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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

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 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.

Authors+Show Affiliations

College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China.State Key Laboratory of New Drug and Pharmaceutical process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China.State Key Laboratory of New Drug and Pharmaceutical process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China.State Key Laboratory of New Drug and Pharmaceutical process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China.State Key Laboratory of New Drug and Pharmaceutical process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China.State Key Laboratory of New Drug and Pharmaceutical process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China.State Key Laboratory of New Drug and Pharmaceutical process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China.State Key Laboratory of New Drug and Pharmaceutical process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China.Shanghai Key Laboratory of Crime Scene Evidence, Shanghai 200437, China.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

33647104

Citation

Li, Lu, et al. "Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl, and the Metabolite Norfentanyl in Rat Urine By LC-MS/MS." Journal of Analytical Toxicology, 2021.
Li L, Yu X, Lyu L, et al. Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl, and the Metabolite Norfentanyl in Rat Urine by LC-MS/MS. J Anal Toxicol. 2021.
Li, L., Yu, X., Lyu, L., Duan, H., Chen, Y., Bian, J., Xu, Z., Liu, L., & Zhang, Y. (2021). Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl, and the Metabolite Norfentanyl in Rat Urine by LC-MS/MS. Journal of Analytical Toxicology. https://doi.org/10.1093/jat/bkab021
Li L, et al. Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl, and the Metabolite Norfentanyl in Rat Urine By LC-MS/MS. J Anal Toxicol. 2021 Feb 27; PubMed PMID: 33647104.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl, and the Metabolite Norfentanyl in Rat Urine by LC-MS/MS. AU - Li,Lu, AU - Yu,Xuejiao, AU - Lyu,Lihong, AU - Duan,Hongbin, AU - Chen,Yao, AU - Bian,Jing, AU - Xu,Zhiru, AU - Liu,Li, AU - Zhang,Yurong, Y1 - 2021/02/27/ PY - 2020/10/26/received PY - 2021/02/24/revised PY - 2021/02/27/accepted PY - 2021/3/1/entrez PY - 2021/3/2/pubmed PY - 2021/3/2/medline KW - LC-MS/MS KW - alpha-methylfentanyl KW - beta-hydroxyfentanyl KW - fentanyl KW - norfentanyl KW - urine JF - Journal of analytical toxicology JO - J Anal Toxicol N2 - 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. SN - 1945-2403 UR - https://www.unboundmedicine.com/medline/citation/33647104/Determination_of_Fentanyl_Alpha_Methylfentanyl_Beta_Hydroxyfentanyl_and_the_Metabolite_Norfentanyl_in_Rat_Urine_by_LC_MS/MS_ L2 - https://academic.oup.com/jat/article-lookup/doi/10.1093/jat/bkab021 DB - PRIME DP - Unbound Medicine ER -
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