Development and Validation of a Stabilized TMPD•+ Spectrophotometric Assay for Total Antioxidant Capacity in Herbal Teas

Abstract

Antioxidant compounds present in plant-based foods and beverages play a crucial role in preventing oxidative stress by neutralizing reactive oxygen species. This study reports the formation and stabilization of the N,N,Nʹ,Nʹ-tetramethyl-p-phenylenediamine radical cation (TMPD^•+) in an aqueous medium and its application as a spectrophotometric probe for total antioxidant capacity (TAC) determination. Unlike the widely used ABTS^•+ and DPPH^•+ assays, which often require organic solvents, the proposed method operates entirely in water, representing a more environmentally friendly alternative.

The chemical parameters affecting TMPD^•+ stability were systematically optimized, including pH, buffer system, solvent effects, and oxidizing agents. The optimal conditions (λ_max = 563 nm) were established at pH 5.0 using Fe(III) 1.0 × 10^–4 M and TMPD 2.0 × 10^–4 M. Standard antioxidants were ranked according to their reducing capacity based on TMPD^•+ consumption, with tannic acid and ascorbic acid showing the highest responses. When applied to herbal teas, TAC values expressed as ascorbic acid equivalents showed a strong correlation with the ABTS^•+ assay (r = 0.900). These findings demonstrate that stabilized TMPD^•+ provides a simple, low-cost, sensitive, and greener approach for TAC determination in plant-based beverages.

Keywords

N,N,Nʹ,Nʹ-Tetramethyl-p-phenylenediamine antioxidant herbal teas polyphenolic compounds vegetable beverages

Introduction

p-phenylenediamines constitute a class of aromatic amines widely recognized for their ability to generate colored compounds via oxidation reactions.¹ Among them, N,N,N′,N′-tetramethyl-1,4-phenylenediamine (TMPD) has attracted considerable attention due to its broad applicability in biological, pharmacological, and analytical contexts.

In biological systems, TMPD has been used as an artificial electron donor to investigate mitochondria-mediated apoptosis,² evaluate the respiratory sensitivity of cytochrome c oxidase in mouse muscle mitochondria,³ and study electron transport during photosynthesis.⁴ In spectrophotometric assays, TMPD serves as a chromogenic probe in cyclooxygenase (COX) inhibition studies^5–8 and has been applied to assess the anti- inflammatory activity of plant extracts such as Plectranthus scutellarioides.⁹

The analytical versatility of TMPD is also evident in methods for quantifying oxidizing and reducing species. The compound has been employed as an artificial electron donor in place of ubiquinol to report enzymatic catalytic activity¹⁰ and as a redox-sensitive substrate in reactions with persulfate, being crucial for the indirect detection of ascorbic acid in commercial tablets,¹¹ nitric oxide (NO) and its decomposition product nitrite (NO₂^–) in biological matrices,¹² as well as benzoyl peroxide (BPO), an oxidizing agent widely used in food products (e.g., flours) and cosmetics.¹³

The analytical versatility of TMPD is associated with its redox behavior. The compound acts as an electron donor, generating the radical cation TMPD^●+ (Equation 1), which participates in colorimetric reactions.^14,15

T M P D^{● +} + e^{-} ⇌ T M P D E 0 = 0.27 V v s N . H . E

(1)

Herbal teas were selected as model samples due to their widespread global consumption, high content of phenolic compounds, and recognized antioxidant-related functional properties. They are extensively used in traditional and complementary medicine practices^16,17 and constitute important dietary sources of bioactive antioxidants.^18–20 Epidemiological evidence suggests that plant-derived antioxidants may contribute to mitigating oxidative stress-related conditions, including diabetes, neurodegenerative, cardiovascular, and hepatic diseases.^21,22

Given this context, methods based on the consumption of ABTS^●+ and DPPH^●+ radicals^23,24 are widely used to assess antioxidant capacity in plant matrices; however, these approaches often rely on organic solvents that are harmful to the environment. The growing awareness of sustainability and environmental impact has driven the need to replace conventional non-aqueous solvents (e.g., hexane, methanol, acetone) with approaches termed eco-extraction or green extraction, which prioritize the use of less aggressive and non-toxic solvents and aim to minimize environmental harm.²⁵

In this context, the present study aims to investigate the formation and stabilization of TMPD^●+ in aqueous medium to evaluate its applicability as a simple, low-cost, and environmentally friendly spectrophotometric method for total antioxidant capacity (TAC) determination in herbal teas. The performance of the proposed method was statistically compared with established TAC and total polyphenol content (TPC) parameters.^23,26

Materials and Methods

Equipment

Absorbance measurements were performed using an Agilent HP UV-8453 spectrophotometer with 1.0 cm path-length glass cuvettes. When required, the solutions were homogenized using an ultrasonic bath (Cristófoli). The pH of the solutions was measured with a microprocessor-controlled potentiometer (Quimis, model Q400 MT). Aqueous solutions were prepared with reverse-osmosis water (Quimis, model Q842-210) unless noted otherwise.

Reagents and Materials

N,N,N′,N′-tetramethyl-p-phenylenediamine solution 2.0 × 10^–3 M (TMPD, C₆H₄[N(CH₃)₂]₂, FW 237.18 g mol^–1, 99%, Sigma). Ferric sulfate pentahydrate solution 2.0 × 10^–3 M (Fe₂(SO₄)₃•5H₂O, FW 489.90 g mol^–1, 97%, Synth). Potassium persulfate solution 1.0 × 10^–3 M (K₂S₂O₈, FW 270.33 g mol^–1, 99%, Synth). Manganese dioxide (MnO₂, FW 86.94 g mol^–1, ≥98%, Reagen) and cobalt(III) hydroxide (Co(OH)₃, FW 109.93 g mol^–1, ≥95%, Merck) were used in solid form.

Ethanol (C₂H₅OH, FW 46.07 g mol^–1, 99%, Merck), acetone (C₃H₆O, FW 58.08 g mol^–1, 99.5%, Neon), 1,4-dioxane (C₄H₈O₂, FW 98.10 g mol^–1, 99%, J. T. Baker), ethylene glycol (C₂H₆O₂, FW 62.07 g mol^–1, 99%, Química Moderna), triethanolamine (C₆H₁₅NO₃, FW 149.19 g mol^–1, 85%, Synth), diethanolamine (C₄H₁₁NO₂, FW 105.14 g mol^–1, ≥98%, Química Moderna), isoamyl alcohol (C₅H₁₂O, FW 88.15 g mol^–1, 99%, Merck), methanol (CH₃OH, FW 32.04 g mol^–1, 99%, Dinâmica Química), ethyl acetate (C₄H₈O₂, FW 88.11 g mol^–1, 99%, Synth), and diethyl ether ((C₂H₅)₂O, FW 74.12 g mol^–1, 99%, Synth).

Buffer solutions of acetate (C₂H₄O₂/C₂H₃NaO₂), biphthalate (C₈H₆O₄/C₈H₅KO₄), and sodium citrate (C₆H₈O₇/Na₃C₆H₅O₇), all adjusted to pH 5.0, were prepared according to procedures described in the literature.²⁷

Ascorbic acid solution 1.0 × 10^–2 M (AA, C₆H₈O₆, FW 176.13 g mol^–1, 99%, Merck). Pyrogallic acid solution 1.0 × 10^–2 M (PA, C₆H₃(OH)₃, FW 126.11 g mol^–1, ≥98%, Synth). Catechol solution 1.0 × 10^–2 M (CT, C₆H₆O₂, FW 110.11 g mol^–1, 99%, Sigma–Aldrich). Tannic acid solution 1.0 × 10^–3 M (TA, C₇₆H₅₂O₄₆, FW 1701.19 g mol^–1, ≥98%, Sigma–Aldrich). 1,2,4-Benzenetriol solution 1.0 × 10^–2 M (1,2,4-B, C₆H₆O₃, FW 126.11 g mol^–1, 99%, Sigma–Aldrich). Gallic acid solution 1.0 × 10^–2 M (GA, C₇H₆O₅•H₂O, FW 188.134 g mol^–1, ≥98%, Celanese). Trolox^→ solution 2.0 × 10^–3 M (TR, C₁₄H₁₈O₄, FW 250.29 g mol^–1, 99%, Sigma).²³ Epigallocatechin gallate stock solution 1.0 × 10^–3 M (EG, C₂₂H₁₈O₁₁, FW 458.37 g mol^–1, ≥98%, Sigma–Aldrich) and working solution 1.0 × 10^–4 M.

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) solution 7.0 × 10^–3 M (ABTS, FW 548.68 g mol^–1, ≥98%, Sigma–Aldrich).²³ Potassium persulfate solution 0.14 M (K₂S₂O₈, FW 270.33 g mol^–1, 99%, Synth).²³

Methods

Optimization of the TMPD^•+ Generation Conditions

All A563 nm measurements were taken every 5 minutes for a total of 30 minutes.

Selection of the Oxidizing Agent

Five hundred microliters (500 µL) of 2.0 × 10^–3 M TMPD solution were added to four 5.0 mL volumetric flasks. In flasks 1, 2, 3, and 4, the following reagents were respectively added: 250 µL of 2.0 × 10^–3 M Fe(III) solution; 250 µL of 1.0 × 10^–3 M K₂S₂O₈ solution; 100 mg of MnO₂; and 100 mg of Co(OH)₃. After complete homogenization, the solutions in flasks 3 and 4 were centrifuged at 15,000 rpm for 15 minutes to remove excess solids.

Evaluation of the TMPD/Oxidant Molar Ratio

Ten 5.0 mL volumetric flasks, aliquots ranging from 100 to 1000 µL of 2.0 × 10^–3 M TMPD solution were added, along with 250 µL of 2.0 × 10^–3 M Fe(III) solution. The volume was completed with water.

Effect of pH on TMPD^•+

Eleven 5.0 mL volumetric flasks, 500 µL of 2.0 × 10^–3 M TMPD solution and 250 µL of 2.0 × 10^–3 M Fe(III) solution were added. From the second flask onwards, 600 µL of buffer solution with pH values ranging from 2.79, 3.07, 3.91, 5.00, 5.89, 6.82, 9.89, 10.0, 11.1, and 12.4 were added. The volume in each flask was completed with water.

Effect of Solvent Composition

The TMPD^●+ solution was prepared in 5.0 mL volumetric flasks by adding 500 µL of TMPD solution (2.0 × 10^–3 M), 250 µL of Fe(III) solution (2.0 × 10^–3 M), and 600 µL of acetate buffer solution (pH 5.0), and completing the volume with non-aqueous solvents (ethanol, acetone, 1,4-dioxane, ethylene glycol, triethanolamine, diethanolamine, isoamyl alcohol, methanol, ethyl acetate, and diethyl ether).

Determination of the RC of Some AOs

As only pure antioxidant compounds (AOs) were analyzed in this section, the measured values are designated as reducing capacity (RC) instead of TAC.

To determine the RC of selected AOs and to evaluate the response of the TMPD^●+ solution in more complex matrices, where the predominant antioxidant is already known, analytical calibration curves were constructed. This was accomplished by separately transferring aliquots of standard solutions into 5.0 mL volumetric flasks containing freshly prepared TMPD^●+ solution, followed by dilution to volume with water: 300–1,500 µL of CT (1.0 × 10^–3 M), 200–1,000 µL of 1,2,4-B (1.0 × 10^–3 M), 125–2000 µL of PA (1.0 × 10^–2 M), 100–500 µL of GA (5.0 × 10^–4 M), 100–500 µL of TA (1.0 × 10^–4 M), 100–500 µL of EG (1.0 × 10^–4 M), 100–500 µL of TR (3.0 × 10^–4 M), or 100–500 µL of AA (3.0 × 10^–4 M). After 15 minutes, absorbance readings were taken at 563 nm (A563 nm). Water was used as the reference.

Assessment of TAC in Herbal Tea Samples Via the TMPD^•+ Assay

Determination of TAC Based on the Consumption of TMPD^•+

The freshly prepared aqueous TMPD^●+ solution was used as a reference in all analyses. A563 nm measurements were taken after 15 minutes, and all sample analyses were conducted in triplicate.

Analytical Curve Using Ascorbic Acid Standard

The analytical curves of AA were obtained by transferring increasing volumes (100–500 µL) of a 3.0 × 10^–4 M AA solution into 5.0 mL volumetric flasks containing 500 µL of TMPD (2.0 × 10^–3 M), 250 µL of Fe(III) (2.0 × 10^–3 M), and 600 µL of acetate buffer solution (pH 5.0). The final volume was adjusted with water. A563 nm measurements resulted in a graph (A563 vs. C_AA), from which the Equation (2) of the line was obtained:

A 563 = a + b \times C A A

(2)

where C_AA represents the concentration of the AA solution (mg mL^–1).

Sample Preparation

The samples were prepared in accordance with the guidelines established by the Brazilian Pharmacopoeia,²⁶ with some modifications.

To prepare the aqueous extract, 0.75 g of the sample was weighed into an Erlenmeyer flask, to which 150 mL of distilled water was added. The mixture was kept in a water bath at 65°C for 30 minutes, stirred every 10 minutes, and then allowed to cool to room temperature.

The extract, along with the sample residues, was quantitatively transferred to a 250 mL volumetric flask, which was filled to the mark with distilled water and homogenized. Subsequently, the extract was filtered through filter paper, discarding the first 25 mL of the filtrate.

Analytical Curves Using Herbal Tea Samples

Analytical curves for teas were obtained by adding aliquots of the aqueous extract (100–1,000 µL) to 5.0 mL volumetric flasks containing 500 µL of TMPD (2.0 × 10^–3 M) and 250 µL of Fe(III) (2.0 × 10^–3 M). The final volume was adjusted with water. Measurements at A563 nm resulted in graphs (A563 vs. C_sample), from which the Equation (3) of the straight line was obtained:

A 563 = a + b \times C s a m p l e

(3)

where C_sample represents the concentration of the sample solution (mg mL^–1).

Preparation of ABTS^•+ and Folin–Denis Reagents

The ABTS^●+ solution and the Folin–Denis reagent (FDR) were prepared according to previously described procedures in the literature.^23,26

Determination of TAC Using the ABTS^•+ Method

The determination of TAC in herbal tea samples was performed using the ABTS^●+ radical cation consumption method, as described in the technical report by the Brazilian Agricultural Research Corporation (EMBRAPA)²³ and in previous studies.^28,29

Determination of TPC Using FDR

The quantification of TPC in herbal tea samples using FDR was performed following the methodology described in previous studies,^26,30 with the substitution of pyrogallic acid by gallic acid as the standard.

Results and Discussion

Optimization of the TMPD^•+ Generation Conditions

Evaluation of Some Oxidizing Agents for the Generation of TMPD^•+

TMPD is an aromatic amine that exhibits a light violet–bluish coloration and, in the presence of an oxidizing agent under acidic conditions, is converted to the purple radical cation TMPD^●+ (Equation 1), characterized by a maximum absorption at 563 nm. All oxidizing agents evaluated (Fe(III), S₂O₈^2–, MnO₂, and Co(OH)₃) were capable of promoting TMPD oxidation, as described by the corresponding half-reactions (Equations 4–7).²⁷

F e (I I I) + e^{-} ⇌ F e (I I) E^{0} = 0.77 V v s . N . H . E .

(4)

S_{2} O_{8}^{2 -} + 2 e^{-} ⇌ 2 S O_{4}^{2 -} E^{0} = 2.01 V v s . N . H . E .

(5)

M n O_{2} + 4 H^{+} + 2 e^{-} ⇌ M n (I I) + H_{2} O E^{0} = 1.23 V v s . N . H . E .

(6)

C o (I I I) + e^{-} ⇌ C o (I I) E^{0} = 1.84 V v s . N . H . E

(7)

As shown in Figure 1A (A563 vs. time), TMPD^●+ formed using Fe(III) or Co(OH)₃ exhibited greater temporal stability compared to systems employing S₂O₈^2– or MnO₂. Although MnO₂ and Co(OH)₃ effectively oxidized TMPD, their low solubility in water would require additional separation steps (e.g., centrifugation or filtration), increasing procedural complexity. The S₂O₈^2– system produced higher absorbance values after 15 minutes; however, its strong oxidizing character may promote secondary reactions that affect long-term radical stability.

Figure 1.

(A) Formation of the TMPD⁺ in the Presence of Oxidizing Agents: ▪ = Co(OH)₃; ● = MnO₂; ▲ = S₂O₈^2– 5.0 × 10^–5 M e ▼ = Fe(III) 1.0 × 10^–4 M. TMPD 2.0 × 10^–4 M in all Solutions. (B) Determination of the Ratio and Stability of the TMPD⁺ Solution: Fe(III) 1.0 × 10^–4 M and TMPD (0.4 – 4.0) × 10^–4 M.

Fe(III) was therefore selected as the oxidizing agent due to its adequate redox potential, high aqueous solubility, operational simplicity, and lower environmental impact. Furthermore, the reduced species formed, Fe(II), does not absorb at 563 nm and does not interfere with the monitored signal under the optimized conditions.

Although different oxidizing agents may generate secondary species during oxidation, no significant spectral interference was observed at 563 nm. Under the selected conditions, potential interference from oxidant byproducts is considered negligible, supporting the suitability of the Fe(III)/TMPD system for controlled radical generation.

Effect of the Molar Ratio Between TMPD and Fe(III) on the Formation of TMPD^•+

The linear working range of TMPD (0.4–4.0 × 10^–4 M) in the presence of Fe(III) (1.0 × 10^–4 M) was evaluated. As shown in Figure 1B, the highest analytical signal (A563 nm) was achieved at a 1:1 ratio. The 1:1 ratio observed in this study is consistent with previous thermodynamic findings demonstrating a single-electron transfer in the reaction between TMPD and oxidizing agents, as well as with the standard reduction potential reported for the TMPD^●+/TMPD redox couple (Equation 1).^14,15 However, the use of a 2:1 [TMPD:Fe(III)] molar ratio, along with the observed excess of TMPD^●+, supports the shift of Equation (1) toward product formation.¹⁵

Thus, a 2:1 molar ratio of [TMPD]:[Fe(III)] was employed to ensure complete consumption of Fe(III) and maximize the formation of TMPD^●+ at 1.0 × 10^–4 M. Since Fe(III) is present at half the concentration of TMPD and is rapidly consumed according to the chemical equilibria described in Equation (1), it is not expected to remain in solution in its free form, thereby minimizing interactions with antioxidants, such as polyphenols, present in the plant matrix.

pH and Buffer Effects on TMPD^•+ Formation and Stabilization

The formation and stability of TMPD^●+ were evaluated over a pH range from 2.5 to 12.4 (Figure 2A). At pH 2.5, although initial radical formation was observed, the signal was not stable over time. This reduced stability under strongly acidic conditions may be attributed to excessive protonation, which can alter electron-transfer equilibria and favor radical recombination or side reactions, thereby decreasing TMPD^●+ persistence.

Figure 2.

(A) Influence of pH on the Formation of the TMPD⁺: TMPD 2.0 × 10^–4 M; Fe(III) 1.0 × 10^–4 M. ▪ = pH 2.5; ● = pH 3; = pH 4.0; = pH 5.0; = 6.0; ◀ = 7.0 and = No Buffer. (B) Influence of Buffer Solution (pH 5.0) on the Formation of the TMPD⁺: TMPD 2.0 × 10^–4 M; Fe(III) 2.0 × 10^–4 M. ▪ = Acetate Buffer and ● = Biphthalate buffer. (C) Influence of Non-aqueous Solvents on the Formation of the TMPD⁺: TMPD 2.0 × 10^–4 M e Fe(III) 1.0 × 10^–4 M. = Ethanol; = Methyl alcohol; ▪ = Ethylene Glycol and ▼ = Deionized Water (Reference Solution).

Above pH 8.0, no purple coloration was observed, likely due to the hydrolysis of Fe(III) and the formation of insoluble iron hydroxide species, which limit its oxidizing capacity and hinder TMPD oxidation.

Buffer solutions at pH 3.9 and 5.0 provided stable TMPD^●+ formation for up to 30 minutes. In the pH range 6.0–7.0, radical formation occurred but without sustained stability, confirming that partial protonation of TMPD (pK₁ = 5.92 ± 0.12)³¹ is required for efficient oxidation and stabilization.

Since improved stability was observed in a mildly acidic medium (pH ≈ 5.0), acetate (pK_a = 4.76), citrate (pK₁ = 3.15; pK₂ = 4.77; pK₃ = 6.40), and biphthalate (pK₁ = 2.9; pK₂ = 5.4) buffer systems were evaluated.²⁷

In citrate buffer, the absorbance at 563 nm decreased significantly, most likely due to strong complex formation between Fe(III) and citrate (log β₁ = 10.25; T = 25°C),^32,33 which reduces the availability of free Fe(III) to oxidize TMPD.

In the acetate buffer, the maximum A563 nm value (0.84) was reached after 20 minutes, followed by a 10% decrease over 30 minutes. A similar profile was observed with biphthalate buffer, with maximum absorbance (0.920) at 15 minutes and a 6% decrease after 30 minutes (Figure 2B). Acetate buffer was selected for subsequent experiments due to its adequate radical stability, low-cost, and widespread laboratory use.

At pH 5.0, TMPD exists predominantly in its protonated form, which favors controlled oxidation to TMPD^●+. Under these conditions, the phenolic groups of polyphenols also remain largely protonated, reducing their tendency to coordinate iron and minimizing Fe(II)/Fe(III) complexation or the formation of iron hydroxide species. This pH, therefore, provides a balanced environment for efficient radical generation and stabilization.

Influence of Selected Solvents on the Stability of the TMPD^•+ Solution

Diethanolamine, triethanolamine, and acetone, when added after the oxidation of TMPD by Fe(III), led to the rapid decolorization of TMPD^●+. The amine-containing solvents may promote reduction or destabilization of the radical species through acid–base interactions or electron donation, shifting the redox equilibrium toward the reduced form. In contrast, isoamyl alcohol, ethyl acetate, and diethyl ether did not exhibit the characteristic purple coloration due to phase separation in the aqueous medium, preventing effective interaction with the oxidized species. In the presence of 1,4-dioxane, a pink coloration was observed without a defined absorption maximum at 563 nm, suggesting altered solvation of the radical cation and possible perturbation of the electronic structure of TMPD^●+. Conversely, ethanol, methanol, and ethylene glycol maintained the purple coloration, allowing absorbance monitoring at 563 nm for up to 30 minutes (Figure 2C).

Ethanol produced the lowest initial absorbance (0.187 ± 0.014), indicating reduced formation or stability of TMPD^●+, followed by gradual signal decay over time.^{31,32,34–36} Methanol exhibited intermediate initial absorbance (0.472 ± 0.036) with slight variation over time, suggesting that solvent–solute interactions may influence radical stabilization and electron-transfer kinetics. Ethylene glycol produced an initial absorbance (1.046 ± 0.002) comparable to that of the aqueous system (0.988 ± 0.011), with minimal variation, indicating effective solvation of the charged species.

The solvent-dependent behavior can be explained by differences in polarity, dielectric constant, and hydrogen-bonding capacity. As TMPD^●+ is a charged species, its stability relies on efficient solvation. Organic solvents may alter redox equilibria and electron-transfer kinetics by modifying the stabilization of ionic intermediates. Protic solvents can engage in hydrogen-bond interactions, whereas less polar or aprotic environments may reduce stabilization of the radical cation, leading to decreased signal stability. These findings reinforce that a pure aqueous medium provides optimal stabilization of TMPD^●+ while minimizing solvent-related perturbations and environmental impact.

Evaluation of RC in AOs

The addition of an AO to the solution containing TMPD^●+ promotes its reduction, leading to a measurable decrease in absorbance at 563 nm. Due to the inability of spectrophotometric techniques to detect intermediate species or specific oxidation products of AOs, the reaction mechanism was generalized using Equation (8),³⁷ which applies to all evaluated standard antioxidants (Table 1). This limitation is inherent to spectrophotometric methods, which, despite being both sensitive and easy to perform, cannot provide detailed insights into reaction intermediates or the specific pathways of antioxidant action.

Table 1.

Reducing Capacity Values Expressed as Ascorbic Acid (RC_AAE) of Reducing Compounds Obtained with the Procedure Based on the Consumption of the TMPD^•⁺ Radical Cation.

Compound	MM	GHL	R₁	R₂	R₃	R₄	R₅	LWR/10^–4	b/10²	RC_AAE
Tannic acid	1701.19	25	–	–	–	–	–	0.002–0.01	–4560 ± 178	17.1
1,2,4-benzenetriol	126.11	3	O H	H	OH	H	H	0.04–0.20	–231 ± 16	0.8640
Epigallocatechin gallate	458.40	8	–	–	–	–	–	0.02–0.10	–80.0 ± 3.50	0.2994
Pyrogallic ácid	126.11	3	OH	H	H	H	OH	0.25–4.0	–8.52 ± 0.39	0.0319
Catechol	110.10	2	OH	H	H	H	H	0.60–3.0	–4.31 ± 0.32	0.0161
Gallic acid	170.12	3	OH	H	COOH	H	OH	2.0–10	–1.52 ± 0.18	0.0057
Trolox^→	250.30	–	–	–	–	–	–	0.8–4.0	–20.3 ± 1.6	0.076
Ascorbic acid	176.13	–	–	–	–	–	–	0.06–0.30	–267 ± 23	1.0000

Note: MM = Molar mass (g mol^–1); HPG = Hydroxyl phenolic groups; FR e b = Linear working range (mol L^–1) and angular coefficient originating from the equation of the line of the analytical curve, respectively; RC_AAE = Reducing capacity expressed in ascorbic acid equivalent (mM AA).

{TMPD}^{● +} + AO ⇌ {AO}^{● +} + TMPD

(8)

In this study, analytical calibration curves generated from ascorbic acid and other standard AOs were employed to quantify their RC, based on the extent of TMPD^●+ consumption as described by Kuss and Compton.³⁷

A typical analytical curve (A563 vs. C_AA, in mg mL^–1), using an AA solution of 3 × 10^–2 mg mL^–1, obtained from the absorption spectra (Figure 3A), is described by the linear equation: A563 = 0.932 − 152 × C_AA (n = 6; r = 0.992, p < .05) for the linear range of (1.06 – 5.28) × 10^–3 mg mL^–1 of AA (Figure 3B). The calculated limits of detection (LOD = 3 × σa/b) and quantification (LOQ = 10 × σa/b) were 2.5 × 10^–4 mg mL^–1 and 8.2 × 10^–4 mg mL^–1, respectively.³⁰ The relative standard deviations (RSD) obtained from nine measurements of C_AA with solutions of 1.06 × 10^–3 mg mL^–1 and 2.11 × 10^–3 mg mL^–1 were 3.6% and 4.3%, respectively.

Figure 3.

(A) Successive Absorption Spectra of Solutions Containing: Fe(III) 1.0 × 10^–4 M + TMPD 2.0 × 10^–4 M + Acetate Buffer (pH 5.0); AA × 10^–3 (mg mL^–¹): a = 0; b = 1.06; c = 2.11; d = 3.17; e = 4.23; f = 5.28. Water is a Reference Solution. (B) Analytical Curve Obtained from the A_563nm Values in (A), Represented by the Equation y = 0.932–151.9 × C_AA (n = 6; r = 0.992, p < .05).

Intermediate precision was evaluated by constructing six independent calibration curves on two separate days under identical experimental conditions. On Day 1, the mean slope (b) was −156 ± 6 (n = 6; RSD = 4.5%), whereas on Day 2, six additional calibration curves yielded a mean slope of −158 ± 8 (n = 6; RSD = 4.8%). Statistical comparison of the slopes using a two-tailed Student’s t-test for independent samples indicated no significant difference between the 2 days (t = 0.49, df = 10, p > .05). These findings demonstrate adequate intermediate precision and confirm the robustness of the method under reproducibility conditions.

These results confirm that AA can be used as a standard compound (Equations 9–11)^14,37 to express the RC of other AO compounds and the TAC of plant-based samples.

T M P D^{● +}_{(b l u e)} + A A ⇌ A A^{● +} + T M P D_{(c o l o r l e s s)}

(9)

A A^{+ *} + A A^{+ *} \to A A + p r o d u c t s

(10)

A A^{+ *} + A A^{+ *} \to A A + A A 2 +

(11)

From the linear equations generated by the analytical curves with the antioxidants (A563 = a + b × [AO]), the slope values (b) were obtained, which correspond to the apparent molar absorptivity (e) of each standard antioxidant investigated.

The calculation of the b value was performed exclusively using analytical curves that exhibited good linearity (r ≥ 0.99).

Figure 4 illustrates the phenol structure as a basis for interpreting the influence of hydroxyl substitution patterns on the reducing activity of polyphenols.

Figure 4.

Basic Structure of Phenol: R₁-R₅.

The average b value obtained from triplicate analytical curves for each AO (A563 = a + b × [AO]) was divided by the average b value (–267) obtained from the analytical curve using the AA solution (A563 = a + b × [AA]), resulting in the individual RC values for each AO (Table 1). The reducing activity is defined as the concentration of AA (in mM) required to exhibit the same RC as a 1.0 mM solution of the analyzed compound and is expressed as RC in Ascorbic Acid Equivalent (RC_AAE).^38,39 For this reason, the study was conducted using concentrations expressed in molarity (M).

Potential interfering compounds commonly found in plant matrices were considered, as summarized in Table 1. The evaluated substances represent typical phenolic and redox-active constituents of herbal samples. Strong reducing agents such as sulfite are unlikely to be naturally present in plant infusions and were therefore not considered relevant interferents in this study.

Tannic acid exhibits the highest RC_AAE value, which can be directly associated with the large number of free hydroxyl phenolic groups (HPGs) distributed across its benzene rings.^39,40 In fact, this compound presented an RC_AAE approximately 17 times higher than that of ascorbic acid. This outstanding response is mainly related to its polymeric structure, which contains a very high density of HPGs (25 groups), enabling multiple and simultaneous electron-transfer events and, consequently, cumulative consumption of the TMPD^●+ radical.

For trihydroxybenzenes, the RC_AAE values follow the order 1,2,4-benzenetriol > pyrogallol > gallic acid. This trend indicates that a higher number of HPGs favors TMPD^●+ consumption and that the meta arrangement of hydroxyl groups, as observed in 1,2,4-benzenetriol, significantly enhances the RC, resulting in an RC_AAE almost 50 times higher than that of pyrogallol, which exhibits an ortho arrangement. In the case of gallic acid, the presence of a carboxyl group, which is partially deprotonated in acidic medium (pKa₁ = 4.7),⁴¹ likely reduces the electron-donating efficiency of the molecule, leading to an RC_AAE value up to 150 times lower than that observed for 1,2,4-benzenetriol.

Although epigallocatechin gallate (EGCG) is a highly hydroxylated polyphenol, its RC determined by the TMPD^●+ consumption assay was notably lower than that of ascorbic acid (RC_AAE = 0.299 vs. 1.000, respectively), corresponding to a reduction of approximately 70% relative to the ascorbic acid standard. This finding contrasts with the common expectation that polyphenols containing multiple HPGs necessarily exhibit superior reducing properties and underscores the strong dependence of antioxidant ranking on the analytical methodology employed.^39,42

Additionally, EGCG is relatively unstable in aqueous solution and may undergo degradation during the analysis, reducing the effective concentration available to react with the radical.⁴³ In contrast, ascorbic acid tends to remain stable and reacts rapidly at the beginning of the measurement, contributing to a higher analytical signal in reduction-based assays. Such instability of polyphenols under certain experimental conditions can significantly lower the measured response in methods that rely on fast and complete redox reactions.⁴⁴

Catechol, in turn, contains only two HPGs located at the R₁ positions, which further reinforces the importance of the number of HPGs in TMPD^●+ consumption and supports observations previously reported in the literature.³⁹

Concerning the data in Table 1, catechol showed a higher RC than gallic acid (RC_AAE = 0.0161 vs. 0.0057), corresponding to an increase of approximately 2.8 times. This difference can be explained by structural and electronic factors, and not only by the number of phenolic hydroxyl groups.³⁹ Although gallic acid contains three hydroxyl groups, the presence of a carboxyl group, which exerts an electron-withdrawing effect, decreases the electron density of the aromatic ring and reduces its efficiency in direct electron-transfer reactions.⁴⁵ Catechol, benefits from intramolecular hydrogen bonds and resonance stabilization of the resulting phenoxyl radical, which facilitates electron donation to the TMPD^●+. Similar behavior has been reported in comparative studies of antioxidants, where phenolic acids with electronegative substituents exhibit lower RC than simpler dihydroxybenzenes in assays governed by fast electron-transfer kinetics.⁴⁶ These results highlight that, in the TMPD^●+ assay, substituent effects and reaction kinetics are more decisive than the total number of hydroxyl groups.

According to Table 1, Trolox^→ exhibited a low RC in the TMPD^●+ consumption assay (RC_AAE = 0.076), corresponding to only 7.6% of the RC of ascorbic acid (RC_AAE = 1.000), indicating markedly lower effectiveness under the experimental conditions. In contrast, ascorbic acid acted as a rapid and efficient electron donor in the TMPD^●+ system, which can be attributed to its small molecular size and high reactivity, allowing fast and quantitative oxidation through a two-electron-transfer mechanism and resulting in immediate radical consumption.^14,37 These findings highlight the strong method dependence of antioxidant ranking and demonstrate that, despite its widespread use in antioxidant assays,²³ Trolox is not the most appropriate reference compound for the proposed TMPD^●+ method. Previous studies have also reported that Trolox often exhibits slower reaction kinetics compared to polyphenolic compounds, reinforcing the suitability of ascorbic acid as a more chemically consistent reference standard for this assay.⁴⁷

It should be emphasized that the observed ranking reflects the relative reducing behavior of the tested compounds within the TMPD^●+ redox system under the optimized experimental conditions. The antioxidant ranking found was: tannic acid > ascorbic acid > 1,2,4-benzenetriol > epigallocatechin gallate > Trolox^→ > pyrogallic acid > pyrocatechol > gallic. Therefore, it is influenced by both kinetic factors and redox potential differences and should not be interpreted as a universal antioxidant hierarchy.

Evaluation of TAC Herbal Teas Using TMPD^•+

The samples analyzed correspond to distinct commercial brands. The addition of sample aliquots promoted the consumption of the TMPD^●+, with A563 nm values showing linearity with respect to the concentration of the sample solution (mg mL^–1). All analyses were performed in triplicate.

Figure 5 presents a typical analytical curve (A563 vs. C_Sample), represented by the equation A563nm = a–b × C_sample. Using the equation obtained from the ascorbic acid (AA) standard solution (A563 = a–b × C_AA), along with the corresponding analytical curves for the samples, the concentrations of each sample equivalent to the antioxidant activity of a 1.0 mg mL^–1 AA solution were calculated. These values were expressed as grams of AA per gram of sample (g AA/g sample), as presented in Table 2.

Figure 5.

Triplicate of the Sample (Chamomile): TMPD 2.0 × 10^–4 M + Fe(III) 1.0 × 10^–4 M + Acetate Buffer (pH 5.0). λ = 563 nm. Sample Concentration (0.18–0.90) mg mL^–1: ▪ Y = 0.5092–0.0011 × X (r = –0.997); ● Y = 0.496–0.0011 × X (r = –0.995) e Y = 0.5005–0.0011 × X (r = –0.996).

Statistical Correlation Among TAC and TPC Values

To evaluate the reliability and agreement between methods, normal distribution and correlation analyses were performed using the Shapiro–Wilk and Spearman tests,^48,49 respectively. The Shapiro–Wilk test was applied to all variables in Table 2 (TPC, TAC_ABTS^●+ and TAC_TMPD^●+). The results indicated that only TPC and TAC_ABTS^●+ exhibited a normal distribution (p > .05). Therefore, the Spearman correlation test was chosen to evaluate the relationships between these variables. Figure 6 illustrates the main observed correlations.

Table 2.

Total Antioxidant Capacity Values Obtained with Methods Based on the Consumption of TMPD^•⁺ and ABTS^•⁺ Radical Cations and Total Polyphenol Content Obtained with the Folin–Denis Reagent of Tea Samples.

Scientific Name	Popular Name		TAC		TPC
Scientific Name	Popular Name	Brand	ABTS^•+a	TMPD^•+ × 10^–4b	FDR^c
Camellia sinensis L.	White tea	Real	909 ± 8	32.4 ± 6.0	128 ± 6
Camellia sinensis L.	Green tea	Matchá	1,576 ± 43	22.3 ± 2.5	139 ± 1
Camellia sinensis L.	Black tea	Dr. Oetker	594 ± 218	11.1 ± 0.2	69 ± 1
Mentha piperita L.	Mint tea	LinTea	442 ± 28	7.8 ± 0.3	74 ± 1
Mentha piperita L.	Mint tea	Dia	219 ± 11	10.5 ± 0.3	64 ± 2
Mentha piperita L.	Mint tea	Dr. Oetker	138 ± 7	8.7 ± 0.5	57 ± 2
Ilex paraguariensis, St. Hil	Herb tea	Ekonômico	436 ± 18	9.4 ± 0.7	73 ± 1
Ilex paraguariensis, St. Hil	Herb tea	Leão	798 ± 16	9.6 ± 0.4	116 ± 1
Matricaria recutita	Chamomile tea	Dr. Oetker	39 ± 1	6.9 ± 0.4	14 ± 1

Notes: (a) TAC analyzed by the ABTS^•+ radical cation consumption method (µM trolox/g sample); (b) TAC analyzed by the TMPD^•+ radical cation consumption method (g AA/g sample); (c) TPC analyzed with Folin–Denis reagent (g AG/100 g sample).

In Figure 6A, a significant positive correlation (r = 0.817, p < .01) was observed between polyphenol concentration and total antioxidant capacity (ABTS^●+), suggesting that polyphenols are the main contributors to the antioxidant capacity of the analyzed samples. In Figure 6B, the TAC (ABTS^●+ and TMPD^●+) values also showed a strong positive correlation with high statistical significance (r = 0.900, p < .01), suggesting that the same compounds quantified by the standard method (ABTS^●+) are also detected and quantified by the proposed method. These results indicate that the reaction proposed here can be used to quantify the TAC of tea samples. These correlations underscore the reliability of the proposed method for quantifying TAC in plant-based samples beyond herbal teas.

Figure 6.

(A) Spearman Correlation of the TAC Values Obtained with ABTS∙+ (µM of trolox/g of Sample) × 10^–4 and the TPC Obtained with FDR (g of GA/100 g Sample) (r = 0.817; p < .01); (B) Spearman Correlation of the TAC Values Obtained with TMPD∙++ (g of AA/g of Sample) × 10^–4 and ABTS∙++ (µM of trolox/g of Sample) (r = 0.900; p < .01).

Conclusion

The results obtained demonstrate that it is possible to form and stabilize the purple solution of the TMPD^●+ radical cation, derived from TMPD in an aqueous medium. This represents an environmental advantage compared to methods that use the DPPH^●+ and ABTS^●+ radical cations, which require organic solvents.

The best experimental condition was achieved in a buffered aqueous solution (pH 5.0; acetate) with TMPD at 2.0 × 10^–4 M and Fe(III) at 1.0 × 10^–4 M. Under these conditions, absorbance measurements at 563 nm were linear with respect to the concentration of AOs and tea samples. This enabled the determination of the RC of AOs and the TAC of the analyzed samples.

The suggested methodology is easy to perform, and the standard AO (ascorbic acid) used to calculate and express the TAC of the samples is readily available and biologically active. Furthermore, the proposed method uses reagents and equipment that are affordable and often already available in chemistry laboratories, making it especially attractive from a socioeconomic perspective.

The findings of this study indicate the potential applicability of the proposed method to other plant-derived samples, including medicinal herb extracts, wines, fruits, and juices.

Footnotes

Acknowledgements

This work was supported by FAPESP (São Paulo Research Foundation).

Authors’ Contribution

H.D.M. conceived and supervised the study. C.S., L.R.M.D., W.E.L.S., L.B.O., and F.F.D.N. performed the experiments and data analysis. All authors contributed to the writing, revision, and approval of the final manuscript.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Not applicable.

Funding

Not applicable.

Informed Consent

Not applicable.

Use of Artificial Intelligence-assisted Tools:

The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.

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Development and Validation of a Stabilized TMPD•+ Spectrophotometric Assay for Total Antioxidant Capacity in Herbal Teas

Abstract

Keywords

Introduction

Materials and Methods

Equipment

Reagents and Materials

Methods

Optimization of the TMPD•+ Generation Conditions

Selection of the Oxidizing Agent

Evaluation of the TMPD/Oxidant Molar Ratio

Effect of pH on TMPD•+

Effect of Solvent Composition

Determination of the RC of Some AOs

Assessment of TAC in Herbal Tea Samples Via the TMPD•+ Assay

Determination of TAC Based on the Consumption of TMPD•+

Analytical Curve Using Ascorbic Acid Standard

Sample Preparation

Analytical Curves Using Herbal Tea Samples

Preparation of ABTS•+ and Folin–Denis Reagents

Determination of TAC Using the ABTS•+ Method

Determination of TPC Using FDR

Results and Discussion

Optimization of the TMPD•+ Generation Conditions

Evaluation of Some Oxidizing Agents for the Generation of TMPD•+

Effect of the Molar Ratio Between TMPD and Fe(III) on the Formation of TMPD•+

pH and Buffer Effects on TMPD•+ Formation and Stabilization

Influence of Selected Solvents on the Stability of the TMPD•+ Solution

Evaluation of RC in AOs

Basic Structure of Phenol: R1-R5.

Evaluation of TAC Herbal Teas Using TMPD•+

Statistical Correlation Among TAC and TPC Values

Conclusion

Footnotes

Acknowledgements

Authors’ Contribution

Consent to Participate

Consent for Publication

Declaration of Conflicting Interests

Ethical Approval

Funding

Informed Consent

Use of Artificial Intelligence-assisted Tools:

References

Optimization of the TMPD^•+ Generation Conditions

Effect of pH on TMPD^•+

Assessment of TAC in Herbal Tea Samples Via the TMPD^•+ Assay

Determination of TAC Based on the Consumption of TMPD^•+

Preparation of ABTS^•+ and Folin–Denis Reagents

Determination of TAC Using the ABTS^•+ Method

Optimization of the TMPD^•+ Generation Conditions

Evaluation of Some Oxidizing Agents for the Generation of TMPD^•+

Effect of the Molar Ratio Between TMPD and Fe(III) on the Formation of TMPD^•+

pH and Buffer Effects on TMPD^•+ Formation and Stabilization

Influence of Selected Solvents on the Stability of the TMPD^•+ Solution

Basic Structure of Phenol: R₁-R₅.

Evaluation of TAC Herbal Teas Using TMPD^•+