Determination of free and protein-bound methadone and its major metabolite EDDP: Enantiomeric separation and quantitation by LC/MS/MS

Objective: To measure free and protein-bound R- and S-enantiomers of methadone and its major metabolite, 2-ethylidine-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP) in serum. Methods: To determine free fraction, samples were filtered using ultrafiltration membranes with a molecular weight cut-off 10,000 Da and extracted using liquid-liquid extraction. The solvent extract was evaporated and reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation was by chiral chromatography. Results: LC conditions resulted in baseline separation of R- and S-EDDP, and 85% resolution of methadone enantiomers. Precision of spiked specimens for both R- and S-methadone and R- and S-EDDP was less than 10% at 100 nM, and did not exceed 20% at 10 nM. Conclusions: Using minimal sample clean-Up and a total instrument run-time of 10 min, a rapid, sensitive and highly specific method was developed for quantitation of free and total R- and S-enantiomers of methadone and EDDP. (C) 2005 The Canadian Society of Clinical Chemists. All rights reserved.