Abstract
Oxidized fatty acids formed via lipid peroxidation are implicated in pathological processes such as inflammation and atherosclerosis. A number of methods may be used to detect specific oxidized fatty acids containing a single or multiple combinations of epoxide, hydroxyl, ketone, and hydroperoxide moieties on varying carbon chain lengths from C8 up to C30. Some of these methods are nonspecific and their use in biological systems is fraught with difficulty. Measures of specific oxidized fatty acid derivatives help in identifying oxidation pathways in pathological processes. We used liquid chromatography coupled with electrospray ionization tandem mass spectrometry as an efficient, selective, and sensitive method for identifying and analyzing multiple specific fatty acid peroxidation products in human plasma and other biological matrices. We then distilled the essential components of a number of these analyses to provide an efficient protocol by which fatty acid oxidation products and their parent compounds can be determined. In this protocol, addition of a synthetic internal standard to the sample, followed by base hydrolysis at elevated temperature and liquid-liquid phase sample extraction with lighter-than-water solvents, facilitates isolation of the oxidized fatty acid species. These species can be identified and accurately quantified using stable-isotope dilution and multiple-reaction monitoring. Use of a coupled multiplexed gradient HPLC system on the front end enables high-throughput chromatography and more efficient use of mass spectrometer time.