by ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RELATED SUBSTANCES 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| Superseded Protocol No. | Nil |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 |
- 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 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 related substances 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 1-(6-methoxynaphthalen-2-yl) ethenone (Imp–L) NMT-0.15 % w/w 6-methoxy-2-Naphthoic acid (Impurity-O) NMT-0.15 % w/w Any other Impurity NMT-0.10% w/w Total Impurity NMT-0.3% w/w |
- Methodology:
Reagent:
Table 1.0: Chemicals/ Reagents
| Sr. No. | Reagent | Grade |
| 1. | Ammonium Acetate | AR Grade |
| 2. | Acetic Acid | AR Grade |
| 3. | Acetonitrile | HPLC Grade |
| 4. | Methanol | HPLC Grade |
| 5. | Water | HPLC grade or Milli Q Water |
| 6. | Filter paper 0.45 µ | NA |
IMPURITIES:
- (2S)-2-(6-hydroxynaphthalen-2-yl) propanoic acid
- Methyl (2S)-2-(6-methoxynaphthalen-2-yl) propanoate
- Ethyl (2S)-2-(6-methoxy naphthalen-2-yl) propanoate
- 1-(6-methoxynaphthalen-2-yl) ethanone
- 2-bromo-6-methoxynaphthalene
- 2-Methoxynaphthalene
- (1RS)-1-(6-methoxynaphthalen-2-yl) ethanol
- 6-Methoxy-2-NaphthoicAcid
- 6-Methoxy-2-Naphthyl acetic acid
CHROMATOGRAPHIC CONDITIONS:
Column : X-Terra RP-18, 150 X 4.6 mm ID, 3.5 µm
Wavelength : 240 nm
Flow rate : 1.0 ml / minute
Column temperature : 40ºC
Run time : Not less than 30 minutes
Injection volume : 20 µl
Pump Mode : Gradient
| Gradient program | |||
| TIME (min) | Mobile phase-A% | Mobile phase-B % | Mobile phase-C% |
| 0.01 | 79 | 13 | 8 |
| 6.00 | 59 | 28 | 13 |
| 20.00 | 34 | 55 | 11 |
| 25.00 | 79 | 13 | 8 |
| 30.00 | 79 | 13 | 8 |
Preparations:
Mobile phase – A : Take 0.77 g of Ammonium acetate into 1000 ml of Milli Q water, Filter through 0.45µm filter paper.
Mobile phase – B : Acetonitrile
Mobile phase – C : Methanol
Diluent: Add 550 ml of Acetonitrile, 450 ml of Milli Q water and 1 ml of acetic acid. Filter through 0.45µm filter paper.
Impurities stock solution preparation: Take about 20 mg of each impurity into 100 ml volumetric flask dissolve and dilute upto mark with diluent.
Resolution solution preparation of Naproxen: Take about 20 mg of Naproxen standard into100 ml volumetric flask, add 1.0 ml of impurities stock solution, dissolve and dilute to mark with diluent.
Sample preparation (Duplicate preparations): Prepare a sample concentration of 0.20 mg/ml in diluent.
Procedure:
Equilibrate the system at typical chromatographic conditions. Inject 20 µL of diluent as a Blank.
Inject 20 µL of resolution solution.
Inject 20 µL of sample in duplicate preparations.
System suitability
The resolution between Naproxen and 6-Methoxy-2-Naphthyl acetic acid should be more than 2.0.Tailing factor for Naproxen peak should not be more than 2.0.
Impurities calculation:
Area % of impurity X RRF
| Impurities | RRT | RRF |
| (2S)-2-(6-hydroxynaphthalen-2-yl) propanoic acid(Impurity-A) | 0.60 | 0.63 |
| Methyl(2S)-2-(6-methoxynaphthalene-2-yl) propanoate (Impurity-E) | 2.69 | 0.51 |
| Ethyl (2S)-2-(6-methoxy naphthalen-2-yl) propanoate (Impurity-F) | 3.01 | 0.54 |
| 1-(6-methoxynaphthalen-2-yl) ethanone (Impurity-L) | 2.16 | 0.38 |
| 2-bromo-6-methoxynaphthalene (Impurity-N) | 3.30 | 0.42 |
| 2-Methoxynaphthalene (Impurity-M) | 2.55 | 1.07 |
| (1RS)-1-(6-methoxynaphthalen-2-yl) ethanol (Impurity-K) | 1.78 | 0.76 |
| 6-Methoxy-2-NaphthoicAcid | 0.75 | 0.24 |
| 6-Methoxy-2-Naphthyl acetic acid (Impurity-I) | 0.81 | 0.59 |
| Each unidentified | — | 1.00 |
Report the sum of all impurities as total 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 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 solutions:
Stock solution of Impurity A:
Weigh accurately about 2.0 mg of Impurity A and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity A:
Dilute 1.0 ml of stock solution of impurity A to 100.0 ml with diluent, mix.
Stock solution of Impurity E:
Weigh accurately about 2.0 mg of Impurity E and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity E:
Dilute 1.0 ml of stock solution of impurity E to 100.0 ml with diluent, mix.
Stock solution of Impurity F:
Weigh accurately about 2.0 mg of Impurity F and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity F:
Dilute 1.0 ml of stock solution of impurity F to 100.0 ml with diluent, mix.
Stock solution of Impurity L:
Weigh accurately about 2.0 mg of Impurity L and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity L:
Dilute 1.5 ml of stock solution of impurity L to 100.0 ml with diluent, mix.
- Stock solution of Impurity N:
Weigh accurately about 2.0 mg of Impurity N and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity N:
Dilute 1.0 ml of stock solution of impurity N to 100.0 ml with diluent, mix.
- Stock solution of Impurity M:
Weigh accurately about 2.0 mg of Impurity M and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity M:
Dilute 1.0 ml of stock solution of impurity M to 100.0 ml with diluent, mix.
- Stock solution of Impurity K:
Weigh accurately about 2.0 mg of Impurity K and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity K:
Dilute 1.0 ml of stock solution of impurity K to 100.0 ml with diluent, mix.
- Stock solution of Impurity O:
Weigh accurately about 2.0 mg of Impurity O and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity O:
Dilute 1.5 ml of stock solution of impurity O to 100.0 ml with diluent, mix.
Stock solution of Impurity I:
Weigh accurately about 2.0 mg of Impurity I and transferred to 100 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity I:
Dilute 1.0 ml of stock solution of impurity I to 100.0 ml with diluent, mix.
Impurities stock solution preparation: Take about 20 mg of each impurity into 100 ml volumetric flask dissolve and dilute upto mark with diluent.
Resolution solution preparation of Naproxen: Take about 20 mg of Naproxen standard into100 ml volumetric flask, add 1.0 ml of impurities stock solution, dissolve and dilute to mark with diluent.
Note: Impurity weight can be reduce to achieve the final concentration same.
Sample preparation: Weight accurately about 20 mg of sample of Naproxen Ph. Eur. into 100 ml of volumetric flask, dissolve and dilute to volume with diluent.
Spike sample preparation: Weight accurately about 20 mg of sample of Naproxen Ph. Eur. into 100 ml of volumetric flask, further add 1 ml of impurities stock solution, dissolve and dilute to volume with diluent.
Procedure: Inject the preparation of blank solution (diluent), resolution solution and identification solution of impurity A, impurity E, impurity F, impurity L, impurity N, impurity M, impurity K, impurity O, impurity I, 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 3.0 and 4.0.
Table 2.0: Sequence
| Solutions | No of Injection to be injected in Sequence |
| Blank solution | 1 |
| Resolution solution | 1 |
| Identification solution of Impurity A | 1 |
| Identification solution of Impurity E | 1 |
| Identification solution of Impurity F | 1 |
| Identification solution of Impurity L | 1 |
| Identification solution of Impurity N | 1 |
| Identification solution of Impurity M | 1 |
| Identification solution of Impurity K | 1 |
| Identification solution of Impurity O | 1 |
| Identification solution of Impurity I | 1 |
| Test solution | 1 |
| Spike test solution | 1 |
| Blank (to avoid carry over) | 1 |
| Resolution solution _Bkt | 1 |
Table 3.0 Specificity data
| Sr. No | Sample | RT (min.) | RRT | Peak purity | ||
| Blank | NA | |||||
| Resolution solution | Impurity A | |||||
| Impurity O | ||||||
| Impurity I | ||||||
| Naproxen | ||||||
| Impurity K | ||||||
| Impurity L | ||||||
| Impurity M | ||||||
| Impurity E | ||||||
| Impurity F | ||||||
| Impurity N | ||||||
| Identification solution of Impurity A | NA | |||||
| Identification solution of Impurity O | NA | |||||
| Identification solution of Impurity I | NA | |||||
| Identification solution of Impurity K | NA | |||||
| Identification solution of Impurity L | NA | |||||
| Identification solution of Impurity M | NA | |||||
| Identification solution of Impurity E | NA | |||||
| Identification solution of Impurity F | NA | |||||
| Identification solution of Impurity N | NA | |||||
| Test Solution | Blank | |||||
| Naproxen | NA | |||||
| Known Impurity | ||||||
| Unknown Impurity | ||||||
Table 4.0 Spiked test solution
| Sr. No | Sample | RT (min.) | RRT | Peak purity |
| Blank | NA | |||
| Naproxen A | ||||
| Known Impurity | ||||
| Unknown Impurity |
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 Resolution solution and known Impurity peaks in Identification solution,
spiked sample solution should be spectrally pure.
- Precision:
- Method Precision:
- 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 entire impurities 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 each 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 5.0 Method precision results by 1st analyst
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.2%) | ||||||||
| Impurity A | Impurity O | Impurity I | Impurity K | Impurity L | Impurity M | Impurity E | Impurity F | Impurity N | Individual Unknown | |||
| 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 0.2%.
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.
- 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.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure and spike entire impurities 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 each 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 Intermediate (ruggedness) precision results by 2nd analyst
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.2%) | ||||||||
| Impurity A | Impurity O | Impurity I | Impurity K | Impurity L | Impurity M | Impurity E | Impurity F | Impurity N | Individual Unknown | |||
| 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 0.2%.
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 7.0 Cumulative results of analyst – I & analyst – II
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.2%) | |||||||||
| Impurity A | Impurity O | Impurity I | Impurity K | Impurity L | Impurity M | Impurity E | Impurity F | Impurity N | |||||
| 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.
Table 8.0: System Suitability Criteria
| Sr. No. | Parameters | System Suitability Criteria | Limit |
| 1. | Resolution | The resolution between Naproxen and 6-Methoxy-2-Naphthyl acetic acid | NMT – 2.0% |
| 2. | Tailing Factor | Tailing factor for Naproxen peak | NMT – 2.0 |
- 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 Naproxen Ph. Eur. by manufacturer 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
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 |
