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A robust, simple and rapid method for chiral separation of the two enantiomers in serum samples using ultra\u2010high performance supercritical fluid chromatography\u2010tandem mass spectrometry (UHPSFC\u2010MSMS) has been developed and validated. Enantiomeric separation was achieved using a Chiralpak IH\u20103 column with a mobile phase consisting of CO<jats:sub>2<\/jats:sub> and 30mM ammonium acetate in methanol\/water (98\/2, v\/v). Runtime was 4\u00a0minutes. Sample preparation was semi\u2010automated using a Hamilton ML Star robot with protein precipitation, and phospholipid removal was carried out using a Waters OSTRO\u2122 96\u2010well plate. The calibration range was 50.0\u20131,500\u2009nM for each enantiomer. The between\u2010assay relative standard deviations were in the range of 1.2\u20133.6%. Matrix effects ranged from 99% to 115% corrected with internal standard. The method has been implemented in our laboratory and has proven to be a robust and reliable method for determining the ratio of R\/S\u2010methadone in authentic patient samples.<\/jats:p>","DOI":"10.1002\/dta.3693","type":"journal-article","created":{"date-parts":[[2024,4,11]],"date-time":"2024-04-11T01:54:00Z","timestamp":1712800440000},"page":"708-716","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Quantitative determination of R\/S\u2010methadone in human serum using ultra\u2010high performance supercritical fluid chromatography\u2010tandem mass spectrometry: A method for routine use"],"prefix":"10.1002","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5009-3802","authenticated-orcid":false,"given":"Miriam","family":"Hansen","sequence":"first","affiliation":[{"name":"Department of Clinical Pharmacology St. Olav University Hospital  Trondheim 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