Development and application of a chiral high performance liquid chromatography assay for pharmacokinetic studies of methadone

Methadone enantiomers and EDDP, the main metabolite of methadone, were sepa rated (R-s = 2.0 for methadone enantiomers) following liquid-liquid extract ion from human serum and urine followed by reverse-phase high-performance l iquid chromatography on a derivatized beta-cyclodextrin column and quantifi ed at therapeutic concentrations with ultraviolet detection. Detector respo nse was linear (r(2) > 0.98) to 1,000 and 2,500 ng.mL(-1) for methadone ena ntiomers and EDDP, respectively. The limit of quantification from a 1-mL bi ological sample was 2.5 and 5 ng.mL(-1) for methadone enantiomers and EDDP, respectively. Interday variation was <13% and intraday variation was <8% f or the analytes of interest. The assay was applied to plasma protein and er ythrocyte binding studies and a 96-h pharmacokinetic study in two healthy f emale volunteers following oral dosing with rac-methadone. The binding of m ethadone to plasma proteins was enantioselective with the active (-)-(R) en antiomer having the highest free fraction (mean +/- SD: 21.2 +/- 7.6% vs. 1 3.3 +/- 6.2% for (+)-(S)-methadone, n = 8). Binding of methadone to erythro cytes was not apparently enantioselective (38.6 +/- 1.3% and 38.1 +/- 1.4% bound for (-)-(R)- and (+)-(S)-methadone, respectively). The pharmacokineti c study revealed enantioselective disposition of methadone in one volunteer but not in the other. EDDP was observed in urine but was only in small or undetectable concentrations in serum. The method is applicable to in vitro and pharmacokinetic studies of rac-methadone disposition in humans. Chirali ty 12:681-687, 2000. (C) 2000 Wiley-Liss. Inc.