title: Quantitative chiral and achiral determination of ketamine and its metabolites by LC-MS/MS in human serum, urine and fecal samples.
abstract: Ketamine (KET) is a widely used anesthetic drug which is metabolized by CYP450 enzymes to norketamine (n-KET), dehydronorketamine (DHNK), hydroxynorketamine (HNK) and hydroxyketamine (HK). Ketamine is a chiral compound and S-ketamine is known to be the more potent enantiomer. Here, we present the development and validation of three LC-MS/MS assays; the first for the quantification of racemic KET, n-KET and DHNK in human serum, urine and feces; the second for the separation and quantification of the S- and R-enantiomers of KET, n-KET and DHNK, and the third for separation and quantification of 2S,6S-hydroxynorketamine (2S,6S-HNK) and 2R,6R-hydroxynorketamine (2R,6R-HNK) in serum and urine with the ability to separate and detect 10 additional hydroxylated norketamine metabolites of racemic ketamine. Sample preparation was done by liquid-liquid extraction using methyl tert-butyl ether. For achiral determination of KET and its metabolites, an isocratic elution with ammonium acetate (pH 3.8; 5mM) and acetonitrile on a C18 column was performed. For the separation of S- and R-enantiomers of KET, n-KET and DHNK, a gradient elution was applied using a mobile phase of ammonium acetate (pH 7.5; 10mM) and isopropanol on the CHIRAL-AGP®column. The enantioselective separation of the HNK metabolites was done on the chiral column Lux®-Amylose-2 with a gradient method using ammonium acetate (pH 9; 5mM) and a mixture of isopropanol and acetonitrile (4:1). The mass spectrometric detection monitored for each analyte 2-3 mass/charge transitions. D4-ketamine and D4-n-KET were used as internal standards. The assays were successfully validated according to current bioanalytical guidelines and applied to a pilot study in one healthy volunteer. Compared to previously published methods, our assays have superior analytical features such as a lower amount of required matrix, faster sample preparation, shorter analytical run time and higher sensitivity (LLOQ up to 0.1ng/ml). Moreover, our assay enables for the first time the enantioselective determination of 2R,6R- and 2S,6S-HNK which were shown to be responsible for the promising antidepressant effects of ketamine.
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald,. Germany.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Hasan
authors.affiliation: Institute of Pharmacy, University of Greifswald, Greifswald, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Hofstetter
authors.affiliation: Institute of Pharmacy, University of Greifswald, Greifswald, Germany.
authors.forename: Georg M
authors.initials: GM
authors.lastname: Fassauer
authors.affiliation: Institute of Pharmacy, University of Greifswald, Greifswald, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Link
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald,. Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Siegmund
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald,. Germany. Electronic address: stefan.oswald@uni-greifswald.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Oswald
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods; D005243:Feces / Q000737:chemistry*; D006801:Humans; D007649:Ketamine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D010865:Pilot Projects; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007649:Ketamine; C033419:norketamine
keywords: Chiral; Dehydronorketamine; Hydroxynorketamine; Ketamine; LC–MS/MS; Norketamine