by RELATED SUBSTANCES METHOD VERIFICATION PROTOCOL OF DICLOFENAC SODIUM BP (API)
| Superseded Protocol No. | Nil |
| Effective Date |
TABLE OF CONTENTS:
| Sr. No. | Subject | Page No. |
| Protocol Approval | ||
| Objective | ||
| Scope | ||
| Responsibility | ||
| Product profile | ||
| Methodology | ||
| Validation 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 Diclofenac sodium by pharmacopeia method is consistent and reliable results within the predetermined acceptance criteria.
Analytical method verification will be performed by considering thefollowing parameters:
| Parameters | Diclofenac Sodium |
| Specificity | yes |
| Precision | |
| System Precision | yes |
| Method Precision | yes |
| Intermediate Precision (Ruggedness) | yes |
| Linearity | yes |
| Stability of Analytical Solution | yes |
| System Suitability | yes |
- Scope :
The scope of this protocol is applicable for the verification of method of related substances of Diclofenac sodium.
- Responsibility of Validation Team:
| Departments | Responsibilities |
| QC | preparation and Review of Validation protocol. |
| Perform the validation as per approved protocol and recording of data. | |
| Compilation and checking of data. | |
| Preparation and review of Validation report. | |
| To impart training of protocol to concerned department/persons. | |
| QA | Review and approval of Validation protocol. |
| Co-ordination with QC to carry out Validation. | |
| Review and approval of Validation report. | |
| Head Quality | Authorization of protocol. |
- Product Profile:
| Category | Active Product Ingredient |
| Reason for Verification | Pharmacopeia method |
| Active Ingredient | Diclofenac sodium |
| Method Reference | British Pharmacopeia |
| Specification Limits | Impurity A : Maximum 0.2 per cent Impurity F : Maximum 0.15 per cent Unspecified Impurity for each impurity : Maximum 0.1 per cent Total Impurity : Maximum 0.4 per cent Reporting threshold : 0.05 percent |
- Methodology:
Reagent:
Table 1.0
| Reagent | Grade |
| Sodium dihydrogen phosphate | AR Grade |
| Phosphoric Acid | AR Grade |
| Methanol | HPLC Grade |
| Water | HPLC grade or Milli Q Water |
Chromatographic conditions (By HPLC)
Instrument : High Performance Liquid Chromatography (UV Visible/DAD Detector)
Column : l = 0.25 m, ø = 4.6mm; Octadecylsilyl silica gel (5µm) or equivalent
Flow rate : 1.0 ml/min
Detector : 254nm
Column Temperature : 25ºC (Ambient)
Injection Volume : 20 µl
Run time : 1.6 times the retention time of Diclofenac sodium
Retention time : About 25 min
Impurity A : 0.4 RRT
Impurity F : 0.8 RRT
Program : Isocratic Method
Preparation of buffer solution for mobile phase:
- 0.5 g/L Phosphoric Acid: Dissolve 0.27 ml of phosphoric acid (88%) into 1000 ml of Water.
- 0.8 g/L Sodium dihydrogen phosphate: Dissolve 0.8 g of sodium dihydrogen phosphate into 1000ml of water.
(Note: Volume of buffer can be prepared based on the requirement provided that the final concentration remains the same.)
Mobile Phase: Mix 34 volumes of a solution containing 0.5 g/L of phosphoric acid R and 0.8 g/L of sodium dihydrogen phosphate R, previously adjusted to pH 2.5 with phosphoric acid R, and 66 volumes of methanol R.
Diluent: Mobile Phase
Reference solution (b): Dissolve the contents of a vial of Diclofenac for system suitability CRS (containing impurities A and F) in 1.0 ml of mobile phase.
Or
System Suitability solution:
Stock solution of Impurity A (20.0 ppm):
Weigh accurately about 2.0 mg of Impurity A and transferred to 100 ml of volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
Stock solution of Impurity F (25.0 ppm):
Weigh accurately about 2.5 mg of Impurity F and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
System suitability solution: Transfer 50.0 mg of Diclofenac sodium CRS/WS in 50.0 ml of volumetric flask, add 30.0 ml of diluent and dissolve further add 5.0 ml of stock solution of impurity A (100 ppm) and 3.0 ml of stock solution of Impurity F into 50 ml with diluent, mix.
Test solution:
Dissolve 50.0 mg of substances to be examined in the mobile phase and dilute to 50.0 ml with mobile phase.
Reference solution (a): Transfer 50.0 mg of Diclofenac sodium CRS/WS in 50.0 ml of volumetric flask, add 30.0 ml of diluent and dissolve, make up the volume to 50.0 ml with mobile phase.
Further dilute 2.0 ml of this solution to 100.0 ml with mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase.
System Suitability:
- Resolution : Minimum 4.0 between the peaks due to impurity F and Diclofenac.
- %,Area RSD : The area % RSD of Diclofenac sodium in reference solution (b) should not more than 5.0%.
Procedure:
Equilibrate the HPLC column with mobile phase; inject the solution as per following sequence or as per the requirement mentioned in Table1.0. Record the chromatogram and calculate the impurities.
Disregard limit:
Disregard the peaks due to blank.
Table 2.0
| Solutions | No of Injection to be injected in Sequence |
| Blank (Mobile Phase) | 1 |
| Reference solution (b) or System Suitability solution | 1 |
| Reference solution (a) | 6 |
| Test Solution | 1 |
| Reference solution (b)+Bracketing | 1 |
Calculation:
Impurity peak area in sample x 0.2 x response factor
% Impurity A & F: ————————————————————————–
Average Area in Reference solution (a)
Impurity peak area in sample x 0.2
% Unspecified Impurity: —————————————————————
Average Area in Reference solution (a)
% Total Impurity: Sum of all known impurities and unknown impurities
Response factor for Impurity A : 0.7
Response factor for Impurity F : 0.3
Dilution Factor : 0.2
- Validation parameters:
The following parameters to be perform for the verification activity.
- Specificity
- Precision
- Stability of solution
- System Suitability
- Linearity
- 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 placebo.
Specificity of test method should be established by separately injecting blank solution (mobile phase), identification solution of Impurity A, Identification solution of Impurity F, spiked test solution and 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 (20.0 ppm):
Weigh accurately about 2.0 mg of Impurity A and transferred to 100 ml of volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
Identification solution of Impurity A (2.0 ppm):
Dilute 5.0 ml of stock solution of impurity F to 50.0 ml with diluent, mix.
Stock solution of Impurity F (25.0 ppm):
Weigh accurately about 2.5 mg of Impurity F and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
Identification solution of Impurity F (1.5 ppm):
Dilute 3.0 ml of stock solution of impurity F to 50 ml with diluent, mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
System suitability solution: Dilute 5.0 ml of stock solution of impurity A (100 ppm) and 3.0 ml of stock solution of Impurity F into 50 ml with diluent, mix.
Test solution:
Dissolve 50.0 mg of substances to be examined in the mobile phase and dilute to 50.0 ml with mobile phase.
Reference solution (a): Transfer 50.0 mg of Diclofenac sodium CRS/WS in 50.0 ml of volumetric flask, add 30.0 ml of diluent and dissolve, make up the volume to 50.0 ml with mobile phase.
Further dilute 2.0 ml of test solution to 100.0 ml with mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase.
Spiked test solution:
Transfer 50.0 mg of Diclofenac sodium test substances into 50 ml of volumetric flask; add 30 ml of diluent to dissolve further add 5.0 ml stock solution of impurity A and 3.0 ml of stock solution of impurity F and mix well, make up the volume to 50 ml with diluent.
Procedure: Inject the preparation of blank solution, reference solution (a), and identification solution of impurity A, identification solution of impurity F, 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 system suitability parameters for all peaks. System suitability criteria should be checked.
Tablet 3.0
| Solution | No of Injection to be injected in Sequence |
| Blank (Mobile Phase) | 1 |
| Reference solution (a) | 6 |
| Identification solution of Impurity A | 1 |
| Identification solution of Impurity F | 1 |
| System suitability solution | 1 |
| Test Solution | 1 |
| Spiked Test Solution | 1 |
| Reference solution (a) + Bracketing | 1 |
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.
Table 4.0 Specificity data
| Sr. No | Sample | RT (min.) | RRT | Peak purity | ||
| 1 | Blank | NA | ||||
| 2 | Reference solution (a) | Diclofenac Sodium | ||||
| 3 | System Suitability solution | Impurity A | ||||
| Impurity F | ||||||
| 4 | Identification solution of Impurity A | |||||
| 5 | Identification solution of Impurity F | |||||
| 6 | Test Solution | Diclofenac Sodium | ||||
| 7 | Unknown Impurity | NA | ||||
Table 5.0 Spiked test solution
| Sr. No | Sample | RT (min.) | RRT | Peak purity |
| 1 | Blank | NA | ||
| 2 | Diclofenac Sodium | |||
| 3 | Impurity A | |||
| 4 | Impurity F | |||
| 5 | Unknown Impurity | NA |
- 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).
Standard solution 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. | Diclofenac Sodium | |
| 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 standard solution injections 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. | Spl. Wt. (mg) | Diclofenac Impurity A (%) | Diclofenac Impurity F (%) | 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 Method precision results spiked sample at limit level
| Sr. No. | Spl. Wt. (mg) | Diclofenac Impurity A (%) | Diclofenac Impurity F (%) | 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. | Spl. Wt. (mg) | Diclofenac Impurity A (%) | Diclofenac Impurity F (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | |
| 1 | 2 | |||||
| RRT | ||||||
| ANALYST I | ||||||
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| RRT | ||||||
| ANALYST II | ||||||
| 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.
- Stability of Analytical solution:
It is essential when validating an analytical method to confirm that the analyte has adequate stability in both the standard and sample solution at room temperature (ambient Temperature) during analytical measurement stages of the testing.
Test Procedure:
Prepared the Blank solution, standard solution and test solution as per method of analysis and spiked the test solution with Impurity A & Impurity F at limit level and analyze the solution at the different time intervals i.e. 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, and 48 hours. Calculate the cumulative RSD of peak area for known Impurity and main peak.
The obtained area of known impurities in test solution after different time interval will be calculated. The obtained area of Reference solution (a) after different time interval will be considered to calculate the area % RSD of initial replicate standard.
Acceptance criteria
Cumulative % RSD of peak area for known Impurities and main peak shall not be more than 10.0 %
- LINEARITY
The linearity of an analytical procedure is its ability (within a given range) to obtained test results which are directly proportional to the concentration levels shall be prepared.
Determine the linearity by preparing and inject the Reference solution (a) and Impurity A and Impurity F in the range of reporting threshold value of 0.05 % of test concentration to 200% of limit level and calculate the correlation coefficient “r”.
Prepare the solution at below threshold value, threshold value, 50%, 100%, 150% and 200%.
Preparation of Linearity solutions:
i) Linearity Stock solution of Impurity A (20.0 ppm):
Weigh accurately about 2.0 mg of Impurity A and transferred to 100 ml of volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
ii) Linearity Stock solution of Impurity F (25.0 ppm):
Weigh accurately about 2.5 mg of Impurity F and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
iii) Stock solution of Diclofenac Sodium:
Transfer 50.0 mg of Diclofenac sodium CRS/WS in 50.0 ml of volumetric flask, add 30.0 ml of diluent and dissolve, make up the volume to 50.0 ml with mobile phase.
Further dilute 2.5 ml of this solution to 100.0 ml with mobile phase.
Table 12: Linearity levels preparation
| Level % | Vol. (mL) of Stock | Volume to be diluted with diluent (ml) | ||
| Impurity A | Impurity F | Diclofenac Sodium | ||
| Below Threshold | 2.0 | 1.8 | 1.6 | 100 |
| Threshold value | 2.5 | 2.0 | 2.0 | 100 |
| 50% | 2.5 | 1.5 | 1.0 | 50 |
| 100% | 5.0 | 3.0 | 2.0 | 50 |
| 150% | 3.0 | 1.8 | 1.2 | 20 |
| 200% | 5.0 | 3.0 | 2.0 | 25 |
Table 13: Linearity of Impurity A
| %Level | Conc. (%) | Theoretical Conc. (ppm) | Actual Conc. (ppm) | Peak Area | Mean peak area |
| Below Threshold | 0.04 | 0.4 | |||
| Threshold | 0.05 | 0.5 | |||
| 50 | 0.10 | 1.0 | |||
| 100 | 0.20 | 2.0 | |||
| 150 | 0.30 | 3.0 | |||
| 200 | 0.40 | 4.0 | |||
| Slope | |||||
| Intercept | |||||
| Corr. coeff.(r) |
Table 14: Linearity of Impurity F
| %Level | Conc. (%) | Theoretical Conc. (ppm) | Actual Conc. (ppm) | Peak Area | Mean peak area |
| Below Threshold | 0.045 | 0.45 | |||
| Threshold | 0.05 | 0.50 | |||
| 50 | 0.75 | 0.75 | |||
| 100 | 0.15 | 1.50 | |||
| 150 | 0.225 | 2.25 | |||
| 200 | 0.30 | 3.00 | |||
| Slope | |||||
| Intercept | |||||
| Corr. coeff.(r) |
Table 15: Linearity of Diclofenac Sodium (Unspecified Impurity)
| %Level | Conc. (%) | Theoretical Conc. (ppm) | Actual Conc. (ppm) | Peak Area | Mean peak area |
| Below Threshold | 0.04 | 0.4 | |||
| Threshold | 0.05 | 0.5 | |||
| 50 | 0.05 | 0.5 | |||
| 100 | 0.10 | 1.0 | |||
| 150 | 0.15 | 1.5 | |||
| 200 | 0.20 | 2.0 | |||
| Slope | |||||
| Intercept | |||||
| Corr. coeff.(r) |
Acceptance criteria
Plot the calibration curve of peak area v/s concentration for the studied peak. The correlation coefficient (r), of known impurities and Diclofenac sodium should not be less than 0.99.
- 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 F and Diclofenac | Minimum 4.0 |
| Area % RSD | The area %RSD of six replicate injection of reference solution (a) | NMT – 5.0% |
- Incident/Deviation:
Any Incident or Deviation observed during Analytical Method validation should be recoded and reported in Validation Report.
- Summary/Conclusion/recommendation:
Final Conclusion should be drawn from analytical method verification for its use to analyze the related substances of Diclofenac sodium by pharmacopeia method and if any differences shall be observed between methodology and experiments than accordingly will be revised and update our methodology.
Abbreviations:
REL : Related substance
VAL : Validation
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 |
