Application of a commercially available derivatization instrument and commonly used reagents to HPLC on-line determination of antioxidants

This study demonstrates the potential of a commercially available derivatization instrument coupled with HPLC for separation and on-line determination of antioxidants detected with widely used screening reagents—DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid)) or FCR (Folin–Ciocalteuʹs phenol reagent). As a result of optimization, key parameters of the analytical procedure were established: concentrations of derivatization reagents 1.5 mmol/L and 2.1 mmol/L in methanol for DPPH and ABTS, respectively, and 40% (v/v) FCR solution in water; reactor temperatures 130 °C for ABTS and FCR and 50 °C in the case of DPPH. In order to validate these procedures, Trolox equivalent antioxidant capacities (TEACs) for standard antioxidants obtained by post-column derivatization (on-line) and typical batch colorimetric methods (off-line) were compared; the corresponding measurements were found to be strongly correlated (Pearson coefficient: 0.973, 0.922, 0.853 for DPPH, ABTS and FCR, respectively). The results obtained for standard antioxidants and real fruit sample—aronia extract confirm the applicability of the proposed system to the on-line detection of antioxidants. Its great advantage compared to current routine methods is that it provides both chromatographic profiles and corresponding fingerprints of antioxidants (including unknown ones) along with quantitative determination of antioxidative potential—total, and those exhibited by individual compounds.