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A study of in vitro metabolism and cytotoxicity of mephedrone and methoxetamine in human and pig liver models using GC/MS and LC/MS analyses

Alshamaileh, Majed; Hussain, Issam; Baron, Mark; Croxton, Ruth; Vetter, Marleen; Gonzalez-Rodriguez, Jose

Authors

Majed Alshamaileh

Issam Hussain

Mark Baron

Ruth Croxton

Marleen Vetter

Jose Gonzalez-Rodriguez



Abstract

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.

Citation

Alshamaileh, M., Hussain, I., Baron, M., Croxton, R., Vetter, M., & Gonzalez-Rodriguez, J. (2020). A study of in vitro metabolism and cytotoxicity of mephedrone and methoxetamine in human and pig liver models using GC/MS and LC/MS analyses. Open Chemistry, 18(1), 1507-1522. https://doi.org/10.1515/chem-2020-0184

Journal Article Type Article
Acceptance Date Nov 16, 2020
Online Publication Date Dec 23, 2020
Publication Date 2020-01
Deposit Date Jan 14, 2021
Publicly Available Date Mar 28, 2024
Journal Open Chemistry
Print ISSN 2391-5420
Publisher De Gruyter Open
Peer Reviewed Peer Reviewed
Volume 18
Issue 1
Pages 1507-1522
DOI https://doi.org/10.1515/chem-2020-0184
Keywords In vitro metabolism; Mephedrone; Methoxetamine; Cytotoxicity; Mass spectrometry; EC50
Public URL https://hull-repository.worktribe.com/output/3693025
Publisher URL https://www.degruyter.com/view/journals/chem/18/1/article-p1507.xml?language=en

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Copyright Statement
© 2020 Majed Alshamaileh et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. BY 4.0





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