DEVELOPMENT AND VALIDATION OF FATTY ACID HYDROPEROXIDE ANALYSIS BY LC-MS: APPLICATION TO THE BEIJING OLYMPICS AIR POLLUTION STUDY
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Introduction: Lipid hydroperoxides are compounds created through free radical attack on poly-unsaturated fatty acids and enzymatic processes. They are a common byproduct of oxidative stress and thus can be used as markers of levels of oxidative stress. A method was developed using LC-MS detection to analyze several lipid hydroperoxides and their derivatives. This method was then applied to the analysis of lipid hydroperoxides response to pollution using Beijing Olympic Air Pollution (BOAP) study samples.Method: The saponification and extraction were optimized by looking at the degree of cholesteryl ester saponification. Different stationary phases were tested for optimal resolution (Alkyl C-18 and C-8, Aq C-18, biphenyl) and a new mobile phase adding propanol and ammonium acetate was tested for improved ionization. The method was partially validated before being applied to 150 complete and 10 incomplete sets of samples. Each set consisted of an individual’s plasma drawn before, during and after reduction in pollution during the Olympics. Results: Saponification was extended to 180min at room temperature to maximize the degree of saponification without creating artifacts. The best resolution was achieved with a UPLC BEH-C18 column with the acetonitrile/propanol gradient with water. The ionization was also improved with ammonium acetate. The hydroxy derivatives 13-HODE and 9-HODE both had significant increases when pollution was reduced and 12-HETE decreased and increased by statistically significant amounts over the three time points. Conclusion: The new HPLC-MS method is both accurate and has high precision for several key hydroxy fatty acids. Analysis of the BOAP samples showed 12-HETE’s concentration following the same pattern as in previous untargeted metabolomics studies.