Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity

• Published in: Talanta (Oxford) Vol. 150; pp. 599 - 606
• Main Authors: Ramos, Inês I., Gregório, Bruno J.R., Barreiros, Luísa, Magalhães, Luís M., Tóth, Ildikó V., Reis, Salette, Lima, José L.F.C., Segundo, Marcela A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2016
• Subjects: Absorbance probe; Antioxidants - analysis; Automation; Coloring Agents - chemistry; Flow injection analysis; Peroxides - chemistry; Peroxides - metabolism; Peroxyl radicals; Pyrogallol - analogs & derivatives; Pyrogallol - chemistry; Reaction kinetics; Reactive Oxygen Species - chemistry; Reactive Oxygen Species - metabolism; Automation; Flow injection analysis; Absorbance probe; Peroxyl radicals; Reaction kinetics
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2015.12.061

An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5µM of PGR and 125mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90±5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8μM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29h−1), low operating costs and low generation of waste. [Display omitted] •Assessment of initial antioxidant reactivity against peroxyl radicals was targeted.•PGR bleaching was monitored within the first minute through a stopped flow protocol.•Programmable flow fostered strict control for in situ generation of radicals.