by ASSAY METHOD VERIFICATION PROTOCOL OF OFLOXACIN TABLETS
| Superseded Protocol No. | Nil |
| Effective Date |
- 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 |
Table of contents :
| Sr. No. | Subject |
| Protocol Approval | |
| Objective | |
| Scope | |
| Responsibility of Validation Team | |
| Product Profile | |
| Methodology | |
| Revision History |
- Objective:
The objective of this method verification is to provide documentary evidence that analytical methodology used in determination of the Ofloxacin in Ofloxacin tablets will yield consistent and reliable results within the predetermined acceptance criteria.
This method verification protocol describes the methodology which will be used in the verification study for the following parameters:
- System Suitability
- Specificity
- Precision
- System Precision
- Method Precision
- Intermediate Precision (Ruggedness)
- Linearity and Range
- Stability of Analytical Solutions
Following parameters will be seen during system suitability:
| Sr. No. | Parameters | Limit |
| 1. | Tailing Factor | Not more than 2.0 |
| 2. | Theoretical plate | Not Less than 2000 |
| 3. | % RSD for peek area of Ofloxacin in Ofloxacin Tablets from 5 replicate injections of standard solution. | Not more than 2.0 |
When the above parameters meet the method, the actual experiment shall be started.
- Scope :
The scope of this method verification protocol is based on SOP and limited to analytical method verification for determination of Ofloxacin in Ofloxacin 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. | |
| QA | Review and approval of protocol. |
| Co-ordination with QC to carryout Verification. | |
| Review of data and summary report. | |
| Head Quality | Authorization of protocol. |
- Product Profile:
| Category (% API Content) | Antibacterial |
| Reason for Verification | 1st Verification |
| Active Ingredient | Ofloxacin USP |
| Strength | Each Film Coated tablet contains: Ofloxacin USP 200 mg |
| Methodology | In House |
| Method Reference | In House |
| Specification Limits (At release) | 95 % -105 % |
| (Shelf Life) | 36 Months |
- Methodology:
Reagents:
Potassium dihydrogen phosphate (AR grade)
Orthophosphoric Acid (AR grade)
Acetonitrile (HPLC grade)
Water (Milli-Q)
Chromatographic conditions:
Column : a stainless steel column 15 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 µm).
Flow Rate : 2 ml per minute
Injection Volume : 10µl
Detection wavelength: 294 nm.
Preparation of Mobile phase:
Prepare a mixture of 92 volumes of buffer solution prepared by dissolving 27.2 g of Potassium Dihydrogen Phosphate in 1000 ml of water and adjust the pH to 2.4 with orthophosphoric acid and 8 volumes of acetonitrile.
Test Preparation:
Weigh and powder 20 tablets. Disperse a quantity of powder containing about 25 mg of Ofloxacin (about 75 mg) in 60 ml of methanol and dilute to 100.0 ml with methanol and filter.
Reference solution:
Take about 25 mg of Ofloxacin RS in 100.0 ml volumetric flask, dissolve in 60 ml of Methanol and dilute to 100.0 ml with Methanol and filter.
Procedure:
Wash the column initially with Methanol at flow rate of 2.0 ml/min for not less than 15 minutes. And then run Mobile Phase for not less than 15 minutes.
Separately inject 10 µl of the Blank in single injection, Standard preparation in five replicate injections and the Test preparation in two replicate injections, record the chromatograms and measure the peak responses. From the peak responses, calculate the amount of Ofloxacin.
Evaluation of System Suitability:
Relative Standard Deviation of five replicate injections of Standard Solution of Ofloxacin peak should not be more than 2.0%.
The Theoretical plates of Ofloxacin peak should not be less than 2000.
The tailing factor of Ofloxacin should not be more than 2.0.
Calculation:
At Ws 100 P
———– x——— x ——– x ———- x Avg. Wt.
As 100 Wt 100
Where:
At: Mean area of the peak due to Ofloxacin obtained in the sample chromatogram.
As: Mean area of the peak due Ofloxacin obtained in the standard chromatogram.
Ws: Weight taken for Standard preparation in mg.
Wt: Weight taken for Test preparation in mg.
P= Potency of Ofloxacin working standard.
Requirement: As per current version of approved specification.
- 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.
Test Procedure:
Prepare and inject blank (diluents), Placebo, Standard solution & test solution. Check the peak purity of main peak.
Documentation:
Print chromatograms.
Acceptance criteria
No peak shall be eluted at the retention time of Ofloxacin in blank and placebo solution.
Peak purity of the main peak should be passes.
- 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).
Prepare and inject separately Blank and Standard solution as per finished product testing procedure and record the area response of main peak in the standard solution. Calculate the % RSD of response for six replicate injections.
Analysis sequence
Blank – 1 injection
Standard solution – 6 injections
Acceptance criteria
The relative standard deviation for peak area counts is not more than 2%.
- Method Precision (Repeatability):
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.
Prepare six individual sample of same batch and analyze as per finish product testing procedure.
Documentation:
Record the % Assay of Ofloxacin in Ofloxacin Tablet on the datasheet. Calculate the mean, standard deviation, and % RSD of six samples.
Acceptance criteria
% RSD shall be not more than 2.
- Intermediate Precision ( Ruggedness ):
Intermediate precision expresses within laboratory variation with different analysts or equipment within the same laboratory using same batch of drug product as specified under precision.
Test Procedure:
The analysis of the same batch will be done in six replicate analyses by using different columns by different analyst, by different system on different day. The mean, standard deviation and relative standard deviation will be calculated.
Documentation:
Calculate the mean, standard deviation, and % RSD for the operators and instruments and record on data sheet.
Acceptance criteria
The relative standard (RSD) of six analyses shall be not more than 2% and overall relative standard deviation shall be not more than 2% when compared with method precision results.
- Linearity and range:
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 standard solution in the range of 80% to 120% of concentration level and calculate the correlation coefficient “r”.
Test Procedure:
Prepare the standard solutions at five concentrations, typically 80, 90, 100, 110 and 120% of target concentration following finished product testing procedure. Duplicate replicates at each concentration will be analyzed.
Documentation:
Record results on a datasheet. Calculate the mean, standard deviation, and Relative Standard Deviation (RSD) for each concentration. Plot concentration (x-axis) versus mean response (y-axis) for each concentration. Calculate the correlation coefficient (r). Record these calculations on the datasheet.
Acceptance criteria
The correlation coefficient (r) shall be not less than 0.999.
- Solution stability:
It is essential when verify an analytical method to confirm that the analyte has adequate stability in both the standard and sample solution during analytical measurement stages of the testing.
Test Procedure:
Prepare the standard solution and test solution as per finish product testing procedure and anlyze the solution at the different time intervals and calculate the % difference for the assay result.
Acceptance criteria
The difference in results shall be not more than 2 from initial results.
- Revision History:
| Revision No. | Details of changes | Reason |
| 00 | New Document | Nil |
