Determination of debrisoquine and 4-hydroxydebrisoquine by high-performance liquid chromatography:: application to the evaluation of CYP2D6 genotype and debrisoquine metabolic ratio relationship

The drug-metabolizing cytochrome P450 (CYP) enzyme CYP2D6 is involved in the metabolism of several clinically important drugs. So far more than 50 different CYP2D6a allelic variants have been described, and thus there is an increased need for routine high-performance liquid chromatography (HPLC) methods for the evaluation of the functional implication of CYP2D6 polymorphism. Debrisoquine is metabolized to 4-hydroxydebrisoquine by CYP2D6, and therefore it has been used widely to determine the hydroxylation capacity of the enzyme. The aim of the present study was to develop a simple, accurate HPLC method with ultraviolet detection for the measurement of debrisoquine and 4-hydroxydebrisoquine in urine for evaluation of the relationship between CYP2D6 enzyme activity and genotypes. For the HPLC determination, a C18 extraction column was used with a flow rate of 0.8 mL/min and detection at 210 nm. The compounds were eluted from the column in less than 10 min. Coefficients of variation at all concentrations were less than 4% for both compounds. The debrisoquine/4-hydroxydebrisoquine ratio (debrisoquine metabolic ratio) was determined in a panel of 16 Caucasian healthy volunteers with zero (poor metabolizers), one, two or more than two (ultrarapid metabolizers) CYP2D6 active genes. Significant correlation (p<0.05) between the number of CYP2D6 active genes and the hydroxylation capacity of the enzyme was found. The present HPLC method was simple, fast and accurate, and thus will be useful for the evaluation of CYP2D6 hydroxylation capacity in pharmacogenetic studies.