Targeting esterified oxylipins by LC–MS - Effect of sample preparation on oxylipin pattern

Publication date: February 2020Source: Prostaglandins & Other Lipid Mediators, Volume 146Author(s): Annika I. Ostermann, Elisabeth Koch, Katharina M. Rund, Laura Kutzner, Malwina Mainka, Nils Helge SchebbAbstractA major part of oxygenated metabolites of polyunsaturated fatty acids – i.e. eicosanoids and other oxylipins – in biological samples is found in the esterified form. Yet, their biological role is only poorly understood. For quantification of esterified oxylipins in biological samples current protocols mostly apply alkaline hydrolysis with or without prior lipid extraction to release oxylipins into their free form which can be subsequently quantified via liquid chromatography-mass spectrometry.Herein, a detailed protocol for precise and reproducible quantification of esterified oxylipins in plasma is presented comprising i) extraction of lipids and removal of proteins with iso-propanol, ii) base hydrolysis with potassium hydroxide to saponify lipids and iii) solid phase extraction of the liberated oxylipins on C8/anion exchange mixed mode material. Unequal extraction of internal standards and lipid classes during lipid extraction before hydrolysis led to distorted concentrations, emphasizing that the choice of solvent used in this step is important to minimize discrimination. Regarding the hydrolysis conditions, at least 30 min incubation at 60 °C is required with 0.1 M KOH in sample. Drying of the SPE cartridges is a critical parameter since autoxidation pr...
Source: Prostaglandins and Other Lipid Mediators - Category: Lipidology Source Type: research