An on-line high performance liquid chromatography-crocin bleaching assay for detection of antioxidants

► Off-line crocin bleaching assay (CBA) was converted to HPLC on-line antioxidant assay. ► It is the first simple on-line antioxidant assay to use a reactive oxygen species (ROS). ► In contrast to DPPH and ABTS on-line assays, a true polyene substrate is used. ► The sensitivity for caffeic acid is 1ng. ► Antioxidants in tea could be detected on-line after RP-HPLC. 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 440nm. 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 7.4ng, depending on the antioxidant.