Quasi-simultaneous determination of antioxidative activities against superoxide anion and nitric oxide by a combination of sequential injection analysis and flow injection analysis with chemiluminescence detection

Abstract

A method that combines sequential injection analysis (SIA), flow injection analysis and chemiluminescence (CL) detection was developed for the quasi-simultaneous determination of antioxidative activities against superoxide anion (O2-) and nitric oxide (NO). The antioxidative activity was expressed as the decrease in luminol CL intensity caused by the quenching of O2- or NO by an antioxidant. The SIA system consisted of two syringe pumps, two selection valves, two holding coils, an HPLC pump to deliver luminol solution, and a CL detector. Operation of the syringe pumps and multiport valves was controlled automatically using a personal computer with appropriate software. A hypoxanthine (HX)-xanthine oxidase (XOD) system was used for the generation of O2-, and (+/-)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1) was employed as NO donor agent. The repeatability of the method was evaluated with 35.2 microg ml(-1) L-ascorbic acid, and the relative standard deviations (RSD) of the antioxidative activities were less than 3.8%. The quasi-simultaneous determination of the antioxidative activities in one sample was completed within 2.0 min. The antioxidative activities of some antioxidants and commercially available supplements containing certain antioxidants were successfully determined using this system. The proposed system is rapid and reproducible, and thus may be useful for the screening of functional foods, supplements and pharmaceutical formulations that exhibit antioxidative activity.