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 t_1/2 of (R)-methadone was significantly longer than for (S)-methadone, and (S)-methadone was more tightly protein bound. The C _max, AUC, C_min, and % protein bound of (S)-EDDP were significantly greater than (R)-EDDP, while the t_1/2 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.