Optimization and validation of an alternative method to evaluate total reactive antioxidant potential

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• The Total Reactive Antioxidant Potential (TRAP) assay is widely used for in vitro antioxidant capacity assessment.
• TRAP relies on luminol-enhanced chemiluminescence inhibition by antioxidants reacting with radicals from AAPH.
• Limitations exist, particularly for samples lacking a lag phase, and TRAP assay validation is underexplored.

Purpose of the Study:

• To optimize and validate the TRAP assay for enhanced reliability.
• To introduce and validate an Area Under the Curve (AUC) method for TRAP analysis.
• To address limitations of the standard TRAP assay, especially for samples without a lag phase.

Main Methods:

• Optimization of the TRAP assay, establishing a system stabilization period of 7000s before antioxidant addition.
• Validation of both standard TRAP and AUC evaluation methods using Trolox as a calibrator (50-250nM).
• Assessment of key validation parameters including specificity, linearity, precision, robustness, and detection/quantitation limits.

Main Results:

• Both TRAP and AUC methods demonstrated satisfactory validation parameters.
• Excellent linearity (r>0.99) and high precision (RSD <5% for intra- and inter-assay) were achieved.
• The AUC method proved advantageous for evaluating antioxidant potential in samples lacking a distinct lag phase.

Conclusions:

• The optimized and validated TRAP assay, particularly with the AUC evaluation method, provides a robust tool for assessing antioxidant capacity.
• The AUC approach enhances the applicability of TRAP assays to a broader range of samples, including those without a lag phase.
• This work establishes a validated methodology for TRAP assay, improving its utility in biochemical and analytical research.