Abstract
AbstractIn 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.
Highlights
In recent years, many designer drugs have appeared on the recreational drug market, where most of them are sold as “legal highs” by street suppliers or via the Internet [1]
Mephedrone was incubated with an in-house prepared pig liver microsomes under the specified conditions and after termination of the metabolic process samples were centrifuged, filtered, and injected onto the liquid chromatography-mass spectrometry (LC-MS) system
gas chromatography-mass spectrometry (GC-MS) was chosen as the analytical technique as it is more common in forensic laboratories, and we aimed to demonstrate the potential versatility of this technique for the identification of novel metabolites in human cell lines
Summary
Many designer drugs have appeared on the recreational drug market, where most of them are sold as “legal highs” by street suppliers or via the Internet [1]. “Designer drugs” are those produced by performing minor alterations to one or more functional groups of a known chemical with specific pharmacological activity to avoid the legal regulations and to produce more effective substances [2]. The therapeutic and toxicological profiles of the designer drugs are not systemically studied by pharmaceutical companies and regulatory authorities as in the case of pharmaceutical drugs. The analysis of designer drugs and their metabolites has recently received increasing interest from academic and governmental researchers [3]. Metabolic studies with regard to the toxicological profiles of designer drugs are one of the most important research areas. The limited opportunities to obtain reference standards for the metabolites of novel psychoactive substances (NPSs) make in vitro production of these metabolites a valuable tool to develop analytical methods for the detection of NPS and their metabolites. As Tice et al suggested [4] the following reasons to observe caution: (i) problems to include xenobiotic metabolism into in vitro assays, (ii) problems to follow interactions between different cell types, (iii) difficulties in extrapolating the results from in vivo doses to in vitro concentrations, and (iv) problems to track long-term exposures in vitro
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
