Determination of total antioxidant capacity of humic acids using CUPRAC, Folin–Ciocalteu, noble metal nanoparticle- and solid–liquid extraction-based methods

• Humic acid antioxidant capacity (TAC) was determined by CUPRAC, FC, and nanoparticle methods.
• QUENCHER extraction involving oxidation-coupled solubilization was effective for TAC measurement.
• QUENCHER-FC and QUENCHER-CUPRAC methods agreed well and had similar precision.
• Highest TAC in QUENCHER-CUPRAC was achieved by mixing DMSO or MeOH with 3-fold water.
• This is the first report on simultaneous solubilization and TAC assay of insoluble humic matrix.

Total antioxidant capacity (TAC) of humic acid (HA) samples was determined using CUPRAC (CUPric Reducing Antioxidant Capacity), FC (Folin–Ciocalteu), QUENCHER-CUPRAC, QUENCHER-FC, Ag-NP (Silver nanoparticle)‒ and Au-NP (Gold nanoparticle)‒based methods. Conventional FC and modified FC (MFC) methods were applied to solid samples. Because of decreased solubility of Folin–Ciocalteu's phenol reagent in organic solvents, solvent effect on TAC measurement was investigated using QUENCHER-CUPRAC assay by using ethanol:distilled water and dimethyl sulfoxide:distilled water with varying ratios. To see the combined effect of solubilization (leaching) and TAC measurement of humic acids simultaneously, QUENCHER experiments were performed at 25 °C and 50 °C; QUENCHER-CUPRAC and QUENCHER-FC methods agreed well and had similar precision in F-statistics. Although the Gibbs free energy change (ΔG°) of the oxidation of HA dihydroxy phenols with the test reagents were negative, the ΔG° was positive only for the reaction of CUPRAC reagent with isolated monohydric phenols, showing CUPRAC selectivity toward polyphenolic antioxidants. This is the first work on the antioxidant capacity measurement of HA having a sparingly soluble matrix where enhanced solubilization of bound phenolics is achieved with coupled oxidation by TAC reagents.

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