Identification and Quantification of Key Radicals in Lipid Oxidation via Spin Trapping and LC–MS/MS–IT–TOF

Accurate qualitative and quantitative determination of free radicals during lipid oxidation, which is the core process of edible oil quality deterioration and essentially a free radical chain reaction, is of crucial importance. To overcome this problem, we developed and validated an analytical method for the specific characterization of key radicals in lipid oxidation. These radicals (R•, RO•, and ROO•) were trapped using N-tert -butyl-α-phenylnitrone under iron-mediated conditions, leading to the formation of stable adducts that were subsequently analyzed by LC–MS/MS–IT–TOF. This method offers good sensitivity with low limits of detection (0.06–0.15 µM) and limits of quantification (0.20–0.50 µM). Density functional theory calculations further confirm that the reaction proceeds readily and yields thermodynamically stable products. When applied to the methyl linoleate oxidation system, the method demonstrated that methyl linoleate alkyl radical (L•) predominated during oxidation, with its concentration peaking at 3.74 × 10 −2 mM (accounting for up to 70% of radicals) at 4 h followed by a decline due to chain termination reactions, whereas methyl linoleate peroxyl radicals (LOO•) concentration stabilized as oxidation progressed (1.46 × 10 −2 mM, 27% of radicals), and methyl linoleate alkoxyl radicals (LO•) remained at a consistently low concentration (1.58 × 10 −3 mM) owing to its preferential conversion via the β-scission pathway. These results were consistent with electron spin resonance (ESR) data, thereby validating the method's reliability. Consequently, this method enables the precise monitoring of lipid oxidation processes, serving as a valuable tool for assessing antioxidant efficacy in lipid science. Practical Applications : We present an analytical method integrating N-tert -butyl-α-phenylnitrone (PBN)-based spin trapping with LC–MS/MS–IT–TOF for direct tracking of key lipid radicals (L•, LO•, and LOO•) throughout the lipid oxidation process. This method addresses a long-standing technical gap in lipid oxidation research, providing a robust, practical tool for both high-throughput screening of high-efficacy antioxidants and rational design of stable lipid-containing food formulations. By bridging fundamental analysis and product development, our technique has clear real-world implications for extending the shelf life of lipid-based foods, offering significant value to food chemistry research while delivering substantial economic and sustainability benefits to the global food industry.