title: Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.
abstract: 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 (540 nm) 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 37.5 µM of PGR and 125 mM 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 8 μM. 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 (29 h(-1)), low operating costs and low generation of waste.
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Inês I
authors.initials: II
authors.lastname: Ramos
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Bruno J R
authors.initials: BJ
authors.lastname: Gregório
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Luísa
authors.initials: L
authors.lastname: Barreiros
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Luís M
authors.initials: LM
authors.lastname: Magalhães
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Ildikó V
authors.initials: IV
authors.lastname: Tóth
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Salette
authors.initials: S
authors.lastname: Reis
authors.affiliation: LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: José L F C
authors.initials: JL
authors.lastname: Lima
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: msegundo@ff.up.pt.
authors.forename: Marcela A
authors.initials: MA
authors.lastname: Segundo
mesh_terms: D000975:Antioxidants / Q000032:analysis*; D004396:Coloring Agents / Q000737:chemistry*; D010545:Peroxides / Q000737:chemistry* / Q000378:metabolism; D011748:Pyrogallol / Q000031:analogs & derivatives* / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000975:Antioxidants; D004396:Coloring Agents; D010545:Peroxides; D017382:Reactive Oxygen Species; D011748:Pyrogallol; C049375:perhydroxyl radical; C058731:pyrogallol sulfonphthalein
keywords: Absorbance probe; Automation; Flow injection analysis; Peroxyl radicals; Reaction kinetics