Analytical Method Development and Validation for Estimation of Spironolactone and Hydrochlorothiazide in Bulk and Tablet Dosage form by High Performance Liquid Chromatography

Abstract

Analytical Method Development and Validation for Spironolactone and Hydrochlorothiazide in bulk and Combined Dosage Form by RP-HPLC. New method was established for simultaneous estimation of Spironolactone and Hydrochlorothiazideby RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Spironolactone and Hydrochlorothiazideby using Inertsil C18 (4.6mm ×250mm, 5µm particle size), flow rate was 1.0ml/min, mobile phase ratio was (55:45% v/v) Methanol: Phosphate buffer pH 4.8 (pH was adjusted with ortho phosphoricacid), 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 Hydrochlorothiazidewas found to be 99.86%. The system suitability parameters for Spironolactone and Hydrochlorothiazidesuch as theoretical plates and tailing factor were found to be 7586, 1.69 and 6235 and 1.58, the resolution were found to be 10.85. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study ofSpironolactone and Hydrochlorothiazidewas 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 Hydrochlorothiaziderespectively. Hence the suggested RP-HPLC method can be used for routine analysis of Spironolactone and Hydrochlorothiazide in API and Pharmaceutical dosage form.