Development of an In-Line Capillary Electrophoresis Assay for Total Antioxidant Capacity
Adam D. Catherman, Emily B Merola, and Timothy G Strein. Bucknell University, Lewisburg, PA
This work focuses on developing a rapid and reliable method to test for antioxidants using capillary electrophoresis. Antioxidants are important in preventing free radical damage to cellular components including DNA. Common antioxidants include ascorbic acid, catechins, and phenolic acids which are found in a variety of food samples and dietary supplements. Due to the difficulty in isolating and quantifying all the individual antioxidant components of a sample, total antioxidant capacity (TAC) has become an important measurement. TAC takes into account all the antioxidants in a sample as well as their relative oxidation capability. Current methods to measure TAC, including FRAP and ABTS, are both lengthy and labor intensive. Moreover, these methods often give divergent results. Capillary electrophoresis (CE) is a relatively recent technology which can be used to mix reactants and/or separate ionic compounds in an automated and rapid analysis, while requiring minimal volumes of sample. In this work, TAC is determined via two sequential redox reactions all within the confines of the CE capillary. The first reaction involves the complete oxidation of the antioxidant(s) in the sample with by use of an excess of a oxidizing agent, either N-bromosuccinimide or 1,4-benzoquinone. The remaining oxidizing agent then reacts with a plug of excess ascorbic acid. The ascorbic acid that remains is detected using absorbance at 265 nm. The analytical performance of this approach has been characterized and this method has been successfully used to determine the TAC of several commercial beverages. However, the TAC values determined with the CE approach are much larger than expected based upon the FRAP method. A critical analysis of the utility of CE-based assays for TAC will be presented, including a discussion of the observed differences between quantitative results with different methods of analysis.