Insights into Phenolamides in Whole Grain Barley: Chemical Profile and Their Levels in Barley-Based Products

Barley (Hordeum vulgare L.) is a nutrient-rich whole grain (WG) with pharmacological potential, partly attributed to its phenolamide content. Using ultrahigh-pressure liquid chromatography coupled with high-resolution electrospray mass spectrometry (UHPLC-HRESI-MS) integrated with Global Natural Products Social (GNPS) molecular networking, we identified 50 phenolamides in WG barley, including 13 agmatines, 14 spermidines, two putrescine conjugates, and 21 hordatines. Notably, we report for the first time two p-coumaroyl hydroxyagmatine derivatives (including a novel cis-isomer), three glycosylated spermidine conjugates, and two putrescine conjugates. Diagnostic MS/MS fragmentation enabled differentiation of 12 spermidine isomers, including CouCaf-Spd [Caf-(Put)-(N¹⁰3AP)-Cou vs. Cou-(Put)-(N¹⁰3AP)-Caf] and CafFer-Spd [Caf-(Put)-(N¹⁰3AP)-Fer vs. Fer-(Put)-(N¹⁰3AP)-Caf]. Targeted quantification of 44 phenolamides across various barley-based products (e.g., alcoholic and nonalcoholic beers, bread, flake, flour, hulled grains, and pearl grains) revealed product-specific distribution patterns, with hordatines and agmatine conjugates enriched in beers and spermidine conjugates more abundant in hulled grains and flours. Our findings expand the known diversity of barley phenolamides and offer a foundation for further investigation into their functional significance.