Quantitative Spin-trapping ESR Investigation of Alkoxyl Radical Derived from AAPH: Development of a Flow-injection Spin-trapping ESR System for the Oxygen Radical Absorbance Capacity Assay

Abstract

A flow-injection electron spin resonance (ESR) system was developed for the quantitative detection of an alkoxyl radical (RO·, R = C(CH3)2–C(+NH2Cl−)NH2) derived from AAPH (2,2′-azobis(2,4-amidinopropane) dihydrochloride) by thermal decomposition. Optimal measurement conditions for the system were examined, and it was found that the control of the radical formation by quenching in an ice-water vessel should be effective to obtain the stable and accurate results. The system was applied for the estimation of the alkoxyl radical-elimination ability and the oxygen radical absorbance capacity values of selected biosubstances, such as Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), caffeic acid, 4-hydroxycinnamic acid, epinephrine, rutin, (+)-catechin, l-tryptophane, and d-mannitol. As the rate constant for the reaction between alkoxyl radical and substrate, k S, could not be unequivocally determined, the defined value, γ 50 = k S/k 1, (k 1, the rate constant for the reaction between alkoxyl radical and spin trap) was used as a reaction parameter of substrate for alkoxyl radical. The flow-injection electron spin resonance system using alkoxyl radical elimination gave valid γ 50 values for biosubstances, and the system should be a valuable method for the evaluation of the antioxidant ability.