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

采用化学发光法(CL)检测高选择性、高灵敏度的过氧化脂质。为此，建立了一种具有在线光反应和过氧草酸化学发光(PO-CL)检测的高效液相色谱(HPLC)系统。PO-CL反应需要过氧化氢、荧光团和二芳基草酸盐才能显示CL。在该体系中，过氧化氢是由过氧化氢在紫外光照射下产生的，因此只有用荧光团标记的过氧化脂才能显示化学发光，这使得该方法具有很高的选择性。使用标准过氧化脂质对体系进行了评价，标准过氧化脂质是油酸在亚甲基蓝存在下光敏氧化制备的，然后用4，5-二苯基-2-(4-肼甲酰基)苯基咪唑(HCPI)或2-(2，3-萘亚氨基)乙基三氟甲烷磺酸盐(NE-OTF)进行荧光标记。结果发现，该方法对过氧化脂质具有选择性，但不像预期的那样灵敏。这是由于过氧化氢在紫外线照射下产生过氧化氢的产率较低。因此，我们尝试用芬顿反应来代替紫外光照射来提高灵敏度。利用Fenton反应观察到化学发光强度增加了15倍。在本研究中，我们选择了含有过氧化氢和荧光团的分析物分子用于PO-CL的检测。然而，荧光团将能够被化学发光团取代。通过检测从过氧化脂质中产生活性氧物种的反应以实现高效的化学发光反应，将提高方法的灵敏度。

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.