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Current Analytical Chemistry


ISSN (Print): 1573-4110
ISSN (Online): 1875-6727

Research Article

Optimization and Validation of Two High-Throughput Methods Indicating Antiradical Activity

Author(s): Graciela Granados-Guzman, Ricardo Salazar-Aranda*, Marsela Garza-Tapia, Rocio Castro-Rios and Noemi Waksman de Torres

Volume 13, Issue 6, 2017

Page: [499 - 507] Pages: 9

DOI: 10.2174/1573411013666170118111516

open access plus


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.

Keywords: ABTS, antiradical activity, DPPH, high-throughput screening methods, natural products, reduction.

Graphical Abstract

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