ANALYTICAL METHOD

VALIDATION PROTOCOL

FOR

(Azithromycin 250 Tablets IP)

 

Product Name	Batch No	Mfg Date	Exp. Date

Pre Approval: 

	PREPARED BY	CHECKED BY	APPROVED BY
NAME
DESIGNATION
SIGNATURE
DATE

CONTENTS

 

S.No.	Table of Content	PAGE
	Report Preparation	1
	Pre Approval	2
	Contents	3
1	Purpose	4
2	Scope	4
3	References	4
4	Responsibility	4
5	Method used for validation	4
5.1	Chromatography  conditions	4
5.2	Procedure for Reference standard and Test Sample	5
6	Batches used for validation	5
7	Validation	6
7.1	Specificity	6
7.2	Linearity	7
7.3	Precision	8-10
7.4	Accuracy	10-11
7.5	Stability in analytical solution	12-13
8	Acceptance criteria	13-14
9	Compilation of Report	14
10	Summary & Conclusion	15
11	Certification	15
12	Post Approval	16

 

1. PURPOSE:

To perform validation of the HPLC method used for the determination of the assay of the active ingredient (Azithromycin) in Azithromycin 250 mg tablet.

 

2. Scope :

This applies to the testing of all Azithromycin tablet manufactured at the Solan plant.

 

3. References :

Text on validation of analytical procedures – ICH Q2A

Validation of analytical procedures: Methodology – ICH Q2B

Validation of compendial methods USP40

 

4. RESPONSIBILITY:

Performing validation: Officer/Executive – QA & QC

Checking validation data:  Head – QC

Approval of validation protocol / report:  Head – QA

 

• Method used for validation:

         ASSAY: (AZITHROMYCIN)

5.1   CHROMATOGRAPHIC CONDITIONS

Instrument                        :  Agilent High Performance Liquid Chromatography

Column                : A Stainless steel Column 25 cm x 4.6 mm, packed with end capped polar      embedded octadecylsilyl amorphous organosilica polymer (5μm)

Wavelength          :   215 nm

Colum Temp        :   70°

Flow Rate             :   1.0 ml/ minute

Injection volume  :   100 μl

Mobile Phase        :   Buffer: Acetonitrile: Water (10:35:55)

Solvent mixture    :  40 volumes of acetonitrile and 60 volumes of water.

System suitability :  Relative standard deviation for replicate injection should be not more                                                                        than 2.0%                

 

        Buffer Preparation: 3.484 per cent w/v solution of dipotassium hydrogen phosphate with the pH adjusted to     6.5 with orthophosphoric acid.

 5.2      PROCEDURE FOR REFERANCE STANDARD AND SAMPLE PREPARATIONS

Following test will be consider during Method validation

1. Identification
2. Related Substances
3. Dissolution Test
4. Assay

Type of Analytical Procedure	Identification	Testing of  impurities		Assay, Dissolution ( Measurement only, Content/Potency)
Characteristics		Quantitative	Limit
Accuracy	No	Yes	NO	YES
Precision
Repeatability	No	Yes	NO	Yes
Interm. Precision	No	Yes	NO	Yes
Specificity (2)	Yes	Yes	Yes	Yes
Detection Limit	No	NO	Yes	No
Quantization Limit	No	Yes	No	No
Linearity	No	Yes	No	Yes
Range	No	Yes	No	Yes

TEST METHOD:

Identification Test:

In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.

Dissolution:

            Apparatus: Paddle

            Medium: 900 ml of a buffer solution prepared by adding to 6 liters of 0.1 M dibasic sodium phosphate about 40 ml of hydrochloric acid, adjusting the pH to 6.0, adding 600 mg of trypsin, and mixing.

            Time: 45 minutes

            Speed: 100 rpm

            Withdraw a suitable volume of the medium and filter through a filter.

 

 

Chromatographic system:

• A stainless steel column 25 cm X 4.6 mm packed with end capped polar embedded octadecylsilyl amorphous organosilica polymer (5µm), (such as waters Xterra),
• Mobile phase: a mixture of 10 volumes of a 3.484 per cent w/v solution of dipotassium hydrogen phosphate with the pH previously adjusted to 6.5 with O.P.A., 35 volumes of acetonitrile and 55 volumes of water,
• Flow rate: 1 ml per minute,
• Column temperature:70°
• Spectrophotometer set at 215 nm,
• Injection volume: 100 µl

Test solution: The filtrate from the dissolution medium suitably diluted with the mobile phase, if necessary.

Reference solution: A solution of Azithromycin reference standard in the dissolution medium suitable diluted with the mobile phase to obtain a solution having the same concentration as that of the test solution.

  Formula:

            Area of test

  —————– X STD Dilution X Test Dilution X Potency of STD X 100

Area of STD

            Limit: N.L.T. 75% + D of labeled claim.

Related substance:

Determined by liquid chromatography.

            Note: Prepare the solution immediately before use.

Solvent mixture: Prepare a 0.173 per cent w/v solution of ammonium dihydrogen phosphate with the pH adjusted to 10.0 with strong ammonia solution. Transfer 350 ml of this solution add 300 ml of acetonitrile and 350 ml of methanol. Mix well.

Test solution: Weigh and powder 20 tablets. Weigh a quantity of the powder containing about 0.2 g Azithromycin, dissolve in the solvent mixture by shaking mechanically, dilute to 25.0 ml with the solvent mixture and filter.

Reference solution (a): A 0.008 per cent w/v solution of Azithromycin reference solution in the solvent mixture.

Reference solution (b): A solution containing 0.01 per cent w/v of Azithromycin reference standard and Azithromycin impurity A reference standard in the solvent mixture.

Chromatographic system:

• A stainless steel column 25 cm X 4.6 mm packed with end-capped octadecylsilyl amorphous organosilica polymer for mass spectrometry (5µm) (such as waters Xterra),
• Column temperature: 60°
• Mobile phase: A. 0.18 per cent w/v solution of anhydrous disodium hydrogen phosphate with the pH adjusted to 8.9 with dilute O.P.A. or with dilute sodium hydrogen solution.

1. a mixture of 25 volumes of methanol and 75 volumes of acetonitrile,

• Flow rate: 1 ml per minute,
• A gradient programme using the conditions given below,
• Spectrophotometer set at 210 nm,
• Injection volume: 50 µl

 

Time (in min.)	Mobile phase( per cent v/v)		Mobile phase ( per cent v/v)
0	50		50
25	45		55
30	40		60
80	25		75
81	50		50
93	50		50
Name		Relative retention time		Correction factor
Azithromycin impurity L1		0.29		2.3
Azithromycin impurity M2		0.37		0.6
Azithromycin impurity E3		0.43		–
Azithromycin impurity F4		0.51		0.3
Azithromycin impurity D5		0.54		–
Azithromycin impurity J6		0.54		–
Azithromycin impurity I7		0.61		–
Azithromycin impurity C8		0.73		–
Azithromycin impurity N9		0.76		0.7
Azithromycin impurity H10		0.79		0.1
Azithromycin impurity A11		0.83		–
Azithromycin impurity P		0.92		–
Azithromycin impurity O12		1.23		–
Azithromycin impurity G13		1.26		0.2
Azithromycin impurity B14		1.31		–

Inject reference solution (b). The chromatogram obtained shows peaks corresponding to Azithromycin and Azithromycin impurity A. The test is not valid unless the resolution between these two peaks is at least 7.0.

Inject reference solution (a) and the test solution. In the chromatogram obtained with the test solution with the test solution, the area of any secondary peak eluting with a relative retention time of about1.3 due to Azithromycin impurity B is not more than twice the area of principal peak in the chromatogram obtained with reference solution (a) (2.0 per cent). The sum of the areas of all the other secondary peaks is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (3.0 per cent). Ignore any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with   reference solution (a) (0.1 per cent); ignore the peaks eluting before Azithromycin impurity L and after Azithromycin impurity B.

11.0)    Assay: Determined by liquid chromatography.

Solvent mixture: 40 volumes of acetonitrile and 60 volumes of water.

Test solution: Weigh and powder 20 tablets. Weigh quantity of the powder containing 0.1 g of Azithromycin dissolve in the solvent mixture by shaking mechanically, dilute to 100 ml with the solvent mixture and filter.

Reference solution (a): A 0.1 per cent w/v solution of Azithromycin reference standard in the solvent mixture.

Reference solution (b): A solution containing 0.01 per cent w/v of Azithromycin reference standard and Azithromycin impurity A reference standard in the solvent mixture.

Chromatographic system:

• A stainless steel column 25 cm X 4.6 mm packed with end capped polar embedded octadecylsilyl amorphous organosilica polymer (5µm), (such as waters Xterra),
• Mobile phase: a mixture of 10 volumes of a 3.484 per cent w/v solution of dipotassium hydrogen phosphate with the pH previously adjusted to 6.5 with O.P.A., 35 volumes of acetonitrile and 55 volumes of water,
• Flow rate: 1 ml per minute,
• Column temperature:70°
• Spectrophotometer set at 215 nm,
• Injection volume: 100 µl

Inject reference solution (b). The chromatogram obtained shows peaks corresponding to Azithromycin and Azithromycin impurity A. The test is not valid unless the resolution between these two peaks is at least 7.0.

Inject reference solution (a) and the test solution.

Calculate the content of Azithromycin in the tablets.

  Formula:

            Area of test

  —————– X STD Dilution X Test Dilution X Potency of STD X Average weight

Area of STD

Acceptance criteria: 90.0%-110.0%

 

 Batches used for validation :

 

Take sample of the Azithromycin 250 Tablets during the method validation.

 

Batch no.	Mfg. date	Exp. date	Claimed content each tablet
			Azithromycin 250 mg

7. VALIDATION

 

Perform validation as per procedure given below. Record all weights and dilutions. Perform calculations   using calculator or Microsoft Excel. Record the results and observations in the given formats. Compare the             results with acceptance criteria.

 7.1      SPECIFICITY

7.1.1   Non-interference from the Excipients

Prepare the placebo granules of the Azithromycin Tablets except the active ingredients. Inject the             blank (diluent), solution of the placebo granules in diluent, standard solution of active ingredients (Azithromycin) and solution of placebo granules with active ingredients in diluent. Record all      the peaks and their corresponding wavelengths.

 

Placebo solution:

Standard solution:

Azithromycin

 

Solutions  details	Wavelength maxima (in nm )	Area
Placebo
Standard 1
Azithromycin Tablets

7.2      LINEARITY

Prepare at least 5 different concentrations of standard solution of the active ingredients with the mean final concentration of 10 ppm for  Azithromycin.(It should be cover 80% to 120 % of  final concentration)  Record the area of each             solution at 215 nm, Record the area against the concentration and plot the graph of area against concentration for each solution. Calculate the correlation coefficient.

          Standard 1 Azithromycin responses:

Level	Azithromycin (%)	Final Conc. (ppm)	Area (in AU)
1
2
3
4
5
Correlation coeff. ( r )

 7.3       PRECISION

7.3.1    Repeatability (Intra – Assay precision)

            Repeatability is the precision under the same operating conditions over a short interval of time.

Repeatability is usually demonstrated by repeated measurement of a single sample ( e.g. use of the     analytical procedure within a laboratory over a short period of time using the same analyst with the same equipment)

Make 5 replicate area of the standard solution at working concentration (i.e. 10 ppm for Azithromycin). Record the area of the standard solution for Azithromycin .and at 215 nm, and Calculate the mean and RSD.

Standard preparation (______ ppm)

Replicate	Time	RS Value	S Value	Assay	RSD
1.
2.
3.
4.
5.
Mean
SD
%RSD

7.3.2    Intermediate Precision

Intermediate precision expressed within laboratory variations: different days,different analyst or equipment,

Intermediate precision is usually demonstrated by repeated measurement of the sample used in the repeatability experiment within the same laboratory.

Make sample solutions & reference standard and analyze by the method at different days, different analyst different equipment. Calculate the assay for each analysis. Calculate the mean and RSD.

Standard preparation –

Mean Area        _____________

Sample preparation –

Calculation –

            Different days:

Time Interval	Wight (mg)		RS Value	S Value	Assay	RSD
	Std.	Sample
Days 1
Days 2
Days 3
RSD: NMT 2 %

Different Analyst

Analyst Details	Wight (mg)		RS Value	S Value	Assay	RSD Value
	Std.	Sample
Analyst- I
Analyst- II
Analyst- III
RSD Value : NMT 2 %

Different Equipment

Equipment ID	Wight (mg)		S Value	Assay	RSD Value
	Std.	Sample
HPLC – I
HPLC – II
RSD Value : NMT 2 %

 7.3.3    Intermediate precision (Ruggedness)

Analysis of a pooled sample to be performed in triplicate by 2 different chemists. Calculate the assay for each analysis. Calculate the mean and RSD.

Standard preparation (Chemist 1) –

Mean Area        _____________

Standard preparation (Chemist 2) –

Mean Area       _____________

Sample preparation (Chemist 1)–       

            Sample preparation (Chemist 2)–

Calculation –

           Component 1: Azithromycin

 

Chemist 1	Assay no.	weight (mg)		Area		Assay (mg)	Assay (%)
		Std.	Sample	Std.	Sample
	1
	2
	3
Chemist 2	4
	5
	6
Mean
SD
RSD

 7.4       ACCURACY

It describes the deviation from the standard or expected result.

The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or as an accepted reference value and the value found.  Spike the placebo at label claim with known amount of the active ingredient at 3 levels each in triplicate,     i.e. 3 x 70%, 3 x 100% and 3 x 130% of labeled amount. analyse each sample. Calculate the assay for each     analysis. From the amount found and the amount added, calculate % recovery. Calculate mean recovery and             its RSD.

Standard preparation –

Mean Area –  _____________

Sample preparation –

             Calculation –

          Azithromycin:

 

Recovery Level	Standard Area	Recovery
		mg	%	Mean	SD	%RSD
70%
100%
130%

 

8.0      Acceptance criteria

 8.1     Specificity

 

Non-interference from the excipients and solvents in the spectrum of the active

Ingredients   with   placebo   &   solvent,    placebo (excipients) and   solvent

(i.e. diluent) do not interfere   with   the active drugs (Azithromycin) peak.

8.2       Wavelength maxima of each active ingredient do not interfere with each other.

8.3       Linearity

Response is linear and correlation coefficient is not less than 0.999

8.4       Precision

8.4.1    System precision

RSD for area of each active ingredient is not more than 2%.

8.4.2    Precision (Repeatability)

RSD for assay values is not more than 2%.

8.4.3    Intermediate precision (Ruggedness)

RSD for assay values is not more than 2%.

8.5       Accuracy

Mean recovery of assay found against added amount is between 98 to 102%. RSD of individual recoveries is not more than 2%.

8.6       Stability in analytical solution

Recovery after 24 hours is between 98 to 102% of the initial value.

9. Compilation of REPORT

Prepare a report based on all above data. Attach linearity graphs and raw data. Based on the results,             comment on the following –

• Specificity of the method
• Suitability of method
• Linearity, precision and accuracy over the analytical range
• Duration for which active ingredient (Azithromycin) is stable in solution
• Pertaining to the proposed HPLC method for routine testing of the Azithromycin Tablet (250 mg)

SUMMARY

 

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CONCLUSION

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CERTIFICATION

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Post Approval:

 

	PREPARED BY	CHECKED BY	APPROVED BY
NAME
DESIGNATION
SIGNATURE
DATE