Analysis of\nIntact <i>In Vivo</i> Peroxidized\nPhospholipids from Bovine Retina via LC-MS/MS and GC-MS/MS Using Autoxidized\nRetina Reference Standards

Highly\nunsaturated fatty acids (HUFAs) are vulnerable to oxygen\nattack, thus making HUFA-rich, high metabolic rate/reactive oxygen\nspecies (ROS)-generating neurological tissue particularly susceptible\nto increased oxidative stress. Lipid oxidation is a putative early\nstage marker of neurodegenerative diseases, suggesting that reliable\nmonitoring of oxidized neural lipids <i>in vivo</i> reveals\nearly pathogenesis. Here, we present a novel methodology to detect\nand quantify intact<i>in vivo</i> ROS-driven peroxidized\nphospholipids (LPOx-PLs) in bovine retina extract. A protocol for\npreparing autoxidized pure phospholipids (PLs) and complex retinal\nextracts served as reference standards and was adapted to enable analytical\nparameter development. Fatty acid profiles of bovine retinas were\nfirst established with routine gas chromatography (GC) methods and\nused to customize mass spectrometry scanning for major HUFA-carrying\nPLs in the retinal extract. Targeted multiple reaction monitoring\n(MRM) scanning via triple quadrupole tandem mass spectrometry detected\nnative (unoxidized) and oxidation-damaged PL regardless of the position\nof the O or O<sub>2</sub> addition along the acyl chains and enabled\nquantification of relative signals from intact native and oxidized\nPL (5%–10% CV). MRM-triggered information-dependent acquisition\n(IDA) spectra confirmed the structure of peroxidized PLs, revealing\nthat peroxidized species (+O–OH) dominated over single O-added\nspecies <i>in vitro</i> and <i>in vivo</i>. Positive\nidentification and relative quantification are reported for 12 selected <i>in vivo</i> native and peroxidized phosphatidylcholines and\nphosphatidylethanolamines. These results enable future studies of\nthe initial peroxidation due to toxins, genetics, or other initiating\nevents influencing <i>in vivo</i> oxidation levels and potentially\nthe effectiveness of strategies to mitigate this mechanism of action.