Strata x 33 m polymeric reversed phase cartridges

10 mL of 80% methanol containing 1% acetic acid was added in 1 g of leaves and homogenized thoroughly, and transferred to a 50 mL tightly capped test tube. The test tubes were placed in a shaker (Scientific Industries Inc., USA) for 15 h at 400 rpm. 0.45 µm filter (MILLEX-HV syringe filter, Millipore Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. The mixture was centrifuged at 10,000 × g for 15 min. Betacyanin components were analyzed from the final filtrate. Betacyanin analysis in the samples could interfere through the precipitation of methanol with the proteins and other insoluble substances in the samples. Strata-X 33 µm Polymeric Reversed-Phase cartridges (Phenomenex, Torrance, CA, USA) were used to purify betacyanin. All extractions were done in triplicate independent samples.

Juices from HS grape homogenate samples (two replicates from each time point) were subjected to strong acid hydrolysis, using methodology similar to that reported by Noestheden and colleagues [15 (link)]. Briefly, aliquots of homogenate (10 g) were centrifuged for 30 min at 3500× g (Universal 320R centrifuge, Andreas Hettich GmBH and Co. KG, Tuttlingen, Germany), and 2 mL of the resulting juice was purified by solid phase extraction (using Strata X 33 µm polymeric reversed phase cartridges, 200 mg/3 mL; Phenomenex, Lane Cove, NSW, Australia). Samples were eluted with 40% acetonitrile in water (2 mL), dried (under nitrogen at 35 °C), reconstituted in 2 mL water and acidified to pH ~1 (via dropwise addition of 1 M hydrochloric acid), before being heated at 100 °C for 4 h in 8 mL PTFE tubes (SPI Supplies, West Chester, PA, USA). Hydrolysates were subsequently cooled to ambient temperature, pH adjusted back to wine pH (i.e., pH 3.0–3.5, via dropwise addition of 1 M aqueous sodium hydroxide) and frozen prior to chemical analysis.

Isoxanthohumol (≥99%) was purchased from Sigma Aldrich GmbH, while xanthohumol (≥98%) and 8-prenylnaringenin (≥ 98%) were purchased from Enzo Life Sciences, Farmingdale, New York. Eriodyctiol (≥ 99%) and 3,4,2’,4’,6’-pentahydroxychalcone(≥99%) were purchased from Extrasynthese Lyon, France. Stock solutions of all reference standards were prepared in HPLC grade methanol (J. T. Baker, Deventer, The Netherlands). The same solvent was used for the extraction. Acetonitrile (ACN) was used as the organic part of the mobile phase (LC-MS grade, J. T. Baker, Deventer, Netherlands). Both formic acid (FA) used for SPE (>98% purity) and FA (LC-MS grade) used as a mobile phase modifier were purchased from Sigma Aldrich (Darmstadt, Germany).MilliQ (18.2 MΩ·cm) water was obtained using a Direct-Q^®Water Purification System (Merck, Darmstadt, Germany). Strata-X 33 µm polymeric reversed phase cartridges (60 mg/3 mL) were purchased from Phenomenex (Torrance, CA, USA). Before injection into the LC-MS system, the samples were filtered through 0.2 µm regenerated cellulose (RC) membrane syringe filters (Phenomenex, Torrance, CA, USA). Hops and beer samples were freeze-dried in a GAMMA 1-16 LSCPlus freeze dryer (Martin Christ Gefrierungstrochnungsanlagen GmbH, Osterode am Harz, Germany).