High-performance liquid chromatographic method to evaluate the hydrogen atom transfer during reaction between 1,1-diphenyl-2-picryl-hydrazyl radical and antioxidants

1,1-Diphenyl-2-picrylhydrazyl (DPPH center dot) is a stable nitrogen centred radical widely used to evaluate direct radical scavenging properties of various synthetic or natural antioxidants (AOs). The bleaching rate of DPPH center dot absorbance at 515 nm is usually monitored for this purpose. In order to avoid the interference of complex coloured natural products used as antioxidant supplements or cosmetics, HPLC systems have been reported as alternative techniques to spectrophotometry. They also rely upon measurement of DPPH center dot quenching rate and none of them permits to identify and measure 1,1-diphenyl-2-picryl-hydrazine (DPPH-H), the reduced product of DPPH center dot resulting from hydrogen atom transfer (HAT), which is the main mechanism of the reaction between DPPH center dot and AOs. We presently report an HPLC method devoted to the simultaneous measurement of DPPH center dot and DPPH-H. Both were fully separated on a C18 column elutad with acetonitrile-10 mM ammonium citrate buffer pH 6.8(70:30, v/v) and detected at 330 nm. Adsorption process of DPPH center dot onto materials of the HPLC system was pointed out. Consequently, the linearity range observed for DPPH center dot was restricted, thus a much lower limit of detection was obtained for DPPH-H than for DPPH center dot using standards (0.02 and 14 mu M, respectively). The method was applied to three commonly used AOs, i.e. Trolox((R)), ascorbic acid and GSH, and compared with spectrophotometry. Further application to complex matrices (cell culture media, vegetal extracts) and nanomaterials demonstrated (i) its usefulness because of higher selectivity than colorimetry, and (ii) its help to investigate the mechanisms occurring with the free radical. (C) 2011 Elsevier B.V. All rights reserved.