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

Abstract

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.

Genesis and development of DPPH method of antioxidant assay

“…7) and NMR method for determining antioxidant polyphenolic acids in the medicinal herb. A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed by Nuengchamnong et al (2005) 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 method This method has been applied to evaluate antioxidant compounds in fruit and vegetables (Pukalskas et al 2002;Kosar et al 2003). This technique would permit the rapid determination of antioxidant activity and provide structural identification of the antioxidant compounds involved (Nuengchamnong et al (2005).…”

On-line HPLC–MS–DPPH assay for the analysis of phenolic antioxidant compounds in fruit wine: Antidesma thwaitesianum Muell.

“…The aglycone of delphinidin, cyanidin, gallic acid, catechin pelargonidin, and caffeic acid showed high activity with the ABTS assay with TEAC value (mM) of 4.44, 4.40, 3.01, 2.40, 1.30, 1.24, respectively (Cai, Sun, Xing, Luo, & Corke, 2006;RiceEvans et al, 1996). In our previous study, it is proved that intensity of antioxidant peaks depend on both types and amounts of the antioxidant compounds present in the sample (Nuengchamnong et al, 2005). Antioxidant compounds in A. thwaitesianum wine can be quantitatively analysed using LC-MS/MS in MRM mode if the standard compounds are available.…”

Excellent combination of HPLC-RSD-DAD-ESI/MS and HSCCC experiments to screen and identify radical scavengers from Neo-Taraxacum siphonanthun

“…10,11 Analytical HPLC was performed on Acquity TM UPLC system (Waters Corp., Milford, MA, USA) with cooling autosampler and column oven enabling temperature control of the analytical column. Separation was performed on a Symmetry® C 18 column (150 mm × 3.9 mm i.d., 5 μm) at 25 °C.…”

“…Furthermore, the HPLC-RSD method coupled online to MS would permit the rapid determination of antioxidant activity and provide structural identification of the antioxidant compounds. 11 However, on most occasions, even for isomeric natural products the MS data were insufficient for the exact structure elucidation of plant secondary products because of the lack of reference standards and MS database, and at this time NMR frequently remained necessary.…”