Kinetic study of CYP3A4 activity on verapamil by capillary electrophoresis

The use of capillary electrophoresis (CE) for the determination of CYP3A4 activity with verapamil as a substrate was investigated. CYP3A4 activity was determined by the quantitation of the product, norverapamil, based on separation by CE. The separation conditions were as follows: capillary, 80.5 cm (75 μm i.d., 72 cm effective length); 50 mM sodium phosphate buffer (pH 8.8); 20 kV (100 μA) applied voltage; UV detection at 200 nm; capillary temperature, 25 °C. With the developed CYP3A4 activity assay and the Lineweaver-Burk equation, the Michaelis-Menten parameters Km and Vmax for formation of norverapamil from verapamil in the presence of CYP3A4 were determined and were 22.8 ± 2.5 μM and 7.67 ± 0.26 pmol/min/pmol (or 983 pmol/min/mg) CYP3A4, respectively.