Advancing profiling of secondary antioxidant metabolites in Allium cepa PDO leaf extract: Online comprehensive two-dimensional liquid chromatography with high-resolution mass spectrometry and pre-column DPPH assay

The food and agricultural processing industries generate significant amounts of phenolic-rich by-products, which hold potential as natural antioxidant sources for a wide range of applications, including functional ingredients and nutraceutical formulations. Allium cepa leaves represent a promising source of bioactive compounds. However, due to the complexity of their chemical composition, advanced analytical techniques are required to fully characterize the secondary metabolite profile and identify specific phytochemical classes or fractions with high nutraceutical potential. In this context, an online comprehensive two-dimensional liquid chromatography (LC×LC) approach was developed and optimized for the in-depth characterization of the phytochemical profile of Allium cepa PDO leaf extract. Key parameters in both dimensions, including flow rate, stationary phase chemistry, and mobile phase composition, were investigated to enhance peak capacity and orthogonality. The optimized method combined reversed phase in both dimension (RP-LC×RP-LC), offering high orthogonality (A0: 70.46 %) and peak capacity (nc: 1788.88). and significantly improving the separation of multiple secondary metabolite classes by effective employment of the 2D separation space. Further hyphenation with high-resolution mass spectrometry (HRMS), enhanced compound annotation compared to mono-dimensional (1D-LC) techniques. A total of 147 compounds were tentatively annotated belonging to multiple classes such as flavonoids, saponins, phenylpropanoids, isoprenoids, terpenes, dipeptides, fatty acids, and lipids. Additionally, the antioxidant activity of Allium cepa leaf extract was assessed by coupling a pre-column 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with the LC×LC-DAD-HRMS platform. This integrated approach enabled the identification of individual contributions of flavonoids, isoprenoids, and phenylpropanoids to radical scavenging activity. The method allowed an in-depth exploration of Allium cepa phytochemical profile, demonstrating to hold significant potential for the standardization of antioxidant biomarkers, with promising applications in the nutraceutical industry.