• oxylipins
• micro-extraction by packed sorbent
• lipidomics
• uhplc-ms/ms

Oxylipins are powerful lipid mediators involved in numerous physiological and pathological processes, generated from both omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) via one or more mono- or dioxygen-dependent reactions. The interest in their profiling has considerably grown during recent years, notably in the active field of biomarker discovery. Mass spectrometry-based metabolomics of oxylipins represents a very interesting tool for various applications in clinical research, but the impact of analytical and biological parameters on the variability of oxylipin patterns needs further understanding. The analytical determination of oxylipins still remains challenging, mostly because of their extremely low concentration levels (ppt range). Therefore, particular attention has to be paid on sample pre-treatment to remove matrix interferents and to concentrate analytes, thus increasing the sensitivity as well as improving the detection limits. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, low-cost and low-solvent extraction, and on-line coupling with analytical instruments. Micro-Extraction by Packed Sorbent (MEPS) presents all these desirable characteristics and, thus, it has emerged as a powerful technique, being much more environment-friendly and less expensive than solid-phase extraction (SPE) and liquid-liquid extraction (LLE) approaches. For the first time in this PhD thesis, the very reliable combination of the off-line semi-automated MEPS technique and liquid chromatography-tandem mass spectrometry was applied to the analysis of oxylipins, as isoprostanes, prostaglandins, epoxyeicosatrienoic and hydroxyeicosatetraenoic acids. The MEPS-UHPLC-MS/MS workflow was fully developed and validated for the targeted and non-targeted profiling of oxylipins in a wide array of biological matrices, as Dried Blood Spots (DBSs), oral fluid and plasma. The novel and fast C18-MEPS procedure guaranteed limits of detection much lower than those reported in literature (5-50 ppts), satisfactory recovery (> 85%) and very good intra- and inter-day precision (RSD < 15%) for most of the oxylipins investigated, by substantially reducing the extraction time (10-20 min), the required volume of both solvents (30-500 μL) and samples (50-500 μL). Furthemore, contrary to the SPE, MEPS cartridge was cost-efficient, being reusable up to 100 times without any loss of extraction efficiency (within ± 10%).
Our results clearly demonstrated the usefulness of the internal standard addition to improve the analytical performances of the methods, especially for the DBS assay. For the first time, the innovative internal standard addition performed directly on the card before DBS sampling has been demonstrated to be extremely useful for the correction of analyte variation during the critical storage step. This strategy led to a substantial improvement of the entire assay, thus ensuring a broad applicability of the proposed workflow to the clinical field.
The validated MEPS-UHPLC-MS/MS platforms were tested within the framework of three exploratory studies aimed at monitoring inflammation status and oxidant injury in preterm newborns suffering from Patent Ductus Arteriosus, in athletes performing an incremental cycle ergometer test and in acute stroke patients. Most of the oxygenated metabolites forming the complex oxylipin network that were detected in our samples (e.g. (F2/E2-isoprostanes, F2-dihomo-isoprostanes and F4-neuroprostanes, epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids) did not match with the standards and remained unknown. Herein, to greatly expand the repertoire of oxylipins assayed, suspect screening profiling by high resolution mass spectrometry was used, trying to extend the spectrum of oxylipins monitored in humans and provide a foundation for new insight into the role of bioactive lipids in mediating health and disease.