Background: The search for new natural or synthetic products with antioxidant activity is
commonly based on methods that involve reduction of either 2,2-diphenyl-1-picrylhydrazyl (DPPH) or
2-2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). However, the reported values of the
effective concentrations are highly variable, even in controls. Herein, we optimize and validate both
methods of determining antiradical activity.
Methods: Optimization was carried out using both a fractionated factorial design and a basic sequential
simplex method, by monitoring the reduction percentage. Quercetin or Trolox were used as positive
control. Furthermore, for each method, linearity, precision, accuracy, robustness, plate uniformity,
signal variability, and Z factor, were established.
Results: The optimized conditions for the DPPH method were: DPPH 280 µM in ethanol and 15 min
of reaction time in the dark. The linear range was between 7 and 140 µM with an R2 value of 0.9987.
The optimized conditions for the ABTS method were: ABTS adjusted to 0.7 absorbance units, 70%
concentration in ethanol, and a reaction time of 6 min in the dark. The linear range was found to be
between 1 and 70% with an R2 = 0.9991. For both methods, the accuracy and precision were within
limits and the Z factor value was higher than 0.89. The applicability of each method was assessed by
analyzing eight plant extracts.
Conclusion: The DPPH and ABTS reduction methods were optimized and validated on a microscale
and could be expected to be implemented in any laboratory.