Separation and Detection of the Antioxidant Flavonoids, Rutin and Quercetin, Using HPLC Coupled on-line With Colorimetric Detection of Antioxidant Activity

Nitra Nuengchamnong; Ank Hermans-Lokkerbol; Kornkanok Ingkaninan

Authors

Nitra Nuengchamnong Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of PharmaceuticalSciences, Naresuan University, Phitsanulok 65000, Thailand
Ank Hermans-Lokkerbol Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of PharmaceuticalSciences, Naresuan University, Phitsanulok 65000, Thailand
Kornkanok Ingkaninan Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of PharmaceuticalSciences, Naresuan University, Phitsanulok 65000, Thailand

Keywords:

antioxidant, flavonoids, rutin, quercetin, on-line separation, activity determination method

Abstract

The HPLC system coupled on-line with colorimetric detection of antioxidant activity was developed in order to separate antioxidants especially the flavonoids, rutin and quercetin, and to determine their activities in a single step. In the system developed, the eluate from the HPLC column was split into two flows at the ratio of 8:2. The major part flowed to the UV-detector set at 220 nm, while the minor part was mixed with a free radical, 2,2-diphenyl-1-picrylhydrazyl, to perform a color reaction with the eluted antioxidants. This reaction occurred in a knitted-shape reactor (70 cm 0.8 mm I.D. PTFE). The peaks indicating antioxidant activity were monitored from the decrease of absorbance at 515 nm. The results showed that the on-line HPLC coupled with antioxidant activity detection developed could separated the flavonoids, rutin and quercetin in plant extracts such as Sophora japonica and Morus alba and simultaneously determine their antioxidant activity. The detection limits for the antioxidant activity determination of these two compounds were 500 and 200 ng, respectively. This method could be applied for rapid determination of rutin, quercetin in complex mixtures.

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