Hydrogen, protons and electrons: Rationalizing in vitro antioxidant assay selection

Antioxidants play a critical role in mitigating oxidative stress by scavenging reactive oxygen species and preventing oxidative damage. Consequently, the accurate assessment of antioxidant activity is essential in pharmaceutical, nutraceutical, food, and biomedical research. Antioxidants neutralize free radicals through various mechanisms, primarily involving the transfer of hydrogen atoms, protons, or electrons. Understanding these mechanistic differences is critical for selecting appropriate in vitro assays that accurately reflect the specific antioxidant activities of compounds under investigation. The use of inappropriate antioxidant assays often results in inconsistent measurements, limited comparability between studies, poor reproducibility, and the generation of false-positive or false-negative results. This article presents a structured, mechanism-driven framework for antioxidant assay selection based on the chemical principles underlying antioxidant–radical interactions. The proposed approach aligns assay chemistry with the structural characteristics, physicochemical properties, and anticipated redox behaviour of antioxidant compounds. By systematically matching antioxidants to suitable in vitro assays, the proposed framework aims to improve the accuracy, reliability, and interpretability of antioxidant activity measurements, supporting robust screening, standardization, and informed drug discovery.