Stereoselective determination of methadone and the primary metabolite EDDP in human plasma by automated on-line extraction and liquid chromatography mass spectrometry.
A sensitive stereoselective bioanalytical liquid chromatographic assay with mass spectrometric detection (LC-MS) was developed and validated for the on-line extraction and quantification of R- and S-methadone and the primary metabolite R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) from human plasma. Deproteinized plasma was injected directly onto a small C8 column, washed and then back-flushed using a column switching valve and a second pump onto an alpha1-acid glycoprotein analytical column, and enantioselective separation achieved using a mobile phase gradient of methanol and ammonium formate. Analytes were validated over a range of 0.1-25 ng/ml R- and S-EDDP and 0.1-100ng/ml R- and S-methadone, respectively. Unweighted standard curves were linear over this concentration range (regression coefficients > 0.999). Quality control samples were evaluated at 1, 5, 12.5 ng/ml R- and S-EDDP and 1, 10, 50 ng/ml R- and S-methadone. Intra- and inter-day accuracy was >95%, and intra- and inter-day coefficients of variation were less than 10% for all analytes and concentrations. This assay represents the only method currently available which combines on-line extraction and achieves chiral separation of both methadone and EDDP from plasma, and offers improvements in sensitivity over existing methods.
Department of Anesthesiology, University of Washington, Box 356540, 1959 NE Pacific St. RR-442, Seattle, WA, USA.
Dale
D
Whittington
Pam
P
Sheffels
Evan D
ED
Kharasch
D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008691:Methadone / Q000097:blood*; D011759:Pyrrolidines / Q000097:blood*; D012680:Sensitivity and Specificity
D011759:Pyrrolidines; D008691:Methadone; C010724:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine