ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF OMEPRAZOLE AND ASPIRIN USING REVERSE PHASE HPLC METHOD IN BULK AND DOSAGE FORM

Objectives: A new simple, accurate, precise and reproducible RP-HPLC method has been developed for the simultaneous estimation of Aspirin and Omeprazole in bulk and pharmaceutical dosage form using C18 column (Agilent, 250 x 4.6 mm, 5μm) in isocratic mode. Methods: The mobile phase consisted of Methanol and 0.1 M Di-potassium Phosphate buffer (pH 3) in the ratio of 60:40 v/v. The detection was carried out at 256 nm. The method was linear over the concentration range for Omeprazole 50-250 μg/ml and for Aspirin 10-50μg/ml. Results: The recoveries of Omeprazole and Aspirin were found to be 100.07 and 100.06% respectively. The validation of method was carried out utilizing ICH-guidelines. The described HPLC method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form. Conclusion: Study concludes that the proposed method is accurate, precise, rapid and selective has advantage of simplicity and convenience for the separation and quantitation of ASP and OMP in the combination which can be used for the assay of their dosage form.


INTRODUCTION
Aspirin (ASP) is chemically 2-(acetyloxy)-benzoic acid ( Figure 1). It is nonselective cyclooxygenase inhibitor used as an antipyretic, analgesic, anti-inflammatory, and antithrombotic agent. omeprazole magnesium (ESO) is S-isomer of omeprazole and proton pump inhibitor. It is magnesium, bis [5- The review of literature revealed that various analytical methods involving spectrophotometry 5-7 , HPLC 8- 11 and HPTLC have been reported for ASP in single form and in combination with other drugs 12 . Several analytical methods have been reported for ESO in single form and in combination with other drugs including spectrophotometry 13,14 , HPLC 15, 16 , and HPTLC 17 .  Methanol in the ratio of 40:60 v/v with the flow rate of 1ml/min. Separation was performed on a Waters C 18 (250x4.6mm i.d, 5μ particle size) analytical column and a pre-column to protect the analytical column from strongly bonded material. Integration of the detector output was performed using the Waters Empower software to determine the peak area. The contents of the mobile phase were filtered through a 0.45µm membrane filter and degassed by sonication before use. Mobile phase was used as diluents. The flow rate of the mobile phase was optimized to 1 ml/min which yields a column back pressure of 110-112kg/cm. The run time was set at 6 min and a column temperature was maintained at 35°C. The volume of injection was 10 µl, prior to injection of the analyte, the column was equilibrated for 30-40 min with the mobile phase. The eluents were detected at 256 nm. The developed method was validated in terms of specificity, linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day precision and robustness for the assay of ASP and OMP as per ICH guidelines. Preparation of standard solutions ASP and OMP were weighed (10mg each) and transferred to two separate 10ml volumetric flasks and dissolved in 5ml of water and make up the volume up to the mark with mobile phase. Working standards of the drugs were prepared from this solution.

Preparation of sample solution
Twenty tablets (Yosprala, Make: Aralez Pharmaceuticals) were weighed. An accurately weighed amount of the finely powdered tablets equivalent to 10mg was made up to 10 ml with mobile phase. The solution was filtered followed by serial dilution to the required concentrations for each experiment.

RESULTS AND DISCUSSION Method Development
Number of mobile phase and their different proportions were tried and finally was selected as 0.1 M Dipotassium Phosphate buffer (pH 3) and Methanol in the ratio of 40:60 v/v appropriate mobile phase which gave good resolution and acceptable system suitability parameters. The results of system suitability parameters were shown in Table 2. The chromatogram of working standard solution is shown in Figure 3. The summary of Chromatographic conditions was given in Table 1.   of drug recovered and percentage recovery were calculated which sense to conformation that the proposed method was accurate. The results were tabulated in Table 3.

Precision
The intraday and inter day precision of the proposed method was determined by analyzing mixed standard solution of OMP and ASP at concentration 150 µg/ml and 30 µg/ml, 3 times on the same day and on 3 different days. The results shown in Table 4 were reported in terms of relative standard deviation.  Figure 4 and Figure 5.

Limit of detection (LOD) and limit of quantitation (LOQ):
The limit of detection (LOD) and limit of quantitation (LOQ) of ASP and OMP were determined by calculating the signal-to-noise (S/N) ratio of 3:1 and 10:1, respectively according to International Conference on Harmonization guidelines.LOD values for ASP and OMP were found to be 3.08and 3.041µg/ml respectively. LOQ values for ASP and OMP were found to be 9.24µg/ml and 10.37µg/ml respectively.

Figure 5: Linearity of Aspirin Assay of the tablet dosage form
The proposed validated method was successfully applied to determine ASP and OMP in tablet dosage form. The result obtained for ASP and OMP were comparable with corresponding labeled amounts. The results were tabulated in Table 4.

CONCLUSION
The proposed method has advantage of simplicity and convenience for the separation and quantitation of ASP and OMP in the combination which can be used for the assay of their dosage form. Also, the low solvent consumption and short analytical run time lead to environmentally friendly chromatographic procedure. The method is accurate, precise, rapid and selective for simultaneous estimation of Aspirin and Omeprazole in tablet dosage form. Hence it can be conveniently adopted for routine analysis.