Development of direct analytical methods for esterified docosahexaenoic acid hydroperoxide in food and biological systems using liquid chromatography-mass spectrometry

Background

    Accumulating evidence highlights the significance of docosahexaenoic acid (DHA) in brain health. Its six double bonds make DHA highly prone to oxidation, compromising nutritional quality and generating potentially harmful products. Dietary DHA is particularly vulnerable to non-enzymatic oxidation during processing, storage, and cooking, where subsets of DHA hydroperoxide (DHA;OOH) isomers are produced depending on the mechanism (radical- and ¹O₂ oxidation). In biological systems, enzymes (e.g., lipoxygenase) also catalyze peroxidation at specific reaction sites. Identifying DHA;OOH isomers is therefore critical for understanding active oxidation mechanisms. However, analytical limitations have persisted for esterified DHA;OOH, despite DHA being predominantly esterified in complex lipids in both food and biological systems. To address this, we developed direct LC-MS/MS methods for esterified DHA;OOH.

Methods

   Target molecular species were determined based on the lipid composition of mackerel and rat whole brains as representative food and biological samples. Multiple reaction monitoring conditions were optimized using a prepared hydroperoxide isomeric mixture for each target. Optimized LC-MS/MS methods were applied to lipid extracts of a mackerel fillet and a whole brain of a Sprague Dawley rat.

Results and discussion

    Based on the LC-MS/MS analyses of mackerel, PC 16:0/22:6 and TG 18:1_18:1_22:6 were targeted due to their abundance. For rat whole brains, PC 16:0/22:6 and CE 22:6 were selected as targets considering the DHA distribution in lipid pools. MS/MS analyses of the mixture references showed all diagnostic product ions for positional isomers, as in free DHA;OOH in our previous report. Both PC 16:0/22:6;OOH and TG 18:1_18:1_22:6;OOH were detected in a raw mackerel fillet, demonstrating the high sensitivity of the developed LC-MS/MS methods. The developed methods were also capable of detecting several isomers of PC 16:0/22:6;OOH and CE 22:6;OOH in the rat whole brain. The constructed LC-MS/MS methods offer potential applicability in exploring DHA oxidation in both food and biological systems.