QualityGuidances

Analytical Method Validation Protocol FOR ASSAY OF Esomeprazole Capsules (40mg)

(As per ICH guidelines)

(BY HPLC)

Quality Control Department

CONTENTS

Sr. No	Contents	Page No.
A	Post-Approval	3
B	Objective	3
C	Method Summary	3
D	Scope	3
E	Responsibility	4
F	Method of Analysis	5
G	Summary Capsule	7
1	Specificity	7
2	Precision-Method Precision	8
	Intermediate Precision	10
3	Solution Stability	13
H	Conclusion	14

 POST APPROVAL:

·  Analytical Method Validation Protocol Number
·            Validation Frequency	Analytical Methods should be re-validated after any significant change in analytical method.
	Designation	Name of the Person	Sign /Date
·  Prepared By
·  Checked By
·  Approved By

 

1. OBJECTIVE: The efficacy & safety of a medicinal product can only be assured by analytical monitoring of its quality. This report is designed to validate the HPLC Assay Method for Esomeprazole capsule Guidelines for routine analysis.
2. METHOD SUMMARY:

The analytical method for assay of Esomeprazole Capsules by HPLC shall be validated as per ICH Guidelines for routine analysis.

1. SCOPE : The Quantity determination of Esomeprazole Capsules by HPLC shall be validated for following analytical and performance parameters as described in ICH guidelines.
2. Specificity
3. Precision
4. Solution Stability
5. RESPONSIBILITY:

Quality Control

 To prepare the analytical method validation Report for the assay of Esomeprazole

To validate the analytical method for the assay of Esomeprazole Capsules

 To prepare analysis record based on the results and observations.

Quality Assurance Department

 To approve the analytical method validation report.

PRODUCT DETAILS:

Product Name	Esomeprazole Capsules
Label Claim	Each hard gelatin capsule Contains : Esomeprazole Magnesium IP Eq. to Esomeprazole          40mg

1. METHOD OF ANALYSIS (BY HPLC):-

Equipment required:

• HPLC system UV/Visible detector model- Agilent 1260 auto sampler , Instrument ID
• Analytical balance, Make RADWAG, Model-AUX220, Instrument ID No.
• Ultrasonic cleaner, Instrument ID No.

Glass wares required:

• Volumetric Flask 100 & 50 ml
• Beaker
• Measuring Cylinder

Reagent required:

• Water (HPLC grade)
• Methanol (HPLC grade)

Working standard: 

• Esomeprazole Magnesium, WS ID No. WS/074 (Valid upto- 13/03/2024), Purity-      45% (on as such basis)

               Assay:

Solvent mixture. Dissolve 5.24 g of trisodium phosphate in water, add 110 ml of 0.5 M di-sodium hydrogen phosphate and make up the volume to 1000 ml with water and adjusted to pH 11.0 with 0.1 M sodium hydroxide solution.

Test solution. Weigh and mix the contents of 20 capsules. Disperse a quantity of the mixed content containing about 20 mg of esomeprazole with 60 ml of the solvent mixture, shake for 20 minutes to dissolve the pellets. Add 20 ml of methanol and mix with the aid of ultrasound and dilute to 100.0 ml with the solvent mixture. Dilute 2.0 ml of the solution to 10.0 ml using water, filter. Store the solution protected from light.

Reference solution. Dissolve 10 mg Omeprazole IPRS in 10 ml methanol, add 40 ml solvent mixture and further dilute with water to obtain a concentration of 0.004 per cent of Eomeprazole.

Chromatographic system

a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porus silica (5 µm).

Mobile phase: a mixture of 350 ml of acetonitrile, 500 ml of buffer solution prepared by mixing 10.5 ml 1.0 M sodium dihydrogen orthophosphate and 60.0 ml 0.5 M disodium hydrogen orthophosphate in 1000 ml of water, adjusted to pH 7.3 and finally diluted to 1000 ml with water,

Flow rate: 1 ml per minute, spectrophotometer set at 302 nm,

Injection volume: 20 ul.

Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injection is not more than 2.0 per cent.

Inject the reference solution and the test solution

Calculate the content of Esomeprazole in the capsules

Inject the reference solution and the test solution.

Spl area     Std. Wt.       2          100             10       Potency

Esomeprazole          ————–X———-X——–X——–X————-X——–XAvg. Fill                                                                                      Std. Area           100           10        Test Wt       2         100

 

=                                      /40 x100=                      %

 

1. SUMMARY CAPSULE:

                                                              Analytical Performance Parameters

Sr. No	Validation Parameters	Acceptance Criteria	Observations
1.	Specificity	There should not be any peaks of Blank, Placebo at main peak retention time
2	Precision
	System Precision
	% Relative standard deviation	Not More Than 2.0
	(i) Method Precision
	% Relative standard deviation	Not More Than 2.0
	(ii) Intermediate Precision
	% Relative standard deviation	Not More Than 2.0
	Overall % Relative standard deviation	Not More Than 2.0
3.	Stability of Solution	The period of time a solution can be held before analysis without compromising accuracy. This delay is beyond that included in the method procedure and anticipates unexpected instrumental delays.

1. SPECIFICITY:

Check the interference of diluents and placebo (as preservatives) by individual injections

Blank: as Methanol

Placebo Preparation: A placebo solution was prepared same as the formulation except for the addition of the active ingredients. Here, the product contains no inactive ingredients. So, here the diluents are used as the placebo solution. Area at 302 nm, Observation Result: Nil

Conclusion for Specificity:

We observed that at wavelength 302 nm there is no significant area for Blank and placebo at main peak of Esomeprazole. And inject the different dilution of Esomeprazole for better clarity to identify the peaks of both. Therefore specificity of the method considered accept capsule.

2.   PRECISION-

Precision in the measure of how close the data value to each other for a number of measurements under the same analytical conditions

2. (i) METHOD PRECISION:

(i) Analyst (I): –

Sample Dilutions: Dissolve 10 mg Esomeprazole RS in 10 ml methanol, add 40 ml solvent mixture and further dilute with water to obtain a concentration of 0.004 per cent of Eomeprazole.

  Test Data Collection:-

Sr. No.		Standards		Area  of Esomeprazole
1.		Standard-1
2.		Standard-2
3.		Standard-3
4.		Standard-4
5.		Standard-5
6.		Mean
7.		%RSD
Acceptance Criteria: RSD is not more than 2.0%
Samples			Sample Area		Mean
			Esomeprazole		Esomeprazole
A	T1
	T2
B	T1
	T2
C	T1
	T2

                        Capsule for Triplicate Assays: Esomeprazole 20mg

Sample Number	Estimated % Amount	Mean	RSD
	Esomeprazole	Esomeprazole	Esomeprazole
Sample A
Sample B
Sample C

 Acceptance Criteria: NMT 2% (% of Relative Standard Deviation)

 Conclusion for precision:

The overall % Relative standard deviation for 1.12% respectively of Esomeprazole in Capsules, there is no significant difference. Therefore Repeatability of the method considered accepts Capsule as it well within 2 % Relative Standard Deviation.

6. (ii) INTERMEDIATE PRECISION:

(Within laboratory variations such as different days, analyst & equipments): 

(ii) Analyst (II): 

Sample Dilutions: Dissolve 10 mg Esomeprazole RS in 10 ml methanol, add 40 ml solvent mixture and further dilute with water to obtain a concentration of 0.004 per cent of Eomeprazole Test Data Collection:-

Acceptance Criteria: RSD is not more than 2.0%
Samples		Sample Area	Sample Area	Mean Mean
		Esomeprazole		Esomeprazole
A	T1
	T2
B	T1
	T2
C	T1
	T2

 

Capsule for Triplicate Assays:

Sample Number	Estimated % Amount Estimated % Amount	Mean		RSD	RSD
	Esomeprazole Ambroxol HCL	Esomeprazole	Ambroxol HCL	Esomeprazole	Ambroxol HCL
Sample A
Sample B
Sample C

Acceptance Criteria: NMT 2% (% of Relative Standard Deviation)

Conclusion for precision:

The overall % Relative standard deviation for 1.34% respectively of Esomeprazole in Capsules, there is no significant difference. Therefore Repeatability of the method considered accepts Capsule as it well within 2 % Relative Standard Deviation.

Capsule for Six Replicate Assays analyst by two different Analysts & days:

Test Data analyst by :-   Standard Data Collection:-

Sr. No.	Standards	Area  of Esomeprazole
1.	Standard-1
2.	Standard-2
3.	Standard-3
4.	Standard-4
5.	Standard-5
6.	Mean
7.	%RSD
Acceptance Criteria: RSD is not more than 2.0%

Capsule for Six Replicate Assays:

Sample Number	Estimated % Amount	Mean	RSD
	Esomeprazole	Esomeprazole	Esomeprazole
Sample A
Sample B
Sample C

 Test Data analyst by :-

Test Data Collection:-

Sr. No.	Standards	Area  of Esomeprazole
1.	Standard-1
2.	Standard-2
3.	Standard-3
4.	Standard-4
5.	Standard-5
6.	Mean
7.	%RSD
Acceptance Criteria: RSD is not more than 2.0%

 Capsule for Six Replicate Assays:

Sample Number	Estimated % Amount	Mean	RSD
	Esomeprazole	Esomeprazole	Esomeprazole
Sample A
Sample B
Sample C

Acceptance Criteria: NMT 2% (% of Relative Standard Deviation).

Conclusion for Intermediate Precision:

The overall % Relative standard deviation of two different analysts is 1.32% & 1.34% respectively of Esomeprazole Capsules there is no significant difference between two analysts within laboratory variations such as different days, analyst & equipments. Therefore reproducibility of the method considered to be accepting Capsule.

7. Stability of Solution:

The robustness of an analytical method should show the reliability of an analysis with respect to deliberate variations in method parameters. The variation used for the study is:

• Change in ratio of the mobile phase. Record the observation in below observation Capsule.
• Change in Flow rate of mobile phase. Record the observation in below observation Capsule.

8. CONCLUSION:

 All the analytical parameter are checked as per the approved validation process and found well within specified acceptance criteria. Hence, it is concluded that, this method is suitable Capsule for accurate & precise results for routine analysis.