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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