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-hydroxydebrisoquine (4-OHD) in urine for the investigation of xenobiotic metabolism by debrisoquine hydroxylase (CYP2D6). Four hundred microl 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 microg/ml). The 5-microm CN-reverse-phase 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 lambdaexcitation = 210 nm and lambdaemission = 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(R), 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: SigmaD + 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.
Pereira
V A
VA
Laboratório de Farmacologia Terapêutica, Unidade 2, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil.
Auler
J O
JO
Carmona
M J
MJ
Mateus
F H
FH
Lanchote
V L
VL
Breimer
D D
DD
Santos
S R
SR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism*; D003647:Debrisoquin / Q000031:analogs & derivatives* / Q000652:urine*; D005260:Female; D005470:Fluorometry / Q000379:methods; D006801:Humans; D006900:Hydroxylation; D008875:Middle Aged; D010641:Phenotype; D012680:Sensitivity and Specificity; D015262:Xenobiotics / Q000378:metabolism
D015262:Xenobiotics; C012359:4-hydroxydebrisoquin; D019389:Cytochrome P-450 CYP2D6; D003647:Debrisoquin