Unit of Forensic Toxicology, Section of Legal Medicine, Department of Biomedical Sciences and Public Health, Marche Polytechnic University, Via Tronto 10/a, Ancona AN 60126, Italy.
Health Service Department, Italian State Police, Piazza Sant'Ambrogio 5, Milan MI 20123, Italy.
J Anal Toxicol. 2023 Jan 24;46(9):e207-e217. doi: 10.1093/jat/bkac065.
From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. β'-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on β'-phenylfentanyl effects and toxicokinetics and its metabolism is yet to be studied. We aimed to investigate β'-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in β'-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. β'-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS-MS) and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation and combinations thereof. We suggest β'-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of β'-phenylfentanyl use. In silico predictions were mostly wrong, and β'-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of new psychoactive substance human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify β'-phenylfentanyl-positive cases to provide authentic samples.
自 2014 年以来，非法芬太尼及其类似物在全球范围内导致了众多中毒和死亡事件，影响了阿片类药物相关过量的人口统计学特征。在临床和法医环境中，鉴定涉及芬太尼类似物的病例对于治疗患者、阐明中毒、解决药物使用障碍和应对药物趋势至关重要。然而，在分析毒理学中，生物基质中芬太尼类似物的浓度较低，因此检测具有挑战性。因此，通常需要鉴定特定的代谢物生物标志物来记录消费情况。β'-苯丙芬太尼（N-苯丙基-N-[1-（2-苯乙基）-4-哌啶基]-苯丙酰胺）是一种芬太尼类似物，于 2017 年在瑞典首次检测到，并于最近重新出现在美国非法毒品市场上。关于β'-苯丙芬太尼的影响和毒代动力学的数据很少，其代谢尚未得到研究。我们旨在研究β'-苯丙芬太尼的人体代谢，以确定潜在的使用生物标志物。为了协助β'-苯丙芬太尼代谢物的鉴定，使用 GLORYx 免费软件通过计算机预测生成了一份可能的反应清单。将β'-苯丙芬太尼与冷冻保存的 10 位供体 pooled 人肝细胞孵育，通过液相色谱-高分辨率串联质谱（LC-HRMS-MS）进行分析，并使用 Compound Discoverer 采用部分自动化的靶向/非靶向方法进行数据处理。我们鉴定出 26 种由 N-去烷基化、氧化、羟化、O-葡萄糖醛酸化、O-甲基化及其组合产生的代谢物。我们建议β'-苯丙基诺芬太尼（N-苯丙基-N-4-哌啶基-苯丙酰胺）和进一步的代谢物 1-氧代-N-苯丙基-N-4-哌啶基-苯丙酰胺和 1-羟基-N-苯丙基-N-4-哌啶基-苯丙酰胺作为β'-苯丙芬太尼使用的主要生物标志物。计算机预测大多是错误的，并且β'-苯丙芬太尼的代谢命运与在相同条件下孵育的密切相关类似物的代谢命运有很大不同，这突出了对新精神活性物质人体代谢进行实验评估的价值。体内数据是必要的，以确认目前的结果。然而，目前的结果可能是必要的，以帮助分析毒理学家鉴定β'-苯丙芬太尼阳性病例，以提供真实样本。
