The Optimisation of Analytical Parameters for Routine Profiling of Antioxidants in Complex Mixtures by HPLC Coupled Post-column Derivatisation

Introduction - The wide application of natural and artificial antioxidants in the food, cosmetic and pharmaceutical industries as well as the recognition of the importance of food antioxidants for supporting human health have created a demand for reliable and industrially applicable methods of determining antioxidative activity. This requirement can be fulfilled with the recently proposed HPLC-post-column derivatisation approach, enabling rapid measurements of antioxidative potential along with profiling antioxidants in complex mixtures. Objective - To employ the commercially available post-column derivatisation device for the on-line detection of antioxidants and to optimise analytical parameters enabling its application for routine profiling of antioxidants in complex mixtures such as those of plant or food origin. Methodology - The mixtures of standards and fruit extracts were resolved using an optimised HPLC method followed by the on-line derivatisation of analytes in a PCX post-column derivatisation instrument. Such parameters as the type of indicator reagent (ABTS, DPPH, Folin-Ciocalteu), its concentration and the temperature of the derivatisation reaction were investigated. The determinations of the Trolox equivalents (TE) values were compared with those obtained by corresponding bulk spectrophotometric tests. Results - The study confirms that the commercial HPLC-coupled post-column derivatisation instruments are suitable for routine on-line detection of antioxidants in complex mixtures and the determination of their TE values. The analytical parameters optimised here appear to represent a ready-to-use toolbox for the food and pharmaceutical industries, enabling the monitoring of bioactive substances along the production line and during storage, and the characterisation of plant material by creating chromatographic profiles supplemented with antioxidant fingerprints. Copyright (C) 2011 John Wiley & Sons, Ltd.