2D-LC-HRMS for Profiling Antioxidants in Allium Cepa Leaves
Researchers from the University of Salerno in Fisciano, Salerno, Italy developed a multidimensional liquid chromatography (LC)-based technique for profiling antioxidant metabolites in Allium cepa leaf extracts and potentially other food byproducts. Their findings were published in the Journal of Chromatography A (1).
Pure organic raw onion | Image Credit: © Veronika Idiyat - stock.adobe.com
Agri-food waste (AFW), such as food peels, seeds, and leaves, are some of the most common by-products generated during fruit and vegetable processing. Food waste is the single most common material landfilled and incinerated in the United States making up 24% and 22% of landfilled and combusted municipal solid waste, according to the United States Environmental Protection Agency (U.S. EPA) (2). AFW is typically used as soil fertilizers, animal food, or biomass for producing energy and fuels. However, AFW is rich in various bioactive components, such as phenolic acids, flavonoids, and saponins, which are of great interest for human health, due to their antioxidant and anti-inflammatory properties, which can help prevent metabolic diseases.
Recovering these high-value components from the matrix requires effective extraction methods. Conventional/advanced techniques (solid-liquid extraction extraction) and green approaches (microwave and supercritical fluid extraction) are applicable. The chemical characterization of AFW is crucial. Liquid chromatography (LC) is the most widely used method for analyzing food and food by-products. When coupled with mass spectrometry (MS), it is the gold standard for profiling secondary metabolites in food matrices, according to the authors. However, one-dimensional LC methods (1D-LC) often fail to resolve the chemical complexity of these matrices, which can cause issues, such as MS overload, due to multiple co-elutions and ion-suppression phenomena, which obscure the detection of minor components. Two-dimensional liquid chromatography (2D-LC), especially in its comprehensive mode (LC×LC), has emerged as an innovative food analysis method, when it is applied to food waste characterization using various stationary phase combinations.
In this study, an online comprehensive two-dimensional liquid chromatography (LC×LC) approach was optimized for in-depth characterization of Allium cepa (onion) Protected Designation of Origin (PDO) leaves (3). 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), significantly improving the separation of multiple secondary metabolite classes by effectively employing the 2D separation space. The 147 compounds belonging to multiple classes, such as flavonoids, saponins, phenylpropanoids, isoprenoids, terpenes, dipeptides, fatty acids, and lipids. Further, the antioxidant activity of Allium cepa leaf extracts were assessed by coupling a pre-column 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with a LC×LC-diode array detector (DAD)–HRMS platform. Using this integrated approach, the identification of individual contributions of flavonoids, isoprenoids, and phenylpropanoids to radical scavenging activity was enabled.
The developed methodologies highlighted the importance of two-dimensional chromatography techniques for valorizing agricultural by-products, enabling detailed characterization of secondary metabolites and the standardization of biomarkers with antioxidant properties. This research could lead to valuable contributions to industrial applications in the food and pharmaceutical sectors, promoting a sustainable means of turning agricultural by-products into high-value-added resources.
References
(1) Aquino, G.; Sommella, E. M.; Salviati, E.; Manfra, M.; et al. 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. J. Chromatogr. A 2025, 1749, 465877. DOI: 10.1016/j.chroma.2025.465877
(2) From Farm to Kitchen: The Environmental Impacts of U.S. Food Waste. EPA 2025. https://www.epa.gov/land-research/farm-kitchen-environmental-impacts-us-food-waste (accessed 2025-4-7)
(3) Onion. ScienceDirect 2019. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/onion (accessed 2025-4-7)
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