Electrochemical Determination of the Antioxidant Capacity, Total Phenolics, and Ascorbic Acid in Fruit and Vegetables by Differential Pulse Voltammetry (DPV) with a p-Toluene Sulfonic Acid Modified Glassy Carbon Electrode (TSA/GCE)

Abstract The determination of antioxidant capacity, total phenolics, and ascorbic acid using accurate, efficient, low cost, and rapid methods has great analytical importance. The antioxidant capacity, total phenolics, and ascorbic acid content of apricots, arugula, banana, cranberries, spinach, and strawberries were investigated with an electrochemical approach and examined for compatibility with conventional methods. The antioxidant activity was determined by ABTS and DPPH assays. The total phenolic content was determined by the Folin Ciocalteu method and ascorbic acid was quantified by high-performance liquid chromatography (HPLC). In order to perform sensitive and simultaneous electrochemical measurements, the surface of a glassy carbon electrode (GCE) was modified by electropolymerization with p-toluene sulfonic acid (TSA). The TSA/GCE modified sensor was used for the first time for the determination of antioxidant capacity and total phenolic content. The surface of the modified sensor was characterized by cyclic voltammetry and scanning electron microscopy. The TSA/GCE was linearly correlated with the differential pulse voltammetry (DPV) for Trolox and gallic acid in 0.1 M NaNO3 and ascorbic acid in pH 7.4 phosphate buffer. Electrochemical methods offer a promising alternative for the determination of antioxidant capacity and total phenolic content due to their simplicity, rapid response, low cost, sensitivity, and reproducibility.


Introduction
Antioxidants represent a wide class of chemical compounds that reduce oxidative processes, prevent possible damage caused by reactive oxygen species, ensure detoxification, are included in the body's defense system, and are present in biological environments and foods (Ames 1983;Diaz et al. 1997;Kaur and Kapoor 2001;G€ uleşci and Ayg€ ul 2016). The human body is equipped with an effective defense system including high and low molecular weight antioxidants and various enzymes. Antioxidants et al. 2001;Dilgin and Nişli 2006;Cui et al. 2009;Mazloum-Ardakani et al. 2010;Fritea et al. 2015;He et al. 2015;Krzyczmonik, Socha, and Andrijewski 2017).
The goal of this study was to determine the antioxidant capacity, total phenolics, and ascorbic acid content of apricots, arugula, bananas, cranberries, spinach, and strawberries cultivated in different seasons using traditional methods and a novel electrochemical approach. No previous work electrochemically determined these parameters in these fruits and vegetables. The electrochemical detection was compared with conventional techniques and the results were shown to agree. A rapid, reliable, and economical approach was developed for the determination of the antioxidant properties of fruits and vegetables.

Samples and chemicals
Apricot (Prunus armeniaca L.), arugula (Eruca sativa L.), bananas (Musa acuminata), cranberries (Cornus mas), spinach (Spinacia oleracea L.), and strawberries (Fragaria x ananassa Duch) were purchased from a local grocer. All samples were obtained 3 times (at least 2 kg) at one-week intervals in 2020. According to the growing season, the sampling times were apricots in July, arugula in October, bananas in August, cranberries in September, spinach in October, and strawberries in April. Unedible parts were discarded. The samples were washed with tap water and homogenized using a T18 Ultra Turrax (Ika, Staufen, Germany). The samples were freeze-dried and stored in polyethylene bags at À18 C until extractions were carried out.

Extraction, antioxidant capacity assays, and total phenolic content
The freeze-dried samples (2 g) were extracted with 50 mL of 80:19:1 methanol/water/ hydrochloric acid in an ultrasonic bath at 20 C for 30 min, centrifuged at 5580 g at 4 C for 6 min, and the supernatant was collected. The pellet was extracted twice with the solvent. Supernatants were combined, passed through a 0.45 mm syringe filter, and used for antioxidant capacity, total phenolic content assays, and electrochemical measurements.
The free radical scavenging capacity of the extracts was determined by DPPH and ABTS assays. ABTS þ cationic radical was produced by a reaction between 7 mM ABTS and 2.45 mM ammonium persulfate in the dark for 12 to 16 h, diluted with ethanol to an absorbance of 0.700 ± 0.020 at 734 nm, and used as the stock solution for the ABTS assay (Re et al. 1999). The ABTS stock (3.8 mL) was added to the extract (200 mL) and incubated for 60 min in the dark, and absorbance was measured at 734 nm using a UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan).
The DPPH radical scavenging assay was performed according to Brand-William, Cuvelier, and Berset (1995). A methanol solution of DPPH (1 g/mL) was mixed with the extract, ncubated for 60 min in the dark, and the absorbance was measured at 520 nm. Calibration graphs were constructed by plotting absorbance versus known concentrations of Trolox for both assays. The results are expressed as mmol Trolox equivalent antioxidant capacity (TEAC)/100 g dry weight (DW).
The total phenolic content (TPC) was determined by the Folin-Ciocalteu method as described in the literature (Kraujalyt_ e et al. 2013). In brief, extract (100 mL), 1 mL of 0.2 N Folin-Ciocalteu reagent, 400 mL of deionized water, and 1 mL of 7% sodium carbonate were sequentially added. The mixture was placed in the dark for 90 min at room temperature and the absorbance was measured at 725 nm. The results are expressed as a gallic acid equivalent (GAE) and mg/100 g of the dry weight.

Extraction and HPLC analysis
Ascorbic acid extraction was performed according to the procedure applied by Fecka et al. (2021). Prior to the homogenization of the freeze-dried sample (10 g), 10 mL of 1% methanolic BHT solution were added and homogenized with a blender. Approximately 5 g of homogenized sample (1 g of sample was used for electrochemical measurement) were extracted with 30 mL of aqueous metaphosphoric acid (2%, m/v) at room temperature in the dark. The obtained solution was centrifuged (5580 g) for 10 min at 4 C, passed through a membrane filter (0.45 mm), and stored at 4 C before HPLC analysis.
The determination of ascorbic acid was performed in triplicate on a Shimadzu HPLC equipped with an autosampler (SIL-20A HT), a column oven (CTO-10AS VP), a degasser (DGU2A 5 R), a gradient pump (LC-20AR), a diode array detector (DAD, SPDM20A), and software for system control and data acquisition (LC Solution). The extract (20 mL) was injected into a Rezex ROA column (300 Â 37.8 mm; Phenomenex, Torrance, CA), which was maintained at 20 C. The flow rate of the mobile phase was 1 mL/min. The mobile phase consisted of aqueous 0.1% (v/v) metaphosphoric acid. The detection was carried out at 245 nm.

Voltammetric procedures
All voltammetric measurements were carried out using a Gamry Interface 1010B potentiostat (Gamry, USA). Voltammetric analyses were performed with a three-electrode system (BASi C3 Cell Stand). The system consisted of a platinum wire auxiliary electrode, a silver-silver chloride reference electrode, and a poly (p-TSA) modified glassy carbon electrode (GCE). Nitrogen was bubbled through each solution for 2 minutes before the voltammetric measurements were carried out.
Cyclic voltammetry (CV) was used to investigate the electrochemical properties of Trolox, gallic acid, and ascorbic acid. Differential pulse voltammetry (DPV) was employed for the determination of Trolox, gallic acid, and ascorbic acid. The Trolox standard solution was prepared in 80% methanol/20% water. Gallic acid and ascorbic acid standards were prepared in aqueous 0.1 M NaNO 3 . The extracts used for spectroscopic and chromatographic measurements were also used for voltammetric measurements. They were diluted ten-fold with supporting electrolyte to perform voltammetric analyses of fruit and vegetable extracts. Voltammetric current responses and calibration graphs were drawn for Trolox, gallic acid, and ascorbic acid and used for the quantitative analysis.
The limits of detection (LOD) and the limits of quantitation of Trolox, gallic acid, and ascorbic acid were determined by DPV using the TSA/GCE modified sensor. The oxidative peak currents corresponding to the concentrations were plotted. The limits of detection (3 x SD/m) and quantification (10 x SD/m) values were determined by dividing the standard deviation (SD) of ten measurements for the lowest signal concentration by the slope of the curve (m) with the appropriate coefficient.

Preparation of the TSA/GCE modified sensor
The GCE was mechanically cleaned with Emery paper with 0.3 and 0.05 mm Al 2 O 3 slurries and cleaned by cyclic voltammetry from À0.7 to þ1.7 V at 0.1 V s À1 in 0.5 M H 2 SO 4 for 10 cycles before each modification, rinsed with ultrapure water, and immersed in 0.1 M NaCl containing 1.0 mM p-toluene sulfonic acid. The electrode was modified by electropolymerization across a potential range of À0.2 to þ2.5 V, scanning speed of 0.05 V s À1 , and applying 5 cyclic voltammetric cycles ( Figure S1). The prepared TSA/GCE was conditioned by differential pulse voltammetry in 0.1 M NaNO 3 before measurements were performed.
The surface properties and morphologies of the TSA/GCE modified sensor were characterized by scanning electron microscopy (SEM, LEO EVO-40xVP).

Statistical analysis
The results were statistically analyzed with one-way analysis of variance (ANOVA) and Tukey multiple range test using the SPSS Statistics 22. Statistical differences with P-values under 0.05 were considered to be significant.

Spectrophotometric and HPLC measurements
The antioxidant capacities, total phenolics, and ascorbic acid contents of the samples evaluated by spectrophotometry and HPLC are shown in Table 1. The results show good agreement between total phenolic and ascorbic acid contents and antioxidant capacity, except for the bananas. While there are significant differences between the ascorbic acid contents of the samples, the ascorbic acid contents of strawberries and arugula were considerably higher than the others (P < 0.05). The total phenolic contents of arugula and spinach are higher than the others (P < 0.05). Despite the lowest total phenolic and ascorbic acid contents, the bananas had the highest antioxidant activity values.
While both total polyphenols and vitamin C are considered the main compounds responsible for the total antioxidant capacity of fruits and vegetables, flavonoids, tocopherols, carotenoids, and even fiber contribute to the scavenging of DPPH and ABTS radicals. (Tiveron et al. 2012;Liu et al. 2014). The antioxidant capacities of all phenolic substances are not the same and the type of phenolic substance is important (Du et al. 2009). There are flavonoids in the composition of bananas, which significantly increase the antioxidant capacity compared to the other phenolics (Bashmil et al. 2021). This explains the contradiction in the banana measurements. The results are also in accordance with the literature carried out with fruits and vegetables (Velioglu et al. 1998;Liu et al. 2014;Maduwanthi and Marapan 2021).

Voltammetric measurements
In order to obtain the highest analytical response in voltammetric studies, optimization of parameters such as electrode modification, film thickness modification, and the supporting electrolyte solution were investigated. First, thin films of the electropolymerized TSA/GCE modified sensor were prepared using cyclic voltammetry from one to ten cycles, and the differential pulse voltammetric responses were investigated for 100 ppm Trolox ( Figure S2). The highest peak current was obtained at the TSA/GCE modified sensor electropolymerized with 5 cycles. Thus, sensors with this film thickness were used in subsequent studies.
Next, the supporting electrolyte was investigated in order to optimize the voltammetric response at the TSA/GCE modified sensor in 0.1 M KCl, LiClO 4 , NaClO 4 , NaCl, NaNO 3 , Na 2 SO 4 , and phosphate buffer saline (PBS) (pH 7.4) with 100 ppm Trolox ( Figure S3). The highest response was obtained in 0.1 M NaNO 3 that was used in subsequent studies.
Scanning electron microscopy (SEM) provides high-resolution morphological details of materials modified on the electrode surface. It is possible to determine whether a material is coated on the electrode surface, as well as the thickness of the coated film. Surface topography of the electropolymerized TSA/GCE modified sensor was examined to observe the structure by SEM (Figure 1).
These images show the polymer structures formed on the surface and that the surface has a hollow and fringed structure. The surface was highly porous, and polymer bundles and catalyst residues are present. The porous and rough structure increased the sensitivity of the modified sensor. In addition, the thickness of the thin film structure on the electrode surface was estimated to be 20.31 mm. Differential pulse voltammetric analyses were performed in 0.1 M NaNO 3 using the TSA/GCE to determine the voltammetric oxidation properties and detection limits for Trolox, gallic acid, and ascorbic acid. The concentrations of Trolox were 0. 083, 0.48, 2.05, 2.60, 3.15, 4.08, 4.41, 5.68, 6.82, 7.38, 8.68, 10.29, 10.94, and 12.00 ppm. A linear curve was obtained with the equation Ip (lA) ¼ 0.8464 C (ppm) þ 1.0954 across this concentration range (Figure 2).
Simultaneous determination of Trolox, gallic acid, and ascorbic acid was performed to characterize the selectivity ( Figure S4). Figure 5 shows the differential pulse     voltammetric responses at the TSA/GCE modified sensor as a function of Trolox concentration in the presence of constant concentrations of gallic acid and ascorbic acid. The anodic peak potentials for ascorbic acid, Trolox, and gallic acid at the modified sensor were at þ0.14, þ0.25, and þ0.41 V, respectively.
The results of simultaneous voltammetric analysis for solutions with increased gallic acid and ascorbic acid concentrations and constant concentrations of other analytes are shown in Figures S5 and S6. These results show that there was no interaction for the electrochemical determination of Trolox, gallic acid, and ascorbic acid. Three analytes were detected sensitively, stably, and with good anodic peak resolution at the TSA/GCE. Hence, the simultaneous determination of these analytes in real samples was performed using the TSA/GCE.
The antioxidant capacity, total phenolics, and ascorbic acid contents of apricot, arugula, banana, cranberry, spinach, and strawberry extracts used for spectrophotometric and chromatographic methods were determined electrochemically by DPV using the TSA/GCE. Voltammetric calibration curves were generated for Trolox, gallic acid, and ascorbic acid. The antioxidant capacity, total phenolic, and ascorbic acid contents were determined by using the calibration relationships. The average values as mmol TE/100 g DW, mg GAE/100 g DW, and mg/g DW based upon three voltammetric measurements are shown in Table 2. Low concentrations of Trolox, gallic acid, and ascorbic acid were determined by the electrochemical protocol.
Regression analysis was performed to correlate the results obtained by conventional methods with electrochemical measurements. Pearson correlation coefficients between conventional techniques (spectrophotometric and HPLC) and voltammetric methods are shown in Table 3. High correlation was observed between voltammetric TEAC value and spectrophotometric DPPH (R 2 ¼ 0.985, p < 0.01) and ABTS (R 2 ¼ 0.983, p < 0.01) assays. The voltammetric total phenolic and ascorbic acid contents also had strong correlations with the spectrophotometric (R 2 ¼ 0.992, p < 0.01) and HPLC (R 2 ¼ 0.995, p < 0.01) results. With good compatibility with standard techniques, the voltammetric approach is a promising alternative to determine the antioxidant capacity, total phenolics, and ascorbic acid content.
Lastly, the developed TSA/GCE was successfully employed for the analysis of fruit and vegetable extracts with high accuracy to demonstrate its applicability and accuracy. The calibration relationships demonstrated high sensitivity of the sensor with low limits of detection and quantification. The results obtained using the TSA/GCE were satisfactory in terms of precision and accuracy.

ÃÃ
Correlation is significant at the 0.01 level.

Ã
Correlation is significant at the 0.05 level.

Conclusion
Electrochemical methods have been used to determine the antioxidant capacity and total phenolic content of food and beverages as an alternative approach by a fast, easy, and economical protocol. Additionally, this approach is attractive because of the high sensitivity. The measurements are based upon applying a potential and measuring the current in an electrochemical cell due to oxidation/reduction on the electrode surface. The surface of a glassy carbon electrodes was modified by electropolymerization with p-TSA to increase the sensitivity. The conditions for the determination of the antioxidant compounds were optimized. Electrochemical analyses were performed on fruit and vegetable extracts of apricots, arugula, bananas, cranberries, spinach, and strawberries with the TSA/GCE. The antioxidant capacity and total phenolic content were calculated from calibration curves for Trolox and gallic acid generated by differential pulse voltammetry. The simultaneous determination of Trolox, gallic acid, and ascorbic acid were performed with the TSA/GCE by DPV. The voltammetric results were demonstrated to be comparable with those obtained using standard procedures.