RP‑HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF NARATRIPTAN AND NAPROXEN IN TABLET DOSAGE FORM

P. Vivek Sagar. et al. © 2012, JPR. All Rights Reserved Jo Vol. 14 RP ‑HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF NARATRIPTAN AND NAPROXEN IN TABLET DOSAGE FORM Palthyapooja*, K. Manisha, J. Swathi, P. Ravi Kiran, P. Vivek Depart ment of Pharmaceutical Analysis, R eceived on: A simple, precise, and accurate reverse (RP ‑HPLC) method was developed and validated for the simultaneous estimation of Naratriptan and Naproxen in combined formulations. Chromatographic separation was achieved on a C18 column (150 containing phosphate buffer (pH adjusted with orthophosphoric acid) and acetonitrile in an optimized ratio. The flow rate, de wavelength, and injection volume were selected to yield symmetrical, well within defined concentration ranges, showing correlation coefficients greater than 0.999. The method exhibited high p (%RSD < 2), accuracy (recovery 98–102 %), and robustness under minor variations of chromatographic parameters. The results confirm that the developed RP ‑HPLC method is reliable and suitable for routine quality control of Naratriptan and Naproxen i pharmaceutical dosage forms. Keywords: Naproxen, RP ‑HPLC, Validation, Linearity, Accuracy, Robustness. INTRODUCTION N aratriptan, a selective serotonin (5 receptor agonist, is used for acute migraine treatment, and Naproxen is a nonsteroidal anti ‑inflammatory drug widely prescribed for pain and inflammation. Their combination formulation requires a reliable analytical technique to quantify both drugs simultaneously for quality control. Reverse‑phase high‑ performance liquid chromatography (RP HPLC) offers a rapid, sensitive, and reproducible approach capable of effectively separating these compounds i run.1‑ 4 Validation as per ICH Q2(R1) guidelines establishes method suitability for routine pharmaceutical analysis Corresponding author: Dr. P. Vivek Sagar Department of Pharmaceutical Analysis Sarojini Naidu Vanita Pharmacy MahaVidyalaya, Tarnaka, Secunderabad, Telangana, India Email: [email protected] DOI: J Pharm Res, 2025; 14(06): 33 https://jprinfo.com/ Jo urnal of Pharma Research Available online through www.jprinfo.com 14 Issues 06, 2025 ISSN: 2319‑5622 Original Article ‑HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF NARATRIPTAN AND NAPROXEN IN TABLET DOSAGE FORM Palthyapooja*, K. Manisha, J. Swathi, P. Ravi Kiran, P. Vivek Sagar ment of Pharmaceutical Analysis, Sarojini Naidu Vanita Pharmacy MahaVidyalaya, Tarnaka, Secunderabad eceived on: 03‑10‑2025 Accepted on: 04‑11‑2025 ABSTRACT simple, precise, and accurate reverse ‑phase high‑performance liquid chromatography ‑HPLC) method was developed and validated for the simultaneous estimation of Naratriptan and Naproxen in combined formulations. Chromatographic separation was achieved on a C18 column (150 × 4.6 mm, 5 µm) using an isocratic mobile phase containing phosphate buffer (pH adjusted with orthophosphoric acid) and acetonitrile in an optimized ratio. The flow rate, de wavelength, and injection volume were selected to yield symmetrical, well ‑resolved peaks for both analytes. Linearity was con within defined concentration ranges, showing correlation coefficients greater than 0.999. The method exhibited high p %), and robustness under minor variations of chromatographic parameters. The results ‑HPLC method is reliable and suitable for routine quality control of Naratriptan and Naproxen i ‑HPLC, Validation, Linearity, Accuracy, Robustness. aratriptan, a selective serotonin (5 ‑HT1) receptor agonist, is used for acute migraine treatment, and ‑inflammatory drug widely prescribed for pain and inflammation. Their combination reliable analytical technique to quantify both drugs simultaneously for quality control. performance liquid chromatography (RP ‑ HPLC) offers a rapid, sensitive, and reproducible approach capable of effectively separating these compounds i n a single 4 Validation as per ICH Q2(R1) guidelines establishes method suitability for routine pharmaceutical analysis . Sarojini Naidu Vanita Pharmacy MahaVidyalaya, Fig 1: Structure of Fig 2: Structure of Naproxen J Pharm Res, 2025; 14(06): 33 ‑36 ‑HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF NARATRIPTAN AND NAPROXEN IN TABLET DOSAGE FORM Sarojini Naidu Vanita Pharmacy MahaVidyalaya, Tarnaka, Secunderabad , India ‑phase high‑performance liquid chromatography ‑HPLC) method was developed and validated for the simultaneous estimation of Naratriptan and Naproxen in combined tablet × 4.6 mm, 5 µm) using an isocratic mobile phase containing phosphate buffer (pH adjusted with orthophosphoric acid) and acetonitrile in an optimized ratio. The flow rate, de tection ‑resolved peaks for both analytes. Linearity was con firmed within defined concentration ranges, showing correlation coefficients greater than 0.999. The method exhibited high p recision %), and robustness under minor variations of chromatographic parameters. The results ‑HPLC method is reliable and suitable for routine quality control of Naratriptan and Naproxen i n Fig 1: Structure of Naratriptan Fig 2: Structure of Naproxen P. Vivek Sagar. et al. J Pharm Res, 2025; 14(06): 33‑36 © 2012, JPR. All Rights Reserved https://jprinfo.com/ MATERIALS AND METHODS Chemicals and Reagents Reference standards of Naratriptan and Naproxen with assay purity >99% were obtained from authorized suppliers. HPLC‑ grade acetonitrile, phosphate buffer reagents, and orthophosphoric acid were of analytical grade. Instrumentation Shimadzu LC system with PDA detector and C18 column (150 × 4.6 mm, 5 µm) was used. Chromatography was controlled using LabSolutions software. Chromatographic Conditions A pH 3.0 phosphate buffer solution with orthophosphoric acid and acetonitrile was used as the mobile phase. For best chromatographic resolution, peak symmetry, and analyte retention, this combination was chosen. At this acidic pH, a phosphate buffer controls analyte ionisation to ensure peak form and selectivity, while acetonitrile balances polarity and elution strength as an organic modifier. Keeping the flow rate at 1.0 mL/min ensured chromatographic run time stability. To maximise Naratriptan and Naproxen absorbance sensitivity, detection was done at 282 nm. To avoid column overload and assure accurate detection, a 20 μL injection volume was utilised. Keeping the column temperature at ambient simplified operation and maintained technique robustness. This mobile phase composition and chromatographic setup produced a reliable, sensitive, and efficient separation for simultaneous quantification in routine pharmaceutical analysis. Materials Required The materials required for the development and validation of the RP‑HPLC method included analytically pure Naratriptan and Naproxen reference standards, pharmaceutical grade excipients, HPLC‑grade acetonitrile, phosphate buffer components, orthophosphoric acid for pH adjustment, and double‑distilled water. Additionally, a calibrated analytical balance, micropipettes, volumetric flasks, and 0.45 µm membrane filters were used to ensure precise and accurate preparation of solutions. Preparation of Standard Solutions For preparation of the standard solutions, accurately weighed quantities of Naratriptan and Naproxen equivalent to 10 mg and 100 mg respectively were separately dissolved in the mobile phase and diluted to 100 mL volumetric flasks to prepare stock solutions. These stock solutions were further diluted appropriately with the mobile phase to yield working standards in the desired concentration ranges for calibration and validation studies. Preparation of Sample Solutions For sample solutions, tablet powders equivalent to the labeled amounts of Naratriptan and Naproxen were accurately weighed and transferred to volumetric flasks. The sample was extracted with the mobile phase via sonication and filtered through 0.45 µm membrane filters to remove particulate matter. The filtrate was then diluted suitably to fit within the calibration range before injection into the HPLC system. METHOD VALIDATION The developed RP‑HPLC method for simultaneous estimation of Naratriptan and Naproxen was validated in accordance with ICH Q2(R1) guidelines to ensure its suitability for routine pharmaceutical analysis.6‑8 The key parameters and their evaluation procedures included: Specificity: Blank, placebo, standard, and sample chromatograms were analyzed to confirm that the method distinctly separates Naratriptan and Naproxen without interference from excipients, impurities, or solvents. Linearity: Calibration curves were prepared by analyzing seven concentration levels covering the expected assay range for both analytes. The linearity was assessed by plotting peak areas against concentrations and calculating the correlation coefficient (R²), which was expected to be >0.999. Accuracy: Accuracy was determined by performing recovery studies through the standard addition method at 50%, 100%, and 150% levels of the target concentration. Percent recoveries close to 100% with low standard deviation indicated accurate quantification without matrix interference. Precision: Repeatability (intra‑day precision) was tested by analyzing three replicates at three concentration levels (50%, 100%, 150%) on the same day. Intermediate precision (inter‑day) was assessed by repeat testing on different days. A %RSD of less than 2 was considered acceptable. Limit of Detection (LOD) and Limit of Quantification (LOQ): These parameters were calculated based on signal‑to‑noise ratios of 3:1 for LOD and 10:1 for LOQ, to determine the smallest detectable and quantifiable concentrations respectively. Robustness: The effect of small deliberate variations such as changes in flow rate (±0.1 mL/min), detection wavelength (±2 nm), and different P. Vivek Sagar. et al. J Pharm Res, 2025; 14(06): 33‑36 © 2012, JPR. All Rights Reserved https://jprinfo.com/ analysts was studied. The peak areas, retention times, and system suitability parameters were monitored. Acceptable %RSD values confirmed the method’s robustness. System Suitability: System suitability tests including theoretical plates, tailing factor, resolution, and repeatability of injections were performed before sample analysis to ensure the chromatographic system was functioning adequately. RESULTS AND DISCUSSION Specificity No interference was observed from excipients or blank solvents at the retention times of Naratriptan and Naproxen, demonstrating method specificity. Linearity The method was linear for both analytes in the concentration range relevant for tablet assay. Calibration points showed proportional peak area response. The results were given in table no. 1, Figure no 3 &4 Table No/. 1: Results for Linearity S.No Naproxen Naratriptan 1 4478 4714 2 40234 18830 3 75704 23510 4 121995 48751 5 169280 74104 6 229862 103374 7 263256 120763 Fig 3. Linearity graph of Naproxen Fig. 4. Linearity graph of Naratriptan Correlation coefficient (R²) values for both Naratriptan and Naproxen were found to be >0.999, confirming excellent linearity. Accuracy Recovery studies using standard addition at 50%, 100%, and 150% concentrations showed excellent accuracy.The results were given in table no. 2 Table no. 2: Results for Accuracy Concentration Naratriptan Mean Area % Recovery Naproxen Mean Area % Recovery 50% 5082.29 101.6 5091.21 101.8 100% 9876.57 98.8 9863.19 98.6 200 % 20041.14 100.2 20045.60 100.2 The grand mean of recovery for both drugs was approximately 100.2% with standard deviations under 2%, indicating precise accuracy. Precision (Intra‑day) Replicate injections at 50%, 100%, and 150% concentration levels exhibited low %RSD values. The results were given in table no. 3 Table No. 3: Results for Intra Assay Precision Level Drug Mean Area Std. Dev. % RSD 50% Naproxen 167239 3049 1.8 Naratriptan 73757 357 0.5 100% Naproxen 111094 875 0.8 Naratriptan 48381 517 1.1 150% Naproxen 55590 2852 5.1 Naratriptan 22573 142 0.6 This shows high method reproducibility with good precision. Robustness Deliberate variations in flow rate (±0.1 mL/min), wavelength (±2 nm), and analyst showed consistent retention times and peak y = 44613x - 49194 R² = 0.993 -50000 0 50000 100000 150000 200000 250000 300000 02468 PEAK AREAS CONCENTRATION Naproxen y = 19851x - 20398 R² = 0.990 -50000 0 50000 100000 150000 0 2 4 6 8 PEAK AREA CONCENTRATION Naratriptan P. Vivek Sagar. et al. J Pharm Res, 2025; 14(06): 33‑36 © 2012, JPR. All Rights Reserved https://jprinfo.com/ areas with % RSD values below 5%, confirming method robustness. RESULTS AND DISCUSSION The developed RP‑HPLC method exhibited efficient and well‑ resolved separation of Naratriptan and Naproxen within 10 minutes. Linear calibration and precise recovery data corroborate the method’s quantitative reliability. The low %RSD values for precision and robustness tests certify its consistent performance across minor operational variations, a critical attribute for routine testing environments. Chromatograms confirmed method specificity and absence of interference. SUMMARY The paper reports successful development and thorough validation of an RP‑HPLC method for simultaneous estimation of Naratriptan and Naproxen in tablet dosage forms. The method demonstrated linearity, specificity, accuracy, precision, and robustness in line with ICH guidelines. The reliable assay is suitable for pharmaceutical quality control and stability assessments. CONCLUSION A rapid, accurate, precise, and robust RP‑HPLC method was developed and validated to simultaneously quantify Naratriptan and Naproxen tablets. All validation parameters confirm compliance with regulatory standards (ICH Q2[R1]). The method’s reproducibility and stability under varied conditions make it ideal for routine pharmaceutical quality assurance. REFERENCES 1. Brunton LL, Lazo JS, Parker KL. Goodman & Gilman's the pharmacological basis of therapeutics. 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