Quantitative determination of R/S‐methadone in human serum using ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry: A method for routine use

Methadone has two enantiomers, which exhibit differences in pharmacological effects, with R‐methadone being the active and S‐methadone the inactive enantiomer. A robust, simple and rapid method for chiral separation of the two enantiomers in serum samples using ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MSMS) has been developed and validated. Enantiomeric separation was achieved using a Chiralpak IH‐3 column with a mobile phase consisting of CO 2 and 30mM ammonium acetate in methanol/water (98/2, v/v). Runtime was 4 minutes. Sample preparation was semi‐automated using a Hamilton ML Star robot with protein precipitation, and phospholipid removal was carried out using a Waters OSTRO™ 96‐well plate. The calibration range was 50.0–1,500 nM for each enantiomer. The between‐assay relative standard deviations were in the range of 1.2–3.6%. 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‐methadone in authentic patient samples.