by 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| Superseded Protocol No. | Nil |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 |
- 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:
- There should be no interference of the diluent, placebo with the main analyte response, no maxima should be obtained at wavelength 265 nm
- 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 |
