Re-evaluation of the 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay for antioxidant activity

Kinetics and stoichiometry of reactions between the 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable radical and 25 antioxidant compounds with different structure, molecular weight, number of -OH groups, and redox potential were investigated by recording the loss of DPPH(•) absorbance at 515 nm continuously for 10 min. A series of antioxidant concentrations was tested to determine linear response ranges and reaction saturation points. The primary feature distinguishing antioxidant activity--rate of initial reaction (<30 s)--was controlled by whether the dominant antioxidant mechanism was electron (very fast) or hydrogen atom (slow) transfer and by impairment of steric accessibility to the DPPH radical site by bulky ring adducts and multiple phenolic rings. Results raise serious questions regarding application of the DPPH assay for ranking antioxidants and natural extracts and suggest possible redirection of this assay to distinguish active reaction mechanisms by comparing reactions rates and patterns in different solvents and in 50% water/methanol mixtures at different pH values.