Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies.

ABSTRACT

An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption.