| Popis: |
In the current study, the metabolism of two novel psychoactive substances (NPSs), mephedrone and methoxetamine (MXE), was studied in vitro in pig liver microsomes to determine potential metabolites by liquid chromatography-mass spectrometry (LC-MS). Later, in vitro studies were performed using HepaRG™ cells to determine the human metabolites of these drugs using gas chromatography-mass spectrometry (GC-MS). The aim of the study was to detect metabolites from the metabolic mixture in the human cell lines using GC-MS, since this is a more readily available technique within forensic laboratories. Microsomes were prepared through a conventional ultracentrifugation method and incubated under optimized conditions with the drugs for 3 h. Subsequently, the samples were investigated using LC-MS. A similar methodology was then applied in the HepaRG™ cells, and the GC-MS conditions were optimized using N,O-bis(trimethylsilyl)trifluoroacetamide as a derivatization agent. The analysis showed two molecules from a successful in vitro metabolism, namely, hydroxytoly-mephedrone and nor-dihydro mephedrone. For MXE, two metabolites are presented produced by the O-demethylation and reduction of the ketone moiety to the corresponding alcohol, respectively. Using the human HepaRG™ cells, only nor-dihydro mephedrone could be identified by GC-MS. Since hydroxytoly-mephedrone and the MXE metabolites are more polar, it is suggested that GC-MS even with derivatization may not be suitable. In addition, cytotoxicity was studied utilizing HepaRG™ cell lines. The drugs show cytotoxic effects causing in vitro cell death, within the specified range of EC50 0.3211 mM (79 μg/mL) and 0.6297 mM (111 μg/mL) for mephedrone and MXE, respectively. These drugs were able to cause 73–84% cell death. |
