LC-ESI-MS/MS Analysis of arachidonic acid and its metabolites prostaglandins, HETEs, thromboxanes and leukotrienes in mammalian cells and tissues

Almost all mammalian cells produce eicosanoids and their oxidation products, which are synthesized rapidly by cyclooxygenase, lipoxygenase or epoxygenase reactions from arachidonic, homogammalinolenic, eicosapentanoic acids. They have physiological effects at trace concentrations, such as local mediation of inflammatory responses (1). Mass spectrometry coupled with electrospray ionization has been widely utilized for trace analysis of eicosanoids (2) combined with HPLC. We describe a LC-ESI-MS/MS method for analysis of arachidonic acid and its metabolites; prostaglandins, HETEs, thromboxanes and leukotrienes, within a single sample run. The method will enable targeted differential profiling and quantitative analysis of these compounds. The compounds of interest are extracted by liquid-liquid or solid phase extraction. The analytes are resolved by reversed phase chromatography, ionized by electrospray in negative ion mode and detected in MRM mode. Our aim is to measure at the order of 50 eicosanoid, HETE, and fatty acid molecular species in parallel at tissue level concentrations in order to evaluate active biochemical pathways in specific biological state. We will describe the analytical method, as well as demonstrate its utility on mouse model tissue samples related to studies of type 2 diabetes and metabolic syndrome.