Comparative Evaluation of Electron Paramagnetic Resonance and Spectrophotometry for DPPH-Based Antioxidant Capacity Determination

Abstract

The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is one of the most widely used methods for determining the radical scavenging capacity (RSC), yet discrepancies between spectrophotometric and electron paramagnetic resonance (EPR) measurements remain insufficiently addressed. In this study, simultaneous RSC measurements were performed by EPR and spectrophotometry to investigate the systematic differences between the methods. The results demonstrate that spectrophotometric measurements can underestimate RSC due to residual absorbance arising from neutralized DPPH and eventual decomposition products, leading to significant differences in IC50 and antioxidant activity index (AAI) values compared with EPR data. To overcome this limitation, three correction approaches are proposed, improving agreement with EPR results and eliminating statistical differences in IC50 and AAI values. These procedures were validated using phenolic standards and further evaluated for pigmented compounds using malvidin-3,5-diglucoside chloride and natural jambolan (Syzygium cumini) juice, an anthocyanin-rich sample. Although pigmentation affects high-concentration measurements, it does not significantly influence IC50 and AAI values after an appropriate absorbance correction. The proposed methodologies enhance the accuracy of the spectrophotometric DPPH assay and enable reliable comparison with EPR-based results, contributing to the improved standardization of RSC measurements.