by RELATED SUBSTANCES METHOD VALIDATION PROTOCOL OF SILDENAFIL CITRATE TABLETS
| Superseded Protocol No. | Nil |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 verify the suitability of HPLC method for related substances test of Sildenafil Citrate in Sildenafil Citrate Tablets by considering thefollowing parameters:
- Precision
- System Precision
- Method Precision
- Intermediate Precision (Ruggedness)
- Specificity
- Force Degradation
- Linearity
- Accuracy
- Limit of detection ( LOD )
- Limit of quantification ( LOQ )
- Solution Stability
- Filter Paper Selection Study
- Robustness
- Scope :
This protocol is applicable for the validation of related substances method of Sildenafil Citrate 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 | For Erectile Dysfunction |
| Reason for validation | First validation |
| Active Ingredient | Sildenafil Citrate |
| Strength | Each film coated tablet contains: Sildenafil Citrate EP Equivalent to Sildenafil 50 mg |
| Methodology | Inhouse |
| Method Reference | Inhouse |
| Specification Limits | Single Impurity——- NMT 0.8% N-Oxide Impurity—- NMT 1.0% Total Impurities—— NMT 2.0% |
- Methodology:
Reagents:
Ammonium acetate (AR Grade)
Triethylamine. (HPLC Grade)
Acetic acid, HPLC Grade)
Methanol (HPLC Grade)
Water Milli-Q or equivalent
Chromatographic system:
Column : 25 cm x 4.6 mm, 5 µm, Zorbax EclipseXDBC18
Column temperature : 40°C
Flow rate : 1ml per minute
Detection Wavelength : 294 nm
Injection volume : 10 µl
Mobile phase:
- a solution containing 3.85 g of ammonium acetate and 1.0 ml of triethylamine in 1000 ml of water, adjusted to pH 5.5 ±0.05 with acetic acid.
2. Methanol,
Solvent mixture:
Mix mobile phase A & mobile phase B in the ratio of 50:50 v/v.
A linear gradient programme using the conditions given below,
| Time (Minutes) | Mobile Phase A | Mobile Phase B |
| 0 | 47 | 53 |
| 20 | 47 | 53 |
| 35 | 10 | 90 |
| 45 | 10 | 90 |
| 50 | 47 | 53 |
| 60 | 47 | 53 |
Test solution:
Crush to fine powder 20 Tablets, and weigh accurately and transfer the powder (Equivalent to 50 mg of Sildenafil) in to 50.0 ml volumetric flask add 35 ml of the solvent mixture, sonicate for 20 minutes and dilute to 50.0 ml with the solvent mixture, Filter through whatman no. 42 or equivalent filter paper.
Placebo preparation:
Weigh and transfer about 295 mg of placebo powder in to 50.0 ml volumetric flask add 35 ml of the solvent mixture, sonicate for 20 minutes and dilute to 50.0ml with the solvent mixture, Filter through whatman no. 42 or equivalent filter paper.
Reference solution (a):
Weigh accurately and transfer 28 mg of sildenafil citrate working standard in to100.0 ml volumetric flask add 75 ml of the solvent mixture, sonicate to dissolve and dilute to 100.0 ml with the solvent mixture, Filter through whatman no. 42 or equivalent filter paper.
Reference solution (b):
Dilute 5.0 ml of reference solution (a) to 100 ml with the solvent mixture.
Procedure:
Wash the column initially with Methanol and water (50:50) at flow rate of 1.0 ml/min for 30 minutes. And then run Mobile Phase for 30 minutes.
Separately inject 10 µl of the solvent mixture in single injection, placebo preparation in single injection.
Inject 10 µl of reference solution (b) in six replicate injections. The test is not valid unless the relative standard deviation for replicate injections is not more than 5.0 per cent.
Inject 10 µl of the solvent mixture and test solution. Append the Bracketing standard from solution (b).
Single Impurity:
In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (b). (NMT 0.8 per cent)
N-Oxide Impurity:
In the chromatogram obtained with the test solution, the area of peak corresponding to N-oxide impurity at relative retention time 0.54 is not more than the area of the principal peak in the chromatogram obtained with reference solution (b). (NMT 1.0 percent)
Total Impurities:
In the chromatogram obtained with the test solution, the sum of areas of all the secondary peaks is not more than 2.0 times the area of the peak in the chromatogram obtained with reference solution (b) (NMT 2.0 per cent).
- Materials and Methods
Analytical Parameters to be verified and acceptance criteria given below:
| Sr. No. | Analytical Performance Parameter | Acceptance Criteria |
| 1. | System Suitability Test | System Suitability Test shall meet the acceptance criteria. |
| 2. | System Precision | The % RSD of Six replicate of reference solution (b) should be NMT 5.0 |
| 3. | Method Precision | For known and other impurities in related substances method, relative standard deviation of six analyses shall be not more than 10%. This criteria is applicable for known impurities above LOQ level and for unknown impurities more than 0.1 %. |
| 4. | Intermediate Precision (Ruggedness) | For known and other impurities in related substances method, relative standard deviation of six analyses shall be not more than 10 % and overall % RSD shall not be more than 10% when compared with method precision results. This criteria is applicable for known impurities above LOQ level and for unknown impurities more than 0.1 %. |
| 5. | Specificity | No peak shall be eluted at the retention time of main peak and known impurities in blank and placebo solution. Peak purity of main peak and known impurities shall be pass. |
| 6. | Force Degradation | Difference between treated sample results and untreated sample shall not be more than 15%. Peak purity of main peak shall be pass. |
| 7. | Linearity | The correlation coefficient shall be not less than 0.99. |
| 8. | Accuracy | The recovery of known impurities of the drug at each spiking level shall be between 85 % and 115 % and RSD from replicate analysis shall be not more than 10 %.The overall average recovery shall be between 85 % to 115 % with RSD of not more than 10%. |
| 9. | LOD | % RSD for six replicates response of LOD Solutions shall be not more than 30.The signal to noise ratio should be more than 3:1. |
| 10. | LOQ | % RSD for six replicates response of LOQ Solutions shall be not more than 10.The signal to noise ratio should be more than 10:1. |
| 11. | Stability of solution | Cumulative % RSD of peak area for known impurities and main peak shall not be more than 10. |
| 12. | Robustness | System suitability criteria should be met under all conditions and result should be complying under all condition. |
| 13. | Filter Paper Selection Study | If the impurity is less than 0.1 %, no comparison shall be made. For known impurities and Total impurities % difference shall be less than ±10 %. |
- System Suitability test
%RSD of 6 replicate of Reference solution (b) shall be not more than 5.0%
- 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 diluents (Blank) and 6 replicate of Reference solution (b). Calculate the % RSD of response for six replicate of Sildenafil Citrate.
Acceptance criteria
The relative standard deviation for peak area counts is not more than 5.0%.
- 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.
Test Procedure:
Sample will be analyzed for six times (single batch) as per described in methodology. Calculate the % RSD of content of each known, unknown impurity and total impurities. Details record on the data sheet. In case any known impurity is not present in specimen sample then spike the known impurity and calculate the % RSD.
Acceptance criteria
For known and other impurities in related substances method, relative standard deviation of six analyses shall be not more than 10 %. This criteria is applicable for known impurities above LOQ level and for unknown impurities more than 0.1 %.
- Ruggedness (Intermediate Precision):
Intermediate precision expresses within laboratory variation with different analysts or equipment or different column on different days using same batch of drug product as per finished product testing procedure.
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.
Note: The result obtained from method precision will be considered as the results of first analyst for comparison in ruggedness.
Acceptance criteria
For known and other impurities in related substances method, relative standard deviation of six analyses shall be not more than 10 % and overall % RSD shall not be more than 10% when compared with method precision results. This criteria is applicable for known impurities above LOQ level and for unknown impurities more than 0.1 %.
- 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.
Preparation of N- oxide impurity solution (10 ppm):
Weigh accurately 5 mg of N – oxide impurity and transfer into a 50.0 ml volumetric flask add about 35 ml of the solvent mixture, sonicate to dissolve and dilute to 50.0 ml with the solvent mixture and mix well. Transfer accurately 1 ml of this solution into 10 ml of volumetric flask. Dilute with the solvent mixture and mix well. Filter it through 0.45 um nylon membrane filter paper.
Procedure:
Inject blank, placebo, standard solution, sample solution spike with known impurity or inject known impurity individually.
Acceptance criteria
No peak shall be eluted at the retention time of n-oxide and Sildenafil Citrate in blank and placebo solution.
Peak purity of main peak and known impurities shall be pass.
- Forced Degradation:
Preparation of mobile phase, solvent mixture (Diluent), reference solution (b), placebo solution, sample solution and system suitability check as per describe in methodology and record on the datasheet.
Stress the sample at the following conditions and evaluate the peak purity and all degradation study procedure shall be recorded in data sheet and mentioned in method validation report.
- Degradation by Hydrochloric Acid
- Degradation by Sodium Hydroxide
- Degradation by hydrogen peroxide 30% (Oxidative degradation)
- Degradation by thermal
- Degradation by hydrolysis
Acceptance criteria
The difference between treated and untreated sample should not be more than 10%.
- 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 (amount) of analyte in the sample.
Determine the linearity by preparing the solution (standard and impurity standard solution) at different levels over a specified range from 1% to 200 % of the specification limit. Each level inject in single. Calculate correlation co-efficient.
Test Procedure:
Prepare and inject the Linearity solutions in the range 1%, 5%, 10%, 50%, 100%, 150% and 200 % concentration level calculate the correlation coefficient.
Acceptance criteria
Plot the calibration curve of peak area v/s concentration for the studied peak. The correlation coefficient (r), after the regression analysis of the results should not be less than 0.99.
- Accuracy:
The accuracy of an analytical procedure express the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. The accuracy shall be established across the specified range of the analytical procedure.
Test Procedure:
Prepare sample solution with spiking known impurities in triplicate at about 40 %, 100 % and 150 % of specification limit and calculate the % overall average recovery for known impurities.
Acceptance criteria
The recovery of known impurities of the drug at each spiking level shall be between 85 % and 115 % and RSD from replicate analysis shall be not more than 10 %.The overall average recovery shall be between 85 % to 115 % with RSD of not more than 10%.
- 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 Sildenafil Citrate and n-oxide will be calculated on hit and trial basis by diluting stock standard. Preparations to such a concentration that the signal to noise ratio will be equal to or greater than 3.
For impurity standard stock solution preparation and Sildenafil citrate standard stock solution refer “linearity and range”. Stock standard solution shall be diluted to such a concentration that the signal to noise ratio will be equal to or greater than 3.
Calculate % RSD for six replicates of LOD Solutions.
Acceptance criteria:
% RSD for six replicates response of LOD Solutions 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 quantified with acceptable precision under the stated experimental condition.
For impurity standard stock solution preparation and Sildenafil citrate standard stock solution refer “linearity and range”. Stock standard solution shall be diluted to such a concentration that the signal to noise ratio will be equal to or greater than 10.
Calculate % RSD for six replicates of LOQ Solutions.
Acceptance criteria:
% RSD for six replicates response of LOQ Solutions shall be not more than 10.The signal to noise ratio should be more than 10:1.
- Solution Stability:
It is essential when validating 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:
Spike sample solution with known impurity at the specification level and standard solution shall be prepared and kept at room temperature. Sample solution and standard solution shall be analyzed initially and at different intervals as 4h, 8h, 12h, 18h, 24h, 30h and 36h. Calculate the system suitability the % cumulative RSD of peak area for known impurities and main peak.
Acceptance criteria:
Cumulative % RSD of peak area for known impurities and main peak shall not be more than 10.
- Filter Paper Selection Study:
Test Procedure:
Prepare sample solution. A portion of sample solution shall be centrifuge and other portion of sample solution shall be filtered with filters (e.g. PVDF and Nylon filter) inject all samples and calculate the % impurity for each sample and calculate the % impurity difference between centrifuges vs. filtered samples.
Acceptance criteria
If the impurity is less than 0.1 %, no comparison shall be made. For known impurities and Total impurities % difference shall be less than + 10 %.
- Robustness:
To establish the robustness of test method and to demonstrate its reliability for minor changes in chromatographic conditions.
Test Procedure:
Robustness of the method shall be investigated by checking the system suitability parameters by deliberately varying the instrumental condition such as
- By changing the flow rate by ±10%
- By changing the column oven temperature by ±5°C
- By changing the pH of mobile phase A by ±0.2 units
- Wave length detection by ± 2nm
Prepare the standard solution and sample solution as per test method by deliberate variations made in the method for each condition as mentioned in protocol and analyze.
Calculate the resolution for known impurities and compare with resolution from method precision experiment.
Acceptance criteria
System suitability criteria should be met under all conditions and result should be complying under all condition.
- Revision History:
| Revision No. | Details of changes | Reason |
| 00 | Nil | New Document |
