ANALYTICAL METHOD VERIFICATION PROTOCOL OF ASCORBIC ACID

nirvesh400

Analytical Method Verification Protocol Cum Report

  ASCORBIC ACID TABLETS IP (500mg)

(As per ICH guidelines)

This document is an exercise on Analytical Method Validation of the various analytical

Methods used in determination of active ingredients in Quality Control Laboratory of

 

CONTENTS

Sr. No Contents Page No.
A Pre-Approval 3
B Objective 3
C Method Summary 3
D Scope 3
E Responsibility 4
F Method of Analysis 5
G Summary Tablet 7
1 Specificity 7
2 System accuracy 8
3 Precision       9
           (i) Method Precision 9
           (ii) Intermediate Precision 10
4 Robustness 13
5 Solution Stability 15
6 Conclusion       15
7 Post-Approval 16

 CONTRIBUTIONS:

This protocol is a team effort of Quality control Laboratory chemists to achieve the objective of validating the analytical methods carried out to estimate the contents of pharmaceutical products manufactured by

 

  1. PRE- APPROVAL:
·           Analytical

Method Validation

Protocol Number

 AMV/QC/018
·            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 Ascorbic acid Tablet Guidelines for routine analysis.
  2. METHOD SUMMARY: The analytical method for assay of Ascorbic acid Tablet by HPLC shall be validated as per ICH Guidelines for routine analysis.
  3. SCOPE: The Quantity determination of Ascorbic acid Tablet by HPLC shall be validated for following analytical and performance parameters as described in ICH guidelines.
  4. Specificity
  5. Precision
  6. Solution Stability
  7. RESPONSIBILITY:

 Quality Control

 To prepare the analytical method validation Report for the assay of Ascorbic acid

To validate the analytical method for the assay of ascorbic acid Tablet

 To prepare analysis record based on the results and observations.

Quality Assurance Department

 To approve the analytical method validation report.

PRODUCT DETAILS:

Product Name

 

 Ascorbic acid Tablet

 
Label Claim

 

 

 

 Each uncoated tablet Contains :

Ascorbic acid IP——-500mg

 

 

 

 

 

  1. METHOD OF ANALYSIS (BY HPLC):

  Equipment required:

  • .HPLC system UV/Visible detector model- Agilent 1260 auto sampler, Instrument ID   SC/QC/038.
  • Analytical balance, Make RADWAG, Model-AUX220, Instrument ID No. SC/QC/002
  • Ultrasonic cleaner, Instrument ID No. SC/QC/010.

Glass wares required:

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

Reagent required:

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

Working standard: 

  • Ascorbic acid Magnesium, WS ID No. WS/309 (Valid upto-……….),Purity-                   39% (on as such basis)

 Assay:

Determine by liquid chromatography.

Test solution: weigh and powdered 20 tablets. Disperse a quantity of powdered containing 25 mg of ascorbic acid in 60 ml  of solvent mixture shake mechanically for 15 min and dilute to 100.0 ml with solvent mixture , filter.

Reference solution: A0.025 per cent w/v solution of ascorbic acid  IPRS in the solvent mixture.

Chromatography Condition

-a stainless steel column  25 cm X 4.6 mm , packed with octadevylsilane bonded to porous silica (5 µl)

-mobile phase: a buffer solution prepared by dissolving 2.04 g of monobasic potassium phosphate in 100 ml water, adjust to pH 3.0 with OPA.

-Flow rate : 1 ml /min

-Spectrophotometer set at 245 nm,

-Injection volume 5 µl

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

Inject the reference solution and test solution

Calculate the content of Vitamin C (Ascorbic acid) in the tablets

                                                                                                                                                            Spl area     Std. Wt.       2          100             10       Potency

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

 

=                                      /500 x100=                      %

 

  1. SUMMARY TABLET:

                                                        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,

2.                  SYSTEM ACCURACY:

(a)               The system precision of the above method was carried out by taking area for six injection of the  standard preparation of exact weight. Text data collection sheet:

Serial No.   Area  of Ascorbic acid
1.
2.
3.
4.
5.
Mean
% RSD

Acceptance Criteria: RSD is not more than 2.0%

Observation Result:

Conclusion for Specificity:

We observed that at wavelength 245 nm there is no significant area for Blank and placebo at main peak of Ascorbic acid. And inject the different dilution of Ascorbic acid for better clarity to identify the peaks of both. Therefore specificity of the method considered accepts Tablet.

3.                  PRECISION:

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

(i)

(ii)               Method precision:

(i) Analyst (I):

Sample Dilutions: Test solution: weigh and powdered 20 tablets. Disperse a quantity of powdered containing 25 mg of ascorbic acid in 60 ml of solvent mixture shake mechanically for 15 min and dilute to 100.0 ml with solvent mixture, filter.

  Test Data Collection:-

Sr. No. Standards   Area  of Ascorbic acid
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
Ascorbic acid Ascorbic acid
A T1
T2
B T1
T2
C T1
T2

                         Tablet for Triplicate Assays: Ascorbic acid 500mg

Sample Number Estimated % Amount Mean RSD
Ascorbic acid Ascorbic acid Ascorbic acid
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 Ascorbic acid in Tablet, there is no significant difference. Therefore Repeatability of the method considered accepts Tablet as it well within 2 % Relative Standard Deviation.

(ii)                   Intermediate precision:

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

 Analyst (II):

Sample Dilutions:

Test solution: weigh and powdered 20 tablets. Disperse a quantity of powdered containing 25 mg of ascorbic acid in 60 ml of solvent mixture shake mechanically for 15 min and dilute to 100.0 ml with solvent mixture, filter

Test Data Collection:-

Acceptance Criteria: RSD is not more than 2.0%
Samples Sample Area Sample Area Mean

Mean
Ascorbic acid Ascorbic acid
A T1
T2
B T1
T2
C T1
T2

 Tablet for Triplicate Assays:

Sample Number Estimated % Amount

Estimated % Amount

 Mean RSD
Ascorbic acid

Ambroxol HCL

 Ascorbic acid Ascorbic acid
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 Ascorbic acid in Tablet, there is no significant difference. Therefore Repeatability of the method considered accepts Tablet as it well within 2 % Relative Standard Deviation.

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

Test Data analyst by:

Standard Data Collection:-

Sr. No. Standards   Area  of Ascorbic acid
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%

Tablet for Six Replicate Assays:

Sample Number Estimated % Amount Mean

 

 RSD

 
Ascorbic acid Ascorbic acid

 

 Ascorbic acid

 
Sample A
Sample B
Sample C

 

Test Data analyst by:

Test Data Collection:-

Sr. No. Standards   Area  of Ascorbic acid
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%

 

 

Tablet for Six Replicate Assays:

Sample Number Estimated % Amount Mean

 

 RSD

 
Ascorbic acid Ascorbic acid

 

 Ascorbic acid

 
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 Ascorbic acid Tablet 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 Tablet.

  1. ROBUSTNESS:

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

Observation Table:-

Change ratio in the mobile phase at 245nm

Change in flow rate at 245nm
Mobile Phase Flow rate (ml/min) System suitability
Buffer Solution   Retention Time Theoretical Plates Tailing Factor
1000 ml 0.8 ml/min
1000 ml 1.0 ml/min
1000 ml 1.2 ml/min

 Acceptance criteria:

Analytical method validation shall be robust (i.e. Theoretical Plates is not less than 2000 & tailing factor is not more than 2.0).

Conclusion for Robustness:

There is no significant difference for Ascorbic Acid for different conditions, such as composition & Flow rate of mobile phase. Therefore Robustness of the method considered acceptable.

  1. 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 Tablet.
  • Change in Flow rate of mobile phase. Record the observation in below observation Tablet.
    1. 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 FOR Tablet for accurate & precise results for routine analysis.

  1. POST- APPROVAL:
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  

 

 

***

Analytical Method Validation