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Phytochem Anal. 2005 Nov-Dec;16(6):422-8.

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba.

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Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand.


A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.

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