ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RESIDUAL SOLVENT OF SILDENAFIL CITRATE Ph. Eur.

ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RESIDUAL SOLVENT 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Superseded Protocol No.	Nil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 verification is to provide documentary evidence that analytical methodology used for residual solvents of Sildenafil citrate Ph. Eur. by GC method is consistent and reliable results within the predetermined acceptance criteria. This verification study is intended to show that the method is suitable for release of residual solvents of manufacturing batches.

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 residual solvents of Sildenafil citrate Ph. Eur. By GC method.

• 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	Dimethyl formamide     :  NMT 500 ppm Methanol :  NMT 1500 ppm Ethyl acetate :  NMT 3000 ppm Toluene :  NMT 500 ppm Acetone :  NMT 3000 ppm Methylene chloride :  NMT 400 ppm

• Methodology:

Solvents:

Table 1.0: Chemical

Sr. No.	Solvents	Grade
	Dimethyl formamide	GC Grade
	Methanol	GC Grade
	Ethyl acetate	GC Grade
	Toluene	GC Grade
	Acetone	GC Grade
	Methylene chloride	GC Grade
	DMSO (Dimethyl sulfoxide)	GC Grade

Residual Solvents (By GC)

Instrument                         :           Gas Chromatography equipped with FID detector

Detector                            :           FID (Flame ionization detector)

Column                             :           30 meters x 0.53 mm fused silica analytical column coated with A 3.0 µm, DB-624 Stationary phase or equivalent

Flow rate                           :           4.0 ml /min

Injector mode                    :           Split (1:5)

Injector Port temp.                        :           180˚C

Detector Port temp.          :           260˚C

Oven Temperature                       :           Start at 40°C. Hold for 8 minutes then rise at a rate of 35°C per min to 240°C and hold for 8 minutes.

Head space parameters:

Equilibration (vial) temperature     :           95°C

Equilibration (vial) time                 :           30 min

Transfer line temperature             :           105°C

Carrier gas                                    :           Nitrogen

Pressurization time                       :           30 seconds

Injection volume                            :           1.0 ml

Blank:

Transfer 5 ml of Dimethyl Sulfoxide into the vial and crimp the vial with PTFE septa and aluminum crimp cap as blank preparation and note observe for interpretations of the unknown peaks if any,

Stock solution preparation:

Take 500 mg of Dimethyl Formamide, 1500 mg of Methanol, 500 mg of Toluene, 3000 mg of Ethyl Acetate, 400 mg of Methylene Chloride and 3000 mg of Acetone into a 100 ml volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Standard Solution preparation:

Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

Test preparation:

Weigh accurately about 500 mg of test sample into a vial; add 5 ml Dimethyl Sulfoxide and crimp cap (100 mg/ml).

Procedure:

Transfer 5 ml of Dimethyl Sulfamide into the vial and crimp the vial with PTFE septa and aluminum crimp cap as blank preparation. Take 5.0 ml of standard solution in 6 different 20 ml head space vial fitted with a septum and crimp camp and seal. Separately inject equal volumes (1.0 ml) of standard solution (six Injections), inject blank to avoid carry over (single) and the test solution (duplicate) into the system. Record the chromatograms and measure the peak responses.

System Suitability:

% RSD    :  Area % RSD for peak due to each solvent content is not more than 15%.

Equilibrate the system and column; inject the solution as per following vial order or as per the requirement mentioned. Record the chromatogram and calculate the solvent content.

Table 2.0: Sequence

Sr. No.	Solutions	No of Injection to be injected in Sequence
1	Blank (DMSO)	1
2	Standard solution (Six vials)	6
3	Blank (DMSO) to avoid carry over	1
4	Test Solution	2
5	Standard Solution +Bracketing	1

                                  At               DS       

     Calculation: =   ———X———– X P X 10⁴

                                  As                 D      

Where,

At   = Solvent peak area in the sample chromatogram

As  = Solvent peak area in the Standard chromatograms

DS =  Standard Dilution (mg/ml)

D   = Sample Dilution (mg/ml)

P    = Solvent Purity (% w/w as is basis)

• Verification parameters:

The following parameters to be perform for the verification activity.

1. Specificity
   1. Precision
   1. System Suitability

• Specificity:

Specificity is the ability to assess unequivocally the Analyte in the presence of components which may be expected to be present.

Specificity of test method should be established by separately injecting blank solution (DMSO), identification solution of Dimethyl Formamide, Methanol, Ethyl acetate, Toluene, Acetone and Methylene chloride, spiked solution and test solution. Spike sample shall be prepared at specification limit level and test solution to be prepared as per method of analysis. sequence shall be prepare as per the sequence mentioned in table no 3.0 and data shall be represent in report as per the table no 4.0 & 5.0 .

Blank: Dimethyl sulfoxide (DMSO)

Preparation of Identification solutions:

Standard stock solution of Dimethyl Formamide :

Transfer 500 mg of Dimethyl Formamide into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Identification solution of Dimethyl Formamide: Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

Standard stock solution of Methanol:

Transfer 1500 mg of Methanol into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Identification solution of Methanol: Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

Standard stock solution of Ethyl acetate:

Transfer 3000 mg of Ethyl acetate into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Identification solution of Ethyl acetate: Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

Standard stock solution of Toluene:

Transfer 500 mg of Toluene into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Identification solution of Toluene: Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

• Standard stock solution of Acetone:

Transfer 3000 mg of Acetone into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Identification solution of Acetone: Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

• Standard stock solution of Methylene chloride:

Transfer 400 mg of Methylene chloride into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Identification solution of Methylene Dichloride: Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

Standard stock solution preparation:

Take 500 mg of Dimethyl Formamide, 1500 mg of Methanol, 500 mg of Toluene, 3000 mg of Ethyl Acetate, 400 mg of Methylene Chloride and 3000 mg of Acetone into a 100 ml volumetric flask. Dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Standard Solution preparation:

Take 1 ml of stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide. Take 5 ml of this solution into a vial fitted with a septum and crimp camp and seal.

Test preparation:

Weigh accurately about 500 mg of test sample into a vial; add 5 ml Dimethyl Sulfoxide and crimp cap (100 mg/ml).

Solvent stock preparation: (for spiked sample)

Take 1 ml of each individual solvent stock solution into a 100 ml volumetric flask, dissolve and dilute up to the mark with Dimethyl Sulfoxide.

Spiked test solution:

Weight accurately about 500.0 mg Sildenafil citrate sample into 20.0 ml of headspace vial and add 5.0 ml of Solvent stock solution.

Tablet 3.0: Sequence

Sr. No	Solution	No of Injection to be injected in Sequence
1	Blank (DMSO)	1
2	Standard Solution (Six vials)	6
3	Identification solution of Dimethyl Formamide	1
4	Identification solution of Methanol	1
5	Identification solution of Ethyl acetate	1
6	Identification solution of Toluene	1
7	Identification solution of Acetone	1
8	Identification solution of Methylene Dichloride	1
9	Blank (DMSO) (to avoid carry over)	1
10	Test Solution	1
11	Spiked Test Solution	1
12	Standard solution + Bracketing	1

Table 4.0 Specificity data

Sr. No	Sample	RT (min.)
1	Blank (DMSO)
2	Standard solution	Dimethyl Formamide
Methanol
Ethyl acetate
Toluene
Acetone
Methylene chloride
3	Identification solution of Dimethyl Formamide
4	Identification solution of Methanol
5	Identification solution of Ethyl acetate
6	Identification solution of Toluene
7	Identification solution of Acetone
8	Identification solution of Methylene chloride
9	Test Solution	Dimethyl Formamide
Methanol
Ethyl acetate
Toluene
Acetone
Methylene chloride
Dimethyl sulfoxide

Table 5.0 Spiked test solution

Sr. No	Sample	RT (minutes)
1	Blank
2	Dimethyl Formamide
3	Methanol
4	Ethyl acetate
5	Toluene
6	Acetone
7	Methylene chloride
8	Dimethyl sulfoxide

Acceptance Criteria:

1. There should be no interference of the blank and solvent content at the same retention time.
2. Solvent peaks should be well resolved from each other and observe the elution of pattern.
   1. Precision:
      1. 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).

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 of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.

Table 6.0 System Precision- Repeatability of Standard Injections

Sr. No.	Dimethyl formamide	Methanol	Ethyl acetate
Peak Area	Retention time (min.)	Peak Area	Retention time (min.)	Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
SD
% RSD
Sr. No.	Toluene	Acetone	Methylene chloride
Peak Area	Retention time (min.)	Peak Area	Retention time (min.)	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 15.0% 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 solvent content of Dimethyl Formamide, Methanol, Ethyl acetate, Toluene, Acetone and Methylene Dichloride at limit level. As such sample will be also analyzed to identify the known solvent content in sample. Obtained known solvent in as such sample will be subtracted in spiked sample to calculate the actually known spiked amount of solvent content.  Record the area on data sheet and calculate the ppm of solvent content. Mean, standard deviation and % relative standard deviation shall be reported.

Table 7.0 Method precision results spiked sample at limit level

Sr. No	Sample wt. (mg)	Dimethyl Formamide	Methanol	Ethyl acetate
Peak Area	Results (ppm)	Peak Area	Results (ppm)	Peak Area	Results (ppm)
1
2
3
4
5
6
Mean
SD
%RSD
Sr. No	Sample wt. (mg)	Toluene	Acetone	Methylene chloride
Peak Area	Results (ppm)	Peak Area	Results (ppm)	Peak Area	Results (ppm)
1
2
3
4
5
6
Mean
SD
%RSD

Acceptance criteria:

Calculate the solvent content in ppm for each analysis and report the mean, SD and % RSD.

% RSD of each of solvent content should be NMT 15.0%.

• 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 substance 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 of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.

Table 8.0 System Precision- Repeatability of Standard Injections

Sr. No.	Dimethyl Formamide	Methanol	Ethyl acetate
Peak Area	Retention time (min.)	Peak Area	Retention time (min.)	Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
SD
% RSD
Sr. No.	Toluene	Acetone	Methylene chloride
Peak Area	Retention time (min.)	Peak Area	Retention time (min.)	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 15.0% and 1.0% respectively.

Test Procedure:

Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known solvent of each content at limit level. As such sample will be also analyzed to identify the known solvent content in sample. Obtained known solvent in as such sample will be subtracted in spiked sample to calculate the actually known spiked amount of solvent content.  Record the area on data sheet and calculate the ppm of solvent content. Mean, standard deviation and % relative standard deviation shall be reported.

Table 9.0 Intermediate precision results spiked sample at limit level

Sr. No	Sample wt. (mg)	Dimethyl Formamide	Methanol	Ethyl acetate
Peak Area	Results (ppm)	Peak Area	Results (ppm)	Peak Area	Results (ppm)
1
2
3
4
5
6
Mean
SD
%RSD
Sr. No	Sample wt. (mg)	Toluene	Acetone	Methylene chloride
Peak Area	Results (ppm)	Peak Area	Results (ppm)	Peak Area	Results (ppm)
1
2
3
4
5
6
Mean
SD
%RSD

Acceptance criteria:

Calculate the solvent content in ppm for each analysis and report the mean, SD and % RSD. % RSD of each of solvent content should be NMT 15.0%.

Table 10.0 Pooled results of analyst – I & analyst – II

Sr. No	Sample wt. (mg)	Dimethyl Formamide	Methanol	Ethyl acetate
Peak Area	Results (ppm)	Peak Area	Results (ppm)	Peak Area	Results (ppm)
Analyst I
1
2
3
4
5
6
Analyst II
1
2
3
4
5
6
Mean
SD
%RSD
Sr. No	Sample wt. (mg)	Toluene	Acetone	Methylene chloride
Peak Area	Results (ppm)	Peak Area	Results (ppm)	Peak Area	Results (ppm)
Analyst I
1
2
3
4
5
6
Analyst II
1
2
3
4
5
6
Mean
SD
%RSD

Acceptance criteria:

The pooled % RSD of each of solvent content of analyst I & analyst II should be NMT 15.0%.

• 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 11.0.

Table 11.0

Parameters	System Suitability Criteria	Limit
Area % RSD	The area %RSD of six replicate injection of each of solvent content in standard solution	NMT – 15.0%

• Incident/Deviation:

Any Incident or Deviation observed during Analytical Method verification shall be recorded and investigate as per the current SOP.

• Summary/Conclusion/recommendation:

Final conclusion should be drawn from analytical method verification for its use to analyze the residual solvent test of Sildenafil citrate Ph. Eur. by GC.

Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.

Abbreviations

RES                :           Residual solvent

            VER                :           Verification

            P                      :           Protocol

            RSD                :           Related standard deviation

            GC                  :           Gas chromatography

            mL                   :           Milliliter

            mg                   :           Milligram

            PPM                :           Parts per million

            min.                 :           Minutes

            QA                   :           Quality Assurance

            QC                  :           Quality Control

            %                     :           Percentage

            ºC                    :           Degree centigrade

            hrs                   :           Hours

            µm                   :           Micrometer

            µl                     :           Microlitre

            EP                   :           European Pharmacopoeia

            RSD                :           Relative standard deviation

            RT                   :           Retention time

            NLT                 :           Not less than

NMT                :           Not more than

Ws                   :           Working standard

Vol                   :           Volume

DMSO             :           Dimethyl sulfoxide

API                  :           Active product ingredient

Revision History :

Revision No.	Details of changes	Reason for change
00	Nil	New Document