Optimizing Oxylipin Analysis with Liquid Chromatography Mass Spectrometry through Bio-Inert Systems and Ion Funnel Adjustments

Abstract

Oxylipins are potent signaling lipids that affect inflammation, vascular tone, and metabolism, making them relevant in many diseases. Oxylipins are measured with liquid chromatography-mass spectrometry (LC-MS), but challenges in quantification arise due to low abundance and rapid degradation. In this study, we optimize LC-MS methods to improve the quantification of oxylipins in human plasma, given growing interest in oxylipins and their impact on clinical research. Plasma samples were obtained from healthy participants, and oxylipins were concentrated by solid-phase extraction. Quantification was performed with a reversed-phase targeted LC-MS/MS method on an Agilent 6495D triple quadrupole with transitions for 248 oxylipin species. Given the rapid degradation of oxylipins with bio-reactive surfaces, we compared both standard and bio-inert (Altura) columns, standard and bio-inert LC setups, and ion funnel voltage. By decreasing voltages applied to ions inside the ion funnel, signal was increased for most oxylipin species while peak quality was maintained. Fully bio-inert setups quantified more compounds and had increased levels of some compounds, but fewer epoxyoctadecadienoic acid (EpODE) species. To explore this further, analytical-grade alpha-linolenic acid (ALA), the direct precursor of EpODEs, was injected and observed to form EpODEs within the instrumentation when using stainless steel columns. These data suggest that oxylipins benefit from fully bio-inert systems and optimized pre-mass analyzer parameters. The stainless-steel components of the column may also contribute to epoxidation reactions of polyunsaturated fatty acids (PUFAs), generating oxylipin species during analysis. Finally, various human sample matrices were assessed for compatibility with oxylipin quantification, including granulocytes, mononuclear cells, erythrocytes, skeletal muscle, and THP-1 cells, a human-derived monocyte cell line.