Hydroxyl radical detection with a salicylate probe using modified CUPRAC spectrophotometry and HPLC

Abstract

Reactive oxygen species (ROS) may attack biological macromolecules giving rise to oxidative stress-originated diseases, so it is important to establish efficient methods to screen hydroxyl radical scavengers for antioxidant therapy. Since OH is very short-lived, secondary products resulting from OH attack to various probes are measured. As a low-cost measurement technique, we used a salicylate probe for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II) + EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 °C-incubated solutions for 2 h. The CUPRAC (cupric ion reducing antioxidant capacity) assay absorbance of the ethylacetate extract due to the reduction of Cu(II)–neocuproine reagent by the hydroxylated probe decreased in the presence of OH scavengers, the difference being proportional to the scavenging ability of the tested compound. Attack by OH radicals upon salicylate produced 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, and 2,5-dihydroxybenzoate as major products. HPLC separation combined with CUPRAC spectrophotometry was used to identify and quantify hydroxylated salicylate derivatives in the presence of synthetic water-soluble antioxidants and green tea infusion. The developed spectrophotometric method for OH detection was validated with HPLC, i.e., the concentrations of dihydroxybenzoates produced by radical attack from the probe were determined by HPLC, and the sum of (concentration × absorptivity) products of these components approximately agreed with the experimentally found CUPRAC absorbances, confirming the validity of Beer's law for the selected system. Statistical comparison of the results found with the proposed methodology and HPLC was made with two-way ANOVA (analysis of variance) test. Under optimal conditions, about 53% of the probe (salicylate) was converted into dihydroxybenzoate isomers in the absence of OH scavengers, and these isomers were more specific markers of hydroxyl radicals than the non-specific malondialdehyde end-product of the TBARS test. Thus, the more costly and less speedy HPLC method could advantageously be substituted with the proposed spectrophotometric assay of OH detection, which was also of much higher yield than the TBARS colorimetric assay.