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

1,1-Diphenyl-2-picrylhydrazyl (DPPHradical 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 DPPHradical 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 DPPHradical dot quenching rate and none of them permits to identify and measure 1,1-diphenyl-2-picryl-hydrazine (DPPH-H), the reduced product of DPPHradical dot resulting from hydrogen atom transfer (HAT), which is the main mechanism of the reaction between DPPHradical dot and AOs. We presently report an HPLC method devoted to the simultaneous measurement of DPPHradical dot and DPPH-H. Both were fully separated on a C18 column eluted with acetonitrile–10 mM ammonium citrate buffer pH 6.8 (70:30, v/v) and detected at 330 nm. Adsorption process of DPPHradical dot onto materials of the HPLC system was pointed out. Consequently, the linearity range observed for DPPHradical dot was restricted, thus a much lower limit of detection was obtained for DPPH-H than for DPPHradical dot using standards (0.02 and 14 μM, respectively). The method was applied to three commonly used AOs, i.e. Trolox®, 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.