by PROTOCOL FOR VALIDATION OF METHOD OF ANALYSIS OF CLEANING SAMPLE OF PARACETAMOL 325 mg AND DICLOFENAC SODIUM 50 mg TABLET
| Superseded Protocol No. | Nil |
| Effective Date |
Table of contents :
| Sr. No. | Subject |
| Protocol Approval | |
| Objective | |
| Scope | |
| Responsibility of validation team | |
| Product profile | |
| Methodology | |
| 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 validation is to validate method of analysis of cleaning samples of Paracetamol and Diclofenac Sodium Tablets by considering the following parameters:
- Specificity
- System Precision
- Linearity
- Recovery Study
- Limit of Detection ( LOD )
- Limit of Quantification ( LOQ )
Following parameters will be seen during system suitability:
| Sr. No. | Parameters | Limit |
| 1 | The Tailing Factor | Not more than 2.0 |
| 2 | % RSD for pack area of Paracetamol and Diclofenac Sodium from 6 replicate injections of standard solution | Not more than 2.0 |
When the above parameters meet the method, the actual experiment shall be started.
- Scope :
This protocol is based on SOP and applicable for the Cleaning validation of Paracetamol and Diclofenac Sodium tablet.
- 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 (% API Content) | Analgesic and anti-inflammatory |
| Reason for Validation | To Validate the analytical method for Cleaning sample analysis. |
| Active Ingredient | Paracetamol and Diclofenac Sodium |
| Strength | Each film coated tablet contains: Paracetamol IP 325 mg Diclofenac Sodium IP 50 mg |
| Methodology | In-House |
| Method Reference | In-House |
- Methodology
Method of analysis:
Reagents:
- Methanol ( HPLC grade )
- Acetonitrile ( HPLC grade )
- Glacial Acetic Acid ( HPLC Grade )
- Milli Q Water
- Preparation of Mobile Phase:
Prepare Mobile Phase of Methanol, water, Acetonitrile and glacial acetic acid in the ratio of 60:25:15:1.Filter through 0.45µm-membrane filters and degas before use.
Standard Solution preparation:
Weigh accurately about 50 mg of Paracetamol working standard and 50 mg Diclofenac Sodium working standard in to a 100.0 ml volumetric flask, and add about 50 ml methanol, sonicate to dissolve and dilute to volume with methanol. Further dilute 2.0 ml of this solution to 100.0 ml with mobile phase.
Chromatographic Condition:
- Column : Nucleodur C 18, (250 x 4.6 mm ,5 µm )
- Flow Rate : 1.0 ml/min.
- Wavelength : 281 nm
- Injection Volume : 20 µl
- Run Time : About 15 minutes
- Column Temperature:25°C
System suitability:
Inject 20 µl of the standard solution in six replicate injections and check the system suitability parameters as mentioned below:
| Sr. No. | Parameters | Limit |
| 1. | Tailing Factor | Not more than 2.0 |
| 2. | Theoretical plate | Not more than 2000 |
| 3. | Resolution | Not less than 2.0 |
| 4. | % RSD for peck area of Paracetamol and Diclofenac Sodium from 6 replicate injections of standard solution. | Not more than 2.0 |
- Parameters to be Validated:
- Specificity
- System Precision
- Linearity
- Recovery Study
- Limit of Detection ( LOD )
- Limit of Quantification ( LOQ )
- Specificity:
Prepare blank, swab blank and standard solution 10 mcg/ml as per procedure given below:
Standard Solution preparation:
Weigh accurately about 50 mg of Paracetamol working standard and 50 mg Diclofenac Sodium working standard in to a 100.0 ml volumetric flask, and add about 50 ml methanol, sonicate to dissolve and dilute to volume with methanol. Further dilute 2.0 ml of this solution to 100.0 ml with mobile phase.
Blank solution: Diluents (Mobile Phase)
Swab Blank: Take 10.0 ml of diluents in a test tube, dip fresh swab stick in test tube and sonicate for 5 minutes.
Procedure: Inject blank, swab blank and standard solution.
Acceptance criteria:
There should not be any interference from blank, swab blank with the analyte peak.
- System Precision:
System precision shall be evaluated bymeasuring the area of 6 replicate injection of standard solution and calculate the relative standard deviation.
Standard Solution preparation:
Weigh accurately about 50 mg of Paracetamol working standard and 50 mg Diclofenac Sodium working standard in to a 100.0 ml volumetric flask, and add about 50 ml methanol, sonicate to dissolve and dilute to volume with methanol. Further dilute 2.0 ml of this solution to 100.0 ml with mobile phase
Acceptance Criteria: % RSD of Six replicate injection should not be more than 2.0%.
- Linearity:
The linearity of the method shall be established between 6 mcg/ml to 14 mcg/ml. The sample area shall be plotted against concentration and correlation coefficient shall be calculated.
Minimum five points shall be covered from the range of 6 mcg/ml to 14 mcg/ml.
Following concentrations shall be prepared and analyzed for linearity: 6 mcg/ml, 8mcg/ml, 10 mcg/ml, 12mcg/ml and 14mcg/ml.
Procedure: Measure the area of sample solution in duplicate. Plot the graph of area vs. actual concentration. Calculate the correlation coefficient from linearity curve.
Acceptance Criteria: Correlation coefficient shall not be less than 0.99.
- Recovery:
Standard Solution preparation:
Weigh accurately about 50 mg of Paracetamol working standard and 50 mg Diclofenac Sodium working standard in to a 100.0 ml volumetric flask, and add about 50 ml methanol, sonicate to dissolve and dilute to volume with methanol. Further dilute 2.0 ml of this solution to 100.0 ml with mobile phase.
Recovery Stock Solution:
Weigh accurately about 50 mg of Paracetamol working standard and 50 mg Diclofenac Sodium working standard in to a 100.0 ml volumetric flask, and add about 50 ml methanol, sonicate to dissolve and dilute to volume with methanol.
- Spiked the 0.16 ml, 0.20 ml and 0.24 ml of recovery stock solution into the area of 10 x 10 cm2 in three different SS Plates for each concentration.
- Allow the solutions to dry in air.
- Take the Swab wipe and moisten with diluents, swab one SS plate in the area of 10 x 10 cm2. Collect the swab in the test tube containing already 10.0 ml of diluents in the test tube and sonicate for 10 minutes with swirling. Squeeze the swab and discard. Filter through 0.45µ filter. Final concentrations of recovery solutions are 8 ppm, 10 ppm, and 12 ppm.
- Spike 0.2 ml of recovery stock solution on two more SS plates for 10 ppm recovery. Keep these two spiked plates for 3rd and for 5th day recovery at room temperature. Repeat the procedure to collect the swab sample at 3rd and 5th day.
- Similarly do the recovery on 10× 10 cm2 apron.
Acceptance criteria:
Recovery at each level should not be less than 70%.
- Limit of Detection (LOD):
Limit of detection is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified under the stated experimental condition. LOD of Paracetamol and Diclofenac Sodium will be calculated on hit and trial basis by diluting standard stock solution to such a concentration that the signal to noise ratio will be equal to or greater than 3.
Prepare the LOD Solution and inject in six replicates. Calculate % RSD for six replicates of Paracetamol and Diclofenac Sodium.
Acceptance criteria:
% RSD for six replicates shall be not more than 30.The signal to noise ratio should be more than 3:1.
- Limit of Quantification (LOQ):
Limit of Quantification is the lowest concentration of analyte in a sample that can be quantify with acceptable precision under the stated experimental condition. LOQ of Paracetamol and Diclofenac Sodium will be calculated on hit and trial basis by diluting standard stock solution to such a concentration that the signal to noise ratio will be equal to or greater than 10.
Prepare the LOQ Solution and inject in six replicates. Calculate % RSD for six replicates of Paracetamol and Diclofenac Sodium.
Acceptance criteria:
% RSD for six replicates shall be not more than 10.The signal to noise ratio should be more than 10:1.
- Revision History:
| Revision No. | Details of changes | Reason |
| 00 | Nil | New |
