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In Vitro and In Vivo Metabolite Identification Studies for the New Synthetic Opioids Acetylfentanyl, Acrylfentanyl, Furanylfentanyl, and 4-Fluoro-Isobutyrylfentanyl

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Abstract

New fentanyl analogs have recently emerged as new psychoactive substances and have caused numerous fatalities worldwide. To determine if the new analogs follow the same metabolic pathways elucidated for fentanyl and known fentanyl analogs, we performed in vitro and in vivo metabolite identification studies for acetylfentanyl, acrylfentanyl, 4-fluoro-isobutyrylfentanyl, and furanylfentanyl. All compounds were incubated at 10 μM with pooled human hepatocytes for up to 5 h. For each compound, four or five authentic human urine samples from autopsy cases with and without enzymatic hydrolysis were analyzed. Data acquisition was performed in data-dependent acquisition mode during liquid chromatography high-resolution mass spectrometry analyses. Data was analyzed (1) manually based on predicted biotransformations and (2) with MetaSense software using data-driven search algorithms. Acetylfentanyl, acrylfentanyl, and 4-fluoro-isobutyrylfentanyl were predominantly metabolized by N-dealkylation, cleaving off the phenethyl moiety, monohydroxylation at the ethyl linker and piperidine ring, as well as hydroxylation/methoxylation at the phenyl ring. In contrast, furanylfentanyl’s major metabolites were generated by amide hydrolysis and dihydrodiol formation, while the nor-metabolite was minor or not detected in case samples at all. In general, in vitro results matched the in vivo findings well, showing identical biotransformations in each system. Phase II conjugation was observed, particularly for acetylfentanyl. Based on our results, we suggest the following specific and abundant metabolites as analytical targets in urine: a hydroxymethoxy and monohydroxylated metabolite for acetylfentanyl, a monohydroxy and dihydroxy metabolite for acrylfentanyl, two monohydroxy metabolites and a hydroxymethoxy metabolite for 4-fluoro-isobutyrylfentanyl, and a dihydrodiol metabolite and the amide hydrolysis metabolite for furanylfentanyl.

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Acknowledgements

The authors thank Advanced Chemistry Development (ACD/Labs) for providing MetaSense™ software as well as Richard Lee and Edward Milton for their help. This research was conducted within the Strategic Research Area Forensic Sciences (Strategiområdet Forensiska Vetenskaper) at Linköping University.

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Authors and Affiliations

  1. Centre for Forensic Science, School of Mathematical and Physical Sciences, University of Technology Sydney (UTS), PO Box 123, Broadway, Sydney, NSW, 2007, Australia

    Shimpei Watanabe

  2. Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185, Linköping, Sweden

    Svante Vikingsson, Henrik Green, Robert Kronstrand & Ariane Wohlfarth

  3. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758, Linköping, Sweden

    Markus Roman, Henrik Green, Robert Kronstrand & Ariane Wohlfarth

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  1. Shimpei Watanabe
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  2. Svante Vikingsson
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Correspondence to Ariane Wohlfarth.

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Electronic Supplementary Material

Supplementary Fig. 1

MSMS spectra and proposed fragmentation pattern of acetylfentanyl and its major metabolites in hydrolyzed human urine samples. (PDF 63 kb)

Supplementary Fig. 2

MSMS spectra and proposed fragmentation pattern of acrylfentanyl and its major metabolites in hydrolyzed human urine samples. (PDF 108 kb)

Supplementary Fig. 3

MSMS spectra and proposed fragmentation pattern of 4-fluoroisobutyryllfentanyl and its major metabolites in hydrolyzed human urine samples. (PDF 111 kb)

Supplementary Fig. 4

MSMS spectra and proposed fragmentation pattern of furanylfentanyl and its major metabolites in hydrolyzed human urine samples. (PDF 94 kb)

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Watanabe, S., Vikingsson, S., Roman, M. et al. In Vitro and In Vivo Metabolite Identification Studies for the New Synthetic Opioids Acetylfentanyl, Acrylfentanyl, Furanylfentanyl, and 4-Fluoro-Isobutyrylfentanyl. AAPS J 19, 1102–1122 (2017). https://doi.org/10.1208/s12248-017-0070-z

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