13] Determination of hydroperoxides with fluorometric reagent diphenyl-1-pyrenylphosphine

Abstract

Publisher Summary Unsaturated fatty acids and their esters are oxidized to produce hydroperoxides at the initial stage of the peroxidation. Recently, hydroperoxides have attracted much attention as one of the factors that might be associated with some diseases and aging. Several methods have been proposed to determine lipid peroxidation products such as iodometries, thiobarbituric acid (TBA) methods, and gas chromatography. In this chapter, diphenyl-1-pyrenylphosphine (DPPP), a fluorescent reagent is used to determine lipid hydroperoxide. This reagent, which has almost no fluorescence, quantitatively produces a strongly fluorescent oxide (DPPP oxide) and corresponding alcohols by a reaction with lipid hydroperoxides. This reaction can be used successfully for a high-performance liquid chromatography (HPLC) postcolumn detection system based on the favorable reaction rates in various organic solvents. The HPLC system can determine specific or classes of hydroperoxides. The lower detection limit of hydroperoxides using a batch method is 200 pmol and using HPLC is just a few picomoles. As the reagent in solution is unstable especially under strong light, care should be taken to use a peroxide-free solvent and not to handle the solution in strong light. Stock solutions can be kept stable for 1 week in a dark refrigerator.