To investigate the antioxidant potential in natural products, radical scavenging tests (ABTS, DPPH, ORAC, etc.) are usually considered as the first approach. In addition to the standard colorimetric assays, methods using separation techniques (on-line and pre-column assays) have been developed in the past decades. Based on the peak area (PA) reductions of compounds monitored by HPLC, the pre-column spiking method allows rapid characterisation of natural matrices avoiding laborious isolation steps. However, available information about the significance of the results produced remains scarce. Here, we report, for the first time, a discussion of the potential of the pre-column DPPH spiking method to pinpoint antioxidant compounds using red maple bark extract (RMBE). First, DPPH spiking was conventionally applied to the galloyl compounds in the extract showing the inadequacy of assessing results by PA reductions. The method was then applied to pure galloyl derivatives, evaluating their molar amount reacted (MAR) for more significance. The comparison with the standard DPPH-HPLC/AE method directly monitoring DPPH• inhibition highlighted the inability to retrieve the respective antioxidant efficiencies (AE) of each compound by using DPPH spiking. Despite its limitations, the DPPH spiking method brought to light an autoxidation phenomenon and a matrix/mixture effect investigated through tertiary mixtures of galloyl compounds. Although restricted to the compounds from one natural matrix, this study questions the validity of the spiking method as usually performed and could serve as a basis for further investigations (explorations of other natural products, kinetics considerations). Graphical abstract Investigation of the pre-column DPPH spiking method through the case of galloyl derivatives.
Keywords: Autoxidation; DPPH spiking; Mixture effect; Pre-column HPLC; Red maple bark extract; Stoichiometry.