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

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 beta-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 (K-m and V-max) have been evaluated for both verapamil enantiomers by non-linear fitting of experimental data obtained under intermediate precision conditions to Michaelis-Menten equation. K-m and V-max estimated values were 51 +/- 91 mu M and 22 +/- 2 pmol min(-1) (pmol CYP)(-1) for S-VER and 47 +/- 9 mu M and 21 +/- 2 pmol min(-1) (pmoICYP)(-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 K-m and V-max for both enantiomers. (C) 2013 Elsevier B.V. All rights reserved.