Chiral on-line solid phase extraction coupled to liquid chromatography–tandem mass spectrometry assay for quantification of (R) and (S) enantiomers of methadone and its main metabolite in plasma

•An enantiomeric separation of methadone is purposed.•A chiral column was used to separate (R), (S) methadone and EDDP.•An online SPE – LC-MS/MS system was used.•We report a complete method validation in accordance with the FDA and EMA guidelines.

The authors aimed at developing a liquid chromatography tandem mass spectrometry (LC-MS/MS) method with online extraction to determine (R)- and (S)- methadone enantiomers and its main metabolite 2-ethylidine-1,5-dimethyl-3,3 diphenylpyrrolidine (EDDP) in plasma.The analysis combined straightforward sample preparation, consisting of protein precipitation with acetonitrile, and an online enrichment by a flush/back-flush cycle before the second dimension chromatography.Using D3-deuterated internal standards allows overcoming significant relative matrix effect. Our method was linear up to 2000 ng/mL. This simple sample preparation provides sensitive (the limit of quantitation is 25 ng/mL for (R,S)-methadone and EDDP and 12.5 ng/mL for (R)- and (S)- methadone), accurate and precise (the intra-day and inter-day imprecision and inaccuracy are lower than 15%) quantification of the plasma concentration of these drugs.We have developed a reliable LC-MS/MS method for both routine therapeutic drug monitoring and pharmacokinetics studies and for toxicology analysis in the setting of methadone treatment or intoxication

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