Polyunsaturated fatty acids (PUFA) are vulnerable to oxidation. This leads to a large number of oxidation products, i.e. oxylipins such as hydro(pero)xy-PUFA and epoxy-PUFA. Epoxy-PUFAs can be formed enzymatically in vegetable oils during seed storage and subsequent pressing. Their formation may also occur via autoxidation during the storage or further processing of oils [J. Agric. Food Chem., 2022, DOI: 10.1021/acs.jafc.2c04987]. Elevated temperatures, such as those used during deep-frying, promote autoxidation and accelerate formation of epoxy fatty acids in the oil [J. Agric. Food Chem., 2023, DOI: 10.1021/acs.jafc.3c00964]. Since polyunsaturated fatty acids are predominantly esterified in triacylglycerols (TAG), which are the main constituents of vegetable oils, the resulting epoxy-PUFA are bound to TAG. Currently, the best way to quantify oxylipins in biological samples is by targeted liquid chromatography tandem mass spectrometry (LC-MS/MS). Esterified oxylipins are indirectly determined as free oxylipins after saponification. However, the information about the lipid class in which the esterified oxylipins were bound is lost.
In this project, TAG bearing oxylipins were analyzed by reversed phase liquid chromatography high resolution mass spectrometry (LC-HRMS). For method optimization, standards with the structure TAG 16:0/epoxy-PUFA/16:0 were synthesized. These were used to characterize the fragmentation pattern and retention times of epoxy-TAG. Moreover, edible oils were treated with meta-chloroperoxybenzoic acid (mCPBA) to generate a large variety of potentially biologically occurring epoxy-TAG. Analyses were carried out using a QExactive HF Orbitrap mass spectrometer operating in Full MS/data dependent MS2 acquisition mode. Epoxy-TAG were analyzed in positive ionization mode (ESI(+)) and precursor ion were detected as ammonium adducts ([M+NH₄]⁺). The bound fatty acids and epoxy-PUFA could be characterized by the product ions of the TAG resulting from neutral loss of the (epoxy-)fatty acids and formation of characteristic epoxy-aldehyde fragments.