Free and bound enantiomers of methadone and its metabolite, EDDP in methadone maintenance treatment: relationship to dosage?

OBJECTIVES: To determine the effects of metabolism and protein binding on the relationship between administered dose, blood levels of R methadone and biological response by measuring the free and protein-bound forms of the R and S enantiomers of methadone and EDDP, its metabolite. DESIGN AND METHODS: To measure free and total drug, trough levels were collected from 45 methadone clients. To measure free methadone, samples were filtered using ultrafiltration with a MW weight cut-off of 10,000 and extracted using liquid-liquid extraction. The solvent was evaporated and samples reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation of methadone and EDDP was by chiral chromatography. RESULTS: The presence of unmetabolized methadone suggested that none of the patients were very fast metabolizers. R and S forms were metabolized at the same rate at all administered doses. Free R methadone levels correlated both with methadone dose and with the total amount of R methadone. The free fraction of R methadone (%free R) was higher at lower doses than at high doses, varied from 5 to 25% and was inversely proportional to the total dose of administered drug in a relationship that was logarithmic and non-linear. CONCLUSIONS: By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. The reason for the large range in dosage may be multifactorial.

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 of 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.