Identification of 1-ethoxyethyl radicals in the reaction of ferrous ions with serums from rats exposed to diethyl ether

Abstract

Using α-(4-pyridyl-1-oxide)- N- tert-butylnitrone (4-POBN) as a spin trap reagent, an electron paramagnetic resonance (EPR) spectrum was observed for the complete reaction mixture of ferrous ions with serums from rats exposed to diethyl ether. The EPR spectrum was not observed for the complete reaction mixture without the serums (or ferrous ions) or with a serum from a rat not exposed to diethyl ether. In order to identify the 4-POBN radical adducts, HPLC–EPR and HPLC–EPR–MS analyses were employed. The HPLC–EPR analysis of the complete reaction mixture showed a prominent peak with a retention time of 25.2 min. The HPLC–EPR–MS analysis of the peak gave ions at m/ z 181 and m/ z 268. The ions m/ z 268 correspond to the protonated molecular ions of 4-POBN/1-ethoxyethyl radical adducts, ( M + H) +. The fragment ions at m/ z 181 correspond to the loss of [(CH 3) 3C(O)N] from the protonated molecular ions. The HPLC–EPR analysis of the Fenton reaction mixture with diethyl ether gave a peak with the same retention time as the one in the reaction of the rat serums with ferrous ions. Thus, the radicals detected in the reaction mixture of ferrous ions with the serums from rats exposed to diethyl ether are identified as 1-ethoxyethyl radicals.