by RELATED SUBSTANCES METHOD VERIFICATION PROTOCOL OF CHLORPHENAMINE MALEATE
| Superseded Protocol No. | Nil |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 verification is to provide documentary evidence that analytical methodology used for related substances of Chlorphenamine Maleate by pharmacopeia method is consistent and reliable results within the predetermined acceptance criteria.
Analytical method verification will be performed by considering thefollowing parameters:
| Parameters | Chlorphenamine Maleate |
| Specificity | |
| Precision | |
| System Precision | |
| Method Precision | |
| Intermediate Precision (Ruggedness) | |
| System Suitability |
- Scope :
The scope of this protocol is applicable for the verification of method of related substances of Chlorphenamine Maleate.
- Responsibility of Verification Team:
| Departments | Responsibilities |
| QC | Preparation and Review of Verification protocol. |
| Perform the verification as per approved protocol and recording of data. | |
| Compilation and checking of data. | |
| Preparation and review of Verification report. | |
| To impart training of protocol to concerned department/persons. | |
| QA | Review and approval of Verification protocol. |
| Co-ordination with QC to carry out Verification. | |
| Review and approval of Verification report. | |
| Head Quality | Authorization of protocol. |
- Product Profile:
| Category | Active Product Ingredient |
| Reason for Verification | Pharmacopeia method |
| Active Ingredient | Chlorphenamine Maleate |
| Method Reference | European Pharmacopeia |
| Specification Limits | Impurity A : NMT 0.2 per cent Impurity B : NMT 0.1 per cent Impurity C : NMT 0.1 per cent Impurity D : NMT 0.1 per cent Unspecified Impurity: NMT 0.10 per cent Total Impurity : Maximum 0.5 per cent Reporting threshold : 0.05 percent |
- Methodology:
Reagent:
Table 1.0
| Reagent | Grade |
| Ammonium Dihydrogen Phosphate | AR Grade |
| Ortho-Phosphoric Acid | AR Grade |
| Acetonitrile | HPLC Grade |
| Water | HPLC grade or Milli Q Water |
Chromatographic conditions (By HPLC)
Instrument : High Performance Liquid Chromatography (UV Visible/DAD Detector)
Column : l = 0.30 m, ø = 3.9mm; Octadecylsilyl silica gel (10µm)
Flow rate : 1.2 ml/min
Detector : 225nm
Column Temperature : 25ºC
Injection Volume : 20 µl
Run time : 3.5 times the retention time of Chlorphenamine
Retention time : about 11.0 minutes (Chlorphenamine)
Program : Isocratic Method
Preparation of buffer solution for mobile phase:
Dissolve 8.57 g of ammonium dihydrogen phosphate into 1000 ml of water. Adjust the pH to 3.0 with phosphoric acid R.
(Note: Volume of buffer can be prepared based on the requirement provided that the final concentration remains the same.)
Mobile Phase: Mix 20 volume of Acetonitrile R and 80 volumes of buffer solution, mix and filter through 0.45 µm membrane filter.
Diluent: Mobile Phase
Test Solution: Dissolve 0.100 g of Chlorphenamine Maleate sample in diluent and dilute to 100.0 ml with Diluent.
Standard solution: Dissolve 0.100 g of Chlorphenamine Maleate standard in diluent and dilute to 100.0 ml with diluent.
Reference Solution (a): Dilute 0.5 ml of the test solution to 100.0 ml with diluent.
Reference Solution (b): Dilute 1.0 ml of the reference solution (a) to 10.0 ml with the diluent.
Reference Solution (c): Dissolve 5.0 mg of Chlorphenamine Impurity C in 5.0 ml of the standard solution and dilute to 50 ml with diluent. Dilute 2 ml of this solution to 20.0mL with the diluent.
Reference Solution (d): Dissolve 5.0 mg of Chlorphenamine Impurity B in 70.0 ml diluent and dilute to volume 100 ml with the diluent.
Reference Solution (e): Dissolve the contents of the vial of Chlorphenamine Impurity A in 2.0 ml of the test solution and sonicate for 5min.
System suitability Solution: Use Reference solution (c)
System Suitability:
- Resolution : Minimum 1.5 between the peaks due to impurity C and Chlorphenamine.
Procedure:
Equilibrate the HPLC column with mobile phase; inject the solution as per following sequence or as per the requirement mentioned in Table 2.0. Record the chromatogram and calculate the % impurities.
Table 2.0: Sequence
| Solutions | No of Injection to be injected in Sequence |
| Blank (Mobile Phase) | 1 |
| Reference solution (c) or System Suitability solution | 1 |
| Reference solution (a) | 6 |
| Reference solution (b) | 1 |
| Reference solution (d) | 1 |
| Reference solution (e) | 1 |
| Blank (avoid carry over) | 1 |
| Test Solution | 1 |
| Reference solution (c)+Bracketing | 1 |
Identify the components in sample preparation with following relative retention times (RRT) as given in table.
Table 3.0: RRT & CF
| Sr. No. | Component Name | Relative Retention Time (RRT) | Correction Factor (CF) |
| 1. | Chlorphenamine | 1.00 | 1.00 |
| 2. | Maleic Acid | 0.2 | 1.00 |
| 3. | Impurity – A | 0.3 | 1.50 |
| 4. | Impurity – B | 0.4 | 1.40 |
| 5. | Impurity – C | 0.9 | 1.00 |
| 6. | Impurity – D | 3.0 | 1.00 |
Disregard limit:
Disregard the peaks due to Blank, Maleic Acid and peaks below 0.05%
Calculation:
Area of impurity A x 1.5 x 0.2
Impurity A = —————————————————
Area of reference solution (a) x 0.4
Limit:
Impurity A: Not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (A) (0.2 per cent).
Area of impurity B x 1.4 x 0.1
Impurity B = ————————————————
Area of reference solution (a) x 0.2
Limit:
Impurities B: For each impurity, not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent).
Area of impurity C x 0.1
Impurity C = ————————————————
Area of reference solution (a) x 0.2
Limit:
Impurities C: For each impurity, not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent).
Area of impurity D x 0.1
Impurity D = ————————————————
Area of reference solution (a) x 0.2
Limit:
Impurities D: For each impurity, not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent).
Area of unspecified impurity x 0.10
Unspecified impurity = —————————————————-
Area of reference solution (a) x 0.2
Limit:
Unspecified impurity: For each impurity, not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent).
Area of Total impurities x 0.5
Total impurities = —————————————————-
Area of reference solution (a)
Limit:
Total impurities: Not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent).
- Verification parameters:
The following parameters to be perform for the verification activity.
- Specificity
- Precision
- System Suitability
- 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 mobile phase.
Specificity of test method should be established by separately injecting blank solution (mobile phase), identification solution of Impurity A, Impurity B, Impurity C, Impurity D, test solution and spiked test solution. Identification solution and spiked test solution will be prepared at specification level. Test solution to be prepared as per method of analysis.
Blank: Mobile Phase
Preparation of Identification solutions:
Stock solution of Impurity A:
Weigh accurately about 2.0 mg of Impurity A and transferred to 50 ml volumetric flask, add 35 ml of diluent and dissolve. Make up the volume to 50.0 ml with diluent and mix.
Identification solution of Impurity A:
Dilute 5.0 ml of stock solution of impurity A to 100.0 ml with diluent, mix.
Stock solution of Impurity B:
Weigh accurately about 2.0 mg of Impurity B and transferred into 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100.0 ml with diluent and mix.
Identification solution of Impurity B:
Dilute 5.0 ml of stock solution of Impurity B into 100.0 ml with diluent, mix.
Stock solution of Impurity C:
Weigh accurately about 2.0 mg of Impurity C and transferred into 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100.0 ml with diluent and mix.
Identification solution of Impurity C:
Dilute 5.0 ml of stock solution of Impurity C into 100.0 ml with diluent, mix.
Stock solution of Impurity D:
Weigh accurately about 2.0 mg of Impurity D and transferred into 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100.0 ml with diluent and mix.
Identification solution of Impurity D:
Dilute 5.0 ml of stock solution of Impurity D into 100.0 ml with diluent, mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
Test Solution: Dissolve 0.100 g of Chlorphenamine Maleate sample in diluent and dilute to 100.0 ml with Diluent.
Standard solution: Dissolve 0.100 g of Chlorphenamine Maleate standard in diluent and dilute to 100.0 ml with diluent.
Reference Solution (a): Dilute 0.5 ml of the test solution to 100.0 ml with diluent.
Reference Solution (b): Dilute 1.0 ml of the reference solution (a) to 10.0 ml with the diluent.
Reference Solution (c): Dissolve 5.0 mg of Chlorphenamine Impurity C in 5.0 ml of the standard solution and dilute to 50 ml with diluent. Dilute 2 ml of this solution to 20.0mL with the diluent.
System suitability Solution: Use Reference solution (c)
Spiked test solution:
Dissolve 0.100 g of Chlorphenamine Maleate sample in diluent, further add 5.0 ml stock solution of impurity A, impurity B, impurity C and impurity D and mix well, make up the volume to 100.0 ml with diluent, mix.
Procedure: Inject the preparation of blank solution, reference solution (c) or system suitability solution, reference solution (a), reference solution (b) and identification solution of impurity A, impurity B, impurity C and impurity D, test solution and spiked test solution on a HPLC system with a Diode array detector (DAD) as follows in Table 3.0. Determine the purity of the individual peaks of interest. Record the retention times and check for system suitability parameters.
Table 3.0: Sequence
| Solutions | No of Injection to be injected in Sequence |
| Blank (Mobile Phase) | 1 |
| Reference solution (c) or System Suitability solution | 1 |
| Reference solution (a) | 6 |
| Reference solution (b) | 1 |
| Identification solution of Impurity A | 1 |
| Identification solution of Impurity B | 1 |
| Identification solution of Impurity C | 1 |
| Identification solution of Impurity D | 1 |
| Test solution | 1 |
| Spiked test solution | 1 |
| Reference solution (c)+Bracketing | 1 |
Table 4.0 Specificity data
| Sr. No | Sample | RT (min.) | RRT | Peak purity | |
| 1 | Blank | NA | |||
| 2 | System Suitability solution | Chlorphenamine | |||
| Impurity C | |||||
| 3 | Reference solution (a) | Chlorphenamine | |||
| 4 | Reference solution (b) | Chlorphenamine | |||
| 5 | Identification solution of Impurity A | ||||
| 6 | Identification solution of Impurity B | ||||
| 7 | Identification solution of Impurity C | ||||
| 8 | Identification solution of Impurity D | ||||
| 9 | Test Solution | Chlorphenamine | |||
| 10 | Unknown Impurity | NA |
Table 5.0 Spiked test solution
| Sr. No | Sample | RT (min.) | RRT | Peak purity |
| 1 | Blank | NA | ||
| 2 | Chlorphenamine | |||
| 3 | Impurity A | |||
| 4 | Impurity B | |||
| 5 | Impurity C | |||
| 6 | Impurity D | |||
| 7 | Unknown Impurity | NA |
Acceptance Criteria:
- There should be no interference of the diluent, impurities at the retention time of analyte peak,
- Impurity peaks should be well resolved from active peak and each other,
- Analyte peak in standard solution and known Impurity peaks in spiked sample solution should be spectrally pure.
- Precision:
- System Precision:
The system precision is the closeness of agreement between the responses of detector. It is usually expressed as the standard deviation (SD) or the relative standard deviation (RSD).
Reference (a) will be prepared as per method of analysis and injected six replicate injections to be injected in sequence and recorded the area response of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.
Table 6.0 System Precision- Repeatability of Standard Injections
| Sr. No. | Chlorphenamine | |
| Peak Area | Retention time (min.) | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD |
Acceptance criteria:
% RSD for peak area and retention time of replicate injections of Reference (a) should be NMT 5.0% and 1.0% respectively.
- 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:
Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known impurity at limit level. Un-spiked sample will be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample will be subtracted in spiked sample to calculate the actually known spiked amount of impurity. Record the area on data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation.
Table 8.0 Method precision results spiked sample at limit level
| Sr. No. | Sample Weight (mg) | Impurity A (%) | Impurity B (%) | Impurity C (%) | Impurity D (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | |
| 1 | 2 | |||||||
| RRT | ||||||||
| 1 | ||||||||
| 2 | ||||||||
| 3 | ||||||||
| 4 | ||||||||
| 5 | ||||||||
| 6 | ||||||||
| Mean | ||||||||
| SD | ||||||||
| % RSD | ` |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below. Reporting threshold value is 0.05%.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
- Intermediate Precision ( Ruggedness ):
Intermediate precision expresses within laboratory variation with different analysts or equipment or different column/same column on different days using same batch of drug product as per method of analysis.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known impurity at limit level. Un-spiked sample will be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample will be subtracted in spiked sample to calculate the actually known spiked amount. Record the area on data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation.
Table 10.0 Intermediate precision results spiked sample at limit level
| Sr. No. | Sample weight (mg) | Impurity A (%) | Impurity B (%) | Impurity C (%) | Impurity D (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | |
| 1 | 2 | |||||||
| RRT | ||||||||
| 1 | ||||||||
| 2 | ||||||||
| 3 | ||||||||
| 4 | ||||||||
| 5 | ||||||||
| 6 | ||||||||
| Mean | ||||||||
| SD | ||||||||
| % RSD | ` |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below. Reporting threshold value is 0.05%.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
Table 11.0 Pooled results of analyst – I & analyst – II
| Sr. No. | Sample Weight (mg) | Impurity A (%) | Impurity B (%) | Impurity C (%) | Impurity D (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | |
| 1 | 2 | |||||||
| ANALYST I | ||||||||
| RRT | ||||||||
| 1 | ||||||||
| 2 | ||||||||
| 3 | ||||||||
| 4 | ||||||||
| 5 | ||||||||
| 6 | ||||||||
| ANALYST II | ||||||||
| RRT | ||||||||
| 1 | ||||||||
| 2 | ||||||||
| 3 | ||||||||
| 4 | ||||||||
| 5 | ||||||||
| 6 | ||||||||
| Mean | ||||||||
| SD | ||||||||
| % RSD |
Acceptance criteria:
Pooled % RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
- System Suitability:
System suitability tests are based on concept that the equipment, electronics, analytical operations and sample to be analyzed, system suitability test provide the added assurance that on specific occasion the method is given accurate and precise results.
The system suitability should be as per below mention criteria in Table 16.0.
Table 16.0
| Parameters | System Suitability Criteria | Limit |
| Resolution | The resolution between the peaks due to impurity C and Chlorphenamine | Minimum 1.5 |
- Incident/Deviation:
Any Incident or Deviation observed during Analytical Method verification should be recoded and reported in Verification Report.
- Summary/Conclusion/recommendation:
Final Conclusion should be drawn from analytical method verification for its use to analyze the related substances of Chlorphenamine Maleate by pharmacopeia method and if any differences shall be observed between methodology and experiments than accordingly will be revised and update our methodology.
Abbreviations
RS : Related substance
VAL : Verification
R : Report
RSD : Related standard deviation
HPLC : High performance liquid chromatography
mL : Milliliter
mg : Milligram
min. : Minutes
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
Imp : Impurity
Ws : Working standard
Vol : Volume
Revision History :
| Revision No. | Details of changes | Reason for change |
| 00 | Nil | New Document |
