Validation of a LC–APCI-MS/MS method for quantification of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) in infant plasma following protein precipitation

A validated, quantitative LC–APCI-MS/MS method for methadone, EDDP and EMDP in 200-μL plasma is presented. Specimen preparation was limited to protein precipitation and centrifugation. Chromatographic separation was achieved on a Synergi Hydro-RP 80A (50 mm × 2.0 mm, 4 μm) column with gradient elution. The assay was linear from 1 to 500 ng/mL, with intra- and inter-assay accuracy ≥87.5% and intra- and inter-assay precision <13.4% R.S.D. and recovery ≥87.5% for all analytes at 40 ng/mL. This analytical method is suitable for the accurate and precise determination of methadone and metabolites in human plasma specimens.