Abstract
This paper presents the development and validation of a new HPLC-UV method for simultaneous quantitative determination of clopidogrel and its hydrolysis product clopidogrel carboxylic acid (CCA) from bulk material and dosage formulations. Development of the chromatographic method is based on a design of experiments (DOE) approach. A Box-Behnken experimental design was used to build the mathematical models and to choose the significant parameters for the optimization by simultaneously taking resolution, capacity factor and peak symmetry as responses. Derringer's desirability function was used for the selection of the optimum experimental conditions in terms of mobile phase composition, column temperature and flow rate. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, robustness and system suitability. The method was further employed for the study of clopidogrel bisulfate hydrolysis kinetics under different pH conditions, with special emphasis on the acidic hydrolysis studies, since different clopidogrel salts are suffering pre-systemic metabolism trough hydrolysis under the acidic pH of the stomach. This effect is generally difficult to quantify, since CCA is also the main circulating metabolite, and no differentiation between the pre-systemic and systemic CCA can be made. In the acidic environment created by a 0.1N HCl solution CLO degradation was slow at room or body temperature (25 and 37°C respectively), less than 5% of the initial CLO amount being hydrolyzed after 48h. Under forced conditions (85ºC) however, 17.8% of CLO was transformed into CCA within 48 hours.