title: Determination of total and free concentrations of the enantiomers of methadone and its metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine) in human plasma by enantioselective liquid chromatography with mass spectrometric detection.
abstract: A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been validated for the determination of total and free plasma concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP, the primary metabolite of Met), using their respective deuterium-labeled compounds as internal standards [(R,S)-d3-Met and (R,S)-d3-EDDP]. For total drug determinations, 1 ml human plasma was extracted, using a cation-exchange solid-phase extraction cartridge; the eluate was evaporated, reconstituted in the mobile phase, and injected into the LC-MS system. The free fractions of Met and EDDP were determined, using 500 microl of plasma, which were placed in an ultrafiltration device and centrifuged at 2000 x g until 250 microl of filtrate was collected. The filtrate was extracted as described above and analyzed. Enantioselective separations were achieved using an alpha1-acid glycoprotein chiral stationary phase, a mobile phase composed of acetonitrile-ammonium acetate buffer [10 mM, pH 7.0] (18:82, v/v), a flow rate of 0.9 ml/min at 25 degrees C. Under these conditions, enantioselective separations were observed for Met (alpha = 1.30) and EDDP (alpha = 1.17) within 15 min. Met, EDDP, [2H3]-Met and [2H3]-EDDP were detected using selected ion monitoring at m/z 310.30, 278.20, 313.30, and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for Met in the range 1.0-300.0 ng/ml, and for EDDP from 0.1 to 25.0 ng/ml with correlation coefficients greater than 0.999, where the lower limit of quantification (LLOQ) was 1 ng/ml for Met and 0.1 ng/ml for EDDP. The relative standard deviation (R.S.D.) expressed as R.S.D. for the intra- and inter-day precision of the method were < 5.3% and the R.S.D. for accuracy was < 5.0%. The method was used to analyze plasma samples obtained from patients enrolled in a Met-maintenance program.
authors.affiliation: Bioanalytical and Drug Discovery Unit, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
authors.forename: Maria Esther
authors.initials: ME
authors.lastname: Rodriguez-Rosas
authors.forename: Juan G
authors.initials: JG
authors.lastname: Medrano
authors.forename: David H
authors.initials: DH
authors.lastname: Epstein
authors.forename: Eric T
authors.initials: ET
authors.lastname: Moolchan
authors.forename: Kenzie L
authors.initials: KL
authors.lastname: Preston
authors.forename: Irving W
authors.initials: IW
authors.lastname: Wainer
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008691:Methadone / Q000097:blood*; D011759:Pyrrolidines / Q000097:blood*; D012015:Reference Standards; D013237:Stereoisomerism
chemical_list: D011759:Pyrrolidines; D008691:Methadone; C010724:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine