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Article

Low-Cost Electrochemical Determination of L-Ascorbic Acid Using Screen-Printed Electrodes and Development of an Electronic Tongue for Juice Analysis

by
Laila El Anzi
1,
María Soledad García
1,*,
Eduardo Laborda
2,
Alberto Ruiz
3 and
Joaquín Ángel Ortuño
1,*
1
Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30100 Murcia, Spain
2
Department of Physical Chemistry, Faculty of Chemistry, University of Murcia, 30100 Murcia, Spain
3
Department of Informatics and Systems, University of Murcia, 30100 Murcia, Spain
*
Authors to whom correspondence should be addressed.
Chemosensors 2024, 12(11), 237; https://doi.org/10.3390/chemosensors12110237
Submission received: 30 September 2024 / Revised: 8 November 2024 / Accepted: 14 November 2024 / Published: 16 November 2024
(This article belongs to the Special Issue Low-Cost Chemosenors for Applications in Environment, Health, Food, and Industry Process Control)
Browse Figures
Versions Notes

Abstract

:
Low-cost electrochemical methodologies for the determination of L-ascorbic acid (vitamin C) and the analysis of juices are developed based on its electro-oxidation on carbon screen-printed electrodes. A novel chronoamperometric methodology is developed for the quantification of L-ascorbic acid in fruit juices. The proposed method stands out for its simplicity and rapidity, demonstrating its efficacy in determining L-ascorbic acid content in various fruit juices. Notably, the results obtained with this chronoamperometric approach are compared with those yielded by chromatography, with no significant differences between the two methods being found. Additionally, an electronic tongue is developed for the differentiation of juices based on the square wave voltammetric signals.

1. Introduction

Screen-printed electrodes (SPEs) are devices created by printing various inks onto substrates such as plastic, ceramic, or paper [1,2,3,4,5,6,7]. SPEs have become essential for advancing and applying electrochemical analytical methods, as well as in developing electrochemical sensors and biosensors. Their low cost and portability provide significant advantages over other electrode types, enabling point-of-use applications as an alternative to sending samples to centralized analytical laboratories [3]. SPEs are commercially available in various working electrode compositions and can also be easily produced in-house with ink printers, allowing for customizable electrode compositions [7].
The development of disposable SPEs for electrochemical applications has been reviewed based on the types of materials used for the working electrode, whether unmodified or modified [2,3,4,5,6], and across various application fields [2,3,4], such as food analysis [1,3,4], biomedical and clinical analysis [3,4], and environmental analysis [3,4,5,6]. Only a limited number of studies have focused on using unmodified SPEs for the determination of analytes of interest [4,6,8]. Carbon materials are preferred due to their simple processing and low cost [4,8].
Vitamin C or L-ascorbic acid (L-AA) is a water-soluble vitamin, well-known as an antioxidant. However, its physiological role is much broader, encompassing a wide range of processes, from facilitating iron absorption and participating in hormone and carnitine synthesis to playing essential roles in epigenetic processes [9]. Humans cannot synthesize L-ascorbic acid endogenously, so it must be obtained through the diet. Numerous foods, primarily fruits and vegetables, are rich in L-AA, with most people deriving a substantial portion of their daily vitamin C intake from fruits and fruit juices [9]. L-AA is degraded by the action of oxygen, with the reaction being enhanced by heat, light, and heavy metal ions. For this reason, it is necessary to control the concentration of L-AA in packaged commercial juices [10].
Several methods for the determination of L-AA using different techniques have been reported. Determination via ultraviolet–visible spectroscopy is possible based on the absorbance in the ultraviolet (UV) region [11]. However, direct spectrophotometric determination of L-AA in the UV region is susceptible to the matrix effect, as numerous organic compounds in complex samples can cause interference. Other types of spectrophotometric methods for the determination of L-AA use a previous chemical reaction. One such method is the spectrophotometric technique based on the oxidation of L-AA, using the Cu(II)–neocuproin complex, which is reduced to Cu(I)–bis(neocuproin) and its absorbance is measured at 450 nm. This technique was applied to a commercial fruit juice, a pharmaceutical preparation, and a red wine [12].
Liquid chromatography is a widely used method for the determination of vitamin C because of its selectivity. High-performance liquid chromatography (HPLC) combined with ultraviolet detection enables the quantification of total L-AA and isoascorbic acid (isoL-AA) in foods [13]. The method is based on the acid extraction of L-AA in the presence of the reducing agent tris [2-carboxyethyl] phosphine (TCEP), which maintains L-AA in its reduced form. The results were validated with a potentiometric titration method using 2,6-dichlorophenol-indophenol as an oxidizing agent. HPLC with electrochemical detection has proved to be a selective and sensitive technique for the evaluation of L-AA in foods and biological fluids [14]. Nevertheless, this methodology demands intricate and frequently costly instrumentation and well-qualified personnel and typically entails time-consuming procedures.
An increasing body of research is devoted to exploring electrochemical methodologies for determining L-AA without the need for preceding separation. This trend is driven by the cost-effectiveness of the required equipment, the simplicity of the procedure, and the ability to avoid sample pretreatment.
Potentiometry using an ion-selective electrode based on a polymeric membrane has been used for the determination of L-AA content in orange juices and pharmaceutical preparations. The sensor was fabricated by modifying a glassy carbon electrode with polypyrrole (PPy), electropolymerized from its monomer in the presence of ascorbate [15].
O’Connell et al. [16] developed an electropolymerized aniline-based L-AA amperometric sensor for pharmaceutical and food analysis. Kumar et al. [17] prepared an amperometric sensor for the determination of L-AA based on a cobalt-hexacyanoferrate-modified electrode which was applied to the determination of L-AA in pharmaceutical preparations. Emran et al. [18] used microporous sulfur-doped carbon spheres as electrodes for the detection of ascorbic acid in food and pharmaceutical products.
Among the reported voltammetric methods for the determination of L-AA, many make use of cyclic voltammetry (CV). Thus, Okiei et al. [19] determined L-AA in tropical fruits via CV using a glassy carbon electrode as the working electrode. The results were compared with those obtained by titration with N-bromosuccinimide. Although the results were generally similar, large differences were obtained in some fruits. CV with a platinum electrode was also assessed [20]. The results of the L-AA determination obtained were compared with those determined by the volumetric method with 2,6-dichloroindophenol. The authors concluded that the voltammetric method can be effectively used as part of quality management within the food industry to evaluate the vitamin C content in natural fruit juices and soft drinks [20]. The use of a pencil lead as a working electrode for the determination of L-AA in commercial orange juices by CV has been proposed. The authors found the results to be comparable with those obtained using a glassy carbon electrode [21]. Square wave voltammetry has also been considered for the determination of L-AA in citrus, based on its electrocatalytic oxidation at zeolite-modified carbon paste electrodes [22].
Several articles highlight the use of screen-printed electrodes for determining L-AA through electrochemical techniques, with the following being particularly noteworthy. With screen-printed electrodes containing a carbon working electrode, Xiang et al. [23] determined L-AA in fresh vegetables and fruits by CV. Ali et al. [24] also employed CV and carbon working electrodes to determine L-AA in orange juices. The results were used to study the stability of the juices. López-Pastor et al. [25] conducted a similar study on multifruit juices using a homemade potentiostat.
Zhao et al. [26] developed a modified electrode by coating the carbon electrode with a poly(o-phenylenediamine) film. The electrochemical behavior of the sensor was investigated by square wave voltammetry and amperometry. The electrode was successfully used for the determination of L-AA in a commercial orange juice.
Fuenmayor et al. [27] determined in situ L-AA in fruits by amperometry employing screen-printed carbon electrodes modified with Nylon-6 nanofibers. The membrane, prepared by electrospinning, acts as a selective barrier against potential interferents, such as phenolic compounds. No sample preparation or dilution is necessary, as the device is applied directly by “puncturing” the electrode into the fruit. A good correlation was obtained between the in situ amperometric method and a reference chromatographic methodology (HPLC-UV), when applied to several fruit samples.
A screen-printed carbon electrode modified with cadmium oxide (CdO) nanoparticles was developed for the detection of L-AA in fruit juices. CV and differential pulse voltammetry (DPV) were used. The L-AA content in commercial fruit juice was determined through standard additions and the results obtained were compared with an iodometric titration method [28].
Jadav et al. [29] constructed their own screen-printed electrodes from polymer-based conductive inks mixed with silver nanoparticles and a carbon working electrode. The determination of L-AA in juices was performed by CV, and the results obtained were similar to those obtained by the indophenol spectrophotometric method.
No chronoamperometric method for the determination of L-AA, either with conventional or screen-printed electrodes, was found in the literature.

2. Materials and Methods

2.1. Materials

2.1.1. Electrodes, Instruments, and Reagents

Screen-printed electrodes (Metrohm-DropSens, Oviedo, Spain) were employed, with carbon-based working and auxiliary electrodes and a silver pseudo-reference electrode. A potentiostat, fabricated in-house at the University of Murcia, was used for chronoamperometric and voltammetric measurements.
A model 1260 Infinity II (Agilent, Santa Clara, CA, USA) HPLC instrument, equipped with a degasser, two binary pumps, an automatic thermostated injector, in this case at 15 °C, and a diode array detector (DAD) in which the wavelength is selected at 245 nm, was used. The column used was Teknokroma C18 (Sant Cugat del Valles, Spain) Brisa of 5 microns, 25 × 0.46 cm, at a constant temperature of 35 °C.
All the reagents used throughout this work were of analytical reagent grade from Panreac (Castellar del Valles, Spain), including L-ascorbic acid (L-AA, C6H8O6) and potassium chloride. All solutions were prepared in 18.2 MΩ cm double-deionized water (Milli-Q water systems, Merck Millipore, Darmstadt, Germany).

2.1.2. Solutions

A 0.1 M KCl solution and 0.02 M L-AA solution in water were used. Working L-AA solutions of different concentrations for chronoamperometric measurements were prepared by diluting appropriate volumes of the 0.02 M L-AA solution with 0.1 M KCl. Working L-AA solutions for high-performance liquid chromatography (HPLC) analysis were prepared by diluting the 0.02 M L-AA solution in water. A 0.01% sulfuric acid solution adjusted to a pH of 2.5 was used as mobile phase.

2.1.3. Commercial Products

Orange juice (Brand 1): orange juice from concentrate (50%). Apple juice (Brand 1): apple juice from apple juice concentrate and acidulant (citric acid). Tropical juice (Brand 1): fruit juice in variable proportions from concentrate (apple, pineapple, orange, tangerine, passion fruit, banana, guava, mango, kiwi, papaya). Peach, apple, and grape juice (Brand 2): peach puree (55%), apple juice from concentrate (30%), and grape juice from concentrate (15%). Orange juice (Brand 2): 100% orange juice from concentrate. Pineapple, apple, and grape juice (Brand 2): pineapple juice from concentrate (55%), apple juice from concentrate (30%), and grape juice from concentrate (15%). Orange juice (Brand 3): orange juice from concentrate (50%). Pineapple, apple, and grape juice (Brand 3): pineapple juice from concentrate (34%), apple juice from concentrate (33%), and grape juice from concentrate (33%). Mediterranean fruit juice and milk (Brand 3): fruit and vegetable juice (15%) (orange, carrot, and peach) partially from concentrate, skimmed milk (10%). Peach, apple, and grape juice (Brand 3): fruit juice partially from concentrate (apple, orange, grape, banana, pineapple, peach, passion fruit, kiwi, guava, and mango). Tropical fruit juice and milk (Brand 3): fruit juice (15%) (pineapple, mango, and apple) partially from concentrate, skimmed milk (10%). Lemon, orange, and mandarin natural juices were obtained by pressing the corresponding fruits.

2.2. Methods

2.2.1. General Procedure for Chronoamperometric Measurements

A drop of the sample solution was placed on the electrodes, ensuring that all three electrodes were fully covered. A constant potential was applied for 10 s and the corresponding current versus time was recorded. All chronoamperometric experiments were performed in triplicate.

2.2.2. Chronoamperometric Calibration Graph for L-AA

A potential of 0.5 V was used in the chronoamperometric study. L-AA solutions of different concentrations, 2 × 10−5 M, 5 × 10−5 M, 1 × 10−4 M, 2 × 10−4 M, 5 × 10−4 M, 1 × 10−3 M, in 0.1 M KCl were employed for the calibration graph. The chronoamperometric response of the 0.1 M KCl solution (blank assay) was also recorded. All chronoamperometric measurements were performed in triplicate, using a fresh sample drop for each measurement. To construct the calibration line, the current at 10 s was considered. The corresponding blank-subtracted values were plotted versus the L-AA concentration.

2.2.3. Chronoamperometric Determination of L-AA in Juices

For juice analysis, no sample pretreatment was necessary, except for a 1:50 dilution of juice with 0.1 M KCl solution. The chronoamperograms and the blank-subtracted currents (at 10 s) were obtained as described in Section 2.2.2. The corresponding L-AA concentration values were obtained from a recent calibration line in the concentration range 2 × 10−5–8 × 10−5 M L-AA standards in 0.1 M KCl.

2.2.4. Square Wave Voltammetric Measurements

The same juice dilution as described in Section 2.2.3 was used. A pulse amplitude of 50 mV, a pulse duration of 0.4 s, a potential step of 10 mV, and a potential range of 0–1 V were employed for the square wave voltammetric recordings.

2.2.5. Determination of L-AA in Juices by HPLC

For juice analysis, a 1:50 dilution of juice with Milli-Q water was used. In the case of thick juices, a prefiltration of the diluted solution through a 0.2 µm Millipore filter was performed. The sample injection volume was 10 µL. Before injection the sample was shaken for 30 s in a vortex at full power. A flow rate of 0.8 mL/min was used.

3. Results

3.1. Chronoamperometric and Voltammetric Study of the Oxidation of L-AA

The chronoamperometric response of the electro-oxidation of L-AA was recorded at different applied potentials E in the range 0–1.0 V, at 0.1 V intervals (Figure 1A), showing the expected increase in the oxidative current as the E-value potential became more positive, up to reaching the limiting current when E ≥ 0.5 V.
The log-transformation of the I-t response shows that the limiting current follows a Cottrellian behavior [30,31], that is, a linear relationship between log(I) and log(t) with a slope close to −0.5. This evidences that, under the conditions of the study, the rate of the electrode reaction is controlled by the semi-infinite linear diffusion towards the electrode surface of a solution-phase reactant (in this case, L-AA).
When considering intermediate values of the applied potential, clear deviations are observed with respect to both the linearity and the slope predicted for the plot log(I) vs. log(t) of a diffusion-controlled response (Figure 1B). This suggests the existence of kinetic limitations related to the electrode reaction and/or to coupled phenomena such as homogeneous chemical reactions or adsorption–desorption processes. This is confirmed via the analysis of the normal pulse voltammetry (NPV) curve obtained from the chronoamperograms (Figure 1A) at different pulse times. As shown in Figure 2A, as the pulse time considered becomes longer, the sigmoidal normalized NPV curve shifts towards less positive potentials and the curve steepness increases. Thus, the value of the half-wave potential (E1/2) decreases with time, as well as the difference E 3 / 4 − E 1 / 4 (the so-called Tomeš criterion [30,32]), where E 3 / 4 and E 1 / 4 are the potential values for which the current is ¾ and ¼, respectively, the limiting current. Both behaviors point to finite electro-oxidation kinetics since, otherwise (i.e., very fast electrochemical and chemical steps), the normalized NPV curves at different pulse times would overlap with E1/2 and E 3 / 4 − E 1 / 4 being time independent [30,31].
The above behavior can be further rationalized attending to the mechanism of electro-oxidation of L-AA. At carbon electrodes in slightly acidic media, the process has been proposed to follow the following reaction scheme [33]:
H 2 AA ⇄ HA A − + H +
HA A − ⇄ H A A • + e −
HA A • ⇄ A A • − + H +
A A • − ⇄ DHA + e −
DHA + H 2 O → 2 , 3 -DKG
where the formation of the radical intermediate has been confirmed by electron paramagnetic resonance [34] and the product of the two-electron, two-proton transfer, dehydroascorbic acid (DHA), undergoes irreversible hydrolysis (step (5) yielding 2,3-diketogulonic acid (2,3-DKG). Taking into account that protonation–deprotonation reactions are typically very fast, the above scheme can be analyzed in terms of an EECi mechanism [30], where each E-step accounts for a one-electron, one-proton transfer and Ci for the irreversible hydrolysis of DHA. As the second electron transfer is thermodynamically much more favorable than the first one according to the data reported for their formal potentials ( E 1 0 ′ ≈ 0.74 V and E 2 0 ′ ≈ 0.22 − 0.24 V   ( v s   NHE ) [35]), if the two electron transfers were reversible, the steepness of the NPV curves would reflect an apparently simultaneous transfer of two electrons, for which E 3 / 4 − E 1 / 4 ≈ 28   mV [32]. This value is significantly smaller than those obtained experimentally (Figure 2B), which again suggests electrode kinetic limitations. Specifically, previous voltammetric studies have reported that the first electron transfer is sensitive to the electrode material [35], non-reversible, and rate determining at carbon electrodes [36,37,38], with the results here obtained being compatible with such scenario.

3.2. Influence of L-AA Concentration

To study the influence of L-AA concentration on the current obtained under the selected experimental conditions, the L-AA concentration was varied from 2 × 10−5 to 1 × 10−3 M in 0.1 M KCl. The chronoamperograms obtained for all the concentrations assayed under limiting current conditions (E = 0.5 V) are shown in Figure 3.
The calibration plot obtained representing the blank-subtracted current sampled at the end of the chronoamperometric measurement (t = 10 s) for each concentration (each in duplicate) versus the corresponding L-AA concentration values is shown in Figure 4. As a slight curvature was observed in the plot, the experimental points were not fitted with a linear plot but the function y = a (1 − e−bx) was employed, obtaining an excellent fitting with the parameter values a = 44.36 and b = 285.6 over the entire range of concentrations studied. The limit of detection was calculated as the concentration of analyte corresponding to three times the standard deviation of the blank signal. A detection limit of 7 × 10−7 M was obtained.

3.3. Repeatability and Reproducibility

In order to evaluate the repeatability of the method, as well as its reproducibility between different SPE units, chronoamperometric signals were measured with a 4 × 10−5 M solution of L-AA using three electrodes from the same batch. For each electrode, chronoamperometric measurements were performed in triplicate, using a fresh drop of sample for each measurement. Repeatability and reproducibility were also studied with a 0.1 M KCl solution (background). The mean current values obtained are shown in Figure 5. The values shown for L-AA are blank subtracted. The coefficients of variation corresponding to the three repeated measurements of 4 × 10−5 M L-AA were 1.4, 0.8, and 1.4% for electrodes A, B, and C, respectively. These values demonstrate that the repeatability of each of the three electrodes is excellent. Moreover, from the mean current values obtained for each electrode, a mean value ± standard deviation of (451.7 ± 7.1) × 10−3 μA was obtained, giving a coefficient of variation between electrodes of 1.6%. This value indicates excellent reproducibility of the chronoamperometric method when using different screen-printed electrodes.

3.4. Chronoamperometric Determination of L-AA in Juices

3.4.1. Square Wave Voltammetry of Juices

To rule out any interference in the chronoamperometric determination of L-AA in juices from other compounds that may be oxidized at the screen-printed carbon electrode, the square wave voltammograms of all juices were recorded following the procedure outlined in Section 2.2.4. The square wave voltammograms obtained are shown in Figure 6. As can be seen, all samples exhibit an initial peak around +0.3 V corresponding to the oxidation of L-AA, along with two or three additional peaks at more positive potentials, which correspond to the oxidation of certain polyphenolic compounds present in the juices. Therefore, a measuring potential of 0.5 V was selected for the chronoamperometric method to determine L-AA, ensuring that these other compounds do not interfere.

3.4.2. Chronoamperometric Study of L-AA Electrochemical Oxidation in Juices

In preliminary studies, the use of undiluted juice was observed to lead to non-reproducible results due to the progressive deterioration of the electrode performance. For this reason, the juice was diluted with the 0.1 M KCl solution. Taking into account the usual contents of L-AA in juices and the determination range of the L-AA calibration graph shown in Figure 4, a dilution of 1:50 was selected.
The chronoamperograms obtained for the pineapple, apple, and grape juice (Brand 2) at the different potentials tested are shown in Figure 7. From these chronoamperograms, the normal pulse voltammogram was obtained by using the current sampled at 10 s. Figure 8 shows the corresponding normalized voltammogram, together with that obtained with an 8 × 10−5 M L-AA solution in 0.1 M KCl. It is observed that the voltammograms corresponding to the juice sample and the L-AA standard almost overlap and, at the selected potential of 0.5 V, the current values are nearly identical. This implies that the calibration with L-AA standards can be applied for the chronoamperometric determination of L-AA in juice samples.
To determine the L-AA content in juices, chronoamperometric measurements were performed on each juice sample in triplicate, following the procedure described in Section 2.2.3. The blank-subtracted chronoamperograms obtained for three representative juices are shown in Figure 9. As observed, the repeatability of the chronoamperograms for the three juices is very good, with the current varying according to the type of juice tested. After obtaining the chronoamperograms of all the juices in triplicate and using the current values at t = 10 s, the concentration of L-AA in each juice was calculated. A calibration line in the concentration range 2 × 10−5–8 × 10−5 M L-AA in 0.1 M KCl, covering all the current values corresponding to the diluted juices, was used for this purpose. The results obtained are given in Table 1, showing that the content of L-AA in commercial juices is very different. The wide concentration range found is remarkable, from 33.4 in apple juice to 712.1 mg L−1 in pineapple, apple, and grape juice (Brand 2). All the tested juices of Brand 2 contain a very high concentration of L-AA. The concentration of L-AA in juices of the same type of fruit varies depending on the brand.

3.4.3. Comparison of the Results with Those Obtained by HPLC

The concentration of L-AA in the juices analyzed was also determined by HPLC as described in Section 2.2.5. The results obtained are shown in Table 1. To compare the results obtained by both techniques, values from the chronoamperometric method were plotted against those obtained by HPLC. The experimental points were then fitted to a linear regression (Figure 10). The values obtained from the linear regression were: intercept a = −21.70; slope b = 1.07; and R2 = 0.9537. Given the significance of random errors in the values of the slope and the intercept, their uncertainties (Sa and Sb) were calculated from the residual standard deviation, Sr, as follows:
S r = ∑ i = 1 n ( y i − y ^ i ) 2 n − 2
S a = S r ∑ i = 1 n x i 2 n ∑ i = 1 n x i − x ¯ 2
S b = S r ∑ i = 1 n x i − x ¯ 2
The values obtained for Sa (=16.50) and Sb (=0.04) were used to estimate the confidence limits for the intercept and the slope, respectively. Thus, the confidence limits for the slope are given by b ± tSb, where the value of t is taken as a desired confidence level and (n − 2) degrees of freedom. Similarly, the confidence limits for the intercept are given by ±tSa. Using the values of t (2.05) for 28 degrees of freedom, and applying the expressions described above, the following 95% confidence intervals are obtained for the intercept (a) and the slope (b):
a = − 21.7 ± 33.8 b = 1.07 ± 0.09
The confidence intervals of the intercept, a (−55.5, 12.1), and of the slope, b (0.98, 1.16), obtained include the theoretical values of 0 and 1, respectively. Therefore, it is concluded that there are no significant differences between the results obtained by the two methods, chronoamperometry and HPLC.

3.5. Comparison with Reported Electrochemical Methods

The proposed method for the determination of L-AA in juices is compared with other reported electrochemical methods for the same purpose in Table 2. As can be seen, our method uniquely employs a commercial unmodified carbon electrode along with the chronoamperometry technique, making this approach highly straightforward and easy to apply. The detection limit of the proposed method is among the lowest reported. In terms of sample application, the proposed method is applied to a wide range of commercial juices from various fruits and fruit mixtures, whereas most reported methods address a more limited number and variety of samples.

3.6. Voltammetric Electronic Tongue for Juices

In addition to the chronoamperometric determination of L-ascorbic acid in juices, the value of the square wave voltammetric signal of different types of juices was assessed as a foundation for the development of an electronic tongue. To achieve this, principal component analysis (PCA) was applied to the full set of experimental square wave signals obtained, revealing that only two principal components are necessary to explain 90% of the total variance of the voltammograms (PC1 explains 62% and PC2 28%). Therefore, the treatment and handling of the voltammograms are drastically simplified by using the two values of the scores of these components of the corresponding voltammograms. Figure 11 shows the values of PC2 versus the corresponding PC1 for the three voltammograms obtained for all the juices. As can be seen, the points corresponding to pineapple, apple, and grape juice (Brand 3), mediterranean fruit juice and milk (Brand 3), peach, apple, and grape (Brand 3), tropical fruit and milk (Brand 3), apple (Brand 1), orange (Brand 1), orange (Brand 2), tropical (Brand 1), and natural lemon are distinctly separated from the points corresponding to any other juice. The PCA score plot indicates that different brands of each type of juice can be discriminated:
  • Orange juices: The four types tested are situated at positive or slightly negative (natural juice) values of PC1, and three of them at positive PC2 values. PC2 allows for Brands 1 and 3 to be distinguished from the natural juice and from Brand 2, which can be discriminated from PC1.
  • Pineapple, apple, and grape juices: Situated at positive PC1 and negative PC2 (unlike most orange juices tested), the two brands can be differentiated by their placement along PC2.
  • Peach, apple, and grape juice: Positioned at negative or slightly positive PC1 values, the two brands studied can be distinguished based on their positions along PC1.

4. Conclusions

The chronoamperometric method using unmodified carbon-based screen-printed electrodes allows for the determination of L-ascorbic acid (L-AA) in a wide range of commercial juices more quickly, affordably, and simply than approaches requiring electrode modifications. Although some of these approaches have achieved lower limits of detection (LODs), the LOD obtained in this study meets the requirements for L-AA analysis in juice. Hence, this method provides reliable and practical chronoamperometric determination of L-AA without sample pretreatment or electrode modification, as confirmed through comparison with HPLC results across various commercial and natural juices.
The developed electronic tongue successfully differentiated commercial brands and even types of juices based on principal component analysis of square wave voltammetric signals.

Author Contributions

Conceptualization, J.Á.O. and E.L.; methodology, M.S.G., E.L., A.R. and J.Á.O.; validation, L.E.A., M.S.G., E.L. and J.Á.O.; formal analysis, L.E.A., M.S.G., E.L., A.R. and J.Á.O.; investigation, L.E.A., M.S.G., E.L. and J.Á.O.; resources, E.L. and J.Á.O.; data curation, L.E.A., M.S.G., E.L., A.R. and J.Á.O.; writing—original draft preparation, L.E.A., M.S.G., E.L. and J.Á.O.; writing—review and editing, M.S.G., E.L. and J.Á.O.; supervision, J.Á.O., M.S.G. and E.L.; project administration, E.L.; funding acquisition, E.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially funded by MCIN/AEI/10.13039/501100011033, NextGenerationEU/PRTR, UE, grants TED2021-129300B-I00, PID2021-122466OB-I00, PID2022-136568NB-I00, and PDC2022-133026-I00.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Dataset available on request from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Chemosensors 12 00237 g001
Figure 1. (A) Experimental background-subtracted current–time curves obtained with 2 × 10−4 M L-AA, 0.1 M KCl solution and (B) their log-transformation for different applied potentials: E (V) = 0 (black curve); 0.1 (red curve); 0.2 (dark blue curve); 0.3 (light green curve); 0.4 (blue curve); 0.5 (pink curve); 0.6 (dark green curve); and 0.7 (grey curve).
Figure 1. (A) Experimental background-subtracted current–time curves obtained with 2 × 10−4 M L-AA, 0.1 M KCl solution and (B) their log-transformation for different applied potentials: E (V) = 0 (black curve); 0.1 (red curve); 0.2 (dark blue curve); 0.3 (light green curve); 0.4 (blue curve); 0.5 (pink curve); 0.6 (dark green curve); and 0.7 (grey curve).
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Figure 2. (A) Experimental background-subtracted normalized current–potential curves obtained with 2 × 10−4 M L-AA, 0.1 M KCl solution (points) and best-fit sigmoid curves (solid lines) for different pulse times: t (s) = 0.4 (black); 0.6 (red); 1 (green); 2 (blue); 5 (pink); and 10 (light blue). Inset: Experimental background-subtracted current–potential curves before normalization. (B) Variation of the experimental half-wave potential, E 1 / 2 (black points), and E 3 / 4 − E 1 / 4 (red points) with the pulse time.
Figure 2. (A) Experimental background-subtracted normalized current–potential curves obtained with 2 × 10−4 M L-AA, 0.1 M KCl solution (points) and best-fit sigmoid curves (solid lines) for different pulse times: t (s) = 0.4 (black); 0.6 (red); 1 (green); 2 (blue); 5 (pink); and 10 (light blue). Inset: Experimental background-subtracted current–potential curves before normalization. (B) Variation of the experimental half-wave potential, E 1 / 2 (black points), and E 3 / 4 − E 1 / 4 (red points) with the pulse time.
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Figure 3. Chronoamperometric current–time signal at E = 0.5 V for different concentrations of L-AA in 0.1 M KCl, 0 M (yellow); 2 × 10−5 M (black); 5 × 10−5 M (green); 1 × 10−4 M (red); 2 × 10−4 M (blue); 5 × 10−4 M (pink); 1 × 10−3 M (purple).
Figure 3. Chronoamperometric current–time signal at E = 0.5 V for different concentrations of L-AA in 0.1 M KCl, 0 M (yellow); 2 × 10−5 M (black); 5 × 10−5 M (green); 1 × 10−4 M (red); 2 × 10−4 M (blue); 5 × 10−4 M (pink); 1 × 10−3 M (purple).
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Figure 4. Calibration plot for the chronoamperometric determination of L-AA.
Figure 4. Calibration plot for the chronoamperometric determination of L-AA.
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Figure 5. Current values for 0.1 M KCl (Blank) and for 4 × 10−5 M L-AA in 0.1 M KCl, obtained with different SPEs: A, B, and C. Error bars correspond to the confidence intervals at 95% level for three determinations.
Figure 5. Current values for 0.1 M KCl (Blank) and for 4 × 10−5 M L-AA in 0.1 M KCl, obtained with different SPEs: A, B, and C. Error bars correspond to the confidence intervals at 95% level for three determinations.
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Figure 6. Square wave voltammograms of the different juices (three repeats).
Figure 6. Square wave voltammograms of the different juices (three repeats).
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Figure 7. Background-subtracted chronoamperograms obtained for the juice from pineapple, apple, and grape (Brand 2) diluted 1:50 with 0.1 M KCl for different applied potentials: E (V) = 0 (black curve); 0.1 (red curve); 0.2 (dark blue curve); 0.3 (light green curve); 0.4 (blue curve); 0.5 (pink curve); 0.6 (dark green curve); 0.7 (grey curve); 0.8 (dark red curve); 0.9 (yellow curve); and 1.0 (light blue curve).
Figure 7. Background-subtracted chronoamperograms obtained for the juice from pineapple, apple, and grape (Brand 2) diluted 1:50 with 0.1 M KCl for different applied potentials: E (V) = 0 (black curve); 0.1 (red curve); 0.2 (dark blue curve); 0.3 (light green curve); 0.4 (blue curve); 0.5 (pink curve); 0.6 (dark green curve); 0.7 (grey curve); 0.8 (dark red curve); 0.9 (yellow curve); and 1.0 (light blue curve).
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Figure 8. Normalized current–potential curve of diluted juice (red) and 8 × 10−5 M L-AA (black), both in 0.1 M KCl. Inset: Experimental background-subtracted current–potential curves before normalization.
Figure 8. Normalized current–potential curve of diluted juice (red) and 8 × 10−5 M L-AA (black), both in 0.1 M KCl. Inset: Experimental background-subtracted current–potential curves before normalization.
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Figure 9. Chronoamperometric records of current–time of three tested juices. A, orange juice (Brand 1); B, orange juice (Brand 3); and C, apple juice (Brand 1).
Figure 9. Chronoamperometric records of current–time of three tested juices. A, orange juice (Brand 1); B, orange juice (Brand 3); and C, apple juice (Brand 1).
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Figure 10. Plot of the L-AA content obtained by the chronoamperometric method (ChA) versus those obtained by the HPLC method. Error bars: mean ± standard deviation.
Figure 10. Plot of the L-AA content obtained by the chronoamperometric method (ChA) versus those obtained by the HPLC method. Error bars: mean ± standard deviation.
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Figure 11. PCA score plot of the square wave voltammetric dataset of the different juices analyzed.
Figure 11. PCA score plot of the square wave voltammetric dataset of the different juices analyzed.
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Table 1. Concentration of L-AA determined in the commercial juices analyzed.
Table 1. Concentration of L-AA determined in the commercial juices analyzed.
JuiceChronoamperometric Method
Mean ± S * (mg L−1)		HPLC
(mg L−1)
Orange (Brand 3)209.0 ± 7.8231.4
Pineapple, apple, and grape (Brand 3)132.6 ± 1.4108.0
Mediterranean fruits and milk (Brand 3)165.1 ± 5.0210.8
Peach, apple, grape, and others (Brand 3)69.2 ± 3.759.3
Tropical fruits and milk (Brand 3)223.5 ± 4.9250.1
Apple (Brand 1)33.4 ± 11.030.9
Orange (Brand 1)462.6 ± 2.8545.2
Orange (Brand 2)593.3 ± 8.8552.9
Pineapple, apple, and grape (Brand 2)712.1 ± 2.9631.9
Peach, apple, and grape (Brand 2)545.4 ± 6.1487.5
* Standard deviation.
Table 2. Characteristics of the reported and proposed electrochemical methods for L-AA determination in juices.
Table 2. Characteristics of the reported and proposed electrochemical methods for L-AA determination in juices.
ReferenceElectrodeTechnique *Range (M)LOD (M)Samples
25SPE-PtCV5 × 10−5–1 × 10−31.25 × 10−7Multifruit juices
16GC–electropolymerized anilineA4 × 10−7–2 × 10−34 × 10−7Commercial fresh and from concentrate juices
FI-A5 × 10−6–1 × 10−42.5 × 10−6
19Glassy carbonCV1 × 10−3–3 × 10−3-Juicy and non-juicy fruits
20Pt diskCV1 × 10−4–1 × 10−29 × 10−5Natural and commercial juices and producers
21Pencil leadCV1.9 × 10−7–4.5 × 10−6-A commercial orange juice
22Zeolite-modified carbon pasteSWV4.0 × 10−7–1.2 × 10−32 × 10−8Citrus fruit juices
23Electrochemically pretreated carbon SPECV1 × 10−4–1 × 10−35 × 10−6One orange and one cabbage
26Molecularly imprinted carbon SPESWV4.5 × 10−7–1.35 × 10−51.1 × 10−7A commercial orange juice
1.35 × 10−5–4.09 × 10−4
27Modified carbon SPE nanofibersA2.8 × 10−4–7.4 × 10−3 Fruits directly in situ
29Carbon SPE (homemade)CV2 × 10−4–1.6 × 10−32 × 10−4Commercial juice from eight different fruits
Proposed methodCarbon SPE (commercial)ChA2 × 10−5–1 × 10−37 × 10−7Commercial juice from different fruits
28Modified carbon SPE-CdO nanoparticlesDPV5 × 10−6–1.5 × 10−45.4 × 10−8A commercial fruit juice
* A, Amperometry; FI, Flow Injection; CV, Cyclic Voltammetry; SWV, Square Wave Voltammetry; DPV, Differential Pulse Voltammetry; ChA, Chronoamperometry.
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El Anzi, L.; García, M.S.; Laborda, E.; Ruiz, A.; Ortuño, J.Á. Low-Cost Electrochemical Determination of L-Ascorbic Acid Using Screen-Printed Electrodes and Development of an Electronic Tongue for Juice Analysis. Chemosensors 2024, 12, 237. https://doi.org/10.3390/chemosensors12110237

AMA Style

El Anzi L, García MS, Laborda E, Ruiz A, Ortuño JÁ. Low-Cost Electrochemical Determination of L-Ascorbic Acid Using Screen-Printed Electrodes and Development of an Electronic Tongue for Juice Analysis. Chemosensors. 2024; 12(11):237. https://doi.org/10.3390/chemosensors12110237

Chicago/Turabian Style

El Anzi, Laila, María Soledad García, Eduardo Laborda, Alberto Ruiz, and Joaquín Ángel Ortuño. 2024. "Low-Cost Electrochemical Determination of L-Ascorbic Acid Using Screen-Printed Electrodes and Development of an Electronic Tongue for Juice Analysis" Chemosensors 12, no. 11: 237. https://doi.org/10.3390/chemosensors12110237

APA Style

El Anzi, L., García, M. S., Laborda, E., Ruiz, A., & Ortuño, J. Á. (2024). Low-Cost Electrochemical Determination of L-Ascorbic Acid Using Screen-Printed Electrodes and Development of an Electronic Tongue for Juice Analysis. Chemosensors, 12(11), 237. https://doi.org/10.3390/chemosensors12110237

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