Improved method for the estimation of hydroxyl free radical levels in vivo based on liquid chromatography with electrochemical detection.

Free radical damage to proteins, lipids, DNA and RNA has been thought to play an important role in many diseases as well as the aging process. One free radical, the hydroxyl free radical (HFR), is extremely reactive and is difficult to measure directly. HFRs were quantified by measuring the hydroxylation products 2,3- and 2,5-dihydroxybenzoic acids (DHBAs) formed as a result of the reaction between HFR and systemically administered salicylate (SAL). DHBAs and SAL concentrations were determined using RP-HPLC with dual coulometric electrode detection. The method has limits of detection of 1 pg for the DHBAs and 100 pg for SAL (signal-to-noise ratio 3:1). A detailed interference study as well as analyte stability and linearity studies were performed. This method was used to determine basal ratios of DHBA/SAL in a variety of tissues and to study the effects of glutamatergic and dopaminergic drugs on DHBA/SAL ratios in brain region homogenates.