A critical examination of the DPPH method: Mistakes and inconsistencies in stoichiometry and IC50 determination by UV-Vis spectroscopy.

The DPPH method has been reported with misconceptions in a large number of studies, thus precluding comparison of results. Attention is drawn to a common mistake in the unit used to express the IC50 of ascorbic acid and other antioxidant substances. Concentration of the antioxidant is widely misused with a total disregard for the DPPH• concentration, while the molar ratio of antioxidant/DPPH• would be the correct choice. Data from 26 studies with widely varying IC50 values were renormalized according to reaction stoichiometry, resulting in values which are more coherent and closer to the ideal one of 0.25 for at least 15 of them. In addition, the model which is currently being used to calculate the DPPH• concentration can lead to an overestimation of around 7%, as it does not take into account the small contribution of the reaction product. In view of that, we present a mathematical model to correct the overestimation of the DPPH• concentration.