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.
Etter
M L
ML
Saskatchewan Health Provincial Laboratory, Regina, SK, Canada.
George
S
S
Graybiel
K
K
Eichhorst
J
J
Lehotay
D C
DC
D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods; D008691:Methadone / Q000097:blood*; D015394:Molecular Structure; D011485:Protein Binding; D011759:Pyrrolidines / Q000097:blood*; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D011759:Pyrrolidines; D008691:Methadone; C010724:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine