A Targeted Mass Spectrometry Approach to detect and quantify Oxidised Phospholipids in plasma samples of Diabetic patients

Phospholipid oxidation by adventitious damage generates a wide variety of products with potentially novel biological activities that can modulate inflammatory processes associated with various diseases such as atherosclerosis, acute Pancreatitis and Type 2 diabetes. To understand the biological importance of oxidised phospholipids (OxPL) and their potential role as disease biomarkers requires precise information about the abundance of these compounds in cells and tissues. There are many chemiluminescence and spectroscopic assays available for detecting oxidised phospholipids, but they all have some limitations. Mass spectrometry coupled with liquid chromatography is a powerful and sensitive approach but its application to complex biological samples remains challenging. The aim of this work was to develop improved methods for detection of OxPLs, specifically by using targeted mass spectrometry approaches (precursor ion [PIS] and neutral loss [NL] scanning), high resolution mass spectrometry and alternative chromatographic approaches. Initial experiments were carried out using oxidation products generated in vitro from a commercially available phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixture in order to optimise the chromatography separation parameters and mass spectrometry parameters. The chromatographic separation of oxidised phosphatidylcholines (OxPCs) and oxidised phosphatidylethanolamines (OXPEs) was evaluated using C8, C18 and C30 reverse phase, polystyrene divinylbenzene based monolithic and mixed mode hydrophilic interaction (HILIC) columns, interfaced with mass spectrometry. Our results suggest that the divinylbenzene based reverse phase monolithic column gave best separation of short chain OxPCs and OxPEs from long chain oxidised and native PCs and PEs. Targeted mass spectrometric approaches for the selective identification of short chain OxPCs using PIS for m/z 184 Da and NL for m/z 34 Da for identification of hydroperoxides were tested on OxPC mixture, it enabled identification of low abundant oxidation products such as: hydroxy alkenals and alkenoates and saturated aldehydes collectively termed as short chain oxidation products such as PONPC, POVPC and HOOA-PC. The combination of these chromatographic and MS methods allowed identification of several oxidised molecular species in plasma of diabetic patients. Quantitative differences in oxidised products were observed in diabetic samples and the trend showed high abundance of oxidised phosphatidylcholine species in diabetic samples, compared to healthy plasma samples. However, the difference in abundance was statistically not significant when the samples were analysed using Progenesis QI software, performing global normalisation and ANOVA analysis because of inherent biological variability observed for OxPC species in samples.