Development and Validation of an On-Line HPLC-DAD-Antioxidant Assay (ORAC)/ESI-HRMS System to Identify Antioxidant Compounds in Complex Mixtures

Abstract

Abstract An online high-performance liquid-chromatography-diode-array detector coupled with detection of antioxidant compounds using oxygen radical absorbance capacity (ORAC) assay and electrospray ionization-high-resolution mass spectrometer (HPLC-DAD-antioxidant assay (ORAC)/ESI-HRMS) was developed for the identification of antioxidant compounds in complex mixtures. The method was validated using quercetin and a mixture of antioxidant compounds with different antioxidant activities (resveratrol, dihydroxymethoxy-dihydrochalcone, ferulic acid, baicalein and luteolin). Accuracy of the system was established by comparing the results from the developed system with those from ORAC microplate assay determination and reveals the ability of the system to determine the respective contribution of antioxidant compounds to the whole activity of complex mixtures. Application of the system to the identification of antioxidants in a commercial Yerba Mate extract (Ilex paraguariensis St. Hil.) reveals the occurrence of seven actives, which were characterized as chlorogenic acids isomers (3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid), dicaffeoylquinic acid isomers (3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid) and rutin based on UV/Vis spectra, HRMS and MS/MS data. This on-line system is able to generate HPLC-DAD fingerprints, UV/Vis spectra, ORAC activity profile and high-resolution mass spectrometric data.