Spectrophotometric determination of free radicals and reactive oxygen species with N,N-dimethyl-p-phenylendiamine

A novel spectrophotometry for the determination of total free radicals and peroxides was developed using N,N,-dimethyl-p-phenylenediamine (DMPD) as a detection reagent. The stability of a DMPD aqueous solution was improved by dissolving DMPD in dimethyl sulfoxide (DMSO). The optimal conditions for the detection of free radicals in human serum were examined with DMPD. DMPD, the sample and 100 mu mol L-1 ammonium iron(II) sulfate were mixed in a 0.1 mol L-1 acetate buffer Solution (pH 5.0) containing 5% DMSO and left standing at 40 C for 10 minutes, followed by a measurement of the absorbance at 550 nm. The calibration curves of free radicals (alkoxyl radical, hydroxyl radical and nitrogen monoxide) showed good linearity over the range of 2.9 nmol L-1 similar to 40 mol L-1 (r(2) = 0.995), 75.2 similar to 500 nmol L-1 (r(2) = 0.992) and 44.5 similar to 300 mu mol L-1 (r(2) = 0.991), respectively. The detection limits of alkoxyl radical, hydroxyl radical and nitrogen monoxide were 0.9 nmol L-1, 22.6 nmol L-1 and 13.3 mu mol L-1, respectively. The recovery of 10 mu mol L-1 alkoxyl radical in human serum was 119%. The average of the total concentration for free radicals and peroxides as the concentration of tert-butyl hydroperoxide in human serum was 7.0 mu mol L-1 (n = 6).