Identification and anti-oxidant capacity determination of phenolics and their glycosides in elderflower by on-line HPLC-CUPRAC method

Introduction Development and application of an on-line cupric reducing anti-oxidant capacity (CUPRAC) assay coupled with HPLC for separation and on-line determination of phenolic anti-oxidants in elderflower (Sambucus nigra L.) extracts for their anti-oxidant capacity are significant for evaluating health-beneficial effects. Moreover, this work aimed to assay certain flavonoid glycosides of elderflower that could not be identified/quantified by other similar on-line HPLC methods (i.e. 2,2-diphenyl-1-picrylhdrazyl and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid). Objective To identify anti-oxidant constituents in elderflower by HPLC and to evaluate their individual anti-oxidant capacities by on-line HPLC-CUPRAC assay with a post-column derivatisation system. Methods The separation and UV detection of polyphenols were performed on a C₁₈-column using gradient elution with two different mobile phase solutions, that is acetonitrile and 1% glacial acetic acid, with detection at 340 nm. The HPLC-separated anti-oxidant polyphenols in column effluent react with copper(II)-neocuproine in a reaction-coil to reduce the latter to copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. Results The detection limits of tested compounds at 450 nm after post-column derivatisation were compared with those of at 340 nm UV-detection without derivatisation. LOD values (μg/mL) of quercetin and its glycosides at 450 nm were lower than those of UV detection at 340 nm. This method was applied successfully to elderflower extract. The flavonol glycosides of quercetin and kaempferol bound to several sugar components (glucose, rhamnose, galactose and rutinose) were identified in the sample. Conclusion The on-line HPLC-CUPRAC method was advantageous over on-line ABTS and DPPH methods for measuring the flavonoid glycosides of elderflower.