[Direct measurement of oxy-radicals using HPLC-ECD]

A direct, sensitive, simple and specific high-pressure liquid chromatographic (HPLC) method was used for the quantitation of hydroxyl radicals by means of oxy-radical trapping of DMPO to form DMPO-OH adducts. The DMPO-OH adduct peak was separated successfully and identified by HPLC-ECD with a Waters ODS reversed phase 10 microns column. The mobile phase composed of citric acid/sodium acetate (citric acid-30 mmol/L-sodium acetate 50 mmol/L-3% acetonitrile, pH 5.1), at a flow rate of 1.2 ml/min and detection potential of 0.6 V with Ag/AgCl as reference electrode. Both EDTA-Fe(2+)-H2O2 (FeSO4 300 mumol/L, EDTA 300 mumol/L, H2O2 180 mumol/L and DMPO 2 mmol/L) and H2O2 photolysis (H2O2 18 mmol/L and DMPO 2 mmol/L photolysis for 6 min) systems were taken to produce hydroxyl free radicals for screening new drugs and studying the mechanism of action. The relative standard deviations were 6.1 and 8.0% respectively. The sensitivity of the method was shown to be similar to that of ESR. The method for detection of superoxide anions with HPLC-ECD was also described.