Characterization of 60Co γ-radiation induced radical products of antipyrine by means of high-performance liquid chromatography, mass spectrometry, capillary zone electrophoresis, micellar electrokinetic capillary chromatography and nuclear magnetic resonance spectrometry

Abstract

Monitoring the amount of oxidative damage, caused by free radicals, is a major problem in free radical and aging research. Antipyrine is proposed as an exogenous marker for the biomolecular monitoring of oxidative stress. In this paper the characterization of the 60Co γ-radiation products of antipyrine is described. Since mainly hydroxyl radicals are generated under these experimental conditions, hydroxylated derivatives can be expected. The reaction kinetics of antipyrine and hydroxyl radicals were examined. The results show pseudo first order reaction kinetics. In vivo hydroxyl radicals are one of the major causes of oxidative stress. The separation of the different derivatives was performed with high-performance liquid chromatography (HPLC) and with micellar electrokinetic capillary chromatography (MECC). Antipyrine and the radiolysis products were baseline separated with MECC and HPLC. The conversion of antipyrine, after exposure to different doses of γ-radiation, was determined with HPLC and with the more customized and cheaper analytical technique MECC. The conversion of antipyrine after exposure was calculated after analysis with MECC and with HPLC. The correlation coefficient between both techniques was 0.9984. The reaction products were characterized with nuclear magnetic resonance (NMR) and mass spectrometry (MS). The dissociation constants (p K a values) of the radiation products were determined by means of capillary zone electrophoresis (CZE). The results identified the radiolysis products as ortho-, meta- and para-hydroxylated antipyrine. The ortho- and meta-hydroxylated isomers are different from the enzymatic metabolites formed in man.