*2.4. Characterization of Iso-*α*-Acids*

The iso-α-acids profile was analyzed as described by Vanhoenacker et al. [33]. The beer samples (~25 mL) were filtered through a polytetrafluoroethylene (PTFE) syringe filter (0.2 µm) and directly injected to an Acquity Ultra-Performance Liquid Chromatography (UPLC, Waters, Milford, MA, USA) coupled to Diode-Array Detector (DAD), monitoring at 270 nm. The chromatographic separation was achieved using a Zorbax Extend C-18 column (100 × 3 mm, 3.5 µm particle size, Agilent, Santa Clara, SA, USA) kept at 35 ◦C. The mobile phase consisted of 5 mM ammonium acetate in 20% ethanol (pH 9.95) as phase A; acetonitrile/ethanol (60:40 v/v) as phase B. The solvent flow rate was 0.4 mL min−<sup>1</sup> , using gradients of 0−3 min, 0% B; 3–4 min, 0−16% B; 4–54 min, 16–40% B; 54–57 min, 40–95% B; 57–65 min, 95%B; 65–67 min, 95–0% B, followed by 20 min re-equilibration. The iso-α-acids quantification was performed with calibration curves of commercial standards of trans-iso-α-acids in dicyclohexylamine (DCHA) obtained from the American Society of Brewing Chemists (Saint Paul, MN, USA). The standards were prepared in methanol (0.05% H3PO4) according to the supplier's recommendations and the ASBC Methods of Analysis [34].
