A Green Approach to the Simultaneous Detection of Vitamin C, Paracetamol, and Caffeine Using an Aspartic Acid-Modified Waste Battery Electrode

Abstract

In this study, a waste dry cell battery (WB) electrode was electrochemically modified with aspartic acid (APA) to fabricate the APA-WB electrode for the simultaneous detection of ascorbic acid (AA), paracetamol (PA), and caffeine (CA). The WB electrode was fabricated, cleaned, and polished, its upper surface was insulated with dye, a copper wire attached for electrical contact, and finally modified with APA using voltammetric techniques. Structural and elemental characterizations of the bare and APA-modified WB electrodes were carried out using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and Fourier-transform infrared (FTIR). The results show that the bare WB electrode is mainly graphite-based and that APA modification forms a poly-APA film on its surface. The detection techniques used in this study included cyclic voltammetry (CV), differential pulse voltammetry (DPV), and UV–vis spectroscopy. Of these techniques, DPV was employed for the simultaneous analysis of the analytes. Among various APA-modified electrodes (APA-GC, APA-Pt, APA-PG, and APA-Au), APA-WB exhibited the highest sensitivity and detection capability. The APA-modified electrode demonstrated excellent analytical performance for the three analytes in phosphate buffer (pH 7.0), exhibiting diffusion-controlled behavior and high selectivity with well-resolved peaks for AA, PA, and CA. Limits of detection were 0.20, 0.15, and 0.33 μmol L^–1, with sensitivities of 54.4, 99.9, and 151.7 μA/mM/cm² for AA, PA, and CA, respectively. Interference studies confirmed reliable detection in complex matrices. The UV–vis spectra were able to detect AA, PA, and CA individually; however, they could not simultaneously detect the presence of two or more compounds. The sensor was successfully applied to pharmaceutical and soft drink samples, achieving recoveries of 98–104%. In this work, we report for the first time the application of an APA-modified WB electrode for the simultaneous detection of AA, PA, and CA. This approach transforms hazardous e-waste into a high-performance, low-cost electrochemical sensor, contributing to green chemistry by enabling resource recycling and reduced environmental pollution.

1. Introduction

In recent years, the food and pharmaceutical industries have experienced a substantial increase in the development of products incorporating bioactive compounds such as AA, PA, and CA. This trend is driven by the growing consumer demand for functional beverages and combination drugs that offer enhanced health benefits, such as improved immunity, pain relief, and stimulation. As a result, these compounds are frequently used in combination within commercial beverages (e.g., energy drinks and soft drinks) and multidrug pharmaceutical formulations to optimize therapeutic efficacy. −

Vitamin C (AA) is a water-soluble antioxidant essential for several physiological functions including hydroxylation reactions, collagen synthesis, neurotransmitter production, and immune response. It is also known to enhance the formation of high-density lipoproteins and reduce the risk of cardiovascular diseases. − Since humans cannot synthesize AA endogenously, it must be obtained through dietary intake. Deficiencies in AA can lead to scurvy, fatigue, mental and physical weakness, and increased susceptibility to infections. , However, excessive intake may cause gastrointestinal disturbances, headaches, and skin flushing. , Due to its functional versatility, AA is extensively used in pharmaceuticals, cosmetics, and nutraceuticals.

Paracetamol (PA), or N-acetyl-p-aminophenol, is widely used for its analgesic and antipyretic properties in treating fever, migraines, backaches, and musculoskeletal pain. − Although generally safe at therapeutic doses, overdose or long-term use can cause hepatotoxicity and nephrotoxicity due to the accumulation of reactive metabolites. − It is often coadministered with caffeine or other drugs (e.g., codeine, tramadol) to enhance analgesic effects, particularly in managing migraines and postpartum pain. −

Caffeine (CA), a naturally occurring alkaloid (1,3,7-trimethylxanthine), acts as a central nervous system stimulant and is a common additive in energy drinks, soft drinks, and over-the-counter medications. − In moderate doses, it improves mental alertness, but excessive intake may cause insomnia, anxiety, tremors, and gastrointestinal issues. − CA has also been reported to increase gastric acid secretion and contribute to bone demineralization and cardiovascular risks upon prolonged consumption. − Given its widespread use, especially in combination with PA or AA, monitoring caffeine levels in consumables is critical. Paracetamol and caffeine are commonly combined in analgesic formulations. The recommended maximum daily intake for adults is 4000 mg for paracetamol, as excessive use may lead to liver toxicity, and 400 mg for caffeine, since higher doses can cause insomnia, anxiety, and increased heart rate. , Similarly, the tolerable upper limit for vitamin C is 2000 mg per day. Maintaining these limits ensures therapeutic efficacy while preventing potential adverse effects.

A wide range of analytical techniques have been employed for the individual and simultaneous quantification of ascorbic acid (AA), paracetamol (PA), and caffeine (CA). These include spectrophotometric methods for PA and CA, − high-performance liquid chromatography (HPLC) for CA − and AA, − liquid chromatography–mass spectrometry (LC–MS) for CA and AA, voltammetric approaches for CA − and AA, − Fourier-transform infrared (FTIR) spectrophotometry for CA − and AA, chemiluminescence for CA, gas chromatography–mass spectrometry (GC–MS) for CA, ion chromatography for CA, capillary electrophoresis for CA, and ultrahigh-performance liquid chromatography (UHPLC) for both CA and AA.

Conventional methods for detecting biomolecules such as AA, PA, and CA typically rely on HPLC, LC–MS, GC–MS, capillary electrophoresis, etc. − ,,,, Although these methods provide excellent sensitivity and selectivity, they involve expensive instrumentation, complex sample preparation, and large volumes of organic solvents, factors that increase both the cost and environmental impact. Moreover, they are time-consuming and unsuitable for rapid or on-site analysis. Similarly, UV–Vis spectroscopy is unable to simultaneously quantify AA, PA, and CA due to overlapping absorption peaks and poor selectivity. −

In contrast, electrochemical detection using modified electrodes offers a cost-effective, sensitive, and rapid alternative. − Techniques such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square-wave voltammetry (SWV) enable direct, real-time monitoring of electroactive species with minimal sample preparation. Surface modification further improves selectivity and sensitivity, allowing the simultaneous detection of multiple analytes in complex matrices. Additionally, electrochemical methods require small sample volumes and produce negligible waste, aligning with green chemistry principles. Thus, developing sustainable, modified electrodes represents a promising approach for efficient, environmentally friendly, and high-performance analytical detection. However, the electrochemical signals of AA and PA often overlap, presenting a major challenge in their concurrent detection on unmodified electrodes. , While some efforts have been made to detect two of these analytes simultaneously, such as AA with PA or PA with CA, − there is limited work addressing the concurrent detection of all three analytes (AA, PA, and CA) in real samples using a single electrode platform. Several studies have been reported the detection of AA, PA, or CA in the presence of other species. −

Graphite rods from waste zinc–carbon batteries can be efficiently repurposed as low-cost electrodes for electrochemical sensing, owing to their excellent conductivity, stability, and surface area. Simple cleaning or functional modification further enhances their electrocatalytic performance toward biomolecule and drug detection. Reusing waste battery electrodes not only reduces experimental costs but also minimizes electronic waste and environmental pollution, supporting sustainable and green analytical practices. Electrodes from waste zinc–carbon batteries have been used for the determination of xanthine and tannic acid. − A recycled waste battery electrode, modified with polyhippuric acid and multiwalled carbon nanotubes, has been employed for the electrochemical detection of serotonin. Electropolymerization of aspartic acid (APA) on electrodes such as glassy carbon, gold, graphite, and carbon paste using a similar cyclic voltammetric method has also been reported. −

In this context, we report for the first time the application of an aspartic acid (APA)-modified waste battery (WB) electrode for the simultaneous determination of AA, PA, and CA. The proposed electrode demonstrates excellent electrocatalytic properties, high sensitivity, well-separated oxidation peaks, and low detection limits for all three analytes. This approach not only provides a high-performance, low-cost electrochemical sensor but also highlights the sustainable reuse of electronic waste, converting hazardous materials into valuable analytical tools. By transforming spent batteries into functional electrodes, this study advances green chemistry principles through resource recycling and reduction of environmental pollution reduction. The developed sensor was successfully applied to real samples from the pharmaceutical industries and commercial beverages in Bangladesh, showing strong agreement with reference spectroscopic methods.

2. Experimental Section

2.1. Chemicals and Instruments

All of these chemicals and reagents used were of analytical grade. Ascorbic acid (99%), acetaminophen (99%), aspartic acid (99%), acetic acid (99.5%), sodium acetate (99%), sodium hydroxide (98%), sodium bicarbonate (99%), and sodium dihydrogen orthophosphate (99%) were purchased from E-Merck, Germany. Caffeine (99%) was obtained from Qualikems Fine Chem Pvt. Ltd., India. Tablet samples (Cevit, Nutrivit-C, Ace, Napa, Ace Plus, and Napa Extra) were collected from a local pharmaceutical shop of Bangladesh. Distilled water was used to prepare all of the solutions. Phosphate buffer, acetate buffer, and carbonate buffer solutions were prepared in ultrapure distilled water and used as supporting electrolytes.

CV and DPV were performed with a Potentiostat (μstat 8000, Metrohm/Dropsens) workstation. In this study, WB and pencil graphite (PG) electrodes were fabricated, while glassy carbon (GC), gold (Au), and platinum (Pt) electrodes were obtained from BASi, USA, to serve as working electrodes. A platinum wire was used as an auxiliary electrode, and a Ag/AgCl (3 M KCl solution, BASi, USA) electrode was used as a reference electrode. The pH of different buffer solutions was measured with an EZDO pH meter (pH 5011, Taiwan). UV–vis spectroscopic measurements were performed by using a computer-controlled spectrophotometer (UV-1800, Shimadzu, Japan) with a pair of quartz cuvettes. Surface morphology was examined by scanning electron microscopy (SEM) using a JCM-7000 microscope (Japan) equipped with an energy-dispersive X-ray spectroscopy (EDS) system. For EDS analysis, the samples were shaped into disks and mounted on carbon tape.

2.2. Preparation of Waste Battery Electrodes

In this study, a dry cell zinc–carbon waste battery was used as the working electrode. The specific battery employed in this experiment was Sunlite, a nonrechargeable (single-use) dry cell commonly available in the local markets of Bangladesh. The used battery was first collected, after which its outer casing was removed to access the inner rod. This rod was polished with sandpaper to achieve a precise electrode shape. Then it was washed with distilled water and dried by air. One part of the rod was then coated with insulating dye, while the tip was intentionally left uncoated to allow it to function as the electrode surface. The exposed tip was further polished against smooth white paper, giving it a shiny, mirror-like appearance. Additionally, the uncoated section of the rod was wrapped in copper wire to facilitate connections to the potentiostat. Figure illustrates the process of fabricating the WBE.

1.

Different steps of fabrication of the waste battery (WB) electrode.

2.3. Preparation of Modified Electrodes

The bare WB electrode was initially polished on white paper, followed by fine polishing with 0.05 μm of alumina powder on a wet polishing cloth. The polished electrode was then rinsed thoroughly with distilled water and dried at room temperature. Different concentrations of APA solution (2–10 mM) were prepared in phosphate buffer solution of pH 7.0 and stirred at room temperature for 30 min using a magnetic stirrer. The electrochemical cell was then assembled by connecting the electrodes to a computer-controlled potentiostat. Several parameters were systematically investigated, including monomer concentration (1–50 mM APA), scan rate (0.05 to 0.3 V/s), potential range (−0.5 to +1.8 V), and number of cycles (10–30). It was observed that after 15 cycles, the current response reached a steady state, indicating that the electrode surface had become saturated with the polymer layer. Based on these findings, the optimal electrodeposition conditions were established as 5 mM APA in 0.1 M phosphate buffer solution (pH 7), using a scan rate of 0.1 V/s for 15 cycles. These optimized conditions are consistent with previous reports. , Figure S1 illustrates the electrochemical modification of the WB electrode with APA, achieved by performing 30 consecutive cyclic voltammetric scans in a 5 mM APA solution (phosphate buffer) over a potential range of −0.5 to +1.6 V at a scan rate of 100 mV/s. The modified electrode was then dried under a nitrogen stream for 10 min. The electrode was then washed with distilled water and referred to as APA-WB. A similar procedure was followed for the preparation of other APA- modified electrodes, such as APA-GC, APA-Pt, APA-PG, and APA-Au.

2.4. Preparation of the Sample Solution

Three targeted analytes of AA, PA, and CA solutions of different concentrations (1–5 mM) were prepared using phosphate buffer solution at pH 7. The phosphate buffer solution of 0.1 M at pH 7 was prepared by using sodium dihydrogen orthophosphate and disodium hydrogen orthophosphate. The modifier solution was prepared by dissolving 30 mg of aspartic acid in 50 mL of 0.1 M phosphate buffer solution (pH 7.0). To determine the total content of the pharmaceutical tablet samples, various sample solutions were prepared. First, the tablets were weighed and then ground into a fine powder by using a mortar. This powder was subsequently dissolved in 0.1 M PBS solution at pH 7 to create the sample solution. The quantitative analysis of the compounds was conducted by using differential pulse voltammetry. The electrochemical behavior and analytical performance of the APA-modified electrodes were studied by using CV and DPV techniques with a potentiostat. Initially, the bare electrode was modified with APA by cyclic voltammetric scanning in a 5 mM APA solution prepared in phosphate buffer. The optimal conditions for electrode modification, such as the potential window, scan rate, pH, and concentration, were determined. After modification, the electrode was rinsed with distilled water and dried under nitrogen. CV studies were carried out in phosphate buffer solution containing AA, PA, and CA, either individually or in combination. The voltammograms were recorded to observe oxidation–reduction peaks, peak potential shifts, and current responses, which provided insights into the redox mechanism and electron transfer properties of the modified electrode. DPV measurements were performed to determine the sensitivity, linearity, and detection limits for AA, PA, and CA at different concentrations. The limit of detection (LOD) was calculated by using a signal-to-noise ratio of 3 (S/N = 3). Furthermore, the applicability of the modified electrode was tested using pharmaceutical tablet samples dissolved in phosphate buffer to verify its practical sensing performance.

3. Results and Discussion

3.1. Surface Features of WB and APA-WB

A graphite rod extracted from a dry cell battery was polished, partially insulated with dye, and connected to a copper wire for electrochemical measurements, as illustrated in Figure . It was modified with APA by cyclic voltammetric scanning (Figure S1). The resulting electrode was designated as the APA-modified WB electrode. The APA-modified pencil graphite (PG) electrode was prepared by using a similar procedure. Commercially available glassy carbon (GC), platinum (Pt), and gold (Au) electrodes were likewise modified with APA under identical electrochemical conditions.

Figure a illustrates an SEM image of the bare WB electrode. The grayish-black color suggests the presence of graphite on the surface of the bare WB electrode. The surface appears rough, with numerous pits and channels. Additionally, the spots of varying sizes are randomly distributed across the surface. Figure b presents the EDS analysis of the bare WBE surface, revealing its elemental composition as 98.09% carbon and 1.91% silicon. This result indicates that the WB electrode mainly consists of a carbon-rich matrix typical of graphite-based materials. Figure a presents the SEM image of the APA-modified WB electrode, where the accumulation of APA appears as a white deposition on the rough graphite surface. Following surface modification with APA (Figure b), elemental analysis reveals a compositional change, with the surface comprising 31.37% carbon, 61.96% oxygen, 2.80% sodium, 1.07% silicon, 1.53% phosphorus, and nitrogen 0.93.

2.

(a) SEM image of the bare WB electrode with individual EDS elemental maps for C and Si shown alongside; (b) EDS spectrum of the analyzed region with the corresponding elemental composition table displayed beside it.

3.

(a) SEM image of the APA-modified WB electrode with individual EDS elemental maps for C, O, Na, P, S, N, and Si shown alongside; (b) EDS spectrum of the analyzed region with the corresponding elemental composition table displayed beside it.

SEM and EDS analyses suggest that the WB electrode mainly consists of a carbon-rich matrix typical of graphite-based materials. Its lead is not pure graphite and contains numerous vacancies, point defects, and a few foreign materials. After modification with poly-APA, the appearance of oxygen, sodium, nitrogen, and phosphorus is consistent with the chemical structure of APA and the phosphate buffer used during electrodeposition. The oxygen and nitrogen content originates from APA’s carboxyl and amine groups, whereas sodium and phosphorus result from the phosphate buffer interaction. These compositional changes, together with the SEM observation of a white layer on the graphite surface, support APA deposition, demonstrating the formation of a polymerized APA layer that is expected to enhance the electrode’s electrochemical performance for the simultaneous determination of AA, PA, and CA.

The EDS color mapping provides clear visual evidence of successful electrode modification and uniform deposition of the poly­(aspartic acid) (APA) film. A uniform color distribution indicates a homogeneous elemental coating across the electrode surface, confirming consistent incorporation of functional groups associated with APA. In the EDS mapping of the bare WB electrode, the high color intensity for carbon indicates carbon-rich regions, reflecting the dominant graphite-like matrix of the electrode. In contrast, the weak signal intensity for silicon suggests that this element is present only in trace amounts (Figure a). For the APA-modified WB electrode, localized bright regions and higher color intensities for carbon and oxygen reveal areas of enhanced elemental concentration, confirming the presence of carboxyl functional groups originating from the poly­(aspartic acid) film. Meanwhile, the weak signals for silicon, sodium, and phosphorus indicate that these elements exist only at low levels on the electrode surface after modification (Figure a).

FTIR analysis provided further evidence for the successful formation of the polymeric film (Figure S2). The spectrum of the electrodeposited layer revealed the appearance of a distinct amide (–CONH–) stretching band near 1650 cm^–1, which is characteristic of peptide linkages formed during polymerization. In contrast, the characteristic absorption peaks corresponding to the free –COOH (∼1700 cm^–1) and –NH₂ (∼3050–3300 cm^–1) functional groups observed in pure aspartic acid were significantly reduced or disappeared. A noticeable change in the FTIR spectra was observed between aspartic acid and the polyaspartic acid film. These spectral changes clearly indicate that aspartic acid molecules underwent electrochemical polymerization through the formation of amide linkages, endorsing the successful conversion of the monomer into polyaspartic acid on the electrode surface. The properties of the newly formed surface of the APA-modified WB electrode (Figure ) would also be influenced by these structural imperfections, thereby affecting its electrochemical performance. −

3.2. Electrochemical Behavior of the APA-Modified WB Electrode

The WB electrode was sequentially polished using sandpaper, alumina slurry, soft cloth, and 80G paper until a smooth, mirror-like surface was obtained. After being polished, the electrode was subjected to continuous CV scanning for different cycles. During the potential sweep, a slow increase in the anodic peak current was observed, indicating the electrodeposition of APA under the optimized conditions. The APA molecules were first oxidized at higher potentials to form α-amino free radicals (Figure S1). These reactive intermediates subsequently coupled through carbon–nitrogen covalent bonds, leading to the formation of a poly­(APA) film on the WB electrode surface, which was visibly noticeable. The observed electrochemical behavior is consistent with previous reports, − which demonstrated similar electropolymerization of aspartic acid on various electrodes such as glassy carbon, gold, graphite, and carbon paste.

3.3. Electrochemical Impedance Spectroscopy Analysis

The EIS Nyquist plot (Figure S3) demonstrates a difference in charge transfer behavior between the bare WB and APA-modified WB electrodes. The bare WB electrode exhibits a larger semicircle at high frequency, indicative of a higher charge transfer resistance (R_ct) and sluggish electron transfer kinetics. In contrast, the APA-modified WB electrode shows a significantly reduced semicircle diameter, reflecting a lower R_ct and enhanced electron transfer. This improvement can be attributed to the presence of the electroactive APA layer, which facilitates faster interfacial charge transfer and better conductivity. The observed decrease in impedance for the modified electrode confirms the successful surface functionalization and suggests improved electrochemical performance, making the APA-modified WB electrode more suitable for sensitive analyte detection.

3.4. Electrochemical Behavior of Ascorbic Acid, Paracetamol, and Caffeine at APA-Modified Electrodes

The sequential cyclic voltammograms (CVs) depict the deposition of an aspartic acid (APA) thin film onto a bare WB in APA solutions, as shown in Figure S1. The process was carried out within a potential window of −0.5 V to +1.6 V for 30 cycles at a scan rate of 0.1 V/s. APA contains an amino group and one-electron oxidation of the amino group turns into its corresponding cation radical, and these cation radicals can form carbon–nitrogen links at the waste battery electrode surface. The oxidation peak currents increase slightly with successive scanning until 15 cycles. This is attributed to passivation of the WB electrode, which is related to grafting of the aspartic acid on the surface of the WB electrode. The electrochemical behavior of aspartic acid is in agreement with the literature. In a similar manner, GC, Pt, Au, and PG electrodes were modified in APA solution by applying a potential range of −0.5 V to +1.8 V for 15 cycles at a scan rate of 0.1 V/s, yielding comparable results. Accordingly, the proposed the EC-modification process is illustrated in Figure S4.

3.5. Electrochemical Behavior of Ascorbic Acid, Paracetamol, and Caffeine at the Bare WB Electrode

Figure illustrates (a) CVs and (b) differential pulse voltammograms (DPVs) of a ternary solution containing ascorbic acid (AA), paracetamol (PA), and caffeine (CA), recorded using bare glassy carbon (GC), platinum (Pt), gold (Au), pencil graphite (PG), and WB electrodes in phosphate buffer solution (pH 7.0) at a scan rate of 0.1 V s^–1. The GC, Pt, and Au electrodes were commercially obtained, whereas the PG and WB electrodes were fabricated in-house. Both CV and DPV measurements revealed two distinct anodic peaks for all electrode types despite the presence of three electroactive species (AA, PA, and CA) in the solution. The oxidation peaks of AA and PA were closely spaced and could not be resolved by using the unmodified electrodes. Among the electrodes tested, the GC and WB electrodes exhibited relatively better-defined peak currents compared with Au, Pt, and PG; however, they still failed to resolve all three analytes. The Pt electrode exhibited particularly weak peak responses. Overall, none of the bare GC, Pt, PG, Au, or WB electrodes were capable of simultaneously detecting AA, PA, and CA in the ternary mixture.

4.

(a) CV of 5 mM AA + 5 mM PA + 5 mM CA ; and (b) DPV of 2 mM AA + 2 mM PA + 2 mM CA at bare waste battery (WB), glassy carbon (GC), gold (Au), platinum (Pt), and pencil graphite (PG) electrodes in phosphate buffer solution of pH 7 at scan rate 0.1 V/s.

The differences in their performance are primarily influenced by variations in surface characteristics, conductivity, mode of interaction, and electroactive area. Among the electrodes tested, GC and WB electrodes exhibited relatively well-defined peak currents for AA, PA, and CA, which can be attributed to their higher electroactive surface area and favorable electron transfer kinetics in a phosphate buffer solution. In contrast, Pt, Au, and PG electrodes displayed weaker peak responses, likely due to slower electron transfer kinetics. These differences are further influenced by variations in surface morphology, available active sites, and interactions with the analytes.

The bare WB electrode exhibited only two anodic peaks at 0.45 and 1.45 V in phosphate buffer solution (pH 7.0), even though the solution contained three electroactive species (AA, PA, and CA). The peak at 0.45 V corresponded to both AA and PA, while the peak at 1.45 V corresponded to CA. The oxidation peaks of AA and PA were too closely spaced to be resolved using the unmodified electrode (Figure ). The APA-modified WB electrode did not exhibit any oxidation peak within this potential region, indicating the absence of interfering signals from the electrode itself.

3.6. Determination of Ascorbic Acid, Paracetamol, and Caffeine by Using Modified Electrodes

Figure illustrates (a) the CV and (b) the DPV of a ternary solution containing AA, PA, and CA, recorded by using aspartic acid (APA)-modified GC, Pt, PG, Au, and WB electrodes. Among these, the APA-modified WB electrode exhibited three well-resolved anodic peaks corresponding to the individual oxidation of AA, PA, and CA, indicating its capability for simultaneous detection. In contrast, the bare WB electrode displayed only two anodic peaks with the oxidation potentials of AA and PA being nearly indistinguishable (Figure ). At the APA-modified WB electrode, distinct oxidation peaks for AA, PA, and CA were observed at 0.20, 0.42, and 1.40 V, respectively, in phosphate buffer solution (pH 7.0). The APA- WB electrode displays a new anodic peak at 0.20 V, which is absent in the bare WB electrode. However, the PBS electrolyte showed no peaks on platinum, gold, glassy carbon, or pencil graphite electrodes, even after modification with APA. Based on these results, the peak potential separations ΔE _pa (AA – PA) = 0.22 V and ΔE _pa (PA – CA) = 0.98 V were sufficient for reliable simultaneous determination of all three compounds. Moreover, the APA-modified WB electrode significantly enhanced the oxidation peak currents of AA, PA, and CA, indicating improved electrocatalytic activity. The larger separation in peak potentials facilitated the clear resolution of the individual analytes in the mixture. These results demonstrate that the APA-WB electrode effectively accelerates the oxidation of AA, PA, and CA, likely due to the presence of abundant edge-plane graphite sites and structural defects. The observed enhancement in peak currents and the reduction in oxidation overpotentials further confirm the strong electrocatalytic properties of the APA-WB electrode toward the oxidation of these compounds.

5.

(a) CV of 5 mM AA + 5 mM PA + 5 mM CA; and (b) DPV of 2 mM AA + 2 mM PA + 2 mM CA at an aspartic acid (APA)-modified waste battery (WB), glassy carbon (GC), gold (Au), platinum (Pt) and pencil graphite (PG) electrodes in PBS (pH 7) at scan rate 0.1 V/s.

The APA-modified GC electrode also showed three distinct anodic peaks corresponding to AA, PA, and CA. In contrast, the APA-modified Pt, PG, and Au electrodes exhibited weak and overlapping peak responses, indicating poor resolution of the individual analytes. The peak intensities observed at the APA-modified WB electrode were sharper and more defined, compared to those recorded with the APA-modified GC electrode. These results indicate that the APA-modified Pt, PG, and Au electrodes are not capable of simultaneously detecting AA, PA, and CA in a ternary mixture. Based on these findings, the APA-modified WB electrode was selected for further studies due to its enhanced performance in the simultaneous detection of AA, PA, and CA.

3.7. CV of AA at Bare and APA-Modified WB Electrodes

Figure a shows the CVs of PBS alone, 5 mM AA at the bare WB electrode, and 5 mM AA at the APA-modified WB electrode, recorded at a scan rate of 0.1 V s^–1. No redox peak was observed for PBS alone, while AA exhibited a well-defined anodic peak in both the bare and modified electrodes. At the bare WB electrode, AA showed an anodic peak at 0.27 V with a current of 83 μA. In contrast, the APA-modified WB electrode displayed a peak at 0.18 V with a current of 117 μA. The increase in current (34 μA) and decrease in oxidation potential (0.09 V) suggest improved electron transfer kinetics and an enlarged electroactive surface area at the APA-modified WB electrode. This enhancement is attributed to the electrocatalytic activity of the APA layer, which introduces active sites and promotes electron transfer. Surface modification increases the active area, introduces functional groups, and optimizes electrode coverage, thereby improving sensor performance. The oxidation of AA involves a two-electron transfer process, consistent with the voltammetric behavior observed in Figure S5a. ,

6.

Cyclic voltammogram (CV) of (a) only 0.5 M PBS (black line), 5 mM AA in a bare WB electrode (blue line), 5 mM AA in an APA-modified WB electrode (red line); (b) only 0.5 M PBS (black line), 5 mM PA in a bare WB electrode (blue line), 5 mM PA in an APA-modified WB electrode (red line); and (c) only 0.5 M PBS (black line), 5 mM CA in a bare WB electrode (blue line), 5 mM CA in an APA-modified WB electrode (red line) in PBS solution at pH 7 and scan rate 0.1 V/s.

3.8. CV of PA at Bare and APA-Modified WB Electrodes

Cyclic voltammograms of only PBS at an APA-modified WB electrode, 5 mM PA in a bare electrode, and 5 mM PA in an APA-modified WB electrode at scan rate 0.1 V/s are shown in Figure b. There is no peak observed for PBS alone, but a distinct and well-defined peak is seen for PA at the bare and modified electrodes in the PBS solution.

At the bare WB electrode, PA exhibits an anodic peak at 0.51 V with a peak current of 155 μA and a cathodic peak at −0.05 V with a peak current of 46 μA. In contrast, when the APA-modified WB electrode is used, PA shows an anodic peak at 0.42 V with a peak current of 222 μA and a cathodic peak at 0.24 V with a peak current of 110 μA. It is evident that the anodic and cathodic peak currents of PA at the APA-modified WB electrode are more pronounced and well-defined compared to those at the bare WB electrode. Additionally, the anodic peak of PA at the APA-modified WB electrode is notably shifted negatively, and the currents are significantly higher than those observed at the bare WB electrode. This indicates that significant enhancement in the rate of electron transfer from PA and the improvement is linked to the increased reversibility of the electron-transfer processes. The oxidation reaction of PA involves a transfer of two electrons, which corresponds with the cyclic voltammetric behavior observed for PA shown in Figure S5b.

3.9. CV of CA at Bare and APA-Modified WB Electrodes

Cyclic voltammograms of only PBS at the APA-modified WB electrode, 5 mM CA in the bare electrode, and 5 mM CA in the APA-modified WB electrode solution are shown in Figure c. There is no peak observed for PBS alone, but a distinct and well-defined peak is seen for CA at bare and modified electrodes in the PBS solution.

At a bare WB electrode, CA produces an anodic peak at 1.44 V with a peak current of 296 μA. In contrast, at the APA-modified WB electrode, CA exhibits an anodic peak at 1.41 V and a peak current of 369 μA. It is evident that the anodic peak for CA at the APA-modified WB electrode is sharper and more distinct compared to that of the bare WB electrode. Although the position of the anodic peak at the APA-modified WB electrode is slightly shifted, the peak currents are significantly greater than those observed at the bare WB electrode. The oxidation reaction of CA involves a transfer of two electrons, which corresponds with the cyclic voltammetric behavior observed for CA, shown in Figure S5c.

The voltammetric responses of AA and CA at both the bare and APA-modified WB electrodes were irreversible, whereas PA exhibited quasi-reversible redox behavior. The WB electrode, primarily graphite-based, exhibits a characteristic carbon electrode behavior. Compared to APA-WB, the unmodified WB electrode shows higher charge transfer resistance and background current for AA, PA, and CA. The local reactivity of graphite and APA, evaluated using the dual descriptor method, revealed distinct nucleophilic and electrophilic regions. Graphite exhibited both types of reactive sites, which were further influenced by surface-renewal- and polishing-induced impurities. In contrast, APA provided fixed reactive sites and its incorporation onto the WB surface likely introduced additional active centers, thereby enhancing electron transfer efficiency.

3.10. Simultaneous Detection of AA and PA at APA-Modified WB Electrode Using CV

Figure a presents the CVs of a mixed solution of AA and PA recorded at the bare WB electrode (blue line) and the APA-modified WB electrode (red line) at a scan rate of 0.1 V/s. At the bare WB electrode, a single broad anodic peak appeared at +0.51 V, indicating the overlap of the oxidation signals of AA and PA. In contrast, the APA-modified WB electrode resolved two distinct anodic peaks at +0.18 V and +0.42 V, corresponding to the oxidation of AA and PA, respectively, along with a cathodic peak at +0.24 V. The improved peak separation (ΔE _pa = 0.24 V) and enhanced peak currents (I _pa) observed with the APA-WB electrode suggest a significant enhancement in selectivity and electrocatalytic activity. These results demonstrate that APA modification effectively enables the simultaneous determination of AA and PA by improving both resolution and sensitivity.

7.

CV of (a) 5 mM AA + 5 mM PA at bare (blue line) and APA-modified WB electrodes (red line); (b) 5 mM PA + 5 mM CA at bare (green line) and APA-modified WB electrodes (red line); and (c) 5 mM AA + 5 mM PA + 5 mM CA at bare (blue line) and APA-modified WB electrodes (red line) in PBS solution of pH 7 at scan rate 0.1 V/s.

At the APA-modified WB electrode, the anodic peaks of AA and PA shift negatively and exhibit significantly higher currents compared with the bare WB electrode, indicating enhanced electron transfer kinetics and reduced overpotential. The aspartic acid modification introduces –NH₂ and –COOH groups, promoting strong electrostatic and hydrogen-bonding interactions with the analytes, which facilitate faster charge transfer. On the bare WB electrode, AA and PA oxidation peaks overlap, resulting in poor resolution. In contrast, the APA modification improves electrocatalytic activity and surface affinity, leading to distinct peak separation and enabling the simultaneous and accurate determination of AA and PA.

3.11. Simultaneous Detection of PA and CA Binary Solution in PBS at an APA-Modified WB Electrode Using CV

Figure b displays the cyclic CVs of a mixed solution of PA and CA at pH 7, recorded at the bare WB electrode (blue line) and the APA-modified WB electrode (red line) at a scan rate of 0.1 V/s. At the bare WB electrode, two anodic peaks were observed at +0.51 V and +1.45 V, with corresponding peak currents of 155 μA for PA and 296 μA for CA. In contrast, the APA-modified WB electrode exhibited anodic peaks at +0.42 V and +1.41 V for PA and CA, respectively, with enhanced peak currents of 207 μA and 365 μA. The APA-modified electrode clearly improved intensity of both peak currents, indicating enhanced electrocatalytic activity.

3.12. Simultaneous Detection of AA, PA and CA Ternary Solution in PBS at a APA-Modified WB Electrode Using CV

Figure c presents the CVs of a ternary mixture containing AA, PA, and CA, recorded on both bare and APA-modified WB electrodes in aqueous solution. The APA-modified WB electrode exhibited three distinct anodic peaks at +0.18 V, +0.42 V, and +1.42 V, with corresponding peak currents of 117 μA, 222 μA, and 369 μA, respectively. In contrast, the bare WB electrode displayed only two anodic peaks at +0.51 V and +1.44 V, indicating overlapping signals for AA and PA and insufficient peak resolution. The enhanced electrochemical performance of the APA-modified WB electrode demonstrated its superior capability for the simultaneous detection of AA, PA, and CA. The clear separation of oxidation peaks observed after modification was attributed to the improved electrocatalytic activity and increased surface area, which promoted faster electron transfer kinetics and minimized peak overlap.

The overlapping peaks observed at the bare WB electrode were likely due to the similar electrochemical behaviors of AA and PA, such as their comparable redox potentials and sluggish electron transfer kinetics. APA modification introduced functional groups capable of facilitating selective interactions with individual analytes, thereby enhancing both the sensitivity and resolution. Moreover, the modified electrode surface may have altered the local electrochemical environment or shifted the electrode kinetics, creating a broader potential window that enabled a distinct peak separation.

These results confirmed that APA modification of the WB electrode not only enhanced its electrocatalytic efficiency but also significantly improved its analytical performance for the simultaneous detection of structurally related compounds in complex mixtures.

3.13. Effect of pH

Figure S6a shows the CVs of the APA-modified WB electrode recorded in buffer solutions with pH values ranging from 3 to 9. It is seen that at pH 3–7, three well-defined oxidation peaks corresponding to AA, PA, and CA are observed, whereas at pH 9, only two peaks appear.

At acidic to neutral pH (3–7), three distinct oxidation peaks of AA, PA, and CA are clearly observed. This occurs because each compound exists in a different protonation state and undergoes oxidation at a separate potential. AA is oxidized most easily due to its hydroxyl groups, PA follows with oxidation of its phenolic –OH group, and caffeine oxidizes last at a higher potential involving its nitrogen-containing ring. The distinct redox potentials of these three species under these conditions allow their oxidation peaks to appear separately and clearly.

At alkaline pH (pH 9), the voltammogram shows only two peaks instead of three. This is because AA and PA become deprotonated, which shifts their oxidation potentials toward more negative values, causing their peaks to move closer together and eventually overlap. As a result, the oxidation signals of AA and PA merge into a single combined peak, while the caffeine peak remains distinct at a higher potential.

As shown in Figure S6b, the anodic peak currents of AA, PA, and CA increased from pH 5 to 7 and then decreased as the pH was raised from 7 to 9, indicating that pH 7 provided the most favorable conditions for oxidation.

The variation in the peak current with pH also follows a clear trend. From pH 5 to 7, the peak currents increase because partial deprotonation enhances electron transfer, and the electrode surface shows optimal charge compatibility with the analytes, promoting faster redox reactions. Beyond pH 7, however, the peak currents decrease as excessive deprotonation leads to electrostatic repulsion between negatively charged species and the electrode surface, and the instability of AA increases at higher pH. Consequently, both the merging of peaks and the reduction in current at higher pH reflect the strong influence of proton-coupled electron transfer processes on the electrochemical behavior of AA, PA, and CA (Figure S5).

In addition, a progressive negative shift in the oxidation peak potentials was observed with increasing pH, suggesting the involvement of protons in the redox reactions. Figure S6c illustrates a linear relationship between the anodic peak potential (E _pa) and pH for all three analytes. The measured slope values of E _pa vs pH for AA, PA, and CA were approximately 29 mV/pH, which closely matched the theoretical Nernstian slope, indicating that equal numbers of protons and electrons participated in the electro-oxidation process, i.e., this electron transfer is accompanied by a simultaneous proton transfer reaction. These results, consistent with previous reports, indicate that the oxidation processes of AA, PA, and CA proceed via a two-proton, two-electron mechanism (Figure S5). The negative shift in peak potential with increasing pH provides further evidence that the electrochemical reactions are governed by a proton-coupled electron transfer (PCET) mechanism, highlighting the involvement of both protons and electrons in the redox process. These observations, consistent with previous reports, indicate that the oxidation of AA, PA, and CA involves the transfer of two electrons and two protons (see Figure S5). Based on considerations such as solubility, peak separation, current response, and detection sensitivity, pH 7 was selected as the optimal condition for the simultaneous electrochemical detection of AA, PA, and CA.

3.14. Effects of Scan Rate

The effect of scan rate on the electrochemical behavior of 5.0 mM ascorbic acid (AA), 5.0 mM paracetamol (PA), and 5.0 mM citric acid (CA), both individually and simultaneously, was investigated at the APA-modified WB electrode using cyclic voltammetry in phosphate buffer solution (pH 7.0) at various scan rates (Figure a–d). The anodic peak currents (I _pa) for AA, PA, and CA, whether analyzed individually or in combination, exhibited a linear relationship with the square root of the scan rate (v ^1/2), as shown in Figure e–h. However, the plots did not pass through the origin, indicating that the electro-oxidation processes were predominantly diffusion-controlled with possible involvement of additional chemical steps or complications. The linear regression equations for AA, PA, and CA individually were I _pa(AA) = 253 ν^1/2 + 28.816 (R ² = 0.9913); I _pa(PA) = 587.07 ν^1/2 + 3.0356 (R ² = 0.9985); I _pa(CA) = 1167.3 ν^1/2 – 27.313 (R ² = 0.9908), respectively. For the simultaneous determination of AA, PA, and CA, the regression equations were I _pa(AA) = 404.13 ν^1/2 + 23.502 (R ² = 0.9960), I _pa(PA) = 652.39 ν^1/2 + 11.755 (R ² = 0.9944), and I _pa(CA) = 1700.6 ν^1/2 – 163.57 (R ² = 0.9953).

8.

CV of an APA-modified WB electrode in buffer solution of pH 7 containing (a) 5 mM AA, (b) 5 mM PA, (c) 5 mM CA, and (d) 5 mM AA + 5 mM PA + 5 mM CA with different scan rates ranging from (a–c) 50 to 400 mV/s and (d) 50 to 250 mV/s. (e–h) The linear relationship of the corresponding peak current vs sq. root of scan rate (ν^1/2).

3.15. Surface Area Study

The effective surface areas of both the bare WB electrode and the APA-modified WB electrode were measured using cyclic voltammetry of potassium ferrocyanide at various scan rates and applying the Randles–Sevcik equation for the analysis.

$$I_{\mathrm{p}}=(2.69\times 105)n^{3/2}A\times C^{*}\times D^{1/2}\times {\upsilon }^{1/2}$$

where I _p refers to the anodic peak current, n is the total number of electron transferred (n = 1), A is the effective surface area of the electrode, D is the diffusion coefficient for K₄[Fe­(CN)₆] = 7.6 × 10^–6 cm² s^–1, C* is the concentration of K₄[Fe­(CN)₆], and υ is the scan rate.

The CVs of 2 mM potassium ferrocyanide at the APA-modified WB electrode were recorded at different scan rates, and the corresponding anodic and cathodic peak currents were plotted against the square root of the scan rate, as illustrated in Figure S7.

The surface areas of the bare WB electrode and the APA-modified WB electrode were measured to be 0.11 cm² and 0.22 cm², respectively. The larger surface area of the APA-modified WB electrode compared with the bare WB electrode suggests that the APA acts as an efficient modifier. It creates a thin polymer layer on the electrode surface, facilitating enhanced electron transfer between the electrode and the solution (Figure ).

The APA-WB electrode significantly enhances the selectivity and, consequently, the simultaneous determination of AA, PA, and CA due to its unique structure and properties. The electroactive surface area of the APA-WB was calculated to be 0.22 cm², over 2 orders of magnitude greater than that of the bare WB electrode. This substantial increase in the electroactive surface area indicates that the APA-WB electrode is promising for electrocatalytic oxidation and electrochemical sensing applications.

3.16. Chronoamperometric Analysis

Chronoamperometric measurements were performed for AA, PA, and CA at the APA-modified WB electrode to evaluate their diffusion-controlled behavior. Upon application of a potential step, the current exhibited a rapid initial decrease followed by a gradual decay with time, which is characteristic of a diffusion-controlled electrochemical process (Figure S8a). The corresponding plots of current (I) versus the square root of the inverse of time (t ^–1/2) showed good linearity (Figure S8b), confirming that diffusion is the dominant mass transport mechanism at the electrode surface. The diffusion coefficients (D) were calculated from the slopes (S) of the linear I vs t ^–1/2 plots using the Cottrell equation,

$$I(t)=\frac{n\mathrm{F}\mathrm{A}{\mathrm{C}}_0\sqrt{D}}{\sqrt{\pi t}}$$

The slope (S) of the line is $S=n\mathrm{F}\mathrm{A}{\mathrm{C}}_0\sqrt{\frac{D}{\pi }}$

$$D={\left(\frac{S}{n\mathrm{F}\mathrm{A}{\mathrm{C}}_0}\right)}^2\pi$$

The obtained D values were 7 × 10^–5 cm² s^–1 for AA, 1 × 10^–5 cm² s^–1 for PA, and 9 × 10^–5 cm² s^–1 for CA, indicating efficient diffusion of these analytes at the modified electrode interface. The relatively higher D values for AA and CA suggest faster diffusion kinetics compared with PA, likely due to differences in their molecular sizes and interactions with the electrode surface.

3.17. Binary Systems

3.17.1. Simultaneous Estimation of AA and PA in PBS at an APA-Modified WB Electrode

Figure a displays the DPV responses for simultaneous determination of AA and PA in a buffer solution of pH 7, with concentrations ranging from 1 to 5 mM. The peak currents of AA and PA in the buffer solution increased linearly with their concentrations. The linear regression equations for AA and PA are Ip (AA) = 7.807_AA + 6.0748 (R ² = 0.9946) and Ip (PA) = 12.715_PA + 23.063 (R ² = 0.9848), respectively (Figure d). The average current increases for AA and PA are 7.8 and 12.96 μA, respectively. The sensitivity of AA and PA are 52.0 μA/mM/cm² and 93.1 μA/mM/cm², respectively. The LOD was estimated using a signal-to-noise ratio of 3. The LOD values of AA and PA are 0.565 μM (±0.06 μM) and 0.411 μM (±0.04 μM), respectively.

9.

DPVs of simultaneous change of (a) AA + PA (1–5 mM), (b) PA + CA (1–5 mM), and (c) AA + CA (1–5 mM); the calibration curve for simultaneous determination of (d) AA (red markers) and PA (blue markers), (e) PA (blue markers) and CA (red markers), (f) AA (green markers) and CA (red markers) in a binary mixture at an APA-modified WB electrode in PBS (pH 7) at a scan rate of 0.1 V/s.

3.17.2. Simultaneous Estimation of PA and CA in PBS at an APA-Modified WB Electrode

Figure b presents the DPVs for simultaneous determination of paracetamol (PA) and caffeine (CA) in buffer solution of pH 7, with concentration ranging from 1 to 5 mM. Peak currents of PA and CA in PBS were increased linearly with the increase of their concentrations. The linear regression equations for PA and CA are Ip (PA) = 15.991_PA + 36.175 (R ² = 0.9985) and Ip (CA) = 13.208_CA + 52.654 (R ² = 0.9977) (Figure e), respectively. The average current increases for PA and CA are 16.05 and 13.08 μA, respectively. The sensitivity was calculated for the binary mixture of PA and CA. The sensitivities of PA and CA were found to be 106.9 μA/mM/cm² and 128.1 μA/mM/cm², respectively. The LOD was determined based on a signal-to-noise ratio of 3 (S/N = 3). The LOD values for PA and CA were 0.370 μM (±0.02 μM) and 0.673 μM (±0.04 μM), respectively.

3.17.3. Simultaneous Estimation of AA and CA in PBS at an APA-Modified WB Electrode

Figure c presents the DPVs for simultaneous determination of AA and CA in buffer solution of pH 7, with concentration ranging from 1 to 5 mM. Peak currents of AA and CA were increased linearly with the increase of their concentrations. The linear regression equations for AA and CA were Ip (AA) = 9.0902_AA + 1.5193 (R ² = 0.9996) and Ip (CA) = 25.308_CA + 40.719 (R ² = 0.9981) (Figure f). The average current increases for AA and CA were 9.04 μA and 25.83 μA, respectively. The sensitivity was calculated for the binary mixture of AA and CA. The sensitivities of AA and CA were 60.2 μA/mM/cm² and 158.6 μA/mM/cm², respectively. LOD was calculated by signal-to-noise ratio (S/N = 3). The LoD of AA and CA were 0.394 μM (±0.02 μM) and 0.750 μM (±0.06 μM), respectively.

3.18. Ternary Systems: Quantitative Determination of AA, PA, and CA in the Ternary Mixture at an APA-Modified WB Electrode

3.18.1. Quantitative Estimation of AA at Constant PA + CA, PA at Constant AA + CA, CA at Constant AA + PA Concentration in PBS at an APA-Modified WB Electrode

To investigate the individual contributions of AA, PA, and CA in a ternary mixture, DPV measurements were performed under three experimental conditions: (i) varying the concentration of one species while maintaining constant concentrations of the other two; (ii) varying the concentrations of two species while keeping the third constant; and (iii) simultaneously varying the concentrations of all three species. These approaches enabled a comprehensive evaluation of the selectivity and interaction effects among the three analytes at different concentration levels.

Figure a–c shows the DPVS of the concentration of the target was varied from 1 to 5 mM, while the concentrations of the other two species were kept constant at 5 mM. Under the optimal conditions, calibration plots for the simultaneous determination of AA, PM and CA at the APA-modified electrode were obtained by DPV. Figure a demonstrates a linear increase in oxidation peak current with increasing AA concentrations (1 to 5 mM), while the concentrations of PA and CA remain constant at 5 mM. Conversely, Figure b shows a linear increase in peak current for PA as its concentration increases from 1 to 5 mM, while AA and CA are kept at 5 mM, and Figure c exhibits a similar linear increase for CA with increasing concentration, as AA and PA remain at 5 mM. The linear regression equations of AA, PA, and CA were I _p = 9.8344 C _AA + 4.5448 (R ² = 0.9979), I _p = 16.298 C _PA + 8.2454 (R ² = 0.9982) and I _p = 14.87 C _CA + 59.894 (R ² = 0.9967), respectively (Figure d–f).

10.

Concentration of DPV of (a) AA was varied from 1 mM to 5 mM, while the concentrations of PA and CA were kept constant at 5 mM; (b) PA was varied from 1 mM to 5 mM, while the concentrations of AA and CA were kept constant at 5 mM; and (c) CA was varied from 1 mM to 5 mM, while the concentrations of AA and PA were kept constant at 5 mM. The concentration of (d) AA, (e) PA and (f) CA were varied linearly within the range of 1 to 5 mM while the concentration other two species were kept constant in a ternary mixture at the APA-modified WB electrode in buffer solution of pH 7 at a scan rate of 0.1 V/s.

3.18.2. Quantitative estimation of AA + PA at Constant CA; PA + CA at Constant AA; CA + AA at Constant PA Concentration in PBS at an APA-Modified WB Electrode

For the determination of the target species simultaneously, we varied the concentrations of the two target compounds in the range of 1–5 mM, keeping the concentrations of the other species at a constant 5 mM (Figure a–c). The concentration of CA was kept constant while the concentrations of AA and PA were varied within the range 1–5 mM (Figure a). Figure d shows the calibration curve for different concentrations of AA and PA while CA was kept constant in a PBS solution. The linear regression equations of AA and PA are I _p (AA) = 8.9262_AA + 2.8865 (R ² = 0.9974) and I _p (PA) = 15.582_PA + 11.807 (R ² = 0.995), respectively.

11.

DPVs of the concentration of (a) AA and PA was varied from 1 mM to 5 mM, while the concentrations of CA were kept constant at 5 mM; (b) PA and CA were varied from 1 mM to 5 mM, while the concentrations of AA were kept constant at 5 mM; (c) CA and AA were varied from 1 mM to 5 mM, while the concentrations of PA were kept at 5 mM. The concentration of (a) AA + PA, (b) PA + CA and (c) CA + AA were varied linearly within the range of 1 to 5 mM while the concentration other species were kept constant in a ternary mixture at an APA-modified WB electrode in buffer solution of pH 7 at a scan rate of 0.1 V/s.

The concentration of AA was kept constant while the concentrations of PA and CA were varied within the range of 1–5 mM (Figure b). Figure e shows the calibration curve for different concentrations of PA and CA while AA was kept constant in the PBS solution. The linear regression equations of PA and CA were I _p (PA) = 17.656_PA + 1.1519 (R ² = 0.9981) and I _p (CA) = 14.883_CA + 70.561 (R ² = 0.9918), respectively.

Similarly, the concentration of PA was kept constant, while the concentrations of CA and AA were varied in a range of 1–5 mM (Figure c). Figure f displays the calibration curve for various concentrations of CA and AA, with PA held constant in a PBS solution. The linear regression equations of AA and CA were I _p (AA) = 10.961_AA + 1.6947 (R ² = 0.9991) and I _p (CA) = 22.86_CA + 35.044 (R ² = 0.9961), respectively.

3.18.3. Simultaneous Determination of AA + PA + CA in PBS at an APA-Modified WB Electrode

Figure a illustrates the simultaneous analysis of AA, PA, and CA using an APA-modified WB electrode in a phosphate buffer solution at pH 7. As the concentrations of AA, PA, and CA increased from 1 to 5 mM, the peak currents exhibited a linear increase, as shown in Figure b. A robust linear relationship between the oxidation peak current and the concentrations of individual analytes in the mixture was observed, as described by the equations. The linear regression equations for AA, PA, and CA were I _p = 8.0478 C _AA + 14.87 (R ² = 0.9975), I _p = 14.813 C _PA + 36.666 (R ² = 0.9972), and I _p = 22.269 C _CA + 41.147 (R ² = 0.9922), respectively (Figure b).

12.

DPVs of the concentration of (a) AA, PA, and CA were varied from 1 mM to 5 mM, concentrations; (b) the concentrations of AA, PA, and CA were varied linearly within the range of 1–5 mM.

The slope of the regression equations for the calibration graph used for the simultaneous detection of AA, PA, and CA is comparable to that obtained when each compound is measured individually while keeping the concentrations of the other compounds constant. This suggests that they do not interfere with the determination of each other. The sensitivity of AA, PA, and CA were 54.4 μA/mM/cm², 99.9 μA/mM/cm², and 151.7 μA/mM/cm², and detection limits (S/N = 3) were 0.2 μM (±0.01 μM), 0.16 μM (±0.02 μM), and 0.33 μM (±0.03 μM), respectively.

At the APA-modified WB electrode, the selective and noninterfering detection of AA, PA, and CA arises from the distinct oxidation potentials of the three analytes and the specific surface interactions provided by the poly-APA layer. Each analyte (AA, PA, and CA) undergoes oxidation at a different potential due to differences in their molecular structures and functional groups. The poly-APA film contains negatively charged carboxylate and amine functional groups that can interact electrostatically or through hydrogen bonding with the analytes. These interactions help to preconcentrate each analyte near its respective oxidation potential region, leading to well-separated peaks in DPV. Furthermore, the surface of the APA-modified electrode facilitates electron transfer in a controlled and selective manner, minimizing cross-interference among the analytes. Since their oxidation potentials are sufficiently separated, the oxidation of one analyte does not affect the electron-transfer kinetics of the others. Therefore, when the concentration of one species changes, its corresponding peak current changes linearly, while the other two remain nearly constant. This ensures that each analyte can be independently detected even in the presence of the others, confirming the electrode’s excellent selectivity and anti-interference capability.

3.19. Repeatability and Stability of the Modified Electrode

Multiple measurements were conducted to assess the repeatability of the electrochemical responses of an APA-modified WB electrode for AA, PA, and CA in a phosphate-buffered solution at pH 7.0. The results from eight consecutive measurements revealed relative standard deviations of 3.65% for AA, 3.32% for PA, and 2.92% for CA, demonstrating that the APA-modified WB electrode exhibits excellent repeatability. The long-term stability of the electrode was also tested by measuring its response to 3.0 mM concentrations of AA, PA, and CA after it was stored in a buffer solution for 14 days. The electrode maintained approximately 96%, 95%, and 98% of its initial activity for AA, PA, and CA, respectively, indicating its high stability.

3.20. Interference Study

Figure S9 presents the DPV results for a ternary solution comprising AA, PA, and CA, along with various potential interfering substances such as aspirin, lysine, arginine, glycine, thiamine (vitamin B1), nicotinamide (vitamin B3), pyridoxine (PD, vitamin B6), glucose, Na⁺, Mg²⁺, Ca²⁺, SO₄ ^2–, and CO₃ ^2–. The voltammogram indicates that the peak potentials for AA (0.2 V), PM (0.45 V), and CA (1.42 V) remain almost unaffected by the presence of interfering substances. The peak positions of AA, PA, and CA remain unchanged, identical with those observed in the absence of interfering substances. In the presence of interfering species, no notable changes in the peak potentials were detected. The tolerance limit for the maximum concentration of interfering substances was established to induce a relative error of approximately ±4.0% in the simultaneous determination of AA, PA, and CA. The anodic peak currents for AA, PA, and CA at the APA-modified WB electrode remained largely consistent even after the electrode was stored at ambient temperature for 14 days.

Therefore, it can be concluded that these species did not have a significant impact on the measurement of AA, PA, and CA, indicating that the APA-modified WB electrode demonstrated good selectivity for detecting AA, PA, and CA.

3.21. Comparison of Electrochemical Data with Reported Literature Values

Table S1 summarizes the analytical performance of various modified electrodes previously reported for the simultaneous electrochemical detection of ascorbic acid (AA), paracetamol (PA), and caffeic acid (CA), highlighting key parameters such as electrode materials, detection limits, pH conditions, and oxidation potentials. ,− While numerous studies have investigated individual or dual-analyte systems, reports on the concurrent determination of all three compounds remained limited. Its well-resolved oxidation peak potentials enable effective simultaneous detection with minimal signal overlap. Notably, the electrode functioned efficiently at a physiological pH (7.0), in contrast to many systems requiring acidic or alkaline conditions, thereby enhancing its suitability for real-sample analysis. Overall, the APA-modified WB electrode represents a promising platform for the selective and sensitive detection of AA, PA, and CA, with applicability in complex matrices such as biological fluids and pharmaceutical products.

3.22. Real Samples Analysis

The applicability of the proposed method was validated for the simultaneous quantification of AA, PA, and CA in soft drinks sample 1 (coca cola) using DPV under optimized experimental conditions. The analytical results are presented in Table . Prior to analysis, soft drink samples were diluted with the supporting phosphate buffer electrolyte (pH 7.0) and spiked with known concentrations of AA, PA, and CA. The corresponding oxidation peaks were observed at potentials nearly identical to those obtained from standard solutions, confirming the reliability and selectivity of the APA-modified WB electrode in complex biological matrices. The calculated recovery values ranged from 98% to 104%, indicating high accuracy of the method, while RSDs ranged from 2.7% to 3.1%, demonstrating good precision. These results suggested that the presence of common endogenous substances in soft drink samples did not significantly interfere with the electrochemical detection of the target analytes. In addition to soft drinks sample analysis, the method showed the potential for broader applications in pharmaceutical quality control and food safety, where simultaneous detection of these analytes may be required.

1. Recovery Test for AA, PA, and CA in Soft Drinks Coca Cola Samples (n = 5) Using an APA-Modified WB Electrode.

sample	analyte	added (μM)	found (μM)	recovery (%)	RSD (%)
1	AA	0.0	-	-	-
		15.0	15.4	102.6	3.1
		20.0	20.3	101.5	2.9
		25.0	25.2	100.7	2.7
		30.0	30.6	102.0	3.1
		35.0	34.8	99.4	2.8
	PA	0.0	-	-	-
		15.0	14.74	98.3	3.2
		20.0	20.40	102.0	3.1
		25.0	25.20	100.8	2.8
		30.0	30.13	100.4	2.6
		35.0	34.67	99.1	2.9
	CA	0.0	-	-	-
		4.0	4.1	102.5	3.2
		6.0	6.1	101.7	2.9
		8.0	7.9	98.1	3.2
		10.0	9.2	99.2	2.8
		12.0	11.8	98.3	3.1

3.23. Determination of AA and PA in Standard and Tablet Samples Using an APA-Modified WB Electrode Sensor

The DPVs obtained for varying concentrations of standard AA and PA in phosphate buffer solution (pH 7.0) are shown in Figures a and a, respectively. Figures b and b present the corresponding calibration curves, which illustrated a linear relationship between the oxidation peak current and the concentration of AA or PA. Standard solutions of AA and PA were analyzed in five replicate measurements, yielding a standard deviation of ±0.4, which demonstrated the excellent repeatability and measurement consistency of the APA-modified WB electrode.

13.

(a) DPVs of different amounts of standard AA in a 30 mL phosphate buffer solution (pH 7) at an APA-modified WB electrode. (b) the calibration curve for AA; (c) DPVs of AA in Nutrivit C (b) (gold line); Cecon (c) (light blue line); Cevit (a) (black line); Vasco (e) (green line); Ascobex (d) (red line) tablets in the same condition.

14.

DPV of (a) different amounts of standard PA in a 30 mL phosphate buffer solution (pH 7) at an APA-modified WB electrode. (b) The calibration curve for PA; (c) DPVs of PA in Napa (d) (green line); Ace (a) (blue line); Xpa XR (f) (violet line); Renova (e) (light blue line); Parapyrol (c) (black line); Longpara (b) (red line) tablets in the same condition.

For the analysis of commercial tablet samples, one-twentieth of each AA or PA tablet was dissolved in 30 mL of phosphate buffer solution. The resulting DPVs of the AA- and PA-containing tablet samples were recorded and are shown in Figures c and c, respectively. The linear regression equation for AA, obtained from the calibration curve (Figure b), was I _p = 1.1349x + 1.3915, where I _p denoted the oxidation peak current and x represented the AA concentration. Similarly, the regression equation for PA, derived from the calibration data (Figure b), was I _p = 2.4768x + 121.43. The quantified amounts of AA and PA in various commercial tablet samples are summarized in Table .

2. Results for AA and PA Determination in Pharmaceutical Samples Obtained under the Optimum Conditions (n = 5).

			methods
tablets	analytes	amount labeled (mg)	DPV of APA-modified WB electrode (mg)	UV–visible (mg)
Cevit	AA	250.0	249.4 ± 0.2	249.5 ± 0.3
Nutrivit C		250.0	247.6 ± 0.3	247.7 ± 0.6
Ascobex		250.0	251.1 ± 0.3	250.8 ± 0.4
Cecon		250.0	248.4 ± 0.4	248.9 ± 0.3
Vasco		250.0	248.0 ± 0.3	248.9 ± 0.2
Xpa XR	PA	665.0	663.8 ± 0.2	662.8 ± 0.7
Ace		500.0	498.6 ± 0.3	497.4 ± 0.5
Napa		500.0	499.0 ± 0.6	499.1 ± 0.4
Renova		500.0	498.2 ± 0.2	498.1 ± 0.3
Parapyrol		500.0	499.0 ± 0.3	499.6 ± 0.2
Longpara		665.0	664.8 ± 0.3	664.6 ± 0.3

3.24. Simultaneous Quantitative Determination of PA and CA in Standard and Combined Tablet Samples Using an APA-Modified WB Electrode

The DPVs recorded for various concentrations of standard PA and CA in 50 mL of phosphate buffer solution (pH 7.0) are shown in Figure a. Figure b,c displays the calibration curves for PA and CA, demonstrating a linear relationship between the oxidation peak current and the respective analyte concentrations. The regression equation for PA is I _pa = 1.902pa + 84.993 (R ² = 0.9899) and of CA is I _ca = 2.0615ca + 18.377 (R ² = 0.9929), where I _pa and I _ca denoted the peak currents, and pa and ca represented the concentrations of PA and CA, respectively. These equations were used to estimate the unknown concentrations of PA and CA in pharmaceutical tablet samples.

15.

(a) DPVs of different amount of standard PA + CA in 50 mL phosphate buffer solution (pH 7) at an APA-modified WB electrode. (b,c) shows the calibration curve for PA and CA. (d) DPVs of PA + CA in Fast plus (b) (blue line); Napa extra (a) (red line); Ace plus (c) (black line); Reset plus (d) (gold line); Feva Plus (e) (light blue line); Tamen X (f) (green line); Nor Plus (g) (dark line) tablets in PBS at an APA-modified WB electrode.

For each analysis, one-fifteenth of a PA + CA tablet was dissolved in 50 mL of phosphate buffer solution (pH 7.0). The DPVs obtained from these tablet samples are presented in Figure d. The inset of Figure b,c includes the corresponding calibration plots used for quantification. Table summarizes the determined concentrations of PA and CA in different commercial PA + CA combination tablets from various pharmaceutical manufacturers, demonstrating the applicability of the method for real-sample analysis.

3. Results for PA and CA in Tablet Samples of Different Pharmaceutical Companies Using an APA-Modified WB Electrode by DPV under the Optimum Conditions (n = 5).

tablet name	analyte	labeled value	PA + CAfound (mg)
Ace Plus	PA	500	497.3 ± 0.3
	CA	65	63.9 ± 0.2
Feva Plus	PA	500	502.3 ± 0.5
	CA	65	62.5 ± 0.4
Fast Plus	PA	500	502.7 ± 0.4
	CA	65	62.8 ± 0.3
Napa Extra	PA	500	501.1 ± 0.4
	CA	65	64.4 ± 0.5
Reset Plus	PA	500	499.7 ± 0.6
	CA	65	65.2 ± 0.2
Nor Plus	PA	500	497.5 ± 0.5
	CA	65	62.3 ± 0.3
Tamen X	PA	500	471.9
	CA	65	47.6

The data presented in Tables and indicate that the results obtained using the APA-modified WB electrode were in good agreement with the labeled contents of AA, PA and concurrent PA and CA in the commercial tablets. This demonstrated the accuracy, reliability, and robustness of the proposed electrochemical method. The relative standard deviation (RSD) values were within acceptable limits, confirming the repeatability of the measurements. High recovery values further validated the method’s precision and suitability for real-sample analysis. The minimal matrix effects observed in the tablet samples suggested that common excipients did not interfere with the simultaneous detection of PA and CA. These findings underscored the potential utility of the APA-modified WB electrode in routine pharmaceutical quality control, offering a simple and rapid approach for the simultaneous determination of AA, PA, and CA in complex commercial formulations.

The LODs obtained for the APA-modified WB electrode, 0.20 μM for AA, 0.16 μM for PA, and 0.33 μM for CA, are comparable to or better than many reported systems listed in Table S1. For example, the LODs achieved here are lower than those reported for GCE/Bi–AgNPs (0.697, 0.155, 2.36 μM) and SWCNT/CCE (3.0, 0.12 μM) and close to high-performance systems such as CuO–graphene nanocomposite and ZnO–Zn₂SnO₄–SnO₂/Gr/CPE. This demonstrates that the APA-modified WBE provides high analytical sensitivity despite being derived from a waste material source, emphasizing its potential as a sustainable and cost-effective electrode.

In terms of real sample relevance, typical concentrations of AA, PA, and CA in pharmaceutical formulations and beverages range from ∼10–1000 μM, ∼50–1500 μM, and ∼10–500 μM, respectively. The low LOD values obtained in this work are thus well below the expected real sample levels, ensuring reliable detection in practical applications.

With respect to regulatory standards, the achieved LODs are sufficiently lower than the pharmacologically active concentration limits and toxicity thresholds (e.g., paracetamol ≤4000 mg/day, caffeine ≤400 mg/day, ascorbic acid ≤2000), confirming that the electrode can accurately detect trace and therapeutic levels. −

Moreover, DPV measurements exhibited superior resolution and sensitivity compared with CV, allowing distinct and well-separated oxidation peaks for AA, PA, and CA, thereby ensuring selective and interference-free quantification.

3.25. Cost–Benefit Analysis

Commercially available electrodes such as glassy carbon electrodes (USD 165-235), platinum electrodes (USD 212-283), and gold electrodes (USD 200-260) are commonly employed in electrochemical sensing but remain prohibitively expensive and are not readily accessible in the local market in Bangladesh. In contrast, WB electrodes offer a highly cost-effective alternative. These electrodes are readily available and locally sourced and require minimal processing, making them highly suitable for widespread use. Furthermore, modification of WB electrodes with aspartic acid (APA) is simple, rapid, and low-cost, as APA is an inexpensive and widely accessible amino acid. The surface modification procedure does not require sophisticated instrumentation or hazardous reagents, contributing to its practicality and environmental friendliness. The affordability, ease of fabrication, and comparable analytical performance of APA-modified WB electrodes highlight their strong potential for use in decentralized laboratories and educational or clinical settings.

3.26. Determination of AA and PA in Tablet Samples Using UV–Vis Spectroscopy

For the quantification of vitamin C (AA), one-sixth of each tablet was dissolved in 100 mL of distilled water. UV–vis spectra were recorded for various concentrations of AA using water as the solvent, as shown in Figure S10a. The calibration curve (inset, Figure S10a) exhibited a linear relationship between absorbance and concentration, described by the regression equation:

A = 0.0403x + 0.3129 (R ² = 0.9928), where A represents absorbance and x is the concentration of AA. This equation was employed to determine AA content in commercial tablet samples. Similarly, for paracetamol (PA), one-fifth of each tablet was dissolved in 100 mL of water. The corresponding UV–vis spectra and calibration curve are shown in Figure S10b. The linear regression equation was: A = 0.0265x + 0.0308 (R ² = 0.9934), used to calculate PA concentrations in real samples.

Quantitative results for both AA and PA in commercial tablets, along with standard deviations (n = 5), are summarized in Table . The values obtained using the UV–vis method were in close agreement with those derived from the APA-modified WB electrode method. This consistency demonstrates the reliability and accuracy of the APA-modified WB sensor for the simultaneous and individual determination of AA and PA in pharmaceutical formulations.

Table summarizes the concentrations of PA and CA determined in commercial tablet samples using the APA-modified WB electrode. The measured values closely matched the labeled contents, confirming the accuracy of the proposed method. In contrast, the UV–vis spectra of PA + CA mixtures exhibited a broad, overlapping absorbance peak with a shoulder (Figure S11), complicating the distinct identification and quantification of each analyte. As shown in Figure S11, while individual spectra of AA, PA, and CA could be clearly recorded, simultaneous detection of all three components in a mixture was not feasible using the UV–vis technique due to spectral overlap.

The APA-modified WB electrode provided a significant advantage by enabling the simultaneous electrochemical detection of AA, PA, and CA, each producing well-resolved oxidation peaks at distinct potentials. This facilitated the selective and quantitative determination of the target analytes in complex pharmaceutical formulations. The results highlight the electrode’s utility as a sensitive, selective, and cost-effective platform for routine quality control of multicomponent preparations.

4. Conclusion

In this work, a simple, cost-effective, and environmentally friendly electrochemical sensor was developed by modifying WB electrodes with aspartic acid (APA) via a straightforward electrochemical approach. The fabricated APA-modified WB electrode demonstrated excellent electrochemical properties, outperforming other APA-modified electrodes, such as APA-GC, APA-Pt, APA-PG, and APA-Au in terms of sensitivity and detection efficiency for the simultaneous determination of ascorbic acid (AA), paracetamol (PA), and caffeine (CA). The sensor exhibited remarkable analytical performance in phosphate buffer solution (pH 7.0) with distinct and well-separated voltammetric signals for the three target analytes. The measured sensitivities were 54.4 μA mM^–1 cm^–2 for AA, 99.9 μA mM^–1 cm^–2 for PA, and 151.7 μA mM^–1 cm^–2 for CA, accompanied by low detection limits of 0.20, 0.15, and 0.33 μmol L^–1, respectively. These values reflect the electrode’s rapid electron transfer kinetics, enhanced surface activity, and high selectivity. Importantly, interference studies confirmed that common biological molecules and inorganic ions did not compromise the accuracy of detection, underscoring the sensor’s robustness and specificity. The practical applicability of the APA-WB sensor was demonstrated through the successful quantification of AA, PA, and CA in a variety of commercial pharmaceutical tablets obtained from Bangladeshi industries. The results showed strong agreement with those obtained from UV–vis spectroscopic analyses, validating the method’s reliability and accuracy. The APA-modified WB electrode presents a sustainable and cost-effective alternative to conventional electrodes, offering ecofriendly fabrication alongside excellent analytical performance. With these advantages, great promise is shown for applications in pharmaceutical quality control, clinical diagnostics, and point-of-care testing, especially in resource-constrained environments.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c08563.

• Cyclic voltammogram of 5 mM Aspartic acid) thin film growth by 30 cycles on the surface of a bare WB electrode at a scan rate of 0.1 V/s in PBS solution (pH 7); FTIR of Aspartic acid and poly aspartic acid (poly APA); poly APA extracted from an APA-modified WB electrode surface; EIS of a bare WB electrode and APA-modified WB electrode in the supporting electrolyte of 0.1 M KCl solution containing 2.0 mM potassium ferrocyanide; polymerization process of aspartic acid; reaction steps of Ascorbic acid; Paracetamol; Caffeine; CV of the ternary mixture of 5 mM AA, 5 mM PA, and 5 mM CA at an APA-modified WB electrode in pH 3, 5, 7, and 9 at a scan rate of 0.1 V/s; plot showing the peak current (μA) vs pH from Figure S6a; plot showing the Ep vs pH from Figure S6a; cyclic voltammograms of 2 mM potassium ferrocyanide on an APA-modified WB electrode at different scan rates of 0.05 V/s, 0.10 V/s, 0.15 V/s, 0.20 V/s, and 0.25 V/s in 1 M KCl as the supporting electrolyte; anodic and the cathodic peak currents of ferrocyanide vs square root of the scan rates; comparison of electrochemical data with reported literature values for the determination of AA, PA, and CA using WBE; chronoamperometric (I–t) responses for 5 mM AA, 5 mM PA, and 5 mM CA obtained by stepping the applied potential to 0.28 V (AA), 0.45 V (PA), and 1.45 V (CA), respectively; corresponding Cottrell plots (I vs t ^–1´²) for 5 mM AA, PA, and CA recorded in PBS (pH 7.0); DPV of the ternary solution of 2 mM of VC, PM, and CA in the presence of 2 mM of aspirin, lysine, arginine, glycine, thiamine (vitamin B1), nicotinamide (vitamin B3), pyridoxine (PD) (vitamin B6), glucose, Na⁺, Mg²⁺, Ca²⁺, SO₄ ^2–, and CO₃ ^2– in PBS (pH 7) at an APA-modified WB electrode; UV–visible spectra of the different concentration (10–50 ppm) of standard AA; UV–vis spectra of different concentrations (10–50 ppm) of standard PA in water; and UV–vis spectra of 20 ppm of AA, PA, CA, PA + CA, and VC + PA + CA in water (PDF)

M.A.M. conceptualized the study and developed its structural framework. M.H. performed most of the measurements and laboratory experiments. M.A.M. and M.A.H.M. supervised the work, carried out some laboratory experiments, data analysis, and contributed to writing the manuscript. M.N.U. and J.A. assisted with data analysis, edited the draft, and corrected the manuscript. All authors reviewed and approved the final version.

The authors declare no competing financial interest.