Offline two-dimensional liquid chromatography-electrochemical detection combined with liquid chromatography-high resolution mass spectrometry for comprehensive characterization and antioxidant activity evaluation of Ilex asprella leaves

Offline LC × LC-ECD coupled with HRMS screens antioxidants in Ilex asprella leaves. • 48 antioxidants were characterized by HRMS, mainly phenolic acids and flavonoid glycosides. • ECD intensity shows strong positive correlation with DPPH and ABTS activities. • LC-ECD fingerprinting reveals distinct geographical differences in sample profiles. Ilex asprella is a traditional medicinal and edible plant with multiple pharmacological activities such as anti-inflammatory properties. Previous studies on its chemical constituents and pharmacology have mostly focused on roots and stems, whereas research on the leaves remains relatively limited. In this study, an offline comprehensive two-dimensional liquid chromatography‑electrochemical detection (LC × LC-ECD) method combined with LC-HRMS was established for the rapid screening and structural identification of antioxidant components in Ilex asprella leaves. LC × LC-ECD achieved efficient separation of components with wide polarity ranges and significant concentration differences. Using LC-HRMS, a total of 48 compounds were identified or annotated, primarily including 19 hydroxycinnamoyl quinic acids, 14 flavonol glycosides, 6 dihydroflavone glycosides, 5 simple phenolic acids and their glycosides, 2 coumarins, and 2 other compounds. In vitro assays indicated that all electrochemically active fractions obtained from the first-dimension separation exhibited varying degrees of antioxidant activity, and their ABTS and DPPH radical scavenging activities were positively correlated with the electrochemical detection response intensity ( R 2 > 0.95). Subsequently, liquid chromatography-electrochemical detection was utilized to compare the antioxidant components among 29 batches of Ilex asprella leaves from different origins. Multivariate statistical analyses, including hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis, consistently demonstrated that the 29 batches of samples could be clearly grouped into three categories based on their antioxidant component profiles, and the classification results were highly correlated with geographical origins, confirming significant variations in the antioxidant component profiles of Ilex asprella leaves from different origins. Download: Download high-res image (207KB) Download: Download full-size image