Fentanyl analogs constitute a particularly dangerous group of new psychoactive compounds responsible for many deaths around the world. Little is known about their metabolism, and studies utilizing liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) analysis of hepatocyte incubations and/or authentic urine samples do not allow for determination of the exact metabolite structures, especially when it comes to hydroxylated metabolites. In this study, seven motifs (2-, 3-, 4- and β-OH as well as 3,4-diOH, 4-OH-3-OMe and 3-OH-4-OMe) of fentanyl and five fentanyl analogs, acetylfentanyl, acrylfentanyl, cyclopropylfentanyl, isobutyrylfentanyl and 4F-isobutyrylfentanyl were synthesized. The reference standards were analyzed by LC–QTOF-MS, which enabled identification of the major metabolites formed in hepatocyte incubations of the studied fentanyls. By comparison with our previous data sets, major urinary metabolites could tentatively be identified. For all analogs, β-OH, 4-OH and 4-OH-3-OMe were identified after hepatocyte incubation. β-OH was the major hydroxylated metabolite for all studied fentanyls, except for acetylfentanyl where 4-OH was more abundant. However, the ratio 4-OH/β-OH was higher in urine samples than in hepatocyte incubations for all studied fentanyls. Also, 3-OH-4-OMe was not detected in any hepatocyte samples, indicating a clear preference for the 4-OH-3-OMe, which was also found to be more abundant in urine compared to hepatocytes. The patterns appear to be consistent across all studied fentanyls and could serve as a starting point in the development of methods and synthesis of reference standards of novel fentanyl analogs where nothing is known about the metabolism.

中文翻译：

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使用LC-QTOF-MS，肝细胞孵育和合成参考标准品阐明芬太尼和5种芬太尼类似物的尿代谢产物的结构。

芬太尼类似物构成一组新型危险的新型精神活性化合物，这些化合物在世界范围内造成许多死亡。对它们的代谢了解甚少，利用液相色谱-四极杆飞行时间质谱（LC-QTOF-MS）分析肝细胞孵育和/或真实尿液样本的研究无法确定确切的代谢物结构，尤其是当涉及羟基化代谢物时。在这项研究中，芬太尼和五个芬太尼类似物的七个基序（2-，3-，4-和β-OH以及3,4-diOH，4-OH-3-OMe和3-OH-4-OMe）合成了乙酰芬太尼，丙烯芬太尼，环丙基芬太尼，异丁酰基芬太尼和4F-异丁酰基芬太尼。参考标准品通过LC–QTOF-MS分析，它可以鉴定在研究的芬太尼肝细胞培养中形成的主要代谢产物。通过与我们以前的数据集进行比较，可以初步确定主要的尿代谢物。对于所有类似物，在肝细胞孵育后都鉴定出β-OH，4-OH和4-OH-3-OMe。在所有研究的芬太尼中，β-OH是主要的羟基化代谢产物，除了乙酰基芬太尼（4-OH含量更高）以外。然而，对于所有研究的芬太尼，尿液样本中4-OH /β-OH的比率高于肝细胞培养中的比率。同样，在任何肝细胞样本中均未检测到3-OH-4-OMe，这表明对4-OH-3-OMe的偏好明显，后者在尿液中的含量也比肝细胞丰富。