METHOD DEVELOPMENT AND VALIDATION OF SIMULTANEOUS DETERMINATION OF FLUTAMIDE AND AZATHIOPRINE BY USING RP-HPLC

The aim of the work is to developing a specific, precise, accuracy, linear, simple, rapid, and validated and cost effective RP-HPLC method for the simultaneous estimation of these drugs in combined dosage forms. For the present study Azathioprine and Flutamide was selected. The separation was carried out by using mobile phase composition selected for the chromatographic separation of Azathioprine and Flutamide was acetonitrile and phosphate buffer of pH 2.3 in the ration of 45:55 v/v. The detection was carried out at 276 nm. The column was ODS Hypersil 5µ C18, 4.6x250mm. The flow rate was selected as 1.0mL/min and Run time 25 Min. Diluent water: acetone (50:50) was used throughout the process. The method was validated according to International Conference on Harmonization guidelines and successfully applied. The percentage of assay for Azathioprine and Flutamide was found to be 101.79% and 104.55% respectively. The percentage mean recovery of Azathioprine and Flutamide was found to be 99.43% and 99.53% respectively. The Developed method will be validated according to ICH guidelines and it can be used for routine analysis. The precision for the developed method was found to be less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0%. The method was linear over the concentration range of 125.35- 376.05 µg/ml for Azathioprine and 125.28-376.83 µg/ml for Flutamide respectively. The method was found to be precise and suitable for the determination of Azathioprine and Flutamide by HPLC. From the study and data it is concluded that, the method is found to be specific, Precise and Linear in the specified range. System suitability is established and recorded. Hence, this method can be used for routine analysis for determination of Azathioprine and Flutamide by RP-HPLC KEYWORDS: HPLC, Azathioprine and Flutamide, Quantitative estimation.