ANALYTICAL METHOD VERIFICATION PROTOCOL OF ASSAY OF CHLORPHENAMINE 4MG TABLETS

ANALYTICAL METHOD VERIFICATION PROTOCOL OF ASSAY OF CHLORPHENAMINE 4MG TABLETS

Superseded Protocol No. Nil
Effective Date  

Table of contents :

Sr. No. Subject Page No.
  Protocol Approval
  Objective
  Scope
  Responsibility
  Product profile
  Methodology
  Verification parameters
  Incident/Deviation
  Summary/Final conclusion/Recommendation
  Abbreviation
  Revision History
  1. Protocol Approval:

Prepared By:

Functional Area Name Designation Signature / Date
Quality Control      

Reviewed By:

Functional Area Name Designation Signature / Date
Quality Assurance      
Head Quality Control      

Approved By:

Functional Area Name Designation Signature / Date
Head QA      

Authorized By:

Functional Area Name Designation Signature / Date
Head Quality      
  • Objective:

The objective of this protocol is to verify the suitability of assay of Chlorphenamine 4 mg tablets by considering thefollowing parameters:

Parameters Chlorphenamine
Specificity  
Precision
Method Precision  
Intermediate Precision  
  • Scope :

This protocol is applicable for the verification of assay of Chlorphenamine 4 mg tablets.

  • Responsibility of Validation Team:
Departments Responsibilities
QC Preparation & Review of Protocol.
Analysis of samples and recording of data.
Compilation and checking of data
Preparation of Summary Report.
To impart training of protocol to concerned department/persons.
QA Review and approval of protocol.
Co-ordination with QC to carryout Validation.
Review of data and summary report.
Head Quality Authorization of protocol.
  • Product Profile:
Category  Antihistamine
Reason for verification First verification
Active Ingredient Chlorphenamine Maleate
Strength Each tablet contains: Chlorphenamine Maleate           4 mg
Method Reference
Specification Limits Assay Each tablet contain: Chlorphenamine Maleate              4 mg Limit : 3.70 mg – 4.30 mg (92.5% to 107.5 %) 
  • Methodology:

 Chemical, reagents and filters:

Table 1.0: Chemical, reagents and filters

Sr. No Material /Chemicals/Filters Grade
1. Sulphuric Acid Merck or equivalent
2. Ether Merck or equivalent
3. Water UV Grade or Milli Q water
4. Sodium Hydroxide Pellets Merck or equivalent
5. Litmus Paper NA

Preparation of 1M Sodium Hydroxide:

Weigh and dissolve 4.0 g of Sodium Hydroxide pallets in 100 ml of water.

Preparation of 0.25M Sulphuric Acid:

Take 13.6 ml of 18.3M Sulphuric Acid and dilute to 1000 ml of water.

Preparation of 0.05M Sulphuric Acid:

Take 2.7 ml of 18.3M Sulphuric Acid and dilute to 1000 ml of water.

Test preparation:

Weigh and powder 20 tablets. Shake a quantity of the power containing 3 mg of Chlorphenamine Maleate with 20 ml of 0.05M Sulphuric acid for 5 minutes, add 20 ml of ether, shake carefully and filter the acid layer into a second separating funnel.  Extract the ether layer with two 10 ml quantities of 0.05M Sulphuric acid, filter each acid layer into the second separating funnel and wash the filter with 0.05M Sulphuric acid.  Make the combined acid extracts and washings just alkaline to litmus paper with 1M Sodium Hydroxide, add 2 ml in excess and extract with two 50 ml quantities of ether.  Wash each ether extract with the same 20 ml of water and extract with successive quantities of 20, 20 and 5 ml of 0.25M Sulphuric acid.  Dilute the combined acid extracts to 50 ml with 0.25 M Sulphuric acid, dilute 10ml to 25 ml with 0.25M Sulphuric acid and measure the absorbance of the resulting solution at the maximum at 265 nm.

Calculate the content of Chlorphenamine Maleate, taking 212 as the value of A (1%, 1cm) at the maximum at 265 nm.

Required powder weight:

         Average wt x Equivalent wt.

=   ———————————————-

                           LC

                       TAbs                50                25            Avg. wt

% Assay = ————— x ————- x ————- x ————- x 1000

                   E1% value         Spl wt.         10              LC

Where,

TAbs: Test absorbance

E1% value: 212

Spl wt.: weight of sample

Avg. wt.: Average weight

LC: Label claim

  • Verification parameters:

The following parameters to be perform for the Verification activity.

      Specificity

      Precision

  • System Precision
  • Method Precision (Analyst I)
  • Intermediate Precision (Analyst II)
    • Specificity:

Specificity of analytical method is its ability to assess unequivocally the analyte in presence of components that may be expected to be present in the blank and placebo.

Specificity of test method should be established by separate titration of blank solution (diluent), Placebo, sample solution and spiked sample solution. Sample solution shall be prepared as per method of analysis given.

Blank Solution:

Repeated the complete titration without adding sample or placebo,  shake a quantity with 20 ml of 0.05M Sulphuric acid for 5 minutes, add 20 ml of ether, shake carefully and filter the acid layer into a second separating funnel.  Extract the ether layer with two 10 ml quantities of 0.05M Sulphuric acid, filter each acid layer into the second separating funnel and wash the filter with 0.05M Sulphuric acid.  Make the combined acid extracts and washings just alkaline to litmus paper with 1M Sodium Hydroxide, add 2 ml in excess and extract with two 50 ml quantities of ether.  Wash each ether extract with the same 20 ml of water and extract with successive quantities of 20, 20 and 5 ml of 0.25M Sulphuric Acid.  Dilute the combined acid extracts to 50 ml with 0.25 M Sulphuric acid, dilute 10ml to 25 ml with 0.25M Sulphuric acid and measure the absorbance of the resulting solution at the maximum at 265 nm.

Placebo Solution:

Weigh and transfer about 95 mg of placebo powder into separating funnel and shake a quantity with 20 ml of 0.05M Sulphuric acid for 5 minutes, add 20 ml of ether, shake carefully and filter the acid layer into a second separating funnel.  Extract the ether layer with two 10 ml quantities of 0.05M Sulphuric acid, filter each acid layer into the second separating funnel and wash the filter with 0.05M Sulphuric acid.  Make the combined acid extracts and washings just alkaline to litmus paper with 1M Sodium Hydroxide, add 2 ml in excess and extract with two 50 ml quantities of ether.  Wash each ether extract with the same 20 ml of water and extract with successive quantities of 20, 20 and 5 ml of 0.25M Sulphuric acid.  Dilute the combined acid extracts to 50 ml with 0.25 M Sulphuric acid, dilute 10ml to 25 ml with 0.25M Sulphuric acid and measure the absorbance of the resulting solution at the maximum at 265 nm.

Sample Solution: as per methodology

Spike Test solution:

Weigh and transfer about 95 mg of placebo powder and 3.0 mg of Chlorphenamine Maleate WS/CRS into separating funnel and shake a quantity with 20 ml of 0.05M Sulphuric acid for 5 minutes, add 20 ml of ether, shake carefully and filter the acid layer into a second separating funnel.  Extract the ether layer with two 10 ml quantities of 0.05 M Sulphuric acid, filter each acid layer into the second separating funnel and wash the filter with 0.05 M Sulphuric acid.  Make the combined acid extracts and washings just alkaline to litmus paper with 1M Sodium Hydroxide, add 2 ml in excess and extract with two 50 ml quantities of ether.  Wash each ether extract with the same 20 ml of water and extract with successive quantities of 20, 20 and 5 ml of 0.25M Sulphuric acid.  Dilute the combined acid extracts to 50 ml with 0.25 M Sulphuric acid, dilute 10ml to 25 ml with 0.25M Sulphuric acid and measure the absorbance of the resulting solution at the maximum at 265 nm.

Procedure: Blank solution, Placebo solution, sample solution and spiked sample solution shall be titrating as per methodology. Record the titration value obtained from the respective solution and check for system suitability parameters for peak obtained.

Table 2.0: Specificity data

Sr. No Sample Maximum Absorbance at about 265 nm
1 Blank solution  
2 Placebo solution  
3 Sample Solution  
4 Spiked  Solution  

Acceptance Criteria:

  1. There should be no interference of the diluent, placebo with the main analyte response, no maxima should be obtained at wavelength 265 nm
  2. The difference in assay should not be more than 2.0% between sample preparation and spike sample.
  • Precision:
    • Method Precision:

The precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatability to multiple samplings of homogenous sample. It is usually expressed as the standard deviation and the relative standard deviation.

Test Procedure for Assay:

Prepare the six sample of same batch and analyze as per method of analysis, record the absorbance on testing data sheet and calculate the % assay, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.

Table 3.0: Method precision results for Assay

Sample No. Sample wt. (mg) Absorbance % Assay % Mean SD % RSD
1            
2      
3      
4      
5      
6      

Acceptance Criteria:

Calculate the assay for each analysis and calculate the mean, SD and % RSD.

% RSD for assay values should be NMT 2.0%.

  • Intermediate Precision ( Ruggedness ):

Intermediate precision expresses within laboratory variation with different analysts or different/same equipment on different days using same batch of drug product as per method of analysis.

Test Procedure:

Prepare the six sample of same batch and analyze as per method of analysis, record the Absorbance on testing data sheet and calculate the % assay, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.

          Table 4.0: Intermediate precision results for Assay

Sample No. Sample wt. (mg) Absorbance Mean % Assay % Mean SD % RSD
1            
2      
3      
4      
5      
6      

Acceptance Criteria:

Calculate the assay for each analysis and calculate the mean, SD and % RSD.

% RSD for assay values should be NMT 2.0%.

Table 5.0: Pooled results of Assay of analyst – I & analyst – II

Sample No. Sample wt. (mg) % Assay
Chlorphenamine Maleate
ANALYST I
1    
2    
3    
4    
5    
6    
ANALYST II  
1    
2    
3    
4    
5    
6    
Mean    
SD    
Pooled % RSD    

Acceptance Criteria:

The pooled % RSD for assay values of Analyst I and Analyst II should be NMT 2.0%.

  • Incident /Deviation:

Any incident or deviation observed during analytical method verification shall be recorded and reported in verification report.

  • Summary/ Conclusion / Recommendation:

Final conclusion should be drawn from analytical method verification for its use to analyze the assay test of Chlorphenamine by UV.

Summary of verification report shall be prepare and accordingly standard testing procedure to be updated.

Abbreviation

ASS                 :           Assay

            VER                :           Verification

            P                      :           Protocol

            SD                   :           Standard deviation

            UV                   :           Ultraviolet–visible

            mL                   :           Milliliter

            mg                   :           Milligram

            min.                 :           Minutes

            nm                   :           Nanometer

            QA                   :           Quality Assurance

            QC                  :           Quality Control

            %                     :           Percentage

            ºC                    :           Degree centigrade

            hrs                   :           Hours

            µm                   :           Micrometer

            µl                     :           Microlitre

            BP                   :           British Pharmacopoeia

            RSD                :           Relative standard deviation

            NLT                 :           Not less than

NMT                :           Not more than

WS                  :           Working standard

Vol                    :           Volume

TAbs                         :               Test Absorbance

Spl.                   :           Sample           

Wt                     :           Weight

M                      :           Molarity          

WS                    :           Working Standard

CRS                  :           Currect Reference Standard             

Revision History:

Revision No. Details of changes Reason  
00 Nil New Document  

Bhanu Pratap Singh

BHANU PRATAP SINGH IS EXPERIENCED IN PHARMACEUTICAL, AUTHOR AND FOUNDER OF PHARMACEUTICAL GUIDESLINE (WWW.PHARMAGUIDESLINE.COM), A WIDELY READ PHARMACEUTICAL BLOG SINCE 2019. EMAIL:- INFO@PHARMAGUIDESLINE.COM

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