Evaluation of sodium 4-hydroxybenzoate as an hydroxyl radical trap using gas chromatography-mass spectrometry and high-performance liquid chromatography with electrochemical detection.

Molecular and tissue damage induced by reactive oxygen species is a serious consequence of the production of free radicals in biological systems. Biological markers produced by reactions with hydroxyl radicals are useful indices of free radical processes in vivo. In this respect, hydroxylation of aromatic compounds such as salicylate (2-hydroxybenzoate) has been used extensively as a measure of hydroxyl radical formation. 4-Hydroxybenzoate will also trap hydroxyl radicals with fewer of the complications for which salicylate has been criticized. We describe two sensitive and specific methods using gas chromatography-ion trap mass spectrometry and high-performance liquid chromatography with electrochemical detection for a number of these aromatic marker compounds in biological fluids. The use of an ion trap mass spectrometer provides enhanced sensitivity along with full mass spectral identification of the compounds of interest. 4-Hydroxybenzoate and salicylate were compared as hydroxyl radical traps (i) by determining relative hydroxyl radical trapping efficiencies in vitro, (ii) by measuring individual dihydroxybenzoate isomers in rat serum following intraperitoneal injection of either 2- or 4-hydroxybenzoate, and (iii) by comparing in vivo hydroxyl radical trapping using intrastriatal microdialysis in the rat. The techniques described have broad applications in the area of free radical biomedical research.