Differentiation of 2-, 3-, and 4-Fluorofuranylfentanyl Using HPLC-MS and Human Liver Microsomes

The structural similarity of positional isomers of fentanyl analogs makes their forensic differentiation difficult, particularly when the parent compounds exhibit similar chromatographic behavior and product-ion spectra. This study evaluated the differentiation of 2-, 3- and 4-fluorofuranylfentanyl (2-, 3- and 4-FFF) using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and human liver microsomes (HLMs). The synthesized positional isomers were analyzed using gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and HPLC-ion trap-time-of-flight mass spectrometry (HPLC-IT-TOF-MS). Although the parent isomers were separated by GC-MS, their retention times and product-ion spectra obtained by HPLC-MS/MS were highly similar under the present conditions. Incubation with HLMs supplemented with NADPH, UDPGA and alamethicin produced three Phase I metabolic categories: an oxidative N-dealkylated metabolite of FFF (M1), monohydroxylated FFF metabolites (M2a and M2b), and an amide hydrolysis product of FFF (M3). Glucuronide conjugates were not detected under the present analytical conditions. Although M1 was the predominant metabolite, M1 did not retain the complete FFF structure. M2a and M2b were putatively assigned as monohydroxylated metabolites that retained the complete FFF structure, and may therefore support the differentiation of FFF positional isomers, although their retention times did not allow the differentiation. M3, which did not retain the complete FFF structure, showed distinct retention times among the three positional isomers, providing chromatographic information useful for metabolite-based differentiation. The in vitro elimination half-lives differed among 2-, 3- and 4-FFF under the present HLM conditions, suggesting possible differences in the pharmacokinetic properties of these positional isomers. These findings indicate that HPLC-MS analysis using HLMs provides useful analytical information for differentiating FFF positional isomers.