Radicals in the reaction between peroxynitrite and uric acid identified by electron spin resonance spectroscopy and liquid chromatography mass spectrometry

Abstract

Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. This article is focused on the identification of free radical intermediates of uric acid formed during its reaction with peroxynitrite. The experimental approach included the ESR spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4 and mass spectrometry studies of the trapped radicals. Using PBN (N-tert-butyl-α-phenylnitrone) as the spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, aN=15.6G and aH=4.4G, revealed the presence of carbon-based radicals. Further structural identification of the PBN–radical adducts was carried out using liquid chromatography–mass spectrometry. After comparison with the control reactions, two species were identified that correspond to the protonated molecules (M+1) at m/z 352 and 223, respectively. The ions of m/z 352 were characterized as the PBN–triuretcarbonyl radical adduct and the m/z 223 ion was identified as the PBN–aminocarbonyl radical adduct. Their mechanism of formation is discussed.