Simultaneous Analysis of Multiple Lipid Oxidation Products In Vivo by Liquid Chromatographic-Mass Spectrometry (LC-MS)

Free radical-induced oxidation of polyunsaturated fatty acid (PUFAs) has been linked to a number of human diseases including atherosclerosis and neurodegenerative disorders. Oxidation of PUFAs generates hydroperoxides and cyclic peroxides that are reduced to lipid alcohol, such as hydroxyeicosatetraenoic acid (HETEs), and isoprostanes (IsoPs) respectively. The IsoPs are isomers of prostaglandins that are generated from autoxidation of arachidonic acid (C20:4). Quantification of F₂ -IsoPs has been regarded as the “gold standard” to assess oxidative stress status in various human diseases. We herein report the protocol of analyzing HETEs and F₂ -IsoPs using a triple quadrupole mass spectrometer coupled to reverse phase liquid chromatography. The selected reaction monitoring (SRM) mode selects the parent ion of interest in the first Quad (m /z 319 for HETE and m /z 353 for F₂ -IsoPs) and fragments it in the second while an ion characteristic of the analyte of interest is monitored in the third Quad. This highly selective technique permits the simultaneous analysis of multiple oxidation products such as the HETEs and F₂ -IsoPs. This LC-MS technique can be applied to study the free radical oxidation mechanism in vitro and assess the oxidative stress status in biological tissues and fluids.