Accurate quantification and antioxidant evaluation of avenanthramides in oat products using authentic reference standards

Avenanthramides are oat-derived phenolic amides. However, prior studies have predominantly focused on C-type avenanthramides. This study aimed to enable accurate determination of A-type avenanthramides in oat products using authentic reference standards. Fifty avenanthramide standards covering A-type and C-type structures were synthesized, characterized, and confirmed to be >98% pure. A quantitative method was developed and analytically validated for representative analytes using LC-MS/MS. Calibration slopes differed substantially between A-type and C-type counterparts, indicating that C-type-based calibration causes systematic underestimation of A-type compounds. Analysis of commercial oat products showed that extrapolation using C-type curves yielded 35.9-80.7% of concentrations obtained with authentic standards, and A-type compounds comprised 29.9-49.0% of total avenanthramides within the quantified panel (six compounds). Antioxidant evaluation using DPPH radical scavenging indicated that while C-type avenanthramides tended to show slightly higher activity on average, multiple A-type avenanthramides exhibited comparable or superior activity, underscoring the biological relevance of A-type structures.