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

Abstract

We describe a new simple, selective and sensitive micromethod based on HPLC and fluorescence detection to measure debrisoquine (D) and 4-hydroxydebrisoquine (4-OHD) in urine for the investigation of xenobiotic metabolism by debrisoquine hydroxylase (CYP2D6). Four hundred µl of urine was required for the analysis of D and 4-OHD. Peaks were eluted at 8.3 min (4-OHD), 14.0 min (D) and 16.6 min for the internal standard, metoprolol (20 µg/ml). The 5-µm CN-reversephase column (Shimpack, 250 x 4.6 mm) was eluted with a mobile 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 l excitation = 210 nm and l emission = 290 nm. The method, validated on the basis of measurements of spiked urine, presented 3 ng/ml (D) and 6 ng/ml (4-OHD) sensitivity, 390-6240 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 using urine of a healthy Caucasian volunteer who received one 10-mg tablet of Declinax ® , po, in the morning after an overnight fast. Urine samples (diuresis of 4 or 6 h) were collected from zero to 24 h. The urinary excretion of D and 4-OHD, Fel (0-24 h), i.e., fraction of dose administered and excreted into urine, was 6.4% and 31.9%, respectively. The hydroxylation capacity index reported as metabolic ratio was 0.18 (D/4-OHD) for the person investigated and can be compared to reference limits of >12.5 for poor metabolizers (PM) and <12.5 for extensive metabolizers (EM). In parallel, the recovery ratio (RR), another hydroxylation capacity index, was 0.85 (4-OHD: SD + 4-OHD) versus reference limits of RR <0.12 for PM and RR >0.12 for EM. The healthy volunteer was considered to be an extensive metabolizer on the basis of the debrisoquine test.

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes

“…Debrisoquine and 4-hydroxydebrisoquine were determined at room temperature, at a flow rate of 0.3 ml/min, using an isocratic mobile phase consisting of 30% methanol, 70% water and 0.1% formic acid. Analytes were monitored by fluorescence detection at excitation and emission wavelengths of 210 nm and 290 nm, respectively (Pereira et al, 2000;Granvil et al, 2002), and 5 ml supernantants were analysed by HPLC without further dilution. Determination of nifedipine and its metabolite oxidized nifedipine (CYP3A4 activity) was carried out at room temperature, at a flow rate of 0.5 ml/min using an isocratic mobile phase consisting of 32% acetonitrile and 68% water.…”

4-Hydroxylation of Debrisoquine by Human CYP1A1 and Its Inhibition by Quinidine and Quinine

“…Microsomal production of 4-hydroxydebrisoquine was determined using an LC/MS/MS method described previously (Corchero et al, 2001) with a minor modification of liquid-liquid extraction (Pereira et al, 2000) rather than solid-phase extraction (Scott et al, 1999). Briefly, to the total microsomal incubations were added 20 l of internal standard solution (phenacetin, 5 g/ml in methanol), 500 l of propan-2-ol, 50 l of 0.4 M sodium hydroxide, and 3 ml of methyl tert-butyl ether.…”

Characterization of the CYP2D6 drug metabolizing phenotypes of the Chilean mestizo population through polymorphism analyses

“…Following liquid-liquid urine extraction, chromatographic analysis was performed using a validated method adapted [28,29]. HPLC (Shimadzu LC-20 AT) separation employed to ODS-3 C18 Inertsil column followed by detection with a fluorescence detector …”