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

A 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 processes of PUFA, which are present in high concentrations after cleavage, lead to artificial formation of epoxy fatty acids. With the developed protocol, inter-day, intra-day and inter-operator variance was <21% for most oxylipins including hydroxy-, dihydroxy-, and epoxy-PUFA. The applicability of the developed methodology is demonstrated by investigating the changes in the oxylipin pattern following omega-3 fatty acid feeding to rats.