Development and Validation of RP-HPLC Method for Simultaneous Determination of Amprolium HCl and Ethopabate in Their Combination Drug

Abstract

In this study a simple, rapid, accurate, sensitive and specific reverse phase-high performance liquid chromatographic (RP- HPLC) method was developed and subsequently validated for simultaneous estimation of Amprolium hydrochloride (AMP) and Ethopabate (ETH) in their combination syrup. The separation of the drugs was carried out using a base deactivated silanol (BDS) C18 (250mm x 4.6mm, 5 μm) column, mobile phase consisting of methanol and purified water in the proportion of 60:40 (v/v) containing 0.5% Heptansulfonic acid sodium at pH of 3.7 and flow rate of 1 ml/min. The influence of the instrument operating conditions on the resolution and retention time were tested. The method was linear over a range of 48-480 μg/ml and 3-30 μg/ml with a correlation coefficient (r2) of 0.99996 for AMP and ETH, respectively. The method validations study revealed excellent accuracy, precision, linearity, specificity, limit of detection (LOD) and limit of quantitation (LOQ) of the proposed method according to the international conference harmonization (ICH) guidelines. Moreover, the stability study revealed that the proposed method can also be used for evaluation of purity and degradation of these drugs in their formulations that arisen due to the temperature, humidity and time.