An on-line stop-flow RPLC × SEC-MS/DPPH radical scavenging activity analysis system and its application in separation and identification of antioxidant peptides

Food-derived antioxidant peptides have become the focus of research due to their high safety and low cost. However, the discovery is suffering from a low efficient and empirical approach, involving multi-step off-line separation and identification. In this work, an on-line stop-flow RPLC  ×  SEC -MS/DPPH radical scavenging activity analysis system was developed. For optimization, the conditions: 10 m reaction loop, 200 μM DPPH radical concentration, 40℃ temperature and 0.06 % formic acid were recommended. The system was fully validated by its application in glutathione analysis. The system was further applied in analysis of complex mixed standards, and the dipeptides GC (Gly-Cys) and CW (Cys-Trp) with relatively strong DPPH radical scavenging activity were validated. Maize protein hydrolysates were used for tests and the peptide AC (Ala-Cys) of high probability with strong DPPH radical scavenging activity was identified, demonstrating a high potential of the system. This would help to facilitate the discovery of antioxidative peptides in the future.