Azithromycin oral suspension

ANALYTICAL METHOD VALIDATION OF AZITHROMYCIN
BY
NON AQUEOUS TITRATION
PRODUCT NAME: Azithromycin Oral Suspension
OBJECTIVE:
The purpose of this protocol is to define the characteristics for consideration when validating
for assay of Azithromycin Oral Suspension
The objectives for validation of an analytical method are as follows:
1. To determine the performance characteristic of the method:
2. To identify that In-house method is suitable for ZEE LABORATORIES Products.
3. To show that the method is suitable for its intended purpose.
SCOPE:
This protocol applies to validation of analytical method for assay of Azithromycin Oral
Suspension manufactured by ZEE LABORATORIES.
REFERENCE:
In-House
RESPONSIBILITIES:
Quality Control / Quality Assurance
Alok Kumar Singh
E.mail:aloksinghlab@gmail.com
Contact No: +91-8950024148
ZEE LABORATORIES
Uchani G.T. Road Karnal-132001 India
Page 1 of 16

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1. SYSTEM SUITABILITY
Definition: System suitability is an integral part of many analytical procedures. The tests are
based on the concept that the equipment, electronics, analytical operations and samples to be
analyzed constitute an integral system that can be evaluated as such. System suitability test
parameters to be established for a particular procedure depend on the type of procedure being
validated.
2. PRECISION
Definition: The precision of an analytical method is a degree of agreement (degree of scatter)
among individual test results when the procedure is applied repeatedly to multiple samplings of
the same homogeneous sample. The precision of an analytical method is usually expressed as the
standard deviation or relative standard deviation (coefficient of variation). Precision may be a
measure of either the degree of reproducibility or of repeatability of the analytical method under
normal operating conditions.
Determination: The precision of an analytical method is determined by assaying a sufficient
number of aliquots of a homogeneous sample to be able to calculate statistically valid estimates of
standard deviation or relative standard deviation (coefficient of variation). Assays in this context
are independent analyses of samples that have been carried through the complete analytical
procedure from sample preparation to final test result. The intermediate precision expresses within
laboratory variation, as on different days.
3. LINEARITY AND RANGE
Definition of Linearity: The linearity of an analytical method is its ability to elicit test results that
are directly, or by a well-defined mathematical transformation, proportional to the concentration
of analyte in samples within a given range. Linearity is usually expressed in terms of the variance
around the slope of the regression line calculated according to an established mathematical
relationship from test results obtained by the analysis of samples with varying concentrations of
analyte.
Definition of Range: The range of an analytical method is the interval between the upper and
lower levels of analyte (including these levels) that have been demonstrated to be determined with
precision, accuracy, and linearity using the method as written. The range is normally expressed in
the same units as these results (e.g. percent, parts per million) obtained by the analytical method.
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 2 of 16

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Determination of Linearity & Range: The linearity of an analytical method is determined by
mathematical treatment of test results obtained by analysis of samples with analyte concentrations
across the claimed range of the method. The treatment is normally a calculation of a regression
line by the method of least squares of test results versus analyte concentrations. In some areas, to
obtain proportionality between assays and sample concentrations, the test data may have to be
subjected to a mathematical transformation prior to the regression analysis. The slope of the
regression line and its variance provide a mathematical measure of linearity; the y-intercept is a
measure of the potential assay bias. Plotting the test results graphically as a function of analyte
concentration on appropriate graph paper may be an acceptable alternative to the regression line
calculation.
The range of the method is validated by verifying that the analytical method provides acceptable
precision, accuracy and linearity when applied to samples containing analyte at the extremes of
the range as well as within the range.
4. ACCURACY
Definition: The accuracy of an analytical method is the closeness of test results obtained by that
method to the true value. Accuracy may often be expressed as percent recovery by the assay of
known, added amounts of analyte. Accuracy is a measure of the exactness of the analytical
method that is true for all practical purposes.
Determination: The accuracy of an analytical method may be determined by applying that method
as samples or mixtures of excipients to which known amounts of analyte have been added both
above and below the normal levels expected in the samples. The accuracy is then calculated from
the test results as the percentage of analyte recovered by the assay.
5. RUGGEDNESS
Definition: The robustness of an analytical method is the degree of reproducibility of test results
obtained by the analysis of the same samples under a variety of condition, such as different
analyst, different days etc.
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 3 of 16

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BY
DETAILS ABOUT VALIDATION EXPERIMENT
SAMPLE: AZITH Suspension
Batch No. : 4865
Date of Mfg.: 03 / 2011
Date of Exp : 02 / 2013
DETAILS OF WORKING STANDARDS:
Name of Working Standard: Azithromycin Dihydrate
Working Standard Reference No: 10
Batch No. : MLAZI-330311
Potency / Assay : 99.23 % w/w
LOD/Water Content : 4.7 % w/w
METHODOLOGY:
Reagents:
1) Chloroform AR Grade
2) Glacial acetic acid AR Grade
3) Methyl Violet Indicator AR Grade
4) Anhydrous Sodium Sulphate EP Grade
5) 0.02M Perchloric Acid volumetric solution
Instrument & Glass ware:
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 4 of 16

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1) Digital Potentiometer
2) Conical Flask -25ml
3) Separating flannel -125ml
4) Pipette-2ml & 25ml
5) Rotary Evaporator
6) Digital Balance
7) Whatman No. 42 Filter Paper
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 5 of 16

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Standardization of Volumetric Solution: 0.02m Perchloric Acid
NAME OF VOLUMETRIC SOLUTION: PERCHLORIC ACID
DATE OF PREPARATION: 16/03/2011 STRENGTH: 0.02 M
DATE OF STANDARDISATION: 18/03/2011 QUANTITY: 500 ml
PREPARATION: Dilute about 100 ml of 0.1M Perchloric Acid to 500 ml with Anhydrous
Glacial Acetic Acid and 10 ml of Acetic Anhydride. Mix well; allow standing for 24 hours.
Standardize the solution in the following manner.
STANDARDISATION: In a dried conical flask taken about 50 ml of Anhydrous Glacial
Acid add 0.1 ml of Methyl Violet and neutralized with 0.02 M Perchloric Acid. Again added
accurately weighed about 50 mg of previously powdered and dried Potassium Hydrogen
Phthalate, Dissolves and titrate with 0.02M Perchloric Acid with potentiometric titrator until
the violet colour change to emerald-green colour. Each ml of 0.02 M Perchloric Acid is
equivalent to 4.0846 mg of Potassium Hydrogen Phthalate.
CALCULATION:
I. Weight of Potassium Hydrogen Phthalate: 50mg Volume Consume:12.3 ml
50 X 99.9 X 0.02
= ----------------------------- =0.01988432458 M
4.0846 X 100 X 12.3
II. Weight of Potassium Hydrogen Phthalate:50.1mg Volume Consume:12.3 ml
50.1X 99.9 X 0.02
= ------------------------------ =0.01992409323 M
4.0846 X 100 X 12.3
0.01988432458 + 0.01992409323
MEAN= --------------------------------------------------------- = 0.0199042089 M
2
PREPARED BY: Deepak Kumar STANDARISED BY: Alok Singh
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 6 of 16

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Procedure:
1)
Weigh Azithromycin and add in 125 ml capacity separating flannel, add 25 ml distilled water
and dissolve it.
2)
Add 25 ml chloroform, shake vigorously dissolve Azithromycin in chloroform layer wait about
10 minute for separation of chloroform layer & water layer, filter the chloroform layer in dried
conical flask with whatman no.42 filter paper through Anhydrous Sodium Sulphate Bed to avoid
the water.
3)
Repeat the above step up to five times with 25 ml chloroform and collect chloroform layer in
Dried Conical flask
4)
Slowly evaporate the collated chloroform by heating with rotary evaporator to dryness and cool
the flask to room temperature
5)
Take 25ml glacial acetic acid in second dried conical flask add methyl violet indicator carried
out blank titration for non aqueous Titration
6)
Transfer this neutralised glacial acetic acid in first conical flask and shake it to dissolve
Azithromycin in neutralised glacial acetic Acid, Titrate with 0.02M Perchloric acid, volumetric
solution determining the end-point Potentiometrically
7)
Factor: Each 1 ml of 0.02 M Perchloric acid volumetric solution is equivalent to 7.4898mg of
Anh. Azithromycin
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 7 of 16

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Calculation:
A) For standard
A X B X C X 100
= Result (in %)
D X E
A= Volume consume (in ml) by Standard
B= Factor
C= Actual Molarity of volumetric solution
D= Defined Molarity of volumetric solution
E= Weight of standard taken
B) For Test
A X B X C X MX100
= Result (in %)
D X E X N
A= Volume consume (in ml) by test
B= Factor
C= Actual Molarity of volumetric solution
D= Defined Molarity of volumetric solution
E= Volume (in ml) of sample taken
M= Volume (in ml) in which label Claim is defined
N= Label claim of Azithromycin
Note:
1) In each step weigh or volume of sample taken and volume consume by sample is note down
and calculation is done according the above formula given for Std and Test
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 8 of 16

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1. SYSTEM SUITABILITY
Procedure:
Standard Azithromycin active 20 mg is taken in separating funnel and assay procedure performed,
assay is calculated this one is repeated with six times (6n) Average and RSD is calculated
Table: System Suitability
S.No. Assay (in mg)
01 19.9
02 20.1
03 20.2
04 20.3
05 19.92
06 19.95
Average Assay (in mg) 20.06167
RSD: 0.82%
Conclusion: Average and Relative standard deviation of six different dilutions was found
20.06167mg and 0.82% respectively. RSD was found within acceptable limits (i.e. 2.0%) hence
the method is suitable and system suitability was established.
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 9 of 16
ACCEPTANCE CRITERIA
RSD of five different analyses (Standard preparation): NMT. 2.0%

BY
2. PRECISION
Sample Preparation:
AZITH Oral Suspension containing Azithromycin active 20 mg is taken in separating funnel and
assay procedure performed, assay is calculated this one is repeated with six times (6n) Average
and RSD is calculated
Table: Precision
S.No. Assay (in mg)
01 20.5
02 20.7
03 21
04 20.6
05 20.8
06 20.6
RSD: 0.86%
Conclusion: Assay and Relative Standard Deviation of six different dilutions was found 20.7mg
and 0.86% respectively. RSD was found within acceptable limits (i.e. 2.0%) hence precision for
test sample was established.
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 10 of 16
ACCEPTANCE CRITERIA
RSD of five different analyses (Sample preparation): NMT. 2.0%

BY
3. LINEARITY AND RANGE:
Procedure: Standard of Azithromycin of 15mg, 20mg, 25mg, 30mg and 35mg were taken in
duplicate assay procedure is performed mean was calculated. The coefficient of correlation of
five standard preparations was calculated and graph for linearity was plotted.
Table: Linearity and Range
S.No. Active Concentration(in mg) Assay(in mg)
1 15mg 14.87
2 15mg 15.1
3. 20mg 19.89
4. 20mg 19.99
5. 25mg 24.86
6. 25mg 25.1
7. 30mg 30.12
8. 30mg 30.15
9. 35mg 35.1
10. 35mg 34.88
Regression Coefficient 0.9999
Slop 1.004x
Linearity Graph:
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 11 of 16
ACCEPTANCE CRITERIA
Coefficient of correlation i.e. r²: NLT. 0.99

BY
Standared Linearity graph of
Azithromycin y = 1.004x - 0.116
R2
= 0.9999
0
20
40
0 10 20 30 40
Active (in mg)
Assay(inmg)
Conclusion: Graph of standard linearity was plotted between Active Concentration (in mg) on x-
axis and average assay (in mg) at y axis. Coefficient of correlation (i.e. r²) was found 0.9999
hence the linearity between concentration range of 15mg to 35mg was established.
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 12 of 16

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04. ACCURACY: (RECOVERY FROM PLACEBO INTERFERENCE):
Procedure:
Five samples standard preparations containing different active concentration of Azithromycin
(15mg, 20mg, 25mg, 30mg, and 35mg) were prepared separately with addition of placebo
(Suspension base) same ratio as suspension.
Based on actual dilution in assay preparation and assay procedure is performed calculated the
assay and percentage recovery.
Table: Accuracy
S. No. Active concentration(in mg) Assay (in mg) Recovery (in %)
1 15mg 15.2 101.3%
2 15mg 14.89 99.27%
3. 15mg 14.86 99.07%
Mean 14.98333 99.89%
4. 20mg 19.92 99.6%
5. 20mg 20.1 100.5%
6. 20mg 20.5 102.5%
Mean 20.17333 100.9%
7. 25mg 24.98 99.9%
8. 25mg 24.86 99.4%
9. 25mg 24.92 99.7%
Mean 24.92 99.7%
10. 30mg 30.12 100.4%
11. 30mg 30.1 100.3%
12. 30mg 30.2 100.7%
Mean 30.14 100.5%
13. 35mg 34.86 99.6%
14. 35mg 34.89 99.7%
15. 35mg 35.15 100.4%
Mean 34.96667 99.9%
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 13 of 16
ACCEPTANCE CRITERIA:
Recovery %: Should be within ± 2.0%

BY
Table: Percentage Recovery
S. No. Conc. of Active(in mg) Assay(in mg) Recovery
1 15mg 14.98333 99.89%
2 20mg 20.17333 100.9%
3 25mg 24.92 99.7%
4 30mg 30.14 100.5%
5 35mg 34.96667 99.9%
Average recovery 100.174%
RSD 0.50%
Conclusion: The individual percentage recovery from various concentrations was found within
the acceptable limit. RSD of The recovery of known amount of Working Reference Standard
solutions were recovered within 0.5% (i.e. within 2.0%) acceptable limits hence the method was
accurate and recovery from placebo and Azithromycin interference was established.
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 14 of 16

BY
5. Ruggedness:
Standard preparation: Same as mention in system suitability.
Sample Preparation: Same as mention in precision.
Procedure:
Above prepared solutions of standard and sample assay procedure performed separately in
triplicate by different analyst and on different day. The assay percent and RSD of standard and
sample solution were calculated separately.
Table: Absorbance Pattern (Different Analyst& Different Day):
S. No. Sample Assay (in mg)
1. Standard 1 20.01
2. Standard 2 19.99
3. Standard 3 20.02
Average assay: 20.01
S. No. Sample Assay (in mg)
1. Sample 1 20.69
2. Sample 2 20.71
3. Sample 3 20.72
Average assay: 20.71
Conclusion: The average Assay percentage of standard and sample solution of different analysts
on different day was found 20.01mg and 20.71mg respectively. Both the assay was found within
the acceptable limits (i.e. 2.0%) hence ruggedness is established.
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 15 of 16

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ACCEPTANCE CRITERIA AND RESULTS
S.No. Parameter Experiment Acceptance criteria Results
1. System Suitability
Parameter
R.S.D. N.M.T. 2.0% 0.82%
2. Precision RSD & Assay NMT. 2.0% (For RSD)
± 10% of labeled amount
0.86%
20.07mg or 103.5%
3. Linearity & Range Coefficient of
Correlation
Slope
Minimum acceptable =
0.99
Record results
1.004x
0.9999
4. Accuracy Recovery
Excipients
Solvent
RSD Within 2.0%
Excipients do not
interfere with assay
Solvents do not interfere
with assay
0.50%
NO
NO
5. Ruggedness Variation in assay NMT. 2.0% Within the limit
Conclusion: The above result shows that the method use for analysis of Azithromycin is accurate
and reproducible hence method is validated for the analysis purpose of Azithromycin oral
Suspension
Alok Kumar Singh
Contact No: +91-8950024148
ZEE LABORATORIES
Page 16 of 16

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