Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl, and the Metabolite Norfentanyl in Rat Urine by LC-MS/MS.J Anal Toxicol. 2021 Feb 27 [Online ahead of print]JA
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 metabolism into alpha-methylfentanyl and beta-hydroxyfentanyl. We conducted in vitro experiments with human liver microsomes (HLM) and rat liver microsomes (RLM) to elucidate the major metabolic pathways of alpha-methylfentanyl and beta-hydroxyfentanyl using UHPLC 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 LC-MS/MS method was developed and validated to determine four analytes in the urine. The lower limit of qualification (LLOQ) 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-hydroxyfentany 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%-74.0% (CV < 15%). Our data indicated that norfentanyl has a higher concentration in rat urine and was detectable for at least three 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.