*4.2. Sampling*

During January and February 2018, 300 Italian volunteers residing in Campania region (southern Italy) provided first-spot morning urine samples. The following exclusion criteria were considered for the study: (i) only one member per family allowed; (ii) people with severe issues in liver, kidney, or bile were not allowed due to potential interferences in the metabolic processes related to mycotoxins; (iii) people exposed to high amounts of mycotoxins in a di fferent way from food, such as farmers and veterinarians, were not allowed. The use of medication was not an exclusion criterion due to the lack of information available about interferences with mycotoxins. Urine was stored in plastic containers at −20 ◦C within 2 h after collection. All volunteers signed informed consent following the Helsinki Declaration on ethical principles for medical research when humans are involved. The present study was accepted by the University of Valencia Institutional human research Committee and the procedures and purposes were properly justified and approved. The numerosity of the sampling (*n* = 300) is in accordance with the International Federation of Clinical Chemists (IFCC) recommendations [32].

Volunteers were asked to specify their age and gender on their own container, in order to classify the sample. The sampling tried to keep the gender parity (male: 45.7%, female: 54.3%). According to the age of participants, three di fferent groups were considered for statistical analysis: ≤30 years old (*n* = 94), from 31 to 59 years old (*n* = 72), and ≥60 years old (*n* = 134). Samples with undetected levels of the analytes of interest were chosen as "blank" and used in spiking and recovery studies. The consumption data were set according to age and gender, following the Guidelines for Healthy Italian Food Supply reported by the National Institute for Food Research and Nutrition [33].

### *4.3. Sample Preparation*

The sample was processed following a previous procedure slightly modified [34]. Briefly, 1.5 mL of sample was placed into a 2 mL Eppendorf Safe-Lock Microcentrifuge tube and centrifuged for 3 min at 4000 rpm. After that, 1 mL of the supernatant was transferred to a 15 mL screw cap test tube with conical bottom and 1 mL of acetonitrile was added. The mixture was vortexed for 30 s and subsequently 30 mg of C18 sorbent and 0.3 g sodium chloride were incorporated to minimize interference from matrix. The solution was vortexed again for 30 s and centrifuged for 3 min at 4000 rpm and 4 ◦C. Then, the upper layer was collected, evaporated under gentle nitrogen stream in a water bath at 45 ◦C, reconstituted with 0.5 mL of MeOH/H2O (70:30 *v*/*v*) and filtered through a 0.2 μm filter before to UHPLC-Q-Orbitrap HRMS analysis.

#### *4.4. UHPLC-Q-Orbitrap HRMS Analysis*

Quantitative and qualitative profiles of Enn B1 and Enn B1 phase I metabolites were acquired through Ultra High Pressure Liquid Chromatograph (UHPLC; Thermo Fisher Scientific, Waltham, MA, USA) equipped with an auto sampler device, a degassing system, a thermostated (T = 30 ◦C) Luna Omega 1.6 μm (50 × 2.1 μm) column, a Dionex Ultimate 3000 a Quaternary UHPLC pump working at 1250 bar.

The eluent consisted of two di fferent phases, both H2O (phase A) and MeOH (phase B) containing 0.1% formic acid and 5 mM ammonium formate. The gradient elution program for LC prior to Orbitrap HRMS analysis was developed as follows: 0–1 min–0% of phase B, 2 min–95% of phase B, 2.5 min–95% of phase B, 5 min–75% of phase B, 6 min–60% of phase B, 6.5 min–0% of phase B, and 1.5 min–0% phase B for equilibrating the column. The flow rate was set at 0.4 mL/min. A total of 5 μL of the sample was injected. Detection was performed using a Q-Exactive mass spectrometer. Data were acquired through full scan in positive mode at a resolving power of 70,000 FWHM at *m*/*z* 200. Ion source parameters in positive (ESI+) mode were: sheath gas (N2 > 95%) 35, auxiliary gas (N2 > 95%) 10, spray voltage 4 kV, capillary temperature 290 ◦C, auxiliary gas heater temperature 305 ◦C, S-lens RF level 50. Data analysis and processing were carried-out using the Xcalibur software, v. 3.1.66.10 (Thermo Fisher Scientific, Waltham, MA, USA). A scan range of *m*/*z* 100–800 was set for the compounds of interest; the injection time was set to 200 ms and the automatic gain control (AGC) was selected at 1 × 106. Scan-rate was set at 2 scans/s.

#### *4.5. Metabolomic Data Processing*

Data processing and data pretreatment were performed to allow the putative identification of significant metabolites. Screening was done by investigating spectral data collected using a mycotoxin spectral library (version 1.1 for Library View Software, AB Sciex, Framingham, MA, USA) containing spectral data for 245 mycotoxins and other fungal/bacterial metabolites and 236 full MS/MS spectral library entries. The features, defined by their m/z and retention time, and their intensities in di fferent samples were used to carry out the statistical analysis. Then, samples were grouped to perform the statistical analysis.

### *4.6. Method Validation*

An in-house validation study was conducted according to the Commission Decision 2002/657/EC [19]. The parameters measured included linearity, matrix e ffect, trueness, precision, LOQ, and LOD. Linearity was evaluated using solvent and matrix-matched calibration curves, analyzing in triplicate six concentration levels ranging between 0.001–5 ng/mL. The matrix-matched calibration curves were prepared spiking aliquots of the corresponding matrices with Enn B1 at similar concentrations than the calibration curve made in solvent. Signal suppression or enhancement e ffect due to matrix co-elution interferences, was evaluated through a comparison between the slope of pure standard curve with the slope of matrix-matched standard curve following the next equation: SSE (%) = Slope matrix-matched calibration/Slope standard in solvent × 100. Trueness and precision were assessed using recovery studies since no suitable reference material was available. Recovery measurements were performed by spiking blank urines with the standard working solution of Enn B1 at the levels of 0.1, 0.5, 1, and 5 ng/mL. Intra-day (RSDr, %) and inter-day precision (RSDR, %) were expressed as the relative standard deviation after repeating three measurements per concentration level on the same day and in three non-consecutive days, respectively. LOD was established as the minimum concentration at which the molecular ion can be identified (mass error < 5 ppm) and the LOQ as the lowest concentration of the analyte at which the concentration can be determined with accuracy and precision ≤20%.
