in general concerned
with events in the present. Quality management is about the ·
future, about prevention, and management's role in improving
the quality system.

The relative relationships between Quality Assurance (QA),

Good Manufacturing Practice (GMP) and Quality Control
(QC) are shown in the following figure.

QA
GMP
QC

The purpose of quality assurance systems is to make certain that

each drug reaching a patient is safe, effective, and of standard
quality.

8
The international organization of standardization (ISO)
developed ISO 9000 family of standards. These standards
represent an international agreement on good management
practices with the aim of ensuring that the organization can ·
deliver the product or services that meet the client's quality
requirements.

The family of standards is generic, business focused standard

which supports the effective management of quality to an
internationally recognized level of best practice. It is flexib1e-
in—that it-specifies what is to· be-achieved but allows each
company freedom to determine, and justify; how these
requirements are achieved. In contrast, GMP is 'an industry-
specific standard prescribing what must be done to. ensure product
safety and efficacy. Thus, ISO 9001 benefits business by ensuring
the quality of the management system, while GMP ensures that
regulatory requirements are met. it is important to understand that
the ISO standards are complementary - not alternative -to GMP.

Quality Assurance:

The requirements and objectives of Quality Assurance are as

follows:

Product designs a development: Medicines are designed

and developed in such a way that they can be produced to
comply with the quality requirements.

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Specifications for Production: Good Manufacturing
Practices are clearly specified and adhered to the production
environment and services to the production operation are
monitored.

Deviations are adequately recorded, investigated, and

responded to Control of starting and packaging materials;

Control of intermediate materials.

Control of finished product: No product is sold or

supplied. until responsible pharmacist has ensured that ea.ch
batch has been produced and controlled in accordance with
legal and · other requirements.

Control of storage and distribution: Medicines are

stored, handled and distributed so that quality is maintained
throughout their shelf life.

Good Laboratory Practices: are clearly specified and

adhered to

Self-inspection programme: The Quality Assurance

system is regularly audited by self-inspection for effectiveness
and applicability.

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Figure: Elements of Quality Control

Quality Control:

Each holder of a manufacturing authorization should have a

quality control department (except for a holder performing
only a fraction of the manufacturing process under a
contract).

The independence of quality control from production is

considered fundamental. The quality control department
should be independent from other departments and under the
authority of a person with· appropriate qualifications and
experience, who has one or several control laboratories at his
disposal.

The quality control department. must have adequate

resources.

This means: Adequate resources, the QC department must have

adequate resources to carry out its responsibilities e.g. Adequate

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space, qualified personnel and approved documented procedures for
all laboratory operations.

Types of quality standards for .laboratories:

Fortunately a number of organizations have already. developed

and published standards for quality systems which are relevant
to laboratories. e,g .

- The international Organization for Standardization (ISO) ·

has produced a range of standard and guidance relevant to
laboratories. ISO 9000 standards, approved in 1987 by ISO
technical committee 176. There are five documents. Published
as the ISO 9000 series contained two guidelines and three
standards. These documents are as follows: -

ISO 9000 :.a guideline for determining which contractual.

Standard to use.

ISO 9001 : the contractual standard for companies that design

and service what they manufacture

ISO 9002 : the contractual standard for companies that

manufacture but do not design what they manufacture.

ISO 9003 : the contractual standard for companies that

assemble and test products designed and manufactured elsewhere.

ISO 9004 : a guideline for a quality system somewhat more

comprehensive than those described in the contractual standards.

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ISO 9003 : ·the contractual standard for Companies that
assemble and test products designed and manufactured ·
elsewhere.

ISO 9004 : a guideline for a quality system somewhat more

comprehensive than those described in the contractual standards

The pursuit of quality is being approached through the concept of

total quality Management (TQM) and continuous improvement ,
whereby .management and labor join .forces to build quality into
Products while helping · to assure the company's financial
success: This changed emphasis is directed toward defect .
Prevention. (Proactive) rather than defect detection (after the fact)

Quality Assurance (QA) and Quality Control (QC) develop

and follow standard Operating Procedures. (SOPs) directed toward
assuring the quality, safety, purity ·and effectiveness of the drug
supply.

Total Quality Management (TQM): it is the aspect of

management function that determines and implements the
quality policy i.e. the overall directions of an organization
regarding quality as formally expressed and authorize by top
management

TQM deals with:

- Establishing .a customer focused organization.

- analyzing
. and improving
. ., work
.. processes to improve efficiency
and reduce waste

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- designing and establishing ·quality in products and
processes .

- Providing a leadership style of management which support

and cultivates the one team approach

- Using statistical methods and other tools to analyze and solve

problems, identify solutions and measure improvement.

-Encouraging new ideas and rewarding, success.

- Creating a- structure and climate which reinforces quality

Improvement and customer service.

Quality Assurance (QA) :

It is all those Planned and systematic actions necessary to

·provide adequate confidence· that a product or – service will
satisfy given requirements for quality. The association of
official analytical chemists (AOAC) proposed an easier
definition, planned activities designed to ensure that the quality
control activities are being properly implemented.

Manufacturing and ·marketing of safe effective products of the

highest quality is the main objective of quality assurance
quality assurance. Quality assurance therefore, incorporates
both good. manufacturing practice. (GMP). quality control and
other factors such as product design and development.

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 The ultimate goals of drug product quality assurance
are to:

1- Assure that the drug product contains the labeled amount of the
active constituent (s), within the accepted limits.
2- Assure that each ingredient (active or inactive) used to
formulate the finished product is of suitable purity (generally
the highest practical purity).
3- Assure that the variation of drug levels between dosage units
is minimized.
4- Assure that each finished dosage form is of the highest possible
purity that is no contaminants have entered during the
manufacturing process·.
5- Assure that the ingredients in the finished dosage form are
stable, both individually and in the presence of each other, under
normal conditions.
6- Assure that finished product is therapeutically effective.

quality Control:(QC)

It is the. operational techniques and activities used to. Fulfill

the requirements for quality Another definition was proposed
by AOAC, QC is planned activities designed to provide .a quality
product.

Quality control, therefore, includes not only the analytical tests of

the finished products, but also the assessment of all operation
beginning with the receipt of raw materials and continuing through

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 the production and packaging operations, finished product testing,
documentation, surveillance and distribution.

Figure (1): Shows the four levels of total quality

management development.

The high quality of pharmaceutical products results

from:

1. Meticulous adherence to written procedures in carrying out all

operations, beginning with research it is at this early point that
quality begins to be designed into a product.

2. Raw materials must be characterized and purchased from

reputable suppliers so that uniform, stable product will result

16
 when these materials are incorporated into the finished dosage form.

3. Facilities must be designed systems installed and the proper

equipment's selected so that the potential of cross
contamination of the product by another is eliminated.

4. Material flow and personnel movements are planned to reduce

the potential for product mix-ups

5. Air and water is adequate in amount and quality for the particular
operation being Performed·

6. Production personnel must be trained Properly and they follow

instructions which must be written approved by responsible
individuals and adhered to strictly.

7. Shipping departments are responsible for safety and purity of

products.

8. .Quality control is over-present, overseeing. each of the latter

operations and giving the final release approval for distribution
only after assessing and being satisfied that each step in this
process has been completed correctly.

Quality control laboratory

The head of the QC, who is responsible for decisions relating to

the acceptability of finished product, should report to someone
other than the person responsible for production will report to
some higher level authority.

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The Quality control function in an organization normally consists
of at least two primary units.

1- Analytical control unit (ACU)

2- Inspection control unit (ACU).
Analytical Control Unit :
The analytical control laboratory is responsible for testing and
approving raw materials, work in process and finished product.
Requirements of analytical control laboratory
1- Staffed with trained personnel experienced to perform
complex analysis required to evaluate the acceptance of a
product.
2- Well equipped with instruments which allow accurate
analysis
3-Detailed specifications of test methods must be available. The
specification must include the limits for acceptance or rejection
for each parameter .

In process control and validation :

Analytical control must be able for in-process control of products

or intermediates.
In. process control is identified at the critical operational steps
The in process limits (alert or action levels) are limits or
specifications which are more restrictive than the final
acceptance level, these levels are giving an early warning of
conditions which could lead to out-of-control situation and

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 allow timely corrective action to be taken before this occurs·
in-process critical testing depends on the dosage form being
manufactured. Sterile products receive the most, critical in
process control and testing in order to ensure a finished product
which is sterile and free of microbial contamination
Validation of operations may be defined as: "Assurance that
production processes are controlled in such a manner that will
perform routinely in the manner in- which they are planned to
Validation of processes, systems and in-processes control gives
increase assurance of finished product lot quality and leading the
way toward reducing eliminating the reliance of end product testing.
Tests and Specifications may be found in several sources such as:
1- Official sources : Compendia E.P., B.P., USP/ NF.
2- None official according to manufacturers approved
standards.

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 Drug Standards

Many. Drugs that are of proven value are described in

international, regional or national pharmacopoeias. Newly
discovered drugs would normally be approved by a national
regulatory authority and be in use before being accepted
for inclusion in a pharmacopoeia. Additionally, there are
drugs. which are combinations of pharmacopoeial and/or non
pharmacopoeial medicinal substances devised by a
manufacturer.This latter group should also be approved· by
the national regulatory authority after evaluation.

Standards of quality for drugs may be defined in terms of

chemical· and physical characteristics and in some cases by
microbiological and/or pharmacological characteristics.

In case of pharmacopoeial drugs, the pharmacopoeial

monograph contains a statement of the minimum standards
with which the individual drug must comply.

In case of non-official drugs the. product license issued by

national regulatory authority should include a statement of
standards i.e. product specification.

Many drugs fail to conform to the required

standards due to:

1. Inadequate product development has led to a production of a

drug which although satisfactory at the time of manufacture, is
unstable.

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2. Properly developed drug with good stability subjected to proper
storage conditions, has been exposed to excessive humidity or
heat.

3. Defective packaging may result in deterioration of the drug.

4. A drug may fail to conform· to required standards through

careless manufacturers· and neglect of a code of ·Good
Manufactory Practices (GMP) such as that published by the
Word Health Organization (WHO).

5. A drug may be produced ·by a licensed manufacturer,

deliberately deficient in active ingredients so as to increase the
profit margin.

6. A drug may contain the correct amount of active ingredient but

this may not be released on administration (not bioavailable).

7. A drug may be produced on unlicensed premises, i.e. either

without or seriously deficient in the stated active ingredient.

In order to reduce the hazards of sub standard drugs to a

very low level, it is necessary for every nation, every factory to
have an effective means to detect sub- standard drugs and to
take effective action either to prevent their production at the
factory level or to remove them from the market.

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Good Laboratory Practice
(G.L.P)

22
Development of the Laboratory:

In setting up a laboratory. the first step must be to ensure

that there .is a pharmacy administration so constituted as to
provide the. required administrative framework. The second
step must ·be to define objectives in terms of the drugs to be
tested and the types of test to be applied, and then to outline
the sampling schemes which will ensure optimal use of
laboratory resources.

The objective must be to establish a laboratory which is

competent, justifiably self-confident and respected. Favorable
reports issued by the laboratory will rarely be questioned. In
contrast, adverse reports are very likely to be disputed either from a
genuine belief that the sample in question is. of good quality or for
less honorable ·reasons.

The wise laboratory manager, therefore, makes every effort

to safeguard the laboratory's reputation· by establishing and
maintaining· routines and discipline designed to avoid errors and
ensure that favorable reports are not issued on sub-standard·
samples and that adverse reports are not issued on satisfactory
samples. Before issuing an unfavorable report he should check and
recheck the entire circumstances of the analysis e.g. instruments,
reference standards and apply the method for comparison to a
sample known to be genuine.

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 The necessary technical competence in all branches of
pharmaceutical analysis as well as managerial competence will be
attained only after several years. It is necessary therefore, that the
laboratory's development be phased, introducing new techniques
and the associated equipment .at a rate which permits a sound
degree of experience to be gained before proceeding to the next
step.

Physico-chemical m e t h o d s u s e d i n quality c o n t r o l ·
laboratories

Amongst the physical methods, spectroscopic methods

rank high and the "UV-VIS method is used more widely to find
the correlation between absorption and structure. UV-VIS
spectrophotometry has certain limitations as it depends on
special- moieties, as for example, all steroids yield absorption
spectra of similar shape. However, in spite of limitations, the
method has very wide application.

In view of the limitation of UV-VIS spectro- photometry,

.IR spectroscopy .was found .to yield more details for rapid
identification of functional groups. AU the functional groups of
a molecule give a characteristic IR band. These spectra serve
as fingerprints for identification of compound by comparison
with standard spectra or by simultaneous recording of the
spectra of a reference standard. Fingerprints could be
obtained in solid state either as suspension in mineral oil or

24
compressed into KBr disc. Another important application of IR
spectroscopy is to determine active and inactive polymorphic
forms of a compound such as· chloramphenicol palmitate in
suspension.

In quality control laboratories different physico- chemical

methods are applied mainly for the following purposes:

i. Assessment of purity

The techniques involved are different types of

chromatography, gel electrophoresis, thermal analysis,
fluorescence, atomic absorption spectroscopy, etc... Thermal
analysis is of special interest for determining absolute purity
of reference standards.

ii.Assay

Once the identity and purity of a compound is

established UV-VIS spectrophotometry could be used in a
wide variety of compounds for determining concentrations.
The method is simple, and both accuracy and sensitivity are
high. A more recent technique of UV-VIS spectroscopy is the
derivative spectra. Computer- controlled instruments. are now
available which record first second and third _derivative of the
absorption curve with reference to standard, thus eliminating
interference from foreign substances.

Other assay methods depend on different types of

titrimetry (acid-base non-aqueous, Karl-Fischer for water

25
determination, nitrite titration for sulfonamides,..etc). Other
assay methods include fluorimetry, GLC, HPLC, and
densitometry for determining spots in the chromatograms.

iii.Physical criteria

In addition to the physiochemic a l tests, some general

tests include disintegration/dissolution rates in tablets and
capsules, weight variation, net content or declared content of
tablets, capsules and injections in solid dosage forms. · ·.

Certain physical tests, could also be applied in determining the

quality of a product by visual inspection · only General requirements
for description of the product should include: color consistency,
clarity, stability, contamination with foreign matter or fungal
growth defects such as chipping of tablets, capping, cracking,
moisture absorption,discoloration of the coating, decomposition,
mottled · appearance and various other characteristic defects which
could be checked by visual inspection. ·

iv.. Stability criteria

Several physical criteria should be taken into

consideration for evaluating quality in respect of stability like:
color changes in drug substances, growth of microorganisms,
hardening of tablets, sedimentation of suspension, changes in·
the viscosity of Oils, crystal growth in suspension or in t.he
surface of tablets, .....etc.

26
 Development of analytical control laboratory

No laboratory can carry out analysis until some

essential equipment chemicals, glassware and books are·
available and accommodation. Provided, with laboratory
furniture and fixtures. In order to accelerate the process of
phased development: the following is suggested:

1- Accommodation:

It is desirable to have at least 500 m2 to house the chemical and

physicochemical testing as follows:

a. Rooms for chief.analyst and consultant

b. General office room and registry.

c. Documentation and sample room.

d. Stores (chemicals, glassware, spares and consumable of

instruments)

e. Library

f. One general laboratory · for routine chemical and preparative

work for instrumental analysis.
g. Instrument rooms (for housing the listed instruments).

h. TLC and fume hood.

i. Distillation, washing and drying units, ovens, water baths,

etc.

j. Balance room. And other ancillary equipment's, e.g. vacuum

oven, disintegration and dissolution units, centrifuge, etc.

27
2. Laboratory furniture and fixture

The indicated space of about 500 m2 will need at least

100 meters length in units of 2 meters of laboratory benches
fitted with cupboards. Some of these benches should include
laboratory sinks. In office, furniture will be needed for all
members of staff.

When ordering equipment it should be kept in mind that

very often the middle. range model of .equipment of a company
could serve the purpose. In order not to delay the establishment
of a laboratory, the analytical work could start with minimum
requirements, and the process of procurement could be phased
for three years accordingly, the requirements for the
equipment are listed in three parts:

First stage: List of equipment's to be obtained before

analysts assures their duties.

Second stage: List of equipment's to be ordered in the first

year.

Third stage: List of equipment's to be ordered in the

second year.

In the third year full utilization of all equipment's

should be achieved. By this time, two UV-VIS
spectrophotometers will be· in the laboratory, one of which is
being used for heavy duty work.

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 Chemicals and glassware

· Immediately on assumption of duties by the consultant

and/or chief analyst, a list of immediate requirements should
be ordered depending on the items selected for testing.

Books and jou rnals

Required books and journals are listed and should be

ordered according to the laboratory work.

All th.e analysts and technicians. Should receive in-

service training during their work with consultant and chief

analyst. In addition, some members of staff recruited initially
could be sent abroad to gain further experience and
qualifications. M.Sc. courses in analytical
·chemistry/pharmaceutical analysis offered by some universities
will be helpful. ·

Staffing and manpower development:

The qualities required of a successful analyst are:

a. A good basic academic background.

b. A critical and inquiring mind.

c. A meticulous attitude· to. presentation of data

d. A enthusiasm to further develop his knowledge and technical

skills ·

A pharmaceutical qualification provides a good general

29
 academic background on which to build.

Ancillary staff

An efficient system of documentation is essential.

Competen.t secretarial staff is needed for maintenance of such
documentation and smooth administrative functioning of the
laboratory.

Problems and constraints

The most common problems and constraints usually

faced in developing a . .Drug quality control laboratory are:

1. Maintenance and use .of equipment's

2. Manpower constraints. ·
3. Non-availability of reference substances.
4. Unproductive use of laboratories
5. Disputed reports.
laboratory organization and administration

In order that the work of a laboratory may proceed smoothly

and efficiently it is· necessary that there- be-· instructions
governing both its internal administration and its relations
with external bodies. All staff members should be. provided
with a copy of the instructions. AU persons working in .the ·
1aboratory should have a clear job description.

30
 SAMPLING

Sampling is one of the most common sources of analytical

error. One that is frequently overlooked · is sampling both
parent-lot sampling· and ·sub-sampling for laboratory analysis.

A sample submitted to the laboratory for analysis should be

representative of the lot from which it was taken. This
seems simple enough, but in practice, drawing a representative
sampl.e from a lot of goods can be quite a challenge. Much
attention has been given to the principles of sampling., .It · has
been recognized that sampling .is at least as important · as, . if
not more important than' the analytical methods used.

In mixed lots of goods or in lots where the analyte to be

detected and estimated is not homogeneously dispersed or is
present in low concentrations, sampling ca11 contribute a
substantial, if not the largest, proportion of the analytical error..

when an organization is responsible for both the collection

and analysis of samples it is essential, as. Part of the quality
assurance program, that laboratory personnel and sample
collectors cooperate in establishing sampling protocols.

Sampling protocol instructions include:

Appropriate attention to size of samples and how they should

be drawn, and to statistical sampling approaches. The protocols
should also include instructions on container type, preservation
of samples, and how they are shipped to the laboratory. Several

31
studies have been conducted to correlate distribution of
sampling and analytical error. Many organizations have· studied
proper sampling and devised programs and schedules based
mainly on statistical approaches.

To ensure a good sample management program, it is essential to

establish procedures for collection. documentation,
contamination control, preservation and shipment of samples to
the laboratory. Deviations from established procedures should
b.e the exception rather than the rule, and Changes should be
justified and documented.

Once the sample is assigned to the analyst and he has

ascertained that the records are in order, that the sample is in
good condition, and that the type of analysis required has been
chosen, the next step is analytical or sub sampling . of the
sample received. The issue to be decided is how to obtain
an analytical portion, or portions, that is representative of the
sample and in turn, representative of the parent lot.

Approaches of analytical sampling

The two chief choices in analytical sampling are the

preparation of composite sample or the examination of
multiple. individual .units. In_ some instances.it may be desirable
or even necessary to use both types. A composite sample is
one in which the individual units, or representative portions of
the units are mixed to form a uniform mixture. Samples are

32
then taken from the composite for analysis, and if the
.composite has been prepared with care, each sample
represents the material composted.

Composting can best- be used when homogeneity is not a

significant problem or when the variability between or among
units is of no great importance. Composting generally saves
analytical time, and in acceptance testing it may be the
procedure of choice or the one specified.

Multiple-unit sampling is indicated, in general, when the

range of values in individual units is large and/or it is
necessary or desirable to establish the variability in the lot
under consideration.

In addition to the type of sample or samples taken for analysis, it

is essential that the sample or samples be prepared to achieve
homogneity and ·treated so as to prevent change from the
original composition. Obviously, failure to achieve
homogeneity· at . this point of sample preparation will affect
the results o.f the. analysis regardless .of the method used.

The result obtained fo·r the proportion of a certain constituent

in a given sample may . form the basis of evaluating a large.
amount of the .commodity .from which the sample was drawn.
In· such cases it is absolutely · essential to be· certain that the
sample used .for analysis is truly representative of the whole.

Dealing with homogeneous liquids, sampling presents minor

33
problems, but if the substance under investigation is a solid .
mixture, it is necessary to combine a . number of .portions
to ensure that a representative.. sample is finally selected for
analysis.

The analyst must therefore be acquainted with the normal

standard sampling · procedures employed for different types of
materials.

The basis of sampling

Sampling is the process of extracting from a large quantity of

material a small portion which is truly representative of the
composition of the whole material. The number and size of
samples is usually determined using statistical calculations
based on sampling standard deviation and allowable error.

This means that although not every item or every part of the
sample is analyzed, the limitation of the selection are
carefully calculated and known in advance.

· Having calculated the deg·ree of acceptable risk or

margin of variation, the sampling plan is then chosen that
will give the maximum· · information and control that is
compatible· with a rapid turn over of samples. For this reason, i
the case of sampling from batches the sel ction of
individual samples is carried out according to special random
tables which ensure that personal factors do not influence the·
choice.

34
Sampling procedure

The sampling procedure. may involve a number of stages prior

to analysis of the material, (figure 2). For the most part,
bulk materials are nonhomogeneous e.g. minerals, sediments
and food stuffs. They may contain particles of different-
composition which are not uniformly distributed within the
material. In this case, a number of increments ,,are taken in a
random mariner from points in the bulk material, so that each
part has an equal chance of being selected. The combination
of these increments then forms the gross sample. The gross
s ample is often· too - large for direct analysis and must be
divided further to produce a sub-sample. The sub-sample may
require treatment as reduction in particle size or through
mixing before the analytical sample can be obtained. The
analytical sample should retain the same composition of the gross
sample.

35
 Figure (2): Sampling Procedure

The reduction in size of gross sample without bias introduction

is possible using the cone and quarter technique. This
involves arranging the . total material into a pile (cone) and then
drawing two vertical lines through the pile a right angles to
each other, producing four identical quarters. Two
opposite quarters are then combined and retained, where
as the other two are discarded. In this way, the mass has
been halved, without bias. There are various sampling
facilities ranging .from the simple spatula and spoon to the
large scoops and measuring buckets (see figure 3).

Zone Powder Sampler: It is used for proper sampling of

powder and other friable materials that may become non
homogenous in · bags. It extracts samples at three levels from
one container. The T-handle is turned to close the sample

36
cells and the steel point is inserted through the bag wall.
When the sampler is deep enough to cover the three cells, a half
·turn of the handle allows samples to enter then return it to
the original position and remove the sampler into the analysis
container.

Glossary of Terms:

Available Sample

Whatever total quantity of sample material is available. .

Batch: A quantity of any drug produced during a given cycle

of manufacture. If the manufacturing process is continuous ·
the batch originates in defined period of time during which the
manufacturing conditions· are stable and have not been
modified.

Combined s a m p l e : Sample resulting from combining

all or parts of two or more samples of the material.

Consignment: Quality of a bulk starting material, or of a·

drug product, made by on manufacturer or supplied by an
agent, that is supplied at one time in response to a particular
request or order. A consignment may comprise one or more
lot-identified packages or containers and may include material
belonging to more than one lot-identified batch.

Final Sample: Sample ready for the application of the test

procedure,

37
Homogeneity: A . material is regarded as homogeneous when
it is all of the same origin (e.g. from the same batch) and as
non- homogeneous when. it is of differing origins.

Original Sample: Sample collected directly from the material.

Random sample: Sample in which the different fractions of

the material have an equal probability of being represented.

Representative Sample: Sample obtained according to a

sampling procedure designed to ensure that the different parts
of a batch or properties of non-uniform material are
proportionately represented.

Sampling Procedure: The complete sampling operations to be

applied to a defined material for a specific purpose. A detailed
written description of the sampling procedure is provided as the
sampling protocol.

Sampling Record: Written record of the sampling operations

carried out on a particular material for defined purpose.

The sampling record must contain the batch number, date and
place of sampling, reference to the sampling protocol used, a
description of the containers and of the materials sampled,
notes on possible abnormalities, together with any other
relevant observations and the name and signature of the
inspector.

Sampling Unit: Discrete part of a consignment such as an

individual. Package, drum or container.

38
Selected Sample:

Sample obtained according to a sampling procedure designed to

select a fraction of the material that is likely to have special
properties. A selected sample that is likely to contain
deteriorated, contaminated, adulterated or otherwise
unacceptable material is known as an extreme sample.

Uniformity:

A starting material may be considered uniform when samples

drawn from different layers do not show significant differences
in. the quality control tests which would result in non-
conformance with specifications,

Retention sample: Sample collected as part of the original

sampling process and reserved for future controls. The size of a
retention sample should be sufficient to allow at least two
confirmatory analyses. In some cases statutory regulations may
require one or more.

Retention samples, each of which must be separately identified,

packaged and sealed.

Sample: A portion of a material collected according to

defined sampling procedure. The size of any sample .
should . be -sufficient to carry out all anticipated test
procedures, including all repetitions and retention. If the
quantity of material available is not sufficient for the intended
analyses and for the retention samples, the inspector must

39
record that the sampled material is the available sample (see
below) and . the evaluation of the results must take account
of the limitations derived from the insufficient sample size.

Sampler: Person responsible for performing the sampling

operations.

Sampling Method: Section of the sampling procedure

dealing with the method prescribed for withdrawing samples.

Sampling Plan: Description of the location, number of units

and/or quantity of material that must be collected and associated
acceptance criteria.

40
Figure 3 : some different sampling devices.

41
Analysis of Pharmaceutical products

Methods of analysis are loosely classified and characterized

in terms that generally indicate their proposed use, as follows:

- Official methods
- Standard, consensus or reference. methods.
- Screening or rapid methods.
- Routine methods
- Automated methods and modified methods.

Official methods are those generally required by law or

regulations of a regulatory agency

Standard, consensus, or reference methods are methods

developed by organization or groups using collaborative
studies or some similar approach.

Screening or rapid methods are used as an expedient way to

determine whether additional testing by a more accurate
method is indicated.

Routine methods are those used on routine basis. They can

be official. or standard, but can be modified to be more
convenient to use.

Automated methods, as the name implies, use automated

equipments, but may also come under the categories of
official,screening, or routine methods.

Modified methods are generally official or standard methods

42
that have been changed to simplify them or to accommodate
the procedures to·different matrices or to remove interfering
substances.

As part of the laboratory's quality . assurance program, and as

a general rule, all methods regardless of their. source or
characterization should be chosen carefully and their
suitability established for their intended use. Methods. should ·
be available in written and approved form before validation
and/or use in the laboratory. ·

The following is a list of information that should typically

be included in a description of an analytical procedure

A. Principle:

A statement of the principle of the analytical procedure

should be included. For example, separation is based on
isocratic reversed phase HPLC with detection by UV.

B. Sampling

The number of samples {e.g., vials, tablets selected how

they are used i.e., as individual· or composite samples), and
the number of replicate analyses per sample should be
described.

C. Equipment and equipment parameters

A listing of all equipment (e.g,., instrument type, detector,

column type & dimensions) should be inqluded, as well as ·a

43
list of equipment parameters (e.g., flow rate, temperatures, run
time, wavelength settings). A. drawing representing the
experimental configuration (e.g, illustrating positions for a
spray pattern analytical procedure) should be provided, when·
appropriate.

D. Reagents

A list of reagents and their grades {e.g., USP/NF, American

Chemical Society. (ACS) Analytical Reagent) should be
included. If inhouse or modified commercial reagents are
used, directions for their preparation should be included.
Unstable or potentially hazardous reagents should be
identified, and storage· conditions, directions for safe use, and
usable shelf life for these reagents should be specified.

E-System suitability testing

System suitability test parameters and acceptance criteria are

based on the concept that the. Equipment, electronics,
analytical operations, and samples to be analyzed constitute
an integrated system. System suitability testing ensures that
the system · is working properly at the time of analysis.
Appropriate system suitability criteria should be defined and
included in the analytical procedure.

. System suitability testing is recommended as a component

9f any analytical -procedure, not just those that involve
chromatographic techniques. Regardless of the type of

44
analyticaJ procedure, testing should be used to confirm that the
system will function · correctly independent of the
environmental conditions. For example, titration analytical
procedures should always include the evaluation of a blank
(commonly referred to as a blank·titration).

F. Preparation of standards

Procedures for the preparation of all standard solutions (e.g.,.

stock, working standard solutions, internal standards) should
be included.

G-Preparation of samples

Sample preparation for individual tests should be clearly

described. Specific details should be provided for unusual
sample preparations (e.g., solid-phase extraction,
derivatization).

H- Procedures

A step-by-step description of the procedure should be

provided. The description should include, where appropriate,
equilibration time, injection sampling sequence, and system
suitability or start-up parameters. Unusual hazard should be
identified.

I. Calculations
Representative calculations. with a tabulation defining all
symbols and numerical factor, and specific instructions for

45
the calculation of degradation products and impurities should
be included. Any mathematical transformation or formulas
used in data analysis should- be described in detail These may
include logarithmic transformations used to obtain a linear
relationship from exponential data, or the use of multiple order
regression analysis.

46
 DOCUMENTATION

Document is the written procedures, instructions,

requirements, registration .files and others that to be needed
in storage, procedures, manufacturing and quality control.

Good documentation is a prime necessity in quality

assurance. Its purposes are to:

- Define the system of control.

- Reduce the number of mistakes inherent in .purely oral

communication.

- Giving clear message on how work should be done and

insure that personnel are instructed in the details of, and follow
the procedures concerned.

- Permit .investigation and tracing of defective products and

the cause of poor quality.

- Tracing which batches. of materials were used in which

batches of product and where the batches were delivered

- Keeping records of how work was done.

Documents should be designed, prepared, reviewed and

distributed. with care. They should comply with the relevant
parts ·of the manufacturing and marketing authorization
dossiers.

Properties of good document

Documents ·should be approved signed and dated by

47
appropriate and autorized persons.

Documents should have unambiguous contents; title, nature

and purpose should be clearly stated. They should be laid out i
an orderly fashion and be easy to check. Reproduced
documents should be clear and legible. The reproduction of
working documents from master documents must riot
allow any error to be introduced through the reproduction
process.

Documents should be regularly reviewed and kept up-to date.

When a document has been revised, systems should be
operated to prevent inadvertent use of superseded documents.

Documents should not. be handwritten; although, where

documents require the entry of data, these entries· may be made
in clear, legible, indelible handwriting. Sufficient space should
be provided for such entries. Data may be recorded by
electronic data processing systems, photographic or other
reliable means.

Any . alteration made to the entry on a document should be

signed and dated;·the alteration should permit the reading of
the original information. Where appropriate, the reason for
the alteration should be recorded.

Types of Documents

Three most important type of documents for quality

assurance are:

48
1. Specification:

Specifications. give the standards of quality that can be

measured and is used by quality department in deciding if a
batch is passed or rejected There should be appropriately
authorized and dated specifications, including tests on identity,
content, purity, and · quality, for starting and packaging
materials and finished· products, where appropriate, they
should also be available for intermediate or bulk
product. Specifications for water, solvents and reagents used in
production should be included. Specifications for raw
materials should include, if applicable:

- The designated name and the internal code reference.

- The reference, if any, to a . pharmacopoeial monograph.·

- Name of final company.

- The approved suppliers and, if possible, the original

procedure of the products.

- .. General specifications (color, taste, odor and form)

2. Records:

- Batch manufacturing records (processing and packaging)

reviewed by quality department.

- Batch analysis records, completed and reviewed by quality

department.

- Logs,.· used in production, engineering and quality

49
department for records not directly related to a batch for e.g.
cleaning, use of equipment, machine maintenance,..etc.

There must be records for test, analysis, approval, rejection .

of raw materials, in-process and final product. These records
should be carefully 'kept and if they are booked, paper serial
number must be given without tear off any paper.

The record must include:

• Date of test.

• Material identification and its code number.

• Supplier's name.

• Batch number from quality control.

• Batch size & Date of batch size.

• Method of analysis,. References & test results.

• Comparison With standard product.

• Name and signature of the analyst.

3. Procedures:

Procedures describe and give instruction on how work must

be carried out. It can be special types of procedure, such as
analytical methods or more general standard operating
procedures (SOP's). . SOP must be us·ed by anybody whose
work can · affect product quality, e.g., production, quality
control department and engineers.

50
 TOTAL ANALYTI CAL VALI DATION.

Quality building and good quality· achievement can only be

expected and obtained when the steps · of development,
optimization and validation approaches are together and t
the same time well considered and regarded. It is necessary
to ·ensure that the analytical system itself is validated or
qualified. Qualification (validation) of an analytical method
is a· subset of the total validation process, with many different
aspects, in TOM-systems. ·

Validation is a process that consists of many steps, such as: .

(1) Tools validation.

(2) Method validation.

(3) System validation or suitability.

TOOLSIN I NSTR UM ENT LABORATORY

A separate instrument room, so-called central instrument lab, is

preferred; otherwise· serious corrosion may spoil the costly
instruments. Regular calibration of all the instruments used
to measure the physical or chemical properties of the drug
substances or their dosage forms is absolutely essential and
a specific schedule should be established for each type of
instrument. Standard operating procedure should be placed
beside the complicated instrument along with the schedule for
calibration.

51
A separate log. book should be maintained for each instrument
which should include: When the instrument was last used,
who had used it and the purpose for which it was used.

All pharmaceutical units need a chemical laboratory for

evaluation of raw material and finished products.

List of equipment's .for central instrument lab:

• Double beam UV-Visible spectrophotometer.

• IR spectrophotometer.

• High Performance Liquid Chromatograph (HPLC).

• HPT'-C scanner with accessories.

• Gas. Liquid Chromatograph (GLC).

• Spectroflourimeter.

• Atomic Absorption Spectrometer (AAS).

• Polarograph.

• Analytical balance.

• pH meter.

• Potentiometeric titrator.

• Computer with ·printer.

All these instruments should be provided with fully

stabilized electric current and housed in air conditioned room.

52
TOOLS (INSTRUMENTS) VALIDATION

The process begins_, with validated (standardized, qualified

and calibrated) tools. In the modern analytical methods,
mostly instrumental, there should be validated software and
hardware as well as a qualified system, where a validated
analytical method using qualified systems can be
developed. Finally, total validation is achieved by stabilizing
system suitability. Prior to the analytical instrument
.(used in the late stages of the analytical
procedure, i.e. measurement) being placed on-line, the
analytical system should be pre validated by verifying the
module and system performance. If the instrument is . not
qualified prior to use, a problem will be difficult to identify.

Vendor's Site User's Site

-
Structural of Functional Validation Qualification Calibration
software Installation operation Performance Maintenance
validation (IQ) (OQ) (PQ) System
Suitability
Before Before Use After use
Purchase
Time

53
(1) Validation begins at:

The vendor's site, as part of a structural stage of validation,

where, the instrument with software is developed and
produced regarding the requirements of good laboratory
practice (GLP), and current good manufacturing practice
(cGMP). Before purchasing the choice qualification insures toe
required specifications of the actual current and future needs.

(2) After purchasing at :

The user's site, It is the functional validation or qualification

phase. It includes two stages:

(a) Before use: the installation qualification (IQ),

operational qualification (OQ), and performance
qualification .(PQ) are performed.

* Installation Qualification (IQ): this can be divided into;

pre-installation and physical instauration. During pre- installation,
all information considers the proper installation, operation ·
and maintenance of the instrument should be reviewed. Site
requirement, receipt of all part, and the user manual, necessary
to perform the installation, are to · be confirmed.
Documentation describing how the instrument was installed,
who performed the installation and serial coding of different
instrument's parts should be archived..

* Operational Qualification (OQ): this process ensures that

the specific modules of the system are operating according the

54
defined specifications for accuracy, linearity ·and precision.
Small or minor clear discrepancies can be overcome by
following the indicated trouble-shooting.

* Performance Qualification (PQ): the PO-testing is conducted

under actual running conditions, however, OQ and PQ are usually ·
performed together particularly for repeatability (linearity . and
precision). Proper documentation supporting the PQ-process should
be archived.

(b} After use: after - placing the instrument on-line in the laboratory,
and after a se.t . period of .use, regulations require maintenance
followed by time to time calibration and standardization; this is
named - maintenance qualification·(MQ) or maintenance procedures. ·

Maintenance and calibration of instruments ·

It is primarily responsibility of ·. the analyst who operates

the instrument· to ensure that the instrument is of
appropriate design, adequate capacity .to · perform intended
functions consistently and precisely. Obviously, ultimate
responsibility for accuracy and precision of an instrument
and data it generates is with the analyst using the equipment.
It is therefore essential that the instruments be inspected,
cleaned and adequately maintained. The need for calibration
and/or standardization of the instrument is well recognized
among scientific community for obtaining analytical data to
be subjected to validation process. The laboratory should

55
establish schedules for each instrument based on the
recommendations of the manufacturer or based on own
laboratory experience.

Note: No instrument will be used until and unless calibrated

or ·checked for · its performance while undertaking the
development of a new method as shown in table.

Calibration: Comparison with standard measurement or

instrument .of. a known accuracy to detect, co-relate or
eliminate by adjustment any variation in the accuracy of the
instrument being compared.

Standardization: Comparison with.. a standard of known

and accepted value· such as use of standard weights in
calibration of analytical balances

The following shows a calibration/validation record of an UV- Visible

spectrophotometer instruments:

56
Comments: On this day, the instrument identified above has been
calibrated/validated and meets the manufacturer' s specifications.

----------------------------------------

(Name and signature of calibrating. analysts)

----------------------------------------

----------------------------------------

Date :... /02 / 2021

57
 Table 1: Schedule for calibration/inspection of some
major instruments

Instrument Interval (Months)

UV-Visible Spectrophotometer 6

Infrared Spectrophotometer 4

NMR Spectrometer 6

Thermo gravimetric analyzer 6

Polarimeter 6

Fluorimeter 4

PH meter 6

Dissolution test apparatus 3

Ultrasonic water-bath 6

Heating baths 4

Analytical balance* 3

*(Fixed weight calibration on daily basis)

This schedule helps in service contract with the

manufacturer distributor.

58
 ANALYTI CAL METHOD VALIDATION

METHOD DEVELOPMENT

The development of an analytical method is usually based on prior art

or existing literature using the same or similar methodology and
instrumentation. Among the reasons for developing new methods of
analysis are:

1- No suitable method for a particular analyte in a specific sample matrix.

2- Existing methods are unreliable (have poor accuracy or precision).,
3- Existing methods are expensive or time consuming or not easily
automatable.
4- No enough sele9tivity or .sensitivity for specific analytes in a
sample.
5- New methods, based on new instrument, may Provide
opportunities for improved identification and quantification
limits.
6- Alternative methods in order to confirm analytical data
originally obtained by existing methods; for scientific purposes.
Instrument selection is based on the methodological
technique, then, the analyte parameters shouldbe determined. To
develop a method, it is necessary. to consider the properties of the
analyte(s). The parameter values include the chromatographic
factors (capacity and retention), U.V..Vis/FL wavelengths, mass/charge-
rati o in mass spectrometri c detection . If the data not available in the
literature, they should be undertaken on highly pure analytical

59
standard(s). Method development should be carried out using only
standards that have been well-identified and characterized, and
whose purity is already known (high). Such precautions will prevent
problems in the future and will remove variables on optimizing or
improving the initial condition during method development.

METHOD OPTIMIZATION

The initial sets of ·conditions (experimental) that have been obtained

from the first stages of development are improved or maximized in terms
of overall ability to quantify specific analyte(s) of interest. Results
obtained during optimization must evaluated against the objectives of
the analysis. Weather the new method offers advantage (s)over
existing methods in term of the objectives of the analysis or not.
Optimization can be made by manual or computerized means.
The manual approach ·involves one experimental variable at
a time, which other factors. are kept constant. · Such
univariate way to an analytical system is·. Slow, expensive and
time- consuming. The · optimization of a new method · must

consider general. criteria, such.as: ·

− Adequate resolution_ (as in chromatography) .

− Lower limit of sample detection.[LOD].
− Lower limit of sample quf1nfification [LQQ].
− Minimal instrument . equilibration . (calibration) before
run and from run to run.
− Minimal sample preparation.

60
 − Identification and interference .minimization of possible
interferent(s)
− Significant reproducibility, accuracy and precision .. .
− Minimal effective costs.

Method Validation

Validation of an analytical method demonstrates that. The

method works with samples of the given analyte (s) with a high
degree of accuracy and precision Validation can occur only
after the method has been developed and optimized. The
validated method can be transferred from one suitably equipped
and staffed laboratory to another. There are many validation
approaches, where the analyst's primary concerns control the
choice of the appropriate validation approach. Validation
approaches include the zero- single- and double-blind spiking
methods, analysis using standard reference materials, inter and
intra laboratory studies, and comparison with a currently
accepted method.

1- Zero – blind Method

This validation approach has a single analyst using the method

to be validated at known levels of analyte to demonstrate
recovery. Accuracy and precision. The method is subject to
analyst bias.

61
2 .Single-blind Method

Such an approach involves one analyst preparing samples at varying

concentration levels to a second analyst, who also analyzes, the
samples. The results of both analysts are then compared. Single-blind
approach of validation is valuable and believable than the zero- blind
one.

3. Double-Blind Method

This validation way involves three analysts, the first prepares samples
at known levels, while, the second does the actual analysis, and the
third analyst compares both sets of data received ."separately from the
first two analysts. The first analyst has no a9cess to the set of data
generated by .the second. It is the ·most objective approach, assuming
no bias on the part of the third analyst.

4- Analysis Using Standard Reference Materials

Standard reference material or. an authenticated sample (authentic) is

an accepted method of validation. The USP, national institute
standards, BP and other private organizations specialize in
preparing, guarantee and marketing standard reference
materials of various chemical substances in different
matrix forms. Analyst bias can be observed, especially
when the, analyst knows the level and m a s s of the
SRMs utilized for validation.

62
5. lnter/laboratory & lntra../laboratory Studies

Collaborative inter-laboratorial method of validation widely

accepted, but is costly and time consuming (can take
years). Th.e method to be validated attached with samples

to be analyzed are to be shipped into several approved

laboratories for analysis, then receiving results,
statistically analyzing and interpreting the results and
verifying the data. Such studies are operator- dependent
(laboratory to laboratory variability). If the results of
various laboratories show values in accordance with known
analyte(s) levels present, the method is generally, accepted
as full validation. If the- same procedure is undertaken in
the samp1e laboratory by various analysts this· is known
as intra-laboratory investigations

6. Comparison with a Currently Accepted Method

This approach is usually done by a single analyst, but it can be
carried out by two analysts using a split samples. Agreement
between results from the currently accepted method as verification
of the new method's results suggests validation. If the analyst can
prove that the currently accepted (reference) method is invalid, he must
then initiate an alternative validation approach to validate the new
method.

In general, the more samples, to be analyzed in any validation

approach, the better, and the ·greater the variety of samples and

63
 variation concentration· range, the better. Inter laboratory studies,
true double-blind study of several sample matrices at wide
concentration range are more acceptable and meaningful than zero-
and single-blind studies.

Outline (step-by-step). of · Method Development,

Optimization and Validation.

Documentation should start at the very beginning of the method

achievement. All data must be recorded in laboratory notebooks or
electronic data base.

1. Standard characterizati on: Physical & chemical

properties, purity, stability.

2. Method requirements: Goals (selectivity, LOD & LOQ, ... le),

instrumentation (type and limitation).

3. Literature Search and Available Methodology

4. Method selection: Suitable· method selection from prior methods

and if there are no available prior method, work from analogy to
show compounds that are similar in structure and properties to the
.analyte.

5. Instrument Setup and .Initial Studies: Instruments should be

prepared ready by using SOPs of the instrument.

6. Optimization: Computer-based optimization software is

available, where one parameter is changed at a time.

64
 7. Demonstration of Analytical Figures of Merit: This includes LOQ,
LOO, linearity, ...etc.

8. Evaluation of Method Development with Actual Samples

(Derivation of Figure of Merit: Considering the
determination of the analyte of interest apart from an other matrix
components.

· 9. Validation of Figures of Merit: The USP- eight steps method of

validation include: precision, accuracy, linearity range, LOO, LOQ,
specificity, ruggedness and robustness.

10. Determination of Percent (%) Recovery.

11. Method Validation: Perform zero- and double-blind studies,

reproducibility.

12. Preparation.of Method Manual {Written .Protocols and

Procedures).

13. Inter-laboratory studies (transfer to outside labs).

14. Comparison and Interpretation of Inter-laboratory Studies.

15. Preparation of Report on Overall Method.

16. Procedures. Results. Article Preparationand

Submission for Publication.

65
 METHOD VALI DATION
(USP/ICH)

method validation, according to the United States Pharmacopeia

(USP), is performed to ensure that an analytical methodology is
accurate, specific, reproducible, and rugged over the specified
range that an: analyte will be analyzed. Method validation provides
assurance of reliability during normal use and is sometime
described as the process of providing documented evidence
that the method does what it is intended to do. Regulated
laboratories must perform method validation in order to be in
compliance with FDA regulations. In a 1987 guideline
(Guideline for Submitting Samples and Analytical Data for
Methods Validation)/ the FDA designated the specifications in the .
current edition of the USP as those legally recognized when
determining compliance with the Federal Food, Drug and Cosmetic
act for method validation, these specifications can be referred to ·as
the "eight steps of method validation, as shown infigure 4.

Figure (4): The USP eight steps of method validation

66
These terms are referred to as "analytical performance parameters"
or sometimes as "analytical figures of · merit." Most of these terms
are familiar and are used daily in the laboratory . .However, some
may mean different things to different people. To avoid
confusion, therefore, it is necessary to have a complete understanding
of the terminology and definitions. Recognizing this, one of the
first harmonization projects taken up by the ICH was the
development of the guideline 'Validation of Analytical Methods:.
Definitions and Terminology." ICH divided the validation ·
characteristics differently, as outlined in figure 5.

Figure (5):ICH method validation parameters

The differences between the USP and ICH terminology is, for the most
part, one of semantics with .one notable exception. ICH treats system
suitability as a part of method validation, whereas the USP treats it in a
separate chapter: Since this guideline has reached step 5 of the ICH

67
process, the FDA has begun to implement it, and it is anticipated
that the ICH definitions and terminology will eventually be
published in the USP. What follows then is a discussion of current USP
definitions of the analytical performance parameters, com- pared
and contrasted to the ICH definitions. Where appropriate,
methodology is also presented according to the ICH methodology
guideline.

Accuracy

Accuracy is the measure of exactness of an analytical method, or

the closeness of agreement between the measured value and the
value that is accepted either as a conventional, true value or an
accepted reference value. Accuracy is measured as the percentage of
analyte recovered by assay, by spiking samples in a blind study.

For the assay of a drug substance: accuracy measurements

are obtained by comparison of the results with those of a standard
reference material, or by comparison to a second, well-characterized
method.

For the assay of-a drug product: accuracy is evaluated by analyzing

sythetic-mixtures spiked with known quantities of components.

for the quantization of impurities: accuracy is determined by

analyzing sample (drug substance or drug. product)-spiked-with-
.known amounts of impurities.

:To document accuracy, the ICH guideline on methodology

recommends-·collecting ,data from minimum of mine determinations over

68
a minimum of three concentration levels covering ;.the Specified range
(for example, three concentrations with three replicates .each). The
data. should be reported as the percent recovery of the known, added
amount, or as the difference between the mean ·and true value with
confidence intervals. Accuracy can be documented.: through, the use of
control charts, an example of which is shown in figure 6.

Precision

Precision is the measure of the degree of repeatability of an analytical

method under normal operation and is normally expressed as the
percent relative standard deviation for a statistically significant
number of samples.

According to the ICH, precision should be performed at three different

levels: repeatability, intermediate precision, and reproducibility.

- Repeatability refers to the results of the method operating over

a short time interval · under the same conditions. It- should be
determined from a minimum of nine determinations covering the
specified range of the procedure (for ·example, three levels with
three repetitions each), or from a minimum of six
determinations at 100% of the test or target
concentration.

- Intermediate precision (intra-laboratory study)

refers to, the results from within-lab variations due to random

events such as differences in experimental periods analysts
equipment, and so forth. In determining intermediate precision,

69
experimental design should be analyzed so that the effects (if any)
of ·the individual variables can be monitored.

- Reproducibility (inter-laboratory study) refers to the results of

collaborative studies a·mong laboratories. Documentation in
support of precision studies should include the standard
deviation, relative standard deviation, coefficient of variation, and
confidence interval. Figure 7 illustrates how custom graphics in the
form of bar charts can be used to document precision.

Run number

Figure (6): Control Charts

70
 Figure (7): Br Chits

Specificity

Specificity is the ability to measure accurately and specifically the

analyte of interest in .the presence of other components that may be
expected to be present in the sample matrix. It is a measure of the
degree of interference from such things as other active ingredients,
additives, impurities, and degradation products, ensuring that A
peak response is due only to a single component; that is that no co-
eluton exists Specificity is measured and documented in a
separation plate count (efficiency), and tailing factor. Specificity
can also be evaluated with modern photodiode array detectors that
compare spectra collected across a peak mathematically as an
indication .of peak homogeneity."

ICH divides the term specificity into two separate categories

identification and assay/impurity tests.

71
− For identification purposes, specificity is demonstrated by the
ability to discriminate ·between compounds of closely related
structures, or by comparison to known reference materials
- For assay/impurity tests, specificity is demonstrated by the
resolution of the two .closest eluting compounds. These
compounds are usually the major .component or active ingredient
and an· impurity. If impurities are available, it ·must be
demonstrated that the assay is unaffected by · the presence of
spiked materials (impurities and/or excipients) If impurities are not
available, the test results are compared to a second well-
Haracterized procedure. For assay tests, the two results are
compared; for impurity tests, the impurity profiles are compared
head to head.

Limit of Detection

The limit of detection (LOD) is defined as the lowest concentration

of an analyte in a sample that can be detected, though not
necessarily quantified. It is a limit test that specifies whether or not
an analyte is above or below a certain value.

Determination of limit of detection

*Signal to noise ratio: It is expressed as a concentration at a specified

signal-to-noise ratio, usually a 2 or 3 to1ratio.

*Visual non instrumental methods may include techniques such as thin-

layer chromatography (TLC) or titrations.

*Calculation method: LODs may also be calculated-based-on the

72
standard deviation (SD) of the response and the slope (S) of the
calibration curve at levels approaching the LOD according to the
formula: LOD = 3.3(SD/S).

The standard deviation of the response can be determined based

on:

a. the standard deviation of the blank

b. the residual standard deviation of the regression line

c. the standard deviation of y-intercepts of regression

lines.

The method used to determine LOD should be documented and

supported, and an appropriate number of samples should be analyzed
at the limit to validate the level.

Limit of Quantitation

The limit of quantitation (LOQ) .is defined as the lowest

.
concentration of an analyte in a sample that can be determined with
acceptable precision and accuracy under the stated operational
conditions of the method.

Like LOD, LOQ is expressed as a concentration, with the precision

and accuracy of the measurement also re-ported The determination.
Of LOQ is a compromise between the concentration and the
required precision and accuracy. That is, as the LQQ concentration
level decreases, the precision decreases. If greater precision is
required, a higher concentration must be reported for LOQ. This

73
compromise is dictated by the analytical method and its intended
use.·

The ICH has recognized methods of determination of LOQ:

*Signal to noise ratio: the use of ·1O-to-1 signal-to- . Noise ratio as

typical determination method.

*Visual non instrumental methods: as for LO.D, ·the same techniques can:
be used to determine LQQ.

*'Calculation method: the calculation method is again based on the

standard deviation (SD) of the response and the :slope (S) of the
calibration curve· according to the formula LOQ= 10(SD/S).

Again, the standard deviation of the response can be determined

based on the standard deviation of the. blank, the residual standard
deviation of the regression line or the standard deviation of y-
intercepts of regression lines. As with LOD, the. method used to
determine;Lo·9.·;should be documented and supported; - and .an·
;appropriate number of samples should be analyzed at the limit to
validate the level

One additional detail should be considered: both the LOQ and the
.
LOO can be· affected by the chromatography. Figure 8, shows how
efficiency and peak shape can affect the signal- to noise ratio.
Sharper peaks result in a higher signal-to-noise ratio, resulting in
1ower LOQs and LOO.. Therefore, the chromatographic determination
of LOO and LOO should take into account both the type and a·ge of the
column .

74
 Figure (8): Effect of chromatographic., separation on signal/noise
ratio.
Linearity and Range

Linearity is the. ability of the method to elicit test results that are
directly proportional to analyte concentration within a given range.
Linearity is generally reported as the variance of the slope of the
regression line. Range is the (inclusive) interval between the upper
and lower levels of analyte that have been demonstrated to be
determined with precision, accuracy, and linearity using the
method. The range is normally expressed in the same units as the
test results obtained by the method. The ICH guidelines specify a
minimum of five concentration levels, along with certain minimum
specified ranges.

− For assay tests, the minimum specified range is 80-120°/o of the target

75
 concentration.

− For impurity tests, the minimum range is from the reporting

level of each impurity to 120o/o of the specification.

− For toxic or potent impurities, the range should be

commensurate with the controlled level.

− For content uniformity testing, the minimum range is 70-130o/o

of the test concentration and for dissolution testing, ± 20% over
the specified range of the test. That is, in the case of an
extended-release product dissolution test, with a Q-factor of
20% dissolved after six hours, and 80% dissolved after 24
hours, the range would be 0-100% Figure 8 is an example of a
linearity (calibration) plot.

Figure (9): A calibration plot.

76
Ruggedness

Ruggedness, according to the USP, is the degree of

reproducibility of the results obtained under a variety of
conditions, expressed as % relative standard deviation (RSD).
These conditions include differences in laboratories, analysts,
instruments, reagents, and experimental periods. In the
guideline on definitions and terminology, the ICH does not
address ruggedness specifically. This apparent omission is
really· a matter of semantics, however, as ICH chooses instead
to cover the topic of ruggedness as part · of precision, as
discussed previously.

Robustness

Robustness is the capacity of a method to remain unaffected by

small deliberate variations in method parameters. The
robustness of a method is evaluated by varying method
parameters such as percent organic solvent, pH, ionic strength,
or temperature, and determining the effect (if any) on the
results of the method. As documented in the . ICH guidelines,
robustness should be considered early in the development of a
method In addition, if the. results of a method or other
measurements are , susceptible to variations in method
parameters, these parameters should be adequately controlled and
a precautionary statement included in the method documentation.

77
Table 2: Potential factors to be examined in robustness
testing.

Method Factors

HPLC pH of mobile phase

Amount of the organic modifier
Buffer concentration, salt concentrations or ionic
strength concentration of additives (ion pairing agents,.
Competing amine)
Flow rate
Column temperature
For gradient elution
ln1tial & final mobile phase composition
Slope of the gradient
Column factors
Batch Of, stationary phase
Manufacturer
Age of the column
Detector factors.
Wavelength (UV or fluorimetric detection)
voltage (electrochemical detection) ·
lntegration factors
Sensitivity

78
Gas injection temperature
Chromatography Column temperature
(GG) . Detection temperature
For Temperature program
Initial & final temperature
Slope of the temperature gradient
Flow .rate of the gas
For flow program
Initial & final flow
Slope of the flow gradient
Split-flow
Type of liner
Column factors
Batch of stationary phase & manufacturer.

79
Table 2: Potential factors to be examined in robustness
testing (continued) ·

TLC Eluent composition

pH of the mobile phase
Temperature
Development distance
Spot shape
Spot size
Batch of the plates
Volume Of sample
Drying conditions (temperature, time)
Conditions of spot visualization (spraying of
reagent, UV detection, dipping into a reagent).

80
 CE and related Electrolyte concentration
techniques Buffer pH
Concentration of ·additives (organic solvents,
chiral selectors, surfactants)
Temperature
Applied voltage
Sample injection time
Sample concentration
Concentration of the liquids Rinse Times
Detector factors.
Wavelength (UV or fluorimetric detection)
Factors related to the capillary
Batch
Manufacturer
integration factors

System Suitability lasting

ICH introduces something termed system suitability, which is

really used after the method has been fully validated, when. validation.
Method is being routinely used to actually analyze real real samples

System suitability samples is run any day that real samples

are being analyzed, always before and always. After the
actual batches of samples, often within that run of batches,
in order to demonstrate that the instrumental system is
performing· properly.

It is used to :show that HPLC column, for example is

yielding the desired peak shapes, plate counts, resolutions

81
and symmetry already demonstrated when the method was
first validatedated

This method is readily used Just to show that the analytical

instrumentation and overall system is performing properly
before real samples are. then run. If the system suitability
sample does not yield chromatograms as expected, it should not
proceed to analyze real samples until the problem is defined and
corrected.

system suitability· samples are run to ensure the . Proper

operation of the instrumental system itself, not necessarily the
entire method including sample preparation and thus such a sample
does not constitute a real sample.

82
 DATA ELEMENTS REQUIRED FOR

ASSAY VALI DATION

Both the USP and the ICH recognize that it is not always
necessary to evaluate every analytical performance
parameter. The type of method and its intended use indicates
which parameters need to be investigated, as illustrated in table 2

The USP divides analytical methods into three categories

1. Quantization of major components or active

ingredients. ,

2. Determination of impurities or degradation products.

3. Determination of performance characteristics.

For assays in category 1, LOD and LOQ evaluations are not

necessary. Because the major component or active
ingredient to be measured is normally present at high levels.
However, since quantitative information is desired, all of the
remaining analytical performance parameters are pertinent. ·
Assays in category 2 are divided into; quantitative and limit
tests. If quantitative information : is desired determination of
LOD is not necessary, but .the remaining ·parameters are
required. The situation is reversed for a limit test. Since
quantization .is not required,· it is sufficient .to measure the
LOD and demonstrate specificity and ruggedness.

Table 3: USP Data Elements Required. For Assay

83
 Validation

Analytical Assay Assay category 2 Assay

Performance category 1 category
Quantitative Limit
Parameter 3
.Tests

Accuracy yes yes * *

Precision yes yes No yes

Specificity yes yes Yes *

LOD No No Yes *

LOQ No yes No *

Linearity yes yes No *

Range yes yes * *

Ruggedness yes yes Yes yes

*·May be required, depending on the nature of the specific test.

84
Table 4 : ICH Validation Characteristics versus type of
analytical procedure

Type of Identification Impurity Testing Assay

Analytical
Quantitative Limit
procedure
.Tests

Accuracy No yes No yes

Precision

Repeatability No yes No yes

Intern No yes No yes

precision

Specificity yes yes Yes yes

LOD No No yes No

LOQ No yes No No

Linearity No yes No yes

Range No yes No yes

The parameters that must be documented for methods in

USP assay category 3 are dependent upon the nature of the
test Dissolution testing for an example falls into this
category

85
 REF ERENCE STANDAR DS

According. to the national Bureau of standards (NBS) of

USA reference standards. are every well characterized
material which can be used for calibration of measuring
systems. It may be a device, a specific system or a substance
for which definite numerical values can be related to specific
characteristics e.g.

Reference methods
Reference data (e.g. specific absorption)
Reference spectra
Calibrated weights, thermometers
Reference solutions e.g. normal solutions
Reference substances e.g. primary standards
Types of Standards·
A reference standard (i:e. primary standard) may be obtained
from the USP/NF or other official sources. If there are
questions on whether a source of a standard would be
considered by FDA to be an official source, applicants
should contact the appropriate chemistry review staff. When
there is no official source, a reference standard should be of
the highest possible purity and be fully characterized.

A working standard · (in _house or secondary standard) is a

standard that is qualified against and used instead of the

86
reference standard.

Certificate of analysis .

A certificate of analysis (COA) for reference standards from

non official sources should be submitted in the section of the
application on analytical procedures and controls; For
standards from. official sources, the user should ensure the
suitability of .the reference standard. The standard should be
·stored correctly and used within the established use
intervaL

Characterization of a reference standard

Reference standards from USP/NF and other official sources

do not require further characterization. A reference standard
that is not obtained from an official . source should be of the
highest purity that can be obtained by reasonable. effort,
and it should · be thoroughly characterized. to ensure its
identity, strength, quality, purity, and potency. The
qualitative and quantitative analytical procedures used to
characterize a reference standard are expected to be different
from, and more extensive than, those used to control the
identity, strength, quality, purity, and potency of the drug
substance or the drug product. Analytical procedures used to
characterize a reference standard should not rely solely on
comparison testing to a previously designated reference
standard.

87
Generally, this characterization information should
include:

A brief description of the manufacture of the reference

standard, if the manufacturing process differs from that of
the drug substance. Any additional purification procedure,
used in the preparation of the reference standard should be
described.

Legible reproductions of the relevant spectra,

chromatograms this layer chromatogram (TLC) photographs
or reproduction. and. other. Appropriate instrumental recording.

Data establishing Purity are data obtained .by using

appropriate tests; such as TLC, gas. chromatography (GC),
'

high pressure liquid chromatography (HPLC), phase

solubility analysis; appropriate thermometric analysis
procedures, and others as necessary.

Appropriate chemical information, such as structural

formula, empirical formula and molecular weights should be
given. Information to substantiate the proof of structure
should include appropriate analytical tests, such as elemental
analysis, infrared spectrophotometry (IR), Ultraviolet
spectrophotometry (UV), nuclear magnetic resonance
spectroscopy (NMR), and mass spectrometry (MS), as well
as applicable functional group analysis. Detailed

88
interpretation of the test data in support of the claimed
structure should be provided

A Physical description of the material, including its color

and physical form should be given. Appropriate physical
constants such as melting range. Boiling range, refractive
index, dissociation constants (PK values), and optical
rotation.

89
 D RUG S T A B I L I T Y

Concept and Objective

Stability studies demonstrate that the necessary critical

Characteristics present at the time of production and release
can be expected to be present when the dosage form is
administered. If safety and efficacy values decline;
Stability studies provide information to determine when and
under what conditions the product should be withdrawn from the
market.

The quality control. department should evaluate the quality and stability.
of finished pharmaceutical products, and when necessary, of
starting materials and intermediate products.

The quality control department should establish expiry dates

and shelf-life specifications on the basis of stability tests
related to storage conditions.

A written programmed for ongoing stability determination

should be developed and implemented to include elements
such as:

a. A complete description of the drug involved in the

study.

b. The complete testing parameters and methods

describing all tests for potency, purity, and (physical
characteristics and documented evidence, that these tests
indicate stability.

90
c. Provision for the inclusion of a sufficient number of
batches

d. Provision of special storage conditions.

f. provision for adequate sample retention

g. A summary of all the data generated, including the

evaluation and the conclusions of the study.

Stability should be determined prior to marketing and

following any significant – change in Processes, equipment,
packaging and materials,....etc.

91
Purpose of Stability Testing

The main objectives of stability testing are:

1. To select adequate (from the viewpoint of stability)

formulations and container closure systems .

2. To determine shelf-life and storage conditions.

3. To substantiate the claimed shelf-life

4. To verify that no changes have been introduced in the formulation

or manufacturing· process that can adversely affect the stability of the
product.

The type of stability studies depends on the different phases of

drug and use.

a. In the development phase:

Accelerated stability studies and forced degradation are

used to predict the stability, shelf-life· and storage condition
of the final formulation. Real time studies have to be started
for confirmation.

b. For the registration dossier:

The results of stability studies from both accelerated and real

time studies for the final dosage form in its final container
and packaging should be submitted to the drug regulatory
authority. Studies at the worldwide climate zone will be
fruitful.

92
 c. In the post-registration period:

Real time stability studies as well as studies at different

climatic zones are used to .substantiate the expiry date and
the storage conditions.

93
 Degradation and Stability of Drugs

(In Bulk or Dosage Forms)

Drug Degradation; since most drugs are mostly organic

molecules; where chemical reactions leading to their
degradation are the opposite of the reactions going to their
synthesis However, they are quite different as illustrated by
the following points:

*Concentration: In drug synthesis high concentrations of

starting materials are usually used, while in drug degradation. The
concentration of drug is much small.

*Reagents: In drug synthesis reactive reagents; such as

sulfony chlorides (-SOCl2) or conc. sulfuric acid are
commonly used, while in drug degradation inert chemicals;
such as H20, air or light (hv), are among the 'Cause of
degradation.

*Condition: In drug synthesis drastic conditions, e.g.,

heating under pressure are usually applied, while in drug
degradation; the drug· is stored at ambient or cooler
temperatures under normal atmospheric pressure.

ROUTES OF DRUG INSTABILITY IN DOSAGE

FORMS

*Chemical Degradation: hydrolysis, oxidation,

decarboxylation, dehydration, photochemical decomposition
and racemization.

94
*Physical degradation: polymorphism, vaporization,
adsorption, particle sedimentation, etc...

*Chemical incompatibilities

CHEMICAL DEGRADATION ROUTES

1- Hydrolysis: Several drugs undergo chemical degradation

through hydrolysis; the following are some illustrative examples

Category Groups Example(s)

Esters RCOOR' e.g., aspirin, clofibrate,

chloramphenicol succinate, etc.

Amides R-CO-NR'R ·e.g., chloramphenicol, etc. .

Lactams e.g., penicillins & cephalosporins

Malonylurea e.g., barbiturates

Sulfonamides R-SO2-NH.R e.g., bactericidal sulfa-drugs

2- Oxidation: It is usually induced via reaction with
atmospheric oxygen under normal temperature. It is
commonly referred to as auto-oxidation. Auto-oxidation
(aerial oxidation) involves mostly free-radical reactions. The
oxidation products are. ·usually, ·more conjugated; thus the

95
 appearance Of or change in the color of dosage from
suggests the occurrence of oxidative degradation
(chromophoric changes).

The following are examples of drugs subjected to autoxidation.

Category Groups Example(s)

*Phenols & e.g., steroids&
catecholes catecholamine's

* Enediol ·e.g.,VitaminC,.

*Thiols R-CH2SH e.g., penicillamine,

(thiohydrils) Captopril

*Ethers & e.g., barbiturates

thioethers

*Aldehydes R.CHO e.g., acetaldehyde

*Nitrites R.NO2 e.g., sulfa-drugs
organo-nitrites
3- Decarboxylation
.
Pyrolytic solid-state
'
or solution degradation through
decarboxylation is not commonly encountered: as relatively
high heat of activation is required However, naproxen and
related drugs such as ibuprofen and ketoprofen can show
decarboxylation..

96
Also p-aminosalicylic acid (PAS) shows decarboxylation to give m-
arninophenol (m-AP). ·

4- Photochemical Degradation

Absorption of light (UV or visible) by the ground state of a

molecule generates electronically excited states
Electronically excited states are electronic isomers of the
ground state, and show a different chemistry. Chemical
changes can occur as a result of dissociation of the,
absorbing molecule into reactive fragments, direct reaction of the
electronically excited species, spontaneous isomerization or other
routes.

97
indomethacin is quite photo-stable in the solid state (7.5°/o
decomposition after 72h irradiation) but ·photo-labile in
solution. The usual decarboxylation is the main process,
when mercury lamps · are used, while daylight irradiation
leads to products conserving the carboxyl group which have·
been rationalized as arising via the acyl radical.

5- Aminosalicylic acid, used for the treatment of chronic

inflammatory bowel diseases, undergoes light accelerated
oxidation and polymerization.

98
Dechlorination occurs, one of the general photochemical
reactions of aromatics. Sequential loss of both chlorine
atoms is followed by ring closure, reasonably via radical
addition, to yield the carbazole-1- acetic acids (i) and (ii) as
the main products.

5- Racemization
It is changes of the optical activity that affect greatly he
.
biological activity Usually, I-form (-) is more
pharmacologically active than d-form (+),
e.g., I-epinephrine is 20 times as active as d- epinephrine.

6·Dehydration

Removal of the water of crystallization may change the crystal

form or give the anhydrous form which would have different
dissolution behavior. e.g. ampicillin trihydrate.

On the other hand, if dehydration creates a double bond, i.e.,

it will change the chemical structure and ·thus the biological
action·, e.g. tetracycline.

7- Chemical _Incompatibility

A typical example of chemical incompatibility is the

99
inactivation of aminoglycoside antibiotic (gentamycin) by
penicillin in admixtures for injection Apparently an inactive
complex (Schiffs base) is formed between these classes of
antibiotics. Drugs containing amino- functional group(s) are
the source of drug-drug chemical incompatibility.

i- In Solutions

Sodium metabisulfite (NaHSO3) is incompatible with

catecholamines (adrenaline & nor-adrenaline) in solutions for
injection.

ii. In Solid Formulations:

Pheylephrine and. codeine· are acetylated when admixed with aspirin.

100
101
 STABILITY Indicting ASSAY

Methods (SIAMs)

To carry out stability-testing, specially chemical stability;

the analyst should develop the analytical methodology
capable to monitor the, concentration of the intact, drug
.(analyte) in pharmaceutical products. This canbe
approached . via Stability- lndicating Assay Methods
(SIAMs). SIAM is defined -as "a procedure that ,would
determine the concentration of the intact drug as well as its
degradation. product(s)".

Most compendial assays for drug substances are relative.ly

non-selective methods. They are chosen for their simplicity
and accuracy however they are supported by other
monograph parts which . Provide enough information on
the drug quality (i.e. purity merits).

SIAMs would be achieved via different approaches as follows:

I- Development of highly selective method for the intact

drug where degradation product(s) do not interfere.

i- Ferric hydroxamate colorimetric method ·

If can be considered SIAM' for lactams (penicillins or

,,

cepfiatosporins), amides (chlorarnphenicol), lactones·

(pilocarpine) and esters (clofibrate).

102
The· hydroxamic acid, derivative gives with Fe3+ in acid
,medium a colored ferric chelate, ·while RCOOH& R'OH, hydrolytic
products, do not react similarly under these conditions.

II- Acid-base titration of p-aminosalicylic acid (PAS), which

contains m-aminophenol as the major degradate on
decarboxylation. The degradate has no -COOH, which can
indicate only the PAS.

III- UV- Spectrophotometric methods

a- Difference Spectrophotometry : Changing the PH- values of the

drug solutions would induce spectral changes as in case of barbiturates,
this can be measured by the ∆A- Method

b- Derivative Spectrophotometry

Such methods would eliminate interference· of degradation

product(s) especially if the sample is not highly degraded.

103
iv- Spectrofluorimetric Methods

These methods are more selective and sensitive than UV-

methods due to the two wavelengths λex.(excitation) &
λem (emission), which makes the λ choice is very 'selective.

II- Development of highly selective method for the assay

of the degradation product.

Aspirin (acetyl salicylic acid) can be determined via its

degradation. It may contain salicylic acid and acetic acid as
degradation products The salicylic acid (in the degraded
sample) as well as that of another completely hydrolyzed
sample is complexed separately with Fe 3+ to give a violet
colored complex .equivalent to the extent of salicylic acid in
both samples. The completely degraded sample gives more
color than the other, where · the difference is due to the
amount of intact aspirin.

104
Ill- Simultaneous separation and quantification of both intact
drug and degradation product(s) by chromatographic
(separation) techniques.

i- Gas Chromatographic (GC) methods

ii- Thin-layer Chromatography TLC/ Densitometry

iii- High Performance Liquid Chromatography (HPLC)

Furosemide, an anthranilic acid derivative, undergoes

degradation in aqueous solutions under the influnce of UV
radiation. Furosemide seems to undergo photo-oxidation,
hydrolysis and dechlorination. At high temperature,
furosemide is hydrolyzed to 4-chloro-5-
sulfamoylanthranilic (CSA)" acid and furfuryl alcohol,
which is quickly converted into levulinic .acid.

Figure 10: Chromatogram of furosemide and its ph otodegradation products .

105
Practical Part

106
 GUIDELINES TO LABORATORY REQUIREMENTS
AND DESCIPLINE

1. The Laboratory work consists of a number of practical periods,

as well as problem -solving sessions.
2. Before beginning the work in the Laboratory, clean the bench
top and your glassware.
3. Never indulge in horseplay· or behavior that .could lead to
injury of others.
4. Food, drink and gum are prohibited in the Laboratory.
5. Upon completion of work, wash and dry all equipment's, your
bench and your clean-up area.
6. Each student will carry out the experiment, collect data and
submit the report of each Laboratory in the format included.
Also he/she should provide any additional experiment
material.
7. The reports will be graded and will be given 5 points.
8. A grade of 25 points will be assigned to the final practical
exam.

107
Lab No. Contents

1 - Colorimetric determination of KMnO4.

2 - Assay of zinc content in Octozinc capsules and

Priceline Zinc eye drops.

3 - Assay of indomethacin. (lndocid Capsules)

- Assay of iron-content. (Pediatric Ferrous Sulfate Oral

Solution)

- Assay of phenol content of an antiseptic

preparation.
4 - Assay of anhydrous theophylline (Quibron tablets).

- Assay of antibiotics (Epicocillin injectable

preparation.

5 - Spectrophotometric justification of purity index.

(illustration).

- Spectrophotometric methods for the assay of

furosemide (Lasix injectable preparation)
6 -Assay of a two component mixture (Tincture of

lodii)

-Assay of calcium-content (Calculim Gluconate

Ampoules)

108
 7 -Assay of sulfadiazine (Veterinary Powder)

-Application of spectrophotometry in the assay of

nalidixic acid (Nalidern Tablets)
8 -Application of potentiometry to the assay of tolbutamide
(Rastinone Tablets)
. 9 -Practical Examination.

109
 Lab.1

Assay of KMnO4 Solution as antiseptic.

A- Constrction of calibration curve and calculations:

-4
1) ) Fonn a standard solution of KMnO4 sulfate (2.5x 10 M), take
0..4,l ,2,3,4,5 & 6.mL, separately into a series of 100-mL

volumetric flasks.

2) Complete to mark with dilute H2SO4 and mix well.

3) Measure the absorbance of each dilution 3 times at 510 nm

against blank (dilute H2SO4).

4) Tabulate the results and plot a calibration curve between the

mean absorbance (A) of each dilution and the concentration gm % (2.5

10-4 M x M.wt. x mL /1000).

5 ) From the curve calculate LOD, LOQ, linearity range & the
regression equation. (A = slope x C ± intercept).

Calibration curve

110
6) Using your regression equation to calculate the found
concentration for each dilution and get the recovery % Recovery % =
(found conc. I claimed cone.) x 100.

7) Calculate SD fro1n the calculator and RSD from the fallowing

formula:

RSD = SD x 100/ X

X (grand mean) = 𝛴 recovery %/n.

8) Determine the ruggedness of method by calculating SD and RSD

between measurements of different students

111
 Calculations :

Ml Claimed conc. A A Found conc. Recovery %

g%

112
 Ruggedness table

Analysts Claimed Absorbance A Found Recovery

conc. A conc. %

113
LOD = ………………………………………………

LOQ = ………………………………………………

Linearity range = ……………………………………

Regression equation = ………………………………

SD of calibration = …………………………………..

RSD % of calibration = ……………………………...

SD of Ruggedness = …………………………………

RSD % of Ruggedness = …………………………….

Recovery % of Ferrofol capsules ……………………

114
 Lab.2

Assay of zinc-content in Octozinc® capsules

Proced ure

1- Transfer the contents of two capsules quantitatively into 100-mL

measuring flask.
2- Add 50 mL distilled water, shake well for 5 min. for complete
extraction, complete to the mark with water, mix and then filter.
3- Discard first 5 mL of the filtrate.
4- Take 10 mL of the filtrate in conical flask + 2 mL NH3 buffer + few
speaks of. EBT (purple), and titrate with 0.01 M EDTA till full blue
color.

Calculation

Each lmL of 0.01 M EDTA equivalent to 0.002874 g =2.874 mg

𝑚𝑙𝑠 ×𝑓×𝐹×100
Found conc. of ZnSO4 (in 2 capsules) = mg%
10

Claimed amount of zinc sulfate in two capsule is 220 mg

𝐹𝑜𝑢𝑛𝑑 𝑐𝑜𝑛𝑐.
Recovery % = X 100 = X100= %
𝑐𝑙𝑎𝑖𝑚𝑒𝑑 220

115
 Report on assay of Prisoline® zinc

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation:

………………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..
……………………. ……………………………..
Chemical Structure(s)

Zn2+ (Zinc)
Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

Experimental Data and Calcula tions

E. No. Start Point (mL) End Point (mL) Volume (mL) Remarks

1…………………………………………………………………

2…………………………………………………………………

3…………………………………………………………………

CONCLUSION
……………………………………………………………

116
 Lab.3

Assay of indom ethacin (Indocid®·capsules)

Procedure

1-Dissolve the content of one capsule (25 mg) in a beaker by the

addition of 10 ml of acetone.
2-Shake then filter into a conical flask and wash with 10 ml of
acetone.
3-Titrate the filtrate with 0.01 N NaOH using ph.ph as an indicator.

F = l ml N/100 NaOH 0;003573 g indomethacin

STUDENT'S REMARKS AND CALCULATIONS

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

117
 Report on assay of indomethacin
Student's Name: ....... Student's No.:………..
Name of pharmaceutical Preparation: ……………….
manufacturer: ...…………………………………
Pharmaceutical Form: ...............………………………………
Active Ingredient(s): Claimed Amount (ug, mg,
g)
……………………. ……………………………..
……………………. ……………………………..
Chemical Structure(s)

Principle of Assay [Chemical Equation(s); whenever

possible]
………………………………………………………………
………………………………………………………………
………………………………………………………………
Experimental Data and Calcula tions
E. No. Start Point {mL) End Point (mL) Volume (mL)
1……………………………………………………………
2……………………………………………………………
3……………………………………………………………
CONCLUSION
…………………………………………………………

118
 Assay of iron-content in Pediatric Ferrous

Sulfate Oral Solution

Procedure

1- Pipette 10 ml of the sample into a conical flask.

2- 2- Add 20 ml of 1.5 N H2SO4
3- Titrate against 0.1N KMnO4 till the appearance of permanent pink
color.

STUDENT'S REMARKS

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

………………………………………………………………………

119
 Report on Assay of Ferrous content

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Fe2+ (Ferrous-Iron)

Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

Experimental Data and Calcula tions

E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks

1…………………………………………………………………

2…………………………………………………………………

3…………………………………………………………………

CONCLUSION
……………………………………………………………

120
 Assay of Phenol Content of an

Antiseptic Preparation.

Procedure

1- Pipette 10 ml sample into a glass stoppered conical flask

(GSCF) and dilute to 25 with distilled water.
2- Pipette 25 ml of 0.05N KBrO3/KBr aqueous solution to the sample
into GSCF.
3- Add 5 mL of conc. HCI and immediately stopper the flask, the
stopper is to be moisten with KI Solution.
4- Allow the mixture to stand for 15 minutes in the dark with
occasional gentle shaking.
5- Add 10 ml of KI solution and shake. Wash the stopper and the
neck of the flask with few mLs of distilled water
6- Add 2 ml CHCL3 (Why?)
7- Titrate the indicator, added near the end point.
8- Carry out a blank experiment by pipetting 25 ml of 0.05N
KBrO3/ KBr-aqueous solution then add 5 ml conc. HCL and
continue the same steps as previously mentioned.

STUDENT'S REMARKS

………………………………………………………………………

………………………………………………………………………

121
 Report on Assay of Phenol

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions

E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks

1…………………………………………………………………

2…………………………………………………………………

3…………………………………………………………………

CONCLUSION
……………………………………………………………

122
 Lab.4

Assay of Theophylline (Quibron TM Tablets)

Procedure

1- Weigh accurately ·10 tablets, find out the average. weight of the tablet;
and then grind in ·a mortar.
2- In a conical flask, place an accurately weighed ·amount
equivalent to 100 mg of anhydrous theophylline.
3- Add 4 mL of 6 N NH3 solution, stir to dissolve the powder.
4- Pipette 25mL of 0.05N AgNO3 into the conical flask.
5- Boil for 15 minutes, cool and filter.
6- Wash the ppt three with three portions of distilled water (10 ML
each) and add the washings to the filtrate.
7- Acidify .with 10 mL .dil..HNO3, cool, add 1mL ferric alum solution.
8- Titrate with 0.05N NH4SCN to the first appearance of red color in the
supernatant liquid. .

STUDENT'S REMARKS:

……………………………………………………………………………

……………………………………………………………………………

……………………………………………………………………………

……………………………………………………………………………

……………………………………………………………………………

……………………………………………………………………………

123
 Report on Assay of Theophylline

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions

E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks

1…………………………………………………………………

2…………………………………………………………………

3…………………………………………………………………

CONCLUSION
……………………………………………………………

124
 Assay of Ampicillin (Epicocillin® Injectable Preparation)

Proced ure

1- Dilute ..an accurately measured sample equivalent to about l 00 mg

ampicillin to 100 mL with distilled water.
2- Transfer 10 mL of this solution by bulb pipette into a glass
stoppered conical flask, add 5 mL O.l N NaOH. Let ·it to stand .for
15 minutes·.
3- Add 5 mL of IN HCl, 20 mL of buffer solution pH 4.6 and pipette
into the· mixture 25 mL of 0.02N iodine solution. Stopper the flask
and stand in the dark for 15 minutes.
4- Titrate against 0.02N Na2S203 Solution in presence of 1.0 mL starch
solution added near the end point. .
5- Carry out a blank experiment, by adding to another 10 mL of the
solution; add 20 mL of the buffer solution and 25 mL of 0.02N I2
solution. Stopper .the flask and let it stand in the dark for 15
minutes and titrate against 0.02N Na2S 2O3 solution using starch
solution as indicator.

N..B.

The difference between the two titrations represents the quantity

of iodine corresponding to the ampicillin content.

125
 Report on Assay of Ampicillin

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions
E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks
1…………………………………………………………………
2…………………………………………………………………
3…………………………………………………………………
CONCLUSION
……………………………………………………………

126
 Lab 5

Spectrophotometric purity index

The presence of impurity can cause increase in the intensity of

absorption. To determine the whether the sample is impure or not,
we choose 2 𝜆 (wavelengths, nm); ·one of them 𝜆 & any other one
and take absorbance (A) at the 2 𝜆 for the test sample and the
reference standard.

1. Impurity index· (I.I.)

𝐴1 𝐴1
I.I = − , 𝑖𝑓
𝐴2 𝐴2

I.I =Zero in pure samples

I.I increases as impurity increase·

Where A' = Light absorbance of test sample.

A = Light absorbance of reference standard

2- Spectrophotometric purity index (S.P.I.)

𝐴1/𝐴2
S.P.I
𝐴1/𝐴2

S.P.I = 1 in pure samples

127
 Assay of Furosemide (Lasix® Injectable
Preparation)

Procedu re

1- Dilute an accurately measured sample equivalent to about 100 mg

furosemide to 100 mL with 0.02 N NaOH.
2- Mix well and allow to stand for 5 minutes
3- Dilute 1mL to 10 mL distilled water.
4- Measure the absorbance of final solution at 271 run, using 1 cm
cell against 0.02 N NaOH solution as a blank.

N.B.:(Al%, lcm) of furosemide at 271 nm = 580.

Ultraviolet Spectrum, (Al%, lcm) in aqueous acid at 235 nm =

1333,at 274 nm = 600; in aqueous alkali.at 271nm = 580.

STUDENT'S REMARKS:

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

128
 Report on Assay of Furosemide

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions
E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks
1…………………………………………………………………
2…………………………………………………………………
3…………………………………………………………………
CONCLUSION
……………………………………………………………

129
 Lab.6

Assay of a Two Component Mixture (Tincture of lodii)

Proced u res

Iodide content:

1- Pipette 10 mL of the preparation into a GSCF.

2- Add 20 mL of distilled water.
3- Titrate against 0.1N Na2S2O3 solution using starch solution as
indicator.

Iodine conten t:

1- Pipette 10 mL of the preparation into a GSCF.

2- Add 10 mL of H20 and 25 mL of cone. HCI.
3- Cool under tap water.
4- Titrate against 0.05 M KLO3 solution using CHCL3 or CCl4 as
indicator. Near the end point shake vigorously till the
disappearance of the purple color in the organic layer (The
aqueous layer is yellow in color.

STUDENT'S REMARKS:

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

130
 Report on Assay of iodin and Iodide

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

I2 (Iodine) & I- (Iodide)

Principle of Assay [Chemical Equation(s); whenever possible]
…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions
E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks
1…………………………………………………………………
2…………………………………………………………………
3…………………………………………………………………
CONCLUSION
……………………………………………………………
……………………………………………………………
……………………………………………………………

131
 Assay of Calcium-content

(Calcium Gluconate Ampoules)

Procedure

1- Pipette 5 mL sample into a beaker.

2- Add 2 mL of · dil. HCI, render alkaline with ammonia solution.

3- Add 20 mL of hot ammonium oxalte solution, while stirring.

4- Boil for three minutes and leave to stand for 30 minutes at room
temperature. ·

5- Filter, wash the ppt with warm distilled water till the filtrate is free
from oxalate (KMnO4 -Test)

6- Transfer the ppt. into a beaker by piercing the filter paper and
dissolving in 50 mL dil, H2SO4

7- Heat to 60°C and titrate against 0.1N KMnO4 till the first
appearance of permanent pink color.

STUDENT'S REMARKS:

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

132
 Report on Assay Calcium-Content

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Ca2+ (Calcium)
Principle of Assay [Chemical Equation(s); whenever possible]
…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions
E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks
1…………………………………………………………………
2…………………………………………………………………
3…………………………………………………………………
CONCLUSION
……………………………………………………………
……………………………………………………………
……………………………………………………………

133
 Lab..7

Assay of Sulfadiazine (Veterina ry Powder)

Procedu re

1- Pipette 10 mL of sample into a beaker.

2- Add 10 mL cone. HCl.
3- cool to 0°C by adding ice cubes into the beaker.
4- Titrate against 0.1M NaNO2 (dip the burette nozzle under the
surface of solution. Why?)
5- After ·every 0.5 mL take a drop of the Solution and test for excess
titrant using KI/ starch paper.
6- End point is achieved.when drop of the Solution gives an
immediate blue color with KI / starch paper.

STUDENT'S REMARKS:

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

134
 Application of Spectrophotome try in assay of Nalidixic acid
(Naliderm® Tablets)

Procedure

1- Weigh and powder 20 tablets.

2- To a quantity of the powder equivalent to SO mg of nalidixic acid,
add 75 mL of t ·N NaOH, shake for three minutes.
3- Dilute to 100 mL with 1 N NaOH & mix well then leave for 15
minutes.
4- Dilute 1 mL to I 00 mL with distilled water then measure the
absorbance of the final Solution at 334 run, using 1 cm cell, against
0.01 N NaOH as a blank.

N.B : The specific absorbance at 334·nm of nalidixic acid is 494.

UV Spectrum, Aqueous acid-257 (Al%,lcm == 1233b), 315nm;

aqueous alkali-258 (Al%,lcm = 1120a),334 nm.

STUDENT'S REMARKS:

…………………………………………………………………

135
 Reporton Assay of Nalidixic acid

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions
E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks
1…………………………………………………………………
2…………………………………………………………………
3…………………………………………………………………
CONCLUSION
……………………………………………………………
……………………………………………………………
……………………………………………………………

136
 Lab. 8

Potentiometric Determination of Wealdy Acidic

Pharmaceuticals

Potentiometric Assay of Tolbutamide (Rastinone® tablets)

Sample solution

Digest an aliquot of the powdered tablets with a measured volume

of suitable solvent. (Instructions will be given in the
Laboratory)

Titrant: ·NaOH of suitable normality as directed in the Laboratory.

Equipments:

- pH meter

- Glass/SCE system or combination electrode.

- Beaker l 00 mL capacity and burette.

- Magnetic stirring set-up.

N.B.: PH meter should be calibrated with buffer pH 7.0

and checked for operation with buffers pH 4.0 and 9.0

Assay: Weigh and powder 20 tablets. To an accurately weighed

quantity of the powder equivalent to about 0.5 g of tolbutamide
add 50 mL of alcohol (95%), warn to dissolve. Cool to room
temperature, add 25 mL of distilled water and titrate with O.1N
NaOH. Determine the end point potentiometrically.

Each mL of 0.1N NaOH is equivalent to 0.02704 g of tolbutamide.

137
 Data and Graphical Presentation:

1- Get the pH of the system as function of the volum e of titrant.

2- Plot the titration curve.

3- Plot the 1st derivative titration curve.

4- Get the equivalence point and calculate the percentage recovery.

5- Get the apparent pKa of the drug.

STUDENT'S REMARKS:

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

…………………………………………………………………

138
 Reporton Assay of Tolbutamide

Student's Name: ....... Student's No.:…………..

Name of pharmaceutical Preparation: ……………….

manufacturer: ...………………………………………

Pharmaceutical Form: ...............……………………………………

Active Ingredient(s): Claimed Amount (ug, mg, g)

……………………. ……………………………..

……………………. ……………………………..

Chemical Structure(s)

Principle of Assay [Chemical Equation(s); whenever possible]

…………………………………………………………………
…………………………………………………………………
…………………………………………………………………
Experimental Data and Calcula tions
E. No. Start Point {mL) End Point (mL) Volume (mL) Remarks
1…………………………………………………………………
2…………………………………………………………………
3…………………………………………………………………
CONCLUSION
……………………………………………………………
……………………………………………………………
……………………………………………………………

139
 Summary

Criterion Evaluation of the criterion

1. Accuracy % Recovery

2. Precision RSD

3. Linearity From Figure

4. LOD From figure

5. LOQ From figure

6. Ruggedness RSD of different analysts. And

different instruments

7. Robustness ……………..

8. Specificity Determined according to the %

recovery, if not within limit,
therefore interference from
additives may occur resulting in
low specificity

140
 REF ERENCES

1. Course notes

''Topics in Chemical Quality-Control in Pharmacy Practice" by staff

of Analytical chemistry department

2. Good Laboratory Practice (GLP), Quality practices for regulated

non-clinical research and development. Geneva, UNDP/World
Bank/WHO, Special Programme for Research and Training in
Tropical Diseases, 2001.

3. The good automated manufacturing practice (GAMP) guide for

validation of automated systems in pharmaceutical manufacture
(GAMP4). ISPE - International Society for Pharmaceutical
Engineering, December 2001.

4. International Conference on Harmonization (ICH) Guidelines.

Tripartite Harmonized Guidelines for Good Clinical Practice, Step 4.
Geneva, ICH Secretariat (IFPMA), 1996.

5. Guidelines for good clinical practice (GCP) for trials on

pharmaceutical products. Geneva, World Health Organization, 1995
(WHO Technical Report Series, No. 850.

6. Directive 2001/20/EC of the European Parliament and the Council,

"approximation of the laws, regulations and administrative
provisions of the Member States relating to the implementation of
good clinical practice in the conduct of clinical trials on medicinal
products for human use". Official Journal of the European
Communities, 1 May 2001;

141
7. Multisource (generic) pharmaceutical products: guidelines on
registration requirernents to establish interchangeability. Geneva,
World Health Organization

8. ation, 2006 (WHO Technical Report Series, No. 937.

142