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 2 S ,6 S -hydroxynorketamine (2 S ,6 S -HNK) and 2 R ,6 R -hydroxynorketamine (2 R ,6 R -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; 5 mM) 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; 10 mM) 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; 5 mM) 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 0.1 ng/ml). Moreover, our assay enables for the first time the enantioselective determination of 2 R ,6 R - and 2 S ,6 S -HNK which were shown to be responsible for the promising antidepressant effects of ketamine.