Pharmaceutical Product Registration

Azithromycin Powder for Oral Suspension 200 mg/5 ml

Advisor
Assoc. Prof. Dr.Thanaporn Amnuaikit
Asst. Prof. Dr.Kasemsiri Chandarajoti
Dr.Paweena Wongwitwichot

Summitted by

6110710020 Ms.Natthakan Watthanathongkul

6110710021 Ms.Nathnicha Yokyong
6110710023 Mr.Nuttapat Pairith
6110710024 Ms.Nattarika Rodjana
6110710025 Ms.Neeranuch Techapanuwat
6110710026 Mr.Dantrai Khansang
6110710028 Mr.Tawesak Jenyongsak
6110710030 Mr.Thana Akkharakunphong
6110710031 Ms.Thisana Yaoduang
6110710033 Ms.Nurwanee Karemka
6110710034 Mr.Burapol Chanrak
6110710035 Mr.Papangkorn Thanissorn
6110710037 Ms.Pajera Chumcheodpattarakul
6110710038 Ms.Piyamon Pimonrat
6110710041 Ms.Pimmada Petchara
6110710042 MS.Penpitcha Soonsim
6110710045 Ms.Manassawee Bairaham

Assurance of Pharmaceutical Product

Faculty of Pharmaceutical sciences
 Acknowledgment
We would like to express our sincere thanks to my advisor , Assoc. Prof. Thanaporn
Amnuaikit , Asst. Prof. Kasemsiri Chandarajoti, Dr. Paveena Wongwitwichot for invaluable help
and constant encouragement throughout the course of this repot, Their suggestion and
instruction have served as the major contributor towards the completion of this project.
finally we would like to thanks Pharmaceutical technology IV course, that provide an
opportunity to learn the process of drug production and drug registration. Also , giving us the
knowledge about the process of drug production and drug registration which is intensely useful
for our project.

Production team

a
 Preface

This report is a part of the Pharmaceutical Technology IV course (581-431). The purpose
of this report is to educate about RA document and to practice in pharmaceutical preparations
development, quality assurance and drug registration. in this report presents "Azithromycin
Powder for Oral Suspension" which is consist of 2 topics as following; Part S Drug substance:
Azithromycin and Part P Drug product: Azithromycin Powder for Oral Suspension We are
hopefully that whoever may concern this report would definitely find it useful and worth
learning.

Production team

b
 Table of content
Contents Page
PART II: Quality Document
Section C: Body of Data
S Drug Substance: Acetaminophen
S1 General Information 1
S2 Manufacture
S3 Characterization
S4 Control of Drug Substance 15
S5 Reference Standard or Materials 80
S6 Closure Container System 91
S7 Stability 92
P Drug Product: Azithromycin oral suspension
P1 Description and Composition 99
P2 Pharmaceutical Development 101
P3 Manufacture 153
P4 Control of excipient 160
P5 Control of finish Product 257
P6 Reference Standards or Materials 334
P7 Container Closure System 337
P8 Stability 354

c
 Part S
Drug Substance
S1 General information
1.1 Nomenclature
International Nonproprietary Names : Azithromycin dihydrate
Compendial name : Azithromycin
Registry Number of CAS : 117772-70-0
IUPAC name : (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-
11-[(2S,3R,4S,6R)-4-(dimethylamino)-3-
hydroxy-6-methyloxan-2-yl]oxy-2-ethyl-
3,4,10-trihydroxy-13-[(2R,4R,5S,6S)-5-
hydroxy-4-methoxy-4,6-dimethyloxan-2-
yl]oxy-3,5,6,8,10,12,14-heptamethyl-1-
oxa-6-azacyclopentadecan-15-
one;dihydrate
1.2 Structure

1.3 General properties

Physiochemical properties
- Appearance : A white or almost white powder.
- pka : Azithromycin has two tertiary amine whose pka value are 8.1 for the ring nitrogen
and 8.8 for the desosaminyl nitrogen

1
- Solubility characteristics: solubility greater than 100 mg/ml in acetone, acetonitrile,
chloroform, dimethylformamide, dimethyl sulfoxide, ethanol, ethyl acetate, isopropanol and
methanol; Azithromycin dihydrate has solubility greater than 100 mg/ml at pH values lower
than 5. The compound degrades under acidic conditions. At pH values above 8, the
solubility of Azithromycin is less than 0.2 mg/ml. Azithromycin is practically insoluble in
water and freely soluble in anhydrous ethanol and methylene chloride.
- Melting behavior: 124 C - 128 C (with decomposition at ca. 250°C). These values have
been determined by DSC.
- Optical rotation: measured in a 2 % w/v solution in absolute ethanol at 20°C, on
anhydrous, solvent-free basis (using sodium D-line), the values being -45° to -49 °.
- Boiling point, enthalpy of vapor, flash point, and vapor pressure: The calculated value
of the boiling point of azithromycin under a pressure of 760 mmHg was 822.1 °C. The
enthalpy of vapor calculated value was 135.99 KJ/mol. The value of flash point was found
to be 451 °C, and the vapor pressure was calculated to be 251 x 10-31 mmHg at 25 °C
- Partition coefficient: The octanol/water partition coefficient (Kow) of azithromycin was
0.65 at 20 °C and pH 7. Adsorption isotherm studies indicated that the thermodynamic data
revealed that the adsorption of azithromycin on the surface of zinc was endothermic,
spontaneous, and consistent with the adsorption model of Langmuir.
- Hygroscopicity The anhydrous form of AZI seemed to be unstable since it converted to
dihydrate on storage at room temperature. On the other hand, monohydrate in the
presence of moisture can convert to the more stable dihydrate form. Therefore, the most
stable form of AZI is dihydrate.

Reference
1. PT_H_1475_002_PAR.pdf [ 28 2021]. Available at:
https://mri.cts-mrp.eu/Human/Downloads/PT_H_1475_002_PAR.pdf

2. Bakheit AHH, Al-Hadiya BMH, Abd-Elgalil AA. Chapter One - Azithromycin. Brittain
HG, Profiles of Drug Substances, Excipients and Related Methodology
[ Academic Press; 2014 [ 20 2021]. 1 40. Available at:
https://www.sciencedirect.com/science/article/pii/B9780128001738000015

2
S2 Manufacture
2.1 Manufacturer

Manufacturer of Azithromycin raw material

Gold Biotechnology®

ADDRESS : Inc.1328 Ashby Rd. St. Louis, MO 63132

PHONE : (314)890-8778
FAX : (314)890-0503
EMAIL : contactgoldbio86@goldbio.com
WEBSITE : https://www.goldbio.com/
*** According to certification document attached in appendix

3
S3 Characterization
To structurally elucidate Azithromycin dihydrate Active Pharmaceutical Ingredient (API)
Batch no S4008044061 manufacturing by, the said substance will be subjective to numeral
spectral analysis which the results have to conform with the existing Azithromycin dihydrate
Reference Standard provided by USP under the same conditions.
3.1) Structure Elucidation
3.1.1) Differential scanning calorimetry (DSC)
The differential scanning calorimetry (DSC) thermogram of Azithromycin dihydrate RS
was recorded on a DSC (Mettler, Toledo DSC 821 Switzerland) using Mettler Star system. The
curve shown in Figure 1.1 and 1.2 were collected from 0 to 250 C with nitrogen purging (80
ml/min) and using a heating rate of 10 C/min. DSC thermograms of Azithromycin dihydrate is
shown in Figure 1

Figure 1 DSC thermogram of Azithromycin dihydrate RS, which was generated by

Mettler Star system using sealed pan.

4
 In the same manner as Azithromycin dihydrate RS, The sample of Azithromycin
dihydrate manufacturing by O-LEAVES Labeled as batch no S4008044061 Also analyzed with
DSC which the thermogram is shown in figure 2

Figure 2 DSC thermogram of Azithromycin dihydrate Batch no S4008044061, Which

was generated by Mettler Star system using sealed pan.
3.1.2) Karl Fisher titration (KFT)
Water content in Azithromycin dihydrate was determined using a Karl Fischer titrimeter
(Metrohm, 716 DMS, Switzerland). Samples (20 25 mg) were accurately weighed and quickly
transferred to the titration vessel containing anhydrous methanol. The water content is shown
in Table 1
Table 1 Thermal analysis and KFT of different from of AZI
Sample name DSC analysis Water content
Temperature range (°C) Heat fusion (J/g) TGA (%) KFT (%)
Azithromycin 139.88 156.31 92.83±2.41 2.472±0.41 2.39±0.79
dihydrate

3.1.3) Elemental analysis

The percentage amount of carbon, hydrogen, nitrogen in Azithromycin Dihydrate
batches were determine by using
5
Table 2 Comparing Elucidation of Elemental analysis of Azithromycin Dihydrate RS from USP
compared with Theoretical value from compendial sources.
Compendial Experimental Value of Azithromycin
Elements
Theoretical value (%) Dihydrate RS
C 60.94 60.82
H 9.69 9.70
N 3.74 3.70
O 25.63 25.78

Table 3 Elucidation of Elemental analysis of Azithromycin Dihydrate from batch no

S4008044061 from manufacturer compared with Theoretical value from compendial sources
and Reference Standard from USP.

Experimental Value Experimental Value

Elements Theoretical value
of Azithromycin RS. of Sample
C 60.94 60.93 60.92
H 9.69 9.70 9.72
N 3.74 3.72 3.73
O 25.63 25.65 25.63

Table 4 Elucidation of Elemental analysis of Azithromycin Dihydrate from batch no

S4008044062 from manufacturer compared with Theoretical value from compendial sources.

Experimental Value Experimental Value

Elements Theoretical value
of Azithromycin RS. of Sample
C 60.94 60.94 60.90
H 9.69 9.68 9.72
N 3.74 3.77 3.75
O 25.63 25.61 25.63
3.1.4) UV spectrum
The ultraviolet spectrum of azithromycin dihydrate in methanol and mobile phase
(methanol: acetonitrile: phosphate buffer pH 6.7: tetrahydrofuran, 15:25:60:2.5, v/v) shown in

6
Figure A, B. The figures were recorded using a double beam Model GBC 916 UV VIS
spectrophotometer (GBC Scientific Equipment Pty Ltd., Melbourne, Victoria, Australia). The
values of wavelength maximum in nanometer ( max) are 201.6 nm on methanol and 199.2
nm on mobile phase. The spectra of azithromycin dihydrate in methanol (Figure A) and mobile
phase (Figure B) were shown below.

Figure 3 : The ultraviolet spectrum of AZI Figure 4 : The ultraviolet spectrum of AZI
dihydrate in methanol. dihydrate in mobile phase.
3.1.5) IR spectrum
The infrared absorption spectrum of azithromycin dihydrate is shown in Figure 5 It was
obtained in a KBr disc using a FT-IR Nicolet® Imoact 410 instrument, infrared
spectrophotometer. The principal peaks were 3561, 3496, 1344, 1282, 1269, 1251, and 1083.

Figure 5.1 : Infrared absorption spectrum of azithromycin dihydrate

7
 Figure 5.2 : Infrared absorption spectrum of azithromycin dihydrate from batch S4008044061
Infrared absorption spectrum of azithromycin dihydrate from our analyze
We have demonstrated that IR spectrum from both drug master file and our analyze
have corresponding principal peaks.

Wavenumber (Cm-1) Functional group

3496.31-3561.88 OH
<3000 C-H stretch
1735 C=O stretch
1251.57, 1282.43 CH3 stretching

3.1.6) Mass spectrum

The mass spectrum of azithromycin dihydrate was obtained utilizing A Thermo
Scientific TSQ Quantum mass spectrometer systems feature HyperQuad quadrupoles.
The MS analysis was performed with electro-spray ionization (ESI) interface in both
negative and positive ion modes.
TSQ Quantum Ultra AM Instrument Conditions: Ionization mode and source: Positive and
negative ESI; electrospray voltage: (þ) 3.5 kV, (") "2.5 kV; sheath gas: 1; auxiliary gas: 0; ion
transfer tube temperature: 270 ºC; ion transfer tube offset: 35 V; tube lens offset: 77 V;
collision energy: 25 eV (famotidine), 23 eV (azithromycin dihydrate); collision pressure:
1.2 m Torr; Q1/ Q3 resolution: 0.1 Da FWHM; accurate mass mode: internal; micro scans:
2.

8
 Figure 6 shows the detected mass fragmentation pattern of azithromycin dihydrate.
The major peaks in the spectrum occur at m/z 749, 591, 574, 434, 158.
For protonated azithromycin dihydrate, elimination of H2O and successive loss of the
two sugar moieties were the major fragmentation pathways. The CID mass spectrum of
the [M"H]" of azithromycin dihydrate was very similar to that of the protonated species
and showed successive loss of the two sugar moieties as the major dissociation
pathways as shown in Figure 7

Figure 6 : Negative ion ESI mass spectra of azithromycin dihydrate

(MW¼748): CID product ion spectra (MS/MS) of [M"H]" at m/z 747 and the
fully exchanged [M(d5)-D] " at m/z 751. Deuteration was achieved by
liquid phase H/D exchange method. MS and MS/MS experiments were
performed on a TSQ Quantum Ultra AM mass spectrometer.

9
 Figure 7 : Proposed CID fragmentation mechanisms for the major fragment ions
from deprotonated azithromycin dihydrate at m/z 747 determined from H/D
exchange patterns, high resolution mass measurements, and MS/MS
experiments. Numbers in parentheses refer to deuterated fragmentations. The
proposed site of deprotonation is based on the most acidic proton of the
lactone ring.
3.1.7) X-ray powder diffraction
The X-ray powder diffraction pattern of azithromycin dihydrate has been measured
using a Bruker D5000 diffractometer (Madison, Wis.) equipped with copper radiation, fixed
slits (1.0, 1.0, 0.6 mm), and a Kevex solid state detector. The pattern obtained is shown in
Figure 8, and Azithromycin dihydrate displays peaks at 7.2, 7.9, 9.3, 9.9, 11.2, 12.0, 12.7,
13.0, 14.0, 15.6, 16.0,16.4, 16.8, 17.5, 18.2, 18.7, 19.1, 19.8, 20.5, 20.9, 21.2, 21.6, 21.8, and
24.0, the data was collected from 3.0 to 40.0 degrees in 2-theta using a step size of 0.04
degrees and a step time of 1.0 seconds.

10
 Figure 8 : An experimental powder X-ray diffraction pattern of
azithromycin dihydrate
3.2) Impurities profile
3.2.1) Synthetic scheme
Provided by manufacturer (Gold Biotechnology, Inc) Azithromycin dihydrate batch no
S4008044061 , S4008044062 were prepared from erythromycin A , by treating the
erythromycin (1) in methanol with hydroxylamine hydrochloride and a base at reflux
temperature for 10 h to form oxime (2). The oxime was isolated, purified, and subjected to
-iminoether) (3) (Scheme 1) in
aqueous acetone in the presence of p-toluenesulfonyl chloride and base for 2 h at 5 C (and
2 h more at room temperature). The iminoether was reduced to the secondary amine (4) with
sodium borohydride in methanol or by catalytic hydrogenation in the presence of platinum
dioxide and acetic acid as solvents. Another alternate synthetic method for azithromycin
dihydrate is reported. The iminoether (3) was prepared in single step from erythromycin A (1),
by treating the erythromycin A (1) solution in acetone with O-mesitylene-

which was treated with an aqueous base (sodium bicarbonate) at 0 C, and then the
intermediary 6-9-
The iminoether (3) was prepared in single step from erythromycin A (1), by treating the
erythromycin A (1) solution in acetone with O-mesitylene-sulfonylhydroxylamine, to form the

11
base (sodium bicarbonate) at 0 C, and then the intermediary 6-9-iminoether (3) was produced
rearrangement. The iminoether (3) was reduced with reductive methylation
using common techniques to obtain azithromycin dihydrate (5)
.1

-iminoether) for
preparation of Azithromycin Dihydrate.
3.2.2 Impurity profiles of Azithromycin
According to United State Pharmacopoeia 41, which providing several possible
impurities from synthesis process of Azithromycin Dihydrate listed as following

USP 42

12
 Impurity Structure Origin
Erythromycin A In process
iminoether impurity

Erythromycin A oxime In process

impurity

Azaerythromycin A In process
impurity
and
Degradati
on
product
from
photolytic
oxidation
3-Deoxyazithromycin In process
(azithromycin B) impurity

13
Reference
1. Bakheit AHH, Al-Hadiya BMH, Abd-Elgalil AA. Chapter One - Azithromycin.

icle/pii/B9780128001738000015

14
S4 Control of Drug Substance
S4.1 Specification and Certificate of analysis
Specification of Azithromycin
Table Specification of Azithromycin
Test Requirement Method
Appearance A white or almost white powder Visual inspection
Identification
A. Infrared absorption A: The spectra of the test specimen Spectroscopic
and corresponding USP reference identification Test USP 42
standard over the range from NF 37 <197K>
about 2.6 µm to 15 µm (3800 cm-1 Page 6520-6522
to 650 cm-1). The IR absorption
spectrum of the preparation of the
test specimen exhibits maxima
only at the same wavelength as
that of a similar preparation of the
corresponding standard.
B. HPLC B. The retention time of the Spectroscopic
Azithromycin peak of the Sample identification Test USP 42
solution corresponds to that of the NF 37 <197K>
Standard solution, as obtained in Page 440
the Assay
Assay 945 1030 µg/mg on the anhydrous Chromatography USP 42
basis NF 37, Page 440
Impurities Residue on ignition
- Residue on ignition NMT 0.3% USP 42 NF 37, Page 440
<281> Page 6587
Organic impurities Chromatography USP 42
- Erythromycin A iminoether NMT 0.5 % NF 37, Page 441-443
- Desosaminylazithromycin NMT 0.3 %
- Erythromycin A oxime NMT 0.5 %
- Azaerythromycin A NMT 1.0 %

15
- Any individual, unidentified NMT 0.2 %
impurities
Total impurities NMT 3.0 %
Specific test USP 42 NF 37
- Optical rotation <781S> Page 6926-6927
- Crystalline form dihydrate-triclinal crystal <776> Page
- pH 9.0 11.0 <791> Page 6923-6926
- Water Determination NMT 2.0% Method Ia <921> Page
7092-7094
- Loss on Drying NMT 4.5% between ambient Page 442
temperature and the inflection
point at about 70°, and 1.8% 2.6%
between the inflection point at
about 70° and the inflection point
at about 130°.

16
 Gold Biotechnology, Inc.
1328 Ashby Rd.
St. Louis, MO 63132
P: (314)890-8778
F: (314)890-0503
E: contactgoldbio86
Physical and chemical property
Batch molecular formula: C38H72N2O12
Batch molecular weight: 749.0
Batch molecular structure:

Product Name: Azithromycin Dihydrate

CAT #: A-900
CAS 117772-70-0
LOT #: S4 8044061
Batch size: 3000 kg
Manufacturing date: 3 January 2019
Analysis date: 10 January 2019
Retest date: 3 January 2020
Expire date: 2 January 2024

17
 Test Specifications Result
Appearance A white or almost white powder A white or almost white powder
Identification
A. Infrared absorption Infrared absorption <197K> Complies

B. HPLC The retention time of the Azithromycin peak Complies

of the Sample solution corresponds to that
of the Standard solution, as obtained in the
Assay

Assay 945 1030 µg/mg on the anhydrous basis 975 µg/mg on the anhydrous
basis
Impurities
- Residue on ignition NMT 0.3 % 0.02 %
Organic impurities
- Erythromycin A iminoether NMT 0.5 % 0.01 %
- Desosaminylazithromycin NMT 0.3 % 0.01 %
- Erythromycin A oxime NMT 0.5 % 0.03 %
- Azaerythromycin A NMT 1.0 % 0.05 %
Any individual, NMT 0.2 % 0.05 %
unidentified impurities
Total impurities NMT 3.0 % 0.05 %
Specific test
- Optical rotation -46.3°
- Crystalline form dihydrate-triclinal crystal dihydrate-triclinal crystal
- pH 9.0 11.0 10.18 (2 mg/mL in methanol
and water)
- Water Determination 4.0 % - 5.0 % 4.55 %
- Loss on Drying NMT 4.5% between ambient temperature 2.3 %
and the inflection point at about 70°,and 1.8
% 2.6 % between the inflection point at
about 70° and the inflection point at about
130°.

18
IR Spectrum

HPLC

19
 Gold Biotechnology, Inc.
1328 Ashby Rd.
St. Louis, MO 63132
P: (314)890-8778
F: (314)890-0503
E: contactgoldbio86
Physical and chemical property
Batch molecular formula: C38H72N2O12
Batch molecular weight: 749.0
Batch molecular structure:

Product Name: Azithromycin Dihydrate

CAT #: A-900
CAS 117772-70-0
LOT #: S4 8044062
Batch size: 3000 kg
Manufacturing date: 3 January 2019
Analysis date: 10 January 2019
Retest date: 3 January 2020
Expire date: 2 January 2024

20
 Test Specifications Result
Appearance A white or almost white powder A white or almost white powder
Identification
A. Infrared absorption Infrared absorption <197K> Complies

B. HPLC The retention time of the Complies

Azithromycin peak of the
Sample solution corresponds to
that of the Standard solution, as
obtained in the Assay
Assay 945 1030 µg/mg on the 975 µg/mg on the anhydrous
anhydrous basis basis
Impurities
- Residue on ignition NMT 0.3 % 0.01 %
Organic impurities
- Erythromycin A iminoether NMT 0.5 % 0.01 %
- Desosaminylazithromycin NMT 0.3 % 0.01 %
- Erythromycin A oxime NMT 0.5 % 0.03 %
- Azaerythromycin A NMT 1.0 % 0.05 %
Any individual, unidentified impurities NMT 0.2 % 0.05 %
- Total impurities NMT 3.0 % 0.05 %
Specific test
- Optical rotation -46.3°
- Crystalline form dihydrate-triclinal crystal dihydrate-triclinal crystal
- pH 9.0 11.0 10.18 (2 mg/mL in methanol
and water)
- Water Determination 4.0 % - 5.0 % 4.55 %
- Loss on Drying NMT 4.5% between ambient 2.5 %
temperature and the inflection
point at about 70°,and 1.8 %
2.6 % between the inflection
point at about 70° and the
inflection point at about 130°.

21
IR Spectrum

HPLC

22
 Gold Biotechnology, Inc.
1328 Ashby Rd.
St. Louis, MO 63132
P: (314)890-8778
F: (314)890-0503
E: contactgoldbio86
Physical and chemical property
Batch molecular formula: C38H72N2O12
Batch molecular weight: 749.0
Batch molecular structure:

Product Name: Azithromycin Dihydrate

CAT #: A-900
CAS 117772-70-0
LOT #: S4 8044063
Batch size: 3000 kg
Manufacturing date: 3 January 2019
Analysis date: 10 January 2019
Retest date: 3 January 2020
Expire date: 2 January 2024

23
 Test Specifications Result
Appearance A white or almost white powder A white or almost white powder
Identification
A. Infrared absorption Infrared absorption <197K> Complies

B. HPLC The retention time of the Azithromycin Complies

peak of the Sample solution corresponds
to that of the Standard solution, as
obtained in the Assay

Assay 945 1030 µg/mg on the anhydrous basis 975 µg/mg on the anhydrous
basis
Impurities
- Residue on ignition NMT 0.3 % 0.01 %
Organic impurities
- Erythromycin A iminoether NMT 0.5 % 0.01 %
- Desosaminylazithromycin NMT 0.3 % 0.01 %
- Erythromycin A oxime NMT 0.5 % 0.03 %
- Azaerythromycin A NMT 1.0 % 0.05 %
Any individual, unidentified NMT 0.2 % 0.05 %
impurities
Total impurities NMT 3.0 % 0.05 %
Specific test
- Optical rotation -46.3°
- Crystalline form dihydrate-triclinal crystal dihydrate-triclinal crystal
- pH 9.0 11.0 10.18 (2 mg/mL in methanol and
water)
- Water Determination 4.0 % - 5.0 % 4.55 %
- Loss on Drying NMT 4.5% between ambient temperature 2.5 %
and the inflection point at about 70°, and
1.8 % 2.6 % between the inflection
point at about 70° and the inflection point
at about 130°
24
IR Spectrum

HPLC

25
 O LEAVES Pharmaceutical Co.,LTD
: 147 Soi Sabaijai Suthisarnwinijchai Rd., HuaiKwang Bangkok Thailand 10310
Tel: 040-523478 FAX: 040-523478 Email: OLEAVESPharmaceutical@oleaves.com

Certification of Analysis

Product name Azithromycin dihydrate Manufacture Date 3 January 2019

Lot Number S4 8044061 Analysis Date 1 February 2019
Batch Quantity 3000 Kg Expire Data 1 May 2024

Test Specifications Result

Appearance A white or almost white powder Complies
Identification
A. Infrared absorption Infrared absorption <197K> Complies

B. HPLC The retention time of the Azithromycin Complies

peak of the Sample solution corresponds
to that of the Standard solution, as
obtained in the Assay

Assay 945 1030 µg/mg on the anhydrous basis 975 µg/mg on the
anhydrous basis
Impurities
- Residue on ignition NMT 0.3 % 0.03 %
Organic impurities
- Erythromycin A iminoether NMT 0.5 % 0.01 %
- Desosaminylazithromycin NMT 0.3 % 0.01 %
- Erythromycin A oxime NMT 0.5 % 0.03 %
- Azaerythromycin A NMT 1.0 % 0.05 %
- Any individual, unidentified NMT 0.2 % 0.05 %
impurity
Total impurities NMT 3.0 % 0.05 %

26
Specific test
- Optical rotation -46.3°
- pH 9.0 11.0 10.18 (2 mg/mL in
methanol and water)
- Water Determination 4.0 % - 5.0 % 4.55 %
- Loss on Drying NMT 4.5% between ambient temperature 2.3 %
and the inflection point at about 70°,and
1.8 % 2.6 % between the inflection point
at about 70° and the inflection point at
about 130°.

27
 O LEAVES Pharmaceutical Co.,LTD
: 147 Soi Sabaijai Suthisarnwinijchai Rd., HuaiKwang Bangkok Thailand 10310
Tel: 040-523478 FAX: 040-523478 Email: OLEAVESPharmaceutical@oleaves.com

Certification of Analysis

Product name Azithromycin dihydrate Manufacture Date 3 January 2019

Lot Number S4 8044062 Analysis Date 1 February 2019
Batch Quantity 3000 kg Expire Data 1 May 2024

Test Specifications Result

Appearance A white or almost white powder Complies
Identification
A. Infrared absorption Infrared absorption <197K> Complies

B. HPLC The retention time of the Complies

Azithromycin peak of the Sample
solution corresponds to that of the
Standard solution, as obtained in the
Assay
Assay 945 1030 µg/mg on the anhydrous 975 µg/mg on the
basis anhydrous basis
Impurities NMT 0.3 % 0.03 %
- Residue on ignition
Organic impurities
- Erythromycin A iminoether NMT 0.5 % 0.01 %
- Desosaminylazithromycin NMT 0.3 % 0.01 %
- Erythromycin A oxime NMT 0.5 % 0.03 %
- Azaerythromycin A NMT 1.0 % 0.05 %
- Any individual, unidentified NMT 0.2 % 0.05 %
impurity
Total impurities NMT 3.0 % 0.05 %
28
Specific test
- Optical rotation -46.3°
- pH 9.0 11.0 10.18 (2 mg/mL in
methanol and water)
- Water Determination 4.0 % - 5.0 % 4.55 %
- Loss on Drying NMT 4.5% between ambient 2.3 %
temperature and the inflection point
at about 70°, and 1.8 % 2.6 %
between the inflection point at about
70° and the inflection point at about
130°.

29
 O LEAVES Pharmaceutical Co.,LTD
: 147 Soi Sabaijai Suthisarnwinijchai Rd., HuaiKwang Bangkok Thailand 10310
Tel: 040-523478 FAX: 040-523478 Email: OLEAVESPharmaceutical@oleaves.com

Certification of Analysis

Product name Azithromycin dihydrate Manufacture Date 3 January 2019

Lot Number S4 8044063 Analysis Date 1 February 2019
Batch Quantity 3000 kg Expire Data 1 May 2024

Test Specifications Result

Appearance A white or almost white powder Complies
Identification
A. Infrared absorption Infrared absorption <197K> Complies

B. HPLC The retention time of the Complies

Azithromycin peak of the Sample
solution corresponds to that of the
Standard solution, as obtained in the
Assay
Assay 945 1030 µg/mg on the anhydrous 975 µg/mg on the
basis anhydrous basis
Impurities NMT 0.3 % 0.03 %
- Residue on ignition
Organic impurities
- Erythromycin A iminoether NMT 0.5 % 0.01 %
- Desosaminylazithromycin NMT 0.3 % 0.01 %
- Erythromycin A oxime NMT 0.5 % 0.03 %
- Azaerythromycin A NMT 1.0 % 0.05 %
- Any individual, unidentified NMT 0.2 % 0.05 %
impurity
30
 Total impurities NMT 3.0 % 0.05 %
Specific test
- Optical rotation 46.3°
- pH 9.0 11.0 10.18 (2 mg/mL in
methanol and water)
- Water Determination 4.0 % - 5.0 % 4.55 %
- Loss on Drying NMT 4.5% between ambient 2.3 %
temperature and the inflection point
at about 70°,and 1.8 % 2.6 %
between the inflection point at about
70° and the inflection point at about
130°.

31
S4.2 Analytical Procedure
Following USP42 Azithromycin

32
33
34
35
1. Appearance
Procedure: Organoleptic method
Observation: A white or almost white powder
2. Identification
2.1 Infrared absorption
following Infrared absorption <197K> USP 42:

A. Infrared Absorption
If a difference appears in the IR spectra of the analyte and the Standard, dissolve equal portions of
the test specimen and the USP Reference Standard in equal volumes of methanol. Evaporate the
solutions to dryness on a water bath, and dry at 80° for 30 min under vacuum. Perform the test on
the residues
The reference 197K in a monograph signifies that the substance under examination is mixed
intimately with potassium bromide and compressed into a transparent pellet.
In each instance, infrared spectra of both the sample and corresponding USP Reference Standard
are obtained using the same sample preparation technique and measurement parameters. Record
and compare the spectra of the sample and the corresponding USP Reference Standard over the
range from 3800 to 650 cm 1, unless otherwise specified in the individual monograph.
The comparison must establish that the IR spectrum of the preparation of the sample exhibits
maxima only at the same wavenumbers as that of the appropriately prepared corresponding USP
Reference Standard. If there are differences between the spectra, and the sample spectrum was
compared with a previously obtained and electronically stored spectrum of the USP Reference
Standard, the comparison must be repeated concomitantly with a freshly prepared USP Reference
Standard.

36
 Differences between the USP Reference Standard spectrum and sample spectrum that may be
observed are sometimes attributable to differences in the solid-state form of the materials, if
a solid-state technique is used (e.g., <197A>, <197K>, or <197M>). If a specific crystal form is not
specified in the monograph, where spectral differences between the sample and USP Reference
Standard are observed, recrystallize both the sample and USP Reference Standard under identical
conditions to produce the same solid-state form, unless specific procedures are provided in
the individual monographs. Dissolve equal portions of the sample and the USP Reference Standard
in equal volumes of a suitable solvent, evaporate the solutions to dryness in similar containers
under identical conditions, and repeat the identification test on the residues. Other techniques for
recrystallizing the sample and USP Reference Standard based on known scientific principles may
be used with appropriate scientific justification.

Instrument
1. Fourier Transform Infrared Spectrophotometer
- Company: SHiMADZU
- Series: AIM-8000
Analytical
Procedure
Standard solution
1. Weight 1 mg of Azithromycin RS to mixing with 99 mg of dried, finely powdered KBr
2. Condensed in the 13-mm die at a pressure of 6 tons for 5 min
3. Pellet method
4. Measure by FT-IR spectrometer
Sample solution
1. Weight 1 mg of Azithromycin to mixing with 99 mg of dried, finely powdered KBr
2. Condensed in the 13-mm die at a pressure of 6 tons for 5 min
3. Pellet method
4. Measure by FT-IR spectrometer

37
Analytical data

FT-IR spectrum of standard solution from USP reference standard

FT-IR spectrum of sample solution

The IR spectrum of USP azithromycin reference standard wave number provided similar as
those of corresponding wavenumber of the azithromycin

38
2.2 High Performance Liquid Chromatography (HPLC)
following USP 42
IDENTIFICATION
B. The retention time of the azithromycin peak of the Sample solution corresponds to
that of the Standard solution, as obtained in the Assay.

Analytical data

Retention time of standard solution

Retention time of sample solution

39
3. Assay
High Performance Liquid Chromatography (HPLC)
following USP 42
Procedure
Solution A: 10 M potassium hydroxide
Solution B: 6.7 g/L of dibasic potassium phosphate, adjusted with Solution A to a pH of 11.0
Solution C: 6.7 g/L of dibasic potassium phosphate, adjusted with phosphoric acid to a pH of 8.0
Mobile phase: Acetonitrile and Solution B (60:40)
Diluent: Acetonitrile and Solution C (60:40)
System suitability solution: 0.5 mg/mL each of USP Azithromycin RS and USP Azaerythromycin A
RS prepared as follows. Dissolve USP Azithromycin RS and USP Azaerythromycin A RS first in
acetonitrile, using 5% of the final volume, and then dilute with Diluent to volume.
Standard solution: 0.53 mg/mL of USP Azithromycin RS prepared as follows. Dissolve USP
Azithromycin RS first in acetonitrile, using 2% of the final volume, and then dilute with Diluent to
volume.
Sample solution: 0.53 mg/mL of Azithromycin prepared as follows. Dissolve Azithromycin first in
acetonitrile, using 2% of the final volume, and then dilute with Diluent to volume.

Chromatographic system
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm x 25-cm;5- m packing L67
Column temperature: 40°
Flow rate: 1 mL/min
Injection volume: 10 µL
System suitability
Samples: System suitability solution and Standard solution
Suitability requirements
Resolution: NLT 3.0 between Azaerythromycin A and Azithromycin, System suitability solution
Tailing factor: 0.8-1.5 for Azithromycin, Standard solution
Relative standard deviation: NMT 1.10% for Azithromycin, Standard solution

40
Analysis
Samples: Standard solution and Sample solution Calculate the quantity, in µg, of azithromycin
(C38H72N2O12) in each milligram of Azithromycin taken:
Result = (rU/rS ) × (CS /CU) × P
rU = peak response from the Sample solution
rS = peak response from the Standard solution
CS = concentration of USP Azithromycin RS in the Standard solution
CU = concentration of Azithromycin in the Sample solution
P = potency of USP Azithromycin RS (µg/mg of azithromycin)
Acceptance criteria: 945 1030 µg/mg on the anhydrous basis

Instumental
1. Tranferpettor Roitive Displacement Pipette
- Company : Combitips Advanced®
- Catalog number : 89232-934
- Type : Analog
- Volume Range : 5 - 500 µL
- Tips : 0.5 ml

2. High Performance Liquid Chromatography (HPLC)

- Company : HITACHI
- Series : Chromater
Analytical Procedure
Standard solution Preparation 0.53 mg/ml of USP Azithromycin
1. Dissolve 265 mg of USP Azithromycin RS in acetonitrile 25 ml in volumetric flak 25 mL
2. Pipet 0.5 ml solution transfer to volumetric flask 10 ml dilute with Diluent to 10 mL
Sample solution Preparation
1. Dissolve 265 mg of Azithromycin in acetonitrile 25 ml in volumetric flak 25 mL
2. Pipet 0.5 ml solution transfer to volumetric flask 10 ml dilute with Diluent to 10 mL
3. replicate 3 flasks

41
Chromatographic system
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm x 25-cm;5- m packing L67
Column temperature: 40°
Flow rate: 1 mL/min
Injection volume: 10 µL

System suitability
Samples: System suitability solution and Standard solution
Suitability requirements
Resolution: NLT 3.0 between Azaerythromycin A and Azithromycin, System
suitability solution
Tailing factor: 0.8-1.5 for Azithromycin, Standard solution
Relative standard deviation: NMT 1.10%

Chromatogram of System suitability

Chromatogram of System suitability Azithromycin

42
Analysis
Samples: Standard solution and Sample solution Calculate the quantity, in µg, of
azithromycin (C38H72N2O12) in each milligram of Azithromycin taken:
Result = (rU/rS ) × (CS /CU) × P
rU = peak response from the Sample solution
rS = peak response from the Standard solution
CS = concentration of USP Azithromycin RS in the Standard solution
CU = concentration of Azithromycin in the Sample solution
P = potency of USP Azithromycin RS (µg/mg of azithromycin)
Acceptance criteria: 945 1030 µg/mg on the anhydrous basis
System suitability
Acceptance criteria
Resolution: NLT 3.0 between Azaerythromycin A and Azithromycin, System
suitability solution
Tailing factor: 0.8-1.5 for Azithromycin, Standard solution
Relative standard deviation: NMT 1.10%

Retention time of System suitability

Table Retention time of System suitability
Standard solution Retention time (min)
1 10.068
2 10.068
3 10.065
4 10.065
5 10.067
6 10.065

43
Result of System suitability
Table Result of System suitability
Standard Area Number of theoretical Tailing factor
(mAU*min) plates (N)
1 25695 4182 1.015
2 25995 4279 1.015
3 26012 3995 1.015
4 26365 4362 1.016
5 25531 4139 1.016
Average 25695 4191 1.015
% RSD 1.1 2.98 0.05

Interpret Result of System suitability

Table Interpret Result of System suitability
System suitability Acceptance Result Interpret
criteria
Tailing factor 0.8 1.5 1.015 Pass
Relative standard NMT 1.10% 0.12% Pass
deviation
Analytical data

Retention time of standard solution

44
 Retention time of sample solution
4. Impurities
4.1 Residual ignition
following Residue on ignition <281> USP 42 : NMT 0.3%, moistening the charred residue with
2 mL of nitric acid and 5 drops of sulfuric acid
RESIDUE ON IGNITION <281>
Procedure:
Ignite a suitable crucible (for example, silica, platinum, quartz, or porcelain) at
600 ± 50° for 30 minutes, cool the crucible in a desiccator (silica gel or other suitable desiccant), and weigh
it accurately. Weigh accurately 1 to 2 g of the substance, or the amount specified in the individual
monograph, in the crucible.
Moisten the sample with a small amount (usually 1 mL) of sulfuric acid, then heat gently at a
temperature as low as practicable until the sample is thoroughly charred. Cool; then, unless otherwise
directed in the individual monograph, moisten the residue with a small amount (usually 1 mL) of sulfuric
acid; heat gently until white fumes are no longer evolved; and ignite at 600 ± 50° unless another
temperature is specified in the individual monograph, until the residue is completely incinerated. Ensure
that flames are not produced at any time during the procedure. Cool the crucible in a desiccator (silica gel
or other suitable desiccant), weigh accurately, and calculate the percentage of residue.
Unless otherwise specified, if the amount of the residue so obtained exceeds the limit specified in
the individual monograph, repeat the moistening with sulfuric acid, heating and igniting as before, using a
30-minute ignition period, until two consecutive weighings of the residue do not differ by more than 0.5 mg
or until the percentage of residue complies with the limit in the individual monograph.
Conduct the ignition in a well-ventilated hood, but protected from air currents, and
at as low a temperature as is possible to effect the complete combustion of the carbon. A muffle furnace
may be used, if desired, and its use is recommended for the final ignition at 600 ± 50°.
Calibration of the muffle furnace may be carried out using an appropriate digital temperature meter and a
working thermocouple probe calibrated against a standard thermocouple traceable to the National Institute
of Standards and Technology.
Verify the accuracy of the measuring and controlling circuitry of the muffle furnace by checking the
positions in the furnace at the control set point temperature of intended use. Select positions that reflect
the eventual method of use with respect to location of
the specimen under test. The tolerance is ± 25° at each position measured.

45
Procedure
1. Ignite a quartz crucible at 600 °C for 30 minutes.
2. Cool the crucible in a silica gel desiccator and weigh it accurately.
3. Weigh accurately 1 g of Azithromycin powder in the quartz crucible.
4. Moistening the charred residue with 2 mL of nitric acid and 5 drops of sulfuric acid
5. Heat gently at 200°C until the sample is thoroughly charred, Cool.
6. Moisten the residue with a 1 mL of sulfuric acid.
7. Heat gently until white fumes are no longer evolved.
8. Ignite at 600 °C until the residue is completely incinerated, ensure that flames are not
produced at any time during the procedure.
9. Cool the crucible in a silica gel desiccator, weigh accurately, and calculate the percentage
of residue.

4.2 Organic impurities

Following USP 42

Organic Impurities
Solution A: 1.8 mg/mL of anhydrous dibasic sodium phosphate in water. Adjust with 1 N sodium
hydroxide or 10% phosphoric acid to a pH of 8.9.
Solution B: Acetonitrile and methanol (3:1)
Solution C: 1.73 mg/mL of monobasic ammonium phosphate. Adjust with ammonia TS to a pH of
10.0 ± 0.05.
Solution D: Methanol, acetonitrile, and Solution C (7:6:7)
Mobile phase: See Table 1.
Table 1
Time (min) Solution A (%) Solution B (%)
0 50 50
25 45 55
30 40 60
80 25 75
81 50 50
93 50 50

46
System suitability solution: 0.0165 mg/mL of USP Azithromycin Related Compound F RS and 0.027 mg/mL of
USP Desosaminylazithromycin RS in Solution D
Standard solution: 86 µg/mL of USP Azithromycin RS in Solution D
Sample solution: 8.6 mg/mL of Azithromycin in Solution D

Chromatographic system
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Column temperature: 60°
Flow rate: 1 mL/min
Injection volume: 50 µL

System suitability
Samples: System suitability solution and Standard solution
Suitability requirements
Peak-to-valley ratio: NLT 1.4, System suitability solution Calculate the peak-to-valley ratio:
Result = HP /HV
HP = height above the baseline of the Desosaminylazithromycin peak
HV = height above the baseline of the lowest point of the curve separating the Desosaminylazithromycin and
azithromycin related compound F peaks

Tailing factor: 0.8-1.5, Standard solution

Analysis
Samples: Standard solution and Sample solution Calculate the percentage of each impurity in the portion of
Azithromycin taken:
Result = (ru/rs)x (Cs/Cu) x P x F1 x (100/F2)
ru = peak response of each impurity from the Sample solution
rs = peak response of azithromycin from the Standard solution
Cs = concentration of USP Azithromycin RS in the Standard solution (mg/mL)
Cu = concentration of Azithromycin in the Sample solution (mg/mL)
P = potency of USP Azithromycin RS (ug/mg of azithromycin)
F1= conversion factor, 0.001 mg/ug.
F2= relative response factor (see Table 2)
47
Acceptance criteria: See Table 2. Disregard peaks eluting before azithromycin N-oxide and after 3-
Deoxyazithromycin (azithromycin B). Disregard peaks with a response less than 0.1 times the
response of the azithromycin peak in the Standard solution (0.1%).
Table 2
Relative
Relative Retention Acceptance Criteria,
Name Response
Time NMT (%)
Factor
Azithromycin N-oxide 0.29 0.43 0.5
-(N-N-Didemethyl)- -N-formyl
0.37 1.7 0.5
azithromycin
Erythromycin A iminoether 0.42 1.0 0.5
-(N,N-Didemethyl)azithromycin
0.43 1.0 0.5
(amino azithromycin)
Azithromycin related compound F 0.51 3.8 0.5
Desosaminylazithromycinb 0.54 1.0 0.3
-N-
Acetylamino)phenyl]sulfonyl)- - 0.55 12 0.15
didemethylazithromycin
N-Demethylazithromycin 0.61 1.0 0.7
Erythromycin A oxime 0.64 1.0 0.5
-O-
0.73 1.0 0.5
demethylazithromycin )
-De(dimethylamino)- -
0.76 1.5 0.5
oxoazithromycin
-N-{[4-
(Aceytylamini)phenyl]sulfonyl}- -
0.79 10 0.5
demethylazithromycin

Azserythromycin A 0.83 1.0 0.5

Azithromycin impurity P 0.92 1.0 .0.2
Azithromycin 1.0
2-Desethyl-2-propylazithromycin 1.23 1.0 0.5
-N-Demethyl- -N-[(4-
1.26 5 0.5
methylphenyl)sulfonyl]azithromycin
3-Deoxyazithromycin (azithromycin B) 1.31 1.0 1.0
Any individual,unidentified impurity 1.0 0.2
Total impurities 3.0 48
Procedure
System suitability solution :
- 0.0165 mg/mL of USP Azithromycin Related Compound F RS in solution D
1. Weight 16.5 mg of USP Azithromycin Related Compound F RS , transfer to 100 mL
Volumetric flask
2. Adjust with solution D to 100 mL
3. Pipette 5 mL, transfer to 50 mL Volumetric flask
4. Sonicate 15 minutes
5. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial
- 0.027 mg/mL of USP Desosaminylazithromycin RS in Solution D
1. Weight 27 mg of USP Azithromycin Related Compound F RS , transfer to 100 mL
Volumetric flask
2. Adjust with solution D to 100 mL
3. Pipette 5 mL, transfer to 50 mL Volumetric flask
4. Sonicate 15 minutes
5. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial

Standard solution: 86 µg/mL of USP Azithromycin RS in Solution D

1. Weight 86 mg of USP Azithromycin Related Compound F RS, transfer to 100 mL
Volumetric flask
2. Adjust with solution D to 100 mL
3. Pipette 5 mL, transfer to 50 mL Volumetric flask
4. Sonicate 15 minutes
5. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial

Sample solution: 8.6 mg/mL of Azithromycin in Solution D

1. Weight 860 mg of USP Azithromycin Related Compound F RS, transfer to50 mL
Volumetric flask
2. Adjust with solution D to 50mL
3. Pipette 5 mL, transfer to 10 mL Volumetric flask
4. Sonicate 15 minutes
5. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial

49
Chromatographic system
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Column temperature: 60°
Flow rate: 1 mL/min
Injection volume: 50 µL

Procedure
1. Equilibrate the column and detector with mobile phase at the specified flow rate
until a constant signal is received.
2. Injection a sample through the injection, or use an auto sample.
3. Begin the gradient program.
4. Record the chromatogram.
5. Analyze as directed in the monograph.

Analysis
Samples: Standard solution and Sample solution Calculate the percentage of each impurity
in the portion of Azithromycin taken:
Result = (ru/rs)x (Cs/Cu) x P x F1 x (100/F2)
ru = peak response of each impurity from the Sample solution
rs = peak response of azithromycin from the Standard solution
Cs = concentration of USP Azithromycin RS in the Standard solution (mg/mL)
Cu = concentration of Azithromycin in the Sample solution (mg/mL)
P = potency of USP Azithromycin RS (ug/mg of azithromycin)
F1= conversion factor, 0.001 mg/ug.
F2= relative response factor (see Table 2)
Acceptance criteria: See Table 2. Disregard peaks eluting before azithromycin N-oxide and
after 3- deoxyazithromycin (azithromycin B). Disregard peaks with a response less than 0.1
times the response of the azithromycin peak in the Standard solution (0.1%).

50
Table 1 Impurities Azithromycin
Relative Relative
Acceptance
Name Retention Response
Criteria, NMT (%)
Time Factor
Erythromycin A iminoether 0.42 1.0 0.5
Desosaminylazithromycin 0.54 1.0 0.3
Erythromycin A oxime 0.64 1.0 0.5
Azaerythromycin A 0.83 1.0 0.5
Any individual, unidentified impurities 1.0 0.2
Total impurities 3.0

51
 5. specific tests
5.1 Optical rotation
Following Optical rotation <781> USP 42

OPTICAL ROTATION
INTRODUCTION
Many pharmaceutical substances are optically active in the sense that they rotate an
incident plane of polarized light so that the transmitted light emerges at a measurable angle to the
plane of the incident light. This property is characteristic of some crystals and of many pharmaceutical liquids
or solutions of solids. Where the property is possessed by a liquid or by a solute in solution, it is
generally the result of the presence of one or more asymmetric centers, usually a carbon atom with four
different substituents.The number of optical isomers is 2n, where n is the number of asymmetric centers.
Polarimetry, the measurement of optical rotation, of a pharmaceutical article may be the only convenient
means for distinguishing optically active isomers from each other and thus is an important criterion of identity
and purity.
Substances that may show optical rotatory properties are "chiral". Those that rotate light in a
clockwise direction as viewed toward the light source are "dextrorotatory", or "(+) optical isomers", and those
that rotate light in a counterclockwise direction are called "levorotatory" or "(-) optical isomers". (The symbols
d- and I-, formerly used to indicate dextrorotatory and levorotatory isomers, are no longer sanctioned owing to
confusion with D- and L-, which refer to configuration relative to D-glyceraldehyde. The symbols R and S, or a
and B, are also used to indicate configuration, the arrangement of atoms or groups of atoms in space.)
The physicochemical properties of nonsuperimposable chiral substances rotating plane-polarized light
in opposite directions to the same extent, "enantiomers", are identical, except for this property and in their
reactions with other chiral substances. Enantiomers often exhibit profound differences in pharmacology and
toxicology, owing to the fact that biological receptors and enzymes themselves are chiral. Many articles from
natural sources, such as amino acids, proteins, alkaloids, antibiotics, glycosides, and sugars, exist as chiral
compounds. Synthesis of such compounds from nonchiral materials usually results in equal amounts of the
enantiomers, i.e., "racemates". Racemates have a net null optical rotation, and their physical properties may
differ from those of the component enantiomers. Use of stereoselective or stereospecific synthetic methods or
separation of racemic mixtures can be used to obtain individual optical isomers. Measurement of optical
rotation is performed using a polarimeter.' The general equation used in polarimetry is:

52
[ ] = specific rotation at wavelength
t = temperature
a = observed rotation in degrees (°)
l = path length (dm)
c = concentration of the analyte (g/100 ml)
Thus, [ ] is 100 times the measured value, in degrees (°) , for a solution containing 1 g in 100
mL, measured in a cell having a path length of 1.0 dm under defined conditions of incident
wavelength of light and temperature. For some Pharmacopeial articles, especially liquids such as
essential oils, the optical rotation requirement is expressed in terms of the observed rotation, a,
measured under conditions defined in the monograph.
Historically, polarimetry was performed using an instrument where the extent of optical
rotation is estimated by visual matching of the intensity of split fields. For this reason, the D-line of
the sodium lamp at the visible wavelength of 589 nm. was most often employed. Specific rotation
determined at the D-line is expressed by the symbol:

Much of the data available are expressed in this form. Use of lower wavelengths, such as
those available with the mercury lamp lines isolated by means of filters of maximum transmittance
at approximately 546, 436, 405, 365, and 325 nm in a photoelectric polarimeter, has been found to
provide advantages in sensitivity with a consequent reduction in the concentration of the test
compound. In general, the observed optical rotation at 436 nm is about double, and at 365 nm,
about 3 times that at 589 nm.2 Reduction in the concentration of the solute required for
measurement may sometimes be accomplished by conversion of the substance under test to one
that has a significantly higher optical rotation. Optical rotation is also affected by the solvent used
for the measurement, and this is always specified. It is now common practice to use other light
sources, such as xenon or tungsten halogen, with appropriate filters, because these may offer
advantages of cost, long life, and broad wavelength emission range, over traditional light sources.

53
PROCEDURES
Specific Rotation
1.Operate the Instrument as per current version of individual SOP
2. Prepare the test solution as per individual testing procedure and measure the optical
rotation Specific optical rotation.
3. Use the same cell for sample and blank.
4. Maintain the same angular orientation of the cell in each reading.
5. Place the cell so that the light passes through it in the same direction each time.
6. Calculate the optical rotation using the formula:

where [ ] is the specific rotation at wavelength, t is the temperature, a is the observed

rotation in degrees, I is the pathlength in decimeters, and c is the concentration of the
analyte in g per 100ml.
7. Unless otherwise mentioned in the individual testing procedure perform the Optical
rotation and specific optical rotation at 589nm and 25°C.
8. Where a photoelectric polarimeter is used, a single measurement, corrected for the
solvent blank, is made.
9. Where a visual polarimeter is used, the average of no fewer than five determinations,
corrected for the reading of the same tube with a solvent blank, is used.
10. Temperature, which applies to the solution or the liquid under test, should be
maintained within ± 0.5°C of the stated value.

54
5.2 Crystallinity
Following Crystallinity <695> USP 42
This test is provided to determine compliance with the crystallinity requirement where stated in
the individual monograph for a drug substance.

A detailed test procedure is described under Optical Microscopy <776>

Use a microscope that is stable and protected from vibration. The microscope magnification
(product of the objective magnification, ocular magnification, and additional magnifying
components) must be sufficient to allow adequate characterization of the smallest particles to be
classified in the test specimen. The greatest numerical aperture of the objective should be sought
for each magnification range. Polarizing filters may be used in conjunction with suitable analyzers
and retardation plates. Color filters of relatively narrow spectral transmission should be used with
achromatic objectives, are preferable with apochromats, and are required for appropriate color
rendition in photomicrography. Condensers corrected at least for spherical aberration should be
used in the microscope substage and with the lamp. The numerical aperture of the substage
condenser should match that of the objective under the conditions of use and is affected by the
actual aperture of the condenser diaphragm and by the presence of immersion oils.

55
5.3 pH
Following pH <791> USP 42

INTRODUCTION
For compendial purposes, pH is defined as the value given by a suitable, properly
calibrated, potentiometric sensor and measuring system.
The measuring system has traditionally been referred to as the "pH meter." While the pH
meter is still in common use, the measuring system can also be embedded inside the pH sensor,
and the pH signal can be transmitted digitally to an external device such as a computer,
Programmable Logic Controller (PLC), Distributed Control System (DCS), data acquisition system,
terminal, or other microprocessor-controlled device. By definition, pH is equal to -log10 [aH+] where
aH+ is the activity of the hydrogen (H+) or hydronium ion (H3O+), and the hydrogen ion activity very
closely approximates the hydrogen ion concentration.
The practical pH scale is defined:
pH= pH5+ [(E-Es)/k]
E = measured potential where the galvanic cell contains the solution under test (pH)
Es = measured potential where the galvanic cell contains the appropriate buffer solution for
calibration (pHs)
K = change in potential/unit change in pH and is derived from the Nernst equation (as follows)
k = loge (10) x (RT/nF)
R = 8.314 J/mole/ °K
T = temperature (°K)
n= moles/half-reaction
F= Faraday constant, 96485 C/mole

56
5.4 Water Determination
Following Water Determination <921> USP 42

Many Pharmacopeial articles either are hydrates or contain water in adsorbed form. As a
result, the determination of the water content is important in demonstrating compliance with the
Pharmacopeial standards. Generally one of the methods given below is called for in
the individual monograph, depending upon the nature of the article. In rare cases, a choice is
allowed between two methods. When the article contains water of hydration, Method I (Titrimetric),
Method II (Azeotropic), or Method III (Gravimetric) is employed, as directed in the individual
monograph, and the requirement is given under the heading Water.

PROCEDURE
Unless otherwise specified, transfer enough methanol or other suitable solvent to the
titration vessel, ensuring that the volume is sufficient to cover the electrodes (approximately
30 40 mL), and titrate with the Reagent to the electrometric or visual endpoint to consume
any moisture that may be present.
(Disregard the volume consumed because it does not enter into the calculations.)
Quickly add the Test Preparation, mix, and again titrate with the Reagent to the
electrometric or visual endpoint.
Calculate the water content of the specimen taken, in mg: SF
in which S is the volume, in mL, of the Reagent consumed in the second titration; and F is
the water equivalence factor of the Reagent.

57
5.5 Loss on Drying
Following loss on drying <731> USP 42

The procedure set forth in this chapter determines the amount volatile matter of any kind that is
driven off under the conditions specified. For substances appearing to contain water as the only
volatile constituent, the procedure given the chapter, Water Determination (921), is appropriate, and
specified the individual monograph.
Unless otherwise directed in the individual monograph, conduct the determination a 1- to 2-
g test specimen. Mix the substance to be tested and, if it is in the form of large particles, reduce the
particle size to about 2 mm by quickly crushing before weighing out the test specimen. Tare an
appropriate glass-stoppered, shallow weighing bottle that has been dried for about 30 minutes
under the same conditions to be employed in the determination and cooled to room temperature
in a desiccator. Put the test specimen in the bottle, replace the cover, and accurately weigh the
bottle and the contents. By gentle, sidewise shaking, distribute the test specimen as evenly as
practicable to a depth of about mm generally, and not more than 10 mm in the case of bulky
materials. Place the loaded bottle in the drying chamber, removing the stopper and leaving it also
in the chamber. Dry the test specimen at the temperature and for the time specified in the
monograph. [NOTE-The temperature specified in the monograph is to be regarded as being within
the range of 2 of the stated figure.]
When "dry to constant weight is specified in a monograph, drying shall be continued until two
consecutive weighings do not differ by more than 0.50 mg per g of substance taken, the second
weighing following an additional hour of drying. Upon opening the chamber, close the bottle
promptly, and allow it to come to room temperature in a desiccator before weighing.

Procedure
1. Conduct the determination on a 1 g test specimen
2. Tare an appropriate glass-stoppered (shallow weighing bottle that has been dried for
about 30 minutes under the same conditions to be employed in the determination
and cooled to room temperature in a desiccator)
3. Put the test specimen in the bottle, replace the 23 covers, and accurately weigh the
bottle and the contents
4. Sidewise shaking

58
5. Distribute the test specimen as evenly as practicable to a depth of about 5 mm
generally
6. Place the loaded bottle in the drying chamber
7. Removing the stopper and leaving it also in the chamber
8. Dry the test specimen at 105 C to constant weight (drying shall be continued until two
consecutive weighing do not differ by more than 0.50 mg per g of substance
taken, the second weighing following an additional hour of drying)
9. Opening the chamber and close the bottle promptly
10. Allow it to come to room temperature in a desiccator before weighing

Analysis
%Loss on dying = Azithromycin powder before dry- Azithromycin powder after dry x 1
Azithromycin powder after dry

59
 S4.3 Validation of analysis procedure
Type of Analytical Procedure to be validated
The discussion of the validation of analytical procedures is directed to the four most
common types of analytical procedures:
- Identification tests.
- Quantitative tests for impurities content.
- Limit tests for the control of impurities.
- Quantitative tests of the active moiety in samples of drug substance or drug product
or other selected component(s) in the drug product.
Table 11 ICHQ2(R1) Common type of Analytical procedures
Type of
TESTING FOR IMPURITIES ASSAY
analytical
IDENTIFICATION - dissolution (measurement only)
procedure
Quantitative Limit test - content / potency
Characteristic
Accuracy - + - +
Precision
- Repeatability - + - +
- inter. Precision - + (1) - + (1)
Specificity (2) + + + +
Detection Limit - - (3) + -
Quantitation Limit - + - -
Linearity - + - +
Range - + - +
- signifies that this characteristic is not normally evaluated
+ signifies that this characteristic is normally evaluated
(1) in cases where reproducibility (see glossary) has been performed, intermediate precision is not
needed
(2) lack of specificity of one analytical procedure could be compensated by other supporting analytical
procedure(s)
(3) may be needed in some cases

All analytical procedures follow USP42/NF37, therefore validation of analytical procedures is

not necessary but analytical procedures need verification.

60
Verification of Analytical Procedure
1. Identification test
Table 1 Method verification of Identification
Test Parameter Method Validation Acceptance criteria
Identification Specificity Test by HPLC method, Injection Negative result no
of the positive result contain peak response
Azithromycin compare with present at the same
negative result do not contain retention time as
Azithromycin positive result.

2. Assay
Table 1 Method verification of Assay Azithromycin
Test Parameter Method Validation Acceptance criteria
Assay Specificity Test by HPLC method, spike azithromycin No interference of
standard, azithromycin sample, solvent peak in the region of
(Acetonitrile). [Check chromatogram of each Azithromycin
individual solution by photodiode array chromatogram
detector]
Linearity Linearity was evaluated by HPLC method, -Linear relationship
and range use five concentration of between peak
test solution in a range of mg/ml. response and
Concentrations as follows: 0.30 0.40 0.53 concentration
0.70 0.80 mg/ml and repeat three times. -Coefficient of
Plot calibration curve determination (R2)
0.999
Accuracy Test by HPLC method, Use the results from 98.0%- 102.0% of
3 concentration/3 replicates each of the each concentration
Azithromycin test Assay (0.40, 0.53, and 0.70
solutions. (9 stimulated sample) Three mg/ml)
concentration as follows: 0.40, 0.53 and 0.70
mg/ml to calculate percent recovery
Precision Test by HPLC method, %RSD 2.0%
Repeatability:
61
 Azithromycin test in three concentrations
(0.40, 0.53 and 0.70 mg/ml) and replicate
three times under the same conditions at
different times in the same day. (0, 6 and 12
hours)
Intermediate precision:
Same procedure as repeatability but test in
day 1, 3 and 6

Specificity
Acceptance criteria: No interference of peak in the region of Azithromycin chromatogram
Result: No interference of peak in the region of Azithromycin chromatogram

Chromatogram of the Azithromycin standard solution

Chromatogram of the Azithromycin standard solution

Chromatogram of the solvent Acetonitrile

Chromatogram of the Azithromycin sample solution

62
Linearity and range
Acceptance criteria :
- Linear relationship between peak response and concentration
- Coefficient of determination (R2) 0.999
Result The peak area of Azithromycin within the concentration 0.30, 0.40, 0.53, 0.70 and 0.80
mg/ml has linear relationship (visual inspection) with coefficient of determination
(R2) = 0.9999
Table 1 Result of Linearity and range assay Azithromycin
Nominal Average
Concentration Peak area
Sample Concentratio of Peak SD % RSD
mg/ml
n (mg/ml) area
1.1 0.30 18540
1.2 0.30 0.30 18641 18744 221 1.2
1.3 0.30 19051
2.1 0.40 20901
2.2 0.40 0.40 21500 21151 254 1.2
2.3 0.40 21054
3.1 0.53 24595
3.2 0.53 0.53 24980 24714 188 0.7
3.3 0.53 24567
4.1 0.70 28585
4.2 0.70 0.70 28964 29031 394 1.4
4.3 0.70 29544
5.1 0.80 31522
5.2 0.80 0.80 31420 31716 349 1.1
5.3 0.80 32207

Linearity of Assay
35000
Peak area (mAU*min)

30000

25000
y = 26291x + 10679
20000 R² = 0.9999

15000
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
63
Concentration (mg/ml)
 Accuracy
Acceptance criteria : %Recovery= 98.0%- 102.0% of each concentration (0.40, 0.53, and 0.70
mg/ml)
%recovery = × 100
Result : Mean of %Recovery of the concentration at 0.40 mg/ml is 100.5%, Mean of
%Recovery of the concentration at 0.53 mg/ml is 99.2% and Mean of %Recovery of the
concentration at 0.7 mg/ml is 99.9%
Table 1 Result of Accuracy assay Azithromycin

Nominal Average
Nominal amount of
Concentration amount of % of %
Sample Concentration azithromycin
(mg/ml) azithromycin Recovery
(mg/ml) (mg) recovery
(mg)
1.1 0.40 200 202 101.0
1.2 0.40 0.40 200 212 100.1 100.5
1.3 0.40 200 201 100.5
2.1 0.53 256 251 98.0
2.2 0.53 0.53 256 254 99.2 99.2
2.3 0.53 256 257 100.4
3.1 0.70 350 352 100.6
3.2 0.70 0.70 350 347 99.1 99.9
3.3 0.70 350 350 100.0
Table: Evaluation data of accuracy for Assay of Drug substance.

Precision
Repeatability Precision
Result: %RSD of Azithromycin with the concentration 0.40, 0.53, 0.70 mg/ml in repeatability
are less than 2%

%RSD = × 100 ; is Average amount found at same Concentration

64
Table 1 Result of Precision assay Azithromycin
Nominal Average
Concentration Peak area
Time Concentratio of Peak SD % RSD
mg/ml
n (mg/ml) area
0 0.40 20901
hours 0.40 0.40 21500 21151 254 1.2
0.40 21054
0.53 24595
0.53 0.53 24980 24714 188 0.7
0.53 24567
0.70 28585
0.70 0.70 28964 29031 394 1.4
0.70 29544
6 0.40 21095
hours 0.40 0.40 21305 21433 340 1.5
0.40 21900
0.53 24670
0.53 0.53 25113 25057 296 1.2
0.53 25390
0.70 28904
0.70 0.70 29700 29502 431 1.5
0.70 29903
12 0.40 20354
hours 0.40 0.40 20995 20784 304 1.5
0.40 21005
0.53 25476
0.53 0.53 24967 25074 294 1.2
0.53 24780
0.70 28996
0.70 0.70 29970
0.70 29752

65
 Intermediate Precision
Result: %RSD of Azithromycin with the concentration 0.40 0.53, 0.70 mg/ml in repeatability
are less than 2%
Table 1 Result of intermediate Precision assay Azithromycin
Nominal
Concentration Peak area Average of Peak %
Day Concentration SD
mg/ml area RSD
(mg/ml)
1 0.40 20901
0.40 0.40 21500 21151 254 1.2
0.40 21054
0.53 24595
0.53 0.53 24980 24714 188 0.7
0.53 24567
0.70 28585
0.70 0.70 28964 29031 394 1.4
0.70 29544
3 0.40 21902
0.40 0.40 22478 22010 347 1.6
0.40 21650
0.53 24850
0.53 0.53 25504 25419 434 1.7
0.53 25904
0.70 29710
0.70 0.70 28540 29032 481 1.7
0.70 28711
6 0.40 21905
0.40 0.40 22051 22227 357 1.6
0.40 22724
0.53 25709
0.53 0.53 24800 25325 384 1.5
0.53 25466
0.70 0.70 29542 28908 1.6

66
 0.70 28641 450
0.70 28541

3. Impurities
Organic Impurities
- Erythromycin A iminoether
- Desosaminylazithromycin
- Erythromycin A oxime
- Azaerythromycin A
Table 1 Method verification of impurities Azithromycin
Test Parameter Method Acceptance
criteria
Impurities Specificity Tested by HPLC method, -No interference of
Spike azithromycin standard peak in the region
solution, azithromycin sample of Azithromycin
solution, organic Impurities, chromatogram
and azithromycin sample
solution with organic
Impurities. [Check
chromatogram of each
individual solution by
photodiode array detector]
Linearity and range Linearity was evaluated by -Linear relationship
HPLC method, use five between peak
concentration of response and
Erythromycin A iminoether, concentration
Desosaminylazithromycin, -Coefficient of
Erythromycin A oximec and determination (R2)
Azaerythromycin A test
solution in a range of 0.05-
0.14 mg/ml.
Concentrations as follows:
0.05, 0.07, 0.09, 0.11 and 0.14
67
 mg/ml and repeat three
times (LOQ=0.05 mg/ml)
And Plot calibration curve
Accuracy Tested by HPLC method, %Recovery 98.0-
Erythromycin A iminoether, 102.0 %
Desosaminylazithromycin,
Erythromycin A oximec and
Azaerythromycin A test
solution three concentration
(0.05, 0.09, 0.14 mg/ml) and
repeat three time
Precision Repeatability:
Erythromycin A iminoether,
Desosaminylazithromycin,
Erythromycin A oximec and
Azaerythromycin A test in
three concentrations (0.05,
0.09, 0.14 mg/ml) and
replicate three times under
the same conditions at
different times in the same
day. (0, 6 and 12 hours)
Intermediate precision:
Same procedure as
repeatability but test in day 1,
3 and 6
Quantitation Limit Test by HPLC method, Base Signal to noise ratio
on Signal-to-Noise (S/N) 10:1
-Prepare a test sample which
contains Erythromycin A
iminoether,
Desosaminylazithromycin

68
 Erythromycin A oximec and
Azaerythromycin A, apply to
HPLC. And repeat ten time

Specificity
Acceptance criteria: No interference of peak in the region of Azithromycin chromatogram
Result: No interference of peak in the region of Azithromycin chromatogram

Chromatogram of the Azithromycin standard solution

Chromatogram of the solvent Acetonitrile

Chromatogram of the Azithromycin sample solution

69
 3

1
4
2

Chromatogram of the azithromycin with organic impurities; (1) Azaerythromycin A, (2)

Erythromycin A iminoether, (3) Azithromycin and (4) Erythromycin A oxime

Linearity and Range

Acceptance criteria: Linearity was evaluated by HPLC method, use five concentration of
Erythromycin A iminoether, Desosaminylazithromycin, Erythromycin A oximec and
Azaerythromycin A test solution in a range of 0.05-0.14 mg/ml. Concentrations as follows:
0.05, 0.07, 0.09, 0.11 and 0.14 mg/ml and repeat three times (LOQ=0.05 mg/ml)
Result: The peak area of Azaerythromycin A within the concentration 0.04, 0.07, 0.09, 0.11
and 0.14 mg/ml has linear relationship (visual inspection) with coefficient of determination
(R2) = 0.9999

Table 1 Result of Linearity and Range impurities Azithromycin

Nominal Average
Concentration Peak area
Sample Concentratio of Peak SD % RSD
mg/ml
n (mg/ml) area
1.1 0.05 20
0.05
1.2 0.05 20 20 0.0 0.0
1.3 0.05 20
2.1 0.05 41
2.2 0.07 0.05 41 41 0.0 0.0
2.3 0.05 41
3.1 0.09 63
3.2 0.09 0.09 63 63 0.0 0.0
3.3 0.09 63

70
 4.1 0.11 84
4.2 0.11 0.11 84 84 0.0 0.0
4.3 0.11 84
5.1 0.14 115
5.2 0.14 0.14 115 115 0.0 0.0
5.3 0.14 115

Linearity of Azaerythromycin A
140
120
Peak area (mAU*min)

100
80
60
y = 1058.2x + 327.54
40
R² = 0.9999
20
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
Concentration (mg/ml)

Accuracy
Acceptance criteria : %Recovery= 98.0%- 102.0% of each concentration (0.40, 0.53, and 0.70
mg/ml)
%recovery = × 100

Result : Mean of %Recovery of the concentration at 0.05 mg/ml is 100.1%, Mean of

%Recovery of the concentration at 0.09 mg/ml is 99.8% and Mean of % Recovery of the
concentration at 0.14 mg/ml is 100.3%

71
 Table Result of Accuracy impurities Azithromycin
Nominal amount Average
Nominal Amount of
Concentration of % of %
Sample Concentration azaerythromycin
(mg/ml) Azaerythromycin Recovery
(mg/ml) A (mg) recovery
A (mg)
1.1 0.05 0.05 25.0 24.8 99.2
1.2 0.04 25.0 25.0 100.0 100.1
1.3 0.05 25.0 25.3 101.2
2.1 0.09 0.09 86.0 86.0 100.0
2.2 0.09 86.0 85.7 99.6 99.8
2.3 0.09 86.0 85.9 99.8
3.1 0.14 0.14 130.0 130.5 100.3
3.2 0.14 130.0 131.1 100.8 100.3
3.3 0.13 130.0 130.0 100.0

Precision
Result: %RSD of Azaerythromycin A with the concentration 0.043, 0.086, 0.129 mg/ml in
repeatability are less than 2.0%

Table 1 Result of Precision impurities Azithromycin

Nominal Average
Concentration
Time Concentration Peak area of Peak SD % RSD
mg/ml
(mg/ml) area
0 0.05 20
Hours 0.05 0.05 20 20 0.0 0.0
0.05 20
0.09 63
0.09 0.09 63 63 0.0 0.0
0.09 63

72
 0.14 115
0.14 0.14 115 115 0.0 0.0
0.14 115
6 0.05 0.05 22
hours 0.05 22 22 0.0 0.0
0.05 22
0.09 0.09 64
0.09 64 64 0.0 0.0
0.09 64
0.14 0.14 110
0.14 110 110 0.0 0.0
0.14 110
12 0.05 0.05 21
hours 0.05 21 21 0.0 0.0
0.05 21
0.09 0.09 66
0.09 66 66 0.0 0.0
0.09 66
0.14 0.14 114
0.14 114 114 0.0 0.0
0.14 114

73
Intermediate Precision
Result: % RSD of Azaerythromycin A with the concentration 0.043, 0.086, 0.129 mg/ml mg/ml
in Intermediate precision not less than 2.0%
Table Result of Intermediate Precision impurities Azithromycin
Day Average
Nominal of Peak
Concentration
Concentration Peak area area SD % RSD
mg/ml
(mg/ml)

1 0.05 20
0.05 0.05 20 20 0.0 0.0
0.05 20
0.09 63
0.09 0.09 63 63 0.0 0.0
0.09 63
0.14 115
0.14 0.14 115 115 0.0 0.0
0.14 115
3 0.05 0.05 23
0.05 23 23 0.0 0.0
0.05 23
0.09 0.09 64
0.09 64 64 0.0 0.0
0.09 64
0.14 0.14 110
0.14 110 110 0.0 0.0
0.14 110
, 0.05 0.05 20
0.05 20 20 0.0 0.0
0.05 20
0.09 0.09 66
0.09 66 66 0.0 0.0

74
 0.09 66
0.14 0.14 113
0.14 113 113 0.0 0.0
0.14 113

Quantitation Limit
Prepare a test sample which contains Erythromycin A iminoether, Desosaminylazithromycin,
Erythromycin A oximec and Azaerythromycin A, apply to HPLC. Repeat ten times. Used
calculate the experiment signal to noise ratio between 10:1 and a concentration of about
0.05 mg/ml will yield a signal to noise ratio of 10:1
Limit of quantification (LOQ) = 0.05 mg/ml

75
 Result of verification
Table Result of method verification analytical procedure
Test Parameter Acceptance criteria Result Interpret
Negative result no peak Negative result no
response present at the peak response
Identification Specificity same retention time as present at the same pass
positive result. retention time as
positive result.
Assay No interference of peak No interference of
in the region of peak in the region of
Specificity pass
Azithromycin Azithromycin
chromatogram chromatogram
Linear relationship The peak area of
between peak response Azithromycin within
and concentration the concentration
Coefficient of 0.30, 0.40, 0.53, 0.70
Linearity and determination (R2) and 0.80 mg/ml has
pass
range 0.999 linear relationship
(visual inspection)
with coefficient of
determination
(R2) = 0.9999
%Recovery at the
%Recovery= 98.0%- concentration 0.40,
Accuracy pass
102.0% 0.53, 0.70 mg/ml is
100.5, 99.2, 99.5
Mean of %RSD in
the concentration
0.40,0.53 and 0.70 of
Precision %RSD 2.0% the 0 hour 6 hour pass
and 12 hour is 1.3 ,
1.4 and 1.9
respectively
76
 Mean of %RSD in
the concentration
0.40,0.53 and 0.70 of
Intermedia
%RSD 2.0% the 0 hour 3 hour pass
precision
and 6 hour is 1.3 ,
1.7 and 1.6
respectively
No interference of peak No interference of
in the region of peak in the region of
Specificity pass
Azithromycin Azithromycin
chromatogram chromatogram
Linear relationship The peak area of
between peak response Azaerythromycin A
and concentration within the
Coefficient of concentration 0.04,
determination (R2) 0.07, 0.09, 0.11 and
Linearity and 0.999 0.14 mg/ml has
pass
range linear relationship
(visual inspection)
with coefficient of
Impurity
determination
(R2) = 0.9999

Mean of %Recovery
of the concentration
at 0.05 mg/ml is
100.1%, Mean of
%Recovery of the
Accuracy %Recovery 98.0-102.0 %
concentration at pass
0.09 mg/ml is 99.8%
and Mean of %
Recovery of the
concentration at
77
 0.14 mg/ml is
100.3%
%RSD of
Azaerythromycin A
with the
Precision %RSD 2.0% concentration 0.043, pass
0.086, 0.129 mg/ml
in repeatability are
0.0
% RSD of
Azaerythromycin A
with the
Intermedia concentration 0.043,
pass
precision 0.086, 0.129 mg/ml
mg/ml in
Intermediate
precision are 0.0
Quantitation 0.05 mg/ml pass
Single to noise ratio 10:1
limit

78
References
United States Pharmacopeial Convention. The United States pharmacopeia the
national formulary official from May , Rockville, MD: United States Pharmacopeial
Convention;
International Conference on Harmonization; "SPECIFICATIONS: TEST PROCEDURES AND
ACCEPTANCE CRITERIA FOR NEW DRUG SUBSTANCES AND NEW DRUG PRODUCTS: CHEMICAL
SUBSTANCES Q A",
Al-Hakkani, M.F. A rapid, developed and validated RP-HPLC method for determination of
azithromycin. SN Appl. Sci. , https://doi.org/ s
A rapid,developed and validated RP-HPLC method for determination of
azithromycin.[internet]. [cited Feb Available from:
https://link.springer.com/content/pdf/ s pdf

79
S5 Reference Standards

Table 2 Reference Standards

Reference Standards Source Lot No.

Azaerythromycin A RS The United States R02440
Pharmacopeial Convention
Azithromycin RS The United States R103C0
Pharmacopeial Convention
Azithromycin Related Compound F RS The United States R08580
Pharmacopeial Convention
Desosaminylazithromycin RS The United States R076H0
Pharmacopeial Convention

80
81
82
83
84
85
86
87
88
89
Reference

1. United State Pharmacopoeia 2019 USP 42- NF 37 pdf [Internet]. Pharmaceuticals Industry -
Web of Pharma. [cited 2022 Feb 24]. Available from:
https://www.webofpharma.com/2021/01/united-state-pharmacopoeia-2019-usp-42.html

2. Azaerythromycin A RS [Internet]. The United States Pharmacopeial Convention. [cited 2022

Feb 24]. Available from: https://store.usp.org/product/1045600

3. Azithromycin RS [Internet]. The United States Pharmacopeial Convention. [cited 2022 Feb
24]. Available from: https://store.usp.org/product/1046056#

4. Azithromycin Related Compound F RS [Internet]. The United States Pharmacopeial

Convention. [cited 2022 Feb 24]. Available from: https://store.usp.org/product/1046045

5. Desosaminylazithromycin RS [Internet]. The United States Pharmacopeial Convention.

[cited 2022 Feb 24]. Available from: https://store.usp.org/product/1046078

90
S6 Closer Container System
Initially drug substance is packed in Low Density Polyethylene (LDPE) bag securely
folded and sealed with white plastic seal. The put into blue colored High Density
Polyethylene (HDPE) drum with screw lid and reinforced with metallic clamp.
Table 2 Specification container

Material Primary: Low Density Polyethylene bag

Secondary: High Density Polyethylene drum

Closure Material Primary: Plastic sealed

Secondary: Metallic clamp

Capacity 50 kg

Reference

1. [Hot Item] High Purity 99% Pure Azithromycin Azithromycina Powder CAS 83905-01-5
From Directly Factory [Internet]. Made-in-China.com. [cited 2022 Feb 24]. Available
from: https://lvyangbiotech.en.made-in-china.com/product/zZWnTMEYRxcA/China-
High-Purity-99-Pure-Azithromycin-Azithromycina-Powder-CAS-83905-01-5-From-
Directly-Factory.html

2. Azithromycin Dihydrate [Internet]. indiamart.com. [cited 2022 Feb 24]. Available from:
https://www.indiamart.com/proddetail/azithromycin-dihydrate-23126342691.html

91
S7 Stability
Stability testing provide evidence on how the quality of a drug substance varies over a given
time period and under the influence of the environment factors throughout its period of
storage and use. The properties and characteristics of the substance are the same as at time
of manufacture.

7.1 Test material

Chemical Name: Azithromycin dihydrate
CAS No.:
Formula: C38H72N2O12 2O
Table 2 Test material
Batch No.
1 2 3
Raw material Azithromycin dihydrate
Source Gold Biotechnology
Lot No. S4008044061 S4008044062 S4008044063
Manufacturing date 15 April 2013 15 May 2013 15 June 2013
Expire date 14 April 2018 14 May 2018 14 June 2018
Batch 50 kg
Packaging Primary LDPE bag
Secondary HPDE drum

7.2 Test design

7.2.1 Sampling plan
The substance was packed in LDPE bag in HPDE drum. The sample was collected by
using sample powder from 3 bags/batch of Azithromycin dihydrate and from 3 part of the
primary packaging (top, middle, bottom).

92
7.2.2 Storage Conditions and Testing Frequency
According to the ASEAN region are the Zone IVb (Hot/Higher Humidity), Long term
study storage condition is 30°C ± C/75% RH ± 5% and Accelerated study condition is 40°C
± C/75% RH ± 5%. Each samples of the package were tested according to following
schedule.
Table 2 storage condition and testing Frequency
Storage condition Testing frequency
Long term 0, 3, 6, 9, 12, 18, 24, 36, 48, 60 months
30°C ± C/75% RH ± 5%
Accelerated 0, 1, 3, 6 months
40°C ± C/75% RH ± 5%

7.2.3 Testing schedule

Table 2 Schedule for stability of Azithromycin
Storage Schedule
Period condition S4008044061 S4008044062 S4008044063
Accelerated
Initial 15/04/2013 15/05/2013 15/06/2013
Long term
1 month Accelerated 15/05/2013 15/06/2013 15/07/2013
Accelerated
3 months 15/07/2013 15/08/2013 15/09/2013
Long term
Accelerated
6 months 15/10/2013 15/11/2013 15/12/2013
Long term
9 months Long term 15/01/2014 15/02/2014 15/03/2014
12 months Long term 15/04/2014 15/05/2014 15/06/2014
18 months Long term 15/10/2014 15/11/2014 15/12/2014
24 months Long term 15/04/2015 15/05/2015 15/06/2015
36 months Long term 15/04/2016 15/05/2016 15/06/2016
48 months Long term 15/04/2017 15/05/2017 15/06/2017
60 months Long term 15/04/2018 15/05/2018 15/06/2018
Remarks Long term : 30°C ± 2°C/75% RH ± 5%
Accelerated : 40°C ± 2°C/75% RH ± 5%

93
7.2.4 Testing and test criteria
Table 2 Testing and test criteria
Test Requirement Method
Appearance A White or almost white powder Visual inspection
Assay 945-1030 µg/mg on the USP 42 NF 37 , Page 440
anhydrous basis
Impurities USP 42 NF 37 , Page 440
- Residue on ignition NMT 0.3% <281> Page 6587
Organic impurities USP 42 NF 37 , Page
- Erythromycin A NMT 0.5% 440-441
iminoethera
- Erythromycin A oximec NMT 0.5%
- Azaerythromycin Ae NMT 1.0%
- Desosaminylazithromycin NMT 0.3%
- Any individual, NMT 0.2%
unidentified impurity
Total impurities NMT 3.0%
Specific test USP 42 NF 37 , Page 442
- Water Determination 4.0% - 5.0% (dihydrate)
- Loss on Drying NMT 4.5% between ambient
temperature and the inflection
point at about 70 , and 1.8% -
2.6% between the inflection
point at about 70 and the
inflection point at about 130

94
 7.2.5 Result of stability test
Table Result of stability of Lot No. S4008044061
Storage Lot No. S4008044061
Period Assay Impurity % Water Loss on
Condition Appearance
mo. % A B C D E F T Determination % Drying %
A White or almost 94.5%- NMT NMT NMT NMT NMT NMT NMT 4.0% - 5.0%
Specification 1.8% - 2.6%
white powder 103.0% 0.3% 0.5% 0.5% 1.0% 0.3% 0.2% 3.0% (dihydrate)
0 Accelerated C 97.5 0.03 % 0.01 % 0.03 % 0.01 % 0.01 % 0.01 % 0.07 % 4.55 % 1.85 %
1 Accelerated C 97.4 0.05 % 0.02 % 0.05 % 0.02 % 0.02 % 0.02 % 0.13 % 4.57 % 1.85 %
3 Accelerated C 97.2 0.07 % 0.03 % 0.07 % 0.03 % 0.03 % 0.03 % 0.19 % 4.59 % 1.87 %
6 Accelerated C 97.0 0.09 % 0.05 % 0.09 % 0.05 % 0.05 % 0.05 % 0.29 % 4.61 % 1.90 %
0 Long term C 97.5 0.03 % 0.01 % 0.03 % 0.01 % 0.01 % 0.01 % 0.07% 4.55 % 1.85 %
3 Long term C 97.3 0.05 % 0.02 % 0.05 % 0.02 % 0.02 % 0.02 % 0.13 % 4.57 % 1.85 %
6 Long term C 97.1 0.07 % 0.03 % 0.07 % 0.03 % 0.03 % 0.03 % 0.19 % 4.59 % 1.87 %
9 Long term C 97.0 0.09 % 0.04 % 0.08 % 0.05 % 0.04 % 0.04 % 0.25 % 4.61 % 1.90 %
12 Long term C 96.9 0.10 % 0.07 % 0.10 % 0.07 % 0.06 % 0.07 % 0.37% 4.63 % 1.92 %
18 Long term C 96.6 0.12 % 0.11 % 0.12 % 0.11 % 0.10 % 0.08 % 0.52% 4.65 % 1.93 %
24 Long term C 96.3 0.15 % 0.13 % 0.15 % 0.13 % 0.13 % 0.09 % 0.63 % 4.68 % 1.95 %
36 Long term C 95.8 0.18% 0.15% 0.17 % 0.14% 0.15 % 0.10% 0.71 % 4.71 % 1.97 %
48 Long term C 95.3 0.20 % 0.17 % 0.19 % 0.17 % 0.18 % 0.13 % 0.84% 4.72 % 2.00 %
60 Long term C 95.0 0.22 % 0.20 % 0.21 % 0.20 % 0.21 % 0.15 % 0.97 % 4.74 % 2.04 %
(C = Complies, NC = Not complies, ND = Not detected)
A: Residue on ignition B: Erythromycin A iminoethera C: Erythromycin A oximec D: Azaerythromycin Ae
E: Desosaminylazithromycin F: Any individual, unidentified impurity T: Total impurities

95
 Table 31 Result of stability of Lot No. S4008044062
Storage Lot No. S4008044062
Period Assay Impurity % Water Loss on
Condition Appearance Determination % Drying %
mo. % A B C D E F T
A White or almost 94.5%- NMT 0.3 NMT 0.5 NMT 0.5 NMT 1.0 NMT 0.3 NMT 0.2 NMT 4.0% - 5.0%
Specification 1.8% - 2.6%
white powder 103.0% % % % % % % 3.0 % (dihydrate)
0 Accelerated C 97.5 0.03 % 0.01 % 0.03 % 0.01 % 0.01 % 0.01 % 0.07 % 4.54 % 1.85 %
1 Accelerated C 97.4 0.05 % 0.02 % 0.04 % 0.02 % 0.02 % 0.02 % 0.12 % 4.56 % 1.85 %
3 Accelerated C 97.2 0.07 % 0.03 % 0.06 % 0.03 % 0.03 % 0.03 % 0.18 % 4.59 % 1.87 %
6 Accelerated C 97.0 0.09 % 0.05 % 0.09 % 0.05 % 0.05 % 0.05 % 0.29 % 4.61 % 1.89 %
0 Long term C 97.5 0.03 % 0.01 % 0.03 % 0.01 % 0.01 % 0.01 % 0.07 % 4.54 % 1.85 %
3 Long term C 97.3 0.05 % 0.02 % 0.05 % 0.02 % 0.02 % 0.02 % 0.13 % 4.56 % 1.85 %
6 Long term C 97.1 0.07 % 0.03 % 0.07 % 0.03 % 0.03 % 0.03 % 0.19 % 4.58 % 1.87 %
9 Long term C 97.0 0.09 % 0.05 % 0.08 % 0.05 % 0.04 % 0.04 % 0.26 % 4.60 % 1.89 %
12 Long term C 96.9 0.10 % 0.07 % 0.10 % 0.07 % 0.06 % 0.07 % 0.37 % 4.62 % 1.92 %
18 Long term C 96.6 0.12 % 0.11 % 0.12 % 0.11 % 0.10 % 0.08 % 0.52 % 4.65 % 1.93 %
24 Long term C 96.4 0.15 % 0.13 % 0.15 % 0.13 % 0.12 % 0.09 % 0.62 % 4.67 % 1.95 %
36 Long term C 95.9 0.18% 0.15% 0.17 % 0.14% 0.14 % 0.10% 0.70 % 4.69 % 1.97 %
48 Long term C 95.5 0.20 % 0.17 % 0.19 % 0.17 % 0.17 % 0.13 % 0.83 % 4.71 % 2.00 %
60 Long term C 95.2 0.22 % 0.20 % 0.22 % 0.20 % 0.21 % 0.15 % 0.98 % 4.73 % 2.03 %
( C = Complies, NC = Not complies, ND = Not detected)
A: Residue on ignition B: Erythromycin A iminoethera C: Erythromycin A oximec D: Azaerythromycin Ae
E: Desosaminylazithromycin F: Any individual, unidentified impurity T: Total impurities

96
 Table 3 Result of stability of Lot No. S4008044063
Storage Lot No. S4008044063
Period Assay Impurity % Water Loss on
Condition Appearance Determination % Drying %
mo. % A B C D E F T
A White or almost 94.5%- NMT NMT NMT NMT NMT NMT NMT 4.0% - 5.0%
Specification 1.8% - 2.6%
white powder 103.0% 0.3% 0.5% 0.5% 1.0% 0.3% 0.2% 3.0% (dihydrate)
0 Accelerated C 97.5 0.03 % 0.01 % 0.03 % 0.01 % 0.01 % 0.01 % 0.07 % 4.55 % 1.85 %
1 Accelerated C 97.4 0.05 % 0.02 % 0.05 % 0.02 % 0.02 % 0.02 % 0.13 % 4.56 % 1.86 %
3 Accelerated C 97.3 0.07 % 0.03 % 0.08 % 0.03 % 0.03 % 0.03 % 0.20 % 4.58 % 1.87 %
6 Accelerated C 97.1 0.09 % 0.05 % 0.09 % 0.05 % 0.05 % 0.05 % 0.29 % 4.60 % 1.91 %
0 Long term C 97.5 0.03 % 0.01 % 0.03 % 0.01 % 0.01 % 0.01 % 0.07 % 4.55 % 1.85 %
3 Long term C 97.3 0.05 % 0.02 % 0.05 % 0.02 % 0.02 % 0.02 % 0.13 % 4.56 % 1.86 %
6 Long term C 97.1 0.07 % 0.03 % 0.07 % 0.03 % 0.03 % 0.03 % 0.19 % 4.58 % 1.87 %
9 Long term C 97.0 0.09 % 0.04 % 0.08 % 0.04 % 0.04 % 0.04 % 0.24 % 4.60 % 1.91 %
12 Long term C 96.9 0.10 % 0.08 % 0.10 % 0.07 % 0.07 % 0.07 % 0.39 % 4.63 % 1.92 %
18 Long term C 96.7 0.12 % 0.11 % 0.12 % 0.11 % 0.10 % 0.08 % 0.52 % 4.65 % 1.93 %
24 Long term C 96.4 0.15 % 0.13 % 0.15 % 0.13 % 0.13 % 0.09 % 0.63 % 4.67 % 1.95 %
36 Long term C 95.9 0.18% 0.15% 0.17 % 0.15% 0.15 % 0.10% 0.72 % 4.70 % 1.98 %
48 Long term C 95.6 0.20 % 0.17 % 0.19 % 0.17 % 0.18 % 0.13 % 0.84 % 4.71 % 2.00 %
60 Long term C 95.2 0.21 % 0.20 % 0.22 % 0.20 % 0.20 % 0.15 % 0.97 % 4.73 % 2.02 %
( C = Complies, NC = Not complies, ND = Not detected)
A: Residue on ignition B: Erythromycin A iminoethera C: Erythromycin A oximec D: Azaerythromycin Ae
E: Desosaminylazithromycin F: Any individual, unidentified impurity T: Total impurities

97
7.2.6 Container closure system
Primary LDPE bag
Secondary HPDE drum
7.2.7 Conclusion
According to Long term study storage condition is 30°C ± C/75% RH ± 5% and
Accelerated study condition is 40°C ± C/75% RH ± 5%, Significant changes in physical and
chemical stability were not observed. All three batches pass the criteria.
Shelf-life: Based on the resulting data the shelf-life has been established for Five years.
Storage Directions: store below 30 C

98
 Part P
Drug Product
P (Drug Product): Azithromycin Powder for Oral Suspension
P1 Description and Composition
Description *** product
Product name: Azinova®
Description: A dry powder White or off-white powder which reconstitutes with water to give a
peppermint and menthol flavoured suspension.
Container and pack size:
Powder for 600 mg or suspension 200mg/5ml.
Packs of powder equivalent to 600 mg azithromycin in a polypropylene container with
child resistant screw cap with or without a tamper evident seal, in a carton box. Pack contains a
double-ended multi-dosing spoon with detachable adaptor. Reconstitute with 9 ml of water to
give 15 ml suspension. *** Reconstitution
Composition
Table 33 Composition of Azithromycin oral suspension 200mg/5ml
Ingredients Unit Formula Content Function Reference
% w/w
1.Azithromycin 0.629 g 5.71 Active ingredient USP42, NF37
dihydrate
2. Lactose Super tab 1.125 g 10.21 Diluent USP42, NF37
11SD
3.Sucrose 8.700 g 78.95 Diluent/ USP42, NF37
Sweetening agent

4.Hydroxypropyl 0.220 g 2.00 Binder USP42, NF37

Cellulose (HPC)

99
 5. Purified water 5 ml - ** Remove during USP42, NF37
manufacturing
process.
6.Xanthan Gum 0.010 g 0.09 Suspending agent USP42, NF37
7.Sodium phosphate 0.085 g 0.77 Buffering agent USP42, NF37
8.Flav.peppermint 0.085 g 0.77 Flavoring agent USP42, NF37
9.Menthol 0.005 g 0.04 Flavoring agent USP42, NF37
10.Silicon dioxide 0.040 g 0.36 Glidant USP42, NF37
(Aerosil®)
11.Sucralose 0.120 g 1.09 Sweetening agent USP42, NF37
Total Weight 11.019 g 100%
- Azithromycin MW. 749 0.600 g equivalent to Azithromycin dihydrate MW. 785 0.629 g

Reference

1. emc. Azithromycin powder for oral suspension. [Internet]. [cited 2022 Feb 24]. Available from:
https://www.medicines.org.uk/emc/product/3006/smpc#PACKAGE
2. ejpmr.QUALITY BY DESIGN APPROACH FOR DEVELOPMENT OF AZITHROMYCIN ORAL
RECONSTITUTABLE SUSPENSION AND ITS COMPARISON WITH MARKETED PRODUCT. [Internet].
[cited 2022 Feb 24]. Available from: https://savaglobal.com/wp-content/uploads/2021/08/Sava-
paper-3.pdf

100
P2 Pharmaceutical Development
P2.2 Component of Drug Product
P 2.2.1 Active ingredient
Azithromycin dihydrate
General properties
Appearance : A white or almost white powder.
pka : Azithromycin has two tertiary amine whose pka value are 8.1 for the ring nitrogen and 8.8
for the desosaminyl nitrogen
Solubility characteristics : solubility greater than 100 mg/ml in acetone, acetonitrile, chloroform,
dimethylformamide, dimethylsulfoxide, ethanol, ethyl acetate, isopropanol and methanol;
Azithromycin dihydrate has solubility greater than 100 mg/ml at pH values lower than 5. The
compound degrades under acidic conditions. At pH values above 8, the solubility of Azithromycin
is less than 0.2 mg/ml. Azithromycin is practically insoluble in water and freely soluble in
anhydrous ethanol and methylene chloride.
Melting behavior : 124oC 128oC (with decomposition at ca. 250oC). These values have been
determined by DSC.
Hygroscopicity : The anhydrous form of AZI seemed to be unstable since it converted to
dihydrate on storage at room temperature. On the other hand, monohydrate in the presence of
moisture can convert to the more stable dihydrate form. Therefore, the most stable form of AZI
is dihydrate
P 2.2.2 Excipients
1. Lactose Supertab 11 SD
Function
Diluent

101
Applications in Pharmaceutical Formulation or Technology
Spray-dried lactose is widely used as a binder, filler-binder, and flow aid in direct
compression tableting.
Description
Lactose occurs as white to off-white crystalline particles or powder. It is odorless and
slightly sweet-tasting. Spray-dried direct compression grades of lactose are generally composed
of 80 90% specially prepared pure a-lactose monohydrate along with 10 20% of amorphous
lactose.
Incompatibility
Lactose is a reducing sugar. The amorphous lactose, which is the most reactive form of
lactose present in spray-dried lactose, will interact more readily than conventional crystalline
grades. Typical reactions include the Maillard reaction with either primary or secondary amines.
Safety
Lactose is widely used in pharmaceutical formulations as a diluent in oral capsule and
tablet formulations. It may also be used in intravenous injections. Adverse reactions to lactose
are largely due to lactose intolerance, which occurs in individuals with a deficiency of the enzyme
lactase.
2. Sucrose
Function
Granulation aid
Applications in Pharmaceutical Formulation or Technology
Sucrose is widely used in oral pharmaceutical formulations. Sucrose syrup, containing
50-67% w/w sucrose, is used in tableting as a binding agent for wet granulation. In the

102
powdered form, sucrose serves as a dry binder (2-20% w/w) or as a bulking agent and
sweetener in chewable tablets and lozenges. that contain large amounts of sucrose may harden
to give poor disintegration.
Description
Sucrose is a sugar obtained from sugar cane (Saccharum officinarum Linne (Fam.
Gramineae), sugar beet (Beta vulgaris Linne (Fam. Chenopodiaceae)), and other sources. It
contains no added substances. Sucrose occurs as colorless crystals, as crystalline masses or
blocks, or as a white crystalline powder; it is odorless and has a sweet taste.
Solubility
Table 34 : Solubility
Solvent Solubility at 20 C unless otherwise started
Chloroform Practically insoluble
Ethanol 1 in 400
Ethanol 95 1 in 170
Propane-2-ol 1 in 400
Water 1 in 0.5
1 in 0.2 at 200 C

Incompatibility
Powdered sucrose may be contaminated with traces of heavy metals, which can lead to
incompatibility with active ingredients, e.g. ascorbic acid. Sucrose may also be contaminated
with sulfite from the refining process. With high sulfite content, color changes can occur in
sugar-coated tablets; for certain colors used in sugar coating the maximum limit for sulfite

103
content, calculated as sulfur, is 1 ppm. In the presence of dilute or concentrated acids, sucrose
is hydrolyzed or inverted to dextrose and fructose (invert sugar). Sucrose may attack aluminum
closures.
Safety
Sucrose is hydrolyzed in the small intestine by the enzyme sucrase to yield dextrose
and fructose, which are then absorbed. When administered intravenously, sucrose is excreted
unchanged in the urine. Although sucrose is very widely used in foods and pharmaceutical
formulations, sucrose consumption is a cause of concern and should be monitored in patients
with diabetes mellitus or other metabolic sugar intolerance. Sucrose is also considered to be
more cariogenic than other carbohydrates since it is more easily converted to dental plaque.
For this reason, its use in oral pharmaceutical formulations is declining. Although sucrose has
been associated with obesity, damage, and a number of other diseases, conclusive evidence
renal linking sucrose intake with some diseases could not be established. It was, however,
recommended that sucrose intake in the diet should be reduced.
LD50 (mouse, IP): 14 g/kg
LD50 (rat, oral): 29.7 g/kg
3. Hydroxy Propyl Cellulose (HPC)
Function
Suspending agent
Applications in Pharmaceutical Formulation or Technology

104
 Hydroxypropyl cellulose is widely used in oral and topical pharmaceutical formulations.
In oral products, hydroxypropyl cellulose is primarily used in tableting as a binder, film-coating,
and extended-release-matrix former. Concentrations of hydroxypropyl cellulose of 2 6% w/w
may be used as a binder in either wet-granulation or dry, direct compression tableting
processes. Concentrations of 15 35% w/w of hydroxypropyl cellulose may be used to produce
tablets with an extended drug release. The release rate of a drug increases with decreasing
viscosity of hydroxypropyl cellulose. The addition of an anionic surfactant similarly increases the
viscosity of hydroxypropyl cellulose and hence decreases the release rate of a drug. Blends of
hydroxypropyl cellulose and other cellulosic polymers have been used to improve wet
granulation characteristics and tableting characteristics, as well as to achieve better control and
manipulation of the rate of drug release. As an alternative technology to wet granulation, dry
granulation and direct compression of hydroxypropyl cellulose formulations have been
reported to exhibit acceptable tableting and flow characteristics for application in extended-
release matrix tablets. Typically, a 5% w/w solution of hydroxypropyl cellulose may be used to
film-coat tablets. Aqueous solutions containing hydroxypropyl cellulose together with an
amount of methyl cellulose or ethanolic solutions have been used. Stearic acid or palmitic acid
may be added to ethanolic hydroxypropyl cellulose solutions as plasticizers. Environmental
concerns have limited the use of ethanol in film coating solutions. A low-substituted
hydroxypropyl cellulose is used as a tablet disintegrant, Low-substituted. Hydroxypropyl
cellulose is also used in microencapsulation processes and as a thickening agent. In topical
formulations, hydroxypropyl cellulose is used in transdermal patches and ophthalmic
preparations. Hydroxypropyl cellulose is also used in cosmetics and in food products as an
emulsifier and stabilizer
Description
Hydroxypropyl cellulose is a white to slightly yellow-colored, odorless and tasteless
powder.
Solubility

105
 Soluble 1 in 10 parts dichloromethane; 1 in 2.5 parts ethanol (95%); 1 in 2 parts
methanol; 1 in 5 parts propan-2-ol; 1 in 5 parts propylene glycol; and 1 in 2 parts water.
Practically insoluble in aliphatic hydrocarbons; aromatic hydrocarbons; carbon tetrachloride;
petroleum distillates; glycerin; and oils.
Hydroxypropyl cellulose is freely soluble in water below 38 oC, forming a smooth, clear,
colloidal solution. In hot water, it is insoluble and is precipitated as a highly swollen floc at a
temperature between 40 and 45oC. Hydroxypropyl cellulose is soluble in many cold or hot
polar organic solvents such as dimethyl formamide; dimethyl sulfoxide; dioxane; ethanol (95%);
methanol; propan-2-ol (95%); and propylene glycol. There is no tendency for precipitation in
hot organic solvents. However, the grade of hydroxypropyl cellulose can have a marked effect
upon solution quality in some organic liquids that are borderline solvents, such as acetone;
butyl acetate; cyclohexanol; dichloromethane; lactic acid; methyl acetate; methyl ethyl ketone;
propan-2-ol (99%); and tert-butanol.
Incompatibility
Hydroxypropyl cellulose in solution demonstrates some incompatibility with substituted
phenol derivatives, such as methylparaben and propylparaben. The presence of anionic
polymers may increase the viscosity of hydroxypropyl cellulose solutions. The compatibility of
hydroxypropyl cellulose with inorganic salts varies depending upon the salt and its
concentration. Hydroxypropyl cellulose may not tolerate high concentrations of other dissolved
materials. The balance of the hydrophilic lipophilic properties of the polymer, which are
required for dual solubility, reduces its ability to hydrate with water and it therefore tends to be
salted out in the presence of high concentrations of other dissolved materials. The precipitation
temperature of hydroxypropyl cellulose is lower in the presence of relatively high
concentrations of other dissolved materials that compete for the water in the system
Safety

106
 Hydroxypropyl cellulose is widely used as an excipient in oral and topical
pharmaceutical formulations. It is also used extensively in cosmetics and food products.
Hydroxypropyl cellulose is generally regarded as an essentially nontoxic and nonirritant
material. It is not absorbed from the gastrointestinal tract and is fully recovered in feces after
oral administration in rats. It does not exhibit skin irritation or skin sensitization. However, the
use of hydroxypropyl cellulose as a solid ocular insert has been associated with rare reports of
discomfort or irritation, including hypersensitivity and edema of the eyelids. Adverse reactions to
hydroxypropyl cellulose are rare. However, it has been reported that a single patient developed
contact dermatitis due to hydroxypropyl cellulose in a transdermal estradiol patch. The WHO
has specified an acceptable daily intake for hydroxypropyl cellulose of up to 1500 mg/kg body-
weight.(28) Excessive consumption of hydroxypropyl cellulose may have a laxative effect.
LD50 (rat, IV): 0.25 g/kg
LD50 (rat, oral): 10.2 g/kg
4. Purified Water
Function
Solvent
Applications in Pharmaceutical Formulation or Technology
Water is widely used as a raw material, ingredient and solvent in the processing, formulation
and manufacture of pharmaceutical products, active pharmaceutical ingredients (API) and
intermediates, and analytical reagents. Specific grades of water are used for particular
applications in concentrations up to 100%
Description
awn direct from the
public supply and is suitable for drinking. Water used in the pharmaceutical industry and related
disciplines is classified as either drinking (potable) water, purified water, sterile purified water,

107
water for injection (WFI), sterile water for injection, bacteriostatic water for injection, sterile
water for irrigation, or sterile water for inhalation. Validation is required for all systems producing
the water indicated, with the exception of potable water. The chemical composition of potable
water is variable, and the nature and concentrations of the impurities in it depend upon the
source from which it is drawn. Water classified as potable water for applications such as some
initial rinsing and API manufacturing operations, must meet the US Environmental Protection

Japan. For most pharmaceutical applications, potable water is purified by distillation, ion
exchange treatment, reverse osmosis (RO),

purified water should be used, e.g. WFI. Water is a clear, colorless, odorless, and tasteless liquid.
Incompatibility
In pharmaceutical formulations, water can react with drugs and other excipients that are
susceptible to hydrolysis (decomposition in the presence of water or moisture) at ambient and
elevated temperatures. Water can react violently with alkali metals and rapidly with alkaline
metals and their oxides, such as calcium oxide and magnesium oxide. Water also reacts with
anhydrous salts to form hydrates of various compositions, and with certain organic materials
and calcium carbide.
Safety
Water is the base for many biological life forms, and its safety in pharmaceutical
formulations is unquestioned provided it meets standards of quality for potability and microbial
content. Plain water is considered slightly more toxic upon injection into laboratory animals

excessive quantities of water can lead to water intoxication, with disturbances of the electrolyte
balance.
WFI should be free from pyrogens.

108
 LD50 (mouse, IP): 25 g/kg
5. XG
Function
Suspending agent
Applications in Pharmaceutical Formulation or Technology
Xanthan gum is widely used in oral and topical pharmaceutical formulations, cosmetics,
and foods as a suspending and stabilizing agent. It is also used as a thickening and emulsifying
agent. It is nontoxic, compatible with most other pharmaceutical ingredients, and has good
stability and viscosity properties over a wide pH and temperature range; Xanthan gum gels show
pseudoplastic behavior, the shear thinning being directly proportional to the shear rate. The
viscosity returns to normal immediately on release of shear stress. Xanthan gum has been used
as a suspending agent for conventional, dry, and sustained-release suspensions. When xanthan
gum is mixed with certain inorganic suspending agents, such as magnesium aluminum silicate, or
organic gums, synergistic rheological effects occur. In general, mixtures of xanthan gum and
magnesium aluminum silicate in ratios between 1:2 and 1:9 produce the optimum properties.
Similarly, optimum synergistic effects are obtained with xanthan gum : guar gum ratios between
3:7 and 1:9.
Description
Xanthan gum occurs as a cream- or white-colored, odorless, free flowing, fine powder.
Solubility
Practically insoluble in ethanol and ether; soluble in cold or warm water.
Incompatibility
Xanthan gum is an anionic material and is not usually compatible with cationic surfactants,
polymers, or preservatives, as precipitation occurs. Anionic and amphoteric surfactants at

109
concentrations above 15% w/v cause precipitation of xanthan gum from a solution. Under highly
alkaline conditions, polyvalent metal ions such as calcium cause gelation or precipitation; this
may be inhibited by the addition of a glucoheptonate sequestrant. The presence of low levels of
borates (<300 ppm) can also cause gelation. This may be avoided by increasing the boron ion
concentration or by lowering the pH of a formulation to less than pH 5. The addition of ethylene
glycol, sorbitol, or mannitol may also prevent this gelation.
Xanthan gum is compatible with most synthetic and natural viscosity-increasing agents,
many strong mineral acids, and up to 30% inorganic salts. If it is to be combined with cellulose
derivatives, then xanthan gum free of cellulase should be used to prevent depolymerization of
the cellulose derivative. Xanthan gum solutions are stable in the presence of up to 60% water-
miscible organic solvents such as acetone, methanol, ethanol, or propane-2-ol. However, above
this concentration precipitation or gelation occurs.
The viscosity of xanthan gum solutions is considerably increased, or gelation occurs, in
the presence of some materials such as Ceratonia, guar gum, and magnesium aluminum silicate.
This effect is most pronounced in deionized water and is reduced by the presence of salt. This
interaction may be desirable in some instances and can be exploited to reduce the amount of
xanthan gum used in a formulation.
Xanthan gum is incompatible with oxidizing agents, some tablet film-coatings,
carboxymethylcellulose sodium, dried aluminum hydroxide gel, and some active ingredients
such as amitriptyline, tamoxifen, and verapamil.
Safety
Xanthan gum is widely used in oral and topical pharmaceutical formulations, cosmetics,
and food products, and is generally regarded as nontoxic and nonirritant at the levels employed
as a pharmaceutical excipient. The estimated acceptable daily intake for xanthan gum has been
set by the WHO at up to 10 mg/kg body weight. No eye or skin irritation has been observed in
rabbits and no skin allergy has been observed in guinea pigs following skin exposure. No adverse

110
effects were observed in long-term feeding studies with rats (up to 1000 mg/kg/day) and dogs
(up to 1000 mg/kg/day). No adverse effects were observed in a three-generation reproduction
study with rats (up to 500 mg/kg/day).
6. Sodium phosphate
Function
Buffering agent
Applications in Pharmaceutical Formulation or Technology
Dibasic sodium phosphate is used in a wide variety of pharmaceutical formulations as a
buffering agent and as a sequestering agent. Therapeutically, dibasic sodium phosphate is used
as a mild laxative and in the treatment of hypophosphatemia. Dibasic sodium phosphate is also
used in food products, for example as an emulsifier in processed cheese.
Description
The USP 42 states that dibasic sodium phosphate is dried or contains, 1, 2, 7, or 12
molecules of water of hydration. Anhydrous dibasic sodium phosphate occurs as a white powder.
The dihydrate occurs as white or almost white, odorless crystals. The heptahydrate occurs as
colorless crystals or as a white granular or caked salt that effloresces in warm, dry air. The
dodecahydrate occurs as strongly efflorescent, colorless, or transparent crystals.
Solubility
Very soluble in water, more so in hot or boiling water; Practically insoluble in ethanol
(95%). The anhydrous material is soluble 1 in 8 parts of water, the heptahydrate 1 in 4 parts of
water, and the dodecahydrate 1 in 3 parts of water.
Incompatibility
Dibasic sodium phosphate is incompatible with alkaloid, antipyrine, chloral hydrate, lead
acetate, pyrogallol, resorcinol and calcium gluconate and ciprofloxacin. Interaction between

111
Calcium and phosphate, leading to the formation of insoluble calcium-phosphate precipitants,
is possible in parenteral admixtures.
Safety
Dibasic sodium phosphate is widely used as an excipient in parenteral, oral, and topical
pharmaceutical formulations. Phosphate occurs extensively in the body and is involved in many
physiological processes since it is the principal anion of intracellular fluid. Most foods contain
adequate amounts of phosphate, making hypophosphatemia (phosphate deficiency virtually
unknown except for certain disease states or in patients receiving total parenteral nutrition.
Treatment is usually by the oral administration of up to mmol of phosphate daily.
Approximately two-thirds of ingested phosphate is absorbed from the gastrointestinal tract,
virtually all of it being excreted in the urine, and the remainder is excreted in the feces.
Excessive administration of phosphate, particularly intravenously, rectally, or in patients with
renal failure, can cause hyperphosphatemia that may lead to hypocalcemia or other severe
electrolyte imbalances. Adverse effects occur less frequently following oral consumption,
although phosphates act as mild saline laxatives when administered orally or rectally.
Consequently, gastrointestinal disturbances including diarrhea, nausea, and vomiting may occur
following the use of dibasic sodium phosphate as an excipient in oral formulations. However,
the level of dibasic sodium phosphate used as an excipient in a pharmaceutical formulation is
not usually associated with adverse effects.

7. Flav.Papermint
Function
Flavoring agent
Applications in Pharmaceutical Formulation or Technology

112
 Peppermint oil is used as a flavouring agent in foods and fragrance in hygienic or cosmetic
products, and as an anti-itch and cooling agent in topical pharmaceutical products. It is also an
active ingredient in topical analgesics for the relief of joint and muscle pain. Peppermint oil can
be applied topically to temporarily relieve tension-type headache. The use of peppermint oil in
the management of irritable bowel syndrome (IBS) has been investigated in many clinical studies
due to its relaxing effects on smooth muscle.
Description
Peppermint oil (Mentha piperita) is a popular essential oil used in aromatherapy for both
external and internal use. Mentha piperita is a hybrid of spearmint (Mentha spicata) and water
mint (Mentha aquatica). Medicinal use of herbal ingredients such as peppermint oil has a long
history of treating digestive disorders and upper respiratory symptoms and cough. There are
various over-the-counter and commercial uses of peppermint oil due to its carminative,
cholagogue, antibacterial, secretolytic, and choleretic actions. Peppermint oil contains pulegone,
which is a naturally-occurring pesticide. Other active constituents of peppermint oil include
Menthol, menthone, cineol, and several other volatile oils.
Solubility
Solubility in alcohol.
Safety
Almost all toxicological data for peppermint oil relate to its use as a therapeutic agent
rather than as an excipient. Overdose may cause severe gastrointestinal symptoms, diarrhea,
rectal ulceration, epileptic convulsions, loss of consciousness, apnea, nausea, disturbances in
cardiac rhythms, ataxia and other CNS problems, probably due to the presence of menthol. In
the event of overdose, the stomach should be emptied by gastric lavage. Observation should be
carried out with symptomatic treatment if necessary. A near fatal case of high dose peppermint
oil ingestion was reported, the overdose was characterized by comatose and reduced heart rate.
LD50 (rat, oral) : 2426 mg/kg

113
 8. Menthol
Function
Flavoring agent
Applications in Pharmaceutical Formulation or Technology
Menthol is widely used in pharmaceuticals, confectionery, and toiletry products as a
flavoring agent or odor enhancer. In addition to its characteristic peppermint flavor, I-menthol,
which occurs naturally, also exerts a cooling or refreshing sensation that is exploited in many
topical preparations. Unlike mannitol, which exerts a similar effect due to a negative heat of
solution, I- d-Menthol has no
cooling effect, while racemic menthol exerts an effect approximately half that of I-menthol. When
used to flavor tablets, menthol is generally dissolved in ethanol (95%) and sprayed onto tablet
granules and not used as a solid excipient. Menthol has been investigated as a skin-penetration
enhancer and is also used in perfumery, tobacco products, chewing gum and as a therapeutic
agent. When applied to the skin, menthol dilates the blood vessels, causing a sensation of
coldness followed by an analgesic effect. It relieves itching and is used in creams, lotions, and
ointments. When administered orally in small doses menthol has a carminative action.
Description
Racemic menthol is a mixture of equal parts of the (1R,2S,5R)- and (1S,2R,5S)-isomers of
menthol. It is a free-flowing or agglomerated crystalline powder, or colorless, prismatic, or acicular
shiny crystals, or hexagonal or fused masses with a strong characteristic odor and
taste. The crystalline form may change with time owing to sublimation within a closed vessel.
The USP 32 specifies that menthol may be either naturally occurring I-menthol or synthetically
prepared racemic or dI-menthol. However, the JP XV and PhEur 6.0, along with other
pharmacopeias, include two separate monographs for racemic and I-menthol.
Solubility

114
 Very soluble in ethanol (95%), chloroform, ether, fatty oils and liquid paraffin. Freely
soluble in glacial acetic acid. Soluble in acetone and benzene. Very slightly soluble in glycerin
and practically insoluble in water.
Incompatibilities
Incompatible with butylchloral hydrate, camphor, chloral hydrate, chromium trioxide,
-naphthol, phenol, potassium permanganate, pyrogallol, resorcinol; and thymol.
Safety
Almost all toxicological data for menthol relate to its use as a therapeutic agent rather
than as an excipient. Inhalation or ingestion of large quantities can result in serious adverse
reactions such as ataxia and CNS depression, hypersensitivity reaction, severe abdominal pain,
nausea, vomiting, vertigo, drowsiness, and coma. Although menthol is essentially nonirritant there
have been some reports of hypersensitivity following topical application. In a Polish study
approximately 1% of individuals were determined as being sensitive to menthol. There have been
reports of apnea and instant collapse in infants after the local application of menthol to their
nostrils. The WHO has set an acceptable daily intake of menthol at up to 0.4 mg/kg body-weight.
LD50 (rat, IM) : 10.0 g/kg
LD50 (rat, oral) : 3.18g/kg
9. Aerosil
Function
Glidant
Applications in Pharmaceutical Formulation or Technology
Colloidal silicon dioxide is widely used in pharmaceuticals, cosmetics, and food products.
Its small particle size and large specific surface area give it desirable flow characteristics that are
exploited to improve the flow properties of dry powders in a number of processes such as

115
tableting and capsule filling. Colloidal silicon dioxide is also used to stabilize emulsions and as a
thixotropic thickening and suspending agent in gels and semisolid with other ingredients of similar
refractive index, transparent gels may be formed. The degree of viscosity increase depends on
the polarity of the liquid (polar liquids generally, require a greater concentration of colloidal
silicon dioxide than nonpolar liquids). Viscosity is largely independent of temperature. However,
changes to the pH of a system may affect the viscosity. In aerosols, other than those for inhalation,
colloidal silicon dioxide is used to promote particulate suspension, eliminate hard settling, and
minimize the clogging of spray nozzles. Colloidal silicon dioxide is also used as a tablet
disintegrant and as a Colloidal adsorbent dispersing agent for liquids in powders. Silicon dioxide
is frequently added to suppository formulations containing lipophilic excipients to increase
viscosity, prevent sedimentation during molding and decrease the release rate. Colloidal silicon
dioxide is also used as an adsorbent during the preparation of wax microspheres as a thickening
agent for topical preparations and has been used to aid the freeze-drying of nano capsules and
nanosphere suspensions.
Description
Colloidal silicon dioxide is a submicroscopic fumed silica with a particle size of
about 15 nm. It is a light, loose, bluish-white-colored, odorless, tasteless, amorphous
powder.
Solubility
Practically insoluble in organic solvent, water, and acid except hydrofluoric acid. Soluble
in hot solution of alkali hydroxide. Forms a colloidal dispersion with water. Solubility in water is

150 mg L at 25 C pH 7
Incompatibilities
Incompatible with diethylstilbestrol preparations.

116
Safety
Colloidal silicon dioxide is widely used in oral and topical pharmaceutical products
and is generally regarded as an essentially nontoxic and nonirritant excipient. However,
intraperitoneal and subcutaneous injection may produce local tissue reactions and/or
granulomas. Colloidal silicon dioxide should therefore not be administered
parenterally.
LD50 (rat, IV): 0.015 g/kg
LD50 (rat, oral): 3.16 g/kg
10. Sucralose
Function
Sweetening agent
Applications in Pharmaceutical Formulation or Technology
Sucralose is used as a sweetening agent in beverages, foods, and pharmaceutical
applications. It has a sweetening power approximately 300-1000 times that of sucrose and has
no aftertaste. It has no nutritional value, is noncariogenic, does not promote dental caries, and
produces no glycemic response
Description
Sucralose is a white to off-white colored, free-flowing, crystalline powder.
Solubility
Freely soluble in ethanol 95 , methanol, and water. Slightly soluble in ethyl acetate.
Safety

117
 Sucralose is generally regarded as a nontoxic and nonirritant material and is approved, in
a number of countries, for use in food products. Following oral consumption, sucralose is
mainly unabsorbed and is excreted in the feces. The WHO has set an acceptable daily intake for
sucralose of up to 15 mg/kg body weight.
LD50 (mouse, oral): > 16 g/kg
LD50 (rat, oral): > 10 g/kg
Compatibility
The first stage of formulation development usually involves excipients compatibility
studies to select excipients that are physically compatible with the API. Azithromycin dihydrate
was mixed with excipients in different ratios. These mixtures were filled in glass vials and

packed properly and exposed to 60 C for closed condition and 30 C 2 C/75%RH 5%

RH, 400 C 2 75% RH 5% RH for an open and closed condition for period of 4 weeks. FTIR
(Jasco, 4100) was used for spectrum determination.
Compatibility studies confirmed that there were no physical interaction between the
drug and excipients

118
 Table 35 Compatibility

Drug Excipient Proportio Observation Conclusion Justification

n ww Initial At the end
of 4th week
Drug 1 White No change Compatible -
Drug lactose supertab 11 SD Drug and lactose
supertab 11 SD
cause maillard
reaction at
temperature of
1 0 25 White brown Compatible 130 - 150 C but
Azinova ®
Manufacturing
underused at
temperature of
130 - 150 C.
Drug caster sugar 11 White No change Compatible -
Drug HPC 11 off white No change Compatible -
Drug Xanthan gum 11 off white No change Compatible -
Drug Sodium phosphate 11 White Compatible Compatible -
Drug Menthol 1 0 25 White No change Compatible -
Drud Flavour peppermint trusil 1 0 25 White No change Compatible -
Drug Aerosil 1 0 25 White No change Compatible -
Drug Sucalose 1 0 25 off white No change Compatible -

119
 P2.3 Finished Product
P2.3.1 Formulation Development
®

the innovator, Zithromax, marketed by Pfizer. Our purpose would be to develop a robust and
®

by Design) as a systemic approach to determine predefined objectives that emphasizes product

quality. Quality risk management was also employed in our developments.
For constructing a strategy to assess quality aspects during manufacturing development,
the following approaches were implemented.
1. Preformulation
2. Setting Quality target product profile of finished Product
3. Determination of Critical quality attribute of Finished product
4. Selection of both type and composition of excipients.
5. Assessing potential impact of formulation and manufacturing process on drug

Lab scale ®

The Pilot batch ®

and will be subjected to further analysis.

Production batch ®

Manufacturing involved with the primary batch will be using the same equipment
described in P 2.4 which after undergoing process optimization, will be used as the condition on
the production batch.

120
Preformulation
Our purpose is to determine the choice of granulation of
Azithromycin from its physical properties to produce Azithromycin Powder for suspension
Solubility
Since Azithromycin is impractically soluble in water ( Reconstitute media ) by conducting
wet granulation which could ensure
dose to patients.
Stability in High temperature

sesquihydrate which spontaneously converted back to Azithromycin dihydrate upon cooling to

temperature of 70 C.
Flowability
Wet granulation was used to make azithromycin dihydrate, which could improve
flowability. Therefore, the angle of repose of azithromycin formulation was 32.1°, which showed
good flowability.

121
 Table 36 : Flowability

Flow Property Angle of Repose

degrees
Excellent 25-30
Good 31-35
Fair aid not needed 36-40
Passable may hang up 41-45
Poor must agitate, vibrate 46-55
Very poor 56-65
Very, very poor >66

Table 37 : Quality target product profile (QTPP) of finished product

Profile Component Target Justification
Dosage form Solid Pharmaceutical equivalence requirement:
same dosage form.
Dosage design Dry powder for oral Design needed to meet label claims.
Suspension
Route of administration Oral Pharmaceutical equivalence requirement:
same route of administration.
Stability At least 24-months Equivalent to or better than marketed
shelf life at room formulation shelf-life.
temperature
Drug product quality Physical Attributes Pharmaceutical equivalence requirement:
attributes Identification Must meet the same compendia or other
Assay applicable (quality) standards (i.e.,
Content Uniformity identity, assay, purity, and quality).
Dissolution
Water Content

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Container closure Container closure Needed to achieve the target shelf-life
system system and during shipping.
qualified as suitable for
this drug product.

Table 38 : Critical Quality Attributes (CQAs) of finished product

Quality Attributes of Target Is this a Justification
the Drug Product COA ?
Appearance Color acceptable to No Color , shape and appearance are
the patient. not directly linked to safety and
efficacy, therefore they are not
critical. The target to ensure
patient.
Dissolution Not more than 75% Yes API is Class II drug; micronization
of the labeled may be required for better
amount of dissolution, hence risk is high.
azithromycin is
dissolved.
Assay not less than 90.0% Yes Assay affect to clinical
and not more than effectiveness - Efficacy
110.0%
Blend uniformity not less than 90.0% Yes Particle size affects the uniform
and not more than distribution of API, hence risk is high.
110.0%

Deliverable volume Volume of more than No Same deliverable volume as

1 containers is less USP42 is prerequisite.
than 95% but not
less than 90% of LV

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 Powder flow Angle of repose 30 Yes Flow of the powder and fill weight
degrees. may vary, risk is high.
Viscosity Not more than 1.25 Yes Suspension must already have a
CPs suitable viscosity.
pH 8.5-11.0 Yes Same pH as USP42 is prerequisite.

Sedimentation 3 hrs. Yes A larger particle settles faster, risk is

medium.
Redispersibility It should be readily Yes Fine particles form smooth and
dispersible on shaking easy to redisperse suspension, risk
so that uniformity of is medium.
dose is assured.
Stability At least 24-months Yes 1. Different temperature
shelf life at room dependent polymorphic
temperature conversion leads to instability, risk
is high.
2. Dihydride form is stable during
stability, risk is high.
3. API is highly prone to oxidative
degradation leading to potency
loss hence risk is high.

Table 39 :
Drug Product CQAs Critical material attributes (CMA)
Sucrose Sodium Phosphate HPC XG Lactose Flavors
supertab
11 SD
Appearance High Low Low Medium High Low
Assay Medium Low Low Low Low Low
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 Dissolution Low Low Low Low Low Low
Blend uniformity High Low High High Low Low
Viscosity & Pourability High Low High High High High
Microbial limit Low Low High High Low High
Suspendability Low Low High High Low High

To prevent millard reaction with lactose, we use temperature control. We also have
annual GMP audits for microbiological limit and in case of viscosity we have prefomulation in
HPC and XG appropriate composition to reduce the risk of HPC and XG on the formulations.

Table 40 :
Drug Product CQAs Manufacturing process variables
Sugar Drying Dry-Mixing Granulation Drying Milling Blending Filling
Appearance Medium Low Low Low Low Low Low
Assay Low High Medium Low Medium High Low
Dissolution Low Low Medium Low Low Low Low
Blend uniformity Low High Low Low Low High High
Moisture absorption Medium Low Low Medium Low Low Low

After we established Quality by design model (Q6D), identifying critical quality attribute
(CQA) and accessing the risk of excipient and manufacturing process to the finished product of
azithromycin powder for suspension we will then manufacturing each formulation in the
quantity of primary batch ( under the same appropriate condition which was implemented by
our experts in this field ). In the end, each formulation will be subject to quality attribute testing
according to USP monograph.

125
 Table 41 : Experimental formulations of Azithromycin powder for suspension
Active Formulation
pharmaceutial
Function
ingredient/Excipi- F1 F2 F3 F4 F5 F6 F7 F8 F9
ents
Azithromycin Active
0.629 0.629 0.629 0.629 0.629 0.629 0.629 0.629 0.629
dihydrate ingredient
Lactose supertab
1.125 1.120 1.125 1.120 1.125 1.120 1.125 1.120 1.125 Diluent
11 SD
Granulation
Sucrose 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5
aid
Suspending
HPC 0.22 0.22 0.22 0.44 0.44 0.44 0.66 0.66 0.66
agent

purified water Q.s. Q.s. Q.s. Q.s. Q.s. Q.s. Q.s. Q.s. Q.s. Solvent

Suspending
XG 0.01 0.03 0.055 0.01 0.03 0.055 0.01 0.03 0.055
agent
sodium Buffering
0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85
phosphate agent
Flavoring
Flav.papermint 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.015
agent
Flavoring
Menthol 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005
agent

126
 Aerosil 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Glient

Sweetening
sucralose 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12
agent

Total 11.514 11.529 11.56 11.73 11.75 11.77 11.95 11.97 11.99

In each of the above formulation, the amount of HPG or XG is different. HPG or XG have
a high impact for product. The amount of HPG in the formulation is 0.22,0.44,0.66 g, whereas
the amount of XG is 0.01,0.03,0.055 g.
Formulation

Table 42 : Demonstration of each formulation quality attribute ( Calculated using Microsoft

Excel ver. 2019 )
Trials Granules Water required for Phase separation Assay Dissolution Blend uniformity
Flow reconstitution (ml) volume (ml) (%) (%) (%)
1 Excellent 9 ±0.57 1 101 ±1.5 94 ±1.5 103.9 ±1.5
2 Excellent 8 ±0.53 5 99.3 ±1.0 89 ±2.0 93.54 ±1.0
3 Excellent 9 ±0.45 4 105 ±1.0 87 ±1.0 101.1 ±1.4
4 Excellent 9 ±0.57 6 105 ±1.0 97 ±2.0 98.59 ±1.0
5 Excellent 7 ±0.57 7 98.2 ±2.0 81 ±1.5 91.23 ±1.0
6 Excellent 7 ±0.54 5 100 ±1.5 97 ±1.0 95.67 ±2.1
7 Excellent 8 ±0.52 8 98.4 ±2.0 90 ±1.5 98.75 ±1.5
8 Excellent 8 ±0.57 2 99.5 ±1.0 92 ±1.5 98.1 ±2.0
9 Excellent 7 ±0.55 6 98.2 ±2.0 90 ±1.5 95.67 ±1.4
127
Formulation selecting
After we separately evaluated and analyzed each formulation of azithromycin dihydrate
according to USP monograph 42, we had established that formulation 1 is the most preferable
since both criteria for Assay and Blend Uniformity have appropriate results, also the said

powder for suspension.

Dissolution test compare of Azinova with Zithromax

Table 43 Characteristics of the tested Azithromycin dihydrate powder for suspension compared
to the Originator (Zithromax).
Originator drug Generic drug
Brand Zithromax Azinova
INN Azithromycin dihydrate Azithromycin dihydrate
Manufacturer Pfizer O-leaves
Pharmaceutical form Powder for suspension Powder for suspension
Declared content 600 mg/15ml 600 mg/15ml
Excipients sucrose; sodium phosphate, Lactose Supertab 11 SD ,
tribasic, anhydrous; Sucrose, Hydroxy Propyl
hydroxypropyl Cellulose (HPC), XG, Sodium
cellulose; xanthan gum; FD&C phosphate, Flav.Papermint ,
Red #40; and spray dried Menthol, Aerosil
artificial cherry, creme de
vanilla and
banana flavors

128
 To test the dissolution rate of Azinova , paddle apparatus was used, adjusting the
following experimental conditions: rotation speed to 50 rpm, dissolution medium temperature
to 37.5 C, and the total test duration to 30 minutes. Sodium phosphate buffer at pH 6 in a
volume of 900 ml was used as the dissolution medium. At intervals of 5, 10, 15, 20, 25 and 30
minutes, 10 ml of each sample was taken using a syringe. Sampling was carried out from a
position halfway between the surface of the dissolution medium and the bottom of the paddle,
and at least 10 mm away from the vessel wall. The volume reduction due to sampling was
subsequently calculated. The test samples were filtered through filter paper and then their
absorbances were measured at 210 nm by spectrophotometric measurement of absorbances
and using the previously obtained calibration equation, the concentration of Azithromycin
dihydrate, dissolution profiles of the studied Azithromycin dihydrate for oral were constructed
(Figure 2)

Figure 1: Dissolution profile of Zithromax Dash line and Azinova Full line
From the figure 2 show that Zithromax and Azinova have the same dissolution profile
from 5 to 30 minute, so this test is corresponding to QTPP that we determined.

129
 Table 44 : Statical results of Azinova comparing with Zithromax. (Retrieved from SPSS
ver 16.0 )

The results have shown that both Zithromax and Azinova consist of insignificant
differences dissolution profile from one another, Which allowed us to declared our product to
be pharmaceutical equivalent to the originatior (Zithromax).

P2.3.2 Overages
There are no overages used in the formulation of Azinova ®
P2.3.3 Physiochemical and Biological Properties
Powder
-Particle size : Very fine particles possess attractive forces such as Van Der
Waals, electrostatics, surface tension etc. and resulting cohesion of powder and poor
flow properties. As particle size increases, flow of the powders also increases and after
certain level works as free flowing. Appropriate blend of coarse and fine particles or in
other words selection of particle size distribution improves the flow properties.
-Hygroscopicity : A hygroscopic powder tends to retain moisture in the air, either
by absorption or adsorption. A powder is not considered to be hygroscopic if its
percentage of hygroscopicity is less than 10%. The excessive moisture content of a

130
 potentially clog transfer equipment.
Suspension
-Viscosity : as an attribute of a solution or a suspension, is sometimes used to
define the scope of
agent such that the resulting solution (or suspension) will have a viscosity of at least 100

is often required.
-Sedimentation: Redispersibility is the major consideration in assessing the
acceptability of a suspension. The measurement of the sedimentation volume and its
ease of redispersion form two of the most common basic evaluative procedures. The
sedimentation volume is the simple ratio of the height of sediment to initial height of
the initial suspension. The larger the value better is the suspendability.
P 2.4 Manufacturing Process Development
After analyzing information from preliminary study we had proposed manufacturing process
suited powder for suspension production, equipment and procedure are described below.
Equipment
1. Fluidized bed dyer Bectochem
2. Double cone blender Karnavati
3. Rapid mixing granulator Karnavati
4. Moisture analyzer (Sartorius, MA35)
5. Viscometer (Brookfield, DV-II+Pro)
6. HPLC (2998, Waters)
7. Infrared spectrophotometer (Jasco, 4100)
131
8. Automatic Auger Filling Weighing Machine (VTOPS-PSH-W)
Manufacturing Process
Step 1 : Screening and Dry-Mixing
Azithromycin dihydrate, lactose supertab 11SD, and sucrose (in a small amount) sifted through
#40 sieve and remaining quantity of sucrose co-sifted with sucralose through #40 sieve. the
above dry mix was added into rapid mixing granulator and Mixing was carried out for 10 min.
Step 2 : Preparing the damp mass
The accurately weighed quantity of HPC was soaked in a required amount of water to get a
clear solution and this solution was used as binder for the granulation. The wet damp mass was
prepared by adding binder into preblended dry mix in Rapid mixing granulator Karnavati .
Step 3 : Granulation
The wet mass was formed into granules
Step 4 : Drying
The wet mass granules were dried in the fluidized bed dryer (FBD)
Step 5 : Dry Screening
further passed the granules through #20 sieve after drying and flow properties of granules
were checked.
Step 6 : Blending of dried granules with drug excipients
The final blending was carried out.
Step 7 : Filling and Packing
Fill powder in a polypropylene container with child resistant screw cap

132
Screening Azithromycin dehydrate, lactose
supertab 11SD, sucrose and sucralose in
small amount #40 sieve

Dry mixing Rapid mixing granulator

10 min

Dry mixing Rapid mixing granulator

Add Soaked HPLC

Wet damp mass

Granulation

Drying fluidized bed dryer

133
 Dry screening #40 sieve

Blending

Filling

Scheme 1 : Manufacturing process developement flow chart

Process optimization
Table 45 : Optimization manufacturing process criteria
Process Parameter Method Acceptance
criteria
Blending - Blending time (15,20,25 mins) -HPLC - 90.0-110.0 %
- Blending speed (30,45,60 RPM) (Assay)

Dry-Mixing - Dry-Mixing time (30,45,60 mins) -HPLC - 90.0-110.0 %

- Blending speed (30,45,60 RPM) (Assay)

134
 Filling - Filling speed ( 1000 , 1500 , 2000 -HPLC Label claim +
unit/hr) 5% (Content
- Powerbed height from the container uniformity)
unit ( 4, 8 , 12 mm )

According to ASEAN common technical requirement ( ACTR ) 2016 edition, we

have performed manufacturing process optimization complied with the said Guideline in
-time, blending-
speed, Mixing-time, and Mixing speed. Conditions on each process optimizations were
carried out by our expertise and preliminary studies associated with the Azithromycin
Dihydrate attribute.
Since ACTR has declared the acceptance criteria for process validation of a
typical solid dosage, The associated criteria will be presented below therefore we had
adapted the information listed in the ACTR Guideline for our manufacturing process
optimization.
Table 46 : Process validation
Stage Sampling plan Test Acceptance Criteria
Final At least 3 samples Blend / Mix Stage 1 Individual
blending/Mixing from at least ten uniformity (Assay) results: Mean +
different location Analyze one 10% ( Absolute )
evenly distributed sample per
throughout the location All individual
mixer. results: RSD < 0.5%
If required In-house
- Flowability
- Density
- Appearance

135
 Filling Stratified sampling Weight uniformity Label claim + 5% (
Absolute)

We have established locations of sampling in blender according to the

diagram below then we were conducting assay under azithromycin dihydrate USP
monograph 42 editions, However in this documentation, we had only reported the
average assays of each location instead.

Figure 2 : Representing sampling plan in Blender from 10 different locations.

136
 Figure 3 : Representing sampling plan in Mixer from 10 different locations.

Table 47/1 : Summary of Blending process parameter resulted from manufacturing optimization

Process
Process Parameter
Parameter Individual results ( + 10% ) %RSD from all samples
Blending speed
Blending time From all locations (< 0.5%)
(RPM)
(Min)
15 30 98.69, Pass 0.41
20 30 100.46, Pass 0.66
25 30 101.22, Pass 1.7
15 45 99.78, Pass 0.49
20 45 100.82, Pass 0.83
25 45 100.8, Pass 1.18

137
 15 60 98.7, Pass 0.53
20 60 100.97, Pass 0.87
25 60 100, Pass 0.87

Process
Process Parameter
Parameter Individual results ( + 10% ) %RSD from all samples
Blending speed
Blending time From all locations (< 0.5%)
(RPM)
(Min)
15 30 98.69, Pass 0.41
20 30 100.46, Pass 0.66
25 30 101.22, Pass 1.7
15 45 99.78, Pass 0.49
20 45 100.82, Pass 0.83
25 45 100.8, Pass 1.18
15 60 98.7, Pass 0.53
20 60 100.97, Pass 0.87
25 60 100, Pass 0.87

138
Table 47/2 : Summary of Mixing process parameter resulted from manufacturing optimization.
Process
Process Parameter Individual results ( + 10% ) %RSD from all samples
Parameter
Mixing speed (RPM) From all locations (< 0.5%)
Mixing time (Min)
30 30 98.69, Pass 0.41
45 30 101.72, Pass 1.25
60 30 101.42, Pass 2.07
30 45 99.78, Pass 0.49
45 45 100.85, Pass 1.86
60 45 99.62, Pass 1.53
30 60 98.70, Pass 0.53
45 60 102.63, Pass 2.30
60 60 100.54, Pass 1.76
Table 48 : Summary of Mixing process parameter resulted from manufacturing optimization

Process Parameter Average content uniformity From each

Process Parameter
(Powerbed height from the sampling plans (9 unit per sampling
(Filling Speed) (unit/hr)
container) (mm) plan)
1000 4 101.0, Pass
1500 4 100.7, Pass
2000 4 101.2, Pass
1000 8 97.7, Pass
1500 8 100.2, Pass
2000 8 99.3, Pass
1000 12 98.7, Pass
1500 12 104.6, Pass
2000 12 100.5, Pass

139
 The results have shown that, Mixing time of 30 minutes corresponding with mixing
speed 30 RPM is the most preferable for the pilot batch that we were experimented. These
implied that the condition mentioned above will be used as the primary condition on the
production batch. As for blending parameter aspect, the most preferable condition was 15
minutes for blending time and 30 RPM for blending speed. Filling also shown consistently
results throughout the experiments , the most preferable condion for Filling speed is 1500
unit/hr and 8 mm Powerbed height from the container.
P2.5 Container Closure System
Every proposed packaging system should be shown to be suitable for its intended use: it
should adequately protect the dosage form; it should be compatible with the dosage form; and
it should be composed of materials that are considered safe for use with the dosage form and
the route of administration. If the packaging system has a performance feature in addition to
containing the product, the assembled container closure system should be shown to function
properly.
Information intended to establish suitability may be generated by the applicant, by the
supplier of the material of construction or the component, or by a laboratory under contract
to either the applicant or the firm. An adequately detailed description of the tests, methods,
acceptance criteria, reference standards, and validation information for the studies should be
provided.
General issues concerning protection, compatibility, safety and performance of packaging
components and/or systems are discussed below.
We will be conductin
choices were selected by pre-formulation study associated with other brand of Azithromycin

140
 dihydrate and/or SAR of macrolide-
for this study that is polypropylene
1. Protection
A container closure system should provide the dosage form with adequate protection
from factors (e.g., temperature, light) that can cause a degradation in the quality of that dosage
form over its shelf life. Common causes of such degradation are: exposure to light, loss of
solvent, exposure to reactive gases (e.g., oxygen), absorption of water vapor, and microbial
contamination. A drug product can also suffer an unacceptable loss quality if it is contaminated
by filth, according to S3
Light protection is typically provided by an opaque or amber-colored container or by an
opaque secondary packaging component (e.g., cartonsor overwrap). The USP test for light
transmission (USP < >) is an accepted standard for evaluating the light transmission properties
of a container.
Water vapor or reactive gases (e.g., oxygen) may penetrate a container closure system
either by passing through a permeable container surface (e.g., the wall of a low density
polyethylene (LDPE) bottle) or by diffusing past a seal. Plastic containers are susceptible to both
routes. Although plastic containers are more effective only if there is a good seal between
the container and the closure.
Table 49 : Light and moisture vapor transmission
Polymer Light transmission Moisture vapor transmission rate
cc 25mm m2 24hr
Polypropylene Practically zero transmittance 10.7

141
 2. Safety and Compatibility
- Compatibility
Packaging components that are compatible with a dosage form will not interact
sufficiently to cause unacceptable changes in the quality of either the dosage form or the
packaging component.
Table 50 : Compatibility of polymer and Product
Polymer Compatibi ®

Polypropylene Compatable

- Extractable
Extractable is chemical compound that can be extracted out of packaging component
and the method of analyze packaging component at
- High-temperatures: to obtain the worst case leachable profile
- Solvent extraction: polar and non-polar solvent to mimic similar properties as drug
product
Table 51 : Extractable study and Extractable procedure
Likelihood of interaction between packaging component and dosage form
Degree of concern
associated with Route
High Medium Low
of Administration
Sterile powders
Inhalation aerosols and solution
Highest Injection powders
Injections and injectable suspensions
Inhalation powders
High Ophthalmic solutions and suspensions

142
 Transdermal ointments and patches
Nasal aerosols and sprays
Topical solutions and suspensions Oral tablets
Topical powders
Low Topical and lingual aerosols Oral hard capsules
Oral powders
Oral solutions and suspensions Oral soft gelatin capsules

Adapted from Guidance for Industry; Container Closure Systems for Packaging Human
Drug and Biologics, US Department of Health and Human Services, Food and Drug
Administration, Rockville, MD, May 1999

Table 52 : Extractable Study & Extraction Procedures

ther N- Acetone propan isoprop Isoprop Aqueo Aqueo Aqueous pH 10 Aqueous pH
mal hexan ol anol anol w us pH us pH 12
e ater 6 8
Reflux - X - - X PC PVC - - - -
only
Soxhle - X - - X - - - - -
t
Sonica - - - - - - X X X X
tion

Since our product have to reconstitute into suspension form. We declared that the
likelihood of interaction between packaging component and dosage form is high and degree of
concern associated with Route of Administration is low.

143
 Total extractable in each polymer's container
Total extractables concentration ( (µg/g)
9
8
7
6
5
4
3
2
1
0
6 8 10 12
pH of aqueous extractable solvents

Polyethylene (PET) Polypropylene (PP) Polyvinyl Chloride (PC) Polystrylene

Figure 4 :
The reason behind not implementing the formulation of Azinova is due to the
very signal to noise ratio resulting in not-feasible experiment, according to USP 42
general chapter <1663> Assessment of extractables associated with pharmaceutical
packaging/delivery system had addressed that using other vehicle of formulation as
extracting media might be implemented instead of formulation, therefore we conducting
a wide range of solvent for mimicking the physical and chemical properties of
Azithromycin dihydrate.
According to the table, polypropylene have lowest total extractables concentration at
pH 8 11 then we decide to use polypropylene as a component of the container

144
Table 53 : Extractable Study : Threshold Levels and Actions

Extractable Level in Component Assignment Category

> 100 µg/g Structure Confirmed
20-100 µg/g Confident
< 20 µg/g Tentative
3. Stability
Regarding stability of Azinova in declared polypropylene container, the nesscery
informations will be available in P8 Stability.
4. Performance
Performance of the container closure system refers to its ability to function in the manner
for which it was designed. A container closure system is often called upon to do more than
simply contain the dosage form. When evaluating performance, two major considerations are
container closure system functionality and drug delivery.
Drug delivery refers to the ability of the packaging system to deliver the dosage form in
the amount or at the rate described in the package insert. Container closure system functionality
and/or drug delivery are compromised when the packaging system fails to operate as designed.
Failure can result from misuse, faulty design, manufacturing defect, improper assembly, or wear
and tear during use. Tests and acceptance criteria regarding dosage form delivery and container
closure system functionality should be appropriate to the dosage form, route of
administration, and design features.
Deliverable volume
The following tests are designed to provide assurance that oral solutions and suspensions

145
will, when transferred from the original container, deliver the volume of dosage form that is
declared on the label of the article. These tests are applicable to products labelled to contain
not more than 250 ml, whether supplied as liquid preparations or liquid preparations that are
constituted from solids upon the addition of a designated volume of a specific diluent. They are
not required for an article packaged in single-unit containers when the monograph includes
"Uniformity of Dosage Units" (Appendix 4.28).
For the determination of deliverable volume, select not fewer than 30 containers, and
Proceed as follows for the dosage form designated.

146
Procedure
The deliverable volume can be determined by weight as follows:
1. Discharge the container contents into a suitable tared container (allowing drainage
for NMT 5 for single-dose containers and NMT 10 min for multiple-unit containers).
2. Determine the mass of the contents.
3. Calculate the volume using the density.
Alternatively, the following by volume procedure may be used:
1 . Under conditions of use or as instructed in the labeling, carefully discharge the
contents of each container into separate dry graduated cylinders of a rated capacity
not exceeding two and a half times the volume to be measured, and calibrated "to
contain" (see Volumetric Apparatus <31 )). Care must be taken to avoid the formation
of air bubbles during the process. In the absence of labeling instructions, support the
containers at about a 30 angle to the horizontal, and gently discharge the contents
into the graduated cylinder.
2. Allow each container to drain for a period not to exceed 10 min for multiple-unit
containers and 5 s for single-unit containers, unless otherwise specified in the
monograph.
3. When free from bubbles, measure the volume of each mixture.
Acceptance criteria
For Multiple-Unit Containers, the average volume of liquid obtained from the 10
containers is NLT 100%, and the volume of no container is less than 95% of the volume
declared
in the labeling. If A, the average volume is less than 100% of that declared in the labeling, but
the volume of no container is less than 95% of the labeled amount, or if B, the average
volume

147
is NLT 100% and the volume of NMT 1 container is less than 95%, but is NLT 90% of the
labeled
volume, perform the test on 20 additional containers. The average volume of liquid obtained
from the 30 containers is NLT 100% of the volume declared in the labeling; and the volume
of
liquid obtained from NMT 1 of the 30 containers is less than 95%, but NLT 90% of that
declared
in the labeling.

Table 54 : %Labeled volume

No. Labeled volumn
1 104
2 102
3 100
4 103
5 102
6 102
7 104
8 104
9 100
10 101
Average 102.2
Pass

After conducting an experiment on Protection, Compatibility, Stability, and Performance

we had found that containers made of Polypropylene have shown extraordinary results
regarding both Protection, Stability, Compatibility and Performance, since our product
comprised of Azithromycin dihydrate as an active pharmaceutical ingredient which has

148
potential for subjecting to photocatalytic oxidation resulting in undesired impurity. Therefore
we had concluded that polypropylene is the most suitable container material for our intended
propose.
P2.6 Microbiological Attributes
According to USP 42 : Microbiological examination for non-sterile product
Powder
Nonaqueous liquids or dry solid dosage forms will not support spore germination or
microbial growth due to their low water activity, Reduced testing. However microbiological
monitoring is required throughout product development , scale-up , process validation , and
routine testing of sufficient marketed product lot to ensure that the product has little or no
potential for microbial contamination
Suspension
Table 55 : Water Activities aw Required to support thr Growth of Representative
Microorganism

Pharmaceutical drug products with water activities well below 0.75 would be
excellent
candidates for reduced microbial limit testing for product release and stability evaluation.
But

149
water activity (aw) of oral suspension is 0.87 according to USP Application of water activity
determination to non-sterile pharmaceutical products.
Table 56 : Microbial Limit Testing Strategy for Representative Pharmaceutical and OTC
Drug Products Based on Water

*TAMC = Total aerobic microbial count; TCYMC = Total combined yeast and mold count

Acceptance criteria for nonsterile pharmaceutical products

Acceptance criteria based upon the total aerobic microbial count (TAMC) and the
total combined yeasts and (TYMC) are given in Tables. Acceptance criteria are based on
individual results or on the average of replicate counts when replicate counts are performed
(e.g., direct plating methods). When an acceptance criterion for microbiological quality is
prescribed, it is interpreted as follows:
101 cfu: maximum acceptable count = 20 ;
102 cfu: maximum acceptable count = 200 ;
103 cfu: maximum acceptable count = 2000; and so forth.

150
Table 57 : Acceptance Criteria for Microbiological Quality of Nonsterile Dosafe Forms

See P5.2 for more information on the procedure and the results of the shelf-life test.
In-use microbial stability
Table 23 : Schedule for in-use microbial stability of Azithromycin Powder for Oral Suspension
200 mg/5mL
Table 58 : Schedule for in-use microbial stability of Azithromycin Powder for Oral
Suspension 200 mg/5mLv
Data of Test : 15/03/2021

Test organism Date Accptance Day 3 Result

Initial Final Criteria
Total Aerobic 102 <10 Pass
Microbial Count 15/03/2021 17/03/2021
cfu/g
151
Total Combined 101 <10 Pass
Yeasts/Molds Count
cfu/g
Specified Absence of Absence of Pass
Microorganisms Escherichia Escherichia
coli 1 ml coli 1 ml

The above test results meet the current USP acceptance criteria for microbiological
quality of nonsterile dosage forms

Reference
1. UCM070551.pdf [Internet]. [cited 2022 Feb 25]. Available from:
http://academy.gmp- compliance.org/guidemgr/files/UCM070551.PDF
2. 4.21 DELIVERABLE VOLUME - [Internet]. Thaiherbal. [cited 2022 Feb 25]. Available
from: https://www.bdn.go.th/tp/ebook/qQScZat1pR9gC3q0GT5gMJq0qT5co3uw
3. General Chapters: <698> DELIVERABLE VOLUME [Internet]. [cited 2022 Feb 25].
Available from: http://www.uspbpep.com/usp29/v29240/usp29nf24s0_c698.html
4. 67.-December-2016-ACTR.pdf [Internet]. [cited 2022 Feb 25]. Available from:
https://asean.org/wp-content/uploads/2017/03/67.-December-2016-ACTR.pdf
5. Permeability of Polymers [Internet]. [cited 2022 Feb 25]. Available from:
https://polymerdatabase.com/polymer%20physics/Permeability.html
6. 4.34 POWDER FLOW - [Internet]. Thaiherbal. [cited 2022 Feb 25]. Available from:
https://www.bdn.go.th/tp/ebook/qQycZUt1pR9gC3q0GT5gMJq0qT5co3uw
7. Opaque-Polymer_Aug-2012.pdf [Internet]. [cited 2022 Feb 25]. Available from:
https://www.paint.org/wp-content/uploads/2021/09/Opaque-Polymer_Aug-2012.pdf
8. download.pdf [Internet]. [cited 2022 Feb 25]. Available from:
https://www.fda.gov/media/70788/download

152
 P3 Manufacture
P3.1 Batch Formula
Product name: Azithromycin Powder for Oral Suspension [Azinova®]
Production Batch: 500,000 bottles
Pilot Batch: 100,000 bottles
Batch size: 5,509.5 kg
Table 59 Batch formula
Quantity
No. Ingredients Per Per Per Function Reference
Unit Production
Pilot
Batch
Batch
Active ingredient
1. Azithromycin dihydrate 0.629 g 62.9 kg kg Active ingredient USP42 NF37
Excipients
1. Lactose 1.125 g 112.5 kg kg Diluent USP42 NF37
(SuperTab® 11SD)
2. Sucrose 8.700 g 870.0 kg 4,350.0 kg Diluent, Sweetening USP42 NF37
agent
3. Hydroxypropyl 0.220 g 22.0 kg 110.0 kg Binder USP42 NF37
cellulose (HPC)
4. Purified water* 5 ml - - Solvent of binder USP42 NF37
5. Xanthan gum 0.010 g 1.0 kg 5.0 kg Suspending agent USP42 NF37
6. Sodium phosphate 0.085 g 8.5 kg 42.5 kg Buffering agent USP42 NF37

153
7. Flavor. Peppermint 0.085 g 8.5 kg 42.5 kg Flavoring agent USP42 NF37
8. Menthol 0.005 g 0.5 kg 2.5 kg Flavoring agent USP42 NF37
9. Silicon dioxide 0.040 g 4.0 kg 20.0 kg Glidant USP42 NF37
(Aerosil®)
10. Sucralose 0.120 g 12.0 kg 60.0 kg Sweetening agent USP42 NF37
Total Weight 11.019 g 1,101.9 kg 5,509.5 kg

*Remove during manufacturing process

154
 P3.2 Manufacturing Process and Process Control

Screening
Screening (#40 sieve)
(#40 sieve)
Remaining sucrose
Azithromycin dihydrate Sucralose
Lactose (supertab® 11SD) Dry-mixing (Rapid mixing granulator)

Sucrose in a small amount

Dry-mixing (Rapid mixing granulator)

Add Soaked HPC

Wet damp mass

Granulation
Xanthan gum
Sodium phosphate
Flavor. Peppermint
Menthol Drying (fluidized bed dryer)
Colloidal silicon dioxide
Sucralose
Sucrose
Dry screening (#40 sieve)

Blending
Filling

Figure 1 Manufacturing process Flow chart

155
Manufacturing Process
Step 1 : Screening and Dry-Mixing
Azithromycin dihydrate, lactose supertab 11SD, and sucrose (in a small amount) sifted
through #40 sieve and remaining quantity of sucrose co-sifted with sucralose through #40
sieve. the above dry mix was added into rapid mixing granulator and Mixing was carried out
for 10 min.
Step 2 : Preparing the damp mass
The accurately weighed quantity of HPC was soaked in 5 ml of water to get a
clear solution and this solution was used as binder for the granulation. The wet damp mass
was
prepared by adding binder into preblended dry mix in Rapid mixing granulator (Karnavati).
Step 3 : Granulation
The wet mass was formed into granules
Step 4 : Drying
The wet mass granules were dried in the fluidized bed dryer (FBD)
Step 5 : Dry Screening
further passed the granules through #20 sieve after drying and flow properties of granules
were checked.
Step 6 : Blending of dried granules with drug excipients
The final blending was carried out.
Step 7 : Filling and Packing
Fill powder in a polypropylene container with child resistant screw cap

Equipment
1. Fluidized bed dyer (Bectochem)
2. Double cone blender (Karnavati)
3. Rapid mixing granulator (Karnavati)
4. Moisture analyzer (Sartorius, MA35)
5. Viscometer (Brookfield, DV-II+Pro)
6. HPLC (2998, Waters)
7. Infrared spectrophotometer (Jasco, 4100)
8. Automatic Auger Filling Weighing Machine (VTOPS-PSH-W)
Table 60 In process control Parameters

156
 Process Parameter Sampling plan Critical quality Acceptance criteria
time attributes
Sifting (Sieving, Sieve size 30 min Assay - 90.0-110.0 %
Milling)
Dry-Mixing Mixing time 20 min Assay - 90.0-110.0 %
Mixing speed Blend uniformity - 90.0-110.0 %
Granulation Mixing time 30 min Assay - 90.0-110.0 %
Mixing speed Dissolution - NLT 75% (Q) of the
Temperature labeled amount of
azithromycin is
dissolved

Drying Drying time 30 min Moisture - 90.0-110.0 %

Temperature absorption
Filling Filling speed 30 min Content uniformity - 95.0-105.0 %
Powerbed
height from the
container unit

157
P3.3 Control of Critical Steps and Intermediates
Table 61 Critical Process Parameter (CPP) and Critical Quality Attribute (CQAs)
Critical step Critical process Critical quality
parameter attributes
Dry-Mixing - Mixing time Assay
- Mixing speed Blend uniformity
Filling - Filling speed Content uniformity
- Powerbed height
from the container
unit

P3.4 Process Validation and/or Evaluation

Manufacturing site at which the validation is carried out
Name of manufacturing: O LEAVES Pharmaceutical Co.,Ltd.
Country: Thailand
Type of validation: Prospective
Number of batches validated: 3 batches
Table 62 Batch Production
Batch Number Date of Production Batch size Batch type
(production/pilot)
AZ200565081 12/02/2021 500,000 production
AZ200565082 12/02/2021 500,000 production
AZ200565083 12/02/2021 500,000 production

158
 Table 63 Testing, Sampling plan and Result of validation
Critical Critical quality Sampling plan Acceptance Result
step attributes criteria
Batch No. Batch No. Batch No.
AZ200565 AZ200565 AZ200565
081 082 083
Dry-Mixing - Assay Take 3 samples 90.0-110.0 95.0 % 96.1 % 95.8 %
- Blend from top, middle %
uniformity and bottom every
20 min (2 g from
each location)
Filling - Content Take 3 samples 95.0-105.0 97.6 % 98.9 % 98.2 %
uniformity from top, middle %
and bottom every
10 min (2 g from
each location)
Reference
1. Sonawane D, Chaudhari DP, Thorat V, Dhavale S. QUALITY BY DESIGN APPROACH FOR
DEVELOPMENT OF AZITHROMYCIN ORAL RECONSTITUTABLE SUSPENSION AND ITS COMPARISON
WITH MARKETED PRODUCT. :10.
2. United State Pharmacopoeia 2019 USP 42- NF 37 pdf [Internet]. Pharmaceuticals Industry -
Web of Pharma. [cited 2022 Feb 24]. Available from:
https://www.webofpharma.com/2021/01/united-state-pharmacopoeia-2019-usp-42.html
3. 67.-December-2016-ACTR.pdf [Internet]. [cited 2022 Feb 24]. Available from:
https://asean.org/wp-content/uploads/2017/03/67.-December-2016-ACTR.pdf
4. Guidanace P. In-process control of oral drug product during manufacturing & Packing -
Pharmaceutical Guidance [Internet]. 2020 [cited 2022 Feb 24]. Available from:
https://pharmaguidances.com/in-process-control-of-oral-drug-product-during-manufacturing-
packing/

159
P4 Control of excipient
P4.1 Speciification
. lactose (super tab 11SD)

Test Acceptance criteria Analytical procedure

Identification
A. Infared Absorption (197K)

B. Thin-Layer The principle spot from the Sample USP 42

Chromatography solution corresponds in appearance NF 37
Indentification Test and R, value to that from Standard Page 5796
solution A.
Assay
Impurity

- Residue on Ignition NMT 0.1% USP 42

NF 37
Page 5796
Specific Test
Clarity and Color of NMT 0.04 USP 42
Solution NF 37
Page 5796
Microbial Enumeration The total aerobic microbial count USP 42
Tests and Tests For does not exceed 1 × 102 cfu/g, the NF 37
Specifide Microorganisms total combined molds and yeasts Page 5796
count does not exceed 5 × 101
cfu/g, and it meets the requirements
of the test for absence of
Escherichia coli

160
 Optical Rotation +54.4o to +55.9o calculated on the USP 42
anhydrous basis, determine at 20o NF 37
Page 5796
Acidity or Alkalinity The solution is colorless, and NMT USP 42
0.4mL of 0.1 N sodium hydroxide VS NF 37
is required to produce a pink or red Page 5796
color.
Loss on Drying Monohydrate: MNT 0.5% USP 42
Monohydrate, modified: NMT 1.0% NF 37
Page 5796
Water Determination 4.5% - 5.5% USP 42
NF 37
Page 5796
Protein and Light- NMT 0.25 in the range of 210 220 USP 42
Absorbing Impurity nm NF 37
And NMT 0.07 in the range of 270- Page 5796
300 nm.

161
162
.Sucrose

Test Acceptance criteria Analytical Procedure

Identification
A. Infared Absorbtion (197K)

Assay

Impurity

- sulfite NMT half the absorbance USP 42

different of the References NF 37
solution Page 6014
Specific tests
- Appearance of Solution Sample solution is the same as USP 42
that of water or its opalescence is NF 37
not more pronounced than that Page 6015
of Reference suspension l.
- Conductivity NMT 35 uS. cm-1 at 20o USP 42
NF 37
Page 6015
- Optical rotation +66.3o to +67.0o at 20o USP 42
NF 37
Page 6015
- Color Value The blue color does not USP 42
disappear completely, ignoring NF 37
any blue color at the air/solution Page 6016
interface.
- Loss on drying NMT 0.1% USP 42
NF 37
Page 6016

163
- Bacterial Endotoxin Test Less than 0.25 IU/mg USP 42
NF 37
Page 6016

164
165
. HPC (Hydroxypropyl Cellulose)

Test Acceptance criteria Analytical Procedure

Identification A thin film is formed USP 42
NF 37
Page
Assay 53.4%-80.5%of hydroxypropoxy USP 42
group NF 37
Page
Impurity
Reudue on Ignition NMT 0.8% USP 42
NF 37
Page
Silica NMT 0.6% USP 42
NF 37
Page
Lead NMT 10 ppm USP 42
NF 37
Page
Specific test
pH 5.0-8.0 USP 42
NF 37
Page
Loss on Drying NMT 5.0% USP 42
NF 37
Page
Viscosity Rotation Methods see Labeling USP 42
NF 37
Page

166
167
. Xanthan gum
Test Acceptance criteria Analytical procedure

Identification A firm, rubbery gel forms with the Sample USP 42

after the temperature drops below 40°, NF 37
but no such gel forms with the Control. Page 6042

Assay 4.2% 5.0% of carbon dioxide on the USP 42

dried basis, corresponding to 91.0% NF 37
108.0% of Xanthan Gum Page 6042
Impurity

- Arsenic NMT 3 µg/g USP 42

NF 37
Page 6042
- Lead NMT 5 µg/g USP 42
NF 37
Page 6042
- Limit of isopropyl alcohol NMT 0.075% USP 42
NF 37
Page 6042
- Pyruvic Acid The absorbance of the Sample solution is USP 42
NLT that of the Standard solution, NF 37
corresponding to NLT 1.5% of pyruvic Page 6043
acid
Specific test

- Microbial Enumeration It meets the requirements of the tests for USP 42

Test and Test For Specified Salmonella species and Escherichia coli. NF 37
Microorganisms Page
- Loss on drying NMT 15.0% USP 42
NF 37

168
 Page

- ASH The weight of the ash is between 6.5% USP 42

16.0%, calculated on the dried basis NF 37
Page
- Viscosity NLT 600 centipoises at 24° USP 42
NF 37
Page

169
170
. Sodium Phosphate
Test Acceptance criteria Analytical procedure

Identification Sodium and Phosphate: A solution (1 in USP 42

20) meets the requirements. NF 37
Page 5960
Assay NLT 97.0% of Na3PO4 on the ignited basis. USP 42
Na3PO4 · 12H2O (dodecahydrate) contains NF 37
NLT 92.0% of Na3PO4 on the ignited basis. Page 5961
Impurity

- Loss on Ignitio The anhydrous form loses NMT 2.0% of USP 42

its weight, the monohydrate loses 8.0% NF 37
11.0% of its weight, and the Page 5961
dodecahydrate loses 45.0% 57.0% of its
weight.
- Arsenic NMT 3 ppm USP 42
NF 37
Page 5961
Specific test

- Insoluble Substances The weight of the residue so obtained USP 42

does not exceed 20 mg (0.2%). NF 37
Page 5961

171
172
. Peppermint oil
Test Acceptance criteria Analytical
procedure
Identification Within 5 min the liquid develops a blue USP 42
color that, on continued heating, NF 37
deepens and shows a copper-colored Page 5
fluorescence and then fades, leaving a
golden-yellow solution.
Assay
Total Ester NLT 5.0% of esters, calculated as C12H22O USP 42
NF 37
Page 5
- Total Menthol NLT 50.0% of C10H20O, free and as esters USP 42
NF 37
Page 5
Impurity
- Limit of Dimethyl Sulfide A white film does not form at the zone of USP 42
contact within 1 min. NF 37
Page 5
Specific test
- Specific Gravity 0.896 0.908 USP 42
NF 37
Page 5866
- Optical Ratation USP 42
NF 37
Page 5866
- Refractive index 1.459 1.465 at 20° USP 42
NF 37

173
 Page 5866
- Solubility in 70 % alcohol One volume dissolves in 3 volumes of USP 42
70% alcohol, with NMT slight NF 37
opalescence Page 5866

174
175
.Menthol

Test Acceptance criteria Analytical procedure

Identification

Identification A The retention time of the major peak of USP42

the Sample NF37
solution corresponds to that of the Page 2735
menthol peak of the
Standard solution, as obtained in the
Assay.

Identification B It meets the requirements in Specific USP42

Tests for Optical NF37
Rotation <781S>, Procedures, Specific Page 2735
Rotation.
Assay 98.0 102.0% USP42
NF37
Page 2735

Impurities Individual impurities: NMT 0.5% for USP42

isomenthol NF37
(relative retention time is 1.08) from Page 2735-2736
synthetic racemic
menthol and 0.3% for all other
impurities from natural
and synthetic menthol

Total impurities : NMT 2.0%

176
Specific tests

Congealing range of dl- dl-Menthol congeals at a USP42

Menthol temperature between 27° and 28°. NF37
Shortly after the Page 2736
temperature has stabilized at the
congealing point, add a
few milligrams of dried dl-menthol to
the congealed
mass, and continue stirring. After a few
minutes, the
temperature of the mass quickly rises to
30.5° 32.0°

Melting range of l- 41°C-44 °C USP42

Menthol NF37
Page 2736

Optical Rotation l-Menthol -45° to 51° USP42

dl-menthol -2° to 2° NF37
Page 2736

177
178
. Silicon dioxide (Aerosil®)

Test Acceptance criteria Analytical procedure

Identification

Identification A A deep yellow color is produced USP42

NF37
Page 5949

Identification B A greenish blue spot is produced USP42

NF37
Page 5949

Assay 99.0 100.5% on the previously ignited USP42

basis NF37
Page 5949

Impurities Inorganic Impurities NMT 8 ppm USP42

NF37
Page 5949

Specific tests

pH 3.5-5.5 USP42
NF37
Page 5949

Loss on Drying It loses NMT 2.5% of its weight USP42

NF37
Page 5949

179
180
.Sucralose

Test Acceptance criteria Analytical procedure

Identification

Identification A Infrared Absorption (197K) USP42

NF37
Page 6013

.
Identification B The retention time of the USP42
principal peak of the NF37
Sample solution Page 6013
corresponds to that of the
Standard solution, as
obtained in the test for
Related Compounds
Identification C The Rf value of the principal USP42
spot of the sample solution NF37
corresponds to that of Page 6013
standard solution A, as
obtained in the test for
related Compounds
Assay 98.0%-102.0% on the USP42
anhydrous sucralose NF37
Page 6013
Impurities

Residue on Ignition NMT 0.7% USP42

NF37
Page 6013-6014

181
 Limit of methanol NMT 0.1% USP42
NF37
Page 6013-6014
Related compounds The Rf value of the principal USP42
spot from the sample NF37
solution correspond to that Page 6013-6014
obtained from standard
solution A, and the color of
any other single spot from
the sample solution is not
more intense than that of
the principal spot from
standard solution B (0.5%)
Limit of Hydrolysis Product The color of the spot from USP42
the sample solution is not NF37
more than that from Page 6013-6014
standard solution B (0.1%)

Specific tests

Optical Rotation +84.0 ° to +87.5 ° at 20 ° USP42

NF37
Page 6014
Water determination NMT 2.0% USP42
NF37
Page 6014

182
183
P4.2 Analytical procedure
. Lactose (super tab 11 SD)

184
Identification
- A. INFRARED ABSORPTION <197K>
Procedure
Sample: Lactose Monohydrate powder
1. Weight about 25 mg of Lactose Monohydrate powder and 225 mg of Potassium
bromide (KBr) and transfer both into the smooth agate mortar.
2. Finely pulverized and put into a pellet-forming die.
3. A force of approximately 10 tons is applied under a vacuum forming a transparent
pellet.
4. Perform degassing to eliminate air and moisture form the KBr powder.
5. Before performing measurements, the background was measured using an empty
pellet holder inserted into the sample chamber.
6. Measure the sample to obtain the infrared spectrum.

- B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST <201>

Diluent: Methanol and water (3:2)
Standard solution A: 0.5 mg/mL of USP Lactose Monohydrate RS in Diluent
Standard solution B: 0.5 mg/mL each of USP Dextrose RS, USP Lactose Monohydrate RS, USP
Fructose RS, and USP Sucrose RS in Diluent
Sample solution: 0.5 mg/mL of Lactose Monohydrate in Diluent
Adsorbent: 0.25-mm layer of chromatographic silica gel
Application volume: 2 µL
Developing solvent system: Ethylene dichloride, glacial acetic acid, methanol, and water
(10:5:3:2)

185
Spray reagent: 5 mg/mL of thymol in a mixture of alcohol and sulfuric acid (19:1)
Analysis
Samples: Standard solution A, Standard solution B, and Sample solution
1. Allow the spots to dry, and develop the plate in a paper lined chromatographic
chamber equilibrated with Developing solvent system for about 1 h prior to use.
2. Allow the chromatogram to develop until the solvent front has moved about three-
quarters of the length of the plate.
3. Remove the plate from the chamber, dry in a current of warm air, and redevelop the
plate in fresh Developing solvent system.
4. Remove the plate from the chamber, mark the solvent front, and dry the plate in a
current of warm air.
5. Spray the plate evenly with Spray reagent.
6. Heat the plate at 130° for 10 min.
System suitability: The test is not valid unless the chromatogram of Standard solution B
shows four clearly discernible spots, disregarding any spots at the origin.
Acceptance criteria: The principal spot from the Sample solution corresponds in appearance
and RF
Assay <197K>
Impurity
- Residue on Ignition <281>
Analysis:
A sample ignited at a temperature of 600 ± 50°
Procedure
1. Ignite a suitable crucible (for example, silica, platinum, quartz, or porcelain) at 600 ±
50° for 30 minutes, cool the crucible in a desiccator (silica gel or other suitable
desiccant), and weigh it accurately.

186
 2. Weigh accurately 1 to 2 g of the substance, or the amount specified in the individual
monograph, in the crucible.
3. Moisten the sample with a small amount (usually 1 mL) of sulfuric acid, then heat
gently at a temperature as low as practicable until the sample is thoroughly charred.
Cool; then, unless otherwise directed in the individual monograph, moisten the
residue with a small amount (usually 1 mL) of sulfuric acid; heat gently until white
fumes are no longer evolved; and ignite at 600 ± 50°, unless another temperature is
specified in the individual monograph, until the residue iscompletely incinerated.
4. Ensure that flames are not produced at any time during the procedure.
5. Cool the crucible in a desiccator (silica gel or other suitable desiccant), weigh
accurately, and calculate the percentage of residue.
6. Unless otherwise specified, if the amount of the residue so obtained exceeds the
limit specified in the individual monograph, repeat the moistening with sulfuric acid,
heating and igniting as before, using a 30-minute ignition period, until two
consecutive weighings of the residue do not differ by more than 0.5 mg or until the
percentage of residue complies with the limit in the individual monograph.
7. Conduct the ignition in a well-ventilated hood, but protected from air currents, and
at as low a temperature as is possible to effect the complete combustion of the
carbon. A muffle furnace may be used, if desired, and its use is recommended for
the final ignition at 600 ± 50°.
8. Calibration of the muffle furnace may be carried out using an appropriate digital
temperature meter and a working thermocouple probe calibrated against a standard
thermocouple traceable to the National Institute of Standards and Technology.
9. Verify the accuracy of the measuring and controlling circuitry of the muffle furnace
by checking the positions in the furnace at the control set point temperature of
intended use. Select positions that reflect the eventual method of use with respect
to location of the specimen under test. The tolerance is ±25° at each position
measured.
Acceptance criteria: NMT 0.1%
Specific tests

187
- Clarity and Color Solution
Sample solution: 1 g in 10 mL of boiling water
Analysis:
1. The Sample solution is clear and nearly colorless.
2. Determine the absorbance of this solution at a wavelength of 400 nm.
Acceptance criteria: The absorbance divided by the path length, in cm, is NMT 0.04.

- Microbial Enumeration Tests <61> and TESTS for Specified Microrganism <62>:
The total aerobic microbial count does not exceed 1 × 102 cfu/g, the total combined
molds and yeasts count does not exceed 5 × 101 cfu/g, and it meets the requirements of
the test for absence of Escherichia coli

- Optical rotation Specific Rotation <781s>

Sample solution:
1. Dissolve 10 g by heating in 80 mL of water to 50°.
2. Allow to cool, and add 0.2 mL of 6 N ammonium hydroxide. Allow to stand for 30
min, and dilute with water to 100 mL.
Procedure
Specific Rotation

signifies that specific rotation is to be calculated from observed optical rotations in the
Test solution or Sample solution obtained as directed therein.
1. Unless otherwise directed, measurements of optical rotation are made in a 1.0-dm
tube at 589 nm at 25°.
2. Where a photoelectricpolarimeter is used, a single measurement, corrected for the
solvent blank, is made.

188
 3. Where a visual polarimeter is employed,the average of no fewer than five
determinations, corrected for the reading of the same tube with a solvent blank, is
used.
4. Temperature, which applies to the solution or the liquid under test, should be
maintained within 0.5° of the stated value.
5. Use the same cell for sample and blank. Maintain the same angular orientation of
the cell in each reading.
6. Place the cell so that the light passes through it in the same direction each time.
7. Unless otherwise specified, specific rotation is calculated on the dried basis where
Loss on Drying is specified in the monograph, or on the anhydrous basis where Water
Determination is specified.
8. Optical rotation of solutions should be determined within 30 min of preparation.
9. In the case of substances known to undergo racemization or mutarotation, care
should be taken to standardize the time between adding the solute to the solvent
and introduction of the solution into the polarimeter tube
Acceptance criteria: +54.4° to +55.9°, calculated on the anhydrous basis, determined at 20°

- Acidity or Alkalinity
Sample solution:
1. Dissolve 6 g by heating in 25 mL of carbon dioxide-free water, cool
2. Add 0.3 mL of phenolphthalein TS.
Acceptance criteria: The solution is colorless, and NMT 0.4 mL of 0.1 N sodium hydroxide VS
is required to produce a pink or red color.

- Loss on Drying <731>

Procedure
1. Conduct the determination on a 1 g test specimen

189
2. Tare an appropriate glass-stoppered (shallow weighing bottle that has been dried for
about 30 minutes under the same conditions to be employed in the determination
and cooled to room temperature in a desiccator)
3. Put the test specimen in the bottle, replace the 23 covers, and accurately weigh the
bottle and the contents
4. Sidewise shaking
5. Distribute the test specimen as evenly as practicable to a depth of about 5 mm
generally
6. Place the loaded bottle in the drying chamber
7. Removing the stopper and leaving it also in the chamber
8. Dry the test specimen at 80° to constant weight for 2 h. (drying shall be continued until
two consecutive weighing do not differ by more than 0.50 mg per g of substance
taken, the second weighing following an additional hour of drying)
9. Opening the chamber and close the bottle promptly
10. Allow it to come to room temperature in a desiccator before weighing
Acceptance criteria:
Monohydrate: NMT 0.5%
Monohydrate, modified: NMT 1.0%

- Water Determination, Method I <921>

Sample solution: Prepare a preparation containing Lactose Monohydrate in a mixture of
methanol and formamide (2:1).
Procedure

190
 1. Unless otherwise specified, transfer enough methanol or other suitable solvent to
the titration vessel, ensuring that the volume is sufficient to cover the electrodes
(approximately 30 40 mL), and titrate with the Reagent to the electrometric or visual
endpoint to consume any moisture that may be present. (Disregard the volume
consumed, because it does not enter into the calculations.)
2. Quickly add the Test Preparation, mix, and again titrate with the Reagent to the
electrometric or visual endpoint.
3. Calculate the water content of the specimen taken, in mg:
SF
in which S is the volume, in mL, of the Reagent consumed in the second titration;
and F is the water equivalence factor of the Reagent.
Acceptance criteria: 4.5% 5.5%

- Protein and Light-Absorbing Impurities (See Ultraviolet-Visible Spectroscopy <857>.

Sample solution: 1% (w/v)
Analysis: Measure the light absorption of the Sample solution in the range of 210 300 nm.
Procedure
1. With few exceptions, compendial spectrophotometric tests and assays call for
comparison against a USP Reference Standard.
2. This helps ensure measurement under identical conditions for the test specimen and
the reference substance.
3. These conditions could include wavelength setting, spectral bandwidth selection,
cell placement and correction, and transmittance levels.
4. Cells that exhibit identical transmittance at a given wavelength may differ
considerably in transmittance at other wavelengths.
5. Appropriate cell corrections should be established and used where required.
6. Comparisons of a test specimen with a reference standard are best made at a peak
of spectral absorption for the compound concerned.

191
 7. Assays that prescribe spectrophotometry give the commonly accepted wavelength
for peak spectral absorption of the substance in question.
8. Different spectrophotometers may show minor variation in the apparent wavelength
of this peak.
9. Good practice demands that comparisons be made at the wavelength at which peak
absorption occurs.
10. Should this differ by more than ±1 nm (in the range 200 400 nm) or ±2 nm (in the
range 400 800 nm) from the wavelength specified in the individual monograph,
recalibration of the instrument may be indicated.
11.
assays involving spectrophotometry indicate that the reference comparator, generally
a USP Reference Standard, should be prepared and observed in an identical
manner for all practical purposes to that used for the test specimen.
12. Usually when analysts make up the solution of the specified reference standard,
they prepare a solution of about (i.e., within 10%) the desired concentration, and
they calculate the absorptivity on the basis of the exact amount weighed out.
13. If a previously dried specimen of the reference standard has not been used, the
absorptivity is calculated on the anhydrous basis.
14.
tests and assays involving spectrophotometry indicate that the absorbances of both
the solution containing the test specimen and the solution containing the reference
specimen, relative to the specified test blank, must be measured in immediate
succession
Acceptance criteria: The absorbance divided by the path length, in cm, is NMT 0.25 in the
range of 210 220 nm and is NMT 0.07 in the range of 270 300 nm value to that from
Standard solution A.

192
2 Sucrose

193
194
Identification
- A. INFRARED ABSORPTION <197K>
Procedure
1. Weight about 25 mg of Sucrose powder and 225 mg of Potassium bromide (KBr) and
transfer both into the smooth agate mortar.
2. Finely pulverized and put into a pellet-forming die.
3. A force of approximately 10 tons is applied under a vacuum forming a transparent pellet.
4. Perform degassing to eliminate air and moisture form the KBr powder.
5. Before performing measurements, the background was measured using an empty pellet
holder inserted into the sample chamber.
6. Measure the sample to obtain the infrared spectrum

Impurity
- Sulfite
Sample solution: 400 mg/mL of Sucrose in freshly prepared distilled water
Sulfite standard solution (80 ppm SO2): 0.1575 mg/mL of anhydrous sodium sulfite in freshly
prepared distilled water
195
Reference solution: Dissolve 4.0 g of Sucrose in freshly prepared distilled water, add 0.5 mL
of Sulfite standard solution (80 ppm SO2), and dilute with freshly prepared distilled water to
10.0 mL.
Blank: Freshly prepared distilled water
Analysis:
1. Determine the sulfite content by a suitable enzymatic method based on the
following reactions.
2. Sulfite is oxidized by sulfite oxidase to sulfate and hydrogen peroxide, which in turn
is reduced by nicotinamide adenine dinucleotide peroxidase in the presence of
reduced nicotinamide adenine dinucleotide (NADH).
3. The amount of NADH oxidized is proportional to the amount of sulfite.
4. Separately introduce 2.0 mL each of the Sample solution, Reference solution, and
Blank in 10 mm cuvettes, and add the reagents as described in the kit instructions.1
5. Measure the absorbance at the maximum at about 340 nm before and at the end of
the reaction time, and subtract the value obtained with the Blank.
Acceptance criteria: The absorbance difference of the Sample solution is NMT half the
absorbance difference of the Reference solution.

Specific tests
- Appearance solution
Sample solution: 500 mg/mL of Sucrose in water.
Hydrazine sulfate solution: 10 mg/mL of hydrazine sulfate in water. Allow to stand for 4 6 h.
Hexamethylenetetramine solution: In a 100-mL ground glass-stoppered flask dissolve 2.5 g of
hexamethylenetetramine in 25.0 mL of water.
Primary opalescent suspension: To the Hexamethylenetetramine solution in the flask add
25.0 mL of Hydrazine sulfate solution. Mix, and allow to stand for 24 h. This suspension is

196
stable for 2months, provided it is stored in a glass container free from surface defects. The
suspension must not adhere to the glass and must be well mixed before use.
Standard of opalescence: Primary opalescent suspension in water (3 in 200). This suspension
is freshly prepared and may be stored for up to 24 h.
Reference suspension I: Standard of opalescence and water (5:95)
Acceptance criteria: The clarity of the Sample solution is the same as that of water or its
opalescence is not more pronounced than that of Reference suspension I

- Conductivity
Sample solution: 313 mg/mL of Sucrose in freshly boiled and cooled water
Apparatus:
1. Use a conductivity meter or resistivity meter that measures the resistance of the
column of liquid between the electrodes of the immersed measuring device.
2. The apparatus is supplied with alternating current to avoid the effects of electrode
polarization. It isequipped with a temperature compensation device or aprecision
thermometer.
Calibration:
1. Choose a conductivity cell that is appropriate for the conductivity of the solution to
be examined.
2. The higher the expected conductivity, the higher the cell constant that must be
chosen so that the value measured, R, is as large as possible for the apparatus used.
3. Commonly used conductivity cells have cell constants on the order of 0.1 cm 1, 1
cm 1, and 10 cm 1.
4. Use a standard solution of potassium chloride that is appropriate for the
measurement.
5. Rinse the cell several times with water that has been previously boiled and cooled
to room temperature and at least twice with the potassium chloride solution used
for the determination of the cell constant of the conductivity cell.
197
 6. Measure the resistance of the conductivity cell using the potassium chloride solution
at 20 ± 0.1°.
The constant, C (in cm-1), of the conductivity cell is given by the expression:
C = RKCL × KKCL
RKCL
KKCL = conductivity of the standard solution of potassium chloride used (µS · cm-1)
The measured constant, C, of the conductivity cell must be within 5% of the given value
Analysis
Sample: Sample solution
1. Measure the conductivity of the Sample solution (C 1), while gently stirring with a
magnetic stirrer, and that of the water used for preparing the Sample solution (C 2).
2. The readings must be stable within 1% over a period of 30 s
3. Calculate the conductivity of the Sample solution from the expression:
Result = C1 2)

C1 = conductivity of the Sample solution

C2 = water used for preparing the Sample solution
Acceptance criteria: NMT 35 µS · cm-1 at 20°

- Optical Rotation, Specific Rotation <781S>

Sample solution: 260 mg/mL
Procedure
Specific Rotation

signifies that specific rotation is to be calculated from observed optical rotations in the
Test solution or Sample solution obtained as directed therein.

198
 1. Unless otherwise directed, measurements of optical rotation are made in a 1.0-dm
tube at 589 nm at 25°.
2. Where a photoelectric polari meter is used, a single measurement, corrected for
the solvent blank, is made.
3.Where a visual polarimeter is employed, the average of no fewer than five
determinations, corrected for the reading of the same tube with a solvent blank, is used.
4.Temperature, which applies to the solution or the liquid under test, should be
maintained within 0.5° of the stated value.
5.Use the same cell for sample and blank. Maintain the same angular orientation of
the cell in each reading.
6.Place the cell so that the light passes through it in the same direction each time.
7.Unless otherwise specified, specific rotation is calculated on the dried basis where
Loss on Drying is specified in the monograph, or on the anhydrous basis where Water
Determination is specified.
8.Optical rotation of solutions should be determined within 30 min of preparation.
9.In the case of substances known to undergo racemization or mutarotation, care
should be taken to standardize the time between adding the solute to the solvent and
introduction of the solution into the polarimeter tube
Acceptance criteria: +66.3° to +67.0° at 20°

- Color Value
Sample solution: Dissolve 50.0 g in 50.0 mL of water. Mix, filter (diameter of pores ,
0.45 µm), and degas.
Analysis:
1. Measure the absorbance at 420 nm, using a cell of at least 4 cm (a cell length of 10
cm or more is preferred).

199
 2. Calculate the Color Value using the expression:
Result = (A × 1000)/(b × c)
A = absorbance measured at 420 nm
b = cell path length (cm)
c = concentration of the solution (g/mL), calculated from the refractive index of the
solution. Use Table 1, and interpolate the values if necessary.
Suitability requirements
Repeatability: The absolute difference between two results is NMT 3
Acceptance criteria: NMT 45 if labeled as parenteral grade; NMT 75 for non parenteral grade
- Dextrins
If intended for use in the preparation of largevolume infusions, it complies with the test for
Dextrins.
Sample solution: Prepare as directed in the test for Appearance of Solution.
Analysis:
1. To 2 mL of the Sample solution add 8 mL of water, 0.05 mL of dilute hydrochloric
acid (73 g/L of HCl), and 0.05 mL of 0.05 M iodine.
Acceptance criteria: The solution remains yellow.
- Reducing sugars
Sample solution: Prepare as directed in the test for Appearance of Solution.
Analysis:
1. To 5 mL of the Sample solution in a test tube, about 150-mm long and 16-mm in
diameter, add 5 mL of water, 1.0 mL of 1 M sodium hydroxide, and 1.0 mL of a 1-g/L
solution of methylene blue.
2. Mix, and place in a water bath.

200
 3. After exactly 2 min, take the tube out of the bath, and examine the solution
immediately.
Acceptance criteria: The blue color does not disappear completely, ignoring any blue color
at the air/solution interface.

- LOSS ON DRYING <731>

Sample: 2.000 g
Analysis: Dry the Sample at 105° for 3 h.
Procedure
1. Conduct the determination on a 1 g test specimen
2. Tare an appropriate glass-stoppered (shallow weighing bottle that has been dried for
about 3the s hours ame conditions to be employed in the determination and cooled to
room temperature in a desiccator)
3. Put the test specimen in the bottle, replace the 23 covers, and accurately weigh the
bottle and the contents
4. Sidewise shaking
5. Distribute the test specimen as evenly as practicable to a depth of about 5 mm generally
6. Place the loaded bottle in the drying chamber
7. Removing the stopper and leaving it also in the chamber
8. Dry the test specimen at 105o C to constant weight (drying shall be continued until two
consecutive weighing do not differ by more than 0.50 mg per g of substance taken, the
second weighing following an additional hour of drying)
9. Opening the chamber and close the bottle promptly
10. Allow it to come to room temperature in a desiccator before weighing
Acceptance criteria: NMT 0.1

201
- Bacteria Endotoxins Test <85>:
The Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from
Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus).
Acceptance criteria: Less than 0.25 IU/mg If intended for use in the preparation of large
volume infusions,it complies with the test for Bacterial Endotoxins.

202
3. HPC (Hydroxypropyl Cellulose)

203
204
Identification
- A. INFRARED ABSORPTION <197K> : Disregard any peak at about 1719 cm-1.
Procedure
1. Weight about 25 mg of Hydroxypropyl Cellulose powder and 225 mg of Potassium
bromide (KBr) and transfer both into the smooth agate mortar.
2. Finely pulverized and put into a pellet-forming die.
3. A force of approximately 10 tons is applied under a vacuum forming a transparent pellet.
4. Perform degassing to eliminate air and moisture form the KBr powder.
5. Before performing measurements, the background was measured using an empty pellet
holder inserted into the sample chamber.
6. Measure the sample to obtain the infrared spectrum.

- B: Sample: 1 g of Hydroxypropyl Cellulose

Analysis:
1. Dissolve the Sample in 100 mL of water.
2. Transfer 1 mL of this solution to a glass plate, and allow the water to evaporate.
Acceptance criteria: A thin film is formed.
Assay
- Hydroxypropoxy Groups
Internal standard solution: Methylcyclohexane in oxylene (1 in 50)
Standard solution:
1. Weigh accurately 60 mg of adipic acid a reaction vial, add 2.00 mL of Internal
standard solution, and 1.0 mL of hydriodic acid.
2. Stopper the vial tightly, and weigh accurately. Inject 25 µL of isopropyl iodide
through the septum, and again weigh accurately.

205
 3. Mix well.
4. After phase separation, pierce through the septum of the vial with a cooled syringe,
and withdraw a sufficient volume of the upper phase as the Standard solution.
Sample solution:
1. Weigh accurately 30 mg of hydroxypropyl cellulose (dried substance), and transfer
toa reaction vial. Add 60 mg of adipic acid, 2.00 mL of Internal standard solution, and
1.0 mL of hydriodic acid.
2. Stopper the vial tightly with the valve, and weigh accurately the reaction vial (total
mass before heating).
3. Place the vial in an oven or heat in a suitable heater with continuous stirring,
maintaining an internal temperature of 115 ± 2° for 70 min. Allow the vial to cool,
and weigh accurately the reaction vial (total mass after heating).
4. If the difference of the total mass before heating to the total mass after heating is
more than 10 mg, prepare a new Sample solution.
5. After phase separation, pierce through the septum of the vial with a cooled syringe,
and withdraw a sufficient volume of the upper phase as the Sample solution.
Chromatographic system (See Chromatography <621>, System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.53-mm × 30-m fused silica capillary, coated with a 3-µm layer of phase G1
Temperatures
Detector: 280°
Injection port: 180°
Column: See Table 1.

206
Table 1

Temperature Ramp Hold Time at Final

Initial Temperature (o) Final Temperature (o)
(o/min) Temperature (min)

40 0 40 3

40 10 100 __

100 50 250 3

Carrier gas: Helium

Linear velocity: 52 cm/s
Injection volume: 2 µL
Injection type: Split; split ratio, 50:1
Run time: 15 min
System suitability
Sample: Standard solution The relative retention time for isopropyliodide
is about 0.8 with reference to methylcyclohexane (retention time about 8 min).]
Suitability requirements
Resolution: NLT 2 between isopropyl iodide and methylcyclohexane
Relative standard deviation: NMT 2.0%, using the response factor calculation (F) for six
injections
Analysis:
Samples: Standard solution and Sample solution
Calculate the response factor (F):

207
 F = (A1 × W1 × C)/(A2× 100)
A1 = peak area of the internal standard from the Standard solution
W1 = weight of isopropyl iodide in the Standard solution (mg)
C = content of isopropyl iodide (%)
A2 = peak area of isopropyl iodide from the Standard solution
Calculate the percentage content (m/m) of the hydroxypropoxy group:
Result = (A4 × F × M1 × 1.15 × 100)/(A3 × W2 × M2 )
A4 = peak area of isopropyl iodide from the Sample solution
F = response factor calculated from above
M1 = molar mass of hydroxypropoxy group, 75.1
1.15 = correction factor to correlate results to previous assay method replaced by this
method
A3 = peak area of the internal standard from the Sample solution
W2 = weight of the sample (dried substance) in the Sample solution (mg)
M2 = molar mass of isopropyl iodide, 170.0
Acceptance criteria: 53.4% 80.5% of hydroxypropoxy groups
Impurities
- Residue on Ignition <281>
Sample: 1.0 g
Analysis: Proceed as directed in the chapter, using a platinum crucible.
Acceptance criteria: NMT 0.8%

208
-Silica
Analysis:
1. If the addition of silica is stated on the label, and if more than 0.2% residue is found
from the Residue on Ignition test, moisten the residue with water, and add 5 mL of
hydrofluoric acid in small portions.
2. Evaporate on a steam bath to dryness, and cool.
3. Add 5 mL of hydrofluoric acid and 0.5 mL of sulfuric acid, and evaporate to dryness.
4. Slowly increase the temperature until all of the acids have been volatilized, and
ignite at1000 ± 25°.
5. Cool in a desiccator, and weigh.
6. The difference between the final weight and the weight of the initially ignited portion
represents the weight of silica.
Acceptance criteria: The weight of silica is NMT 0.6%

- Lead <251>:
Sample: Prepare a Test Preparation as directed in the chapter
Control: Use 5 mL of Diluted Standard Lead Solution (5 µg of Pb).
Test Preparation or Sample Solution
Where the monograph does not specify preparation of a solution, prepare a Test preparation
or Sample solution as follows.
1. Transfer 1.0 g of the substance under test to suitable flask
2. Add 5 mL of sulfuric acid a few glass beads, and digest on a hot plate in a hood until
charring begins.
3. Other suable means of heating may be substituted.
4. Add, dropwise and with caution, 30% hydrogen peroxide, allowing the reaction to
subside and heat between drops.

209
5. Add the first few drop very slowly, mix carefully to prevent a rapid reaction.
6. Discontinue heating if foaming becomes excessive.
7. Swirl the solution in flask to prevent unreacted substance from caking on the walls
of the flask.
8. Continue the digestions until the substance is completely destroyed, copious fumes
of sulfur trioxide are evolved, and the solution is colorless.
9. Cool, and cautiously add 10 mL of water,
10. Evaporate until sulfur trioxide again is evolved, add cool
11. Repeat this procedure with another 10 ml of water to remove any traces of
hydrogen peroxide
12. Cautiously dilute with 10 mt of water, and cool
Analysis
Analysis: Proceed as directed in the chapter.
1. Transfer the Test preparation or Sample solution
2. Rinsing with 10mL of water, or the volume of the prepared sample specified in
monograph to a separator.
3. Add 6 mL of Ammonium citrate solution and 2 mL of Hydroxylamine hydrochloride
solution.
4. Add 2 drops of phenol red TS
5. Make the solution just alkaline (red color) by the addition of ammonium hydroxide.
6. Cool the solution if necessary, and add 2 mL of Potassium cyanide solution.
7. Immediately extract the solution with 5 mL portions of Dithizone extraction solution
8. Draining off each extract in to another separator, until the dithizone solution retains its
green color.
210
9. Shake the combined dithizone solutions for 30 s with 20 mL of dilute nitric acid
10. Discard the chloroform layer
11. Add to the acid solution 5.0 ml of Standard dithizone solution and 4 ml of Ammonia-
cyanide solution
12. Shake for 30 s.
Acceptance criteria: NMT 10 ppm
Specific tests
- pH <791>: 5.0 8.0, in a solution (1 in 100), prepared by evenly distributing the powder into
boiling carbon dioxide-free water and stirring the mixture with a magnetic stirrer.

- Loss on Drying <731>

Analysis: Dry at 105° for 4 h.
Procedure
1. Conduct the determination on a 1 g test specimen
2. Tare an appropriate glass-stoppered (shallow weighing bottle that has been dried for
about 4 hours under the dame conditions to be employed in the determination
and cooled to room temperature in a desiccator)
3. Put the test specimen in the bottle, replace the 23 covers, and accurately weigh the
bottle and the contents
4. Sidewise shaking
5. Distribute the test specimen as evenly as practicable to a depth of about 5 mm generally
6. Place the loaded bottle in the drying chamber
7. Removing the stopper and leaving it also in the chamber

211
8. Dry the test specimen at 105 oC to constant weight (drying shall be continued until two
consecutive weighing do not differ by more than 0.50 mg per g of substance taken, the
second weighing following an additional hour of drying)
9. Opening the chamber and close the bottle promptly
10. Allow it to come to room temperature in a desiccator before weighing
Acceptance criteria: NMT 5.0%

- VISCOSITY- ROTATIONAL METHODS < 912> :

Determine the apparent viscosity at the concentration and temperature specified on the
label with a suitable rotational viscometer (see Labeling).

212
4. Xanthan gum

213
Identification A
Sample: Prepare a dry blend of 1.5 g of Xanthan Gum and 1.5 g of locust bean gum.
Control: 3.0 g of Xanthan Gum
Analysis
Samples: Sample and Control
1. To two separate 400-mL beakers add 300 mL of water, and heat to 80°. Stir rapidly
by mechanical means.
2. Add the Sample to one of the beakers and the Control to the other beaker at the
point of maximum agitation.
3. Stir until the mixtures dissolve, and then continue stirring for 30 min longer.
4. Do not allow the temperature of the mixtures to drop below 60° during the stirring.
5. Discontinue stirring, and allow the mixtures to cool at room temperature for NLT 2 h.
Acceptance criteria: A firm, rubbery gel forms with the Sample after the temperature
drops below 40°, but no such gel forms with the Control.

Assay
Sample: 1.2 g
Analysis: Proceed as directed in Alginates Assay <311>.
Acceptance criteria: 4.2% 5.0% of carbon dioxide on the dried basis, corresponding to
91.0% 108.0% of Xanthan Gum

Impurity
- Arsenic, Method II <211>
Standard Preparation:

214
1. Pipet 3.0 mL of Standard Arsenic Solution into a generator flask
2. add 2 mL of sulfuric acid, mix
3. add the total amount of 30 percent hydrogen peroxide used in preparing the Test
preparation.
4. Heat the mixture to strong fuming, cool
5. add cautiously 10 mL of water, and again heat to strong fumes.
6. Repeat this procedure with another 10 mL of water to remove any traces of
hydrogen peroxide.
7. Cool, and dilute with water to 35 mL
Test Preparation: Unless otherwise directed in the individual monograph, transfer to a
generator flask the quality, in g, of the test substance calculated by the formula:
3.0/L
L = limit of arsenic (ppm)
Acceptance Criteria: NMT 3 µg/g

- Lead <251>
Sample: Prepare a Test Preparation as directed in the chapter
Control: Use 5 mL of Diluted Standard Lead Solution (5 µg of Pb).
Test Preparation or Sample Solution
Where the monograph does not specify preparation of a solution, prepare a Test preparation
or Sample solution as follows.
1. Transfer 1.0 g of the substance under test to suitable flask
2. Add 5 mL of sulfuric acid a few glass beads, and digest on a hot plate in a hood until

215
charring begins.
3. Other suable means of heating may be substituted.
4. Add, dropwise and with caution, 30% hydrogen peroxide, allowing the reaction to
subside and heat between drops.
5. Add the first few drop very slowly, mix carefully to prevent a rapid reaction.
6. Discontinue heating if foaming becomes excessive.
7. Swirl the solution in flask to prevent unreacted substance from caking on the walls
of the flask.
8. Continue the digestions until the substance is completely destroyed, copious fumes
of sulfur trioxide are evolved, and the solution is colorless.
9. Cool, and cautiously add 10 mL of water,
10. Evaporate until sulfur trioxide again is evolved, add cool
11. Repeat this procedure with another 10 ml of water to remove any traces of
hydrogen peroxide
12. Cautiously dilute with 10 mt of water, and cool
Analysis
Analysis: Proceed as directed in the chapter.
1. Transfer the Test preparation or Sample solution
2. Rinsing with 10mL of water, or the volume of the prepared sample specified in
monograph to a separator.
3. Add 6 mL of Ammonium citrate solution and 2 mL of Hydroxylamine hydrochloride
solution.
4. Add 2 drops of phenol red TS

216
5. Make the solution just alkaline (red color) by the addition of ammonium hydroxide.
6. Cool the solution if necessary, and add 2 mL of Potassium cyanide solution.
7. Immediately extract the solution with 5 mL portions of Dithizone extraction solution
8. Draining off each extract in to another separator, until the dithizone solution retains its
green color.
9. Shake the combined dithizone solutions for 30 s with 20 mL of dilute nitric acid
10. Discard the chloroform layer
11. Add to the acid solution 5.0 ml of Standard dithizone solution and 4 ml of Ammonia-
cyanide solution
12. Shake for 30 s.
Acceptance criteria: NMT 5 µg/g

- Limit of Isopropyl Alcohol

Internal standard solution: 1mg/mL of tertiary butyl alcohol
Standard stock solution: 1 mg/mL of isopropyl alcohol
Procedure
Standard solution:
1. Pipet 4 mL of the stock solution and 4 mL volumetric flask
2. dilute with water to volume.
Sample solution:
1. Disperse 1 mL of a suitable antifoam emulsion in 200 mL of water contained in a
1000 mL, round-bottom distilling flask having a 24/40 standard taper ground joint.
2. Add 5 g of Xanthan Gum, and shake for 1 h on a wrist-action mechanical shaker.

217
3. Connect the flask to a fractionating column
4. distill 100 mL, adjusting the heat so that foam does not enter the column
5. Add by pipet 4 ml of the Internal standard solution.
Chromatographic system
Mode: GC
Detector: Flame ionization
Column: 3.2-mm x 1.8-m stainless steel column packed with 80- to 100-mesh surface-
silanized packing S3, or equivalent
Temperatures Column: 165°
Detector: 200°

Injection volume: 4-5 l.

Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of isopropyl alcohol in the portion of Xanthan Gum taken:
Result = (Ru/Rs)x(Cs/Cu)x100
Ru= peak response ratio isopropyl alcohol to tertiary butyl alcohol from the Sample solution
Rs= peak response ratio of isopropyl alcohol to tertiary butyl alcohol from the Standard
solution
Cs= concentration of isopropyl alcohol in the Standard solution (mg/ml)
Cu= concentration of the Sample solution (mg/mL)
Acceptance criteria: NMT 0.075 %

218
- Pyruvic Acid
Solution A: 5 mg/mL of 2,4-dinitrophenyl hydrazine in 2 N hydrochloric acid Standard stock
solution:
1. Transfer 10.0 mL of the Standard stock solution to a glass-stoppered, 50-ml flask
2. Add 20.0 mL of 1 N hydrochloride acid, weigh the flask, and reflux for 3 h
3. Taking precaution to prevent loss of vapors. Cool, and add water to make up for any
weight loss during refluxing
4. Transfer 2.0 mL of this solution to a 30-mL separator containing 1.0 mL of this solution A.
5. Mix, and allow to standard for 85 min. Extract the mixture with 5 mL of ethyl acetate
6. Discard the aqueous layer
7. Extract the hydrazine from the ethyl acetate with three 5 mL portions of sodium
carbonate flask
8. Dilute with sodium carbonate TS to volume. S
ample stock solution: 6 mg/mL of Xanthan Gum
Sample solution: Transfer 10.0 mL of the Sample stock solution to a glass-stoppered, 50 mL
flask. Proceed as directed in the Standard solution, beginning with "Add 20.0 mL of 1

Blank: Sodium carbonate TS

Instrumental conditions
Mode: Spectrophotometry
Analytical wavelength: 375 nm
Cell: 1 cm

219
Analysis
Samples: Standard criteria: The absorbance solution, corresponding to NLT 1.5% of pyruvic
acid

Specific test
- MICROBIAL ENUMERATION TESTS <61> and TESTS FOR SPECIFIED MICROORGANISMS
<62>: The total aerobic microbial count does not exceed 1000 cfu/g, and the total
combined molds and yeasts count does not exceed 100 cfu/g. It meets the requirements of
the tests for absence of Staphylococcus aureus and Pseudomonas aeruginosa and for the
absence of Escherichia coli and Salmonella species.

- LOSS ON DRYING <731>

Procedure
1. Conduct the determination on a 1 g test specimen
2. Tare an appropriate glass-stoppered (shallow weighing bottle that has been dried for
about 30 minutes under the same conditions to be employed in the determination
and cooled to room temperature in a desiccator)
3. Put the test specimen in the bottle, replace the 23 covers, and accurately weigh the
bottle and the contents
4. Sidewise shaking
5. Distribute the test specimen as evenly as practicable to a depth of about 5 mm
generally
6. Place the loaded bottle in the drying chamber

220
7. Removing the stopper and leaving it also in the chamber
8. Dry the test specimen at 105 °C to constant weight for 2.5 h. (drying shall be continued
until two consecutive weighing do not differ by more than 0.50 mg per g of substance taken,
the second weighing following an additional hour of drying)
9. Opening the chamber and close the bottle promptly
10. Allow it to come to room temperature in a desiccator before weighing
Acceptance criteria: NMT 15.0%

- ASH
Sample: Weigh 3 g in a tared crucible.
Analysis: Incinerate the Sample at 650° until free form carbon. Cool the crucible and its
contents in a desiccator, and weigh.
Acceptance criteria: The weight of the ash is between 6.5% -16.0%, calculated on the dried
basis.

- VISCOSITY-ROTATION METHODS <921>

Sample: Prepare a dry blend of 3.0g of Xanthan Gum and 3.0 of potassium chloride.
Acceptance criteria: NLT 600 centipoises at 24°

221
5. Sodium Phosphate

222
223
Identification A:
Sodium <191>: A solution (1 in 20) meets the requirements
1. Unless otherwise specified in an individual monograph, prepare a solution to contain
0.1 g of the sodium compound in 2 mL of water.
2. Add 2 mL of 15% potassium carbonate, and heat to boiling. No precipitate is
formed.
3. Add 4 mL of potassium pyroantimonate TS, and heat to boiling.
4. Allow to cool in ice water and, if necessary, rub the inside of the test tube with a
glass rod. A dense precipitate is formed
Phosphate <191>: A solution (1 in 20) meets the requirements
1. Use the tests for orthophosphates, unless the instructions specify the use of the
pyrophosphate tests or indicate that the product is to be ignited before performing
the test.
Assay
Sample: 5.5 g of Tribasic Sodium Phosphate, on the anhydrous basis
Blank: 100.0 mL of 1 N hydrochloric acid, accurately measured
Titrimetric system
Mode: Residual titration
Titrant: 1 N sodium hydroxide VS
Endpoint detection: Potentiometric
Analysis:
1. Transfer the Blank to a 400-mL beaker, and titrate with the Titrant to the endpoint at
a pH of 7.0.
2. Record as the volume consumed, and designate as A.
3. Transfer the Sample to a 400-mL beaker, add 100.0 mL of 1 N hydrochloric acid, and
stir until dissolved.

224
 4. Pass a stream of carbon dioxide-free air, in fine bubbles, through the solution for 30
min to expel carbon dioxide, covering the beaker loosely to prevent any loss by
spraying.
5. Wash the cover and sides of the beaker with a few mL of water.
6. Titrate the excess acid potentiometrically with the Titrant to the inflection point at a
pH of 4. Record the buret reading, and designate as B.
7. Protect the solution from carbon dioxide absorbed from the air, and continue the
titration with 1 N sodium hydroxide VS to the inflection point at a pH of 8.8.
8. Record the buret reading, and designate as C.
9. Calculate the amount of Titrant consumed by the Sample to the first inflection
point, correcting for the Blank (V1
the Sample between the two inflection points (V2 1 is equal to or greater
than 2V2 , calculate the amount of Na3PO4 in the portion of Sample taken:
D = V2 × N × F
V2 = volume of Titrant consumed between the two inflection points (mL)
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 163.9 mg/mEq
If V1 is less than 2V2, calculate the amount of Na3PO4 in the portion of Sample taken:
D = (V1 2) ×N×F
V1 = volume of the Titrant consumed to the first inflection point, correcting for the
Blank (mL)
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 163.9 mg/mEq
Calculate the percentage of Na3PO4 on the ignited basis in the portion of Tribasic Sodium
Phosphate taken:
Result = [10/(100 L)] × (D/W)
L = percentage calculated in the test for Loss on Ignition
D = amount of Na3PO4 found (mg)
225
W = weight of the Sample (g)
Acceptance criteria: NLT 97.0% of Na3PO4 on the ignited basis. Na3PO4 · 12H2O
(dodecahydrate) contains NLT 92.0% of Na3PO4 on the ignited basis.

Impurity
- Loss on Ignition <733>
Sample: 2 g
Analysis:
1. Perform the test on finely powdered material, and break up lumps, if necessary, with
the aid of a mortar and pestle before weighing the specimen.
2. Weigh the specimen to be tested without further treatment, unless a preliminary
drying at 110° for 5 h, conduct the ignition at 800° for 30 min in a suitable muffle
furnace or oven that is capable of maintaining a temperature within 25° of that
required for the test, and use a suitable crucible, complete with cover, previously
ignited for 1 hour at the temperature specified for the test, cooled in a desiccator,
and accurately weighed.
3. Transfer to the tared crucible an accurately weighed quantity, in g, of the substance
to be tested, about equal to that calculated by the formula:
10/L
in which L is the limit (or the mean value of the limits) for Loss on ignition, in
percentage.
4. Ignite the loaded uncovered crucible, and cover at the temperature (±25°) and for
the period of time designated in the individual monograph.
5. Ignite for successive 1-hour periods where ignition to constant weight is indicated.
Upon completion of each ignition, cover the crucible, and allow it to cool in a
desiccator to room temperature before weighing.
Acceptance criteria: The anhydrous form loses NMT 2.0% of its weight, the monohydrate
loses 8.0% 11.0% of its weight, and the dodecahydrate loses 45.0% 57.0% of its weight.

226
- ARSENIC
Standard Preparation: Pipet 3.0 mL of Standard Arsenic Solution into a generator flask, and
dilute with water to 35 ml.
Test preparation: Dissolve a portion equivalent to 1.0 g of anhydrous tribasic sodium
phosphate in 35 mL of water.
Analysis: Method I
1. Treat the Standard Preparation and the Test Preparation similarly as follows.
2. Add 20 mL of 7 N sulfuric acid, 2 mL of potassium iodide TS, 0.5 mL of stronger acid
stannous chloride TS, and 1 mL of isopropyl alcohol, and mix. Allow to stand at
room temperature for 30 minutes.
3. Pack the scrubber tube with two pled gets of cotton that have been soaked in
saturated lead acetate solution, freed from excess solution by expression, and dried
in vacuum at room temperature, leaving a 2-mm space between the two pled gets.
4. Lubricate the joints with a suitable stopcock grease designed for use with organic
solvents, and connect the scrubber unit to the absorber tube.
5. Transfer 3.0 mL of silver diethyldithiocarbamate TS to the absorber tube. Add 3.0 g
of granular zinc (No. 20 mesh) to the mixture in the flask, immediately connect the
assembled scrubber unit, and allow the evolution of hydrogen and the color
development to proceed at room temperature for 45 minutes, swirling the flask
gently at 10-minute intervals.
6. Disconnect the absorber tube from the generator and scrubber units, and transfer
the absorbing solution to a 1-cm absorption cell.
7. Any red color produced by the Test Preparation does not exceed that produced by
the Standard Preparation.
8. If necessary or desirable, determine the absorbance at the wavelength of maximum
absorbance between 535 and 540 nm, with a suitable spectrophotometer or
colorimeter, using silver diethyldithiocarbamate TS as the blank. Interfering
Chemicals: Metals or salts of metals, such as chromium, cobalt, copper, mercury,

227
 molybdenum, nickel, palladium, and silver, may interfere with the evolution of
arsine.
9. Antimony, which forms stibine, produces a positive interference in the color
development with silver diethyldithiocarbamate TS; when the presence of antimony
is suspected, the red colors produced in the two silver diethyldithiocarbamate
solutions may be compared at the wavelength of maximum absorbance between
535 and 540 nm, with a suitable colorimeter, since at this wavelength the
interference due to stibine is negligible
Acceptance criteria: NMT 3 ppm

Specific test
- Insoluble substances
Sample solution: Dissolve a portion equivalent to 10.0 g of anhydrous tribasic sodium
phosphate in 100 mL of hot water.
Analysis:
1. Filter the Sample solution through a tared filtering crucible. (Do not use glass)
2. Wash the insoluble residue with hot water, and dry at 105° for 2 h.
Acceptance criteria: The weight of the residue so obtained does not exceed 20 mg (0.2%)

228
6. Peppermint oil

Identification A
Sample: 6 drops of Oil
Analysis:
229
 1. Place the Sample in a dry test tube and mix with 5 mL of a 1-in-300 solution of nitric
acid in glacial acetic acid,
2. Place the tube in a beaker of boiling water.
Acceptance criteria: Within 5 min the liquid develops a blue color that, on continued
heating, deepens and shows a copper-colored fluorescence and then fades, leaving a
golden-yellow solution.
Assay
- Total Ester
Sample: 10 g of Oil
Analysis:
1. Place the Sample in a 250-mL conical flask, add 10 mL of neutralized alcohol and
2 drops of phenolphthalein TS, then add, dropwise, 0.1 N sodium hydroxide until a faint pink
color appears.
2. Add 25.0 mL of 0.5 N alcoholic potassium hydroxide VS, connect the flask to a
reflux condenser, and heat on a boiling water bath for 1 h.
3. Allow the mixture to cool, add 20 mL of water, and add phenolphthalein TS.
Titrate the excess alkali with 0.5 N hydrochloric acid VS.
4. Perform a blank determination, disregarding the 0.1 N sodium hydroxide (Residual
Titrations).
5. Each mL of 0.5 N alcoholic potassium hydroxide consumed in the saponification is
equivalent to 99.15 mg of total esters calculated as C12H22O2 .
Acceptance criteria: NLT 5.0% of esters, calculated as C12H22O.

- Total Menthol
Sample: 10 mL of Oil
Analysis:

230
 1. Place the Sample in an acetylation flask of 100- mL capacity, and add 10 mL of
acetic anhydride and 1 g of anhydrous sodium acetate.
2. Boil the mixture gently for 1 h, accurately timed, cool, disconnect the flask from the
condenser, transfer the mixture to a small separator, rinsing the acetylation flask with
three 5-mL portions of warm water, and add the rinsings to the separator.
3. When the liquids have completely separated, discard the water layer, and wash the
remaining oil with successive portions of sodium carbonate TS, diluted with an equal
volume of water, until the last washing is alkaline to phenolphthalein TS.
4. Dry the resulting oil with anhydrous sodium sulfate, and filter. Transfer 5 mL of the
dry acetylated oil to a tared, 100-mL conical flask, and weigh.
5. Add 50.0 mL of 0.5 N alcoholic potassium hydroxide VS, connect the flask to a reflux
condenser, and boil the mixture on a steam bath for 1 h, accurately timed.
6. Allow the mixture to cool, and add 10 drops of phenolphthalein TS.
7. Titrate the excess alkali with 0.5 N hydrochloric acid VS. Perform a blank
determination (Residual Titrations).
8. Calculate the percentage of total menthol in the Oil tested:

A = result obtained by subtracting the number of mL of 0.5 N hydrochloric acid required in

the above titration from the number of mL of 0.5 N hydrochloric acid required in the
residual titration blank
E = percentage of esters calculated as menthyl acetate (C12H22O2 )
W = weight of acetylated Oil taken (g)
Acceptance criteria: NLT 50.0% of C10H20O, free and as esters

Impurity
- Limit of Dimethyl Sulfide
Analysis:

231
 1. Distill 1 mL from 25 mL of Oil, and carefully superimpose the distillate on 5 mL of
mercuric chloride TS in a test tube.
Acceptance criteria: A white film does not form at the zone of contact within 1 min.

Specific tests
- Specific Gravity <841>
Procedure: Method I
1. Select a scrupulously clean, dry pycnometer that previously has been calibrated by
determining its weight and the weight of recently boiled water contained in it at 25°.
2. Adjust the temperature of the liquid to about 20°, and fill the pycnometer with it.
3. Adjust the temperature of the filled pycnometer to 25°, remove any excess liquid,
and weigh. When the monograph specifies a temperature different from 25°, filled
pycnometers must be brought to the temperature of the balance before they are
weighed. Subtract the tare weight of the pycnometer from the filled weight.
4. The specific gravity of the liquid is the quotient obtained by dividing the weight of
the liquid contained in the pycnometer by the weight of water contained in it, both
determined at 25°, unless otherwise directed in the individual monograph
Acceptance criteria: 0.896 0.908

- Optical-lotation, Angular Rotation <781A>

Procedures:
1. Unless otherwise directed, that the optical rotation of the neat liquid is measured in
a 1.0-dm tube at 589 nm at 25°, corrected for the reading of the dry, empty tube

- Reflective Index <831>

Although the standard temperature for Pharmacopeial measurements is 25°, many of
the refractive index specifications in the individual monographs call for determining this
value at 20°. To achieve the theoretical accuracy of ±0.0001, it is necessary to calibrate the
232
instrument against a standard provided by the manufacturer and to check frequently the
temperature control and cleanliness of the instrument by determining the refractive index of
distilled water, which is 1.3330 at 20° and 1.3325 at 25°
Acceptance criteria: 1.459 1.465 at 20°

- Solubility in 70 % alcohol: One volume dissolves in 3 volumes of 70% alcohol, with NMT
slight opalescence

233
7. Menthol

234
Identification A
The retention time of the major peak of the Sample solution corresponds to that of the
menthol peak of the Standard solution, as obtained in the Assay.
Identification B
It meets the requirements in Specific Tests for Optical Rotation <781S> , Procedures, Specific
Rotation.

- Optical rotation
Following Optical rotation <781> USP 42
OPTICAL ROTATION
INTRODUCTION

235
Many pharmaceutical substances are optically active in the sense that they rotate an
incident plane of polarized light so that the transmitted light emerges at a measurable angle
to the plane of the incident light. This property is characteristic of some crystals and of
many pharmaceutical liquids or solutions of solids. Where the property is possessed by a
liquid or by a solute in solution, it is generally the result of the presence of one or more
asymmetric centers, usually a carbon atom with four different substituents. The number
of optical isomers is 2n, where n is the number of asymmetric centers. Polarimetry, the
measurement of optical rotation, of a pharmaceutical article may be the only convenient
means for distinguishing optically active isomers from each other and thus is an important
criterion of identity and purity.
Substances that may show optical rotatory properties are "chiral". Those that rotate light in
a clockwise direction as viewed toward the light source are "dextrorotatory", or "(+) optical
isomers", and those that rotate light in a counterclockwise direction are called "levorotatory"
or "(-) optical isomers". (The symbols d- and I-, formerly used to indicate dextrorotatory and
levorotatory isomers, are no longer sanctioned owing to confusion with D- and L-, which
refer to configuration relative to D-glyceraldehyde. The symbols R and S, or a and B, are
also used to indicate configuration, the arrangement of atoms or groups of atoms in space.)
The physicochemical properties of nonsuperimposable chiral substances rotating
plane-polarized light in opposite directions to the same extent, "enantiomers", are identical,
except for this property and in their reactions with other chiral substances. Enantiomers
often exhibit profound differences in pharmacology and toxicology, owing to the fact that
biological receptors and enzymes themselves are chiral. Many articles from natural sources,
such as amino acids, proteins, alkaloids, antibiotics, glycosides, and sugars, exist as chiral
compounds. Synthesis of such compounds from nonchiral materials usually results in equal
amounts of the enantiomers, i.e., "racemates". Racemates have a net null optical rotation,
and their physical properties may differ from those of the component enantiomers. Use of
stereoselective or stereospecific synthetic methods or separation of racemic mixtures can be
used to obtain individual optical isomers. Measurement of optical rotation is performed
using a polarimeter.' The general equation used in polarimetry is:

236
[ ] = specific rotation at wavelength
t = temperature
a = observed rotation in degrees (°)
l = path length (dm)
c = concentration of the analyte (g/100 ml)

Thus, [ ] is 100 times the measured value, in degrees (°) , for a solution containing 1
g in 100 mL, measured in a cell having a path length of 1.0 dm under defined conditions of
incident wavelength of light and temperature. For some Pharmacopeial articles, especially
liquids such as essential oils, the optical rotation requirement is expressed in terms of the
observed rotation, a, measured under conditions defined in the monograph.
Historically, polarimetry was performed using an instrument where the extent of
optical rotation is estimated by visual matching of the intensity of split fields. For this
reason, the D-line of the sodium lamp at the visible wavelength of 589 nm. was most often
employed. Specific rotation determined at the D-line is expressed by the symbol:

Much of the data available are expressed in this form. Use of lower wavelengths,
such as those available with the mercury lamp lines isolated by means of filters of
maximum transmittance at approximately 546, 436, 405, 365, and 325 nm in a photoelectric
polarimeter, has been found to provide advantages in sensitivity with a consequent
reduction in the concentration of the test compound. In general, the observed optical
rotation at 436 nm is about double, and at 365 nm, about 3 times that at 589 nm.2
Reduction in the concentration of the solute required for measurement may sometimes be
accomplished by conversion of the substance under test to one that has a significantly

237
higher optical rotation. Optical rotation is also affected by the solvent used for the
measurement, and this is always specified. It is now common practice to use other light
sources, such as xenon or tungsten halogen, with appropriate filters, because these may
offer advantages of cost, long life, and broad wavelength emission range, over traditional
light sources.
PROCEDURES
Specific Rotation
1.Operate the Instrument as per current version of individual SOP
2. Prepare the test solution as per individual testing procedure and measure the optical
rotation Specific optical rotation.
3. Use the same cell for sample and blank.
4. Maintain the same angular orientation of the cell in each reading.
5. Place the cell so that the light passes through it in the same direction each time.
6. Calculate the optical rotation using the formula:

where [ ] is the specific rotation at wavelength, t is the temperature, a is the observed

rotation in degrees, I is the pathlength in decimeters, and c is the concentration of the
analyte in g per 100ml.
7. Unless otherwise mentioned in the individual testing procedure perform the Optical
rotation and specific optical rotation at 589nm and 25°C.
8. Where a photoelectric polarimeter is used, a single measurement, corrected for the
solvent blank, is made.
9. Where a visual polarimeter is used, the average of no fewer than five determinations,

238
corrected for the reading of the same tube with a solvent blank, is used.
10. Temperature, which applies to the solution or the liquid under test, should be
maintained within ± 0.5°C of the stated value.

Assay
Procedure
Standard solution: 10 mg/mL of USP Menthol RS in hexanes
Sample solution: 10 mg/mL of Menthol in hexanes
Chromatographic system
Mode: GC
Detector: Flame ionization
Column: 0.18-mm × 20-m fused silica; coated with a
0.18-µm layer of G16 stationary phase
Temperatures
Injection port: 250°
Detector: 260
Arier gas: Hydrogen
Flow rate: 0.9 mL/min
Injection volume: 0.5 µL
Injection type: Split ratio, 50:1
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0% for the menthol peak in replicate injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of menthol (C10H20O) in the portion of Menthol taken

239
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of menthol (C10H20O) in the
portion of Menthol taken:
Result = (rU/rS) × (CS/CU) × 100
rU = peak area of menthol from the Sample solution
rS = peak area of menthol from the Standard solution
CS = concentration of USP Menthol RS in the Standard solution (mg/mL)
CU = concentration of Menthol in the Sample solution (mg/mL)

Impurities
- Limit of nonvolatile residue
Analysis: Evaporate 2 g, accurately weighed, in a tared
open porcelain dish on a steam bath, and dry the residue at 105° for 1 h.

- Related compound
Standard solution, Sample solution, Chromatographic
system, and System suitability: Proceed as directed in the Assay.

Analysis
Sample: Sample solution
Calculate the percentage of each individual impurity in
the portion of Menthol taken:
240
 Result = (rU/rT) × 100
rU = peak area of each impurity from the Sample
solution
rT = sum of the peak areas from the Sample
solution

Specific tests
- Congealing range of dl-Menthol
Sample: 10 g of dl-menthol, previously dried in a
desiccator over silica gel for 24 h
Analysis: Place the Sample in a dry test tube having an internal diameter of 18 20 mm, and
melt the contents at a temperature of about 40°. Suspend the test tube in
water having a temperature of 23° 25°, and stir the contents of the tube continually with a
thermometer, keeping the bulb of the thermometer immersed in the liquid
- Congealing temperature <651>
The temperature at which a substance passes from the liquid to the solid state upon
cooling is a useful index to purity if heat is liberated when the solidification takes place,
provided that any impurities present dissolve in the liquid only, and not in the solid. Pure
substances have a well-defined freezing point, but mixtures generally freeze over a range of
temperatures
For many mixtures, the congealing temperature, as determined by strict adherence to
the following empirical methods, is a useful index of purity. The method for determining

and 150°, the range of the thermometer used in the bath.

241
8. Silicon dioxide (Aerosil®)

242
Identification A
1.Transfer 5 mg to a platinum crucible,
2. Mix with 200 mg of anhydrous potassium carbonate
3.Heat the crucible to a red color with the aid of a Bunsen burner for
10 min, and cool
4.Dissolve the melt in 2 mL of freshly distilled water, warming if necessary
5.slowly add 2 mL of ammonium molybdate TS to the solution
Identification B
1.Place 1 drop of the yellow silicomolybdate solution from Identification test A on
a filter paper, evaporate the solvent
2.Add 1 drop of a saturated solution of o-tolidine in glacial acetic acid
3.place the paper over ammonium hydroxide.
Assay
Sample: 500 mg
Analysis:
1. Ignite the Sample in a tared platinum crucible at 1000 ± 25° for 2 h, cool in a
desiccator, and weigh.
2. Add 3 drops of sulfuric acid, and add enough alcohol to just moisten the sample
completely.
3. Add 15 mL of hydrofluoric acid, and in a well-ventilated hood evaporate on a hot
plate to dryness, using medium heat (95° 105°) and taking care that the sample does
not spatter as dryness is approached. Heat the crucible to a red color with the aid of
a Bunsen burner.
4. Ignite the residue at1000 ± 25° for 30 min, cool in a desiccator, and weigh.
5. If a residue remains, repeat the Analysis, beginning with

ignited at 1000 ± 25°, represents the weight of SiO2 in the portion taken

243
Impurities
- Loss on Ignition:
Analysis
1. Ignite the portion of Colloidal Silicon Dioxide, retained from the test for Loss on
Drying, at 1000 ± 25° to constant weight: the previously dried
Colloidal Silicon Dioxide loses
Acceptance criteria : NMT 2.0% of its weight.

- Arsenic, Method 1
Sample solution:
1. To 2.5 g add 50 mL of 3 N hydrochloric acid, and reflux for 30 min using a water
condenser. Cool, filter with the aid of suction, and transfer the filtrate to a 100-mL
volumetric flask.
2. Wash the filter and flask with several portions of hot water, and add the washings to the
flask. Cool, and dilute with water to volume.
Analysis:
1. A 15.0-mL portion of Sample solution, to which 3 mL of hydrochloric acid has been
added, meets the requirements of the test, the addition of the 7 N sulfuric
acid being omitted.
Specific tests
- pH
Following pH <791> USP 42
INTRODUCTION
For compendial purposes, pH is defined as the value given by a suitable, properly
calibrated, potentiometric sensor and measuring system.

244
 The measuring system has traditionally been referred to as the "pH meter." While the
pH meter is still in common use, the measuring system can also be embedded inside the pH
sensor, and the pH signal can be transmitted digitally to an external device such as a computer,
Programmable Logic Controller (PLC), Distributed Control System (DCS), data acquisition system,
terminal, or other microprocessor-controlled device. By definition, pH is equal to -log10 [aH+]
where aH+ is the activity of the hydrogen (H+) or hydronium ion (H3O+), and the hydrogen ion
activity very closely approximates the hydrogen ion concentration.
The practical pH scale is defined:
pH= pH5+ [(E-Es)/k]
E = measured potential where the galvanic cell contains the solution under test (pH)
Es = measured potential where the galvanic cell contains the appropriate buffer solution for
calibration (pHs)
K = change in potential/unit change in pH and is derived from the Nernst equation (as follows)
k = loge (10) x (RT/nF)
R = 8.314 J/mole/ °K
T = temperature (°K)
n= moles/half-reaction
F= Faraday constant, 96485 C/mole

- Water Determination

Following Water Determination <921> USP 42

Procedure
1. Unless otherwise specified, transfer enough methanol or other suitable solvent to
the titration vessel, ensuring that the volume is sufficient to cover the electrodes
(approximately 30 40 mL), and titrate with the Reagent to the electrometric or visual
endpoint to consume any moisture that may be present.

245
 2. (Disregard the volume consumed, because it does not enter into the calculations.)
Quickly add the Test Preparation, mix, and again titrate with the Reagent to the
electrometric or visual endpoint.
3. Calculate the water content of the specimen taken, in mg:
SF
in which S is the volume, in mL, of the Reagent consumed in the second titration; and F is
the water equivalence factor of the Reagent.

- Loss on drying
Following loss on drying <731> USP 42
Procedure
1. Conduct the determination on a 1 g test specimen
2. Tare an appropriate glass-stoppered (shallow weighing bottle that has been dried for
about 30 minutes under the same conditions to be employed in the determination
and cooled to room temperature in a desiccator)
3. Put the test specimen in the bottle, replace the 23 covers, and accurately weigh the
bottle and the contents
4. Sidewise shaking
5. Distribute the test specimen as evenly as practicable to a depth of about 5 mm
generally
6. Place the loaded bottle in the drying chamber
7. Removing the stopper and leaving it also in the chamber
8. Dry the test specimen at 105 C to constant weight (drying shall be continued until two
consecutive weighing do not differ by more than 0.50 mg per g of substance
taken, the second weighing following an additional hour of drying)

246
9. Opening the chamber and close the bottle promptly
10. Allow it to come to room temperature in a desiccator before weighing

9. Sucralose

247
248
Identification A
- Infrared Absorption (197K)
Procedure
Sample: Sucralose powder
1. Weight about 25 mg of Sucralose powder and 225 mg of Potassium bromide (KBr)
and transfer both into the smooth agate mortar.
2. Finely pulverized and put into a pellet-forming die.
3. A force of approximately 10 tons is applied under a vacuum forming a transparent
pellet.
4. Perform degassing to eliminate air and moisture form the KBr powder.
5. Before performing measurements, the background was measured using an empty
pellet holder inserted into the sample chamber.
6. Measure the sample to obtain the infrared spectrum.
Identification B
The retention time of the principal peak of the Sample solution corresponds to that
of the Standard solution, as obtained in the Assay
Procedure : The procedure was performed as directed in the Assay section.
Identification C
The Rf value of the principal spot of the sample solution corresponds to that of
Standard solution A, as obtained in the test for Related Compounds.
Procedure: The procedure was performed as directed in the Related Compound section.

249
Assay
Mobile phase preparation
Mobile phase: Acetonitrile and water (17:3) prepared 1000 mL.
1. Transfer 150 mL of Acetonitrile into a 1000 mL beaker
2. Add 850 mL of water and mix
3. Filtrate through 0.22micron membrane.
Standard and Sample preparation
Standard Solution
1. Accurately weight 50 mg of USP Sucralose RS into volumetric flask 50 mL.
2. Dissolve and dilute with mobile phase to volume and mix to obtained
concentration of 1 mg/mL Standard solution.
3. Filter the solution through 0.45micron membrane into 1.5 mL vial.
Sample solution
1. Accurately weight 50 mg of Sucralose raw material into volumetric flask 50 mL
2. Dissolve and dilute with mobile phase to volume and mix to obtained
concentration of 1 mg/mL concentration
3. Filtrate the solution through 0.45micron membrane into 1.5 mL vial.
4. Repeat the procedure 3 times to obtained 3 samples.
Apparatus and software
Model : SHIMADZU HPLC Nexera-i
Software : ACD/Spectrus Processor
Chromatographic system
Mode : LC

250
 Detector : Refractive Index
Column : 8-nm x 10-cm; packing L1 Flow rate: 1.5 mL/min
Injection size : 20 microLite
System suitability
Sample : Standard solution Suitability requirement:
Relative standard deviation: NMT 2.0%
1. Automatically inject the Standard solution into the HPLC, repeat 5 times.
2. Calculate the average relative standard deviation of the5 peak responds
obtained.
Analysis
Test solution : Standard solution and Sample solution
1. Automatically Inject the Standard solution into the HPLC, repeat 3 times.
2. Automatically inject the first Sample solution into the HPLC, repeat 3 times.
3. Repeat the same procedure with another 2 Sample solutions.
4. Calculate the percentage of Sucralose (C12H19Cl3O8) in the portion of Sucralose
taken
with The following formula;
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru= Peak response from the Sample solution
Rs= Peak response from the Standard solution
Cs= Concentration of USP Sucralose RS in the Standard solution (mg/mL)
Cu= Concentration of the Sample solution (mg/mL)

Impurities
- Residue on ignition
The procedure was proceed as directed in USP Residue on Ignition <281>
1. Accurately weight silica crucible and ignite the silica crucible at 600± 50 in muffle

251
 furnance for 30 minutes.
2. Cool the crucible in silica gel desicator.
3. Accurately 1 g of Sucralose in the crucible and moisten the sample with 1mL of
sulfuric Acid.
4. Heat until sample is thoroughly charred
5. Cool and moisten the residue with 1 mL of sulfuric acid.
6. Heat gently until no longer white fumed. (Ensure that the flames are not produce
during the procedure).
7. Cool the crucible in silica-gel desicator.
8. Weight accurately and calculate the percentage of residue as directed in the
following
Formula ;% Residue on Ignition = (residue weight /sample weight) X 100%
9. If the calculated residue is exceed0.7%, Repeat moistening with sulfuric acid,
heating,
igniting ass before until two consecutive weighing of residue do not differ by more

- Limit of methanol
Internal standard solution: 0.1 µL/mL of n-propyl alcohol in pyridine
Standard solution: 0.2 µL/mL of methanol in Internal standard solution
Sample solution: 0.2 g/mL of Sucralose in Internal standard solution
Chromatographic system (See Chromatography á621ñ, System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 4-mm × 2-m glass column; packed with 80- to 100-mesh silanized support S6
Temperature
Column: 150°
Detector: 250°

252
Injector: 200°
Carrier gas: Helium
Flow rate: 20 mL/min
Injection size: 1 µL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution Calculate the percentage of methanol in
the portion of Sucralose taken:
Result = (R U/R S ) × [(C S /C U) × F 1 ] × F 2 × 100
RU= peak response ratio of methanol to n-propyl
alcohol, from the Sample solution
RS= peak response ratio of methanol to n-propyl
alcohol, from the Standard solution
CS=concentration of methanol in the Standard
solution (µL/mL)
CU= concentration of Sucralose in the Sample
solution (g/mL)
F1= conversion factor from µL to mL
F2= specific gravity of methanol, 0.79 g/cm3
Acceptance criteria: NMT 0.1%

253
- Related Compound
Adsorbent: 0.20-mm layer of octadecylsilanized
chromatographic silica gel. The thin-layer
chromatographic plate also has a preadsorbent zone.
Detection reagent: Sulfuric acid in methanol (3 in 20)
Standard solution A: 10.0 mg/mL of USP Sucralose RS in methanol
Standard solution B: 0.5 mL Standard solution A diluted to 10.0 mL with methanol
Sample solution: 100.0 mg/mL of Sucralose in methanol
Developing solvent system: Acetonitrile and sodium chloride solution (1 in 20) (3:7)
Application volume: 5 µL
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Proceed as directed under Chromatography <621>, Thin Layer Chromatography. Spray
the plate with Detection reagent. Heat the plate for 10 min at 125°.
Acceptance criteria: The RF value of the principal spot from the Sample solutioncorresponds
to that obtained from Standard solution A, and the color of any other single spot from the
Sample solution is not more intense than that of the principal spot from Standard solution B
(0.5%).

- Limit of Hydrolysis
Adsorbent: 0.25-mm layer of chromatographic silica gel
Spray reagent: 12.3 mg/mL of p-anisidine and 16.6

254
mg/mL of phthalic acid in methanol. Store the solution in
the dark and refrigerate to prevent discoloration. Discard
if the solution becomes discolored.
Standard solution A: 100 mg/mL of mannitol
Standard solution B: 0.4 mg/mL of fructose and 100
mg/mL of mannitol
Sample solution: 250 mg/mL of Sucralose in methanol
Application volume: 5-µL portions separately applied in 1-
µL increments, allowing the plate to dry between
applications
Analysis
Samples: Standard solution and Sample solution
Proceed as directed under Chromatography <621>, Thin-Layer Chromatography.
1. Spray the plate with Spray reagent, and heat the plate at 100 ± 2° for 15 min.
2. If the spot from Standard solution A has darkened, repeatthe test, heating for a
shorter period of time.
3. Immediately after heating, view the plate against a dark background.
Acceptance criteria: The color of the spot from the Sample solution is not more intense
than that from Standard solution B (0.1%).

Specific tests
- Optical lotation specific Rotation <781S>: +84.0° to +87.5° at 20°
Sample solution: 10 mg/mL of Sucralose
Procedures:

255
 1. Unless otherwise directed, that the optical rotation of the neat liquid is measured in
a 1.0-dm tube at 589 nm at 25°, corrected for the reading of the dry, empty tube

- Water Determination, Method I <921>:

procedure
1. Unless otherwise specified, transfer enough methanol or other suitable solvent to the
titration vessel, ensuring that the volume is sufficient to cover the electrodes (approximately
30 40 mL), and titrate with the Reagent to the electrometric or visual endpoint to consume
any moisture that may be present.
2. (Disregard the volume consumed, because it does not enter into the calculations.) Quickly
add the Test Preparation, mix, and again titrate with the Reagent to the electrometric or
visual endpoin.
3. Calculate the water content of the specimen taken, in mg:
SF
in which S is the volume, in mL, of the Reagent consumed in the second titration; and F is
the water equivalence factor of the Reagent.
Acceptance criteria: NMT 2.0%

256
 P5 Control of finished Product
P5.1 Specification and Certificate of Analysis
Finished Product Specification
Table 64 Finished Product Specification

Test Requirement Method

Appearance A dry powder (White or off- Visual inspection
white powder)
Identification
A. HPLC A. The retention time of the Chromatography USP
azithromycin peak of the 42 NF 37
Sample solution corresponds Page 448
to that of the Standard
solution, as obtained in the
Assay.
Assay Chromatography USP
90.0%-110.0%
42 NF 37, Page 448
Performance tests
- Deliverable volume The average volume of liquid Deliverable volume
obtained from the 10 USP 42 NF 37 <698>,
containers is NLT 100%, and Page 6862
the volume of no container is
less than
95% of the volume declared
in the labeling.
- Dissolution Apparatus 2 Each unit is not less than Q + USP 42 NF 37
- (Paddle Apparatus) 5% within 15 minutes. Dissolution <711>,
Page 6879

Organic impurities
- Azithromycin N-oxide NMT 0.50%

257
 - -(N-N-Didemethyl)- -N-formyl NMT 0.50% Chromatography USP
azithromycin 42 NF 37 ,
- - N.N-Dimethyl NMT 0.50% Page 449-450
azithromycin aminoazithromycin
- Azithromycin related compound F NMT 0.50%

- Desosaminylazithromycin NMT 0.30%

- N-Demethylazithromycin NMT 0.50%
- -O- -
demethylazithromycin)
- -De(dimethylamino)- - NMT 0.50%
Oxoazithromycin
- Specified unidentified impurity -
- Azithromycin -
- 2-Desethyl-2-propylazithromycin -
- -N-Demethyl- -N-[(4- -
methylphenyl)sulfonyl]azithromycin
- 3-Deoxyazithromycin (azithromycin -
B)
- Any individual, unidentified impurity NMT 0.20%
- Total degradation products NMT 3.5%
Specific test
- pH 8.5 11.0 USP 42 NF 37 ,
<791> Page 450
- Microbiological test
A. Microbial enumeration test : -Total aerobic microbial Pour-plate method
Enumeration method count less than 102 cfu/ml USP 42 NF 37 , <61>
-Total combined Yeast/Molds Page 6387-6393
count less than 10 cfu/ml
B. Test for specified microorganism: Absence of Escherichia coli Pour-plate method
Bile-tolerant gram-negative USP 42 NF 37, <62>
bacteria Page 6393-6402

258
- Uniformity of dosage units
- A. Weight variation The requirements for dosage Weight variation USP
uniformity are met if the 42 NF 37, <905>
acceptance value of the first Page 7079-7082
10 dosage units is less than
or equal to L1%. If the
acceptance value is > L1%,
test the next 20 units, and
calculate the acceptance
value.

259
 O LEAVES Pharmaceutical Co.,LTD
: 147 Soi Sabaijai Suthisarnwinijchai Rd., HuaiKwang Bangkok Thailand 10310Tel:
040-523478 FAX: 040-523478 Email: OLEAVESPharmaceutical@oleaves.com

Certification of Analysis

Product name Azithromycin powder oral Manufacture Date

suspension 200 mg/5 ml (15 ml) 3 February 2019
Azinova®
Lot Number AZ200565081 Analysis Date 10 February 2019
Batch Quantity 500,000 bottles Expire Data 2 February 2022

Test Specifications Result

Appearance A dry powder (White or off-white
Complies
powder)
Identification
A. HPLC A. The retention time of the
azithromycin peak of the
Sample solution corresponds to Complies
that of the Standard
solution, as obtained in the
Assay.
Assay 90.0%-110.0% 98.9%
Performance tests
- Deliverable volume The average volume of liquid Average volume: 109.96%
obtained from the 10 containers No container is less than 95%
is NLT 100%, and the volume of
no container is less than 95% of

260
 the volume declared in the
labeling.

- Dissolution Apparatus 2 Each unit is not less than Q+5% Sample 1 :99 %
(Paddle Apparatus) within 15 minutes. Sample 2 :101%
Sample 3 :101%
Sample 4 :104%
Sample 5 :102%
Sample 6 :100%
Organic impurities
- Azithromycin N-oxide NMT 0.50% 0.01%
- -(N-N-Didemethyl)- -N-formyl 0.01%
NMT 0.50%
azithromycin
- - N.N-Dimethyl NMT 0.50% 0.01%
azithromycin aminoazithromycin
- Azithromycin related compound F NMT 0.50% 0.02%
- Desosaminylazithromycin NMT 0.30% 0.03%
- N-Demethylazithromycin NMT 0.50% 0.01%
- -O- - Not found
demethylazithromycin)g
- -De(dimethylamino)- - NMT 0.50% 0.01%
Oxoazithromycin
- Specified unidentified impurity - Not found
- Azithromycin - Not found
- 2-Desethyl-2-propylazithromycin - Not found
- -N-Demethyl- -N-[(4- - Not found
methylphenyl)sulfonyl]azithromycin
- 3-Deoxyazithromycin (azithromycin B) - Not found
- Any individual, unidentified impurity NMT 0.20% 0.03%
- Total degradation products NMT 3.5% 0.13%
Specific test
- pH 8.5 11.0 9.7

261
 (200 mg/5 ml in water)

- Microbiological test
A. Microbial enumeration test : -Total aerobic microbial count <10 cfu/ml
Enumeration method less than 102 cfu/ml
-Total combined Yeast/Molds <10 cfu/ml
count less than 10 cfu/ml
B. Test for specified microorganism: Absence of Escherichia coli Absence per mL
Bile-tolerant gram-negative bacteria
- Uniformity of dosage units
A. Weight variation The requirements for dosage Acceptance Value = 2.7
uniformity are met if the L1=15.0)
acceptance value of the first 10
dosage units is less than or
equal to L1%. If the acceptance
value is > L1%, test the next 20
units, and calculate the
acceptance value.

262
 O LEAVES Pharmaceutical Co.,LTD
: 147 Soi Sabaijai Suthisarnwinijchai Rd., HuaiKwang Bangkok Thailand 10310
Tel: 040-523478 FAX: 040-523478 Email: OLEAVESPharmaceutical@oleaves.com

Certification of Analysis

Product name Azithromycin powder oral Manufacture Date

suspension 200 mg/5 ml (15 ml) 3 February 2019
Azinova®
Lot Number AZ200565081 Analysis Date 10 February 2019
Batch Quantity 500,000 bottles Expire Data 2 February 2022

Test Specifications Result

Appearance A dry powder (White or off-white
Complies
powder)
Identification
A. HPLC A. The retention time of the
azithromycin peak of the Complies
Sample solution corresponds to
that of the Standard
solution, as obtained in the Assay.
Assay 90.0%-110.0% 98.9%

263
Performance tests
- Deliverable volume The average volume of liquid Average volume: 109.96%
obtained from the 10 containers is No container is less than 95%
NLT 100%, and the volume of no
container is less than 95% of the
volume declared in the labeling.

- Dissolution Apparatus 2 Each unit is not less than Q + 5% Sample 1 :101%

(Paddle Apparatus) within 15 minutes. Sample 2 :102%
Sample 3 :100%
Sample 4 :105%
Sample 5 :102%
Sample 6 :100%
Organic impurities
NMT 0.50% 0.01%
- Azithromycin N-oxide
- -(N-N-Didemethyl)- -N-formyl 0.02%
NMT 0.50%
azithromycin
- - N.N-Dimethyl
NMT 0.50% 0.01%
azithromycin aminoazithromycin

- Azithromycin related compound F NMT 0.50% 0.03%

- Desosaminylazithromycin NMT 0.30% 0.01%
- N-Demethylazithromycin NMT 0.50% 0.01%
- -O-
- Not found
demethylazithromycin)
- -De(dimethylamino)- -
NMT 0.50% 0.03%
Oxoazithromycin
- Specified unidentified impurity - Not found
- Azithromycin - Not found
- 2-Desethyl-2-propylazithromycin - Not found

264
 - -N-Demethyl- -N-[(4-
- Not found
methylphenyl)sulfonyl]azithromycin
- 3-Deoxyazithromycin (azithromycin B) - Not found
- Any individual, unidentified impurity NMT 0.20% 0.01%
- Total degradation products NMT 3.5% 0.13%
Specific test
- pH 8.5 11.0 9.7
(200 mg/5 ml in water)

- Microbiological test
- Total aerobic microbial count
A. Microbial enumeration test : <10 cfu/ml
less than 102 cfu/ml
Enumeration method
- Total combined Yeast/Molds
<10 cfu/ml
count less than 10 cfu/ml
B. Test for specified microorganism: Absence of Escherichia coli Absence per mL
Bile-tolerant gram-negative bacteria

- Uniformity of dosage units The requirements for dosage Acceptance Value = 2.6
A. Weight variation uniformity are met if the L1=15.0)
acceptance value of the first 10
dosage units is less than or equal
to L1%. If the acceptance value is
> L1%, test the next 20 units, and
calculate the acceptance value.

265
 O LEAVES Pharmaceutical Co.,LTD
: 147 Soi Sabaijai Suthisarnwinijchai Rd., HuaiKwang Bangkok Thailand 10310
Tel: 040-523478 FAX: 040-523478 Email: OLEAVESPharmaceutical@oleaves.com

Certification of Analysis

Product name Azithromycin powder Manufacture Date

oral suspension 200
3 February 2019
mg/5 ml (15 ml)
Azinova®
Lot Number AZ200565081 Analysis Date 10 February 2019
Batch Quantity 500,000 bottles Expire Data 2 February 2022

Test Specifications Result

Appearance A dry powder (White or off-white
Complies
powder)
Identification
A. HPLC A. The retention time of the
azithromycin peak of the
Sample solution corresponds to Complies
that of the Standard
solution, as obtained in the
Assay.
Assay 90.0%-110.0% 98.9%

266
Performance tests
- Deliverable volume The average volume of liquid Average volume:
obtained from the 10 containers 109.96%
is NLT 100%, and the volume of No container is less
no container is less than than 95%
95% of the volume declared in
the labeling.

- Dissolution Each unit is not less than Q+5% Sample 1 :101%

Apparatus 2 within 15 minutes. Sample 2 :103%
(Paddle Apparatus) Sample 3 :100%
Sample 4 :104%
Sample 5 :102%
Sample 6 :102%
Organic impurities
- Azithromycin N-oxide NMT 0.50% 0.01%
- -(N-N-Didemethyl)- -N-formyl 0.01%
NMT 0.50%
azithromycin
- - N.N-Dimethyl 0.01%
NMT 0.50%
azithromycin aminoazithromycin

- Azithromycin related compound F NMT 0.50% 0.01%

- Desosaminylazithromycin NMT 0.30% 0.03%

- N-Demethylazithromycin NMT 0.50% 0.01%
- -O- Not found
-
demethylazithromycin)
- -De(dimethylamino)- -
NMT 0.50% 0.01%
Oxoazithromycin
- Specified unidentified impurity - Not found
- Azithromycin - Not found
- 2-Desethyl-2-propylazithromycin - Not found

267
 - -N-Demethyl- -N-[(4- Not found
-
methylphenyl)sulfonyl]azithromycin
- 3-Deoxyazithromycin (azithromycin B) - Not found
- Any individual, unidentified impurity NMT 0.20% 0.02%
- Total degradation products NMT 3.5% 0.11%
Specific test
- pH 8.5 11.0 9.5
(200 mg/5 ml in water)
- Microbiological test
- Total aerobic microbial count
A. Microbial enumeration test : <10 cfu/ml
less than 102 cfu/ml
Enumeration method
- Total combined Yeast/Molds
<10 cfu/ml
count less than 10 cfu/ml
B. Test for specified microorganism:
Absence of Escherichia coli Absence per mL
Bile-tolerant gram-negative bacteria

- Uniformity of dosage units

A. Weight variation The requirements for dosage Acceptance Value = 2.8
uniformity are met if the
acceptance value of the first 10
dosage units is less than or
equal to L1%. If the acceptance
value is > L1%, test the next 20
units, and calculate the
acceptance value.

268
P5.2 Analytical Procedure
Following USP42 Azithromycin

269
270
1. Appearance
Method: visual examination
Acceptance criteria: A dry powder (White or off-white powder)
2. Identification
A. The retention time of the azithromycin peak of the Sample solution corresponds
to that of the Standard solution, as obtained in the Assay
3.Assay
Following USP42 Azithromycin
Procedure
Solution A : Dissolve 8.7 g of dipotassium hydrogen phosphate anhydrous in 1000 mL of water and
adjust with potassium hydroxide or dilute orthophosphoric acid to a pH of 8.2
Solution B : Acetonitrile
Mobile phase : Solution A and Solution B (30:70)
Diluent : Acetonitrile, methanol, and water (40:40:20)
Standard solution: 0.6 mg/mL of USP Azithromycin RS in Diluent. Sonicate in cool water to
dissolve as needed.
Sample solution : Nominally 0.6 mg/mL of azithromycin in Diluent prepared as follows. Transfer
an accurately measured portion of the constituted suspension to a suitable volumetric flask. Add
Diluent equal to 50% of the volume of the flask, and sonicate for 20 min with shaking in cool
water. Dilute with Diluent to volume. Pass a portion of this solution through a suitable filter of
0.45-µm pore size.
Chromatographic system
Mode : LC
Detector : UV 210 nm
Column : 4.6-mm x 25-cm; 5-µm packing L1
Temperatures
Autosampler : 10o
Column : 30o
Flow rate : 2 mL/min

271
 Injection volume : 50 µL
Run time : NLT 2 times the retention time of azithromycin

System suitability
Sample : Standard solution
Suitability requirements
Tailing factor : NMT 2.0
Relative standard deviation : NMT 2.0%
Analysis
Samples : Standard solution and Sample solution Calculate the percentage of the labeled
amount of azithromycin (C38H72N2O12) in the portion of Azithromycin for Oral Suspension taken:
Result = (ru/rs) x (Cs/Cu) x P x F X100

ru = peak response of azithromycin from the Sample solution

rs = peak response of azithromycin from the Standard solution
Cs = concentration of USP Azithromycin RS in the Standard solution (mg/mL)
Cu = nominal concentration of azithromycin in the Sample solution (mg/mL)
P = potency of USP Azithromycin RS (ug/mg)
F = conversion factor, 0.001 mg/ug
Acceptance criteria : 90.0%-110.0%

Procedure

Mobile Phase Preparation

Mobile Phase A : buffer solution and Acetonitrile (30:70)
Solution A (buffer solution)
1. Dissolve 8.7 g of dipotassium hydrogen phosphate anhydrous in 1000 mL
of water in Volumetric flask 1000 mL
2. adjust with potassium hydroxide or dilute orthophosphoric acid to a pH
of 8.2
3. Filtrate with filter paper pore size 0.45 m under vacuum and transfer to
a solvent reservoir

272
 4. Sonicate 15 min
Solution B (Acetonitrile)
1. Measure 1000 mL of Acetonitrile with a 10 mL of Volumetric flask
2. Filtrate with filter paper pore size 0.45 pm under vacuum and transfer to
a solvent reservoir
3. Sonicate 15 min
Diluent preparation
1. Measure 500 mL Acetonitrile, 500 mL methanol in 500 mL of volumetric
flask and Measure 250 mL water in 250 mL of volumetric flask
2. Mix 500 mL Acetonitrile, 500 mL methanol and 250 mL water
Standard solution Preparation: 0.6 mg/ml of USP Azithromycin RS
Table 65 Weigh of USP Azithromycin RS
List Weight
USP Azithromycin RS preparation 60 mg
Range weight of USP Azithromycin RS 57-63 mg
which can used for preparation (±5%)
USP Azithromycin RS 60.3 mg

1. To weigh 60 mg of Azithromycin RS with weighing boat into volumetric

flask 100 mL
2. Add about 30 ml of diluent and sonicate 20 min to until completely
dissolved.
3. Dilute with Diluent to volume 100 mL
4. Filtrate with filter membrane 0.45 µm
5. Standard solution 0.6 mg/mL
*Note: Concentration of standard solution = 60.3 mg /100 mL = 0.603 mg/mL

Sample solution Preparation: 0.6 mg/ml of USP Azithromycin

Table 66 Weigh of Sample Preparation
Weight
List
Sample 1 Sample 2 Sample 3
USP Azithromycin oral suspension preparation 180 mg 180 mg 180 mg

273
 Range weight of USP Azithromycin oral
suspension which can used for preparation 171-189 mg 171-189 mg 171-189 mg
(+5%)
USP Azithromycin oral suspension 181.5 mg 179.8 mg 180.5 mg

1 Random and pooled 3 bottles of Azithromycin oral suspension

2 Weight 180 mg of Azithromycin powder for oral suspension, transfer to mL
in Volumetric flask and adjust with water to 15 mL
3 Pipette 5 mL of Azithromycin oral suspension, transfer to 100 mL
Volumetric flask
4 Add about 30 ml of diluent and sonicate 20 min to until completely
dissolved.
5 Dilute with Diluent to volume 0 mL
6 Filtrate with filter membrane 0.45 µm
7 Sample solution 0.6 mg/mL
8 replicate 3 flasks
*Note: Concentration of sample solution = 180.6 mg x (15 ml /5 mL) x (1/100 ml) =
0.602 mg/mL
Chromatographic system
Mode : LC
Detector : UV 210 nm
Column : 4.6-mm x 25-cm; 5-µm packing L1
Temperatures
Autosampler : 10o
Column : 30o
Flow rate : 2 mL/min
Injection volume : 50 µL
Run time : NLT 2 times the retention time of azithromycin

274
System suitability
Sample : Standard solution
Suitability requirements
Tailing factor : NMT 2.0
Relative standard deviation : NMT 2.0%

Chromatogram of System suitability

Chromatogram of System suitability

Result of System suitability

Table 67 Result system suitability
Peak Area
Standard Tailing factor (Asym)
(mAU*min)
1 34421 1.
2 34774 1.

275
 3 34703 1.
4 34353 1.
5 34206 1.
Average 34491.4
SD 239.9 0.09
% RSD 0.70 7.31
Interpret Result of System suitability
Table 5 Interpret result system suitability
System suitability Acceptance criteria Result Interpret
Tailing factor NMT 2.0 1. Pass
Relative standard
NMT 2.0% 0.7% Pass
deviation

Analysis
Samples: Standard solution and Sample solution Calculate the percentage of the
labeled amount of azithromycin (C38H72N2O12) in the portion of Azithromycin for Oral
Suspension taken:
Result = (ru/rs) x (Cs/Cu) x P x F X100
ru = peak response of azithromycin from the Sample solution
rs = peak response of azithromycin from the Standard solution
Cs = concentration of USP Azithromycin RS in the Standard solution (mg/mL)
Cu = nominal concentration of azithromycin in the Sample solution (mg/mL)
P = potency of USP Azithromycin RS (µg/mg)
F = conversion factor, 0.001 mg/µg
Acceptance criteria : 90.0% -110.0%
Calculation example
Result = (ru/rs) x (Cs/Cu) x P x F X100

276
 = (34550 mAU*min /34505 mAU*min) x (0.603 mg/ml /
0.602mg/ml) x 947 µg/mg x 0.001 mg/µg x 100
= 95.0%
4. Performance tests
4.1 Deliverable volume
Following Deliverable volume <698> USP42
Deliverable volume<698>: Meets the requirement

For Multiple-unit Containers

Procedure of Density determination
1. Tare a 100 ml volumetric flask containing 50.0 ml of water
2. Add approximately 25 g of well-shaken product
3. gently swirl the content to mix
4. Reweigh the flask
5. From a burette, add an accurately measured amount of water to bring the flask
contents to volume while gently swirling the content of the flask.
6. Record the volume taken from the burette
7. Calculate the density of the sample.
Weight of sample=24.9208 g
The volume taken from burette of sample = 27.90 ml
The density of sample = 22.10 ml

density = 1.1276 g/ml

Procedure of Deliverable volume test

277
 1. Shake the contents of 10 containers individually
2. Discharge the container content into a the 100 graduated beaker
3. Drainage for NMT 10 min
4. Determine the mass of the contents
5. The average volume of liquid obtain from 10 container NLT 100% and the volume of
no container is less than 95% of the volume declared in the labeling

4.2 Dissolution
Following Dissolution <711> USP42
Dissolution<711>
NOTE Solution containing azithromycin are stable up to 12 h at 10
Medium: Sodium phosphate buffer at pH of 6.0 ; 900ml
Apparatus 2: 50 rpm
Time: 30 min
Solution A : Dissolve 8.7 g of dipotassium hydrogen phosphate anhydrous in 1000 mL of water
and adjust with potassium hydroxide or dilute orthophosphoric acid to a pH of 8.2
Solution B: Acetonitrile
Mobile phase: Solution A and Solution B 35:65
Standard stock solution: 0.55 mg/ml of USP Azithromycin RS prepare as follows. Transfer an
accurately weighed amount of USP Azithromycin RS to a suitable volumetric flask. Add
acetonitrile to fill 5% of the volume of the flask and sonicate in cool water for 5 min to dissolve
completely. Dilute with Medium to volume
Standard solution
For Azithromycin for oral Suspension labeled to contain 100 mg/5 ml: 0.11 mg/mL of USP
Azithromycin RS in Medium from standard stock solution
For Azithromycin for oral Suspension labeled to contain 200 mg/5 ml: 0.22 mg/mL of USP
Azithromycin RS in Medium from standard stock solution
Sample solution : Pass portion of the solution under test through a suitable filter
Chromatographic system
Mode: LC
Detector : UV 210 nm

278
 Column : 4.6-mm x 25-cm; 5-um packing L1
Temperatures
Autosampler : 10o
Column : 30o
Flow rate : 2 mL/min
Injection volume : 100 uL
Run time : NLT 2 times the retention time of azithromycin

System suitability
Sample : Standard solution
Suitability requirements
Tailing factor : NMT 2.0
Relative standard deviation : NMT 2.0%
Analysis
Samples : Standard solution and Sample solution Calculate the percentage of the
labeled amount of azithromycin (C38H72N2O12) dissolve
Result= ru/rs × (Cs/L × D × d/W × V × 100
ru = peak response of azithromycin from the Sample solution
rs = peak response of azithromycin from the Standard solution
Cs= concentration of USP Azithromycin RS in the standard solution
L = label claim of Azithromycin RS in the standard solution mg/ml
D = dilution factor, necessary only if the sample solution requires dilution ml/ml
D = density of sample solution g/ml
W= weight of Azithromycin for oral suspension taken g
V = volume of medium,900 mL
Tolerance: NLT 75 %(Q) of the labeled amount of azithromycin (C38H72N2O12) is dissolve
Medium : pH 6.0 Sodium phosphate buffer
Apparatus 2 Paddle apparatus : 50 rpm
Time: 30 min
Temperature: 37±0.5 °C

279
Mobile phase: Solution A : Solution B = 35 : 65
Solution A : 1.Weigh 8.7 g of dipotassium hydrogen phosphate anhydrous, transfer to
1000 mL Volumetric flask and adjust with water to 1000 mL
2. adjust with potassium hydroxide to pH 8.2
Solution B: Acetonitrile
Standard stock solution
1.Weight 0.0250 g of Azithromycin RS in volumetric flask 100 ml
2. Add Acetonitrile 5 ml
3. Sonicate in cool water 5 min
4. pipette 11 ml
4. Dilute to 100 ml with medium until concentration 0.55 mg/ml
Standard solution
For Azithromycin for oral suspension labeled to contain 200 mg/ 5 ml
1. Pipette stock solution 4 ml and transfer to volumetric flask 10 ml
2. Adjust volume to 10 ml with Medium
Sample solution : Pass a portion of the solution under test through a suitable
filter
Chromatographic system
Mode: LC
Detector : UV 210 nm
Column : 4.6-mm x 25-cm; 5-um packing L1
Temperatures
Autosampler : 10o
Column : 30o
Flow rate : 2 mL/min
Injection volume : 100 uL
Run time : NLT 2 times the retention time of azithromycin

280
 System suitability
Sample : Standard solution
Suitability requirements
Tailing factor : NMT 2.0
Relative standard deviation : NMT 2.0%
Chromatogram of system suitability

Retention time of System suitability

Table 68 Retention time of System suitability
Standard solution Retention time (min)
1 7.321
2 7.384
3 7.343
4 7.319
5 7.318

Result of System suitability

Table 69 Result of System suitability
Standard Area Tailing factor (Asym)
(mAU*min)
1 165477 1.12
2 166899 1.10
3 163439 1.08
4 168767 1.14
5 162231 1.08

281
 Average 165362.6 1.10
% RSD 1.4 2.37

Table 70 Interpret Result of System suitability

System suitability Acceptance criteria Result Interpret
Tailing factor NMT 2 1.1 Pass
Relative standard deviation NMT 2 1.4 Pass

Analysis
Samples : Standard solution and Sample solution Calculate the percentage of the
labeled amount of azithromycin (C38H72N2O12) dissolve
Result= ru/rs × (Cs/L × D × d/W × V × 100
ru = peak response of azithromycin from the Sample solution
rs = peak response of azithromycin from the Standard solution
Cs= concentration of USP Azithromycin RS in the standard solution
L = label claim of Azithromycin RS in the standard solution mg/ml
D = dilution factor, necessary only if the sample solution requires dilution ml/ml
D = density of sample solution g/ml
W= weight of Azithromycin for oral suspension taken g
V = volume of medium; 900 mL

Tolerance: NLT 75 % Q of the labeled amount of azithromycin (C38H72N2O12) is

dissolve
Result =

282
 = 93.0 %
Procedure
1. Place the stated volume of the Dissolution medium (+1%) in the vessel of the specified
apparatus.
2. Assemble the apparatus, equilibrate the Dissolution medium to 37 + 0.5~ then remove the
thermometer
3. Place 6 dosage unit in the each apparatus (Stage 1) and each unit is NLT 93 +5 % (Q), if
stage 1 is not pass. Place 6 dosage unit in the each apparatus (Stage 2) and average of 12 units
(S1+S2) is < 93(Q), and no unit is < 93(Q) - 15%, taking care to exclude air bubbles from the
surface of the dosage unit
4. Immediately operate the apparatus at the specified rate.
5. Within the 30 min, withdraw a specimen from a zone midway between the surface
of the Dissolution medium and the top of the rotating blade, NLT 1 cm from the
vessel wall.
6. Perform the analysis, using a suitable assay method.

Interpretation:
Immediate-Release Dosage Forms
Unless otherwise specified, the requirements are met if the quantities of active ingredient
dissolved from the dosage units tested conform to Acceptance Criteria of Dissolution.
Continue testing through the three stages unless the results conform at either S1 or S2. The
quantity, Q, is the amount of dissolved active ingredient, expressed as a percentage of the
labeled content of the dosage unit; the 5%, 15%, and 25% values in Acceptance Criteria of

283
Dissolution are percentages of the labeled content so that these values and Q are in the
same terms.
Acceptance criteria
Table 71 Immediate release dosage form
Stage Number Tested Acceptance criteria
S1 6 Each unit is NLT Q+5%
S2 6 Average of 12 units S1+S2
and no unit is < Q 15 %
S3 12 Average of 12 units S1+S2
NMT 2 unit are < Q 15 % and
no unit is < Q 25 %

5. Organic impurities
Following USP42 Azithromycin
Organic Impurities
Solution A: Dissolve 1.8 g of disodium hydrogen phosphate dihydrate in 1000 mL of
water and adjust with dilute phosphoric acid to a pH of 8.9.
Solution B: Acetonitrile and methanol (75:25)
Mobile phase: See Table 1
Table 1
Time (min) Solution A (%) Solution B (%)
0 50 50
25 45 55
30 40 60
80 25 75
81 50 50
93 50 50
Buffer: Dissolve 1.73 g of ammonium dihydrogen phosphate in 1000 mL of water and
adjust with ammonia solution to a pH of 10.0 ± 0.05.
Diluent: Buffer, methanol, and acetonitrile (35:35:30)

284
 System suitability solution: 0.015 mg/mL of USP Azithromycin Related Compound F
RS and 0.025 mg/ml of USP Desosaminylazithromycin RS in Diluent
Standard solution: 0.04 mg/mL of USP Azithromycin RS in Diluent. Sonicate in cool
water to dissolve as needed.
Sample solution: Nominally 4.0-mg/mL solution of azithromycin in Diluent prepared
as follows. Transfer a portion of the constituted suspension, equivalent to about
400.0 mg of azithromycin, to a 100-mL volumetric flask. Add 70 mL of Diluent and
sonicate in cool water for about 15 min. Dilute with Diluent to volume. Pass a portion
of this solution through a suitable filter of 0.45-um pore size
Chromatographic system
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm x 15-cm; 5-um packing L1
Temperatures
Autosampler: 10o
Column: 60o
Flow rate: 0.9 mL/min
Injection volume: 100 uL
Run time: NLT 2 times the retention time of azithromycin
System suitability
Samples: System suitability solution and Standard solution
Suitability requirements
Resolution: NLT 1.5 between desosaminylazithromycin and azithromycin
related compound F, System suitability solution
Relative standard deviation: NMT 5.0%, Standard solution
Analysis
Samples: Standard solution and Sample solution Calculate the percentage of each
impurity in the portion of Azithromycin for oral suspension taken:
Result = (ru/rs)x (Cs/Cu) x P x F1 x (1/F2) x 100
ru = peak response of each impurity from the Sample solution
rs = peak response of azithromycin from the Standard solution
Cs = concentration of USP Azithromycin RS in the Standard solution (mg/mL)
Cu = concentration of Azithromycin in the Sample solution (mg/mL)

285
P = potency of USP Azithromycin RS (ug/mg of azithromycin)
F1= conversion factor, 0.001 mg/ug.
F2= relative response factor (see Table 2)
Acceptance criteria: See Table 2. Disregard peaks at relative retention times before
0.29 and after 1.31
Table 2
Relative Relative Acceptance
Name Retention Response Criteria,NMT
Time Factor (%)
Azithromycin N-oxide 0.29 0.43 0.50
-(N-N-Didemethyl)- -N-formyl
0.37 1.7 0.50
azithromycin
- N.N-Didemethyl
0.43 1.0 0.50
azithromycin aminoazithromycin c
Azithromycin related compound Fd 0.51 3.8 0.50
Desosaminylazithromycine 0.54 1.0 0.30
N-Demethylazithromycin 0.61 1.0 0.50
-O-
0.73 --- ----
demethylazithromycin )
-De(dimethylamino)- -
0.76 1.5 0.50
Oxoazithromycin
Azserythromycin Ah,j 0.83 --- ---
Specified unidentified impurityh,k 0. --- ---
Azithromycin 1.0
2-Desethyl-2-propylazithromycinh,I 1.23 --- ---
-N-Demethyl- -N-[(4-
1.26 --- ---
methylphenyl)sulfonyl]azithromycinh,m
3-Deoxyazithromycin (azithromycin B) 1.31 --- ---
Any individual,unidentified impurity 1.0 0.20
Total degradation products 3.5

286
Procedure
Mobile phase
Solution A
1.Weigh disodium hydrogen phosphate dihydrate 1.8 g
2.transfer to volumetric flask 1,000 ml
3.Add water to 1,000 ml
4.dissole the solution completely
5.Adjust with dilute phosphoric acid to pH 8.9
6. Filtrate with filter paper pore size 0.45 µm under vacuum
7. Sonicate 15 min

Solution B
1.Measure acetonitrile 750 ml in volumetric flask 750 ml
2.Measure methanol 250 ml in volumetric flask 250 ml
3. Mix acetonitrile and methanol in solvent reservoir
4. Filtrate with filter paper pore size 0.45 µm under vacuum
5. Sonicate 15 min

Buffer
1.Weigh ammonium dihydrogen phosphate dihydrate 1.73 g
2.transfer to volumetric flask 1,000 ml
3.Add water to 1,000 ml
4.dissolve the solution completely

287
5.Adjust with dilute phosphoric acid to pH 10.0 0.05
6.Filtrate with filter paper pore size 0.45 µm under vacuum

Diluent
1. Measure acetonitrile 300 ml in volumetric flask 300 ml
2. Measure methanol 350 ml in volumetric flask 350 ml
3. Measure buffer 350 ml in volumetric flask 350 ml
4. Mix acetonitrile methanol and buffer in solvent reservoir

System suitability solution :

- 0.015 mg/mL of USP Azithromycin Related Compound F RS and 0.025 mg/ml of USP
Desosaminylazithromycin RS in Diluent
1. Weight 0.015 g of USP Azithromycin Related Compound F RS , transfer to 100 mL
Volumetric flask
2. Adjust with diluent to 100 mL
3. Pipette 1 ml transfer to 10 ml volumetric flask
4. Adjust to 10 ml with diluent
5. Sonicate 15 minutes
6. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial
- 0.025 mg/mL of USP Desosaminylazithromycin RS in Diluent
1. Weight 0.025 g of USP Azithromycin Related Compound F RS , transfer to 100 mL
Volumetric flask
2. Adjust with diluent to 100 mL
3. Pipette 1 ml transfer to 10 ml volumetric flask
4. Adjust to 10 ml with diluent
5. Sonicate 15 minutes
6. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial
Standard solution : 0.04 mg/mL of USP Azithromycin RS in Solution

288
 1. Weight 40 mg of USP Azithromycin RS , transfer to 100 mL Volumetric flask
2. Adjust with solution to 100 mL
3. Pipette 1 mL, transfer to 10 mL Volumetric flask
4. Sonicate 15 minutes
5. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial
Sample solution : Nominally 4.0 mg/mL solution of Azithromycin in Solution
1. Weight 2 g of Azithromycin powder for oral suspension, transfer to 50 mL in
Volumetric flask and adjust with water to 50 mL
2. Pipette 10 mL of Azithromycin oral suspension, transfer to 100 mL Volumetric
flask
3. Add diluent to 70 mL
4. Sonicate 15 minutes
5. Adjust with diluent to 100 mL
6. Centrifuge 15 minutes, filter the supernatant liquid transfer to vial
Chromatographic system
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Column temperature: 60°
Flow rate: 1 mL/min
Injection volume: 50 µL

289
Procedure
1. Equilibrate the column and detector with mobile phase at the specified flow rate
until a constant signal is received.
2. Injection a sample through the injection, or use an auto sample.
3. Begin the gradient program.
4. Record the chromatogram.
5. Analyze as directed in the monograph.

Retention time of impurities

a = Azithromycin N- -(N-N-Didemethyl)- -N- - N.N-
Dimethyl azithromycin aminoazithromycin , d = Azithromycin related compound F, e =
Desosaminylazithromycin, f = N-Demethylazithromycin,, g = Azithromycin

290
System suitability
Samples: System suitability solution and Standard solution
Table 72 System suitability solution and Standard solution
Name Suitability requirement Result
Resolution NLT 1.5 between 1.6
desosaminylazithromycin
and azithromycin related
compound F, System suitability
solution

Relative standard deviation NMT 5.0%, Standard solution 2.0%

Analysis
Samples: Standard solution and Sample solution Calculate the percentage of each
impurity in the portion of Azithromycin for oral suspension taken:
Result = (ru/rs)x (Cs/Cu) x P x F1 x (1/F2) x 100
ru = peak response of each impurity from the Sample solution
rs = peak response of azithromycin from the Standard solution
Cs = concentration of USP Azithromycin RS in the Standard solution (mg/mL)
Cu = concentration of Azithromycin in the Sample solution (mg/mL)
P = potency of USP Azithromycin RS (ug/mg of azithromycin)
F1= conversion factor, 0.001 mg/ug.
F2= relative response factor (see Table 2)
Acceptance criteria: See Table 11. Disregard peaks at relative retention times before 0.29
and after 1.31

291
Table 73 Organic impurities Azithromycin oral suspension
Relative Relative
Acceptance
Name Retention Response
Criteria, NMT (%)
Time Factor
Azithromycin N-oxide 0.29 0.43 0.50
-(N-N-Didemethyl)- -N-formyl
0.37 1.7 0.50
azithromycin
- N.N-Dimethyl
0.43 1.0 0.50
azithromycin aminoazithromycin
Azithromycin related compound F 0.51 3.8 0.50
Desosaminylazithromycin 0.54 1.0 0.30
N-Demethylazithromycin 0.61 1.0 0.50
-O-
0.73 --- ----
demethylazithromycin )
-De(dimethylamino)- -
0.76 1.5 0.50
oxoazithromycin
Azserythromycin A 0.83 --- ---
Specified unidentified impurity 0. --- ---
Azithromycin 1.0
2-Desethyl-2-propylazithromycin 1.23 --- ---
-N-Demethyl- -N-[(4-
1.26 --- ---
methylphenyl)sulfonyl]azithromycin
3-Deoxyazithromycin (azithromycin B) 1.31 --- ---
Any individual,unidentified impurity 1.0 0.20
Total degradation products 3.5

292
Calculate
Result = (ru/rs)x (Cs/Cu) x P x F1 x (1/F2) x 100

= 0.27

6. Specific test
6.1 pH
Following pH <791> USP42

INTRODUCTION
For compendial purposes, pH is defined as the value given by a suitable, properly
calibrated, potentiometric sensor and measuring system.
The measuring system has traditionally been referred to as the "pH meter." While the
pH meter is still in common use, the measuring system can also be embedded inside the pH
sensor, and the pH signal can be transmitted digitally to an external device such as a computer,
Programmable Logic Controller (PLC), Distributed Control System (DCS), data acquisition
system, terminal, or other microprocessor-controlled device. By definition, pH is equal to -log10
[aH+] where aH+ is the activity of the hydrogen (H+) or hydronium ion (H3O+), and the hydrogen
ion activity very closely approximates the hydrogen ion concentration.
The practical pH scale is defined:
pH= pH5+ [(E-Es)/k]
E = measured potential where the galvanic cell contains the solution under test (pH)
Es = measured potential where the galvanic cell contains the appropriate buffer solution for
calibration (pHs)
K = change in potential/unit change in pH and is derived from the Nernst equation (as follows)
k = loge (10) x (RT/nF)
R = 8.314 J/mole/ °K
T = temperature (°K)
293
For a solid packaged in multiple-unit containers
Sample: The suspension constituted as directed in the labeling

Acceptance criteria: 8.5-11.0

Procedure
1. Calibration pH sensor
2. Rinse the pH sensor with water, then with a few portion of sample 3 sample 3 time
3. Record the pH value

6.2 Microbiological test

6.2.1 Micro enumeration test
Following Micro enumeration test <61> USP 42
Procedure

Preparation of Test Strains (90 cfu)

1. Prepare inoculum (Table1) and buffered sodium chloride-peptone solution pH 7.0 to
make a suspension
2. Compare the suspension to the McFarland turbidity standard (no.3 McFarland
turbidity standard)
3. Prepare the sample with serial dilution method (1 in 10 dilution) using buffered
sodium chloride-peptone solution pH 7.0 as diluent
4. Repeat 6 times to make 90 cfu/mL
5. Suspension (use within 2 hours or within 24 hours if stored between 2o to 8o

294
 Negative control
1. Soybean-casein digest broth and soybean-casein digest agar or sabouraud
dextrose agar
2. Using a separate portion/plate of medium for each
3. Incubate as same as table 1
4. Check colonies on each medium
Acceptance criteria : No growth of microorganisms

Growth Promotion of the Media

1. Soybean-casein digest broth and soybean-casein digest agar
2. Add microorganisms (Table 1) 90 cfu
2. Using a separate portion/plate of medium for each
3. Compare number of colonies on previously approved medium to number on new
Acceptance criteria : Growth obtained must not differ by a factor greater than 2 from the
calculated value for a standardized inoculum that must growing between 45 -180 cfu

Suitability of the Counting Method in Presence of product

1. Add to the sample prepared as directed in Nonfatty product insoluble in Water and
to a control a volume of the microbial suspension to obtain an 90 cfu inoculum.

295
 2. Recovery of microorganism of product perform pour-plate method in duplicate (two
petri-dishes).
3. Incubate the plate as indicate in Table 1 preparation using of test microorganisms
and use mean count as the result.
Acceptance criteria : Microorganisms grow in both control and in the presence of product.
A mean count on test organisms not differing by a factor greater than 2 from the value of
the control. Therefore, there are no effect of antimicrobial activity interfere in the
procedure.

Testing of product
Use 10 mL of the product (Azithromycin oral suspension 200g/5ml) to be examined.
Product is diluted to overcome antimicrobial properties and tested by plate count method.
Sample Serial dilution preparation
1. Prepare the sample with serial dilution method (1 in 10 dilution) using Soybean-
Casein Digest Broth as diluent.
2. The procedure began when 1 ml of the sample is added to 9 ml of diluent and
mixed (Creating 10-1 dilution)
3. Then, 1 mL from that mixture is added to 9 mL of the same diluent, and it is mixed
(Creating 10-2 dilution).
4. Continue the procedure as plate-count methods.

Plate-count method: Pour-plate method

Prepare for each medium at least two petri dishes for each level of dilution. Take the
arithmetic mean of the counts per medium, and calculate the number of cfu.
Determination of Total aerobic microbial count (TAMC)
1. For Petri dishes 9 cm in diameter, add to the dish 1 ml of the diluted sample.
Fill the plate with 20 ml of Soybean-Casein Digest Agar.
2. Incubate the plates of Soybean-Casein Digest Agar at 30o-35o C for 3 to 5
days.
3. Select the plates corresponding to a given dilution and showing the highest

296
 number of colonies less than 250 for TAMC.
4. Take the arithmetic mean per culture medium of the counts and calculate
the number of cfu per ml of product.
Determination of Total combined yeasts and molds count (TYMC)
1. For Petri dishes 9 cm in diameter, add to the dish 1 ml of the diluted sample.
Fill the plate with 20 ml of Sabouraud Dextrose Agar.
2. Incubate the plates of Sabouraud Dextrose Agar at 20 o-25 o for 5 to 7 days.
3. Select the plates corresponding to a given dilution and showing the highest
number of colonies less than 50 for TYMC.
4. Take the arithmetic mean per culture medium of the counts and calculate
the number of cfu per ml of product.
Acceptance criteria : Total Aerobic Microbial Count less than 102 cfu/g or cfu/mL
Total Combined yeasts/Molds Count less than 10 cfu/g or cfu/ml

6.2.1 Test for specified microorganisms

Following Test for specified microorganisms <62> USP 42
Procedure

Preparation of Test Strains (100 cfu)

1. Prepare inoculum (E.coli ATCC 8739) and add Buffered Sodium Chloride-Peptone
Solution pH 7.0 to make a suspension.
2. Compared the suspension to the McFarland turbidity standard (no.3 McFarland
turbidity standard)
3. Prepare the sample with serial dilution method (1 in 10 dilution) using add Buffered
Sodium Chloride-Peptone Solution pH 7.0 as diluent.
4. Repeat 6 time to make 100 cfu/mL
5. Suspension (use within 2 hour or within 24 hours if store between 2o to 8o C)
Growth Promotion of the Media
1. Soybean-casein digest broth or MacConkey broth and MacConkey agar
2. Add Escherichia coli (ATCC 8739) 100 cfu

297
 3. Using a separate portion/plate of medium for each
- Soybean-casein digest broth: incubate at 30o - 35o C for 18-24 hr.
- MacConkey broth: incubate at 42o 44o C for 24-48 hr.
- MacConkey agar: incubate at 30o - 35o C for 18-72 hr.
4. Compare number of colonies on previously approved medium to number on new
medium.
Acceptance criteria : Growth obtained must not differ by a factor greater than 2 from the
calculated value for a standardized inoculum that must growing between 50-200 cfu

Negative control
- Soybean-casein digest broth: incubate at 30 o - 35 o C for 18-24 hr.
- MacConkey broth: incubate at 42 o - 44 o C for 24-48 hr.
- MacConkey agar: incubate at 30 o - 35 o C for 18-72 hr.
Acceptance criteria : No growth of microorganisms

Positive control
1. Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be
examined as described in Microbiological Examination Of Nonsterile Product:
Microbial Enumeration Tests <61>. Under Sample Serial dilution preparation
2. Use 1 ml of the diluted sample, Add microorganisms (E.coli ATCC 8739) from Flow
chart and mix with Soybean-Casein Digest Broth 20 ml to inoculate a suitable
amount (determined as described under Suitability of the Test Method)
3. Incubate at 30 o-35 o C for 18-24 hr.
4. Shake the container, transfer 1 mL of Soybean-Casein Digest Broth to 100 mL of
MacConkey Broth
5. Incubate at 42 o-44 o C for 24-48 hr.
6. Subculture on a plate of MacConkey Agar and Incubate at 30 o-35 o C for 18-72 hr.
Acceptance criteria : Under ambient light, Escherichia coli should appear as pink to red
colonies. And If pink colonies are seen that fluoresce blue-green under UV light (366 nm),
it is a presumptive identification for Escherichia coli.

298
 Testing of product
1. Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be
examined as described in Microbiological Examination of Nonsterile Product:
Microbial Enumeration Tests <61>. Under Sample Serial dilution preparation
2. Use 1 ml of the diluted sample and mix with Soybean-Casein Digest Broth 20 ml to
inoculate a suitable amount (determined as described under Suitability of the Test
Method)
3. Incubate at 30 o-35 o C for 18-24 hr.
4. Shake the container, transfer 1 mL of Soybean-Casein Digest Broth to 100 mL of
MacConkey Broth
5. Incubate at 42 o-44 o C for 24-48 hr.
6. Subculture on a plate of MacConkey Agar and Incubate at 30 o- 35 o C for 18-72 hr.
Acceptance criteria : Absence of Escherichia coli.
Table 74 Result of microbiological test
Acceptance Result day 5
Test Pour-plate method Remarks
Criteria (cfu/ml) (cfu/ml)
Total Aerobic Microbial Incubate the plates of 102 <10 Pass
Count (TAMC) Soybean-Casein Digest
Agar at 30o-35o C for 5
days.
Total Combined Incubate the plates of 101 <10 Pass
yeasts/Molds Count Sabouraud Dextrose
(TYMC) Agar at 20 o-25 o 5
days.
Specified
Microorganisms:
- Escherichia coli Incubate the plates of Absence of Absence Pass
MacConkey Agar at 30 Escherichia coli
o- 35 o C for 18-72 hr. (1 ml)

299
 The result of microbiological test of 5 day are less than 102 cfu/mL of Total Aerobic
Microbial, less than 10 cfu/ml of Total Combined yeasts/Molds and Absence of Escherichia
coli. Based on the resulting data the shelf life established for five day.

The reasons for not testing the microbial in the dry powder form are our factory has
passed GMP standards, An assure the products are quality , safety compliant defined
standards. And the products are consistent in every production lot. Having control and
maintaining cleanliness at every step of production.
6.3 Uniformity of dosage unit
Following Uniformity of dosage unit <905> USP 42
Uniformity of dosage unit <905>
Weight variation
Carry out an assay for the drug substance(s) on a representative sample of the batch
using an appropriate analytical method. This value is result A, expressed as percentage of
label claim (see Calculation of Acceptance Value). Assume that the concentration (weight of
drug substance per weight of dosage unit) is uniform. Select not fewer than 30 dosage units,
and proceed as follows for the dosage form designated
Hard Capsules
Accurately weigh 10 capsules individually, taking care to preserve the identity of each
capsule. Remove the contents of each capsule by a suitable means. Accurately weigh the
emptied shells individually, and calculate for each capsule the net weight of its contents by
subtracting the weight of the shell from the respective gross weight. Calculate the drug
substance content of each capsule from the net weight of the individual capsule content and
the result of the Assay. Calculate the acceptance value
Solid Dosage Forms Other Than Tablets and Capsules
Proceed as directed for Hard Capsules, treating each unit as described therein.
Calculate the acceptance value

300
Procedure
1. Select Azithromycin oral suspension not fewer than 30 bottle.
2. Then, select 10 bottle from 30 bottle.
3. Accurately weigh 10 bottle individually and accurately record the weight of each bottle.
4. Remove the contents of each capsule by a suitable means.
5. Accurately weigh the emptied shells individually and calculate for each bottle the net
weight of its contents by subtracting the weight of the empty bottle from the respective gross
weight.
6. Calculate the drug content of each bottle from the net weight of the individual bottle
content and the result of the Assay. Calculate the acceptance value.
Acceptance criteria : The requirements for dosage uniformity are met if the
acceptance value of the first 10 dosage units is less than or equal to L1%. If the acceptance
value is > L1%, test the next 20 units, and calculate the acceptance value.

301
P5.3 Verification of analysis procedure
Type of Analytical Procedure to be validated
The discussion of the validation of analytical procedures is directed to the four most
common types of analytical procedures:
- Identification tests.
- Quantitative tests for impurities content.
- Limit tests for the control of impurities.
- Quantitative tests of the active moiety in samples of drug substance or drug product
or other selected component(s) in the drug product.

Table 76 ICHQ2(R1) Common type of Analytical procedures

Type of TESTING FOR ASSAY

analytical IMPURITIES - dissolution
IDENTIFICATION
procedure (measurement only)
Quantitative Limit test - content / potency
characteristic
Accuracy - + - +
Precision
- Repeatability - + - +
- inter. precision - + (1) - + (1)
Specificity (2) + + + +
Detection Limit - - (3) + -
Quantitation Limit - + - -
Linearity - + - +
Range - + - +
- signifies that this characteristic is not normally evaluated
+ signifies that this characteristic is normally evaluated

302
(1) in cases where reproducibility (see glossary) has been performed, intermediate precision is not
needed
(2) lack of specificity of one analytical procedure could be compensated by other supporting analytical
procedure(s)
(3) may be needed in some cases

All analytical procedures follow USP42/NF37, therefore validation of analytical

procedures is not necessary but analytical procedures need verification.
1. Identification test
Table 77 Method verification of Identification test Azithromycin oral suspension
Test Parameter Method Validation Acceptance criteria
Identification Specificity Test by HPLC method, Run Negative result no
placebo, Injection of the positive peak response
result contain Azithromycin present at the same
compare with negative result do retention time as
not contain Azithromycin positive result.

2. Assay
Table 78 Method verification of assay Azithromycin oral suspension
Test Parameter Method Validation Acceptance criteria
Assay Specificity Test by HPLC method, spike azithromycin RS No interference of peak in
with placebo, Azithromycin oral suspension, the region of
[Check chromatogram of each individual Azithromycin chromatogram
solution by photodiode array detector]
Linearity Linearity was evaluated by HPLC method, use - Linear relationship
and range five concentrations of test solution and spike between peak response and
azithromycin RS with placebo in a range of concentration
0.30-0.80 mg/ml. Concentrations as follows: - Coefficient of
0.30, 0.40, 0.60, 0.70, 0.80 mg/ml and repeat determination (R2) 0.999
three times plot calibration curve

303
 Accuracy Test by HPLC method, Use the results from 3 %Recovery= 98.0%- 102.0%
concentration/3 replicates each of the
Azithromycin sample solution, spike
azithromycin RS with placebo. (9 stimulated
sample) Three concentration as follows:
0.40,0.60 and 0.80 mg/ml to calculate
percent recovery
Precision Test by HPLC method,
Repeatability:
Azithromycin test, spike azithromycin RS with
placebo in three concentrations (0.40,0.60,
0.80 mg/ml) and replicate three times under
the same conditions at different times in the
same day. (0, 6 and 12 hours)
Intermediate precision:
Same procedure as repeatability but test in
day 1, 3 and 6

Specificity
Result: No interference of peak in the region of Azithromycin chromatogram

Chromatogram of Azithromycin RS with placebo

304
 Chromatogram of Azithromycin oral suspension
Linearity and range
Acceptance criteria : Linearity was evaluated by HPLC method, use five concentrations of
test solution in a range of mg/ml. Concentrations as follows: 0.30 0.40 0.60, 0.70 0.80 mg/ml
and repeat three times
Result: The peak area of Azithromycin within the concentration 0.30, 0.40, 0.60, 0.70 and
0.80 mg/ml has linear relationship (visual inspection) with coefficient of determination
(R2) = 0.9999
Table 79 Result of Linearity and Range assay Azithromycin oral suspension

Nominal Average
Concentration Peak area
Sample Concentration of Peak SD % RSD
mg/ml
(mg/ml) area
1.1 0.30 100547
1.2 0.30 0.30 101055 101395 866 0.9
1.3 0.30 102584
2.1 0.40 117208
2.2 0.40 0.40 119854 117642 1656 1.4
2.3 0.40 115864

305
 3.1 0.60 148022
1864
3.2 0.60 0.60 152514 150031 1.2
3.3 0.60 149557
4.1 0.70 169541
4.2 0.70 0.70 167922 167531 1822 1.1
4.3 0.70 165130
5.1 0.80 185224
5.2 0.80 0.80 181250 184006 1953 1.1
5.3 0.80 185544

Linearity of Assay
200000
Peak area (mAU*min)

150000

100000
y = 166771x + 50662
50000 R² = 0.9999

0
0 0.2 0.4 0.6 0.8 1
Concentration (mg/ml)

Accuracy
Acceptance criteria: %Recovery 98.0%- 102.0% of each concentration (0.40, 0.60, and 0.80
mg/ml)

%recovery = × 100
Result : Mean of %Recovery of the concentration at 0.40 , 0.60 , 0.80 mg/ml is 100.3%,
100.2%, 100% respectively
Table 80 Result of Accuracy assay Azithromycin oral suspension

Nominal Average
Nominal amount of
Concentration amount of % of %
Sample Concentration azithromycin
(mg/ml) azithromycin Recovery
(mg/ml) (mg) recovery
(mg)

306
1.1 0.40 120 119 99.2
1.2 0.40 0.40 120 120 100.0 100.3
1.3 0.40 120 122 101.7
2.1 0.60 180 180 100.0
2.2 0.60 0.60 180 180 100.0 100.2
2.3 0.60 180 181 100.5
3.1 0.80 240 240 100.0
3.2 0.80 0.80 240 240 100.0 100.0
3.3 0.80 240 240 100.0

Precision
Repeatability Precision
Result: %RSD of Azithromycin with the concentration 0.40, 0.60, 0.80 mg/ml in repeatability
are less than 2%

%RSD = × 100 ; is Average amount found at same

Concentration

307
Table 81 Result of Repeatability Precision assay Azithromycin oral suspension

Nominal Average of
Concentration Peak area
Time Concentration Peak area SD % RSD
mg/ml
(mg/ml)
0 0.40 117208
hours 0.40 0.40 119854 101395 866 0.9
0.40 115864
0.60 148022
0.60 0.60 152514 117642 1656 1.4
0.60 149557
0.80 185224
0.80 0.80 181250 184006 1953 1.1
0.80 185544
6 0.40 113541
hours 0.40 0.40 115479 114855 929 0.8
0.40 115544
0.60 152495
0.60 0.60 155247 154519 1450 0.9
0.60 155814
0.80 185732
0.80 0.80 189504 187137
1684 0.8
0.80 186174
12 0.40 113824
hours 0.40 0.40 115500 115155 977 0.8
0.40 116142
0.60 158854
0.60 0.60 157249 157050 1561 0.9
0.60 155047
0.80 183800
0.80 0.80 187204 183800 1908 1.0
0.80 188276

308
 Intermediate Precision
Result:%RSD of Azithromycin with the concentration 0.40 0.60, 0.80 mg/ml in repeatability are
less than 2%
Table 82 Result of Intermediate Precision assay Azithromycin oral suspension

Nominal
Concentration Peak area %
Day Concentration Average of Peak area SD
mg/ml RSD
(mg/ml)
1 0.40 117208
0.40 0.40 119854 101395 866 0.9
0.40 115864
0.60 148022
0.60 0.60 152514 117642 1656 1.4
0.60 149557
0.80 185224
0.80 0.80 181250 184006 1953 1.1
0.80 185544
3 0.40 115502
0.40 0.40 117577 115929 1210 1.0
0.40 114707
0.60 152277
0.60 0.60 157703 155662 1534 0.9
0.60 154005
0.80 189327
0.80 0.80 185207 186602 1927 1.0
0.80 185272
6 0.40 116520
0.40 0.40 115507 116711 1070 0.9
0.40 118107
0.60 155403
0.60 0.60 152078 153186 1568 1.0
0.60 152076
0.80 0.80 183707 185595 1686 0.9

309
 0.80 187801
0.80 185277
2. Performance Test
Table 83 Method verification of Performance Test Azithromycin oral suspension
Test Parameter Method Validation Acceptance criteria
Performance Specificity Test by HPLC method, spike Azithromycin RS No interference of peak in
Test <711> with placebo, Azithromycin oral suspension. the region of
Dissolution [Check chromatogram of each individual Azithromycin
solution by photodiode array detector] chromatogram
Accuracy Test by HPLC method, Use the results from 3 %Recovery= 98.0%-
concentration/3 replicates each of the 102.0%
Azithromycin sample solution, spike
azithromycin RS with placebo. (9 stimulated
sample) Three concentration as follows:
0.11,0.22 and 0.33 mg/ml in Sodium
phosphate buffer 900 ml, calculate percent
recovery
Linearity Linearity was evaluated by HPLC method, - Linear relationship
and range use five concentrations of test solution and between peak response
spike azithromycin RS with placebo in a range and concentration
of 0.11- 0.33 mg/ml. Concentrations as - Coefficient of
follows: 0.11, 0.15, 0.22, 0.27, 0.33 mg/ml and determination (R2) 0.999
repeat three times and plot calibration curve
Precision Test by HPLC method,
Repeatability:
Azithromycin test, spike azithromycin RS with
placebo in three concentrations (0.11,0.22
and 0.33 mg/ml) in Sodium phosphate buffer
900 ml and replicate three times under the
same conditions at different times in the
same day. (0, 6 and 12 hours)
Intermediate precision:

310
 Same procedure as repeatability but test in
day 1, 3 and 6

Specificity
Result: No interference of peak in the region of Azithromycin chromatogram

Chromatogram of Azithromycin RS with placebo

Chromatogram of Azithromycin oral suspension

Linearity and range
Acceptance criteria: Linearity was evaluated by HPLC method, use five concentrations of

311
test solution in a range of mg/ml. Concentrations as follows: 0.11, 0.15, 0.22, 0.27 and 0.33
mg/ml and repeat three times
Result: The peak area of Azithromycin within the concentration 0.11, 0.15, 0.22, 0.27 and
0.33 mg/ml has linear relationship (visual inspection) with coefficient of determination
(R2) = 0.9999
Table 84 Result of Linearity and Range performance test Azithromycin oral suspension

Nominal Average
Concentration Peak area
Sample Concentration of Peak SD % RSD
mg/ml
(mg/ml) area
1.1 0.11 86204
1.2 0.11 0.11 86204 85987 375 0.4
1.3 0.11 85553
2.1 0.15 99739
2.2 0.15 0.15 100544 100180 407 0.4
2.3 0.15 100257
3.1 0.22 126208
3.2 0.22 0.22 126854 126462 344 0.3
3.3 0.22 126324
4.1 0.27 145022
4.2 0.27 0.27 144514 144561 439 0.3
4.3 0.27 144147
5.1 0.33 167041
5.2 0.33 0.33 167922 167531 448 0.3
5.3 0.33 167630
200000
Linearity of Dissolution
Peak area (mAU*min)

150000
100000
y = 370691x + 44875
50000 R² = 0.9999
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
Concentration (mg/ml)

312
 Accuracy
Acceptance criteria: %Recovery 98.0%- 102.0% of each concentration (0.11, 0.22, and 0.33
mg/ml)

%Recovery = × 100
Result : Mean of %Recovery of the concentration at 0.11 , 0.22 , 0.33 mg/ml is 100%
Table 85 Result of Accuracy performance test Azithromycin oral suspension

Nominal Average
Nominal amount of
Concentration amount of % of %
Sample Concentration azithromycin
(mg/ml) azithromycin Recovery
(mg/ml) (mg) recovery
(g)
1.1 0.11 0.6 0.6 100.0
1.2 0.11 0.11 0.6 0.6 100.0 100.0
1.3 0.11 0.6 0.6 100.0
2.1 0.22 1.2 1.2 100.0
2.2 0.22 0.22 1.2 1.2 100.0 100.0
2.3 0.22 1.2 1.2 100.0
3.1 0.33 1.8 1.8 100.0
3.2 0.33 0.33 1.8 1.8 100.0 100.0
3.3 0.33 1.8 1.8 100.0

Precision
Repeatability Precision
Result: %RSD of Azithromycin with the concentration 0.11 0.22, 0.33 mg/ml in repeatability
are less than 2%

313
%RSD = × 100 ; is Average amount found at same
Concentration

Table 86 Result of Repeatability Precision performance test Azithromycin oral suspension

Nominal Concentratio Average

Peak area
Time Concentratio n of Peak SD % RSD
n (mg/ml) mg/ml area
0 0.11 86204
hours 0.11 0.11 86204 85987 375 0.4
0.11 85553
0.22 126208
0.22 0.22 126854 126462 344 0.3
0.22 126324
0.33 167041
0.33 0.33 167922 167531 448 0.3
0.33 167630
6 0.11 86504
hours 0.11 0.11 86854 86779 246 0.3
0.11 86980
0.22 113541
0.22 0.22 115479 114855 929 0.8
0.22 115544
0.33 152495
0.33 0.33 155247 154519 1450 0.9
0.33 155814
12 0.11 86040
hours 0.11 0.11 86570 86440 354 0.5
0.11 86712
0.22 0.22 113824 115155 977 0.8

314
 0.22 115500
0.22 116142
0.33 158854
0.33 0.33 157249 157050 1561 0.9
0.33 155047

Intermediate Precision
Result:%RSD of Azithromycin with the concentration 0.11 0.22, 0.33 mg/ml in repeatability are
less than 2%
Table 87 Result of Intermediate Precision performance test Azithromycin oral suspension

Nominal
Concentration Peak area %
Day Concentration Average of Peak area SD
mg/ml RSD
(mg/ml)
1 0.11 86204
0.11 0.11 86204 85987 375 0.4
0.11 85553
0.22 126208
0.22 0.22 126854 126462 344 0.3
0.22 126324
0.33 167041
0.33 0.33 167922 167531 448 0.3
0.33 167630
3 0.11 85716
0.11 0.11 86435 85752 664 0.7
0.11 85107
0.22 115502
0.22 0.22 117577 115929 1210 1.0
0.22 114707
0.33 152277
0.33 0.33 157703 155662 1534 0.9
0.33 154005

315
6 0.11 86547
0.11 0.11 86504 86172 611 0.7
0.11 85467
0.22 115020
0.22 0.22 115207 11524 200 0.2
0.22 115507
0.33 15403
0.33 0.33 152078 153185 1567 1.0
0.33 152076
3. Impurities
Organic impurity see on table 1 organic impurities
Table 88 Method verification of Impurities Azithromycin oral suspension

Test Parameter Method Acceptance criteria

Impurities Specificity Tested by HPLC method, - No interference of
Spike Azithromycin standard peak in the region of
with placebo, Azithromycin Azithromycin
sample, solvent, organic chromatogram
Impurities [Check
chromatogram of each
individual solution by
photodiode array detector]

Linearity Linearity was evaluated by - Linear relationship

and range HPLC method, prepare between peak
organic impure test solution, response and
spike Azithromycin RS with concentration
placebo use five - Coefficient of
concentrations in a range of determination (R2)
0.02-0.06 mg/ml. 0.999
Concentrations as follows:
0.02, 0.03, 0.04, 0.05, 0.06
mg/ml and repeat three

316
 times and plot calibration
curve

Accuracy Tested by HPLC method, %Recovery 98.0-

prepare organic impure 102.0 %
Azithromycin test solution,
spike Azithromycin RS with
placebo three concentration
(0.02, 0.04, 0.05 mg/ml) and
repeat three time
Precision Repeatability:
Organic impurities
Azithromycin oral suspension
test solution, Azithromycin
RS with placebo in three
concentrations (0.02, 0.04,
0.05 mg/ml) and replicate
three times under the same
conditions at different times
in the same day. (0, 6 and 12
hours)
Intermediate precision:
Same procedure as
repeatability but test in day
1, 3 and 6
Quantitation Test by HPLC method, Base Signal to noise ratio
Limit on Signal-to-Noise (S/N) 10:1

317
 -Prepare a test sample which
contains organic impure
, apply to HPLC. Repeat ten
times
Specificity
Result: No interference of peak in the region of Azithromycin oral suspension
chromatogram

Chromatogram of the Azithromycin oral suspension standard solution with placebo

Chromatogram of the Azithromycin oral suspension sample solution

318
 b
c
a de f

Chromatogram of the Azithromycin oral suspension with organic impurities; a = Azithromycin

N- -(N-N-Didemethyl)- -N- - N.N-Dimethyl
azithromycin aminoazithromycin , d = Azithromycin related compound F, e =
Desosaminylazithromycin, f = N-Demethylazithromycin,, g = Azithromycin

Linearity and range

Acceptance criteria: Linearity was evaluated by HPLC method, use five concentrations of
test solution in a range of mg/ml. Concentrations as follows: 0.02 0.03 0.04, 0.05 0.06 mg/ml
and repeat three times
Result: The peak area of Azithromycin within the concentration 0.02 0.03 0.04, 0.05 and 0.06
mg/ml has linear relationship (visual inspection) with coefficient of determination
(R2) = 0.9992
Table 89 Result of Linearity and Range impurities Azithromycin oral suspension

Nominal Average
Concentration Peak area
Sample Concentration of Peak SD % RSD
mg/ml
(mg/ml) area
1.1 0.02 0.02 31 31 0.0 0.0

319
1.2 0.02 31
1.3 0.02 31
2.1 0.03 41
2.2 0.03 0.03 41 41 0.0 0.0
2.3 0.03 41
3.1 0.04 52
3.2 0.04 0.04 52 52 0.0 0.0
3.3 0.04 52
4.1 0.05 61
4.2 0.05 0.05 61 61 0.0 0.0
4.3 0.05 61
5.1 0.06 71
5.2 0.06 0.06 71 71 0.0 0.0
5.3 0.06 71

Linearity of Impurity
80
Peak area (mAU*min)

60

40
y = 1000x + 11.2
20
R² = 0.9992
0
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
Concentration (mg/ml)

320
 Accuracy
Table 90 Result of Accuracy impurities Azithromycin oral suspension

Sample Nominal Concentration Nominal amount of % Recovery Average

Concentration (mg/ml) amount of organic of %
(mg/ml) organic impurities recovery
impurities (mg)
(mg)
1.1 0.02 20 20 100
1.2 0.02 0.02 20 20 100 101.6
1.3 0.02 20 21 105
2.1 0.04 40 40 100
2.2 0.04 0.04 40 40 100 98.3
2.3 0.04 40 38 95
3.1 0.05 50 50 100
3.2 0.05 0.05 50 50 100 101.3
3.3 0.05 50 52 104
Precision
Result: %RSD of organic impurities with the concentration 0.02, 0.04 and 0.05 mg/ml in
repeatability are less than 2.0%
Table 91 Result of Precision impurities Azithromycin oral suspension

Nominal Average
Concentration Peak area
Time Concentration of Peak SD % RSD
mg/ml
(mg/ml) area
0 0.02 31
hours 0.02 0.02 31 31 0.0 0.0
0.02 31
0.04 52
0.04 0.04 52 52 0.0 0.0
0.04 52

321
 0.05 61
0.05 0.05 61 61 0.0 0.0
0.05 61
6 0.02 31
hours 0.02 0.02 32 32 0.47 1.5
0.02 32
0.04 52
0.04 0.04 50 51 0.94 1.8
0.04 52
0.05 63
0.05 0.05 62 63 0.47 0.8
0.05 63
12 0.02 31
hours 0.02 0.02 32 31 0.47 1.5
0.02 31
0.04 52
0.04 0.04 53 53 0.47 0.9
0.04 53
0.05 62
0.05 0.05 63 63 0.74 0.8
0.05 63

322
 Intermediate Precision
Result: %RSD of organic impurities with the concentration 0.02, 0.04 and 0.05 mg/ml in
Intermediate are less than 2.0%
Table 92 Result of Precision impurities Azithromycin oral suspension

Nominal Concentratio
Peak area Average of Peak %
Day Concentratio n SD
area RSD
n (mg/ml) mg/ml
1 0.02 31
0.02 0.02 31 31 0.0 0.0
0.02 31
0.04 52
0.04 0.04 52 52 0.0 0.0
0.04 52
0.05 61
0.05 0.05 61 61 0.0 0.0
0.05 61
3 0.02 35
0.02 0.02 34 35 0.47 1.4
0.02 35
0.04 55
0.04 0.04 55 54 0.94 1.7
0.04 53
0.05 63
0.05 0.05 63 63 0.74 0.8
0.05 64
6 0.02 35
0.02 0.02 35 34 0.47 1.4
0.02 34
0.04 55
0.04 0.04 55 55 0.47 0.9
0.04 56

323
 0.05 63
0.05 0.05 64 64 0.81 1.3
0.05 65

Quantitation Limit
Test by HPLC method, Base on Signal-to-Noise (S/N), Prepare a test sample which contains
organic impure , apply to HPLC. Repeat ten times. Used calculate the experiment signal to
noise ratio between 10:1 and a concentration of about 0.02 mg/ml will yield a signal to
noise ratio of 10:1
Limit of quantification (LOQ) = 0.02 mg/ml

324
Result of verification
Table 93 Result of verification Azithromycin oral suspension
Interpret
Test Parameter Acceptance criteria Result

Negative result no Negative result no peak Pass

peak response response present at the
Identification Specificity present at the same same retention time as
retention time as positive result
positive result.
Assay No interference of No interference of peak in Pass
peak in the region of the region of Azithromycin
Specificity
Azithromycin chromatogram
chromatogram
Linear relationship The peak area of
between peak Azithromycin within the
response and concentration 0.30, 0.40,
concentration 0.60, 0.70 and 0.80 mg/ml
Linearity
and coefficient of has linear relationship Pass
and range
determination (R2) (visual inspection) with
0.999 coefficient of
determination
(R2) = 0.9999
Mean of %Recovery of the Pass
%Recovery= 98.0%-
Accuracy concentration at 0.11 , 0.22
102.0%
, 0.33 mg/ml is 100%
Mean of %RSD in the Pass
concentration 0.4,0.6 and
Precision 0.8 of the 0 hour 6 hour
and 12 hour are 1.1 , 0.8
and 0.9 respectively
Mean of %RSD in the Pass
Intermedia
concentration 0.4,0.6 and
precision
0.8 of the day 1 , 3 and 6

325
 are 1.1 , 1.0 and 0.9
respectively
Performance Negative result no Negative result no peak Pass
test peak response response present at the
dissolution Specificity present at the same same retention time as
retention time as positive result.
positive result.
Linear relationship The peak area of
between peak Azithromycin within the
response and concentration 0.11, 0.15,
concentration 0.22, 0.27 and 0.33 mg/ml
Linearity
and coefficient of has linear relationship Pass
and range
determination (R2) (visual inspection) with
0.999 coefficient of
determination
(R2) = 0.9999
%Recovery= 98.0%- Mean of %Recovery of the Pass
Accuracy 102.0% concentration at 0.11 , 0.22
, 0.33 mg/ml is 100%
Mean of %RSD in the Pass
concentration 0.11,0.22
and 0.33 of the 0 hour 6
Precision
hour and 12 hour are 0.3 ,
0.7 respectively

Mean of %RSD in the Pass

concentration 0.11,0.22
Intermedia
and 0.33 of the day 1 , 3
precision
and 6 are 0.3 , 0.9 and 0.6
respectively
Impurities No interference of No interference of peak in Pass
peak in the region of the region of Azithromycin
Specificity
Azithromycin chromatogram
chromatogram

326
 The peak area of Pass
Linear relationship Azithromycin within the
between peak concentration 0.02 0.03
response and 0.04, 0.05 and 0.06 mg/ml
Linearity
concentration has linear relationship
and range
-Coefficient of (visual inspection) with
determination (R2) coefficient of
0.999 determination
(R2) = 0.9992
Mean of %Recovery of the Pass
concentration at 0.02
mg/ml is 101.6%, Mean of
%Recovery of the
%Recovery 99.0-
Accuracy concentration at 0.04
101.0 %
mg/ml is 98.3% and Mean
of % Recovery of the
concentration at 0.05
mg/ml is 101.3%
Mean of %RSD of organic Pass
impurities with the
concentration 0.02, 0.04,
Precision
0.05mg/ml of the 0 hour 6
hour and 12 hour are 0.0
,1.4 and 1.1 repeatability
Mean of %RSD of organic Pass
impurities with the
Intermedia concentration 0.02, 0.04,
precision 0.05mg/ml of the day 1,3
and 6 are 0.0 ,1.3 and 1.2
repeatability
Quantitation Pass
0.02 mg/ml 0.02 mg/ml
limit

327
 P 5.4 Batch analysis
Batch analysis
Table 94 Result of Batch analysis Azithromycin oral suspension
Date of Site of
Drug name Batch number Batch size Batch type
production production
O LEAVES
AZ200565081 500,000 bottles Production 10/02/2019
Azithromycin Pharmaceutical
powder for O LEAVES
AZ200565082 500,000 bottles Production 10/02/2019
oral Pharmaceutical
Suspension O LEAVES
AZ200565083 500,000 bottles Production 10/02/2019
Pharmaceutical

Batch analyzed
Table 95 Result of Batch analyzed Azithromycin oral suspension

Result
Test Specification
AZ200565081 AZ200565082 AZ200565083
White or off-white
Appearance Complies Complies Complies
crystalline powder
The retention time of
the Azithromycin peak
of the
Sample solution
Identification by HPLC Complies Complies Complies
corresponds to that of
the Standard
solution, as obtained in
the Assay.
Assay by HPLC 90.0%-110.0% 98.9% 101.2% 102.5%

328
 The average volume of
Performance test
liquid obtained from
- Deliverable Average volume:
the 10 containers is NLT Average volume: Average volume:
volume 109.96% No
100%, and the volume 112.17% No 110.47% No
container is less
of no container is less container is less container is less
than 95%
than than 95% than 95%
95% of the volume
declared in the
labeling.
- Dissolution Each unit is not less
Apparatus 2 than Q + 5% within 15 99-104 % 100-105 % 100-104 %
(Paddle Apparatus) minutes. (Q=93%)
Impurities See on table 33 Complies Complies Complies

Specific test 8.5-11.0 9.5 9.3 9.2

- pH (200 mg/5 ml in water) (200 mg/5 ml in water) (200 mg/5 ml in water)

- microbial limit -Total aerobic microbial <10 cfu/ml <10 cfu/ml <10 cfu/ml
A. Microbial count less than 102
enumeration test : cfu/ml <10 cfu/ml <10 cfu/ml <10 cfu/ml
Enumeration method -Total combined
Yeast/Molds count less
than 10 cfu/ml
B. Test for specified Absence of Escherichia Absence per mL Absence per mL Absence per mL
microorganism : Bile- coli
tolerant gram-negative
bacteria

329
Table 96 organic impurities Azithromycin oral suspension
Relative Relative
Acceptance
Name Retention Response
Criteria, NMT (%)
Time Factor
Azithromycin N-oxide 0.29 0.43 0.50
-(N-N-Didemethyl)- -N-formyl
0.37 1.7 0.50
azithromycin
- N.N-Didemethyl
0.43 1.0 0.50
azithromycin aminoazithromycin
Azithromycin related compound F 0.51 3.8 0.50
Desosaminylazithromycine 0.54 1.0 0.30
N-Demethylazithromycin 0.61 1.0 0.50
-O-
0.73 --- ----
demethylazithromycin )
-De(dimethylamino)- -
0.76 1.5 0.50
Oxoazithromycin
Azserythromycin A 0.83 --- ---
Specified unidentified impurity 0. --- ---
Azithromycin 1.0
2-Desethyl-2-propylazithromycin 1.23 --- ---
-N-Demethyl- -N-[(4-
1.26 --- ---
methylphenyl)sulfonyl]azithromycin
3-Deoxyazithromycin (azithromycin B) 1.31 --- ---
Any individual, unidentified impurity 1.0 0.20
Total degradation products 3.5

330
P5.5 Characterization of impurities
Following USP 42 or the procedure follows P5.2
Result
According to the criteria in the Acceptance criteria specified that the peakarea
of organic impurities in the sample solution must not be greater than the peak area
of the standard solution and when considering the chromatogram of all 5 sample
solutions, it was found that during the retention time period of 2.5 to 13.5 , found
of organic impurities peak was observed not more than the criteria. Therefore, it can
be concluded that meet the standard requirements of USP42

Chromatogram of Standard solution

331
 P 5.6 Justification of specification
Table 97 Justification of specification

Specification Justification
Azithromycin oral suspension will be examined by visual
Appearance
inspection such as color
The identification test identified by using HPLC method.
The requirements are show in the USP41 monograph is
Identification
used for identification active pharmaceutical ingredient is
Azithromycin
%Labelled amount of Azithromycin tablets is defined in
the USP 42 monograph is used for verify active
Assay
pharmaceutical ingredient consist Azithromycin in range
90.0-110.0%
Performance test
The acceptance value of deliverable volume is defined in
- Deliverable volume the USP41 general chapter <698> to confirm that oral
liquid, when transferred from the original container,
deliver the volume of dosage form that is declared on the
- dissolution label claim.
The acceptance limit of dissolution test is defined in the
USP 42 general chapter <711>
The acceptance limit of organic impurities is defined in the
monograph Azithromycin oral USP 42 to confirm that the
Impurities
product contains organic impurities that is of Azithromycin
not more than the criteria
Specific test
The range of pH is defined in the USP41 monograph <791>
- pH
to confirm pH of the product that important form and
solubility of Azithromycin.
microbial limit

332
A. Microbial enumeration test The acceptance criteria described in USP general chapter
Microbial Enumeration test <61> to ensure that a
preparation complies with pre-set specifications
for microbiological quality. The USP <61> test is a full
quantitative analysis of a product to determine the Total
Aerobic Microbial Count (TAMC)
and Total Yeast and Mold Count (TYMC) present in
sample.
B. Test for specified microorganism The acceptance criteria described in USP general chapter
Test for specified microorganism <62> to determine that
any microorganisms that may be present in a product are
not specific pathogenic microorganisms of particle concern

References
1. United States Pharmacopeial Convention. The United States pharmacopeia 42: the
national formulary 37: official from May 1, 2019. Rockville, MD: United States Pharmacopeial
Convention; 2018.
2. International Conference on Harmonization; "SPECIFICATIONS: TEST PROCEDURES AND
ACCEPTANCE CRITERIA FOR NEW DRUG SUBSTANCES AND NEW DRUG PRODUCTS: CHEMICAL
SUBSTANCES Q6A", 1995
3. Okaru AO. DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHIC METHOD
FOR THE ANALYSIS OF AZITHROMYCIN. [online]. 2013 [cited 2022 Jan 20]; [135 screen].
Available from: URL: http://erepository.uonbi.ac.ke/handle/11295/62628?show=full.

333
P6 Reference Standards
Table 98 Reference Standards
Reference Standards Source Lot No.
Azithromycin RS The United States R103C0
Pharmacopeial Convention
Azithromycin Related Compound F RS The United States R08580
Pharmacopeial Convention

Desosaminylazithromycin RS The United States R076H0

Pharmacopeial Convention

334
Reference
335
1. United State Pharmacopoeia 2019 USP 42- NF 37 pdf [Internet]. Pharmaceuticals Industry -
Web of Pharma. [cited 2022 Feb 24]. Available from:
https://www.webofpharma.com/2021/01/united-state-pharmacopoeia-2019-usp-42.html
2. Azithromycin RS [Internet]. The United States Pharmacopeial Convention. [cited 2022 Feb
24]. Available from: https://store.usp.org/product/1046056#
3. Azithromycin Related Compound F RS [Internet]. The United States Pharmacopeial
Convention. [cited 2022 Feb 24]. Available from: https://store.usp.org/product/1046045
4. Desosaminylazithromycin RS [Internet]. The United States Pharmacopeial Convention.
[cited 2022 Feb 24]. Available from: https://store.usp.org/product/1046078

P7 Container Closure system

336
 According to USP42 NF37 in Packaging and storage section of Azithromycin for Oral
Suspension monograph that require to preserve in tight containers. Therefore, the primary
packaging is polypropylene container with child resistant screw cap with a tamper evident
seal, the secondary packaging use kraft paper container and the tertiary packaging is carton
box.
The primary packaging
Primary packaging is the material that first envelops the product and holds it. This
usually is the smallest unit of distribution or use and is the package which is in direct
contact with the content. Polypropylene is an economical material that is suitable for
packaging liquid oral dosage form and that shape retention and fatigue resistance properties.
Polypropylene (PP) bottles are excellent barrier properties totally exclude moisture, solvent
and micro-organisms.
Weight: 13 mg
Height: 6 cm + 2 cm (cap)
Diameter: 3.5 cm

337
 Design of the primary packaging

Table 99 Primary packaging specification

Test Requirement Method

Appearance White and no foreign particles Visual inspection

Barrier properties The polypropylene bottle cannot be Water vapor and oxygen
approved the oxygen and moisture moisture. transmission test

Identification - Determine the infrared spectrum from - Infrared spectrophotometry

3800 cm-1 to 650 cm-1 (2.6-15 µm).
The specimen exhibits an absorption
spectrum that is substantially
equivalent to that of the USP
Homopolymer
Polypropylene RS. Substantial, as
opposed to exact, equivalence allows
for minor spectral differences arising
from the
natural compositional and/or physical

338
 variation among polymers of this
class. Substantial equivalence is
achieved
when all differences between the
sample and RS spectra can be
explained in the context of such
natural compositional and/or physical
variations.

- The melting peak temperature in the

thermal analysis curve does not differ
from that of USP Homopolymer
Polypropylene RS by more than 12.0 °.

- Differential scanning
calorimetry

339
Physicochemical - Absorbance (Solution S1): Maximum - Ultraviolet-Visible
Tests absorbance is 0.2 Spectroscopy

- Titration

- Acidity/alkalinity: NMT 1.5 mL of 0.01

N sodium hydroxide is required to
change the color of the indicator to
blue. NMT 1.0 mL of 0.01 N
hydrochloric acid is required to reach
the beginning of the color change of
the indicator from yellow to orange.

- Total organic carbon: The difference

between the sample and blank TOC
concentrations is NMT 5 mg/L.

- Total organic carbon test

Extractable metal - Aluminum: Solution S3 contains NMT Extractable metal testing
0.4 mg/L (ppm), corresponding to 1
ug/g.
- Arsenic, cadmium, lead, mercury,
cobalt, nickel, and vanadium: Report
the measured value in Solution S3 at
values above 0.01 mg/L (ppm),
corresponding to 0.025 ug/g. If the
measured values are below these
values, report the result as less than
0.01 mg/L (ppm), corresponding to
less than 0.025 ug/g.

340
- Chromium: Solution S3 contains NMT
0.02 mg/L (ppm), corresponding to
0.05 ug/g.
- Titanium: Solution S3 contains NMT
0.4 mg/L(ppm), corresponding to 1
ug/g.
- Zinc: Solution S3 contains NMT 0.4
mg/L (ppm), corresponding to 1 ug/g.

341
342
343
344
The secondary packaging : Kraft Paper Container
Natural brown kraft and white abs chipboard carton materials can print various
designs, beautiful, durable and able to support the weight well. The folding boxes are easy
to assemble and fold flat for easy shipping and storage.
The labeling will show
1. Product name
2. Dosage form
3. Name of Active Ingredients
4. Strength of Active Ingredients
5. Batch Number
6. Manufacturing date
7. Expiration date
8. Route of Administration
9. Storage condition

11. Name and address of Marketing Authorization Holder

12. Name and address of manufacturer
13. Special labelling (e.g. external use only, dangerous drug)
14. Recommended Daily Allowance (Vitamins and minerals)
15. Warning as indicated by Ministerial Announcement)
16. Pack sizes

345
Pack sizes
Weight: 200 g
Width: 4 cm
Length: 4 cm
Height: 8.5 cm
Design of the secondary packaging

346
Table 101 Secondary packaging specification

Test Requirement Method

Appearance Kraft Paper Visual inspection

Paper Grade KA125 Density measurement

Thickness 0.5 mm Micrometer measurement

Basic Weight (g/m2) 125 ± 5% Weighing test

Burst strength (KPa) 390-400 Burst strength test

Moisture (%) 6-9 Moisture test

347
348
349
The tertiary packaging : Carton box (Cardboard box)
Brown paper, good strength, good for boxes that need strength, suitable for export
products durability and higher burst strength.
Package size:
Width: 18 inches
Length: 12 inches
Depth: 14 inches
Capacity: 50 boxes (Kraft Paper Container)

Design of the tertiary packaging

Table 102 Tertiary Packaging Specification

Test Requirement Method

Appearance Brown paper container Visual inspection

Paper Grade TA150 Density measurement

Type A flute Visual inspection

350
 Thickness 1 mm ± 0.02 Micrometer measurement

Basic Weight (g/m2) 150 ± 5% Weighing test

Burst strength (KPa) 350-375 Burst strength test

Moisture (%) 6-9 Moisture test

351
352
Reference
1. United State Pharmacopoeia 2019 USP 42- NF 37 pdf [Internet]. Pharmaceuticals
Industry - Web of Pharma. [cited 2022 Feb 24]. Available from:
https://www.webofpharma.com/2021/01/united-state-pharmacopoeia-2019-usp-
42.html
2. Zithromax Powder for Oral Suspension - Summary of Product Characteristics
(SmPC) - (emc) [Internet]. [cited 2022 Feb 24]. Available from:
https://www.medicines.org.uk/emc/product/3006/smpc
3. Azithromycin Oral Suspension - FDA prescribing information, side effects and uses
[Internet]. Drugs.com. [cited 2022 Feb 24]. Available from:
https://www.drugs.com/pro/azithromycin-oral-suspension.html
4. SKS Bottle & Packaging - 30 cc 30 cc Plastic Bottles, Natural HDPE Wide Mouth
Pharmaceutical Round Bottles w/ White Lined Screw Caps [Internet]. [cited 2022 Feb
24]. Available from: https://www.sks-bottle.com/product/2256.html
5. Printed Paperboard Folding Chipboard Cartons [Internet]. Big Valley Packaging
Corporation. [cited 2022 Feb 24]. Available from:
https://bigvalleypackaging.com/folding-cartons
6. QUALITY CHECK PROCESS) [Internet].
2019 [cited 2022 Feb 24]. Available from:
https://hongthaipackaging.com/blog/quality-check-process/

353
P8 Stability
8.1 Protocol of Stability Study
8.1.1 Objective
To evaluate stability of product and provide evidence of quality of product in
primary container under the influence of the environment factors and to determine the
shelf-life and storage directions of the product.
8.1.2 Test design
8.1.2.1 Test material
Selection of batch
Drug product: Azithromycin Powder for Oral Suspension
Strength: 200 mg/5 ml
Volume/unit: ml
Production by following minimum pilot scale 100,000 units per 1 production lot.
Table 103 Test material
Date of
Date of expire
Lot Number Packaging type manufacture Batch size
(DD/MM/YY)
(DD/MM/YY)
AZ200565081 3/02/2019 2/02/2022 500,000 bottles
AZ200565082 PP bottle 3/02/2019 2/02/2022 500,000 bottles
AZ200565083 3/02/2019 2/02/2022 500,000 bottles

8.1.2.2 Testing plan

1. Storage condition and sampling intervals
Azithromycin Powder for Oral Suspension 200 mg/5 ml is filled in a polypropylene
(PP) bottle.

354
 Table 104 Storage condition and sampling intervals
Storage condition Sampling interval
Long term 0, 3, 6, 9, 12, 18, 24, 36 months
30°C ± °C/75% RH ± 5%
Accelerated 0, 1, 3, 6 months
40°C ± °C/75% RH ± 5%

Table 105 Schedule for stability of Azithromycin Powder for Oral Suspension 200 mg/5mL
Storage Schedule
Period condition AZ200565081 AZ200565082 AZ200565083
Accelerated
Initial 3/02/2019 3/02/2019 3/02/2019
Long term
1 month Accelerated 3/03/2019 3/03/2019 3/03/2019
Accelerated
3 months 3/05/2019 3/05/2019 3/05/2019
Long term
Accelerated
6 months 3/08/2019 3/08/2019 3/08/2019
Long term
9 months Long term 3/11/2019 3/11/2019 3/11/2019
12 months Long term 3/02/2020 3/02/2020 3/02/2020
18 months Long term 3/08/2020 3/08/2020 3/08/2020
24 months Long term 3/02/2021 3/02/2021 3/02/2021
36 months Long term 3/02/2022 3/02/2022 3/02/2022
Remarks Long term : 30°C ± 2°C/75% RH ± 5%
Accelerated : 40°C ± 2°C/75% RH ± 5%
2. Testing and test criteria
Table 106 Testing and test criteria

Test Requirement Method

Appearance A dry powder (White or off-white Visual inspection
powder)
355
Assay 90.0%-110.0% USP 42 NF 37, Page 448
Performance tests
- Dissolution Apparatus 2 (Paddle Each unit is not less than Q* + USP 42 NF 37 <711>,
Apparatus) 5% within 15 minutes. Page 6879
Organic impurities
- Azithromycin N-oxide NMT 0.50% USP 42 NF 37,
- -(N-N-Didemethyl)- -N-formyl NMT 0.50% Page 449-450
azithromycin
- - N.N-Dimethyl NMT 0.50%
azithromycin aminoazithromycin c
- Azithromycin related compound Fd NMT 0.50%
- Desosaminylazithromycine NMT 0.30%
- N-Demethylazithromycin NMT 0.50%
- -O- -
demethylazithromycin )
- -De(dimethylamino)- - NMT 0.50%
Oxoazithromycin
- Specified unidentified impurityh,k -
- Azithromycin -
- 2-Desethyl-2-propylazithromycinh,I -
- -N-Demethyl- -N-[(4- -
methylphenyl)sulfonyl]azithromycinh,m
- 3-Deoxyazithromycin (azithromycin B) -
- Any individual,unidentified impurity NMT 0.20%
- Total degradation production NMT 3.50%
Specific test
- pH 8.5 11.0 USP 42 NF 37, Page 450
- Microbiological test -Total aerobic microbial count USP 42 NF 37 , <61>
C. Microbial enumeration test : less than 102 cfu/ml Page 6387-6393
Enumeration method -Total combined Yeast/Molds
count less than 10 cfu/ml
Test for specified microorganism : Bile- Absence of Escherichia coli USP 42 NF 37 , <62>
tolerant gram-negative bacteria Page 6393-6402
356
* Q = 93%

8.1.3 Number of Samples (of on batch/ storage condition)

Accelerated test
-Appearance : 0 bottles
-Assay : 3 bottles
-Dissolution : 10 bottles
-pH : 10 bottles
-Microbiological test : 10 bottles
Total = 33 bottles rounded to 40 bottles
Number of testing : 4 times
Quality needed = 4*40 = 160 bottles
Long term stability test
- Appearance : 0 bottles
- Assay : 3 bottles
- Dissolution : 10 bottles
- pH : 10 bottles
- Microbiological test : 10 bottles
Total = 33 bottles rounded to 40 bottles
Number of testing : 8 times
Quality needed = 8*40 = 320 bottles
In-use stability test
- Appearance : 0 bottles
- Assay : 3 bottles
- Dissolution : 10 bottles
- pH : 10 bottles
- Microbiological test : 10 bottles
Total = 33 bottles rounded to 40 bottles

357
8.2 Stability report data
8.2.1 Responsibility
Table 107 Responsibility
Person in charge Site/Department Responsibility
Nattha S. R&D Physical and chemical test
Nattha S. R&D Microbiological test
2 Summary
Shelf-life: The product has a shelf-life of three years
Storage Directions: The finish product have to preserve in a polypropylene
(PP) bottle.
8.2.3 Objective
The objective of present study on Azithromycin Powder for Oral Suspension
200 mg/5 ml is assessment of the stability profile for storage under long term and
accelerated conditions.
8.2.4 Test material
8.2.4.1 Starting material
Table 108 Starting material
Product Batch No.
Material Source
#01 #02 #03
Azithromycin Gold
dihydrate as S4008044061 S4008044062 S4008044063 Biotechnology
anhydrous base
Lactose 16L23-H02-00272 16L23-H02-00272 16L23-H02-00273 Fagron
Hydroxypropyl Sigma-Aldrich®
MKBX9273V MKBX9273V MKBX9273V
cellulose
Sucrose EMPROVE®
0000181993 0000181993 0000181993
ESSENTIAL

358
 xanthan gum 1AL0717 1AL0717 1AL0717 Spectrum®
Sodium phosphate C33055A C33055A C33055A Fagron
Flav.pappermint 15G23-H04-00145 15G23-H04-00145 15G23-H04-00145 Fagron
Menthol 20190910 20190910 20190910 CHEMIPAN
Aerosil 1KE0856 1KE08576 1KE0856 Spectrum®
Sucralose Z114121005 Z114121005 Z114121005 CHEMIPAN
Drug product
Table 109 Drug product
Manufacturing Batch size
Dosage Lot No. Scale
Date Site (Unit)
Azithromycin Powder for Oral
AZ200565081 3/02/2019 Bangkok Production 500,000
Suspension 200 mg/5mL
Azithromycin Powder for Oral
AZ200565082 3/02/2019 Bangkok Production 500,000
Suspension 200 mg/5mL
Azithromycin Powder for Oral
AZ200565083 3/02/2019 Bangkok Production 500,000
Suspension 200 mg/5mL
8.2.5 Composition
Table 110 Composition
No. Ingredients Unit formula %w/w Function Reference
1. Azithromycin dihydrate 1.258 g 5.71 g Active ingredient USP42 NF37
2. Lactose USP42 NF37
2.25 g 10.21 g Diluent
(SuperTab® 11SD)
3. Sucrose 17.4 g 78.98 g Diluent USP42 NF37
4. hydroxypropyl cellulose (HPC) 0.44 g 1.99 g Suspending agent USP42 NF37
5. Purified water water for USP42 NF37
Q.s. Q.s.
reconstitution
6. Xanthan gum 0.02 g 0.09 g Suspending agent USP42 NF37

359
7. Sodium phosphate 0.17 g 0.77 g Buffering agent USP42 NF37
8. Flav.pappermint 0.17 g 0.77 g Flavoring agent USP42 NF37
9. Menthol 0.01 g 0.045 g Flavoring agent USP42 NF37
10. Aerosil 0.08 g 0.36 g glidant USP42 NF37
11. Sucralose 0.24 g 1.08 g Sweetening agent USP42 NF37
Total 22.03 g 100

2 Packaging
polypropylene (PP) bottle.
8.2.7 Storage conditions and testing intervals
Table 111 Schedule for storage condition and sampling intervals
Months
Storage condition
0 1 3 6 9 12 18 24 36
Long term / - / / / / / / /
30°C ± °C/75% RH ± 5%
Accelerated / / / / - - - - -
40°C ± °C/75% RH ± 5%
2. Analytical procedures
Table 112 Analytical procedures for stability of Azithromycin Powder for Oral Suspension
200 mg/5mL

Test Requirement Method

Appearance A dry powder (White or off-white Visual inspection
powder)
Assay 90.0%-110.0% USP 42 NF 37, Page 448

360
Performance tests
Dissolution Apparatus 2 (Paddle Apparatus) Each unit is not less than Q* + USP 42 NF 37 <711>,
5% within 15 minutes. Page 6879

Organic impurities
- Azithromycin N-oxide NMT 0.50% USP 42 NF 37 ,
- -(N-N-Didemethyl)- -N-formyl NMT 0.50% Page 449-450
azithromycin
- - N.N-Dimethyl NMT 0.50%
azithromycin aminoazithromycin c
- Azithromycin related compound Fd NMT 0.50%
- Desosaminylazithromycine NMT 0.30%
- N-Demethylazithromycin NMT 0.50%
- -O- -
demethylazithromycin )
- -De(dimethylamino)- - NMT 0.50%
Oxoazithromycin
- Specified unidentified impurityh,k -
- Azithromycin -
- 2-Desethyl-2-propylazithromycinh,I -
- -N-Demethyl- -N-[(4- -
methylphenyl)sulfonyl]azithromycinh,m
- 3-Deoxyazithromycin (azithromycin B) -
- Any individual,unidentified impurity NMT 0.20%
- Total degradation production NMT 3.50%
Specific test
8.5 11.0
- pH USP 42 NF 37, Page 450
- Microbiological test -Total aerobic microbial count USP 42 NF 37 , <61>
A. Microbial enumeration test : less than 102 cfu/ml Page 6387-6393
Enumeration method -Total combined Yeast/Molds
count less than 10 cfu/ml

361
 D. Test for specified microorganism : Absence of Escherichia coli USP 42 NF 37 , <62>
Bile-tolerant gram-negative Page 6393-6402
bacteria
* Q = 93%
2 Reference standard
Table 113 Reference standard
Reference Standards Source Lot
No.
Azithromycin RS The United States Pharmacopeial Convention R103C0
Azithromycin Related The United States Pharmacopeial Convention R08580
Compound F RS
Desosaminylazithromycin RS The United States Pharmacopeial Convention R076H0

8.2.10 In-use stability

Storage condition 200mg/5ml and sampling intervals Azithromycin Powder for Oral
Suspension 200 mg/5 ml. Reconstitute with 9 ml of water to give 15 ml suspension.

Table 114 Schedule for in-use stability of Azithromycin Powder for Oral Suspension 200 mg/5mL

Date
Number of Batch size
Lot No. Storage condition
Initial Final Sample (Unit)

AZ200565081 30°C ± °C 3/02/2019 5/02/2019 40 500,000

AZ200565082 30°C ± °C 3/02/2019 5/02/2019 40 500,000

AZ200565083 30°C ± °C 3/02/2019 5/02/2019 40 500,000

362
 2.11 Analytical procedures
Table 115 Analytical procedures for in-use stability of Azithromycin Powder for Oral
Suspension 200 mg/5mL

Test Requirement Method

Appearance After reconstitution a white to Visual inspection
off white colored/ opaque
Assay 90.0%-110.0% USP 42 NF 37 ,
Page 448
Performance tests
Dissolution Apparatus 2 (Paddle Apparatus) Each unit is not less than Q* + USP 42 NF 37
5% within 15 minutes. <711>, Page 6879
Organic impurities
- Azithromycin N-oxide NMT 0.50% USP 42 NF 37 ,
- -(N-N-Didemethyl)- -N-formyl NMT 0.50% Page 449-450
azithromycin
- - N.N-Dimethyl NMT 0.50%
azithromycin aminoazithromycin c
- Azithromycin related compound Fd NMT 0.50%
- Desosaminylazithromycine NMT 0.30%
- N-Demethylazithromycin NMT 0.50%
- -O- -
demethylazithromycin )
- -De(dimethylamino)- - NMT 0.50%
Oxoazithromycin
- Specified unidentified impurityh,k -
- Azithromycin -
- 2-Desethyl-2-propylazithromycinh,I -
- -N-Demethyl- -N-[(4- -
methylphenyl)sulfonyl]azithromycinh,m
- 3-Deoxyazithromycin (azithromycin B) -
- Any individual,unidentified impurity NMT 0.20%

363
 - Total degradation production NMT 3.50%
Specific test
- pH 8.5 11.0 USP 42 NF 37, Page
450
- Microbiological test -Total aerobic microbial count USP 42 NF 37 ,
A. Microbial enumeration test : less than 102 cfu/ml <61>
Enumeration method -Total combined Yeast/Molds Page 6387-6393
count less than 10 cfu/ml
B. Test for specified microorganism : Absence of Escherichia coli USP 42 NF 37 ,
Bile-tolerant gram-negative <62>
bacteria Page 6393-6402
* Q = 93%
8.2.12 Result
Physical stability
The physical stability of Azithromycin Powder for Oral Suspension 200 mg/5 ml
proved to be unchanged after storage up to 36 months at long term condition
30°C ± °C/75% RH ± 5% and after 6 months accelerated condition at 40°C ± °C/75% RH ±

not changed significantly

Chemical stability
Stability under Long term conditions
Storage under Long term conditions 30°C ± °C/75% RH ± 5% for 36 months
had no significant effect on the chemical stability of drug product. With regard to the item
- N.N-Dimethyl azithromycin aminoazithromycin c
Azithromycin related compound Fd
Desosaminylazithromycine
N-Demethylazithromycin

364
 -O-demethylazithromycin )
-De(dimethylamino)- -Oxoazithromycin
Specified unidentified impurityh,k
Azithromycin
2-Desethyl-2-propylazithromycinh,I
-N-Demethyl- -N-[(4-methylphenyl)sulfonyl]azithromycinh,m
3-Deoxyazithromycin (azithromycin B)
Any individual,unidentified impurity
Total degradation production
The content of Azithromycin did not significantly changed compared to the initial
batches.
Stability under accelerated conditions 40°C ± °C/75% RH ± 5%
Storage under accelerated conditions 40°C ± °C/75% RH ± 5% for 6 months
did not effect the chemical stability. The content of Azithromycin was not significantly
changed compared to the initial batches.
Microbiological test
- Total aerobic microbial count and Total combined Yeast/Molds count are NMT
acceptance criteria.
- Absence of Escherichia coli
8.2.13 Conclusion
Storage under long term testing conditions causes insignificant change of assay
results of Azithromycin. Significant changes in physical and chemical stability were not
observed. Since the long term data and accelerated data show little or no change over time
and little variability, a statistical analysis is considered unnecessary.
Shelf life: Based on the resulting data the shelf-life established for three years.

365
 In-use stability after reconstitution: Based on the resulting data the In-use stability
established for three day.
Storage direction °C

366
 Table 116 Result of stability of Azithromycin Powder for Oral Suspension
Product: Azithromycin Powder for Oral Suspension 200 mg/5 ml Lot No. AZ200565081
Packaging: polypropylene (PP) bottle. Experimental Start Date: 3/02/2019
Storage Lot No. AZ200565081
Microbial Test
Period Assay Impurity (%)
Condition Appearance pH Dissolution* (cfu/ml)
(mo.) (%)
A B C D E F H N T TAMC TYMC
A dry powder
90.0%- NMT NMT NMT NMT NMT NMT NMT NMT NMT 8.5
specification (White or off- <102 <10
110.0% 0.50% 0.50% 0.50% 0.50% 0.30% 0.50% 0.50% 0.20% 3.50% 11.0
white powder)
0 Accelerated C 101.9 0.03 0.04 0.03 0.03 0.04 0.02 0.01 0.00 0.20 9.5 101.2-102.1 <10 <10
1 Accelerated C 101.3 0.05 0.06 0.04 0.05 0.06 0.05 0.02 0.00 0.33 9.5 101.3-102.2 <10 <10
3 Accelerated C 98.8 0.08 0.10 0.08 0.09 0.08 0.09 0.05 0.01 0.58 9.4 101.4-102.3 <10 <10
6 Accelerated C 97.6 0.14 0.13 0.15 0.14 0.12 0.16 0.11 0.01 0.96 9.3 101.4-102.5 <10 <10
0 Long term C 101.9 0.03 0.04 0.03 0.03 0.04 0.02 0.01 0.00 0.20 9.5 101.2-102.2 <10 <10
3 Long term C 101.5 0.05 0.07 0.07 0.06 0.07 0.05 0.03 0.00 0.40 9.5 101.3-102.3 <10 <10
6 Long term C 100.9 0.09 0.10 0.13 0.11 0.10 0.08 0.07 0.00 0.68 9.4 101.4-102.3 <10 <10
9 Long term C 100.4 0.14 0.13 0.15 0.14 0.12 0.12 0.10 0.01 0.91 9.2 101.4-102.4 <10 <10
12 Long term C 100.0 0.16 0.17 0.17 0.18 0.15 0.17 0.15 0.01 1.16 9.1 101.5-102.4 <10 <10
18 Long term C 99.2 0.21 0.24 0.24 0.23 0.19 0.24 0.19 0.01 1.55 8.9 101.6-102.5 <10 <10
24 Long term C 98.3 0.32 0.32 0.33 0.34 0.23 0.32 0.24 0.02 2.12 8.8 101.6-102.5 <10 <10
36 Long term C 97.1 0.41 0.40 0.43 0.43 0.26 0.40 0.28 0.02 2.63 8.7 101.7-102.6 <10 <10
( C = Complies, NC = Not complies, ND = Not detect )

367
 *Dissolution : The results shown are the ranges dissolution of 6 samples. Each sample is not less than Q+5%. (Q=93%)
Table 117 Result of stability of Azithromycin Powder for Oral Suspension
Product: Azithromycin Powder for Oral Suspension 200 mg/5 ml Lot No. AZ200565082
Packaging: polypropylene (PP) bottle. Experimental Start Date: 3/02/2019
Storage Lot No. AZ200565082
Microbial Test
Period Assay Impurity (%)
Condition Appearance pH Dissolution* (cfu/ml)
(mo.) (%)
A B C D E F H N T TAMC TYMC
A dry powder
90.0%- NMT NMT NMT NMT NMT NMT NMT NMT NMT 8.5
specification (White or off- <102 <10
110.0% 0.50% 0.50% 0.50% 0.50% 0.30% 0.50% 0.50% 0.20% 3.50% 11.0
white powder)
0 Accelerated C 101.3 0.05 0.06 0.04 0.05 0.06 0.05 0.02 0.00 0.33 9.5 101.3-102.3 <10 <10
1 Accelerated C 98.8 0.09 0.11 0.08 0.10 0.08 0.10 0.05 0.01 0.62 9.4 101.2-102.2 <10 <10
3 Accelerated C 97.6 0.15 0.13 0.14 0.16 0.12 0.17 0.10 0.01 0.98 9.3 101.1-102.1 <10 <10
6 Accelerated C 101.9 0.03 0.04 0.03 0.03 0.04 0.02 0.01 0.00 0.20 9.5 101.4-102.5 <10 <10
0 Long term C 101.5 0.06 0.07 0.07 0.06 0.07 0.05 0.03 0.00 0.41 9.5 101.2-102.1 <10 <10
3 Long term C 100.9 0.12 0.10 0.11 0.11 0.09 0.08 0.07 0.00 0.68 9.4 101.1-102.3 <10 <10
6 Long term C 100.4 0.14 0.13 0.15 0.14 0.12 0.12 0.10 0.01 0.91 9.2 101.3-102.2 <10 <10
9 Long term C 100.0 0.17 0.18 0.17 0.19 0.15 0.16 0.15 0.01 1.18 9.1 101.3-102.2 <10 <10
12 Long term C 99.2 0.24 0.26 0.24 0.24 0.21 0.24 0.19 0.01 1.63 8.9 101.2-102.3 <10 <10
18 Long term C 98.3 0.31 0.34 0.32 0.33 0.25 0.31 0.22 0.02 2.10 8.8 101.5-102.3 <10 <10
24 Long term C 97.1 0.39 0.40 0.42 0.42 0.28 0.39 0.27 0.02 2.59 8.7 101.4-102.3 <10 <10
36 Long term C 97.0 0.41 0.41 0.42 0.42 0.28 0.39 0.27 0.02 2.62 8.7 101.3-102.2 <10 <10
368
 ( C = Complies, NC = Not complies, ND = Not detect )
*Dissolution : The results shown are the ranges dissolution of 6 samples. Each sample is not less than Q+5%. (Q=93%)
Table 118 Result of stability of Azithromycin Powder for Oral Suspension
Product: Azithromycin Powder for Oral Suspension 200 mg/5 ml Lot No. AZ200565083
Packaging: polypropylene (PP) bottle. Experimental Start Date: 3/02/2019
Storage Lot No. AZ200565083
Microbial Test
Period Assay Impurity (%)
Condition Appearance pH Dissolution* (cfu/ml)
(mo.) (%)
A B C D E F H N T TAMC TYMC
A dry powder
90.0%- NMT NMT NMT NMT NMT NMT NMT NMT NMT 8.5
specification (White or off- <102 <10
110.0% 0.50% 0.50% 0.50% 0.50% 0.30% 0.50% 0.50% 0.20% 3.50% 11.0
white powder)
0 Accelerated C 101.3 0.05 0.06 0.04 0.05 0.06 0.05 0.02 0.00 0.33 9.5 101.3-102.1 <10 <10
1 Accelerated C 98.8 0.09 0.11 0.08 0.10 0.08 0.10 0.05 0.01 0.62 9.4 101.4-102.2 <10 <10
3 Accelerated C 97.6 0.15 0.13 0.14 0.16 0.12 0.17 0.10 0.01 0.98 9.3 101.4-102.3 <10 <10
6 Accelerated C 101.9 0.03 0.04 0.03 0.03 0.04 0.02 0.01 0.00 0.20 9.5 101.5-102.5 <10 <10
0 Long term C 101.5 0.06 0.07 0.07 0.06 0.07 0.05 0.03 0.00 0.41 9.5 101.3-102.2 <10 <10
3 Long term C 100.9 0.12 0.10 0.11 0.11 0.09 0.08 0.07 0.00 0.68 9.4 101.3-102.2 <10 <10
6 Long term C 100.4 0.14 0.13 0.15 0.14 0.12 0.12 0.10 0.01 0.91 9.2 101.3-102.2 <10 <10
9 Long term C 100.0 0.17 0.18 0.17 0.19 0.15 0.16 0.15 0.01 1.18 9.1 101.2-102.3 <10 <10
12 Long term C 99.2 0.24 0.26 0.24 0.24 0.21 0.24 0.19 0.01 1.63 8.9 101.5-102.5 <10 <10
18 Long term C 98.3 0.31 0.34 0.32 0.33 0.25 0.31 0.22 0.02 2.10 8.8 101.6-102.5 <10 <10
369
 24 Long term C 97.1 0.39 0.40 0.42 0.42 0.28 0.39 0.27 0.02 2.59 8.7 101.6-102.6 <10 <10
36 Long term C 97.0 0.41 0.41 0.42 0.42 0.28 0.39 0.27 0.02 2.62 8.7 101.6-102.5 <10 <10
( C = Complies, NC = Not complies, ND = Not detect )
*Dissolution : The results shown are the ranges dissolution of 6 samples. Each sample is not less than Q+5%. (Q=93%)
Table 119 Result of in-use stability of Azithromycin Powder for Oral Suspension
Product: Azithromycin Powder for Oral Suspension 200 mg/5 ml. Reconstitute with 9 ml of water to give 15 ml suspension.
Experimental Start Date: 3/02/2019
Storage Test
Microbial
Date Assay Impurity (%)
Lot No. Appearance pH Dissolution* Test (cfu/ml)
(Initial/Final) (%)
A B C D E F H N T TAMC TYMC
a white to
off white 90.0%- NMT NMT NMT NMT NMT NMT NMT NMT NMT 8.5
Specification <102 <10
colored/ 110.0% 0.50% 0.50% 0.50% 0.50% 0.30% 0.50% 0.50% 0.20% 3.50% 11.0
opaque
3/02/2019 C 102.0 0.03 0.04 0.03 0.03 0.04 0.02 0.01 0.00 0.20 9.5 101.3-102.1 <10 <10
AZ200565081
5/02/2019 C 101.8 0.03 0.04 0.04 0.03 0.05 0.02 0.01 0.00 0.22 9.5 101.4-102.2 <10 <10
3/02/2019 C 99.7 0.05 0.04 0.03 0.04 0.03 0.02 0.02 0.01 0.24 9.4 101.2-102.3 <10 <10
AZ200565082
5/02/2019 C 99.5 0.05 0.04 0.03 0.04 0.04 0.02 0.02 0.01 0.25 9.3 101.4-102.5 <10 <10
AZ200565083 3/02/2019 C 101.4 0.03 0.04 0.03 0.03 0.04 0.02 0.01 0.00 0.20 9.5 101.5-102.5 <10 <10

370
 5/02/2019 C 101.6 0.03 0.04 0.03 0.03 0.05 0.02 0.01 0.00 0.21 9.4 101.6-102.6 <10 <10
( C = Complies, NC = Not complies, ND = Not detect )
*Dissolution : The results shown are the ranges dissolution of 6 samples. Each sample is not less than Q+5%. (Q=93%)
A : Azithromycin N-oxide -De(dimethylamino)- -Oxoazithromycin T : Total degradation production
-(N-N-Didemethyl)- -N-formyl azithromycin I : Specified unidentified impurityh,k
-(N.N-Dimethyl) azithromycin(aminoazithromycin)c J : Azithromycin
D : Azithromycin related compound Fd K : 2-Desethyl-2-propylazithromycinh,I
E : Desosaminylazithromycine -N-Demethyl- -N-[(4-methylphenyl)sulfonyl]azithromycinh,m
F : N-Demethylazithromycin M : 3-Deoxyazithromycin (azithromycin B)
-O-demethylazithromycin ) N : Any individual,unidentified impurit

371
372
Reference
67.-December-2016-ACTR.pdf [Internet]. [cite 25 DEC 2021]. Available at:
https://asean.org/wp-content/uploads/2017/03/67.-December-2016-ACTR.pdf

373