Stereoselective CZE method for analysis of verapamil and norverapamil in human plasma.

A stereospecific capillary zone electrophoresis (CZE) method was developed for the determination of verapamil (VER) and its main metabolite norverapamil (NOR) in human plasma. Optimal temperature, cyclodextrin selectors (CDs), pH of background electrolyte (BGE) and voltage were established to obtain complete separation of the chiral analytes and clopidogrel internal standard (I.S.) during one analytical run. Successful resolution of analytes was obtained in silica capillary filled with BGE consisting of heptakis 2,3,6-tri-O-methyl-beta-CD in phosphate buffer, pH 2.5 at 15 degrees C of capillary temperature. The calculated electrophoretic parameters of the analytes were as follows: apparent electrophoretic mobility, for VER enantiomers: micro(ap(+)-R) = 1.1 x 10(-4), micro(ap(-)s) = 1.06 x 10(-4) cm2/Vs and for NOR enantiomers: micro(ap(+)-R) = 1.09 x 10(-4), micro(ap(-)s) = 1.04 x 10(-4) cm2/Vs, resolution factors, R(s) = 5.4-6.6. Liquid extraction was applied for isolation of the analytes. The calibration curves ware linear in the range 0.25-10 microg/mL for VER and NOR enantiomer concentrations. The validation parameters were also established. The precision and accuracy of intra- and inter-day analysis were less than 15%. The lower limit of detection and limit of quantification for single enantiomers were 0.1 and 0.2 microg/mL, respectively. Recovery of the enantiomers from plasma was in the 91-103% range. To evaluate analytical applicability of the proposed method, plasma sample from patient suffering from arterial hypertension treated with 80 mg of commercial tablets was analyzed.