Analytical Method Development and Validation for Simultaneous Determination of Sumatriptan and Naproxen by RP - HPLC

About Author: Rajesh Nuni
Department of Pharmaceutical Analysis,
Vels School of Pharmaceutical Sciences,
Vels University, Pallavaram,
Chennai, Tamilnadu, India

Abstract
A reverse phase HPLC method is developed for the determination of Sumatriptan and naproxen in pharmaceutical dosage forms. Chromatography was carried out on a C8 column [4.6 x 150mm, 3.5mm, Make: XTerra] using a mixture of potassiumdi hydrogen ortho phosphate buffer and acetonitrile (50:50 v/v) as the mobile phase at a flow rate of 0.7ml/min. Detection was carried out at 285 nm. The retention time of the drug Naproxen and sumatriptan was 2.24 minand 5.871 min. The method produced linear responses in the concentration range of 60 to 100μg/ml of Sumatriptan and naproxen. The LOD values for HPLC method for naproxen and sumatriptan were found to be 3.20 and 3.36 ng/ml. The LOQ for Naproxn and Sumatriptan were foud to be 9.86 and 9.90 ng/ml respectively. The method was found to be applicable for determination of the drug in tablets.

Reference ID: PHARMATUTOR-ART-1116

Introduction
Sumatriptan is a salt of 1-{3-[2-(dimethylamino)ethyl]-1H-indol-5-yl}-N-methylmethanesulfonamide. It is used in the treatment of migraine disorder. Naproxen is a salt of 2-(6-methoxynaphthalen-2-yl)propanoic acid used in the treatment of rheumatoid arthritis and other rheumatic or musculoskeletal disorders, dysmenorrhea, and acute gout. As no HPLC method have been reported for the determination of Sumatriptan and Naproxen an attempt was made to report a simple, reliable and reproducible RP-HPLC method which was duly validated by statistical parameters precision, accuracy, linearity, LOD, LOQ, Robustness and Ruggedness. The method has been satisfactorily applied to the determination of Sumatriptan and Naproxen in pharmaceutical preparations.

Materials & Methods:
Equipments and Apparatus:
Different kinds of equipments viz Analytical weighing balance (shimadzu AUX 200), High performance liquid chromatography(waters, separation module 2695) equipped with Auto Sampler and UV detector. Column Symmetry C8 (4.6 x 150mm, 3.5mm, Make: XTerra),pH meter, Vacuum filter pump (model XI 5522050 of Millipore), Millipore filtration kit, mobile phase reservoir, Water bath, Sample filtration assembly and glassware’s were used throughout the experiment.

Chemicals and solvents:
Potassiumdi hydrogen ortho phosphate and orthophosphoric acid (AR grade, Qualigens) were used for preparing the buffer. HPLC grade acetonitrile (Qualigens) was used for diluent preparation. Pure sample of Sumatriptan and Naproxen was a gift sample from a local pharmaceutical industry. Commercial samples of tablets containing the drug zinc carnosine were purchased from the local pharmacy.

Chromatographic Parameters
Equipment : High performance liquid chromatography equipped with Auto Sampler and UV detector

Column : Symmetry C8 (4.6 x 150mm, 3.5mm, Make: XTerra)

Flow rate : 0.7 mL per min

Wavelength : 285 nm

Injection volume : 20 ml

Column oven : Ambient

Run time : 8min

Preparation of mobile phase
Mix a mixture of above buffer 500 mL (50%) and 500 mL of Acetonitrile HPLC (50%) and degas in ultrasonic water bath for 5 minutes. Filter through 0.45 µ filter under vacuum filtration.

Diluent Preparation:
Use the Mobile phase as Diluent

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Preparation of standard solution:
Accurately weigh and transfer 10 mg of Sumatriptan and Naproxenworking standard into a 10mL clean dry volumetric flask add about 7mL of Diluent and sonic ate to dissolve it completely and make volume up to the mark with the same solvent.

Further pipette 5ml of Sumatriptan&Naproxenthe above stock solution into a 50ml volumetric flask and dilute up to the mark with diluent.

Further pipette 8ml of Sumatriptan&Naproxenthe above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

Preparation of sample solution:
Accurately weigh and transfer equivalent to 10 mg of Sumatriptan and Naproxensample into a 10mL clean dry volumetric flask add about 7mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent.

Further pipette 5ml of Sumatriptan&Naproxenof the above stock solution into a 50ml volumetric flask and dilute up to the mark with diluent.

Further pipette 8ml of Sumatriptan&Naproxenthe above stock solution into a10ml volumetric flask and dilute up to the mark with diluent

Method validation
The proposed method was validated as per ICH guidelines. The drug solutions were prepared as Per the earlier adopted procedure given in the experiment.

Linearity study
Linearity was performed by taking from stock solution aliquots of 6, 7, 8, 9 and 10 ml were taken in 10ml volumetric flasks and dilutedupto the mark with diluent such that the finalconcentration of Sumatriptan and Naproxen in the range of 60to 100 μg/ml. Volume of 20 μl of each sample was injected in six times for each concentration level and calibration curve was constructed by plotting the peak area versus the drug concentration. The observations and calibration curve is shown in Table 1, 2,3

Accuracy as recovery
I t was done by recovery study. Sample solutions were prepared by spiking at about 50 %, 100% and 150 % of specification limit to Placebo and analyzed by the proposed HPLC method. Results are shown in Table 5,6..

System precision
Precision is the measure of how close the data values are to each other for a number of measurements under the same analytical conditions. Standard solution of (80 ppm) were prepared as per test method and injected for 5 times. Results are shown in Table 7.

Limit of detection and limit of quantitation:
The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation.

Robustness:
As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.Results are shown in Table 8,9.

Ruggedness:
To evaluate the Ruggedness of the method, ruggedness was performed on different day by using different make column of same dimensions. Results are shown in Table 10.

Results and discussions
Sumatriptan is an serotonin agonist that acts selectively at 5HT1 receptors and Naproxen An anti-inflammatory agent with analgesic and antipyretic properties. These both are combindly used mainly to treat acute migraine. A simple reverse phase HPLC method was developed for the determination of Sumatriptan and Naproxen. Symmetry C8 (4.6 x 150mm, 3.5mm, Make: XTerra) in an isocratic mode with mobile phase Acetonitrile:Phosphate buffer PH3(50:50) was used. The flow rate was 0.7ml/ min and effluent was monitored at 285 nm. The retention time for Sumatriptan is 2.2min and Naproxen 5.8min.

From the linearity Table 1,2,3 it was found that the drug obeys linearity within the concentration range of 60-100mg/ml for Sumatriptan and Naproxen. From the results shown in accuracy Table 5, 6 it was found that the percentage recovery values of pure drug were in between 99.8 to 101.9, which indicates that the method was accurate and also reveals that the commonly used excipients and additives present in the pharmaceutical formulations were not interfering the proposed method. From the results shown in precision Tables 7, It was found that % RSD is less than 2%; which indicates that the proposed method has good reproducibility. The system suitability parameters also reveal that the values were within the specified limits for the proposed method. The results of robustness were shown in tables 8,9 it was found that the results are within the limits. The results of ruggedness were shown in tables 10. It was found that the results are within the limits, the proposed method is found to be rugged.

Summary and Conclusion
In the present work, an attempt was made to provide a newer, sensitive, simple, accurate and low cost RP-HPLC method. It is successfully applied for the determination of Sumatriptan and Naproxenin pharmaceutical preparations without the interferences of other constituent in the formulations.

In HPLC method, HPLC conditions were optimized to obtain, an adequate separation of eluted compounds. Initially, various mobile phase compositions were tried, to get good optimum results. Mobile phase and flow rate selection was based on peak parameters (height, tailing, theoretical plates, capacity factor), run time etc. The system with Buffer : acetonitrile (50:50 v/v) with 0.7 ml/min flow rate is quite robust.

The optimum wavelength for detection was 285 nm at which better detector response for drug was obtained. The average retention time for Sumatriptan and Naproxenwere found to be 5.87 and 2.24min. System suitability tests are an integral part of chromatographic method. They are used to verify the reproducibility of the chromatographic system. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions. The calibration was linear in concentration range of 60 – 100 mg/ml. The low values of % R.S.D. indicate the method is precise and accurate. The mean recoveries were found in the range of 99.0 – 101.0 %.

Sample to sample precision and accuracy were evaluated using, three samples of five and three different concentrations respectively, which were prepared and analyzed on same day. Day to day variability was assessed using three concentrations analyzed on three different days, over a period of three days. These results show the accuracy and reproducibility of the assay.

Ruggedness of the proposed methods was determined by analysis of aliquots from homogeneous slot by different analysts, using similar operational and environmental conditions; the % R.S.D. reported was found to be less than 2 %.The proposed method was validated in accordance with ICH parameters and the results of all methods were very close to each other as well as to the label value of commercial pharmaceutical formulation. Therefore, there is no significant difference in the results achieved by the proposed method.

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Acknoledgements
The authors are grateful to the Management of school of pharmaceutical sciences, VELS University, Chennai, for their continuous support and encouragement and for providing the necessary facilities.

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