Table 2.

Illustration of the main principles and detection mechanisms in antioxidant activity measurement.

Method for antioxidant capacity assay	Principles underlying the analytical techniques	Detection modes	Ref.
Chromatographic techniques
Thin layer chromatography	The stationary phase is a thin layer of silica gel, aluminium oxide, or cellulose which covers a support of glass, plastic, or aluminium foil. The mobile phase moves by capillarity.	Migration of analytes takes place at different rates due to various repartition coefficients	[91]
High performance thin layer chromatography	It relies on the same principle as conventional TLC but uses a stationary phase with smaller particle size.	Separation performed with improved resolution versus TLC	[93, 94]
Gas chromatography	Separation is based on the repartition between a liquid stationary phase and a gas mobile phase.	Flame ionization, thermal conductivity, or mass spectrometry detection	[124]
Liquid chromatography	Separation is based on the repartition between a solid stationary phase and a liquid mobile. phase	Mass spectrometry or electrochemical detection	[106]
High performance liquid chromatography	Separation is based on the repartition between a solid stationary phase and a liquid mobile phase with distinct polarities at high flow rate and pressure of the mobile phase.	UV-VIS (diode array), fluorescence, mass spectrometry, or electrochemical detection	[108]
Spectrometric techniques
DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging method	Antioxidant reaction with the nitrogenated radical, followed by absorbance diminution at 515–518 nm.	Photocolorimetry	[125, 126]
TEAC (Trolox Equivalent Antioxidant Capacity) method	Antioxidant reaction with ABTS∙+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid cation radical) generated by K2S2O8, followed by blue solution absorbance diminution at 734 nm.	Photocolorimetry	[127]
DMPD (N,N-dimethyl-p-phenylenediamine) method	Reduction of DMPD∙+ by antioxidants, in the presence of FeCl3, with subsequent absorbance decrease at 505 nm.	Photocolorimetry	[128]
FRAP (ferric reducing antioxidant power) method	Reduction of the Fe3+-TPTZ (2,4,6-tripyridyl-s-triazine) complex, by sample antioxidants, with absorbance taken at 593 nm.	Photocolorimetry	[129]
PFRAP (potassium ferricyanide reducing power) method	Reduction of potassium ferricyanide by antioxidants, yielding potassium ferrocyanide. The latter reacts with ferric trichloride, and the resulted ferric ferrocyanide blue colored complex is measured at maximum absorbance of 700 nm.	Photocolorimetry	[130]
CUPRAC (cupric reducing antioxidant capacity) method	Cu(II)-neocuproine complex reduction to Cu(I) – bis (neocuproine) chelate, with absorbance recorded at 450 nm.	Photocolorimetry	[131, 132]
Phosphomolybdenum assay	Mo (VI) is reduced Mo (V) by the antioxidants in the sample with generation of a green phosphate/Mo (V) complex at acidic pH, determined at 695 nm.	Photocolorimetry	[133]
Lipid peroxidation activity assay	Antioxidants delay lipid hydroperoxide generation caused by lipoxygenase. The absorbance is measured at 234 nm.	UV absorbance	[106, 134]
	Antioxidants delay radical-induced malonyl dialdehyde generation, as decomposition product of endoperoxides of unsaturated fatty acids, in the presence of thiobarbituric acid. The absorbance is measured at 535 nm.	Photocolorimetry	[106, 135]
	Antioxidants delay conjugated dienes generation as a result of peroxidation of lipid components. The absorbance is measured at 234 nm.	UV absorbance	[85]
Superoxide radical scavenging activity assay	Antioxidants are subject to reaction with a substrate solution containing xanthine sodium salt and 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride. Xanthine oxidase is used as biocatalyst and the absorbance increase was monitored at 505 nm.	Photocolorimetry	[136]
	Superoxide anions are generated in a solution containing nitroblue tetrazolium, NADH and phenazine methosulfate. The absorbance taken at 560 nm decreases in the presence of antioxidants, pointing towards superoxide anion scavenging activity.	Photocolorimetry	[137]
Beta carotene bleaching method	Linoleic acid is oxidized by reactive oxygen species. The generated oxidation products such as lipid peroxyl radicals initiate β-carotene oxidation and, consequently, its decolorization. Antioxidants delay the discoloration rate, with absorbance measured at 434 nm.	Photocolorimetry	[138, 139]
Xanthine oxidase inhibition assay	Xanthine is used as substrate that yields uric acid as product of XOD-catalyzed reaction. Allopurinol is used as xanthine oxidase inhibitor. Absorbance is measured at 293 nm.	Photocolorimetry	[140]
Superoxide dismutase method	It is assessed in an erythrocyte lysate in the presence of pyrogallol. The enzyme inhibits the autooxidation of the hydroxylated compound, with absorbance read at 420 nm.	Photocolorimetry	[141]
Catalase activity assay	It is measured in an erythrocyte lysate in the presence of H2O2. The rate of H2O2 decomposition is assessed at 240 nm.	Photocolorimetry	[142]
Ferrous ion chelating activity assay	Antioxidants react with ferrous salt (e.g., FeCl2). Ferrozine as Fe(II) chelator yields a violet complex with absorbance read at 562 nm. The reaction is hindered in the presence of antioxidants that act by chelation, and the result is a decrease of the color of the ferrozine-Fe2+ complex, as chelators other than ferrozine act as competing agents for the metal ion.	Photocolorimetry	[143, 144]
ORAC (Oxygen Radical Absorbance Capacity) assay	Antioxidants scavenge the peroxyl radicals, induced by 2,2′-azobis-(2-amidino-propane) dihydrochloride (AAPH) decomposition, slowing the fluorescent decay of fluorescein or phycoerythrin.	Fluorimetry	[145–147]
HORAC (Hydroxyl Radical Antioxidant Capacity) assay	Antioxidants quench OH radicals formed in a Fenton-like system.	Fluorimetry	[148]
TRAP (Total Radical Trapping Antioxidant Parameter) assay	The rate of peroxyl radical generation by 2,2′-diazobis-2-amidinopropane dihydrochloride (ABAP) is quantified through the fluorescence diminution of the protein R-phycoerythrin.	Fluorescence	[149, 150]
Horseradish peroxidase-luminol-hydrogen peroxide chemiluminescent assay	Horseradish peroxidase catalyses luminol oxidation by H2O2 with light emission. Light emission is quenched by antioxidants.	Chemiluminescence	[151]
Electrochemical techniques
Cyclic voltammetry	The potential is linearly swept in a triangular waveform.	The analytical signal is represented by the intensity of the cathodic/anodic peak	[152, 153]
Differential pulse voltammetry	Potential voltage pulses are superimposed on the potential scan, which is performed linearly or stairstep-wise.	First current sampling before applying the pulse and the second towards the end of the pulse period	[154, 155]
Square-wave voltammetry	A square wave is superimposed on the potential staircase sweep.	Current intensity recorded at the end of each potential change	[155, 156]
Amperometry	The potential of the working electrode is maintained at a constant value versus the reference electrode.	Current intensity generated by the oxidation/reduction of an electroactive analyte	[157]
Biamperometry	The reaction of the antioxidant with the oxidized form of a reversible indicating redox couple in an electrochemical cell containing two identical electrodes.	The current flowing between two identical working electrodes at a constant small applied potential difference	[158–160]
Potentiometry	The analytical signal represented by the potential change is the result of the variation of an ionic species concentration. The antioxidants react with the oxidized form of a redox couple, altering the concentration ratio between the oxidized form and the reduced form.	Potential change after reaction of antioxidants with an indicating redox couple	[161]