A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the...

A capillary electrophoresis (CE) method has been developed for the determination of quinidine sulfate (QS) and (3S)-3-hydroxyquinidine (3-OHQ) by direct injection of microsomal incubation mixtures. 3-OHQ is the CYP 3A4 metabolite of QS and hence useful for metabolism screening studies. The method was validated analytically and tested for its effectiveness as a metabolic inhibition model. A linear calibration was found to provide the best fit for the standard curve with an r of 0.9966 and all residuals less than 12%. The percent relative standard deviations (RSDs) of the two controls, 2 and 8 microg/ml were 5.27 and 2.90% and the percent difference from normal (% DFN) were -12.58 and -0.31% respectively. The limit of quantitation (LOQ) in the incubation matrix was 0.5 microg/ml. 3-OHQ formation complied with Michaelis-Menten kinetics and the mean values+/-S.D. of Km and Vmax were 36.98+/-4.62 microg/ml and 321.39+/-3.88 ng/mg/h respectively. Preliminary inhibition studies suggest that the method has adequate sensitivity to screen for high and medium inhibitors of the CYP 3A4 isozyme. The lack of sample preparation coupled with the small sample size capability of CE would enable the direct injection technique to aid in miniaturized high-throughput screening.