CYP2D6-Dependent Bufuralol 1-Hydroxylation Assayed by Reversed-Phase lon-Pair High-Performance Liquid Chromatography with Fluorescence Detection

CYP2D6 is an enzyme that is subject to genetic polymorphism 1 . This P450 is absent in individuals with the poor metabolizer CYP2D6 phenotype 2 , 3 ), resulting in a substantially reduced capacity to metabolize a large number of clinically useful drugs, including metoprolol, propafenone, haloperrdol, dextromethorphan and codeine (1 ). Quinidine competrtively inhibits CYP2D6, with a K₁ of 3-30 mM (3 –5 ). Because of its preferential inhibition of CYP2D6 (6 7 ), quinidine is frequently used as an inhibitory chemical probe of this polymorphically expressed P450. Diagnostic catalytic monitors of human hepatic CYP2D6 include debrisoquine 4-hydroxylase (4 ), dextromethorphan Odemethylase (8 ), and bufuralol l’-hydroxylase activities (2 –4 ). Advantages of using bufuralol as a CYP2D6-selective substrate include the high sensitivity of the assay owing to the highly fluorescent l’-hydroxybufuralol metabohte and the fact that the use of radiolabeled substrate is not required. This chapter describes a modification of a reversed-phase ion-pair high-performance liquid chromatographic (HPLC) assay (9 ) with fluorescence detection for the determmation of bufuralol l’-hydroxylation activity. Methods for other CYP2D6 assays such as debrisoquine 4-hydroxylase (9 ) and dextromethorphan O-demethylase (9 , 10 ) can be found in the cited references.