Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5138110 | Journal of Pharmaceutical and Biomedical Analysis | 2017 | 35 Pages |
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; 5Â 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; 10Â 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; 5Â 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 0.1Â ng/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.
Keywords
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Mahmoud Hasan, Robert Hofstetter, Georg M. Fassauer, Andreas Link, Werner Siegmund, Stefan Oswald,