A Novel Stoichio-Kinetic Model for the DPPH• Assay: The Importance of the Side Reaction and Application to Complex Mixtures

• Main Authors: Lucrezia Angeli, Sebastian Imperiale, Yubin Ding, Matteo Scampicchio, Ksenia Morozova
• Format: Article
• Language: English
• Published: MDPI AG 2021-06-01
• Series: Antioxidants
• Subjects: DPPH assay; kinetic model; phenolic compounds; side reaction; high-resolution mass spectrometry; antioxidant activity
• Online Access: https://www.mdpi.com/2076-3921/10/7/1019

The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay is widely used to determine the antioxidant activity of food products and extracts. However, the common DPPH• protocol uses a two-point measurement and does not give information about the kinetics of the reaction. A novel stoichio-kinetic model applied in this study monitors the consumption of DPPH• by common antioxidants following the second order reaction. The fitting of such decay yields the rate constant k₁, which describes the main reaction between antioxidants and DPPH•, and the rate constant k₂, which is attributed to a slower side reaction considering the products generated between the transient radicals (AO•) and another molecule of DPPH•. The model was first applied to antioxidant standards. Sinapic acid, Trolox and ascorbic and chlorogenic acids did not show any side reaction. Instead gallic, ferulic and caffeic acids achieved the best fitting with k₂. The products of the side reaction for these compounds were confirmed and identified with high-resolution mass spectrometry. Finally, the kinetic model was applied to evaluate the antioxidant activity of eight herbal extracts. This study suggests a new kinetic approach to standardize the common DPPH• assay for the determination of antioxidant activity.