ANALYTICAL METHOD VERIFICATION PROTOCOL FOR IMPURITY E (BY TLC) OF SILDENAFIL CITRATE Ph. Eur.

ANALYTICAL METHOD VERIFICATION PROTOCOL FOR IMPURITY E (BY TLC) 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Superseded Protocol No.	Nil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

1. 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 Impurity E by TLC (Thin Layer Chromatography) of Sildenafil Citrate Ph. Eur. by considering thefollowing parameters:

Parameters	Chlorphenamine
Specificity	yes
Precision
Method Precision	yes
Intermediate Precision	yes
System Suitability	yes

• Scope :

This protocol is applicable for the verification of Impurity E 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
Specification Limits	Maximum 0.1% (Compare the intensities of any zone due to impurity E is not more intense than the principal zone in the chromatogram obtained with reference solution (b).

• Methodology:

Chemical, reagents and filters:

Table 1.0: Chemical, reagents and filters

Sr. No	Material /Chemicals/Filters	Grade
1.	Conc. Ammonia	Merck or equivalent
2.	Methanol	Merck or equivalent
3.	Ethanol	Merck or equivalent
4.	Ethyl Acetate	Merck or equivalent
5.	Dichloromethane (MDC)	Merck or equivalent
6.	Imidazole (Impurity E)	European Pharmacopoeia
7.	TLC silica gel F254 plate R (2-10µm)	Merck or equivalent

Solvent mixture:

Prepare a mixture of concentrated Ammonia, Water and Methanol in reagent bottle with the ratio of 5: 25: 75 v/v/v.

Test Solution:

Transfer about 35.0 mg of test solution to be examined in 2.0 ml of solvent mixture and dissolve with the aid of ultra sound if necessary.

Reference solution (a):

Weigh accurately about 7.0 mg of Imidazole CRS (Impurity E) in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture.

Reference solution (b):

Dilute 5.0 ml of reference solution (a) to 10.0ml with the solvent mixture.

Reference solution (c):

Mix 1.0 ml of the test solution and 1.0 ml of reference solution (a).

Mobile Phase:

Prepare a mixture of concentrated Ammonia, Ethanol (96 %), Ethyl Acetate and Methylene Chloride in the ratio of 1: 20: 30: 50 v/v/v/v. Close the stopper and mix well. Transfer the mobile phase in to TLC chamber which is lined with filter paper on three sides. Wet the paper with mobile phase, close the lid and keep the chamber for half an hour for saturation.

Procedure:

Separately apply 10 µl of the test solution, reference solution (b) and reference solution (c) as a bands of 6 mm. Place the plate in the chromatographic chamber and develop the chromatograms in the mobile phase until the solvent front has moved about 15 cm from the origin. Remove the plate from the developing chamber, mark the solvent front and drying 100°C for about 15 minutes. Expose to iodine vapour until the plate is light brown. Examine the plate under short-wavelength UV at 254nm.

Retardation factor: Citrate = about 0; impurity E = about 0.25; Sildenafil = about 0.4

System Suitability: Reference solution (c)

The chromatogram shows 2 clearly separated zones.

Limits:

Impurity E:

Compare the intensities of any zone due to impurity E is not more intense than the principal zone in the chromatogram obtained with reference solution (b) (0.1%).

• Verification parameters:

The following parameters to be perform for the verification activity.

      Specificity

      Precision

• Method Precision (Analyst I)
• Intermediate Precision (Analyst II)

        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 and placebo.

Specificity of test method shall be established by separate injecting of blank solution (solvent mixture), reference solution (b) and test solution and on TLC plate. Sample solution shall be prepared as per method of analysis given in section 6.0.

Blank Solution:

Apply separately to the plate10 µl of Solvent mixture.

Test Solution:

Transfer about 35.0 mg of test solution to be examined in 2.0 ml of solvent mixture and dissolve with the aid of ultra sound if necessary.

Reference solution (a):

Weigh accurately about 7.0 mg of Imidazole CRS (Impurity E) in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture.

Reference solution (b):

Dilute 5.0 ml of reference solution (a) to 10.0ml with the solvent mixture.

Reference solution (c):

Mix 1.0 ml of the test solution and 1.0 ml of reference solution (a).

Procedure:

Separately apply 10 µl of the test solution, reference solution (b) and reference solution (c) as a bands of 6 mm. Place the plate in the chromatographic chamber and develop the chromatograms in the mobile phase until the solvent front has moved about 15 cm from the origin. Remove the plate from the developing chamber, mark the solvent front and drying 100°C for about 15 minutes. Examine the plate under short-wavelength UV at 254nm.

Acceptance Criteria:

1. There shall be no interference of the solvent mixture at zone of Imidazole region.
2. Compare the intensities of any zone due to impurity E is not more intense than the principal zone in the chromatogram obtained with reference solution (b) (0.1%).
3. Retardation factor: Citrate = about 0; impurity E = about 0.25; Sildenafil = about 0.4

• Precision:
  • 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 by visual observation in UV cabinet.

Test Procedure:

Prepare the six sample of same batch and analyze as per method of analysis, record the observation as given below in table.

Table 2.0: Method precision results for TLC

Sample no.	Sample wt. (mg)	Observation
1
2
3
4
5
6

Acceptance Criteria:

Compare the intensity is of any zone due to impurity E is not more intense than the principal zone in the chromatogram obtained with reference solution (b) (0.1%).

• Intermediate Precision (Ruggedness):

Intermediate precision expresses within laboratory variation with different analysts or different/same equipment or different days using same batch of drug substance as per method of analysis.

Test Procedure:

Prepare the six sample of same batch and analyze as per method of analysis, record the record the observation as given below in table.

Table 3.0: Intermediate precision results for TLC

Sample no.	Sample wt. (mg)	Observation
1
2
3
4
5
6

Acceptance Criteria:

Compare the intensities of any zone due to impurity E is not more intense than the principal zone in the chromatogram obtained with reference solution (b) (0.1%).

Table 4.0: Pooled Observation of analyst – I & analyst – II

Sample No.	Sample wt. (mg)	Impurity E
Observation
ANALYST I
1
2
3
4
5
6
ANALYST II
1
2
3
4
5
6

Acceptance Criteria:

Compare the intensities of any zone due to impurity E is not more intense than the principal zone in the chromatogram obtained with reference solution (b) (0.1%).

• 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 5.0: System Suitability Criteria

Sr. No.	System Suitability Parameter	Observation
1.	The chromatogram shows 2 clearly separated zones in reference solution (c).	Complies

• Incident /Deviation:

Any incident or deviation observed during analytical method verification shall be recorded and reported as per SOP.

• Summary/ Conclusion / Recommendation:

Final conclusion should be drawn from analytical method verification for its use to analyze the Impurity E by TLC method.

Summary of verification report shall be prepared and accordingly standard testing procedure to be updated if required.

Abbreviation:

REL                 :           Related substance

            VER                :           Verification

            P                      :           Protocol

            TLC                 :           Thin Layer Chromatography

            ml                    :           Milliliter

            mg                   :           Milligram

            min.                 :           Minutes

            QA                   :           Quality Assurance

            QC                  :           Quality Control

            %                     :           Percentage

            hrs                   :           Hours

            µm                   :           Micrometer

            µl                     :           Microlitre

            Ph.Eur.            :           European Pharmacopoeia

            NLT                 :           Not less than

NMT                :           Not more than

Vol                    :           Volume

Revision History:

Revision No.	Details of changes	Reason
00	Nil	New Document