Dual-injection single-run LC-MS/MS analysis for the simultaneous determination of (lipo)phenolic compounds in...

The olive oil industry is rapidly expanding, driven by the worldwide recognition of olive oil as a nutritious food and a rich source of health-promoting compounds. However, its production generates substantial by-products, including olive pomace, mill wastewater, and leaves. If not properly managed, these residues can pose environmental risks due to their phytotoxic and antimicrobial properties. Nevertheless, their high content of bioactive, antioxidant-rich compounds makes them promising for the development of eco-friendly and economically valuable co-products. In this study, a dual-injection single-run approach was applied for a comprehensive determination of (lipo)phenolic compounds in industrial olive oil by-products by liquid chromatography coupled with high-resolution mass spectrometry. The method involves two consecutive injections combined into a single run using a binary gradient program, integrating an online focusing step for trapping lipophenols, enabling efficient separation of these analytes from phenolic compounds with high-throughput workflows. A baseline separation of the target analytes was obtained in <25 min; considering the column conditioning step, the total analysis time for a single run is 37 min. Once optimized, the method was applied for both targeted and untargeted profiling of olive by-products from Olea europaea cultivars. Molecular characterization was carried out by processing MS and MS/MS spectra. >40 compounds were identified in olive pomace samples, including several molecules showing MS² fragment ions at 281.25 and 279.23 m/z, which were attributed to metabolites derived from oleic acid and linoleic acid, respectively.