**3. Conclusions**

In this study, the occurrence of the *Fusarium* mycotoxin Enn B1 and its phase I metabolites was evaluated in 300 human urine samples throughout ultrahigh-performance liquid chromatography high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). Results confirmed the presence of Enn B1 in 94.3% of samples ranging from 0.007 to 0.429 ng/mL (mean value: 0.065 ng/mL). Furthermore, the occurrence of Enn B1 metabolites previously found in in vitro and in vivo analysis was evaluated for the first time in human urine samples. In accordance with literature, demethylated and oxidated metabolite (5.3% samples, mean content = 0.035 ng/mL), hydroxylated metabolites (78.0% samples, mean content = 0.069 ng/mL), carbonylated metabolites (66.0% samples, mean content = 0.196 ng/mL), and carboxylated metabolites (26.3%, mean content = 0.066 ng/mL) were tentatively identified. Statistical analysis confirmed hydroxylated and carbonylated products as the most prevalent metabolites of Enn B1 in human urine. Di fferences observed between this work and in vitro Enn B1 biotransformation assays stand as additional evidence of potential pre-systemic metabolism. The characterization of metabolites derived from food contaminants is an important issue when performing safety and risk evaluations, and according to the here-obtained results a frequent exposure to Enn B1 is highlighted in Italian population.
