Quantitation of the hydroxyl radical adducts of salicylic acid by micellar electrokinetic capillary chromatography: Oxidizing species formed by a Fenton reaction

Article abstract of DOI:10.1007/s002040050093

There has been controversy concerning the products formed by a Fenton reaction. We determined the hydroxyl radical (^.OH) generated in a Fenton reaction system with no iron chelator using micellar electrokinetic capillary chromatography (MECC). The hydroxyl radical generated in this Fenton system attacked salicylic acid to produce major products of 2,3- and 2,5-dihydroxybenzoic acid (DHB), 2,3-DHB being prominent. Hydroxyl radical scavengers, such as mannitol, ethanol, thiourea and a ferric chelator, Desferal, significantly diminished the peaks for DHBs, showing production of ^.OH. We compared the MECC method with the electron paramagnetic resonance (EPR) spin trapping technique. The quantity of DHBs obtained by MECC increased dose-dependently up to 1 μM Fe²⁺ at a fixed concentration of H₂O₂, whereas that of the spin adduct by EPR showed a bell-shaped curve. This quantitation of ^.OH adducts by MECC supports the proposal that the oxidizing species formed by a Fenton reaction with no chelator is ^.OH. The EPR spin trapping method appears to be erroneous, particularly when iron is present at a higher concentration than hydrogen peroxide. The application of this method to the paraquat effect in vitro is demonstrated, and the possibility for analysis of ^.OH in vivo is also discussed.