High-performance liquid chromatographic method for the direct quantitation of oxy radicals in myocardium and blood by means of 1,3-dimethylthiourea and dimethyl sulfoxide.

A direct, sensitive (50-200 ng), simple and specific high-performance liquid chromatographic (HPLC) method is described for the quantitation of oxy radicals by means of the consumption of dimethyl sulfoxide (DMSO) by the hydroxy radical and dimethylthiourea (DMTU) by hydrogen peroxide. The specific scavengers catalase and L-methionine were used to quantitate hydrogen peroxide and OH, respectively. The DMSO and DMTU peaks were separated and identified by HPLC on a Waters C18 Resolve 10-microns Radial-Pak column with an isocratic mobile phase (5% aqueous methanol) at 2 ml/min with UV detection (DMSO, 214 nm; DMTU, 240 nm). The OH concentrations were extrapolated by a luminol chemiluminescence technique. A linear relationship was obtained for DMTU consumption by hydrogen peroxide in the range 0.25-0.40 mM with a coefficient of variation (C.V.) of 8.8 +/- 2.1% and for DMSO consumption by hydroxy radicals in the range 0.1-3.2 microM OH, with a C.V. of 9.6 +/- 3.6%. The limits of detection for this method were 50 ng of hydrogen peroxide for DMTU and 200 ng of OH for DMSO. Hydrogen peroxide averaged 10.5 +/- 3.6 nmol/ml in blood and 56.4 +/- 5.3 mumol/g wet weight in left ventricular (LV) tissue. The hydroxy radical concentration was 0.1 microM in blood and 0.3 microM in LV tissue.