Development of on-line HPLC-FRAP antioxidant activity determination method and application to some natural products

A new on-line HPLC-antioxidant activity method (on-line HPLC-FRAP) was added to the existing methods in literature, and the method was applied to plant samples. Antioxidant activities of plant extracts (sugar/sugar-free aqueous herbal tea bag extracts, methanolic Rhododendron extracts, Thymus praecox extracts in different solvents) known to have high activity were determined by spectrophotometric (off-line) methods. Then the developed method (on-line HPLC-FRAP) was optimized (gradient conditions, gradient pump and syringe pump flow rates and the reaction temperature and waiting time of FRAP) for the determination of 15 standards, extract components, and their antioxidant activities simultaneously. LOD and LOQ values of six standards were in 0.4 ? 14.7 ppm range. The antioxidant activities of the components of plant extracts were determined with the new method; the results were compared with spectrophotometric FRAP data. On-line -ABTS and -DPPH methods were also tested using the same system, and the results were compared with FRAP data. The developed method showed linearity in 0-1000 ?M range for all the standards. The elapse time (1,5 minutes) in reaction coil in on-line system was found sufficient based on the kinetic study. The on-line and off-line total activities for plant extracts were in agreement (R2 = 0.89). The values obtained by summing the antioxidant activities of individual constituents in on-line system revealed that the compounds in extracts could demonstrate antagonism or synergy. Activity changes between sugar/sugar-free herbal tea extracts were found similar in both methods, with differing behaviour from plant to plant. The developed on-line HPLC-FRAP method was found to be superior to the on-line HPLC-ABTS and -DPPH methods adapted from literature with respect to noise and antioxidant response, and the method appeared to be highly suitable for application to the determination of components and antioxidant activities of food and plants.