Simultaneous enantioselective quantification of methadone and of 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine in oral fluid using capillary electrophoresis

A capillary electrophoresis method was developed for the enantioselective quantification of methadone (MTD) and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP). The enantiomers of MTD and EDDP were resolved by CE in 5min using 0.2% highly sulphated gamma-cyclodextrins as chiral selectors and a 50mM phosphate solution at pH 4.5 as background electrolyte. The optimized method was applied and validated for oral fluid testing. Linear relationships were obtained for MTD enantiomers in the range of 8.1-625ng/mL and in the range of 7.6-500ng/mL for EDDP enantiomers. The detection limits ranged from 2.3 to 2.4ng/mL, whereas the limits of quantification ranged from 7.6 to 8.1ng/mL. Intra- and inter-assay precision and accuracy were acceptable, respectively. The method was applied to the analyses of 60 oral fluid specimens obtained from patients enrolled in a MTD maintenance programme. Our data pointed out that higher concentrations of (R)-MTD and the enantioselective excess of (S)-EDDP in OF may reflect the free fraction of MTD and EDDP enantiomers in plasma.