2.2.4. High-Performance Liquid Chromatography (HPLC) Analysis of Xanthohumol (XN) and Isoxanthohumol (IXN) Content

The content of XN and IXN was analyzed using the method by Jurková et al. [18] with modifications. The analysis consisted of solid-phase extraction (SPE) using the Strata C18-E, 500 mg/6 mL column (Phenomenex, Torrance, CA, USA). For the analytes separation from beer samples, we used: No.1—methanol, No. 2—water:H₃PO₄ (100:0.2 v/v), No. 3—methanol:water:H₃PO₄ (20:80:0.2 v/v/v) and No.4—methanol:H₃PO₄ (100:0.2 v/v). Columns were conditioned by passing 10 mL of No. 1, and next 10 mL of No. 2. The acidified beer samples (25 mL) were applied to the SPE columns, treated with 10 mL of No. 3 and allowed to “dry” for 10 min under vacuum. The analytes were washed out with 5 mL of No. 4 solution into 2.5 mL measuring tubes to the level just before the mark, filled with the same solution and mixed. Samples were filtered through a 0.45 Pm PTFE filter and analyzed by HPLC.

High-performance liquid chromatograph (Waters 2690) with a diode array detector (Waters 996) was used. The analyzes were performed on a C-18 reverse phase column (Kinetex 5u XB-C18 100A). The column was thermostated at 28 °C and the analyzed samples at 12 °C. The following solutions were used as eluent: A- 1% vol. formic acid in water; B-1% vol. formic acid in acetonitrile. Elution program: 65% A-10 min., 65% A-10% A-8 min., 10% A-4 min., 10% A-65% A-1 min., 65% A-7 min. The flow rate was 1.5 mL/min. The XN and IXN external standard (0.535 mg/mL of XN and 0.496 mg/mL of IXN) was used in the analysis. The analyses were performed in triplicate. Results were presented in mg of XN or IXN per liter of beer.