Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl and the Metabolite Norfentanyl in Rat Urine by LC–MS-MS

作者：Lu Li, Xuejiao Yu, Lihong Lyu, Hongbin Duan, Yao Chen, Jing Bian, Zhiru Xu, Li Liu, Yurong Zhang · 发表于：Journal of Analytical Toxicology · 年份：2021 · DOI：10.1093/jat/bkab021 · 被引用次数：13 · 研究领域：Forensic Toxicology and Drug Analysis、Opioid Use Disorder Treatment、Pain Management and Opioid Use

Fentanyl and its analogs are potent synthetic opioids with a high potential for abuse and dependence. They have become major contributors to opioid deaths. This study aimed to determine whether the metabolites of fentanyl, alpha-methylfentanyl and beta-hydroxyfentanyl, excreted in the urine, can demonstrate historical drug exposure. Fentanyl is primarily metabolized via CYP3A4 into norfentanyl, although there is little research on its metabolization into alpha-methylfentanyl and beta-hydroxyfentanyl. We conducted in vitro experiments with human liver microsomes (HLMs) and rat liver microsomes (RLMs) to elucidate the major metabolic pathways of alpha-methylfentanyl and beta-hydroxyfentanyl using ultra-high-performance liquid chromatography coupled with mass spectrometry. The results showed that both alpha-methylfentanyl and beta-hydroxyfentanyl were predominantly metabolized into norfentanyl in HLM and RLM. Urine samples were collected at different intervals from 0 h to 72 h after intravenous administration of alpha-methylfentanyl and beta-hydroxyfentanyl (20 μg/kg) to Sprague-Dawley rats. We prepared the samples by liquid-liquid extraction, and the internal standard (IS) was cariprazine. A sensitive, rapid liquid chromatography-tandem mass spectrometry method was developed and validated to determine four analytes in the urine. The lower limit of qualification in urine was 2 pg/mL for fentanyl, 5 pg/mL for alpha-methylfentanyl, 10 pg/mL for beta-hydroxyfentanyl and 40 pg/mL fo...