title: An on-line high performance liquid chromatography-crocin bleaching assay for detection of antioxidants.
abstract: An on-line HPLC (high performance liquid chromatography) method for the rapid screening of individual antioxidants in mixtures was developed using crocin as a substrate (i.e. oxidation probe) and 2,2'-azobis(2-amidinopropane dihydrochloride (AAPH)) in phosphate buffer (pH 7.5) as a radical generator. The polyene structure of crocin and AAPH-derived peroxyl radicals resemble the lipidic substrates and radicals found in true food more closely than the popular, albeit artificial, DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS(+) (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)) do. After separation by a C18 (octadecyl silica) column and UV (ultraviolet) detection, antioxidative analytes react with peroxyl radicals at 90°C and the inhibition of crocin oxidation (i.e. bleaching) is detected as a positive peak by an absorbance detector at 440 nm. The method is simple, uses standard instruments and inexpensive reagents. It can be applied for isocratic HPLC runs using mobile phases containing 10-90% organic solvent in water, weak acids or buffers (pH 3.5-8.5). With baseline correction, gradient runs are also feasible. The radical scavenging activity of several natural antioxidants and a green tea extract was studied. After optimisation of conditions such as reagent concentrations and flows, the limit of detection varied from 0.79 to 7.4 ng, depending on the antioxidant.
authors.affiliation: Laboratory of Organic Chemistry, Natural Products Chemistry and Microfluidics Group, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands.
authors.forename: Olga
authors.initials: O
authors.lastname: Bountagkidou
authors.forename: Elbert J C
authors.initials: EJ
authors.lastname: van der Klift
authors.forename: Maria Z
authors.initials: MZ
authors.lastname: Tsimidou
authors.forename: Stella A
authors.initials: SA
authors.lastname: Ordoudi
authors.forename: Teris A
authors.initials: TA
authors.lastname: van Beek
mesh_terms: D000975:Antioxidants / Q000032:analysis*; D002338:Carotenoids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007700:Kinetics; D057230:Limit of Detection; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000975:Antioxidants; D002338:Carotenoids; C029036:crocin