An Electrochemical Sensing Platform Based on a Carbon Paste Electrode Modified with a Graphene Oxide/TiO₂ Nanocomposite for Atenolol Determination ^†

Abstract

Atenolol is a medication belonging to the class of drugs known as beta-blockers, used to treat high blood pressure (hypertension) and irregular heartbeats (arrhythmia). Their presence in the environment has serious impacts on humans, animals, and the water ecosystem. In this context, the aim of this study was to develop a simple voltammetric method for the determination of atenolol (ATN) using carbon paste electrodes modified with the nanomaterials TiO₂ and rGO/TiO₂. The analytical performance of the modified sensor was evaluated using square wave voltammetry and cyclic voltammetry in 0.1 mol L⁻¹ acid sulfuric solution (H₂SO₄), pH 2. The nanocomposite electrode CPE/rGO/TiO₂ exhibited excellent electrocatalytic activity towards ATN oxidations at 0.1 mol L⁻¹ H₂SO₄ compared with unmodified carbon paste electrodes CPEs and those modified with titanium oxide, CPE/TiO₂. Different experimental and conditional parameters were optimized, such as supporting electrolytes, pH, amplitude, frequency, etc. Under optimal conditions, linear calibration curves were obtained, ranging from 1.7 to 23.2 µmol L⁻¹ for ATN with detection limits of 0.05 μmol L⁻¹. The modified nanocomposite CPE/rGO/TiO₂ sensor showed good sensitivity and good repeatability (RSD ≤ 0.61%) for ATN determination. The proposed sensor is mechanically robust and presented reproducible results and a long useful life. In order to verify the usefulness of the developed methods, the nanocomposite sensor CPE/rGO/TiO₂ was applied for the detection of atenolol in real samples (pharmaceutical tablets without any pre-treatment). The excipients present in the tablets did not interfere in the assay. Recoveries ranging from 97.7% to 106% were obtained. The results showed that the CPE/rGO/TiO₂ voltammetric sensor could be successfully applied in the routine quality control of ATN in complex matrices.

1. Introduction

The β-blocker drug atenolol (I), designated chemically as 4-(2-hydroxy-3-isopropylaminopropoxy) phenylacetamide, is a hydrophilic, β1-selective (cardioselective) adrenoceptor antagonist [1]. The tremendous increase in the use of antihypertensive medications such as beta-blockers points toward the increasing number of hypertension cases in the last decade. Atenolol is one of the most widely used β-blockers in the treatment of various cardiovascular disorders [2]. The use of nanomaterials for the determination of a variety of compounds is attracting attention in recent electroanalytical research. In order to modify and improve the sensitivity and selectivity of working electrodes, nanomaterials have been shown to be suitable materials [3]. Electroanalytical techniques can be easily adopted to solve many problems of fundamental importance with a high degree of accuracy, precision, sensitivity, and selectivity, often in a spectacularly reproducible way. In these techniques, the electrode surface itself is a powerful tool for the quantification of an analyte. By controlling the potential, the electrode can be used as a variable free energy source (or sink) of electrons. In addition, electrons crossing the electrode–solution interface can be determined with great sensitivity by measuring current. Voltammetry is considered an important electrochemical technique for electroanalytical chemistry because it has low cost, sensitivity, and precision, as well as accuracy, simplicity, and rapidity. Voltammetric techniques, such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), etc., have been proven to be very sensitive for the determination of organic molecules including drugs and related molecules in pharmaceutical dosage forms and biological fluids. Therefore, simpler and faster methods for the analyses of different receptor blocking agents are interesting for the quality of pharmaceutical formulations that contain them and also for therapeutic drug monitoring purposes. Despite their high use and application, little work has been conducted on the determination of cardiovascular drugs for electrochemical and related sensing devices [2,4,5,6]. In the present work, we introduced a highly sensitive electrochemical method, which is simple and selective. The determination of ATN has been reported on by utilizing carbon paste sensors nanomodified with nanoparticles TiO₂/CPE and reduced graphene oxide decorated with nanoparticles rGO/TiO₂/CPE. Two nanostructure modifiers, TiO₂ and rGO/TiO₂, are effective in composite sensors for β-blocker (ATN) detection because these materials are easy to prepare, have good biocompatibility, and can transfer electrons quickly, making them ideal for sensitive and selective ATN detection. In the pharmaceutical industry, electroanalysis is an effective analytical method that is becoming more useful [5,6,7,8,9,10]. Square wave voltammetry (SWV) is a technique that is supposed to be used with samples in order to investigate analytes in their pharmaceutical formulations.

2. Materials and Methods

2.1. Experimental Section

2.1.1. Reagents and Chemicals

All experimental materials and reagents were of analytical grade (Sigma and Merck, by Merck, KGaA Darmstadt, Germany). ATN was in powder form, pure standard form (series 00336). Double-distilled water was used to prepare each solution. Merck (99% Merck) provided the H₂SO₄ needed to create the supporting electrolyte. Synthetic graphite powder (90–71 μm particle size) was obtained from Alfa Aesar (99.9% Alfa Aesar, by Thermo Scientific Chemicals, MA, USA), and paraffin oil (Olio di Vaselina, by Humanitas, Milano, Italia) was supplied by Zeta Farmaceutici (Sandrigo VI, Italy). The TiO₂ synthetic powder was of analytical grade, 99%. Sodium chloride, sodium hydrogen phosphate, acetic acid, and all other salts were obtained from Aldrich, except for sodium hydroxide and hydrochloric acid, which were supplied by Merck. A freshly made stock solution containing 10 mmol L⁻¹ of ATN was diluted in 0.1 mol L⁻¹ of H₂SO₄ solution and refrigerated at 4 °C.

2.1.2. Apparatus

PalmSens4 (PalmSens, De Indruk, The Netherlands), potentiostat–galvanostat, connected via Bluetooth to a computer running three-electrode system software, was used for all electrochemical measurements. A platinum wire was the counter-electrode, silver–silver chloride Ag/AgCl/KCl (3M) was the reference electrode, and the CPE/rGO/TiO₂ and CPE/TiO₂ sensors were developed as working electrodes for this study. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were used to electrochemically detect ATN. SW voltammograms were first recorded in an electrochemical cell with 15 mL of 0.1 mol L⁻¹ H₂SO₄ serving as a supporting electrolyte and then by adding different concentrations of ATN beta-blockers at a potential between 1.0 and 1.7 V (vs SCE), with a frequency of 30 Hz, an amplitude of 50 mV, and a scan rate of 100 mVs⁻¹. Voltammetric measurements were performed at 26 ± 0.5 °C in an unstirred electrochemical cell with 15 mL of 0.1 mol L⁻¹ H₂SO₄ as the supporting electrolyte. Pulsed ultrasonic bath model: QC, capacity: 2.5 L, tank cleaning power: 375 w, cleaning frequency: 49 Khz, timer adjustment: 1–30 min, quiet operation, tank size internal: (mm) 240 × 130 × 100 deep (inches) 9.5 × 5 × 4 deep. A scheme of the ATN oxidation mechanism is shown in Figure 1.

2.2. Synthesis of Carbon-Based Modified Nanocomposite Sensors

2.2.1. Preparation of CPE/TiO₂ Sensor

CPE/TiO₂ was obtained by mixing 1.00 g of carbon powder, 0.100 g of TiO₂, and 300 µL of paraffin for 30 min to prepare a homogenous paste. Firstly, paraffin and graphite powder (particle size, 90–72 μm) were mixed, and a TiO₂ modifier was added and mixed with a mortar and pestle until a homogeneous paste was achieved. Before measurement, the produced composite material was stored for 24 h at 4 °C in a refrigerator. The produced paste was packed into a plastic syringe with an 8 mm inner diameter containing a 9.5 mm copper wire as the external electric contact. Preparing the unmodified CPE involved identical steps but without adding a modifier (TiO₂): 1.00 g of graphite powder and 300 µL of paraffin. Before the measurements, the electrode surface was smoothed on a glass surface. After removing a portion of the paste and polishing the glass surface, the electrode’s surface was renewed.

2.2.2. Preparation of CPE/rGO/TiO₂ Sensor

• Preparation of rGO/TiO₂ Composite: In total, 1.00 g of graphene oxide, 0.143 g of TiO₂, and 15 mL of distilled water were mixed to create the composite. After the paste was mixed, it remained in an ultrasonic bath for 4 h and was then centrifuged at 5000 RPM.
• CPE/rGO/TiO₂: The composite electrode material was synthesized by mixing 1.00 g of carbon powder with particle sizes ranging from 70 to 90 μm, 0.100 g of reduced graphene oxide (rGO) decorated with titanium dioxide (TiO₂), and 300 µL of paraffin. The mixture was thoroughly mixed for 30 min to obtain a uniform paste. First, the graphite powder and paraffin were mixed, and then the rGO/TiO₂ mixture modifier was added and mixed again with a mortar and pestle until a consistent paste was formed. The resulting paste was filled into a plastic syringe with an internal diameter of 8 mm and an exterior diameter of 9.5 mm. The syringe also contained a copper wire as the external electric contact.

3. Results and Discussion

3.1. Electrochemical Characterization of Nanocomposite Sensors

The electrochemical characterization of the nanocomposite sensor CPE/TiO₂ was carried out by cyclic voltammetry, using K₃Fe(CN)₆ as a redox probe in acetate buffer pH 4.9, from −0.5 to +0.5 V at scan rate 100 mV/s. Typical cyclic voltammograms registered with bare CPEs and modified electrodes are presented in Figure 2. It can be observed that the incorporation of TiO₂ nanoparticles in the composite CPE showed an improvement in the electrochemical response of CPE/TiO₂, manifested by an increase in the peak current and a well-defined peak (oxidation and reduction peak) compared to the CPE. This can be related to the increase in the active surface area due to the TiO₂ nanoparticles and its electrocatalytic effects [2,3,7].

The voltammetric behavior of the nanocomposite sensor CPE/TiO₂ was studied using K₃Fe(CN)₆ as a redox probe in cyclic voltammetry at a 100 mVs⁻¹ scan rate. The Randles–Sevcik equation was used to calculate the effective surface area [11].

I = (2.69 × 10⁵) ACD1/2 n 3/2 ʋ1/2

(1)

where the effective surface area of the electrode in cm² is represented by A; n is the number of electrons taking part in the charge transfer process; D is the diffusion coefficient of the analyte in the solution; and C is the K₃Fe(CN)₆ solution concentration. The values of n and D for K₃Fe(CN)₆ are 1 and 7.6 × 10⁻⁶ cm² s⁻¹, respectively. The surface areas of the electrodes were calculated using the Randles–Sevcik equation [12]; the calculated surface areas of the bare CPE and CPE/TiO₂ were 0.14 cm² and 0.32 cm², respectively (Figure 2), indicating that CPE/TiO₂ has the largest surface area and electroactive site for analyte detection.

3.2. Electrochemical Behavior of ATN

Our previous research demonstrated that using ilmenite natural nanomaterials incorporated as nanocomposites into CPEs resulted in a catalytic impact during the electro-oxidation of atenolol [2,3]. In the present study, we tested the effect of adding nanostructure modifiers such as TiO₂ and reduced graphene oxide decorated with TiO₂ nanoparticles to CPE composite sensors. We tested the peak intensity and potential values using an unmodified carbon paste electrode and a modified carbon paste (CPE/TiO₂ and CPE/rGO/TiO₂). The best modifier should provide a low background current, the highest current value, and a resolution. The square wave voltammetric method was used to investigate the oxidation behavior of ATN (930 µM). The voltammetric-modified sensors (CPE/TiO₂ and CPE/rGO/TiO₂) showed good electrocatalytic activity toward ATN electrochemical oxidation at a potential of 1.42 V in a 0.1 mol L⁻¹ H₂SO₄ solution (pH 2) compared with the bare electrode (CPE). The maximum current was observed with the CPE/rGO/TiO₂ sensor (Figure 3) [2,13,14,15].

The results show that a well-defined anodic peak and the highest signal were obtained using CPE/rGO/TiO₂, while for the other electrodes, a smaller anodic peak appeared.

3.3. Study of pH and Supporting Electrolyte

The influence of pH values on the oxidation peak current for ATN was investigated using the square wave technique, employing different electrolytes of pH 4.9–7.0 (Figure 4). In the ion exchange reactions, the response of the nanomodified sensor CPE/rGO/TiO₂ was strongly affected by the supporting electrolyte, pH, or ionic strength. Concretely, the effect of pH and the supporting electrolyte on the sensor response was tested in 1500 µM ATN solutions prepared with an acetate buffer (pH 4.5), a phosphate buffer (pH 7), and a 0.1 mol L⁻¹ H₂SO₄ solution (pH 2). Figure 4 and its inset show the effect of the electrolyte and the pH of the preconcentration solution on the anodic current response in detecting ATN, revealing that pH strongly influences the oxidation process. These pH values were investigated because ATN provides better electrochemical behavior at 0.1 mol L⁻¹ in H₂SO₄ (pH 2.0), as previously shown by N. Broli et al., 2022 [2,3,13]. The highest anodic peak current and a well-defined peak were observed at a pH of 2.0 in a sulfuric acid solution, and this was selected for further experiments.

3.4. Optimization of Experimental Parameters and Analytical Curves

We optimized the influence of SWV parameters like square wave frequency (f) and pulse amplitude (a) on the electro-oxidation reaction of 1500 µmol L⁻¹ ATN at CPE/TiO₂ in a 0.1 mol L⁻¹ H₂SO₄ solution with a pulse width of 30 mS (Figure 5). The ranges studied were 10–60 Hz for the square wave frequency and 10–100 mV for the pulse amplitude; the registered SW voltammograms are shown in Figure 5 A, B. The oxidation peak currents (Ipa) of ATN for the pulse amplitude ranging from 10 to 100 mV indicated that a well-defined peak and a greater peak current were obtained using a 50 mV pulse amplitude. The impact of frequency was also assessed within a range of 10 to 60 Hz. The peak current intensity was positively correlated with the increasing frequency, reaching a distinct maximum at 30 Hz. The optimal peak definition for ATN detection was achieved by utilizing a pulse amplitude of 50 mV and a frequency of 30 Hz.

3.5. Analytical Performance of Nanocomposite Sensors for ATN Determination

Square wave voltammetry was employed for the quantitative measurements of atenolol, owing to its higher sensitivity than cyclic voltammetry. Thus, SWV was employed to study the electrochemical response of the CPE/TiO₂ and CPE/rGO/TiO₂ nanocomposite sensors toward ATN. Under optimized conditions, the applicability of the proposed voltammetric sensors (CPE/TiO₂ and CPE/rGO/TiO₂) for ATN detection was examined by measuring the peak current as a function of the analyte (ATN) concentration. Figure 6 illustrates the SW voltammograms obtained with CPE/TiO₂ sensors for different concentrations of ATN in 0.1 mol L⁻¹ H₂SO₄ at a pH of 2.0.

Square wave voltammograms obtained with increasing amounts of ATN showed that the oxidation peak current increased linearly with increasing concentration, as shown in Figure 6. Under the optimal working conditions described above, linear calibration curves were obtained for ATN in a range of 1.7 to 23.2 µmol L⁻¹. The linear equation was Ip (µA) = 1.734C (µM) + 10.9 (R2 = 0.990). A deviation from linearity was observed for more concentrated solutions, owing to the adsorption of its oxidation product on the nanoelectrode surface. Related statistical data on the calibration curves were obtained from three different calibration curves. The limit of detection (LOD) was calculated using the equation LOD = 3 s/m, where s is the standard deviation of the peak currents of the blank (four runs), and m is the slope of the calibration curve resulting from 0.68 µM. The LOD sample solutions recorded after 48 h did not appreciably change the assay values. The electrochemical behavior of the nanocomposite sensor (CPE/rGO/TiO₂) was also evaluated for ATN detection in the same optimal conditions as the CPE/TiO₂ sensor. SW voltammograms obtained after successive ATN standard solution additions are shown in Figure 7.

Figure 7 shows that the height of this peak increases with increasing analyte concentrations. The calibration graph of the peak current versus concentration was constructed using data from these measurements, and the least squares were evaluated using the linear regression method. The oxidation peak currents exhibited a linear relationship with the ATN concentrations of 6.6 to 26.6 µM. The inserts in this figure depict the work zone and the analytical curve obtained for ATN (r = 0.9954), with a corresponding best-fit regression equation of Iap (µA) = 5.89C (µM) + 1.0.0 (R2 = 0.9954), where Iap is the anodic peak current, and C is the ATN concentration in mol L⁻¹, with an LOD of 0.05 µM. The

Table 1. The quantitative analytical results for the investigated sensors using the SWV technique.

Sensors	Dynamic Range (µM)	Correlation Coefficient R2	Sensitivity (µA/µM)	LOD (µM)	RSD (%)
CPE	36–587	0.998	0.3	12.72	5.0
CPE/TiO2	1.7–3185	0.990	1.7	0.8	1.0
CPE/rGO/TiO2	6.6–909	0.995	5.9	0.1	0.6

summarizes the analytical parameters of bare CPE and modified nanocomposite sensors, evaluated using the SWV technique in the 0.1 M H₂SO₄ solution (pH 2).

The results show that the proposed modified nanocomposite sensors are preferable for atenolol detection, where CPE/rGO/TiO₂ resulted in a higher sensitivity of 5.89 µA/µM. Moreover, these modified electrodes are simple to prepare and use, have a low cost, and have very high stability; thus, they can be alternatively used to detect ATN in pharmaceutical formulations.

3.6. Real Sample Analysis

The high-performance carbon paste electrode modified with rGO/TiO₂ was successfully applied to measure ATN in pharmaceutical preparations (100 mg per tablet) using SWV and CV. The ATN tablets were ground into powder, dissolved in H₂SO₄ 0.1 mol L⁻¹ solution, filtered, and then diluted so that the ATN concentration was in the working range. A standard addition method was applied to measure accuracy.

Table 2. ATN detection in pharmaceutical formulations using the proposed SWV and CV methods with CPE/rGO/TiO₂.

Sample ATN (mg Tablet)	Label Value	SWV ATN (mg Tablet)	RSD (%)	CV	RSD (%)
1	100.0	107	1.3	125.0	4.2
2	100.0	102.5	1.8	127.8	3.8
3	100.0	84.3	2.7	121.3	4.5

summarizes the data from the examined samples utilizing the functionalized CPE/rGO/TiO₂ nano-composite sensors. The results agreed well with the content marked on the label. The detected contents were 97.9 ± 6.0 mg and 124.7 ± 6.0 mg per tablet, respectively, for SWV and CV.

An ATN recovery test ranging from 65 µmol L⁻¹ to 130 μmol L⁻¹ was performed using square wave voltammetry. The recoveries in different samples ranged from 97.7% to 106%, with an RSD of 1.58%. These results indicate that the proposed method employing the developed nanosensors are suitable for practical applications.

4. Conclusions

In the current work, stable and durable modified carbon paste nanocomposite sensors based on CPE/TiO₂ and CPE/rGO/TiO₂ sensitively detected atenolol. We studied the electrochemical performance of the examined nanomodifier (using TiO₂ nanoparticles and graphene oxide functionalized with TiO₂) based on factors such as high electrical conductivity, high surface area, and good adsorptive characteristics. The CPE/rGO/TiO₂ nanocomposite carbon paste sensor showed improved ATN detection in terms of its linearity range, detection limit, mean recovery percentage, and precision. The nanocomposite sensors successfully detected ATN in the pharmaceutical tablets. The sensors showed good capabilities, and the analytical data show that they can quickly detect ATN in routine control analyses.