We aimed to purify polyphenols from distiller’s grain extract using macroporous resins and to identify its polyphenolic components. The influence of operational parameters on purification efficiency was investigated. The polyphenolic composition was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and then quantified by UPLC-MS using authenticated standards. The results showed that the optimal purifying conditions were D101 resin with a dosage of 3 g, four hours adsorption, three hours desorption time, and 60% ethanol as the eluent, producing the highest purification rate of 51%. The purified distiller’s grain extract exhibited stronger antioxidant activity than the unpurified extracts, which was assessed using DPPH and ABTS methods (IC50 DPPH = 34.03 and 16.21 μg/mL, respectively; IC50 ABTS = 20.31 and 5.73 μg/mL, respectively). UPLC-MS results indicated that (−)-epicatechin is the major compound found in distiller’s grain extract which was quantified as 562.7 μg/g extract, followed by ferulic acid (518.2 μg/g), p-hydroxybenzoic acid (417.7 μg/g), caffeic acid (217.1 μg/g), syringic acid (158.0 μg/g) and quercetin (147.8 μg/g). Two compounds, vanillic acid (66.5 μg/g) and gallic acid (41.4 μg/g), were found in lower concentrations. The findings of this study suggest that purification of polyphenolic compounds from distiller’s grain by macroporous resins is feasible, providing a new and effective method for the secondary use of distiller’s grain resources.
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