Barley (Hordeum vulgare L.; Poaceae) is a nutrient-dense cereal rich in phenolamides, yet their chemical profile and distribution across barley products and germinated barley remain underexplored. This study identified 47 phenolamides, including two previously undescribed Cou-Agm derivatives (cis- and trans-p-coumaroyl agmatine diene, cis-CADe and trans-CADe) in whole-grain barley using GNPS molecular networking and UHPLC-HR-ESI-MS. GNPS analysis enabled the detection of sugar-containing spermidine conjugates, while MS² fragmentation patterns facilitated the rapid differentiation of spermidine isomers and the construction of a comprehensive molecular network. To enable quantification, hordatines A and C (HA, HC) were synthesized, while hordatine B (HB) and glycosylated hordatines A and B (HAG, HBG) were isolated from a standardized hordatine-rich fraction (HRF) developed from germinated barley. Quantitative analysis of barley products (alcoholic and non-alcoholic beers, flakes, flour, bread, hulled and pearl grains) revealed that beer products were enriched in hordatines, while hulled grains and flour contained higher spermidine conjugate levels. The impact of germination on phenolamide concentrations was assessed over 9 days under light and dark conditions, showing that darkness enhances hordatine aglycone accumulation, particularly HA and HB. HAG-HCG were abundant in early germination (Days 0-1), whereas aglycones dominated in later stages (Days 5-9). Notably, HA and Cou-Put were identified as shoot- and root-specific phytochemicals, respectively, with increased methylation and glycosylation in roots. These findings provide new insights into the phenolamide composition of barley and the impact of germination on their distribution, offering valuable information for functional food development.

Weixin Wang

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