Development and Validation of a High-Throughput Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Approach for Screening of Oxylipins and Their Precursors

Abstract

Lipid mediators, highly bioactive compounds synthesized from polyunsaturated fatty acids (PUFAs), have a fundamental role in the initiation and signaling of the inflammatory response. Although extensively studied in isolation, only a limited number of analytical methods offer a comprehensive coverage of the oxylipin synthetic cascade applicable to a wide range of human biofluids. We report the development of an ultrahigh-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UHPLC-MS) assay to quantify oxylipins and their PUFA precursors in 100 μL of human serum, plasma, urine, and cell culture supernatant. A single 15 min UHPLC run enables the quantification of 43 oxylipins and 5 PUFAs, covering pro and anti-inflammatory lipid mediators synthesized across the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) pathways. The method was validated in multiple biofluid matrixes (serum, plasma, urine, and cell supernatant) and suppliers, ensuring its suitability for large scale metabonomic studies. The approach is accurate, precise, and reproducible (RSD < 15%) over multiple days and concentrations. Very high sensitivity is achieved with limits of quantification inferior to picograms for the majority of analytes (0.05-125 pg) and linear range spanning up to 5 orders of magnitude. This enabled the quantification of the great majority of these analytes at their low endogenous level in human biofluids. We successfully applied the procedure to individuals undergoing a fasting intervention; oxylipin profiles highlighted significantly altered PUFA and inflammatory profiles in accordance with previously published studies as well as offered new insight on the modulation of the biosynthetic cascade responsible for the regulation of oxylipins.

Longitudinal analysis of serum oxylipin profile as a novel descriptor of the inflammatory response to surgery

“…There were no differences in the procedures, underlying pathologies or development of post-operative complications between the two groups. However, patients undergoing laparoscopic surgery had a significantly shorter length of stay (7 [6–10] vs. 10 [8–15], p  = 0.047).…”

“…Recent developments in analytical platforms allowed us to develop an assay for the simultaneous analysis of numerous lipid mediators in biofluids [10]. In the present investigation we applied this oxylipin assay to adult patients undergoing elective colorectal surgery to demonstrate that oxylipin profiling is feasible in a clinical context and that it has the potential to provide novel insights into the systemic inflammatory response.…”

“…Sample preparation and the quantification of lipid mediators by ultra high-performance liquid chromatography (UPLC)-mass spectrometry followed the previously published method of Wolfer et al [10], resulting in the quantification of 21 oxylipins and PUFAs (Table 1). The procedure is described in detail in the Additional file 1 and summarised below.…”

“…We have previously described a novel, highly sensitive assay for the detection in biofluids of a panel of oxylipins and PUFAs spanning the major PUFA precursors and the three principal biosynthetic enzyme pathways (CYP450, COX and LOX) [10]. In the present study, we applied this assay to longitudinally quantify the serum levels of key oxylipins in patients undergoing colorectal surgery.…”

Lipidomic profiling of targeted oxylipins with ultra-performance liquid chromatography-tandem mass spectrometry

“…Yang et al [44] quantified 39 oxylipins originating from AA and LA including EETs and 20-HETE that regulate vascular function [45, 46]. Wolfer et al [47] profiled 43 oxylipins generated from LA, EPA, DHA, and AA, including 8-isoPGF2α, a biomarker for oxidative stress. A few methods papers have targeted over 100 oxylipins for quantitative lipidomic investigation [3, 48, 49].…”