Fully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach

In this study, a fully automatized in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an in-line bioreaction in the presence of CYP1A1 supersomes and NADPH as co-factor, giving rise to hydroxycoumarin, the product that was assayed. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The results obtained in optimal conditions were compared not only to those achieved after off-line metabolization but also with liver microsomes. Finally, inhibition studies were conducted showing an important decrease of hydroxycoumarin formation using apigenin as CYP1A1 potent inhibitor. This study demonstrates the usefulness of our in-line system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors.