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

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.

Correlations between metabolism and structural elements of the alicyclic fentanyl analogs cyclopropyl fentanyl, cyclobutyl fentanyl, cyclopentyl fentanyl, cyclohexyl fentanyl and 2,2,3,3-tetramethylcyclopropyl fentanyl studied by human hepatocytes and LC-QTOF-MS

“…Multi-donor human hepatocytes have proven to be a useful system to study the metabolism of fentanyls and identified metabolites correlate well with those found in authentic urine samples (Watanabe et al 2017). …”

Fentanyl, fentanyl analogs and novel synthetic opioids: A comprehensive review

“…Minor metabolites are also created through various other pathways. For example, despropionylfentanyl is formed by carboxamide hydrolysis, while both hydroxyfentanyl and hydroxynorfentanyl metabolites are hydroxylated at the propionyl moiety (Watanabe et al, 2017). All metabolites are inactive, and only a small amount of fentanyl (8-10%) is renally and fecally cleared (McClain & Hug, 1980;Mather, 1983;Poklis, 1995).…”

“…Like fentanyl and other fentanyl analogs, acetylfentanyl, acrylfentanyl, and 4-fluoro-isobutylfentanyl each have a major inactive nor-metabolite produced by N-dealkylation (Watanabe et al, 2017). In addition, each also has a hydroxyethyl and hydroxymethyoxy metabolite, similar to fentanyl.…”

“…Another study of a set of presumptive heroin-positive urine specimens demonstrated a dihydrodiol metabolite and fentanyl precursor, 4-ANPP, and a sulfate metabolite (Goggin et al, 2017). The difference in the metabolism of furanylfentanyl is attributed to its structure; while acetylfentanyl, acrylfentanyl, 4-fluoroisobutylfentanyl contain a different substituent at the amide group, furan is an aromatic, heterocyclic system known to undergo characteristic reactions (Watanabe et al, 2017) Remifentanil is the only analog found to be 95% metabolized in the blood and tissues by non-CYP enyzmes (Burkle et al, 1996). This is attributed to an easily accessible ester group allowing for rapid hydrolysis by circulating blood esterases.…”

“…In recent years, LC-HRMS has been used for the detection of synthetic opioids such as butyrfentanyl, 4-fluorobutyrfentanyl, acetylfentanyl, 4-methoxybutyrfentanyl, furanylfentanyl, acrylfentanyl, 4Cl-iBF, 4F-iBF, THF-F, cyclopentylfentanyl, AH-7921, MT-45, and U-47700 in individual cases, case series, outbreaks, and epidemiological surveillance efforts (Backberg et al, 2015;Wohlfarth et al, 2016;Helander et al, 2016;Schneir et al, 2017;Armenian et al, 2017;Breindahl et al, 2017;Jones et al, 2017;Steuer et al, 2016;Vo et al, 2017;Fleming et al, 2017;Guerrieri et al, 2017;Helander et al, 2017;Goggin et al, 2017;Watanabe et al, 2017;Fels et al, 2017). It has also been used for the elucidation of the metabolic pathways of emerging synthetic opioids, such as, AH-7921, butyrfentanyl, carfentanil, U-47700, furanylfentanyl, acetylfentanyl, acrylfentanyl, and 4F-iBF Jones et al, 2017;Steuer et al, 2016;Feasel et al, 2016;Fleming et al, 2017;Goggin et al, 2017;Watanabe et al, 2017). LC-HRMS has clearly emerged as the predominant method for the detection of novel synthetic opioids, however, this technology is not readily available in most routine clinical and forensic laboratories.…”