Chemical Recognition of Oxidation-Specific Epitopes in Low-Density Lipoproteins by a Nanoparticle Based Concept for Trapping, Enrichment, and Liquid Chromatography-Tandem Mass Spectrometry Analysis of Oxidative Stress Biomarkers

Monitoring bioactive oxidized phospholipids (OxPLs), such as 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine (PONPC), is of major interest as they play a crucial role in a variety of age related diseases, e.g., in the development and progression of atherosclerosis. Since they are in low abundance in samples like oxidized low-density lipoproteins (OxLDL) and human plasma, respectively, their analysis as risk biomarkers requires the combination of an efficient selective sample preparation with highly sensitive detection methods, such as liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). In this study, a nanoparticle-based strategy for successful trapping and enrichment of aldehyde-containing oxidized phospholipids is presented. The concept involves a derivatization step with a bifunctional reagent containing both a hydrazide group for hydrazone formation with carbonyl-containing PLs and a thiol moiety for subsequent trapping on GNPs. After washing, the trapped analytes are quantitatively released from the nanoparticles' surface by transimination with hydroxylamine. The released oxime-derivatives of the carbonylated-OxPLs are subsequently analyzed by LC-ESI-MS/MS in the selected reaction monitoring scan mode. Several parameters of this workflow were optimized. With the optimized nanoparticle-based extraction and enrichment step, very clean extracts of these biomarkers can be obtained and the detection limits can be significantly decreased from 2.76 and 2.65 nM for PONPC and POVPC, respectively, to 0.17 and 0.44 nM. The applicability of this nanoparticle-based sample preparation concept was demonstrated by successful extraction of oxidized phospholipids from biological samples, such as human plasma, MDA-modified LDL and Cu2+-oxidized LDL.