Quantification of Prenylflavonoids in Beer Intervention Samples by LC-MS/MS

Qualitative and quantitative analyses of prenylflavonoids in beer samples were carried out according to the method of Quifer-Rada et al. (2013), with some modifications [26]. Briefly, the beer foam from AB and NAB was removed by agitation and ultrasonication. Then, the alcohol content from alcoholic beer was evaporated under a gentle stream of N₂ and was refilled with water. Samples were filtered through a 0.45-µm polytetrafluoroethylene filter and 600 ng/mL of taxifolin was added as an internal standard prior to the analysis. The identification and quantification of the selected analytes (IX, XN, 6-prenylnaringenin (6-PN), and 8-PN) was carried out using an Acquity UHPLC system equipped with a Waters binary pump (Waters, Milford, MA, USA). The UHPLC separation was performed with a Luna C18 column, 50 mm × 2.0 mm i.d., 5 μm (Phenomenex, Torrance, CA, USA), directly interfaced to an API 3000™triple quadrupole mass spectrometer (Sciex, Concord, ON, Canada) with a turbo ion spray source working in negative mode. The mobile phases used were 5 mM of ammonium bicarbonate buffer adjusted to pH 7.0 and acetonitrile and methanol (1:1), at a constant flow rate of 600 µL/min and a column temperature of 40 °C. Sample injection volume was 10 µL. Multiple reaction monitoring mode was used to identify and quantify the analytes. Calibration curves from 0 to 1000 ppb were prepared adding standards to pure water containing 20 mg/L of ascorbic acid. The reagents, materials, and MS/MS parameters were the same as reported in Quifer-Rada et al. (2013) [18].