high performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: application to a bioequivalence study

a sensitive and rapid method is described for determination of clopidogrel carboxylic acid (cca), the inactive metabolite of the antiplatelet agent clopidogrel in human serum. the analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. a mobile phase consisting of 0.05 m phosphate buffer containing triethylamine (0.5 ml/l; ph 5.7) and acetonitrile (56:44; v/v) was used and chromatographic separation was achieved using a c18 analytical column at detector wavelength of 220 nm. the calibration curves were linear over a concentration range of 0.05-10 μg/ml of cca in human serum. the total run time of analysis was 5.5 min. and the lower limits of detection (lod) and quantification (loq) were 0.02 and 0.05 μg/ml, respectively. the method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. the validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.