Formation of oxidized triacylglycerols during frying-like heating of trilinolein: Isotope-tracing evidence for the roles of oxygen and water in oil deterioration

Oxidized triacylglycerols (OxTGs) are major oxidation products generated during thermal oxidation of edible oils, but the respective roles of molecular oxygen and water in their formation during frying remain unclear. In this study, trilinolein was heated at 180 °C for 4 h under frying-like conditions, and hydrogen–oxygen isotope tracing using D₂O, H₂ 18 O, and 18 O₂ was combined with UHPLC–HRMS/MS, GC–MS, and 1 H NMR to investigate OxTG formation and its relationship with oil deterioration. During heating, total polar compounds increased markedly and reached 23.95–31.35 g/100 g oil at 4 h, while total OxTGs accumulated to 12.97–14.61 g/100 g oil. Concomitantly, secondary oxidation products also accumulated continuously, and total glycerol-core aldehydes reached 20.86 mg/g, with 9-oxo being the predominant aldehydic product. Isotopologue analysis showed pronounced 18 O enrichment in major OxTGs only in the 18 O₂ treatment, whereas incorporation from H₂ 18 O was negligible, indicating that oxygen incorporated into major OxTGs originated predominantly from molecular oxygen rather than water. By contrast, D₂O suppressed the accumulation of OxTGs and aldehydic products, supporting the involvement of water primarily in hydrogen-transfer and exchange processes rather than direct oxygen incorporation. Hydroperoxy OxTGs were more characteristic of early oxidation, whereas hydroxylated OxTGs were more closely associated with prolonged heating and the formation of secondary carbonyl products. These results provide isotope-based evidence for the formation pathways of OxTGs during frying-like heating and support the combined use of OxTGs, total polar compounds, and carbonyl-related markers for evaluating oxidative deterioration in heated oils.