title: Development and application of a chiral high performance liquid chromatography assay for pharmacokinetic studies of methadone.
abstract: Methadone enantiomers and EDDP, the main metabolite of methadone, were separated (R(s) = 2.0 for methadone enantiomers) following liquid-liquid extraction from human serum and urine followed by reverse-phase high-performance liquid chromatography on a derivatized beta-cyclodextrin column and quantified at therapeutic concentrations with ultraviolet detection. Detector response was linear (r(2) > 0.98) to 1,000 and 2,500 ng x mL(-1) for methadone enantiomers and EDDP, respectively. The limit of quantification from a 1-mL biological sample was 2.5 and 5 ng x mL(-1) for methadone enantiomers and EDDP, respectively. Interday variation was <13% and intraday variation was <8% for the analytes of interest. The assay was applied to plasma protein and erythrocyte binding studies and a 96-h pharmacokinetic study in two healthy female volunteers following oral dosing with rac-methadone. The binding of methadone to plasma proteins was enantioselective with the active (-)-(R) enantiomer having the highest free fraction (mean +/- SD: 21.2+/-7.6% vs. 13.3+/-6.2% for (+)-(S)-methadone, n = 8). Binding of methadone to erythrocytes was not apparently enantioselective (38.6+/-1.3% and 38.1+/-1.4% bound for (-)-(R)- and (+)-(S)-methadone, respectively). The pharmacokinetic 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.
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: D W
authors.initials: DW
authors.lastname: Boulton
authors.forename: C L
authors.initials: CL
authors.lastname: Devane
mesh_terms: D000284:Administration, Oral; D001798:Blood Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D006801:Humans; D008691:Methadone / Q000032:analysis / Q000737:chemistry* / Q000493:pharmacokinetics*; D011485:Protein Binding; D011759:Pyrrolidines / Q000032:analysis; D013237:Stereoisomerism
chemical_list: D001798:Blood Proteins; D011759:Pyrrolidines; D008691:Methadone; C010724:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine