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.

中文翻译：

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使用 GC/MS 和 LC/MS 分析在人和猪肝脏模型中研究甲氧麻黄酮和甲氧塞胺的体外代谢和细胞毒性

摘要 在目前的研究中，两种新型精神活性物质 (NPS) 甲氧麻黄酮和甲氧塞胺 (MXE) 在猪肝微粒体中的体外代谢研究，以通过液相色谱-质谱法 (LC-MS) 测定潜在代谢物。后来，使用 HepaRG™ 细胞进行了体外研究，以使用气相色谱-质谱法 (GC-MS) 确定这些药物的人体代谢物。该研究的目的是使用 GC-MS 检测人体细胞系代谢混合物中的代谢物，因为这是法医实验室中更容易获得的技术。通过常规超速离心方法制备微粒体，并在优化条件下与药物一起孵育 3 小时。随后，使用LC-MS研究样品。然后将类似的方法应用于 HepaRG™ 细胞，并使用 N,O-双（三甲基甲硅烷基）三氟乙酰胺作为衍生剂优化 GC-MS 条件。分析表明，成功的体外代谢产生了两种分子，即羟基甲氧麻黄酮和去甲二氢甲氧麻黄酮。对于 MXE，分别通过 O-去甲基化和酮部分还原为相应的醇产生两种代谢物。使用人 HepaRG™ 细胞，GC-MS 只能鉴定去甲二氢甲氧麻黄酮。由于羟基甲氧麻黄酮和 MXE 代谢物的极性更大，因此建议即使衍生化也可能不适合 GC-MS。此外，还使用 ​​HepaRG™ 细胞系研究了细胞毒性。这些药物显示出导致体外细胞死亡的细胞毒性作用，在 EC50 0 的指定范围内。甲氧麻黄酮和 MXE 分别为 3211 mM (79 μg/mL) 和 0.6297 mM (111 μg/mL)。这些药物能够导致 73-84% 的细胞死亡。