*2.5. Hordenine Determination by UPLC-MS*/*MS*

Hordenine sample preparation was performed as described by Sommer et al. [35] with slight modifications. Beer samples were centrifuged for 15 min at 12,000× *g* and 4 ◦C; two dilution steps were followed. *Dilution I (Dil. I):* 50 µL of degassed beer were added to 450 µL of 0.1% formic acid. *Dilution II (Dil. II)*: 20 µL of *Dil. I* were added to 980 µL of 0.1% formic acid. The solutions obtained after *Dil. II* were passed through a PVDF filter (0.2 µm, Thermo Scientific™, Waltham, MA, USA) prior to the analysis. For quantification, a calibration curve with a range of 0–0.1 ppm was developed using a stock solution (2 mg mL−<sup>1</sup> ) of a hordenine commercial standard (Sigma-Aldrich, St. Louis, MO, USA) prepared in formic acid (0.1%).

Hordenine separation and quantification were conducted in a Quattro Premier XE Micromass UPLC-MS/MS system (Waters, Milford, MA, USA) equipped with a triple quadrupole mass spectrometer (QQQ-MS) connected to an Acquity UPLC (Waters, Milford, MA, USA) with electrospray ionization (ESI) source in positive mode. Hordenine was analyzed in the multiple reaction monitoring (MRM) mode of m/z 165.95:121. Masslynx 4.1 software (Waters, Milford, MA, USA.) was used for data

acquisition and instrument control. Hordenine separation was performed using a high strength silica (HSS T3 C18) column (2.1 mm × 100 mm, 1.8 µm particle size) coupled with a VanGuard HSS T3 C18 column (2.1 × 5 mm, 1.8 µm) maintained at 50 ◦C. Mobile phases were 0.1% formic acid in water (solvent A) and 0.1% formic acid in 70% acetonitrile and 30% methanol (solvent B) with 6.6 min total gradient solution as follows: 0–1 min, 5–15% B; 1–2 min, 15–40% B; 2–3 min, 40–70% B; 3–3.5 min, 70–100% B; 3.5–5 min, 100% B; 5–5.2 min, 100–5% B, followed by 1.4 min re-equilibration. The flow rate was kept constant at 0.5 mL min−<sup>1</sup> with an injection volume of 10 µL. Nitrogen was used as the desolvation gas (400 L/h). The selected ion monitoring conditions were set as capillary voltage 2.5 kV, source temperature 120 ◦C, and desolvation temperature 400 ◦C. All determinations were conducted in triplicates.
