title: Solutions to decrease a systematic error related to AAPH addition in the fluorescence-based ORAC assay.
abstract: Oxygen radical absorbance capacity (ORAC) assay in 96-well multi-detection plate readers is a rapid method to determine total antioxidant capacity (TAC) in biological samples. A disadvantage of this method is that the antioxidant inhibition reaction does not start in all of the 96 wells at the same time due to technical limitations when dispensing the free radical-generating azo initiator 2,2'-azobis (2-methyl-propanimidamide) dihydrochloride (AAPH). The time delay between wells yields a systematic error that causes statistically significant differences in TAC determination of antioxidant solutions depending on their plate position. We propose two alternative solutions to avoid this AAPH-dependent error in ORAC assays.
authors.affiliation: Departamento de Estrés Abiótico, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas 52, 37008 Salamanca, Spain. Electronic address: elena.mellado@irnasa.csic.es.
authors.forename: Elena
authors.initials: E
authors.lastname: Mellado-Ortega
authors.affiliation: Departamento de Estrés Abiótico, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas 52, 37008 Salamanca, Spain. Electronic address: i.zabalgo@irnasa.csic.es.
authors.forename: Iñigo
authors.initials: I
authors.lastname: Zabalgogeazcoa
authors.affiliation: Departamento de Estrés Abiótico, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas 52, 37008 Salamanca, Spain. Electronic address: beatriz.dealdana@irnasa.csic.es.
authors.forename: Beatriz R
authors.initials: BR
authors.lastname: Vázquez de Aldana
authors.affiliation: Departamento de Estrés Abiótico, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas 52, 37008 Salamanca, Spain. Electronic address: juan.arellano@irnasa.csic.es.
authors.forename: Juan B
authors.initials: JB
authors.lastname: Arellano
mesh_terms: D000578:Amidines / Q000737:chemistry*; D000975:Antioxidants / Q000032:analysis*; D001205:Ascorbic Acid / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D002839:Chromans / Q000737:chemistry; D005453:Fluorescence; D005707:Gallic Acid / Q000737:chemistry; D016877:Oxidants / Q000737:chemistry; D000072257:Oxygen Radical Absorbance Capacity; D017382:Reactive Oxygen Species / Q000737:chemistry
chemical_list: D000578:Amidines; D000975:Antioxidants; D002839:Chromans; D016877:Oxidants; D017382:Reactive Oxygen Species; D005707:Gallic Acid; C046728:2,2'-azobis(2-amidinopropane); D001205:Ascorbic Acid; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
keywords: 96-Well plate reader; AAPH; ORAC; Peroxyl radical; Total antioxidant capacity; Trolox