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 microm i.d., 72 cm effective length); 50 mM sodium phosphate buffer (pH 8.8); 20 kV (100 microA) applied voltage; UV detection at 200 nm; capillary temperature, 25 degrees 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 microM and 7.67+/-0.26 pmol/min/pmol (or 983 pmol/min/mg) CYP3A4, respectively.
Zhang
Jie
J
Laboratory of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmaceutical Sciences, K.U. Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium.
Thi Thanh Ha
Pham
P
Lou
Yijia
Y
Hoogmartens
Jos
J
Van Schepdael
Ann
A
D002021:Buffers; D002121:Calcium Channel Blockers / Q000032:analysis; D002626:Chemistry, Pharmaceutical / Q000379:methods; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000737:chemistry*; D004345:Drug Industry / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D008956:Models, Chemical; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors; D014700:Verapamil / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology
D002021:Buffers; D002121:Calcium Channel Blockers; D003577:Cytochrome P-450 Enzyme System; C016904:norverapamil; D014700:Verapamil; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human