Dual-Injection LC–HRMS Strategy Enables Comprehensive Profiling of Phenolic and Lipophenolic Compounds in Olive Oil By-Products

Driven by the worldwide recognition of olive oil as a nutritious food and a rich source of health-promoting compounds, the olive oil industry is quickly expanding. The production of olive oil, however, production, however, 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. Despite this, their containing a high amount of bioactive, antioxidant-rich compounds makes them a promising foundation for the development of eco-friendly and economically valuable co-products. This potential has inspired a study by researchers at the University of Foggia, the University of Messina, and the University of Calbria (all in Italy) where 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 (LC-HRMS). A paper based on their efforts was published in the Journal of Chromatography A.¹

Established chromatographic methods for the analysis of industrial olive oil and its by-products mostly focus on the determination of polyphenolic compounds.^2-4 In contrast, studies on lipophenols (hybrid compounds combining polyphenols with fatty acids to enhance bioavailability and stability) are scarce in the literature, which primarily focuses on their bioactive properties,^5,6 or limited to the chromatographic detection of a single representative of lipophenolic compounds such as hydroxytyrosyl oleate, tyrosyl oleate, or homovanillyl oleate, generally analyzed by LC-tandem mass spectrometry (MS/MS) alongside polyphenols.^6-10 “The markedly different solubility properties and chemical structures of lipophenols and polyphenols (a compound containing more than one phenolic hydroxyl group),” write the authors of the paper,¹ “pose a significant analytical challenge for their simultaneous determination, especially in selecting solvents that are compatible with both solubilization processes and chromatographic eluents.”

The researchers describe their method as involving 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 less than 25 minutes; considering the column conditioning step, the total analysis time for a single run is 37 minutes. 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 tandem mass spectrometry (MS/MS) spectra. Over 40 compounds were identified in olive pomace samples, including several molecules showing MS² fragment ions at 281.25 and 279.23 mas-to-charge ratio (m/z), which were attributed to metabolites derived from oleic acid and linoleic acid, respectively.¹

“The proposed method,” write the authors of the study, “represents a significant advance over conventional chromatographic approaches, which are typically designed exclusively for the determination of polyphenols or hydroxytyrosyl esters. Studies are currently going on in our laboratories to extend this method to other compound classes with different solubility, polarity, and chemical properties.”¹

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References

1. Marchesiello W. M. V.; Nardiello, D.; Cacciola, F. et al. Dual-Injection Single-Run LC-MS/MS Analysis for the Simultaneous Determination of (Lipo)phenolic Compounds in Industrial Olive Oil By-Products. J Chromatogr A 2026, 1776, 466944. DOI: 10.1016/j.chroma.2026.466944
2. Bartella, L.; Mazzotti, F.; Napoli, A. et al. A Comprehensive Evaluation of Tyrosol and Hydroxytyrosol Derivatives in Extra Virgin Olive Oil by Microwave-Assisted Hydrolysis and HPLC-MS/MS. Anal Bioanal Chem. 2018, 410 (8), 2193-2201. DOI: 10.1007/s00216-018-0885-1
3. Vergara-Barberán, M.; Navarro-Huerta, J. A.; Torres-Lapasió, J. R. et al. Classification of Olive Leaves and Pulp Extracts by Comprehensive Two-Dimensional Liquid Chromatography of Polyphenolic Fingerprints. Food Chem. 2020, 320, 126630. DOI: 10.1016/j.foodchem.2020.126630
4. Grigoletto, I.; García Salas, P.; Valli, E. et al. HPLC-MS/MS Phenolic Characterization of Olive Pomace Extracts Obtained Using an Innovative Mechanical Approach. Foods 2024, 13 (2), 285. DOI: 10.3390/foods13020285
5. Benincasa, C.; Cersosimo, A.; Perri, E. et al. Homovanillyl Oleate, a Novel Lipophenol from Olive Plant with Antioxidant Properties. ACS Food Sci.Technol. 2024, 4, 1570-1578. DOI: 10.1021/acsfoodscitech.4c00175
6. Plastina, P. Hydroxytyrosol and Hydroxytyrosyl Fatty Esters: Occurrence and Anti-Inflammatory Properties in Olives and Olive Oil in Health and Disease Prevention (Second Edition); Elsevier, (2021), 547-555. DOI: 10.1016/B978-0-12-819528-4.00010-9
7. Plastina, P.; Benincasa, C.; Perri, E. et al. Identification of Hydroxytyrosyl Oleate, a Derivative of Hydroxytyrosol with Anti-Inflammatory Properties, in Olive Oil By-Products. Food Chem. 2019, 279, 105-113. DOI: 10.1016/j.foodchem.2018.12.007
8. Benincasa, C.; La Torre, C.; Fazio, A. et al. Identification of Tyrosyl Oleate as a Novel Olive Oil Lipophenol with Proliferative and Antioxidant Properties in Human Keratinocytes. Antioxidants (Basel) 2021, 10 (7), 1051. DOI: 10.3390/antiox10071051
9. Benincasa, C.; La Torre, C.; Plastina, P. et al. Hydroxytyrosyl Oleate: Improved Extraction Procedure from Olive Oil and By-Products, and In Vitro Antioxidant and Skin Regenerative Properties. Antioxidants (Basel) 2019, 8 (7), 233. DOI: 10.3390/antiox8070233
10. Medina, S.; Auñón, D.; Lehoux, J. et al. Hydroxytyrosol Fatty Acid Esters as New Candidate Markers for Detecting Olive Oil Inadequate Storage Conditions by UHPLC-QqQ-MS/MS. Microchem. J. 2022,181, 107656. DOI: 10.1016/j.microc.2022.107656