title: Study of the antioxidant mechanisms of Trolox and eugenol with 2,2'-azobis(2-amidinepropane)dihydrochloride using ultra-high performance liquid chromatography coupled with tandem mass spectrometry.
abstract: The study of antioxidant mechanisms is a difficult task that involves the monitoring and identification of unknown intermediate and final products. Most of the time, the lifetime of intermediates is too short to allow their isolation and subsequent identification by nuclear magnetic resonance (NMR). The developments of ultra-high performance liquid chromatography (UHPLC) coupled with the advances in the acquisition rates of mass spectrometry could facilitate the research on antioxidant mechanisms. This work is based on the reaction involved in the Oxygen Radical Antioxidant Capacity (ORAC) and Total Radical trapping Antioxidant Parameter (TRAP) assays. Hence, the reaction between 2,2'-azobis(2-amidinepropane)dihydrochloride (AAPH) radicals and an antioxidant was carried out in the thermostatized autosampler of a chromatographic device. Then, the reaction media were injected every six minutes, and the compounds were separated by UHPLC and detected by mass spectrometry in scan mode. Nine consecutive injections were registered in a unique file, then the evolution of the reaction for one hour in a single run was monitored. In this way, the reaction mechanisms of Trolox and eugenol with AAPH were studied, leading to the detection of nine and thirteen different compounds, respectively. An exhaustive analysis of the spectra obtained in product ion scan mode led to the identification of the compounds. Most of them were species previously found in the literature, but others have never been reported, so tentative structures were suggested. All this allowed the proposal of several steps within the antioxidant mechanisms of Trolox and eugenol, showing the great performance of UHPLC-MS/MS to complement the use of NMR in antioxidant mechanistic studies.
authors.affiliation: Departamento de Química Analítica, Instituto de Investigación en Ingeniería de Aragón (I3A), CPS-Universidad de Zaragoza, María de Luna 3, E-50018 Zaragoza, Spain.
authors.forename: Karim
authors.initials: K
authors.lastname: Bentayeb
authors.forename: Carlos
authors.initials: C
authors.lastname: Rubio
authors.forename: Cristina
authors.initials: C
authors.lastname: Nerín
mesh_terms: D000578:Amidines / Q000737:chemistry*; D000975:Antioxidants / Q000737:chemistry* / Q000378:metabolism; D002839:Chromans / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005054:Eugenol / Q000737:chemistry* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D000578:Amidines; D000975:Antioxidants; D002839:Chromans; D017382:Reactive Oxygen Species; D005054:Eugenol; C046728:2,2'-azobis(2-amidinopropane); C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid