Development for separation analysis of micro free radical materials and its application for disease elucidation

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The roles of free radicals in the body have been investigated in the relationship with various diseases and the detection methods are attracting much attention from various medical fields. Many kind of free radicals in various chemical and biological systems have been successfully detected and identified by an electron spin resonance (ESR) spectroscopy. Carboxylic acids are important as nutritional substrates and metabolites in living organisms. It was found that phenacyl esters, which are derived from phenacyl bromide and carboxyl acids, give rise to the free radicals under the alkaline condition, The p-nitrophenacyl esters derived from p-nitrophenacyl bromide and carboxylic acids were determined by a high-performance liquid chromatography with ESR and ECD as detection. The p-nitrophenacyl bromide was used as a pre-column reagent to derivative the carboxylic acids. The p-nitrophenacyl esters of carboxylic acid gave free radicals by hydrolysis with an alkaline and then the free radicals were detected with ESR and ECD.The ECD peak height was related to the concentration with high linearity in the range of 10-500 pmol for acetic acid, 50-500 pmol for propionic acid, 50-1000 pmol for butyric acid, 0.1-1nmol for valeric and 0.1- 1nmol for hexanoic acid.The radical chromatography using ESR for HPLC detector was carried out for simultaneous detection of free radicals derived from similar spin adducts of g value.