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

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 μl of plasma, which were placed in an ultrafiltration device and centrifuged at 2000 × g until 250 μl of filtrate was collected. The filtrate was extracted as described above and analyzed. Enantioselective separations were achieved using an α1-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 °C. Under these conditions, enantioselective separations were observed for Met (α = 1.30) and EDDP (α = 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.