Looking in Depth at Oxidized Cholesteryl Esters by LC–MS/MS: Reporting Specific Fragmentation Fingerprints and Isomer Discrimination

Cholesteryl esters (CE) are prone to oxidation under increased oxidative stress conditions, but little is known about oxidized CE species (oxCE). To date, only a few oxCE have been identified, however, mainly based on the detection of molecular ions by mass spectrometry (MS) or target approaches for specific oxCE. The study of oxCE occurring from radical oxidation is still scarcely addressed. In this work, we made a comprehensive assessment of oxCE derivatives and their specific fragmentation patterns to identify detailed structural features and isomer differentiation using high-resolution C18 HPLC-MS- and MS/MS-based lipidomic approaches. The LC-MS/MS analysis allowed us to pinpoint oxCE structural isomers of long-chain and short-chain species, eluting at different retention times (tR). Data analysis revealed that oxCE can be modified either in the fatty acyl moiety or in the cholesterol ring. The location of the hydroxy/hydroperoxy group originates characteristic fragment ions, namely the unmodified cholestenyl cation (m/z 369) for the isomer with oxidation in the fatty acyl chain or ions at m/z 367 and m/z 385 (369 + 16) when oxygenation occurs in the cholesterol ring. Additionally, we identified CE 18:2 and 20:4 aldehydic and carboxylic short-chain products that showed a clear fragmentation pattern that confirmed the modification in the fatty acyl chain. Specific fragmentation fingerprinting allowed discrimination of the isobaric short-chain species, namely carboxylic short-chain products, from hydroxy aldehyde short-chain products, with a hydroxycholesterol moiety. This new information is important to identify different oxCE in biological samples and will contribute to unraveling their role in biological conditions and diseases such as cardiovascular disease.