The FC method is a SET assay based on the reaction of phenolic compounds with the FC reagent in alkaline conditions. This reagent is a mixture of phosphomolybdate and phosphotungstate, yellow in color, which, when reduced by the phenolic groups, gives rise to an intense blue complex, with a broad absorption band in the 600–800 nm range. Diverse wavelengths in this range have been reported for the spectrophotometric analysis. The measured absorbance is proportional to the polyphenol content in the sample, which is usually quantified based on a calibration curve of gallic acid, and the results are expressed in terms of gallic acid equivalent (GAE) concentration. The assay is not specific for polyphenols, and other reducing compounds (e.g., carotenoids, vitamin E, vitamin C, reducing sugars, certain amino acids) interfere [13,67]. However, it is assumed that the reaction of the FC assay with agri-food products is mostly due to polyphenols, and therefore the FC index is a good estimation of polyphenols content for such samples, as evidenced by the correlations reported for FC results and those obtained by other techniques such as LC-UV [66,68].

There are different studies on the analysis of TPC by the FC method in residues of olive oil production. For instance, Elkacmi et al. [69] determined the TPC in olive mill wastewater, obtaining values of 2.1–6.43 g GAE L⁻¹. For olive pomace, Alu’datt et al. [45] and Razek et al. [47] reported TPC values of 0.06–4.37 mg GAE g⁻¹ dry weight (dw) and 69.66 mg GAE g⁻¹ dw, respectively. There are also many studies for TPC determination in residues of wine production by the FC method, which is dependent on the extraction procedure used. As a matter of example, some values reported for TPC are 3.22 mg GAE g⁻¹ dw in grape skin [55], 42.10–188.21 mg GAE g⁻¹ wet weight (ww) [70], 50.96–219.23 mg GAE kg⁻¹ dw [57], and 196.2 mg GAE g⁻¹ dw [58] in grape pomaces, 63.46 mg GAE g⁻¹ ww in grape steams [70]_, 188.21 mg GAE g⁻¹ ww in grape seeds [70], and 26.1 g GAE g⁻¹ dw [71], 9.84–23.17 mg GAE g⁻¹ dw [60] in wine lees_.

The DPPH assay is a mixed-mode HAT/SET, used to determine the antioxidant capacity. DPPH is a free radical that has a deep purple color; it is one of the few stable organic radicals. The assay is based on the reaction of the DPPH radical with an antioxidant, e.g., phenolic compounds, which generates a reduced molecular form of the radical, and results in the loss of the purple color. Thus, the decrease in absorbance at 515 nm depends on the concentration of the antioxidant. Trolox is used as standard in calibration and the results of antioxidant capacity are expressed in terms of Trolox equivalent (TEAC) concentration [72,73,74,75]. Tournour et al. [76] evaluated the antioxidant capacity by DPPH in ethanol/water extracts and aqueous suspensions of grape pomaces from four different Portuguese grape varieties. The highest DPPH values were found in Touriga Nacional grape cultivar with 1.09 ± 0.13 and 1.12 ± 0.04 mmol TEAC g⁻¹ dry residue, in ethanol/water extracts and aqueous suspensions, respectively.

In this method, the 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic) acid (ABTS) is used. Antioxidants reduce the cation radical (ABTS^·+), throughout mixed-mode HAT/SET mechanisms, and a change of color occurs from a bluish-green solution to a decolorized one. The absorbance diminution at 743 nm depends on the concentration of antioxidants in the sample. Trolox is commonly used as a standard in calibration, and the ABTS results are expressed in terms of Trolox equivalent concentration [72,73,74,75,77]. Poveda et al. [70] applied the ABTS assay to the analysis of winery by-products, such as grape seeds, pomace, and stems. The highest antioxidant capacity values were found in grape seeds extracts (0.061–0.065 mmol Trolox g⁻¹). Similarly, Abdel-Razek et al. [47] applied the ABTS assay to evaluate the antioxidant activity of olive oil production by-products (olive pomace, olive leaves, and pomace olive oil). The highest antioxidant capacity was observed in olive pomace samples.

FRAP assay is a colorimetric method that measures the antioxidant capacity of a sample based on a redox reaction. The reaction involves a SET mechanism and consists of the reduction of ferric tripyridyl triazine complex to ferrous form at low pH (3.6), which has an intense blue color and can be monitored by measuring the change in absorption at 593 nm. The increase of absorption is related to the concentration of antioxidants in the sample. Trolox is used as standard and results are expressed in Trolox equivalent concentration [72,75,77]. Jurčević et al. [60] evaluated the potential of wine lees polyphenols as novel functional bioactive compounds in the protection against oxidative stress and hyperlipidemia. The antioxidant capacity of wine lees determined by FRAP was 45.7 ± 1.05 mM of Trolox equivalents per 100 g, respectively.

The ORAC method is a HAT assay used to determine antioxidant activity. It is based on the quenching of the fluorescence of fluorescein by peroxyl radicals. In the presence of antioxidants, which react with peroxyl radical, the decrease in the fluorescence signal is slowed down. The fluorescence decay over time is recorded, and the area under the curve is related to the antioxidant capacity. Trolox is habitually used as the standard in calibration, and results are reported as Trolox equivalent concentration [72,73,77]. Antoniolli et al. [58] determined the antioxidant capacity by ORAC of grape pomace extracts of cv. Malbec, and reported 2756 µmol TEAC g⁻¹. Pasten et al. [78] analyzed the antioxidant activity of Arbequina olive residues. The antioxidant capacity of fresh samples was 66.81, 101.75, and 283.5 µmol TEAC g⁻¹ dw, for DPPH, FRAP, and ORAC, respectively.

It is not evident what is the equivalence of the results provided by the different assays. It is not just a question of the units used to express the results; quite often, there is not a clear correlation between the results from different assays. However, as a general trend, when comparing results from different samples measured by several assays, samples having high results in one assay usually show high values in the others (See Figure 2).

Finally, it should be mentioned that, although less applied than spectrophotometric assays, electrochemical assays can also be used to determine the antioxidant capacity of samples, with cyclic voltammetry, differential pulse voltammetry, and square wave voltammetry being the most widely used techniques [79]. In this sense, electrochemical detection combined with molecularly imprinted polymers (MIPs) is an innovative approach for the quick detection of antioxidants [80].