Validation of Microplate Methods for Total Phenolic Content and Antioxidant Activity on Honeys, and Comparison with Conventional Spectrophotometric Methods

Conventional spectrophotometric methods used for determining total phenolic content and antioxidant activity are typically time-consuming, labor-intensive, and require large amounts of reagents. In the context of sustainable development and green chemistry, minimizing the use of hazardous substances and reducing reagent consumption have become key priorities. The implementation of microplate-based methods offers significant advantages, including reduced reagent volumes and shorter analysis times compared with traditional methods. Therefore, the aim of this study was to validate the Folin–Ciocalteu and DPPH microplate methods and compare their performance with conventional protocols. The limits of detection (LOD) for the microplate methods were lower than those for the conventional approaches, being approximately 0.7 µg/mL and 4.1 µg/mL for TPC, and 0.015 µg/mL and 0.081 µg/mL for DPPH, respectively. The relative standard deviation (RSD) of repeatability and reproducibility for both microplate methods was ≤6%. The accuracy ranged from 95.0% to 97.7% for TPC and from 95.3% to 98.7% for DPPH. Overall, the results confirm that the microplate and conventional methods are statistically equivalent at the 95% confidence level, demonstrating that microplate assays represent a reliable and environmentally friendly alternative for assessing total phenolic content and antioxidant activity.