ANALYTICAL METHOD VERIFICATION PROTOCOL OF ENANTIOMERIC PURITY OF NAPROXEN Ph. Eur.

ANALYTICAL METHOD VERIFICATION PROTOCOL OF ENANTIOMERIC PURITY OF NAPROXEN 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Superseded Protocol No.	Nil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

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 enantiomeric purity of Naproxen Ph. Eur. by pharmacopeia method is consistent and to provide the reliable results within the predetermined acceptance criteria.

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 of analysis of enantiomeric purity of Naproxen 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	Nonsteroidal Anti-inflammatory Drug (NSAID)
Reason for Verification	1st verification
Active Ingredient	Naproxen Ph. Eur.
Method Reference
Specification Limits	Related substance   Impurity G      :       NMT – 2.5%

• Methodology:

Reagent:

Table 1.0: Chemicals/ Reagents

Sr. No.	Reagent	Grade
1.	Glacial Acetic acid	HPLC Grade
2.	Acetonitrile	HPLC Grade
3.	2-Propanol	HPLC Grade
4.	Hexane	HPLC Grade
5.	Tetrahydrofuran	HPLC Grade

Precaution to be taken during conversion from reverse phase to normal phase and vice versa:

Degasser must be off during analysis. Glassware should be dried before use and there shall be no contamination of water. HPLC System shall be purge with methanol thoroughly to remove water content present in HPLC. Further system flushing shall be performed as mention below.

Table 2.0 : HPLC System saturation to Normal Phase

Sr. No.	Solvent	Flow Rate	Flushing Time
	Methanol	2.0 ml	A : 1 hour, B : 1 hour, C : 1 hour, D : 1 hour
	2-Propanol	1.0 ml	Overnight flushing & Port ratio shall be A : 25 %, B : 25 %, C : 25 %, D : 25 %
	Hexane	1.0 ml	A : 30 minutes , B : 30 minutes
	Mobile Phase	1.0 ml	A : 30 minutes

Above given system flushing is limited to this verification activity. After system flushing attached column and saturate with mobile phase till the constant back pressure (Min: 15min)

Liquid chromatography:

Protect solutions from light

Test solution:

Dissolve 25.0 mg of the substance to be examined in tetrahydrofuran R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of the solution to 20.0 ml with the mobile phase.

Reference solution (a):

Dilute 2.5 ml of the test solution to 100.0 ml with the mobile phase.

Reference solution (b):

Dissolve 5 mg of racemic naproxen CRS in 10.0 ml of tetrahydrofuran R and dilute to 100.0 ml with the mobile phase.

Column:

Size: L = 0.25 m, φ = 4.6 mm;

Stationary phase: Silica gel π-acceptor/π-donor for chiral separations R (5 µm) (S,S);

Temperature: 25°C

Mobile phase:

Glacial acetic Acid R, Acetonitrile R, 2-propanol R, hexane R (0.5:5:10:84.5 v/v/v/v)

Flow rate: 2 ml/min

Detection: Spectrophotometer at 263 nm

Injection: 20 µl

Run time: 1.5 times the retention time of naproxen

(Retention time = about 5 min)

System suitability: Reference solution (b):

Resolution: minimum 3 between the peaks due to Impurity G and Naproxen.

• 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 diluent.

Specificity of test method should be established by separately injecting blank solution (diluent), resolution solution, identification solution of impurities, spike test solution with impurities shall be prepared as per specification limit level and test solution. Test solution shall be prepared as per method of analysis.

Blank: Diluent

Preparation of Identification solution:

Stock solution of Impurity G:

Weigh accurately about 2.5 mg of Impurity G and transferred to 50 ml volumetric flask. Dissolve and dilute to volume with tetrahydrofuran, mix. Further dilute 5 ml of this solution to 50 ml with mobile phase and mix.

Identification solution of Impurity G:

Dilute 5.0 ml of stock solution of impurity G to 20.0 ml with diluent, mix.

Reference solution (a):

Dilute 2.5 ml of the test solution to 100.0 ml with the mobile phase.

Reference solution (b):

Dissolve 5 mg of racemic naproxen CRS in 10.0 ml of tetrahydrofuran R and dilute to 100.0 ml with the mobile phase.

Note: Impurity weight can be reduce to achieve the final concentration same.

Test solution:

Dissolve 25.0 mg of the substance to be examined in tetrahydrofuran R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of the solution to 20.0 ml with the mobile phase.

Spike test solution:

Dissolve 25.0 mg of the substance to be examined in tetrahydrofuran R, and dilute to 50.0 ml with the same solvent. Further pipette 2.0 ml of the solution to 20 ml of volumetric flask and dissolve in mobile phase. Also spike 5 ml of stock solution of impurity G in this solution before make up and mix.

Procedure: Inject the preparation of blank solution (mobile phase), reference solution (a) reference solution (b) and identification solution of impurity G,  test solution and spike test solution on a HPLC system with a Diode array detector (DAD) as follows in table 2.0. 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 4.0 and 5.0.

Table 3.0: Sequence

Solutions	No of Injection to be injected in Sequence
Blank solution	1
Resolution solution (b)	1
Resolution solution (a)	6
Identification solution of Impurity G	1
Test solution	1
Spike test solution	1
Blank (to avoid carry over)	1
Resolution solution (a)_Bkt	1

Table 4.0 Spiked test solution

Sr. No.	Sample	RT (min.)	RRT	Peak purity
	Blank			NA
	Naproxen
	Impurity G

Acceptance Criteria:

1. There should be no interference of the diluent, impurity at the retention time of Analyte peak.
2. Impurity peaks should be well resolved from active peak and each other.
3. Analyte peak and known impurity peak in identification solution, spiked sample solution should be spectrally pure.   
   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 % RSD for Area and Retention time of main analyte peak.

Table 5.0 System Precision- Repeatability of Standard Injections

Sr. No.	Naproxen
Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
SD
% RSD

Acceptance Criteria:

% RSD for peak area and retention time of naproxen in six replicate injection of reference solution (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 spike impurity G at specification limit level. Un-spiked sample shall be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample to be subtracted in spiked sample to calculate the actually spiked known amount of impurity. Records the area response of impurity obtained in test solution and calculates the % impurity, mean, standard deviation and % relative standard deviation of six samples in tabular manner as given below.

Table 6.0 Method precision results by 1^st analyst

Sr. No.	Sample wt. (mg)	Impurity G
Peak Area	Impurity %
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.

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.

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 shall 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 % RSD for area and retention time.

Table 7.0 System Precision- Repeatability of Standard Injections

Sr. No.	Naproxen
Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
SD
% RSD

Acceptance Criteria:

% RSD for peak area and retention time of naproxen in six replicate injection of reference solution (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 spike impurity G at specification limit level. Un-spiked sample shall be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample to be subtracted in spiked sample to calculate the actually spiked known amount of impurity. Records the area response of impurity obtained in test solution and calculates the % impurity, mean, standard deviation and % relative standard deviation of six samples in tabular manner as given below.

Table 8.0 Intermediate (ruggedness) precision results by 2^nd analyst

Sr. No.	Sample wt. (mg)	Impurity G
Peak Area	Impurity %
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 0.2%.

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 9.0 Cumulative results of analyst – I & analyst – II

Sr. No.	Sample wt. (mg)	Impurity G
Peak Area	Impurity %
Analyst I
1
2
3
4
5
6
Analyst II
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.

Table 10.0: System Suitability Criteria

Sr. No.	System Suitability Criteria	Limit
1.	The resolution between Impurity G and Naproxen.	Minimum 3.0
2.	Area % RSD of Naproxen in reference solution (a)	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 enantiomeric purity of Naproxen Ph. Eur. by European pharmacopoeia procedure.

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

Abbreviations:

REL                 :           Related substance

            VERP              :           Verification 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/Ph.Eur       :           Europeian Pharmacopoeia

            RSD                :           Relative standard deviation

            NLT                 :           Not less than

NMT                :           Not more than

Revision History :

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