by ANALYTICAL METHOD VERIFICATION PROTOCOL OF RELATED SUBSTANCES OF SILDENAFIL CITRATE Ph. Eur.
| Superseded Protocol No. | Nil |
| Effective Date |
TABLE OF CONTENTS:
| Sr. No. | Subject | Page No. |
| Protocol Approval | ||
| Objective | ||
| Scope | ||
| Responsibility of validation team | ||
| 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 verification is to provide documentary evidence that analytical methodology used for related substances of Sildenafil Citrate Ph. Eur. by pharmacopeia method is consistent and reliable results within the predetermined acceptance criteria.
Analytical method verification will be performed by considering thefollowing parameters:
| Parameters | Sildenafil Citrate Ph. Eur. |
| Specificity | |
| Precision | |
| System Precision | |
| Method Precision | |
| Intermediate Precision (Ruggedness) | |
| System Suitability |
- Scope :
The scope of this protocol is applicable for the verification of method of related substances 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 | 1st verification |
| Active Ingredient | Sildenafil Citrate Ph. Eur. |
| Method Reference | European Pharmacopeia |
| Specification Limits | Impurity A : NMT 0.15 % Impurity D : NMT 0.15 % Unspecified Impurity: NMT 0.10 % Total Impurity : NMT 0.5 % |
- Methodology:
Reagent:
Table 1.0: Chemicals/ Reagents
| Sr. No. | Reagent | Grade |
| 1. | Potassium Dihydrogen Phosphate | LR Grade |
| 2. | Potassium Hydroxide | LR Grade |
| 3. | Methanol | HPLC Grade |
| 4. | Acetonitrile | HPLC Grade |
| 5. | Water | HPLC grade or Milli Q Water |
Chromatographic conditions (By HPLC)
Instrument : High Performance Liquid Chromatography (UV Visible/DAD Detector)
Column : 250 mm x 4.6 mm, end-capped Octadecylsilyl silica gel for chromatography R (5µm)
Flow rate : 1.5 ml/min
Detector : 230 nm
Column Temperature : 25ºC
Injection Volume : 10 µl
Run time : 50 minutes
Retention time : Sildenafil – about 16 minutes
Pump mode : Gradient Method
Relative Retention time : Impurity A – about 1.25
: Impurity D – about 0.15
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 à 30 | 25 à 70 |
| 26 – 38 | 30 | 70 |
| 38 – 45 | 75 | 25 |
| 45 – 50 | 75 | 25 |
Preparation of buffer solution:
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.
Solvent mixture:
Acetonitrile for chromatography R, mobile phase A (10: 90 v/v).
Preparation of Mobile Phase:
Mobile phase A: Acetonitrile for chromatography R, buffer solution (20: 80 V/V)
Mobile phase B: Buffer solution, Methanol R1, Acetonitrile for chromatography R (20: 20: 60 V/V/V)
Test Solution (a):
Dissolve an accurately weighed 25.0 mg of test substance 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 (a):
Dissolve an accurately weighed 2.0 mg of Sildenafil impurity A CRS in test solution (a) and dilute to 10.0 ml with test solution (a). Dilute 1.0 ml of the solution to 20.0 ml with the solvent mixture.
Reference Solution (b):
Dissolve 5.0 mg of Sildenafil for peak identification CRS (containing impurity D) in the solvent mixture and dilute to 10.0 ml with the solvent mixture.
Reference Solution (c):
Dilute 1.0 ml of test solution (a) to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture.
Reference Solution (d):
Dissolve 25.0 mg of Sildenafil Citrate CRS / WS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Dilute 2.0 ml of the solution to 50.0 ml with the solvent mixture.
System suitability Solution:
Reference solution (a) or system suitability
- Resolution : Minimum 5.0 between the peaks due to Sildenafil and Impurity A.
- % RSD : Relative standard deviation should not more than 5.0 % for the peak of
Sildenafil citrate peak for reference solution (c).
Calculation of percentage contents:
For each impurity, use the concentration of Sildenafil in reference solution (c).
Procedure:
Equilibrate the HPLC column with mobile phase; inject the solution as per following sequence or as per the requirement mentioned in Table 2.0. Record the chromatogram and calculate the % impurities.
Table 2.0: Sequence
| Solutions | No of Injection to be injected in Sequence |
| Blank (Solvent mixture) | 1 |
| Reference solution (a) or System Suitability solution | 1 |
| Reference solution (b) | 1 |
| Reference solution (c) | 6 |
| Test Solution (a) | 1 |
| Blank (to avoid carry over) | 1 |
| Reference solution (c) Bracketing | 1 |
Identify the components in sample preparation with following relative retention times (RRT) as given in table.
Table 3.0: RRT & CF
| Sr. No. | Component Name | Relative Retention Time (RRT) | Correction Factor (CF) |
| 1. | Sildenafil | 1.0 | 1.0 |
| 2. | Impurity – A | 1.25 | 1.0 |
| 3. | Impurity – D | 0.15 | 0.70 |
| 4. | Unspecified Impurity | NA | 1.0 |
Disregard limit:
Disregard the peaks due to blank, citrate and peaks below 0.05 %.
Calculation:
At Wt 1 1 50
Impurity A = ———–x ———–x———–x———x———x C.F. x 100
As 50 100 10 Wt
At Wt 1 1 50
Impurity D = ———–x ———–x———–x———x———x C.F. x 100
As 50 100 10 Wt
At Wt 1 1 50
Any Unspecified Impurity = ———–x ———–x———–x———x———x C.F. x 100
As 50 100 10 Wt
Total Impurities: % Impurity A + % Impurity D + % Total unspecified impurities
- 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 solvent mixture.
Specificity of test method should be established by separately injecting blank solution (Solvent mixture), Reference solution (a), Reference solution (c), identification solution of Impurity A and Impurity D, test solution and spiked test solution. Identification solution and impurities spiked test solution will be prepared at specification level. Test solution to be prepared as per method of analysis.
Blank: Solvent Mixture
Preparation of Identification solutions:
Stock solution of Impurity A:
Weigh accurately about 2.5 mg of Impurity A and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100.0 ml with diluent and mix.
Identification solution of Impurity A:
Dilute 1.5 ml of stock solution of impurity A to 50.0 ml with diluent, mix.
Stock solution of Impurity D:
Weigh accurately about 2.5 mg of Impurity D and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100.0 ml with diluent and mix.
Identification solution of Impurity D:
Dilute 1.5 ml of stock solution of impurity D to 50.0 ml with diluent, mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
Reference Solution (a):
Dissolve an accurately weighed 2.0 mg of Sildenafil impurity A CRS in test solution (a) and dilute to 10.0 ml with test solution (a). Dilute 1.0 ml of the solution to 20.0 ml with the 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.
Reference Solution (c):
Dilute 1.0 ml of test solution (a) to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture.
Spike Test Solution (a):
Dissolve an accurately weighed 25.0 mg of test solution in 50 ml volumetric flask. Add 1.5 ml of stock solution of impurity A and stock solution of impurity D and make up the volume with solvent mixture.
Procedure: Inject the preparation of blank solution (solvent mixture), reference solution (a) or system suitability solution, reference solution (c) and identification solution of impurity A, impurity D, test solution (a) and spiked test solution (a) on a HPLC system with a Diode array detector (DAD) as follows in table 4.0. Determine the purity of the individual peaks of interest. Record the retention times and check for system suitability parameters. Obtained specificity data shall be reported in tabular manner with reference of table 5.0 and 6.0.
Table 4.0: Sequence
| Solutions | No of Injection to be injected in Sequence |
| Blank (Solvent mixture) | 1 |
| Reference solution (a) or System suitability solution | 1 |
| Reference solution (c) | 6 |
| Identification solution of Impurity A | 1 |
| Identification solution of Impurity D | 1 |
| Test solution (a) | 1 |
| Spike test solution (a) | 1 |
| Blank (to avoid carry over) | 1 |
| Reference solution (c) bracketing | 1 |
Table 5.0 Specificity data
| Sr. No | Sample | RT (min.) | RRT | Peak purity | |
| 1 | Blank (Solvent mixture) | NA | |||
| 2 | Reference solution (a) | Sildenafil | |||
| Impurity A | |||||
| 3 | Reference solution (c) | Sildenafil | |||
| 4 | Identification solution of Impurity A | ||||
| 5 | Identification solution of Impurity D | ||||
| 6 | Test Solution (a) | Sildenafil | |||
| 7 | Unknown Impurity | NA |
Table 6.0 Spiked test solution
| Sr. No | Sample | RT (min.) | RRT | Peak purity |
| 1 | Blank | NA | ||
| 2 | Sildenafil | |||
| 3 | Impurity A | |||
| 4 | Impurity D | |||
| 5 | Unknown Impurity | NA |
Acceptance Criteria:
- There should be no interference of the diluent, impurities at the retention time of Analyte peak,
- Impurity peaks should be well resolved from active peak and each other.
- Analyte
peak in standard solution and known Impurity peaks in spiked sample solution
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 (c) will be prepared as per method of analysis and six replicate injections to be injected in sequence and recorded the area response of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.
Table 7.0 System Precision- Repeatability of Standard Injections
| Sr. No. | Sildenafil | |
| 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 injections of Reference (a) should be NMT 5.0% 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.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known impurity at limit level. Un-spiked sample will be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample will be subtracted in spiked sample to calculate the actually known spiked amount of impurity. Record the area on data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation.
Table 8.0 Method precision results spiked sample at limit level
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | ||
| Impurity A | Impurity D | 1 | 2 | |||
| RRT | ||||||
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| Mean | ||||||
| SD | ||||||
| % RSD |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below. Reporting threshold value is 0.05%.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
- 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.
Reference (c) will be prepared as per method of analysis and six replicate injections to be injected in sequence and recorded the area response of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.
Table 9.0 System Precision- Repeatability of Standard Injections
| Sr. No. | Sildenafil | |
| 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 injections of Reference (a) should be NMT 5.0% and 1.0% respectively.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known impurity at limit level. Un-spiked sample will be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample will be subtracted in spiked sample to calculate the actually known spiked amount. Record the area on data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation.
Table 10.0 Intermediate (ruggedness) precision results spiked sample at limit level
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | ||
| Impurity A | Impurity D | 1 | 2 | |||
| RRT | ||||||
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| Mean | ||||||
| SD | ||||||
| % RSD |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below. Reporting threshold value is 0.05%.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
Table 11.0 Pooled results of analyst – I & analyst – II
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | ||
| Impurity A | Impurity D | 1 | 2 | |||
| Analyst I | ||||||
| RRT | ||||||
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| Analyst II | ||||||
| RRT | ||||||
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| Mean | ||||||
| SD | ||||||
| % RSD |
Acceptance criteria:
Pooled % RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
- System Suitability:
System suitability tests are based on concept that the equipment, electronics, analytical operations and sample to be analyzed, system suitability test provide the added assurance that on specific occasion the method is given accurate and precise results.
The system suitability should be as per below mention criteria in Table 12.0.
Table 12.0
| Sr. No. | Parameters | System Suitability Criteria | Limit |
| 1. | Resolution | The resolution between the peaks due to Sildenafil and Impurity A. | Minimum 5.0 |
| 2. | % RSD | Peak area due to sildenafil obtained from 6 replicate injections of reference solution (c) | NMT 5.0 % |
- Incident/Deviation:
Any Incident or Deviation observed during Analytical Method verification should be recorded and investigate as per SOP.
- Summary/Conclusion/recommendation:
Final Conclusion should be drawn from analytical method verification for its use to analyze the related substances of Sildenafil Citrate Ph. Eur. by pharmacopeia method.
Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.
Abbreviations:
REL : Related substance
VER : Verification
P : Protocol
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 for change |
| 00 | Nil | New Document |
