DEVELOPMENT AND VALIDATION OF A RP - HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF SPIRONOLACTONE AND HYDROCHLOROTHIAZIDE IN PURE AND PHARMACEUTICAL DOSAGE FORM

New method was established for simultaneous estimation of Spironolactone and Hydrochlorothiazide by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Spironolactone and Hydrochlorothiazide by using Inertsil C18 (4.6mm ×250mm, 5µm particle size), flow rate was 1.0 ml/min, mobile phase ratio was (55:45% v/v) Methanol: Phosphate buffer pH 4.8 (pH was adjusted with ortho phosphoric acid), detection wavelength was 282nm. The instrument used was WATERS Alliance 2695 separation module, Software: Empower 2, 996 PDA detector. The retention times were found to be 1.688mins and 3.282mins. The % purity of Spironolactone and Hydrochlorothiazide was found to be 99.86%. The system suitability parameters for Spironolactone and Hydrochlorothiazide such as theoretical plates and tailing factor were found to be 7586, 1.69 and 6235 and 1.58, the resolution was found to be 10.85. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study of Spironolactone and Hydrochlorothiazide was found in concentration range of 100µg-500µg and 30µg-70µg and correlation coefficient (r2) was found to be 0.999 and 0.999, % recovery was found to be 100.112% and 100.16%, %RSD for repeatability was 0.1702 and 0.043 respectively. The precision study was precise, robust, and repeatable. The LOD value was found to be 2.1µg/ml and 1.28µg/ml, and LOQ value was 6.3µg/ml and 3.84µg/ml for Spironolactone and Hydrochlorothiazide respectively. The results of study showed that the proposed RP‐HPLC method is a simple, accurate, precise, rugged, robust, fast and reproducible, which may be useful for the routine estimation of Spironolactone and Hydrochlorothiazide in pharmaceutical dosage form.

Keywords: Spironolactone, Hydrochlorothiazide, RP‐HPLC, Simultaneous estimation.