Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes (HHeps) over three hours, to identify unique and abundant metabolites using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation, and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), whilst the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.

中文翻译：

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使用人肝细胞、LC-QTOF-MS 和合成参考标准品分析合成大麻素受体激动剂 ADB-HEXINACA 的代谢特征

合成大麻素受体激动剂（SCRA）仍然是一个主要的公共卫生问题，其使用与世界范围内的中毒和药物相关死亡有关。增加我们对 SCRA 代谢的系统了解支持临床和法医毒理学案例工作，促进及时识别毒理学筛选程序和验证分析的分析目标。随着非法药物市场上不断出现新的 SCRA，这一点尤为重要。在这项工作中，ADB-HEXINACA（ADB-HINACA，N-[1-氨基-3,3-二甲基-1-氧代丁-2-基]-1-己基-1H-吲唑-3-甲酰胺）的代谢，使用体外技术对这种疾病在英国和其他司法管辖区的患病率有所增加进行了调查。ADB-HEXINACA 的 (S)-对映体与混合的人肝细胞 (HHeps) 一起孵育三个小时，使用液相色谱-四极杆飞行时间质谱 (LC-QTOF-MS) 鉴定独特且丰富的代谢物。总共鉴定了 16 种代谢物，它们来自单羟基化、二羟基化、酮形成（单羟基化然后脱氢）、羧酸形成、末端酰胺水解、二氢二醇形成、葡萄糖醛酸化及其组合。大部分代谢发生在己基尾部，形成酮和单羟基化产物。主要代谢物是 5-氧代-己基产物 (M9)，而最重要的单羟基化产物是 4-羟基-己基产物 (M8)，这两种产物均通过与内部合成参考标准品的比较得到证实。5-羟基己基 (M6) 和 6-羟基己基 (M7) 代谢物未通过色谱分离，5-羟基己基产物是第二大单羟基化代谢物。通过与合成参考标准品以及 4-氧代己基代谢物 (M11) 的比较，还确认了不含（M16，第三大代谢物）和含 5 位酮（M13）的末端酰胺水解产物的结构。建议将 5-氧代-己基和 4-羟基-己基代谢物作为 ADB-HEXINACA 消耗的生物标志物。以及 4-氧代己基代谢物 (M11)。建议将 5-氧代-己基和 4-羟基-己基代谢物作为 ADB-HEXINACA 消耗的生物标志物。以及 4-氧代己基代谢物 (M11)。建议将 5-氧代-己基和 4-羟基-己基代谢物作为 ADB-HEXINACA 消耗的生物标志物。