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.