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

Abstract 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