Phospholipids that contain polyunsaturated fatty acid are easily oxidized by free radicals or oxidants and can yield numerous oxidation species, including positional and structural isomers. However, it is difficult to separate these oxidation products for structural analysis. In this study, a high-resolution separation analytical system based on supercritical fluid chromatography with tandem mass spectrometry (SFC/MS/MS) was established for the separation and identification of oxidized phosphatidylcholine (PC) isomers derived from esterified linoleic acid or arachidonic acid. Separation of oxidatively modified PC containing hydroxy, epoxy and hydroperoxy groups was achieved by SFC. Positional isomers of hydroxides and epoxides were identified based on MS/MS fragment information. To investigate whether this method is applicable to biological samples, we then analyzed oxidized PC isomers from mouse liver. Oxidized isomers, such as hydroxides, hydroperoxides and epoxides, were simultaneously observed. This method may be a powerful tool for providing further insight into how oxidized phospholipids are produced and are correlated with various diseases.
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