The potential pitfalls of using 1,1-diphenyl-2-picrylhydrazyl to characterize antioxidants in mixed water solvents.

Approaching living systems, aqueous solutions are appropriate to characterize antioxidants, whereas the frequently used standard 1,1-diphenyl-2-picrylhydrazyl (DPPH) is insoluble in water. Therefore, mixed water-ethanol solvents were investigated using the electron paramagnetic resonance (EPR) spectroscopy. Two forms of DPPH were identified: at higher ethanol ratios a quintet spectrum characteristic of solutions, and at lower ratios, a singlet spectrum typical for solid DPPH, were found. Mixed solvents with 0-50% (v/v) water reproduced the same antioxidant equivalent points well and the reaction rate between DPPH and the antioxidant may increase considerably with increasing water ratios, as demonstrated using vitamin E as an antioxidant. But at still higher water ratios (70-90% (v/v)) the antioxidant activities dropped, since a part of the DPPH in the aggregated form does not react sufficiently with the antioxidants. Characteristics of the most common antioxidants were determined in ethanol or its 50% (v/v) aqueous solution.