title: Cytochrome P450 bio-affinity detection coupled to gradient HPLC: on-line screening of affinities to cytochrome P4501A2 and 2D6.
abstract: Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC-EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column.
authors.affiliation: LACDR-Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
authors.forename: Sebastiaan M
authors.initials: SM
authors.lastname: van Liempd
authors.forename: Stefan
authors.initials: S
authors.lastname: Harmsen
authors.forename: Joran
authors.initials: J
authors.lastname: Beckman
authors.forename: Danny
authors.initials: D
authors.lastname: van Elswijk
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
authors.forename: Hubertus
authors.initials: H
authors.lastname: Irth
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000032:analysis / Q000737:chemistry*; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D013237:Stereoisomerism