In-line capillary electrophoretic evaluation of the enantioselective metabolism of verapamil by cytochrome P3A4

•EMMA for metabolic reaction and PFT for chiral separation joined for the 1st time.•In vitro evaluation of CYP3A4 enantioselective metabolism in less than 35 min.•Simultaneous chiral separation of verapamil and norverapamil enantiomers.•Very low consumption of enzymes, substrate and chiral selector.

In this paper a methodology for the in-line evaluation of enantioselective metabolism by capillary electrophoresis has been developed and applied to the study of verapamil metabolism by cytochrome P3A4. The developed methodology comprises an in-capillary reaction step carried out by electrophoretically mediated microanalysis and a separation step in which highly sulfated β-cyclodextrin with partial filling technique has been employed as chiral selector for verapamil and norverapamil enantiomers resolution, joining the advantages of both methodologies in a unique assay. Kinetic parameters of the enzymatic reaction (Km and Vmax) have been evaluated for both verapamil enantiomers by non-linear fitting of experimental data obtained under intermediate precision conditions to Michaelis–Menten equation. Km and Vmax estimated values were 51 ± 9 μM and 22 ± 2 pmol min−1 (pmol CYP)−1 for S-VER and 47 ± 9 μM and 21 ± 2 pmol min−1 (pmol CYP)−1 for R-VER. Consequently, slight enantioselectivity was found for the CYP3A4 metabolism of verapamil. However, since confidence intervals of estimates overlap, we cannot assure a significant enantioselectivity. Intrinsic clearance values were also estimated from Km and Vmax for both enantiomers.