ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RESIDUAL SOLVENT OF NAPROXEN Ph. Eur.

ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RESIDUAL SOLVENT OF NAPROXEN 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Superseded Protocol No.	Nil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 Naproxen 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 batches of Naproxen Ph. Eur.

Analytical method verification will be performed by considering thefollowing parameters:

Parameters	Naproxen Ph. Eur.
Specificity	yes
Precision
System Precision	yes
Method Precision	yes
Intermediate Precision (Ruggedness)	yes
System Suitability	yes

• Scope :

The scope of this protocol is applicable for the verification of method for residual solvents of Naproxen 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	Non-steroidal Anti-inflammatory Drug (NSAID)
Reason for Verification	1st verification
Active Ingredient	Naproxen Ph. Eur.
Method Reference
Specification limits	Residual Solvent Methanol NMT-300 ppn Toluene NMT-250 ppm Acetic acid covered by loss on drying test

• Methodology:

Residual Solvents (By GC)

Solvents:

Table 1.0: Chemical

Sr. No.	Solvents	Grade
	Dimethyl acetamide (DMA)	GC Grade
	Methanol	GC Grade
	Toluene	GC Grade

Chromatographic conditions:

GC Parameters:

Column	:	Rtx-624: 30m x 0.53 mm ID, 3.0 µm or equivalent
Carrier gas	:	Helium
Carrier pressure	:	5 PSI
Injector type	:	Split
Split flow	:	25 ml/min.
Injector temperature	:	140°C
Detector	:	FID
Detector temperature	:	250°C
Oven programme	:	40°C initial temperature, hold for 6.0 min, ramp at a rate of 30°C/min up to 200°C hold for 4.0min
Run time	:	Not less than15.33 min.
Recording & Integration time	me     :	15.00 min.

Head space auto sampler parameters:

Oven temperature                                :           85°C

Needle temperature                             :           100°C

Transfer line temperature                     :           115°C

GC cycle time                                      :           25 min.

Thermostat time                                   :           20 min.

Pressurization time                               :           3.0 min.

Inject time                                             :           0.05 min.

Withdrawal time                                    :           0.5 min.

Carrier pressure                                   :           12.0 PSI (approximately)

Standard stock solution preparation:

Weigh accurately about 500 mg of methanol and 625 mg of toluene into a 100 mL volumetric flask half filled with DMA. Dissolve and dilute to volume with DMA, and mix.

Standard solution preparation:

Further transfer 1.0 ml of standard stock solution to a 100 mL volumetric flask, half filled with DMA. Dilute to volume with DMA, and mix. The concentration of the preparation is approximately 50 ppm of methanol and 62.5 ppm of toluene. (200 ppm of methanol and 250 ppm of toluene with respect to sample concentration)

Standard vial preparation:Transfer 1.0 ml of standard solution to a head space vial and tightly close the vial with butyl rubber septa and crimp cap.

Sample preparation: Weigh accurately 0.25 g (250 mg) of Naproxen sample into a head space vial. Dissolve in 1.0 ml of DMA and tightly close the vial with butyl rubber septa and crimp cap, and mix.

Blank vial preparation: Transfer 1.0-mL of DMA to a head space vial and tightly close the vial with butyl rubber septa and crimp cap.

Sequence of injections:

• Inject DMA as blank
• Inject six standard injections
• Inject sample
• Inject tailing standard

System suitability:

Inject 6 standard injections into gas chromatograph. Calculate the %RSD of the each solvent peak area responses. Report the tailing factor of toluene for 1st standard injection.

The %RSD of the each solvent peak area response shall not be more than 15.0. Tailing factor of toluene for 1st standard injection shall not be more than 1.5. Theoretical plate count per meter for toluene peak for 1st standard injection not less than 7500.

Restandardization: After every 6 sample injections and at the end of session run the restandardization injection and calculate %RSD of each solvent area and average area from standard injections. The % RSD shall not be more than 15.0.

Sample analysis:

Prepare and analyze the sample. Calculate each solvent in the sample as below.

Area response of solvent in sample             Conc. of solvent in standard (ppm)

—————————————————- x ———————————————–            =        ppm

Average area response of solvent in           Weight of sample taken in g

                  Standard injections    

• Verification parameters:

The following parameters to be perform for the verification activity.

1. Specificity
   1. Precision
   1. System Suitability
   1. 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 (DMA), identification solution of methanol and toluene, 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 prepared as per the sequence mentioned in table no 2.0 and data shall be represented in report as per the table no 3.0 & 4.0.

Blank: Dimethyl acetamide (DMA)

Preparation of Identification solutions:

Standard solvent stock solution of Methanol:

Transfer 750 mg of Methanol into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl acetamide.

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 acetamide. Take 1 ml of this solution into a vial fitted with butyl rubber septa and crimp cap.

Standard solvent stock solution of Toluene:

Transfer 625 mg of Toluene into 100 ml of volumetric flask. Dissolve and dilute up to the mark with Dimethyl acetamide.

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 acetamide. Take 1 ml of this solution into a vial fitted butyl rubber septa and crimp cap.

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 acetamide.

Sample preparation: Weigh accurately 0.25 g (250 mg) of Naproxen sample into a head space vial. Dissolve in 1.0 ml of DMA and tightly close the vial with butyl rubber septa and crimp cap, and mix.

Spiked test solution:

Weigh accurately 0.25 g (250 mg) of Naproxen sample into a head space vial. Dissolve in 1.0 ml of solvent stock preparation and tightly close the vial with butyl rubber septa and crimp cap, and mix.

Tablet 2.0: Sequence

Sr. No	Solution	No of Injection to be injected in Sequence
	Blank (DMA)	1
	Standard Solution (Six vials)	6
	Identification solution of Methanol	1
	Identification solution of Toluene	1
	Blank (DMA) (to avoid carry over)	1
	Test Solution	1
	Spiked Test Solution	1
	Standard solution + Bracketing	1

Table 3.0 Specificity data

Sr. No	Sample	RT (min.)
	Blank (DMA)
	Standard solution	Dimethyl acetamide
Methanol
Toluene
	Identification solution of Methanol
	Identification solution of Toluene
	Test Solution	Dimethyl acetamide
Methanol
Toluene

Table 4.0 Spiked test solution

Sr. No	Sample	RT (minutes)
	Blank
	Dimethyl acetamide
	Methanol
	Toluene

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 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 5.0 System Precision- Repeatability of Standard Injections

Sr. No.	Methanol	Toluene
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 methanol and toluene in six 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 Methanol and Toluene 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 6.0 Method precision results spiked sample at limit level

Sr. No.	Sample wt. (mg)	Methanol	Toluene
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 7.0 System Precision- Repeatability of Standard Injections

Sr. No.	Methanol	Toluene
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 8.0 Intermediate precision results spiked sample at limit level

Sr. No.	Sample wt. (mg)	Methanol	Toluene
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 9.0 Cumulative results of analyst – I & analyst – II

Sr. No	Sample wt. (mg)	Methanol	Toluene
Peak Area	Results (ppm)	Peak Area	Results (ppm)
Method Precision
1
2
3
4
5
6
Intermediate Precision
1
2
3
4
5
6
Mean
SD
%RSD

Acceptance criteria:

The cumulative % RSD of each of solvent content of method precision & intermediate precision 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.

Table 10.0

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%
Tailing Factor	Tailing factor of toluene for 1st standard injection	NMT – 1.5
Theoretical  plates	Theoretical plate count per meter for toluene peak for 1st standard injection	NLT – 7500

• 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 Naproxen Ph. Eur. by GC.

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

Abbreviations

RES                :           Residual solvent

            VERP              :           Verification Protocol

            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

            Ph.Eur/EP       :           European Pharmacopoeia

            RSD                :           Relative standard deviation

            RT                   :           Retention time

            NLT                 :           Not less than

NMT                :           Not more than

DMA                :           Dimethyl acetamide

Revision History :

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