Validation and Peak Purity Assessment of RP-HPLC Method for Simultaneous Estimation of Amitriptyline HCl, Chlordiazepoxide and Comparison with Spectrophotometry

Abstract

Purpose : The study was performed with the aim to design a Simple, precise and economical RP-HPLC method have been developed for simultaneous estimation of amitriptyline HCl and chlordiazepoxide in pharmaceutical formulation. Approach : RP-HPLC was developed using WATERS C18 250 mm × 4.6 mm (5 μm) column, utilizing a mobile phase of methanol: acetonitrile: 0.2 M orthophosphoric acid, pH 4.5 (adjusted with triethylamine) 56:24:20 ml (with a flow rate of 1ml/min) and U.V detection was carried out at 240nm. Optimization of this method was carried out as a function of mobile phase composition, pH and flow rate. Peak purity was assessed by spectral comparison method. Findings : RP-HPLC method shows linearity in the range of for 0.05-1.6 μg/ml and 0.01-0.32μg/ml amitriptyline HCl and chlordiazepoxide respectively. The LOD and LOQ values for amitriptyline HCl were 0.0250μg/ml and 0.0759 μg/ml and 0.0045 μg/ml and 0.0135 μg/ml for chlordiazepoxide. The results of % purity was found to be 99.97 ± 0.58 % w/w and 99.29 ± 0.59 % w/w for HPLC method and 98.87 ± 0.03 % w/w and 98.81 ± 1.04 % w/w for first derivative spectrophotometric method for amitriptyline HCl and chlordiazepoxide respectively. Conclusion : Results of descriptive statistics studies conclude that RP-HPLC method found to be more sensitive and convenient than first derivative spectrophotometric method.