Identification of all classes of spin-trapped carbon-centered radicals in soybean lipoxygenase-dependent lipid peroxidations of ω-6 polyunsaturated fatty acids via LC/ESR, LC/MS, and tandem MS

Using the combined techniques of on-line high performance liquid chromatography/electron spin resonance (LC/ESR) and mass spectrometry (MS), we previously identified spin-trapped adducts of all expected carbon-centered lipid-derived radicals (•Ld) formed in linoleic acid peroxidation. In the present study, spin trapped lipid-derived carbon-centered radicals formed from the reactions of two ω-6 polyunsaturated fatty acids (PUFAs: linoleic and arachidonic acids) with soybean lipoxygenase in the presence of α-[4-pyridyl 1-oxide]-N-tert-butyl nitrone (POBN) were identified using a combination of LC/ESR and LC/MS. All expected lipid-derived carbon-centered radicals in lipoxygenase-dependent peroxidations of linoleic acid and arachidonic acid were detected and identified by the combination of LC/ESR and LC/MS with confirmation by tandem mass spectrometry (MS/MS). The five classes of •Ld formed from both ω-6 PUFAs including lipid alkyl radicals (L•), epoxyallyic radicals (OL•), dihydroxyallyic radicals (•L(OH)2), and a variety of R• and •RCOOH from β-scission of lipid alkoxyl radicals, gave distinct retention times: POBN/•L(OH)2 4–6 min, POBN/R• and POBN/•RCOOH 8–22 min, POBN/L• and PBON/OL• 25–36 min. The major β-scission products in peroxidations of ω-6 PUFAs were the pentyl radicals. The ratio of β-scission products, however, varied significantly depending on pH, [PUFA], as well as [O2].