by PROTOCOL FOR VALIDATION OF METHOD OF ANALYSIS FOR CONTAMINATION STUDY OF PARACETAMOL
| Superseded Protocol No. | Nil |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 contamination samples study of Paracetamol 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. | % RSD for peak absorbance of Paracetamol from 6 replicate 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 validation of method of analysis of contamination samples study of Paracetamol.
- 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 |
| Reason for Validation | To Validate the analytical method for Cleaning sample analysis. |
| Active Ingredient | Paracetamol |
| Methodology | In-House |
| Method Reference | In-House |
- Methodology
Method of analysis:
Reagents:
- Methanol
- Diluents: Methanol
- Instrument : UV-VIS Spectrophotometer
Standard Solution preparation:
Weigh accurately about 50 mg of Paracetamol 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 methanol.
Determine the maxima in the range of 400 nm to 200 nm by UV-VIS Spectrophotometer using diluents as a blank.
- 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 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 methanol.
Blank solution: Diluents
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: Take the absorbance ofblank, swab blank and standard solution at maxima in the range of 400 nm to 200 nm.
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 absorbance of 6 replicate of standard solution and calculate the relative standard deviation.
Standard Solution preparation:
Weigh accurately about 50 mg of Paracetamol 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 methanol.
Acceptance Criteria: % RSD of Six replicate 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 absorbance 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 absorbance of sample solution in duplicate. Plot the graph of absorbance 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 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 methanol.
Recovery Stock Solution:
Weigh accurately about 50 mg of Paracetamol 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.
Based on the standard deviation of the response of swab blank and the slope of linearity curve, LOD shall be calculated by the following equation:
LOD (mcg/ml) = 3.3 x σ
S
Where σ = Standard deviation of the response of swab blank.
S = Slope of Linearity Curve.
- Verification of Limit of Detection (LOD):
Prepare the LOD Sample and measure the absorbance of LOD sample in six replicates. Calculate % RSD for six replicates of Paracetamol.
Acceptance criteria:
% RSD for six replicates shall be not more than 30.
- 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.
Based on the standard deviation of the response of swab blank and the slope of linearity curve, LOQ shall be calculated by the following equation:
LOD (mcg/ml) = 10 x σ
S
Where σ = Standard deviation of the response of swab blank.
S = Slope of Linearity Curve.
- Verification of Limit of Quantification (LOQ):
Prepare the LOQ Solution and measure the absorbance of sample in six replicates. Calculate % RSD for six replicates of Paracetamol.
Acceptance criteria:
% RSD for six replicates shall be not more than 10.
- Revision History:
| Revision No. | Details of changes | Reason |
| 00 | Nil | New |
