Method development and validation for quantitative determination of methadone enantiomers in human plasma by liquid chromatography/tandem mass spectrometry.
A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.
Liang
H R
HR
Tandem Labs, A Division of NWT Inc, 1121 East 3900 South, Salt Lake City, UT 84124, USA. hairui@tandemlabs.com
Foltz
R L
RL
Meng
M
M
Bennett
P
P
D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008691:Methadone / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013237:Stereoisomerism
D008691:Methadone