Analytical Method Verification Report for Azithromycin Tablets USP 500mg

TABLE OF CONTENTS

• Introduction
• Purpose
• Scope
• Responsibility
• Methodology
• Equipment, Chemicals, Glasswares, Documents
• Working Standard Details & Verification Sample Details
• Method of Analysis
• Description of Verification Parameters
• Verification parameters and acceptance criteria

1. Specificity
2. Linearity
3. Accuracy
4. Precision

• Summary
• Conclusions

INTRODUCTION

• Assessing selected analytical performance characteristics to generate appropriate, relevant data rather than repeating the validation process.
• Method verification consists of partial validation. It should be performed for already validated analytical methods under the following circumstances:
• when an already validated method is used on a product for the first time (e.g. in case of a change in active pharmaceutical ingredient (API) supplier, change in the method of synthesis or after reformulation of a drug product).
• when an already validated method is used for the first time in a laboratory (in some cases, method transfer may be preferable).

ALSO READ: SOP for Transfer of Analytical Procedure

PURPOSE

• This document established the method selection & procedures for verification of standard method performance. The laboratory shall confirm that it can properly operate standard methods before introducing the tests or calibrations. If the standard method changes, the verification shall be repeated. 
• Verification that a laboratory can adequately operate a standard method requires that the laboratory provide objective evidence the performance parameters specified in the test method have been met with the matrices to which the method is being applied. Most often, the critical requirements are accuracy and precision (generally accepted as repeatability and reproducibility) which are reflected in the measurement uncertainty. The objective evidence is the accuracy and precision obtained from actual lab data.

SCOPE

• The scope of verification of the analytical procedure for Accuracy, Precision, Specificity & Linearity is to demonstrate that the method is suitable for the analysis of Azithromycin Tablets USP 500 mg.

RESPONSIBILITY

• It is the responsibility of the validation officer to ensure that the finished product analysis method is verified as per U.S. Pharmacopoeia.
• It is the responsibility of the QC Executive to follow the verified method for routine analysis.
• The Manager - Q.C, should ensure that methods of analysis are available and that the equipment to be used for analysis is properly working and calibrated.
• The Q.A. Manager should ensure that the method verified for the finished product should meet the acceptance criteria and maintain records for further activity.

METHODOLOGY

Equipment
HPLC	:
Instrument ID & Calibration Date
Analytical Balance	:	An analytical balance is sufficiently sensitive to detect changes of 0.1 mg.
Instrument ID & Calibration Date
pH Meter	:
Instrument ID & Calibration Date
Chemicals
Methanol (HPLC Grade)			:
Acetonitrile (HPLC Grade)			:
Dipotassium Hydrogen Orthophosphate			:
Sodium 1 - Octane Sulfonate			:
Monobasic Ammonium Phosphate			:
Glassware
Volumetric Flasks	:
Pipettes	:
Beakers	:
Other Glassware	:
Documents
Standard Operating Procedure	:
Calibration Documents	:
Guidelines	:	(1226) Verification of Compendial Procedures

Working Standard Used
Azithromycin Dihydrate USP
Reference No.	:
LOD	:
Purity (as is basis)	:
Prepared on	:
Valid up to	:
Sample Taken for Verification
Name of Sample	:	Azithromycin Tablets USP 500 mg
Batch Number	:
Manufacturing Date	:
Expiry Date	:

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Method of Analysis

Chromatographic Conditions:

Column: 250 mm × 4.6 mm, C18, 5 µm

Flow Rate: 1.5 mL/minute

Column Temperature: 50°C

Detection Wavelength: 210 nm

Injection Volume: 50 µL

Mobile Phase: Buffer : Acetonitrile : Methanol (40 : 45 : 15, v/v)

Diluent: Solution A : Acetonitrile : Methanol (35 : 30 : 30, v/v)

Buffer Preparation:

Dissolve 4.4gm of dipotassium hydrogen orthophosphate & 0.5gm of sodium 1-octane sulfonate in 1000mL of water, adjust pH of solution to 8.20 ± 0.05 with dilute phosphoric acid.

Solution A Preparation:

Dissolve about 1.7gm of monobasic ammonium phosphate in 1000mL of water, adjust pH of solution to 10.00 ± 0.05 with ammonium hydroxide

Standard Preparation:

Transfer about 40 mg of Azithromycin dihydrate SRS, accurately weighed, to a 100 mL volumetric flask. Add 70 mL of diluent, sonicate to dissolve & dilute with diluent to volume.

Sample Preparation:

Transfer about 53 mg of tablet powder (equivalent to 40mg of Azithromycin dihydrate), to a 100 mL volumetric flask. Add 70 mL of diluent, sonicate to dissolve & dilute with diluent to volume.

Procedure:

Inject standard preparation in five replicates & record the chromatogram. Inject sample preparation in duplicate, record the chromatogram & calculate the percent of the assay by using the following formula:

DESCRIPTION OF VERIFICATION PARAMETERS:

Accuracy

The accuracy is the degree of agreement of test results with the true value, or the closeness of the results obtained by the procedure to the true value. It is normally established on samples of the material to be examined that have been prepared to quantitative accuracy. Accuracy should be established across the specified range of the analytical procedure.

Precision

Precision is the degree of agreement among individual results. The complete procedure should be applied repeatedly to separate, identical samples drawn from the same homogeneous batch of material. It should be measured by the scatter of individual results from the mean (good grouping) and expressed as the relative standard deviation (RSD).

Precision may be performed as system precision, repeatability and intermediate precision

1. System precision: To check system suitability criteria
2. Repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure, e.g. three concentrations/three replicates each, or a minimum of six determinations at 100% of the test concentration.
3. Intermediate precision expresses within laboratory variations (usually on different days, different analysts and different equipment).

Specificity

Specificity (selectivity) is the ability to measure unequivocally the desired analyte in the presence of components such as excipients and impurities that may also be expected to be present. An investigation of specificity should be conducted during the verification of identification tests, the determination of impurities and assay.

Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

VERIFICATION PARAMETERS AND ACCEPTANCE CRITERIA:

Specificity

Interference study

Prepared the following solutions for interference study :

Preparation of Blank: Used diluent as blank.

Preparation of Placebo:

Transferred_________ mg (53) mg of placebo powder to a 100 mL volumetric flask, added _______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

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Standard Preparation:

Transferred_________ mg (40) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Sample Preparation:

Transferred_________ mg (53) mg of tablet powder to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Procedure:

Injected blank, Placebo, standard solution and sample solution and checked the interference due to blank and Placebo.

Acceptance criteria

No interference should be observed at a retention time of the Azithromycin peak from Blank and Placebo.

Linearity

The linearity study for Assay of Azithromycin tablets was performed at various concentrations level: 80%, 90%, 100%, 110%, and 120% of the standard concentration.

Prepared the following solutions for the linearity study:

Preparation of Blank: Used diluent as blank.

Linearity solution at 80% level

Transferred_________ mg (32) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Linearity solution at 90% level

Transferred_________ mg (36) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Linearity solution at 100% level

Transferred_________ mg (40) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Linearity solution at 110% level

Transferred_________ mg (44) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Linearity solution at 120% level

Transferred_________ mg (48) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Procedure:

Inject blank, Placebo, five replicates injection of standard solution and checked the acceptance criteria for system suitability, injected linearity solutions in triplicate and calculated correlation coefficient.

Serial Number	Sample Identification	Result
1.	80%
2.	90%
3.	100%
4.	110%
5.	120%
Correlation Coefficient (R2)

Accuracy

The accuracy of the analytical method for the determination of the assay of Azithromycin in Azithromycin Tablets was studied in terms of recovery. A recovery study was performed for the content of Azithromycin at 80%, 100% and 120% of test concentration.

Preparation of Blank: Used diluent as blank.

Standard Preparation:

Transferred_________ mg (40) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Linearity solution at 80% level

Transferred_________ mg (32) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Linearity solution at 100% level

Transferred_________ mg (40) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Linearity solution at 120% level

Transferred_________ mg (48) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

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Precision

System Precision

Preparation of Blank: Used diluent as blank.

Preparation of Placebo

Transferred_________ mg (53) mg of placebo powder to a 100 mL volumetric flask, added _______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Standard Preparation:

Transferred_________ mg (40) mg of Azithromycin dihydrate SRS, to a 100 mL volumetric flask, added_______ ml (70 mL) of diluent, sonicated to dissolve and diluted with diluent to volume.

Acceptance criteria: Acceptance criteria for system suitability should pass.

Observation

RSD of replicate injections for standard preparation is _________ (NMT - 2.0%)

Theoretical plates for standard peak ______________ (NLT 1000)

Tailing factor for standard peak___________________ (NMT 2.0).

Repeatability

Method: Refer Method of Analysis

Acceptance Criteria: Relative Standard Deviation, Not More Than 2.0%.

Preparation – 1:- ___________ mg Dissolve in 100 ml

Preparation – 2:- ___________ mg Dissolve in 100 ml

Preparation – 3:- ___________ mg Dissolve in 100 ml

Preparation – 4:- ___________ mg Dissolve in 100 ml

Preparation – 5:- ___________ mg Dissolve in 100 ml

Preparation – 6:- ___________ mg Dissolve in 100 ml

Calculation:

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Result:

Sr. No.	1	2	3	4	5	6
Result
Average
% RSD

Intermediate Precision

Method: Refer Method of Analysis

Acceptance Criteria: Relative Standard Deviation, Not More Than 2.0%.

Preparation – 1:- ___________ mg Dissolve in100 ml

Preparation – 2:- ___________ mg Dissolve in 100 ml

Preparation – 3:- ___________ mg Dissolve in 100 ml

Preparation – 4:- ___________ mg Dissolve in 100 ml

Preparation – 5:- ___________ mg Dissolve in 100 ml

Preparation – 6:- ___________ mg Dissolve in 100 ml

Calculation:

Result:

Sr. No.	1	2	3	4	5	6
Result
Average
% RSD

Details of Analysts who did the Precision test study

Details	Repeatability	Intermediate Precision
Analyst
Date

Result of Repeatability & Intermediate Precision:

% Assay of Azithromycin
Repeatability
Intermediate Precision
AVERAGE
SD
% RSD

SUMMARY

CONCLUSIONS

________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

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