Lipidomic analysis of polyunsaturated fatty acids and their oxygenated metabolites in plasma by solid-phase extraction followed by LC-MS

Abstract

The present work describes the development of a robust and sensitive targeted analysis platform for the simultaneous quantification in blood plasma of lipid oxygenated mediators and fatty acids using solid-phase extraction (SPE) and high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The concurrent analysis of these lipid mediators is challenging because of their instability, differences in solubility, and the frequent occurrence of isobaric forms with similar fragmentation patterns. Results demonstrated that the reduction of SPE temperature to 4 °C is a critical parameter for preserving the hydroperoxy derivatives. Polymeric HLB cartridges increased 40-50 % ARA, EPA, and DHA sensitivity compared to C18 sorbent and also provided higher global performance for most hydroxides and other oxidation products. The proposed method for the two tested mass analyzers yields high sensitivity, good linearity, and reproducibility, with detection limits ranging 0.002-7 ng/mL and global recoveries as high as 85-112 %. However, the additional advantage of the linear ion trap (LIT) mass analyzer working in full scan product ion mode, compared to the triple quadrupole (QqQ) operating in multiple reaction monitoring (MRM), should be noted: the full scan product ion mode provides the full fragmentation spectra of compounds that allowed the discrimination of coeluting isomers and false positive identifications without additional chromatography development. The proposed lipidomic procedure demonstrates a confident, simple, and sensitive method to profile in plasma a wide range of lipid eicosanoid and docosanoid mediators, including innovatively the analysis of hydroperoxy congeners and nonoxidized PUFA precursors.

Oxidative Lipidomics Coming of Age: Advances in Analysis of Oxidized Phospholipids in Physiology and Pathology

“…Fully targeted MRM-based methods have been in use for a number of years and there are a substantial number of publications related to physiology and disease, especially for nonesterified lipid oxidation products (oxylipins) (19,121,147,154), but the semi-targeted methods are newer and currently there are fewer applications. As oxidized phospholipids are formed in inflammatory situations, many of the applications have been to cardiovascular conditions, such as ischemia-reperfusion injury and atherosclerosis, or infectious diseases, leading to systemic or chronic inflammation.…”

Recent developments in fatty acids profile determination in biological samples - a review

“…The extraction and/or a pre-concentration step is needed because the oxygenated PUFA metabolites show low physiological levels and a large number of isomers, which can harden the analytical determination (30). Because of their low concentration, the identification and separation of FAE remains problematic (24).…”

“…The LIT system had the advantages of lower matrix effect and higher global recovery than in the case of the QqQ system. Although the QqQ assays were highly sensitive, when applied to rat plasma samples, they gave several false identifications, because many isomers with similar MRM transitions were co-eluted (30).…”

“…It is important to elucidate the relationship between odd number FA and bacterial infections in meibomian gland dysfunction patients, because bacteria can produce an odd number of FAs (25). On the other hand many hydroxyl-derived compounds of EPA and DHA are involved in the regulation of vascular tone, arteriosclerosis and are considered markers of lipid peroxidation (30).…”

“…However, liquid-chromatography (4,17,(23)(24)(25)(26)(27)(28)(29)(30)(31) has been presented by several authors and also, there are few articles which deal with other techniques such as capillary electrophoresis or infrared (IR) and Raman spectroscopy (16,31,32).…”