An On-Line HPLC Method for Detection of Radical Scavenging Compounds in Complex Mixtures

Abstract

A rapid on-line method for screening of complex mixtures for radical scavenging components was developed using a methanolic solution of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) stable free radical. The HPLC-separated analytes react postcolumn with the DPPH solution, and the induced bleaching is detected as a negative peak by an absorbance detector at 517 nm. An optimized instrumental setup is presented. The method is suitable for both isocratic and gradient HPLC runs with mobile-phase compositions ranging from 10 to 90% organic solvent in water or buffer (pH 3-6). The method is simple, has a broad applicability, and uses common instruments, inexpensive and stable reagents, and a time-saving and nonlaborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The limits of detection were 0.33-94 microg/mL, depending on the compound tested.

Genesis and development of DPPH method of antioxidant assay

“…In HPLC-FIA method, the separated analytes react postcolumn with DPPH• (Koleva et al 2000) and the induced scavenging is detected as a negative peak. These methods have an advantage over batch methods, as bioassay-guided fractionation of natural or food samples is a time consuming and labour-intensive process and the loss of activity of antioxidants due to decomposition during the isolation and purification procedures is avoided (Luıs et al 2006).…”

“…All solvents used for the dissolution of standards, extract samples and dilution of stock solutions, DPPH reagent, and SIA carrier stream were degassed by 10-min sonication in Sonorex Super 10P (Bandelin) ultrasound bath (sonication level 10) Yamaguchi et al (1998) developed a DPPH-HPLC method and compared the antioxidant activities of known standard antioxidants and commercial beverages with that of colorimetric method. Koleva et al (2000) presented an on-line HPLC method whereby analytes separated by HPLC, were reacted post-column with DPPH. A specific HPLC method was developed by Chandrasekar et al (2006) for evaluating the DPPH free radical scavenging activity of commercial polyherbal formulations using a LiChrospher® 100 RP-18e column.…”

Recent developments in the rapid analysis of plants and tracking their bioactive constituents

“…A typical example developed in our group is online radical scavenging detection (Dapkevicius et al 1999;Koleva et al 2000;Koleva et al 2001;Dapkevicius et al 2001;Niederländer et al 2008). Phenolic antioxidants can terminate radical chain processes in lipid rich foods by reacting with reactive oxygen species (ROS) forming more stable resonance-stabilised radicals or quinone-like structures (Fig.…”

Therapeutic effect of umbelliferon-α-D-glucopyranosyl-(2I→1II)-α-D-glucopyranoside on adjuvant-induced arthritic rats

“…Many of the antioxidant protect molecules exposed the free radical and reactive oxygen species, which have been reduced the oxidative stress and protect the food from the deterioration and spoilage (Koleva et al 2000). Lots of the synthetic antioxidant available in the market such as BHT (butylated hydroxyl toluene), BHA (butylated hydroxyl anisole) and PG (propyl gallate).…”