azithromycin oral suspension

ANALYTICAL METHOD VALIDATION OF AZITHROMYCINBY

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 SinghE.mail:[email protected] No: +91-8950024148ZEE LABORATORIESUchani G.T. Road Karnal-132001 India

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1. SYSTEM SUITABILITYDefinition: 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.


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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. RUGGEDNESSDefinition: 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.


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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:


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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


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Standardization of Volumetric Solution: 0.02m Perchloric AcidNAME 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 + 0.01992409323MEAN= --------------------------------------------------------- = 0.0199042089 M 2

PREPARED BY: Deepak Kumar STANDARISED BY: Alok Singh


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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


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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= FactorC= Actual Molarity of volumetric solutionD= Defined Molarity of volumetric solutionE= 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 testB= FactorC= Actual Molarity of volumetric solutionD= Defined Molarity of volumetric solutionE= Volume (in ml) of sample takenM= Volume (in ml) in which label Claim is definedN= 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


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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 SuitabilityS.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.


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ACCEPTANCE CRITERIA RSD of five different analyses (Standard preparation): NMT. 2.0%




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: PrecisionS.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.


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ACCEPTANCE CRITERIA RSD of five different analyses (Sample preparation): NMT. 2.0%




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 RangeS.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:


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ACCEPTANCE CRITERIA Coefficient of correlation i.e. r²: NLT. 0.99




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 (

in m

g)

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.


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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%


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ACCEPTANCE CRITERIA: Recovery %: Should be within ± 2.0%




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.


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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.


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ACCEPTANCE CRITERIA AND RESULTS

S.No. Parameter Experiment Acceptance criteria Results 1. System Suitability

ParameterR.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

0.99994. 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


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azithromycin oral suspension

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