Universidade de Sao Paulo,Faculdade de Ciencias Farmaceuticas de Ribeirao Preto,Departamento de Analises Clinicas,Toxicologicas e Bromatologicas,Avenida do Cafe s/n,Campus da USP,14040-903 Ribeirao Preto,SP,Brazil;
Paula Macedo Cerqueira;
Debrisoquine (D), an anunypertenslve arug metaboUZea to 4-nyaroxyaeDnsoqume ( 4-UttlJ ) Dy L y y LU(), IS commonly used as an in vivo probe of CYP2D6 activity and can be used to phenotype individuals as either extensive (EMs) or poor metabolizers (PMs) of such drugs as 13-adrenergic blockers, tricyclic antidepressants, and class IC antiarrhythmics. This report describes reversed-phase HPLC systems by which D and 4-0HD or S-( + ) and R-( -)-4-0HD in urine are more selectively quantified without the need for derivatization techniques. We also studied the urinary excretion of R-( -)- and S-( + )-4-hydroxydebrisoquine in EM hypertensive patients in order to determine weather 4-0HD formation exhibits enantioselectivity. Twelve patients with mild to severe essential hypertension were admitted to the study. They received a single tablet of Declinax containing 10 mg debrisoquine sulfate. All the urine excreted during the following 8 h was collected. The debrisoquine metabolic ratio (DMR) was calculated as % of dose excreted as D/% of dose excreted as 4-0HD and the debrisoquine recovery ratio (DRR) was calculated as % of dose excreted as 4-0HD/% of dose excreted as D+4-0HD. Debrisoquine and its metabolite were determined in urine by HPLC using a reversed-phase Select B LiChrospher column, a mobile phase of 0.25 N acetate buffer, pH 5-acetonitrile (9:1, vlv) and a fluorescence detector. The limit of quantitation was determined to be 25.0 ng/ml for D and 18.75 ng/ml for 4-0HD. Intra- and inter-day relative standard deviations (RSDs) were less than 10%. All hypertensive patients studied showed a DMR of less than 12.6 or a DRR higher than 0.12 and were classified as EMs. Direct enantioselective separation on chiral stationary phase involved resolution of S-( + )-4-0HD and R-( -)-4-0HD on a Chiralcel OD-R column with a mobile phase of 0.125 N sodium perchlorate, pH 5-acetonitrile-methanol (85:12:3, vlv/v). The quantitation limit of each enantiomer was 3.75 ng/ml of urine. Intra- and inter-day RSDs were less than 10% for each enantiomer. A high degree of enantioselectivity in the 4-hydroxylation of D favouring the S-( + ) enantiomer was observed, resulting in R-( -)-4-0HD not detected in the urine of the EM hypertensive natients studied.
Paula Macedo Cerqueira; Universidade de Sao Paulo,Faculdade de Ciencias Farmaceuticas de Ribeirao Preto,Departamento de Analises Clinicas,Toxicologicas e Bromatologicas,Avenida do Cafe s/n,Campus da USP,14040-903 Ribeirao Preto,SP,Brazil; Journal of Chromatography B:Biomedical Sciences and Applications.; V.749 N.2 2000; 153-161