by ANALYTICAL METHOD VERIFICATION PROTOCOL FOR ASSAY OF SILDENAFIL CITRATE Ph. Eur.
| Superseded Protocol No. | Nil |
| Effective Date |
Table of contents :
| Sr. No. | Subject | Page No. |
| Protocol Approval | ||
| Objective | ||
| Scope | ||
| Responsibility | ||
| Product profile | ||
| Methodology | ||
| Verification 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 |
- Objective:
The objective of this protocol is to verify the suitability of Sildenafil citrate Ph. Eur. by considering thefollowing parameters:
| Parameters | Sildenafil citrate Ph. Eur. |
| Specificity | yes |
| Precision | |
| System Precision | yes |
| Method Precision | yes |
| Intermediate Precision | yes |
| System Suitability | yes |
- Scope :
This protocol is applicable for the verification of assay of Sildenafil citrate Ph. Eur.
- 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 of protocol. |
| Co-ordination with QC to carryout Verification. | |
| Review of data and summary report. | |
| Head QA | Approval of Protocol |
- Product Profile:
| Category | Erectile dysfunction |
| Reason for Verification | First Verification |
| Active Ingredient | Sildenafil citrate Ph. Eur. |
| Method Reference | In House |
| Specification Limits | Assay (On anhydrous basis) 98.0 to 102.0% of C28H38N6O11S |
- Methodology:
- Assay
Chemical, reagents and filters:
Table 1.0: Chemical, reagents and filters
| Sr. No | Material /Chemicals/Filters | Grade |
| 1.0 | Potassium dihydrogen phosphate (KH2PO4) | AR Grade |
| 2.0 | Potassium hydroxide | HPLC Grade |
| 3.0 | Water | HPLC Grade or Milli Q water |
| 4.0 | Methanol | HPLC Grade |
| 5.0 | Acetonitrile | HPLC Grade |
Chromatographic conditions (By HPLC):
Instrument : High Performance Liquid Chromatography (UV Visible/DAD Detector)
Column : 250mm x 4.6mm,(5µ) end-capped Octadecylsilyl silica gel for chromatography
Flow rate : 1.5 ml/min
Detector : 230 nm
Column Temp : 25°C
Injection Volume : 10 µl
Run time : 50 minutes (Sildenafil RT Approximately 16.0 min)
Program : Gradient Method
Mobile Phase A : Acetonitrile: Buffer solution (20:80 V/V) mix well and filtered.
Mobile Phase B : Buffer solution: Methanol: Acetonitrile (20:20:60 V/V/V) mix well and filtered.
Solvent mixture : Acetonitrile: Mobile phase A (10:90 V/V)
Preparation of Phosphate Buffer:
Take 2.72 g of Potassium Dihydrogen Phosphate R in 900 ml of water to dissolve and adjust to pH 6.5 with a 120 g/l solution of Potassium Hydroxide R in water and then dilute to 1000 ml of water.
Gradient program:
| Time (min) | Mobile phase A(Per cent V/V) | Mobile phase B (Per cent V/V) |
| 0 – 3 | 75 | 25 |
| 3 – 26 | 75 →30 | 25 →70 |
| 26 – 38 | 30 | 70 |
| 38 – 45 | 75 | 25 |
| 45 – 50 | 75 | 25 |
Test Solution (a):
Dissolve an accurately weighed 25.0 mg of test solution in 50 ml volumetric flask and make up the volume with solvent mixture.
Test Solution (b):
Dilute 2.0ml of the test solution (a) to 50.0ml with the solvent mixture.
Reference Solution (d):
Dissolve an accurately weighed 25 mg of Sildenafil Citrate CRS / WS in the solvent mixture in a 50 ml volumetric flask and then make up the vole with solvent mixture. Transferred 2 ml of this solution in to 50.0 ml volumetric flask and then dilute up to mark with solvent mixture.
Evaluation of System Suitability:
- Area % RSD : Should not be more than 2.0 % for peak area due to Sildenafil from six replicate injections of Reference solution (d).
Procedure:
Equilibrate the HPLC Column with mobile phase. Inject as per the below sequence or as per requirement.
Sequence of Injections (Assay):
Table 2.0: Sequence of Injection for assay
| Preparation | No. of Injections | Action |
| Blank Solution | 1 | Record the blank Chromatograms |
| Reference solution (d) | 6 | Check for system suitability Criteria (i) |
| Test Solution (b) | 2 | Inject the solution and calculate assay for mean injection and report |
| Standard solution (Bracketing standard) | 1 | %, Area RSD of total 7 injections ≤ 2.0% (6 Injections + 1 injection of bracketing standard) |
Calculation for Assay
Calculate the percentage of Sildenafil citrate Ph. Eur. taken by the formula:
% Assay on as in basis
At Ws 2 50 50 P
% Sildenafil citrate = ——- x ——- x —— x —— x —— x —— x 100
As 50 50 Wt 2 100
% Assay on Anhydrous basis
Assay on as is basis
% Sildenafil citrate = ———————————– x 100
(100 – Water content)
Where,
As = Average peak area due to Sildenafil obtained from six replicate injections of
Reference solution (d)
At = Average peak area due to Sildenafil obtained from Test solution (b)
Ws = Weight of Sildenafil citrate Ph. Eur. RS/WS taken in mg.
Wt = Weight of sample taken in mg.
P = Potency of Sildenafil citrate Ph. Eur. RS/WS in %, on as such basis.
- Water content:
Equipment: Karl Fischer Apparatus
Procedure: Introduce into the titration vessel add methanol. Where applicable for the apparatus used, eliminate residual water from the measurement cell or carry out a pre-titration. Introduce 0.200 g of the substance to be examined rapidly and carry out the titration, stirring for the necessary extraction time.
Limit: Maximum 2.5 %
- Verification parameters:
The following parameters to be perform for the verification activity.
Specificity
Precision
System Suitability
- 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 blank.
Specificity of test method should be established by separately injecting blank solution (solvent mixture), reference solution (d) and test Solution (b). Test Solution (b) to be prepared as per method of analysis.
Blank: Solvent mixture
Test Solution (a):
Dissolve an accurately weighed 25.0 mg of test solution in 50 ml volumetric flask and make up the volume with solvent mixture.
Test Solution (b):
Dilute 2.0ml of the test solution (a) to 50.0ml with the solvent mixture.
Reference Solution (d):
Dissolve an accurately weighed 25 mg of Sildenafil Citrate CRS / WS in the solvent mixture in a 50 ml volumetric flask and then make up the vole with solvent mixture. Transferred 2 ml of this solution in to 50.0 ml volumetric flask and then dilute up to mark with solvent mixture.
Procedure: Inject the preparation of blank solution (solvent mixture), Reference solution (d), and test solution (b) on a HPLC system with a Diode array detector (DAD) as follows in Table 4.0. Determine the purity of the individual peak of interest. Record the retention times of all peaks obtained from the respective solution and check for system suitability parameters for sildenafil peak obtained.
Table 3.0: Sequence of Injection (Specificity)
| Solution | No of Injection to be injected in Sequence |
| Blank (solvent mixture) | 1 |
| Reference solution (d) | 6 |
| Test Solution (b) | 1 |
| Standard Solution + Bracketing | 1 |
Table 4.0: Specificity data
| Sr. No | Sample | RT (min.) | Peak purity | |
| 1 | Blank | NA | ||
| 2 | Reference solution (d) | Sildenafil | ||
| 3 | Test Solution (b) | Sildenafil |
Acceptance Criteria:
- There should be no interference of the solvent mixture at the retention time of analyte peak.
- Blank peak should be well resolved from active peak and each other,
- Analyte peak in Reference solution (d)
and test solution (b) should be spectrally pure.
- Precision:
- System Precision:
- 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). Reference solution (d) will be prepared as per method of analysis and six replicate injections to be injected in sequence and recorded the area response and retention time of main analyte peak. Calculate the % area RSD and % RT RSD of main analyte peak.
Table 5.0: System Precision- Repeatability of Standard Injections
| Sr. No. | Sildenafil Peak | |
| 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 2.0% 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. Check for system suitability criteria.
Test Procedure for Assay:
Prepare the six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % assay (on anhydrous), mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 6.0: Method precision results for Assay
| Sample No. | Sample wt. (mg) | Peak area | Mean % Assay | % Mean | SD | % RSD |
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
Acceptance Criteria:
Calculate the assay for each analysis and calculate the mean, SD and % RSD.
% RSD for assay values should be NMT 2.0%.
- 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.
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 and retention time of main analyte peak. Calculate the % area RSD and % RT RSD of main analyte peak.
Table 7.0: Repeatability of Standard Injections
| Sr. No. | Sildenafil Peak | |
| 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 2.0% and 1.0% respectively.
Test Procedure:
Prepare the six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % assay (on anhydrous), mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 8.0: Intermediate precision results for Assay
| Sample No. | Sample wt. (mg) | Peak area | Mean % Assay | % Mean | SD | % RSD |
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
Acceptance Criteria:
Calculate the assay for each analysis and calculate the mean, SD and % RSD of assay (on anhydrous) values should be NMT 2.0%.
Table 9.0: Pooled results of Assay of analyst – I & analyst – II
| Sample No. | Sample wt. (mg) | % Assay |
| Sildenafil citrate Ph. Eur. | ||
| ANALYST I | ||
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| ANALYST II | ||
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| Pooled % RSD |
Acceptance Criteria:
The pooled % RSD for assay values of Analyst I and Analyst II should be NMT 2.0%.
- System Suitability
To ensure that during each analysis, the analytical procedure is giving accurate and precise results, system suitability parameters have been set. The set limits are given below. The data obtained will be summarized in Table.
Table 10.0: System Suitability Criteria
| Sr. No. | System Suitability Parameter | Set Limits |
| 1. | RSD for the peak area of the five replicate injections of Sildenafil. | Not more than – 2.0% |
- Incident /Deviation:
Any incident or deviation observed during analytical method verification should be recorded and reported in verification report.
- Summary/ Conclusion / Recommendation:
Final conclusion should be drawn from analytical method verification for its use to analyze the assay test of Sildenafil citrate Ph. Eur. by HPLC.
Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.
Abbreviation
ASS : Assay
VER : Verification
P : Protocol
T : Tablets
SD : Standard deviation
HPLC : High performance liquid chromatography
DAD : diode-array detector
RT : Retention Time
mL : Milliliter
mg : Milligram
min. : Minutes
QA : Quality Assurance
QC : Quality Control
% : Percentage
ºC : Degree centigrade
µl : Microlitre
EP : Europeian Pharmacopoeia
RSD : Relative standard deviation
NLT : Not less than
NMT : Not more than
Ws : Working standard
Wt : Weight
Vol : Volume
As : Standard Area
At : Test Area
Revision History:
| Revision No. | Details of changes | Reason |
| 00 | Nil | New Document |
