Critical Re-Evaluation of DPPH assay: Presence of Pigments Affects the Results

A limitation of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability (DRSA) due to the presence of pigments and colors in the extracts of plant-based foods was addressed. The pigments present in the test samples absorb in the same wavelength region as the DPPH radicals; this interferes with the elaborate absorbance readings of the DPPH radicals. In this contribution, electron paramagnetic resonance (EPR) spectroscopy for DPPH assay is proposed in order to avoid this limitation. In the EPR-DPPH assay, the underestimation of the radical scavenging abilities of four different pigment-containing extracts was corrected and this reflected up to a 16.1% change compared to the original values. Thus, the EPR-DPPH assay eliminated interference from the pigments coexisting in the reaction medium and improved the accuracy of the DPPH radical scavenging potential of such extracts. The principle of the proposed solution might also be employed in other assays which suffer from pigment interference.