An on-line high performance liquid chromatography-crocin bleaching assay for detection of antioxidants
Bountagkidou, O. ; Klift, E.J.C. van der; Tsimidou, M.Z. ; Ordoudi, S.A. ; Beek, T.A. van - \ 2012
Journal of Chromatography. A, Including electrophoresis and other separation methods 1237 (2012). - ISSN 0021-9673 - p. 80 - 85.
radical scavenging compounds - chemiluminescence detection - natural antioxidants - complex-mixtures - identification - capacity - extracts - inhibition - plant
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. --------------------------------------------------------------------------------
Antioxidant activity assays on-line with liquid chromatography
Niederländer, H.A.G. ; Beek, T.A. van; Barsatute, A. ; Koleva, I. - \ 2008
Journal of Chromatography. A, Including electrophoresis and other separation methods 1210 (2008)2. - ISSN 0021-9673 - p. 121 - 134.
radical scavenging compounds - coulometric array detection - flow-injection system - dpph screening method - dad-spe-nmr - phenolic-compounds - electrochemical detection - natural-products - plant-extracts - chemiluminescence detection
Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper both electrochemical and chemistry-based assays are reviewed and discussed. The focus is on the mechanisms involved and differences between the assays, rather than on the matrix or analytes. With 45 applications high resolution antioxidant screening has now become an almost routine tool for the rapid identification of antioxidants in plant extracts, foods and beverages. The methods based on true reactive oxygen species (ROS) provide the most realistic measure of antioxidant activity. Unfortunately these methods are difficult to set up and control and have not been applied since they were reported. The methods based on electrochemical detection are more practical, but have still received only limited attention for practical screening purposes. The methods based on a single relatively stable reagent such as DPPH and ABTS+ have become most popular, because of their simple set-up and ease of control. The methods have been combined with on-line DAD, MS and NMR detection for rapid identification of active constituents.