title: Determination of free and protein-bound methadone and its major metabolite EDDP: enantiomeric separation and quantitation by LC/MS/MS.
abstract: 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.
authors.affiliation: Saskatchewan Health Provincial Laboratory, Regina, SK, Canada.
authors.forename: M L
authors.initials: ML
authors.lastname: Etter
authors.forename: S
authors.initials: S
authors.lastname: George
authors.forename: K
authors.initials: K
authors.lastname: Graybiel
authors.forename: J
authors.initials: J
authors.lastname: Eichhorst
authors.forename: D C
authors.initials: DC
authors.lastname: Lehotay
mesh_terms: 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
chemical_list: D011759:Pyrrolidines; D008691:Methadone; C010724:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine