Simultaneous detection of superoxide anion, hydroxyl radical, and methyl radical by use of high performance liquid chromatography electron spin resonance

Electron spin resonance (ESR) spectroscopy has been used to investigate free radical formation in chemical and biochemical reaction. Spin trapping technique has been used to detect short-lived radicals. The radical chromatography using ESR for HPLC detector was carried out for simultaneous detection of free radicals of similar spin adducts g value. Free radical mixtures, such as O-2(-), . OH and . CH3, were prepared by mixing DMSO, H2O2, NaOH and ferric ions in the presence of 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-t-butyl-phenylnitrone (PBN) as spin trapping agents. DMPO or PBN adducts of O-2(-), . OH and . CH3 were well separated on Inertsil ODS-2 column with citrate buffer (pH 7.0) /acetonitrile (80/20) or HEPES buffer (pH 7.0) /acetonitrile (50/50) as a mobile phase. When the sample solution of mixed free radicals was loaded on the column, three peaks due to the spin adducts appeared in the ESR chromatogram. Corresponding ESR spectra of each peak was recorded. This specific and direct detection method permitted the simultaneous quantitation of mixed free radicals in the sample, and it is applicable for various samples including biological materials.