Abstract

In the current study, the metabolism of two novel psychoactive substances (NPS), mephedrone and methoxetamine (MXE), was studied in vitro using 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 be able to detect metabolites from the metabolic mixture in the human cell lines using a GC-MS, since this is a more readily available technique within forensic laboratories
Microsomes were prepared through a conventional ultracentrifugation method and incubated under optimised conditions with the drugs for 3 h. Subsequently, the samples were investigated using LC-MS. A similar methodology was then applied for the HepaRG™ Cell and GC-MS conditions were optimised by using N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) as a derivatisation agent.
The analysis showed two molecules from a successful in vitro metabolism, namely hydroxytoly-mephedrone and nor-dihydro mephedrone. For Methoxetamine (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 derivatisation 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 for mephedrone and MXE, respectively. These drugs were able to cause 73-84% cell death.