Evaluation of oxidized phospholipids analysis by LC-MS/MS

Spiro Khoury, Corinne Pouyet, Bernard Lyan, Estelle Pujos-Guillot

Phospholipids (PLs) represent a class of metabolites of interest for evaluating the relationship between diet and the development of several metabolic diseases. Given that PLs are rich in unsaturated fatty acids, they can be oxidized. Because of their structure and reactivity, oxidized phospholipids (PLs-Ox) are increasingly recognized as markers of oxidative stress and of various diseases associated with inflammation. Therefore, there is a growing interest in studying PLs-Ox in lipidomics. Because of their limited commercial availability, very little information is currently available in databases to identify these molecules. The aim of this study is to acquire new knowledge about PLs-Ox in order to propose an analytical strategy for their analyses. For this purpose, a synthesis method of PLs-Ox, in auto-oxidation, has been developed and applied on phosphatidylcholine and phosphatidylethanolamine molecular species with various chain lengths, degree, and position of unsaturations. An analysis method based on mass (MS) and tandem mass spectrometry coupled to electrospray ionization was then developed and enabled the identification of a great diversity of long- and short-chain oxidation products. Formation kinetics of oxidation products was evaluated. Results showed that the formation of oxidized compounds was largely influenced by the degree of unsaturation on fatty acid chains. Oxidation time promotes the formation of some biologically important oxidation products. Coupling the MS method with liquid chromatography in flow injection analysis mode enabled the development of a full analytical strategy. Structural analysis of PLs-Ox allowed the enrichment of databases with important information to identify these molecules in biological matrices.