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.

Direct measurement of total antioxidant capacity of cereals: QUENCHER-CUPRAC method.

Polyphenols in cereal samples are distributed as free, soluble-esterified, and insoluble-bound forms either esterified or etherified to cell wall constituents. In the evaluation of total antioxidant capacity (TAC) of cereals, rather difficult and time-consuming acid, alkaline and enzymatic treatments of residue have been applied to complete the extraction of bound phenolic compounds. Thus, this work is aimed to measure the TAC of cereals (i.e. barley, wheat, rye, oat) by the 'QUENCHER procedure' (involving forced solubilization of bound phenolics by oxidizing TAC reagents) with the direct use of copper(II)-neocuproine (Cu(II)-Nc) reagent of the CUPric Reducing Antioxidant Capacity (CUPRAC) assay. In this novel 'QUENCHER-CUPRAC' method, reaction time and solvent composition parameters were optimized, and the method was applied to cereal samples with CUPRAC reagent dissolved in 1:1 (v/v) ethanol-water mixture. The antioxidant capacities of soluble and insoluble fractions were simultaneously measured to give a hierarchic TAC order of cereals as: barley>rye>oat>wheat. The TAC values of cereals measured by QUENCHER-CUPRAC were higher than those of original QUENCHER method using ABTS(•+) and DPPH reagents. Polyphenolic mixtures in a cellulose matrix gave additive TAC values with QUENCHER-CUPRAC. The proposed method correlated linearly with QUENCHER-ABTS(•+) (r=0.956) and QUENCHER-DPPH (r=0.976).

Determination of biothiols by a novel on-line HPLC-DTNB assay with post-column detection.

A novel on-line HPLC-DTNB method was developed for the selective determination of biologically important thiols (biothiols) such as L-cysteine (Cys), glutathione (GSH), homocysteine (HCys), N-acetylcysteine (NAC), and 1,4-dithioerythritol (DTE) in pharmaceuticals and tissue homogenates. The biothiols were separated on C18 column using gradient elution, reacted with the postcolumn reagent, DTNB in 0.5% M-ß-CD (w/v) solution at pH 8, to form yellow-colored 5-thio-2-nitrobenzoic acid (TNB), and monitored with a PDA detector (λ=410 nm). With the optimized conditions for chromatography and the post-column derivatization, 40 nM of NAC, 40 nM of Cys, and 50 nM of GSH can be determined. The relative standard deviations of the recommended method were in the range of 3.2-5.4% for 50 µM biothiols. The negative peaks of biothiol constituents were monitored by measuring the increase in absorbance due to TNB chromophore. The detection limits of biothiols at 410 nm (in the range of 0.04-0.58 µM) after post-column derivatization with DTNB+M-ß-CD were much lower than those at 205 nm UV-detection without derivatization, and were distinctly lower than those with post-column DTNB alone. The method is rapid, inexpensive, versatile, nonlaborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of biothiol constituents of biological fluids and pharmaceuticals.

Differences in responsivity of original cupric reducing antioxidant capacity and cupric-bathocuproine sulfonate assays to antioxidant compounds.

The cupric reducing antioxidant capacity (CUPRAC) method using bathocuproine sulfonate (BCS) as cuprous-chelating ligand claims to be an efficient substitute for the conventional CUPRAC assay using neocuproine; however, differences in their physical chemistry give rise to significantly varying responsivities to antioxidant compounds. The antioxidant capacity due to ß-carotene (in acetone or dichloromethane) was fully measurable by conventional CUPRAC but not at all by cupric-BCS assay. Furthermore, CUPRAC exhibits distinctly stronger responses than cupric-BCS to naringenin, α-tocopherol, and bilirubin.