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

Maria Esther Rodriguez-Rosas, Juan G. Medrano, David H. Epstein, Eric T. Moolchan, Kenzie L. Preston, Irving W. Wainer

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)237-248
Number of pages12
JournalJournal of Chromatography A
Volume1073
Issue number1-2
DOIs
StatePublished - May 6 2005

Keywords

  • (R)-Methadone
  • (S)-Methadone
  • AGP-chiral stationary phase
  • Chiral chromatography

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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