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 for norfentanyl. The analytical range was 0.002–2 ng/mL for fentanyl, 0.005–5 ng/mL for alpha-methylfentanyl, 0.01–10 ng/mL for beta-hydroxyfentanyl and 0.04–40 ng/mL for norfentanyl. All analytes demonstrated good linearity (R2 > 0.99). The extraction recoveries were in the 67.8%–92.1% range, and the IS-normalized matrix effects were between 55.5% and 74.0% (coefficient of variance < 15%). Our data indicated that norfentanyl has a higher concentration in rat urine and was detectable for at least 3 days after exposure to these compounds. This developed method may be useful in various fields, including forensic analysis, workplace drug testing and monitoring drug abuse.

中文翻译：

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LC-MS-MS法测定大鼠尿液中的芬太尼、α-甲基芬太尼、β-羟基芬太尼和代谢物去甲芬太尼

芬太尼及其类似物是强效合成阿片类药物，具有很高的滥用和依赖潜力。它们已成为阿片类药物死亡的主要贡献者。本研究旨在确定尿液中排泄的芬太尼、α-甲基芬太尼和β-羟基芬太尼的代谢物是否可以证明药物暴露史。芬太尼主要通过 CYP3A4 代谢为去甲芬太尼，尽管对其代谢为 α-甲基芬太尼和 β-羟基芬太尼的研究很少。我们使用超高效液相色谱与质谱联用对人肝微粒体 (HLM) 和大鼠肝微粒体 (RLM) 进行了体外实验，以阐明 α-甲基芬太尼和 β-羟基芬太尼的主要代谢途径。结果表明，在 HLM 和 RLM 中，α-甲基芬太尼和 β-羟基芬太尼主要代谢为去甲芬太尼。在向 Sprague-Dawley 大鼠静脉注射 α-甲基芬太尼和 β-羟基芬太尼 (20 μg/kg) 后，在 0 小时至 72 小时的不同时间间隔收集尿液样本。我们通过液-液萃取制备样品，内标（IS）为卡利拉嗪。开发并验证了一种灵敏、快速的液相色谱-串联质谱法，用于测定尿液中的四种分析物。尿液中的定性下限为芬太尼 2 pg/mL、α-甲基芬太尼 5 pg/mL、β-羟基芬太尼 10 pg/mL 和去甲芬太尼 40 pg/mL。芬太尼的分析范围为 0.002–2 ng/mL，α-甲基芬太尼的分析范围为 0.005–5 ng/mL，0。β-羟基芬太尼为 01–10 ng/mL，去甲芬太尼为 0.04–40 ng/mL。所有分析物都表现出良好的线性（R2＞0.99）。萃取回收率在 67.8%–92.1% 范围内，IS 归一化基质效应在 55.5% 和 74.0% 之间（方差系数 < 15%）。我们的数据表明，去甲芬太尼在大鼠尿液中的浓度较高，并且在接触这些化合物后至少 3 天可以检测到。这种开发的方法可能在各个领域都有用，包括法医分析、工作场所药物测试和监测药物滥用。我们的数据表明，去甲芬太尼在大鼠尿液中的浓度较高，并且在接触这些化合物后至少 3 天可以检测到。这种开发的方法可能在各个领域都有用，包括法医分析、工作场所药物测试和监测药物滥用。我们的数据表明，去甲芬太尼在大鼠尿液中的浓度较高，并且在接触这些化合物后至少 3 天可以检测到。这种开发的方法可能在各个领域都有用，包括法医分析、工作场所药物测试和监测药物滥用。