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 reversedphase 10 μm 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—Fe²⁺-H₂O₂ (FeSO₄300 μmol/L, EDTA 300 μmol/L, H₂O₂180 μmol/L and DMPO 2 mmol/L) and H₂O₂ photolysis (H₂O₂18 mmol/L and DMPO 2 mmol/L photolysis for 6 min) systems were taken to produce hydroxyl free radicals for screening new drugs and studyins 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.