title: Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection.
abstract: A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 microM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples.
authors.affiliation: Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey.
authors.forename: Saliha Esin
authors.initials: SE
authors.lastname: Celik
authors.forename: Mustafa
authors.initials: M
authors.lastname: Ozyürek
authors.forename: Kubilay
authors.initials: K
authors.lastname: Güçlü
authors.forename: Reşat
authors.initials: R
authors.lastname: Apak
mesh_terms: D000975:Antioxidants / Q000032:analysis*; D028241:Camellia sinensis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D005419:Flavonoids / Q000032:analysis*; D007202:Indicators and Reagents / Q000737:chemistry; D027541:Mentha / Q000737:chemistry; D031341:Origanum / Q000737:chemistry; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000737:chemistry*; D010636:Phenols / Q000032:analysis*; D010936:Plant Extracts / Q000032:analysis*; D059808:Polyphenols
chemical_list: D000975:Antioxidants; D005419:Flavonoids; D007202:Indicators and Reagents; D010618:Phenanthrolines; D010636:Phenols; D010936:Plant Extracts; D059808:Polyphenols; D003300:Copper; C002701:neocuproine