Enantioselective quantification of metha- done and its major metabolite (EDDP) in oral fluid by capillary electrophoresis

Methadone is a synthetic analgesic widely used in methadone maintenance programmes. Methadone (MTD) is generally administered orally as a racemate, however the therapeutic effect is almost exclusively mediated by the (R)-enantiomer since its affinity for the μ-opioid receptor and its analgesic effect is up to 8 and 50 times, respectively, greater than for the (S)-enantiomer. Because of important inter-individual variability and in order to prevent withdrawal symptoms, an individual dose adjustment is often required. To the best of our knowledge, only two analytical procedures using both liquid chromatography-mass spectrometry have been reported for the enantioselective quantification of MTD and/or its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP) in oral fluid. The purpose of the study was to develop a more rapid and reliable assay for the simultaneous quantification of MTD and EDDP in oral fluid using capillary electrophoresis (CE). Oral fluid specimens were collected before the daily MTD administration using Salivette devices (Sarstedt) and then centrifuged. After the addition of the internal standard, (R)-phenylethylamine, the mixture was alkalinized with 0.2 mL of an ammonia solution (25%), and extracted with 3 mL of cyclohexane. After centrifugation, the upper organic layer was evaporated to dryness. Baseline separations of MTD and EDDP enantiomers were obtained in 5 min using 0.2% highly sulphated γ-cyclodextrin as chiral selector and a 50 mM phosphate solution as background electrolyte. The extraction yields were between 77.4 and 92.9%, whereas the limits of detection ranged from 2.3 to 2.4 ng/mL. Intraand inter-assay precision respectively accuracy were acceptable. The method was used for the analysis of oral fluid specimens obtained from patients enrolled in a MTD maintenance programme. Results showed MTD R vs. S ratios > 1 and EDDP R/S ratios < 1, confirming previous studies and/or plasma results.