Trace determination of hydroxyl radical in biological systems

A simple and highly sensitive method to quantify the rates of production of OH in biological systems is described. This method employs the reaction between OH and dimethyl sulfoxide to generate quantitatively a methyl radical, which then reacts with a fluorescamine-derivatized nitroxide to produce the stable O-methylhydroxylamine. This O-methylhydroxylamine is separated by reversed-phase high-performance liquid chromatography and quantified fluorometrically, The estimated detection limit of the O-methylhydroxylamine is 3.5 nM for a 50 mu L injection at a signal to noise ratio of 2. The method is applied to the determination of the rates of OH production in a biologically relevant model system and in a mouse epidermal cell line treated with a quinone anticancer compound.