An enantiomer-selective liquid chromatography-tandem mass spectrometry method for methadone and EDDP validated for use in human plasma, urine, and liver microsomes

A liquid chromatography-electrospray ionization-tandem mass spectrometry method has been developed and validated to detect (R)- and (S)-methadone and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human plasma with cross-validation to urine and liver microsomes. Use of deuterated internal standards and liquid-liquid extraction coupled with chiral separation provided baseline separation with a lower limit of quantitation (LLOQ) of 2.5 ng/mL. The LLOQ was established from comparison of signal in blanks from six different sources per matrix with the same sources fortified at the LLOQ (none exceeded 19% of LLOQ) and precision and accuracy at the LLOQ determined in the same six sources per matrix. The assay was precise (% coefficients of variation within 13.8%) and accurate (% targets within 15%) in all three matrices. No interference was seen from addition of other psychoactive drugs. Stability was determined in plasma (24 h at room temperature, 321 days at -20°C, 3 freeze-thaw cycles); processed plasma samples (5 days at -20°C, 12 days on autosampler); urine (24 h at room temperature); and stock solutions (20 h at room temperature, 61 days at -20°C). Applications of varying degree are presented for each matrix. Plasma from five subjects maintained on 100 mg oral methadone per day permitted comparison of the pharmacokinetics of the enantiomers. The t1/2 of (R)-methadone was significantly longer than for (S)-methadone, and (S)-methadone was more tightly protein bound. The C max, AUC, Cmin, and % protein bound of (S)-EDDP were significantly greater than (R)-EDDP, while the t1/2 of (R)-EDDP was significantly greater than (S)-EDDP. In spot urines, (R)- was higher than (S)-methadone, and (S)- was generally higher than (R)-EDDP. (R)- and (S)-EDDP production was detected after incubation of therapeutic concentrations of racemic methadone with human liver microsomes, and (S)-EDDP production was twofold greater than (R)-EDDP in three human placental microsomes incubated with racemic methadone.