Automatic in Vitro Determination of Hypochlorous Acid Scavenging Capacity Exploiting Multisyringe Flow Injection Analysis and Chemiluminescence

Abstract

In the present work, a chemiluminometric automatic flow methodology for the in vitro determination of hypochlorous acid scavenging capacity, under pH and concentration conditions similar to those found in vivo, is proposed. As the pH found in physiological conditions (7.4) and the pH required for the chemiluminescence detection reaction (>10) are different, the multisyringe flow injection analysis features were exploited to perform the in-line reaction of HOCl and the scavenger molecule at physiological pH prior to reaction of the remaining HOCl with luminol at alkaline conditions. These two reactions were carried out in about 3 s, allowing the determination of fast reacting antioxidants, in a time frame closer to in vivo generation of HOCl when compared to previously described methods. The developed method was applied to nonsteroidal antiinflammatory drugs of different chemical families, and positive controls (cysteine, gallic acid, lipoic acid). The HOCl scavenging capacity was evaluated at pH 7.4 and 10.0; different results were found for oxicam derivatives, providing evidence that the pH of in vitro methods should be carefully selected to allow assumptions about putative in vivo effects.