Validation of two bio-analytical assays for the measurement of hydrophilic antioxidant in several food and beverage commodities in accordance with ISO 17025 regulatory guidelines

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology in the Faculty Health and Wellness Sciences at the Cape Peninsula University of Technology, 2013The accurate and consistent measurement of antioxidants is crucial to evaluating their biological role in the prevention and delay of cancer and other pathological conditions. Hence, the performance of the analytical method utilized should be evaluated for acceptable levels of accuracy, precision and other performance parameters according to internationally accepted standards. Additionally, the measure and influence of existing errors should be evaluated and the method optimized to reduce such errors. In furtherance of this vital aim, this research project sought out to optimize and validate two bio-analytical assays for the measurement of total antioxidant capacity and L-ascorbic acid (L-AA), respectively in food commodities. The validation procedure was performed in accordance with ISO 17025 international standard. The first study in this thesis evaluated, optimized and validated the hydrophilic oxygen radical absorbance capacity (H-ORACFL) assay using fluorescein for total antioxidant capacity in various food and beverage products. The assay demonstrated good results with regard to accuracy, precision, linearity, specificity, limits of detection (LOD) and quantification (LOQ) and robustness. The extraction solvent (60% ethanol) recovered excellent antioxidant yields for most samples tested. The optimization of the method in terms of temperature and sample usage on the micro-plate significantly (p