High standard deviations in antioxidant assays may result from inconsistent sample preparation, reagent quality, or environmental factors. To improve reliability, standardize protocols, increase sample sizes, and use multiple controls to minimize variability.

Thank you very much. We will try to use more sample sizes and maybe also multiple controls

• Elisabeth García High standard deviations in the DPPH assay, even in controls, often result from issues such as pipetting errors, light sensitivity, temperature variability, or instrument inconsistencies. To reduce variability, ensure precise pipetting with calibrated tools, thoroughly mix samples, and conduct the assay in controlled lighting to prevent DPPH degradation. Maintain a stable temperature, validate the solubility of DPPH and sample components, and regularly calibrate the spectrophotometer, ensuring proper blanking. Using multiple replicates and carefully monitoring control conditions can further improve reliability.

It’s also important to consider potential color interference from colored compounds and the effects of complex sample matrices, such as plant extracts, which may interact unpredictably with DPPH. Sample purity is a critical factor to monitor. Additionally, keep in mind that the DPPH assay has inherent limitations, including the lack of universal standards, which complicates comparisons across studies. It also measures only free radical scavenging activity, excluding other antioxidant mechanisms like metal chelation or singlet oxygen quenching. To strengthen your study, consider complementing the DPPH assay with other methods for a more comprehensive assessment of antioxidant activity.

Thanks Janson, we will try this for the next time. Thanks once again