Abstract 618: Novel High-Resolution Mass Spectroscopy Methodologies to Study Anti-inflammatory Nitro Fatty Acids

Abstract

It is widely hypothesized that the protective effects of certain polyunsaturated fatty acids involves the enzymatic or non-enzymatic conversion to biologically active metabolites. Accordingly, definitive identification and quantitation of bioactive fatty acid derivatives is essential for both evaluating the beneficial effects of these nutrients and identifying biological markers to monitor the therapeutic efficacy of fatty acid administration. Nitro-fatty acids are a class of fatty acid metabolites that are formed by chemical nitration of the double bonds of unsaturated fatty acids, and in mouse models, nitro-fatty acids decrease oxidative stress and inflammation suggesting that these could be biologically relevant mediators. However, due to inherent limitations of currently used analytical approaches for the detection and quantitation of free and esterified nitro-fatty acids, many questions remain to be elucidated regarding their abundance and clinical efficacy. Thus, we hypothesize that using an AMMP derivatization extraction procedure coupled with the use of HR electrospray ionization tandem mass spectrometry and stable isotope dilution will lead to a more accurate profile of nitro-fatty acids in biological samples. To accomplish this we generated methyl pyridinium derivatives of nitro-fatty acids which ionize strongly in positive mode resulting in increased sensitivity of more than 1000-fold over underivatized fatty acids monitored in negative ionization mode. We next developed methods for analysis of methyl pyridinium derivatives of nitro-fatty acids using an AB Sciex 5600 quadrupole TOF MS. Together, these methods allow us to identify the ions of the parent nitro-fatty acid derivative at high resolution and confirm their identities by other techniques such as mass defect and isotopomer ratio pattern analyses. To validate these methods, plasma was collected from C57BL/6 mice and levels of nitro-oleic acid were successfully determined using TOF product ions of 386.31 and 433.31 as key identifiers. In conclusion, we have developed and validated novel analytical methodologies to study nitro-fatty acids which may aide in advancing research of nitro-fatty acids as a therapeutic agent against inflammation and atherosclerosis.