The diffusion of new psychoactive substances (NPS) is highly dynamic and the available
substances change over time, resulting in forensic laboratories becoming highly engaged in NPS
control. In order to manage NPS diffusion, efficient and innovative legal responses have been
provided by several nations. Metabolic profiling is also part of the analytical fight against NPS,
since it allows to identify the biomarkers of drug intake which are needed for the development of
suitable analytical methods in biological samples. We have recently reported the characterization
of two new analogs of fentanyl, i.e., 4-fluoro-furanylfentanyl (4F-FUF) and isobutyrylfentanyl (iBF),
which were found for the first time in Italy in 2019; 4F-FUF was identified for the first time in
Europe and was notified to the European Early Warning System. The goal of this study was
the characterization of the main metabolites of both drugs by in vitro and in vivo experiments.
To this end, incubation with mouse hepatocytes and intraperitoneal administration to mice were
carried out. Samples were analyzed by means of liquid chromatography-high resolution mass
spectrometry (LC–HRMS), followed by untargeted data evaluation using Compound Discoverer
software with a specific workflow, designed for the identification of the whole metabolic pattern,
including unexpected metabolites. Twenty metabolites were putatively annotated for 4F-FUF, with
the dihydrodiol derivative appearing as the most abundant, whereas 22 metabolites were found for
iBF, which was mainly excreted as nor-isobutyrylfentanyl. N-dealkylation of 4F-FUF dihydrodiol
and oxidation to carbonyl metabolites for iBF were also major biotransformations. Despite some
differences, in general there was a good agreement between in vitro and in vivo samples