Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products.

Evidence is presented for a sensitive method useful for the detection of hydroxyl free radical generation in various systems. The methodology employs high pressure liquid chromatography with electrochemical detection (LCED) for the quantification and identification of the hydroxylation products from the reaction of OH with both phenol and salicylate. A detection limit of less than 1 pmol for the hydroxylation products has been achieved with electrochemical detector responses linear over at least three orders of magnitude. Detection and quantitation of the hydroxylation products obtained and formed during OH generation from biologically meaningful systems have been demonstrated. The three systems utilized were ADP/FE(II)/H2O/, hypoxanthine/xanthine oxidase plus chelated iron, and UV photolysis of H2O2.