Development and Validation of a High-Throughput Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Approach for Screening of Oxylipins and Their Precursors.
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.
Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London , Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom.
Arnaud M
AM
Wolfer
Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London , Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom.
Mathieu
M
Gaudin
Department of Medicine, Imperial College London , St. Mary's Campus, London, United Kingdom.
Simon D
SD
Taylor-Robinson
Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London , Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom.|MRC-NIHR National Phenome Centre, Imperial College London , IRDB Building, Hammersmith Hospital, London W12 0NN, United Kingdom.
Elaine
E
Holmes
Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London , Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom.|MRC-NIHR National Phenome Centre, Imperial College London , IRDB Building, Hammersmith Hospital, London W12 0NN, United Kingdom.
Jeremy K
JK
Nicholson
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005231:Fatty Acids, Unsaturated / Q000032:analysis*; D057166:High-Throughput Screening Assays; D006801:Humans; D013058:Mass Spectrometry; D054883:Oxylipins / Q000032:analysis*
D005231:Fatty Acids, Unsaturated; D054883:Oxylipins