DEVELOPMENT OF HIGHLY SELECTIVE AND SENSITIVE DETERMINATION METHOD FOR LIPID PEROXIDES AND ITS APPLICATION TO BIOLOGICAL SAMPLES

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Highly selective and sensitive determination of lipid peroxides was examined by the use of chemiluminescence (CL) detection. For this purpose, a high-performance liquid chromatographic (HPLC) system with on-line photoreactor and peroxyoxalate chemiluminescence (PO-CL) detection was developed. The PO-CL reaction requires hydrogen peroxide, fluorophore and diaryloxalate in order to show CL. In the proposed system, hydrogen peroxide is generated from lipid peroxide under UV radiation, and thus only the lipid peroxide labeled with fluorophore is expected to show CL, which makes the method very selective.The system was evaluated by using standard lipid peroxides, which were prepared by photosensitized oxidation of oleic acid in the presence of methylene blue and followed by fluorescence (FL) labeling with 4,5-diphenyl-2-(4-hydrazinocarbonyl)phenyl imidazole (HCPI) or 2-(2,3-naphthalimino)ethyltrifluoromethane sulfonate (NE-OTf). As a result, the method was found to be selective for lipid peroxides but was not as sensitive as expected. This was due to the low generation yield of hydrogen peroxide from hydroperoxide by UV irradiation. We therefore attempted to increase the sensitivity by using Fenton reaction instead of UV irradiation. Fifteen-fold increase in CL intensity was observed by using the Fenton reaction. This sensitivity will further be improved by optimizing the Fenton reaction.In this research, we selected the analyte molecule having hydroperoxide and fluorophore for PO-CL detection. However, the fluorophore will be able to replace with chemiluminophore. The sensitivity of the method will be improved by examining the reaction that generates reactive oxygen species from lipid peroxides for the efficient CL reaction.