β′-Phenylfentanyl Metabolism in Primary Human Hepatocyte Incubations: Identification of Potential Biomarkers of Exposure in Clinical and Forensic Toxicology

Abstract

From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders, and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. β’-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on β’-phenylfentanyl effects and toxicokinetics, and its metabolism is yet to be studied. We aimed to investigate β’-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in β’-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. β’-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography–high-resolution tandem mass spectrometry (LC–HRMS-MS), and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation, and combinations thereof. We suggest β’-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of β’-phenylfentanyl use. In silico predictions were mostly wrong, and β’-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of NPS human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify β’-phenylfentanyl-positive cases to provide authentic samples.

In vitro metabolite identification of acetylbenzylfentanyl, benzoylbenzylfentanyl, 3‐fluoro‐methoxyacetylfentanyl, and 3‐phenylpropanoylfentanyl using LC‐QTOF‐HRMS together with synthesized references

“…The MSMS spectra and proposed fragmentation of the metabolites are also shown in the Supporting Information. The most abundant metabolite (nor‐metabolite; P17) was formed through N ‐dealkylation with the fragment ions of m/z 84.0808 and 105.0699, which agrees with the findings reported by Brunetti et al 5 …”

“…P23 was identified as β‐OH‐phenylpropanoylfentanyl ( 5h ) based upon matching RTs (Figure 7) and MSMS spectra of metabolite P23 and reference 5h (fragment ions at m/z 105.0700, 107.0845, 134.0960, and 188.1438; Supporting Information). Hence, the use of synthesized references could confirm the previous findings by Brunetti et al 5 Previous studies have shown that β‐hydroxylation at the phenethyl moiety is a common metabolic pathway for fentanyl analogs 21,22 . It has also been detected in authentic urine samples 21 .…”

“…It is believed that this metabolite is formed from metabolite P24 and/or P23 and that the carbonyl group therefore is located at the β position. The metabolites P6, P8, P16, and P25 have been reported previously 5 …”

“…Synthesis and analysis of reference substances enabled the identification of the exact chemical structures of these three metabolites (P4, P7, and P9). The formation of these metabolites has been reported previously, 5 although the exact position of the hydroxy group could not be determined. No N ‐dealkylated metabolites with the hydroxy group at the ortho ( 8f ), meta ( 8e ), and para ( 8d ) positions at the N ‐phenyl moiety could be found.…”

“…However, Brunetti et al recently studied the metabolism of 3‐phenylpropanoylfentanyl and reported the formation of 26 metabolites. A number of these metabolites were monohydroxylated compounds, although the exact position of the hydroxy group could not be identified 5 . Metabolism experiments of other fentanyl analogs have shown how human hepatocytes can be used in the identification of metabolic pathways and important key metabolites for individual fentanyl analogs 6–8 .…”