Screening of Phenolic Antioxidant Components in Polygonum perfoliatum L. Using LC×LC-ECD and UPLC-Orbitrap-MS/MS

Polygonum perfoliatum L. is a medicinal plant rich in phenolic and flavonoid compounds, but severe peak overlap in one-dimensional liquid chromatography (1D-LC) hinders the separation of antioxidants. We developed an offline comprehensive two-dimensional liquid chromatography-electrochemical detection (LC×LC-ECD) platform with high orthogonality and sensitive electrochemical response for screening of antioxidants. Identification was performed using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-Orbitrap-MS/MS), and quantification was performed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). A total of 63 antioxidants were annotated, mainly phenolic acids and flavonoids, with caffeoyl and quercetin derivatives most abundant. The first-dimension fractions exhibited radical scavenging capacity, and their peak areas were positively correlated with the activity, with correlation coefficients of 0.9697 (p < 0.001) for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 0.9688 (p < 0.001) for 1,1-diphenyl-2-picrylhydrazyl (DPPH). The 11 major compounds were quantitatively analyzed. Tartroylcaffeic acid and quercetin 3-O-β-D-glucuronide predominated, with average contents of 5101.7-29,296.2 µg/g and 3876.9-21,535.2 µg/g, respectively. Multivariate statistical analysis indicated that geographical origin influenced compound profiles, highlighting the need for source-specific evaluation. This offline LC×LC-ECD strategy overcomes the separation bottlenecks of 1D-LC in complex plant extracts and provides a new paradigm for systematic antioxidant screening and activity-guided separation.