A micromethod for quantitation of debrisoquine and 4-hydroxydebrisoquine in urine by liquid chromatography

We describe a new simple, selective and sensitive micromethod based on HPLC and fluorescence detection to measure debrisoquine (D) and 4-hydroxydebris oquine (4-OHD) in urine for the investigation of xenobiotic metabolism by d ebrisoquine hydroxylase (CYP2D6). Four hundred ul of urine was required for the analysis of D and 4-OHD. Peaks were eluted at 8.3 min (4-OHD), 14.0 mi n (D) and 16.6 min for the internal standard, metoprolol (20 mu g/ml). The 5-mu m CN-reverse-phase column (Shimpack, 250 x 4.6 mm) was eluted with a m obile phase consisting of 0.25 M acetate buffer, pH 5.0, and acetonitrile ( 9:1, v/v) at 0.7 ml/min with detection at lambda(excitation) = 210 nm and l ambda(emission) = 290 nm. The method, validated on the basis of measurement s of spiked urine, presented 3 ng/ml (D) and 6 ng/ml (4-OHD) sensitivity, 3 90-6240 ng/ml (D) and 750-12000 ng/ml (4-OHD) linearity, and 5.7/ 8.2% (D) and 5.3/8.2% (4-OHD) intra/interassay precision. The method was validated u sing urine of a healthy Caucasian volunteer who received one 10-mg tablet o f Declinex(R), po, in the morning after an overnight fast. Urine samples (d iuresis of 4 or 6 h) were collected from zero to 24 h. The urinary excretio n of D and 4-OHD, Fel (0-24 h), i.e., fraction of dose administered and exc reted into urine, was 6.4% and 31.9%, respectively. The hydroxylation capac ity index reported as metabolic ratio was 0.18 (D/4-OHD) for the person inv estigated and can be compared to reference limits of >12.5 for poor metabol izers (PM) and <12.5 for extensive metabolizers (EM). In parallel, the reco very ratio (RR), another hydroxylation capacity index, was 0.85 (4-OHD: Sig ma D + 4-OHD) versus reference limits of RR <0.12 for PM and RR >0.12 for E M. The healthy volunteer was considered to be an extensive metabolizer on t he basis of the debrisoquine test.