Highly Sensitive Detection of Phenylbutazone with Metallic Particle-Based Electrochemical Sensors

Abstract

Nonsteroidal anti-inflammatory drugs such as phenylbutazone (PBZ) are among the most widely used medications globally due to their effectiveness in relieving pain and reducing inflammation. This study aims to detect PBZ with metallic particle-based electrochemical sensors using cyclic voltammetry (CV) in the presence of catechol as a redox probe. The approach focuses on evaluating the electrochemical behaviour of PBZ under different experimental conditions and optimizing the detection parameters to develop a simple, rapid, and cost-effective analytical method suitable for this pharmaceutical compound in lab practice. CV was performed using four types of screen-printed electrodes, each modified with different transitional metal particles, in potassium ferrocyanide/potassium ferricyanide, catechol, and catechol-PBZ solutions to study the electrochemical response and detection capability for PBZ. The best performance characteristics were obtained for the sensor modified with Ir particles that detect PBZ, with a linearity range of 0.01 to 1.00 μM and a detection limit of 1.53 nM. Additionally, Fourier-transform infrared spectroscopy (FT-IR) was used to characterize the PBZ in pharmaceuticals. The method using an iridium-modified sensor developed in this study allows the accurate detection of PBZ in pharmaceuticals with a relative error lower than 4%.

1. Introduction

Phenylbutazone (PBZ) (Figure 1), first introduced in 1949, has been used in human medicine to manage acute and chronic inflammatory pain, especially in different forms of arthritis. It has been used as a first-line therapeutic option in the treatment of arthritis for three decades. PBZ is a nonsteroidal anti-inflammatory drug (NSAID). The first pharmaceutical product with PBZ had an initial recommended dosage of 400–600 mg/day, divided into multiple doses; later, the recommended dosage was reduced to 200–300 mg/day, with maximum recommended doses up to 1 g/day [1]. The half-life of PBZ ranges from 50 to 105 h, this variation depending on genetics, dose, and time of administration. After oral administration, it is largely absorbed into plasma and binds mainly to albumin, while 10% of the drug is converted to metabolites in the bile and approximately 1% of PBZ is eliminated renally [2]. The use of PBZ is associated with common adverse reactions such as skin rash, edema, nausea, and gastrointestinal problems (peptic ulcer, stomatitis), as well as severe hematological complications, including anemia, leukopenia, thrombocytopenia, and agranulocytosis [3,4]. The administration of high doses, especially those that reached or exceeded 1600 mg daily, was largely correlated with severe adverse effects. Such elevated doses were administered at the time, which led to a very high incidence of severe adverse reactions and highlighted the need for rigorous dose control and treatment monitoring [3,4]. PBZ began to be withdrawn from the market in the USA in the 1980s, but that did not stop the drug from being used in veterinary medicine, especially for dogs and horses [1]. Even so, the continued availability of PBZ in topical forms in different European countries (e.g., Romania) increases the risk of environmental leaching and accidental human exposure, requiring sensitive detection methods to monitor compliance with international health bans. Also, its use in veterinary medicine poses a persistent risk of accidental entry into the human food supply chain [5], and in the environment, where its stability and slow degradation lead to the contamination of soil and aquatic ecosystems, potentially affecting non-target species [6]. Considering that PBZ remains in use, despite significant adverse reactions, there is a need to develop an efficient method for its detection in pharmaceutical products and biological fluids.

In the literature, a variety of advanced techniques have been used for the detection of PBZ. Among the most common techniques are chromatographic techniques (thin-layer chromatography—TLC [7], liquid chromatography coupled with mass spectrometry, LC-MS/MS [8], high-performance liquid chromatography—HPLC [9,10,11], gas chromatography—GC [12], which is often coupled with mass spectrometry—GC-MS [13]) and spectrometric techniques (IR spectrometry [14], UV-Vis spectroscopy [15]). Usually, these classical methods have very good accuracy, but they are usually expensive, laborious, and time-consuming and involve complex sample preparation processes with relatively high consumption of reagents and solvents. An appropriate analytical approach to overcome these issues involves the use of electrochemical sensors, as they require minimal sample preparation and enable rapid and accurate analysis. Electrochemical techniques have emerged as valuable tools in drug analysis, largely due to their simplicity, rapid analysis time, and lower cost compared to other methods [16]. These techniques offer a viable alternative to other instrumental methods, demonstrating considerable efficiency in detecting PBZ in different types of samples. A series of electrochemical sensors for PBZ detection has been reported in the scientific literature (

Table 1. Main sensors used in the detection of PBZ from the literature.

Sensor/Sensitive Material	Method	Linearity Range (μM)	LOD (nM)
MWCNT/CPE [17]	CV, LSV, SWV	0.1–10	50
CTN-Fe3O4/g-C3N4@GCE [18]	CV, SWV	0.6–10	1.28
SPGE [16]	DPV	0.081–32.43	32
Ir/CSPE—this work	CV	0.01 to 1.75	1.53

MWCNT/CPE = multi-walled carbon-nanotube-modified paste electrode; CTN-Fe₃O₄/g-C₃N₄@GCE = glassy carbon electrode modified with Citretten-decorated Fe₃O₄ nanoparticles and graphitic carbon nitride (g-C₃N₄); SPGE = screen-printed graphite electrodes.

).

Although recently developed voltammetric sensors have achieved low detection limits for PBZ, most still face challenges related to their applicability in real samples, including matrix interferences and signal instability under variable experimental conditions. To address these limitations, this study introduces commercially available screen-printed electrodes (SPEs) modified with transition metal particles, used here for the first time to catalyze the response.

The primary objective is to develop a rapid and reliable electroanalytical method for the selective and sensitive determination of PBZ in complex matrices. SPEs are widely used due to their simple fabrication process [19], low production cost, ease of use, and excellent reproducibility.

Carbon-based screen-printed electrodes (CSPEs) are particularly versatile owing to the diverse carbon inks available for screen-printing and the numerous possibilities for surface modification [20]. Transitional metals such as rhodium, palladium, platinum, and iridium are successfully used for the manufacture of modified SPEs for the detection of various organic compounds due to their favourable physicochemical properties, such as high melting point, corrosion resistance, and excellent conductivity, but also due to their stability in electrochemical processes and high catalytic activity [21]. These features make metal particle-modified SPEs a feasible alternative to fabricate devices with high detection limits, high sensitivity, high electrode surface resistance, as well as high reproducibility [22,23,24,25,26], properties necessary for chemically modified electrochemical sensors to be an alternative to conventional analysis methods.

The approach proposed in this work was to detect PBZ involving a catechol-mediated electrooxidation process. Catechol can be oxidized electrochemically to o-quinone, and the o-quinone can participate in a coupling reaction with different nucleophilic compounds to form a C–C or C–O bond [27]. o-Quinone is a reactive intermediary compound, which acts as a Michael acceptor to react with several nucleophilic compounds [28,29]. This type of reaction could be taking place because PBZ is a nucleophilic compound [30]. The electrochemical process of the adduct formed between catechol and PBZ could be facilitated by the presence of metallic particles on the SPE surface [31,32]. The modalities of electrochemical process enhancement include increasing the active surface area, facilitating electron transfer, and catalytic effects [33,34,35]. The catechol was selected as a redox probe due to its susceptibility to oxidation, which is related to its antioxidant properties. The electro-oxidation process results in the formation of o-quinone, which is reactive and electron-deficient, and it can act as a reactive species in different chemical reactions with nucleophilic compounds such as cycloheptylamine, aniline, sulfanilic acid, phenyl-Meldrum’s acid, 2-thiobarbituric acid, proline, etc. [29].

The catechol exhibits rapid charge transfer kinetics; the redox process involves the transfer of two electrons and two protons, providing a robust signal, and it allows amplification of currents through redox cycling processes, providing increased sensitivity for analyte detection [36,37].

In this paper, a novel application of CSPEs modified with rhodium, palladium, platinum, or iridium particles for PBZ detection is presented. The novelty of this work also consists of the use of catechol as a redox probe, which facilitates the redox process of PBZ and increases the sensitivity. To the best of our knowledge, there are no studies on the electrochemical oxidation of catechol in the presence of PBZ.

2. Materials and Methods

All compounds used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA) and were of analytical grade. The 10⁻¹ M KCl solution, used in the preliminary studies, was prepared by dissolving a quantity of potassium chloride in ultrapure water. The 10⁻³ M potassium ferro/ferricyanide solution (5 × 10⁻⁴ M potassium ferrocyanide + 5 × 10⁻⁴ M potassium ferricyanide) and the 10⁻⁴ M catechol solution were prepared by dissolving substances in appropriate amounts in a 10⁻¹ M KCl solution. The same catechol–KCl solution was used as the supporting electrolyte in the studies for the detection of PBZ. For the preparation of the 10⁻⁴ M PBZ solution, the required (calculated) amount of pure substance was dissolved in 10⁻¹ M KCl—10⁻⁴ M catechol solution.

Electrochemical experiments were performed at 20 ± 2 °C with a Biologic Instruments SP 150 potentiostat/galvanostat (BioLogic Science Instruments, Seyssinet-Pariset, France), connected to the EC-LAB Express software Version 5.52. The experiments were performed in a 50 mL electrochemical cell (Princeton Applied Research, Oak Ridge, TN, USA) equipped with a three-electrode system: an Ag/AgCl reference electrode, a platinum counter electrode, and a screen-printed carbon working electrode modified with transitional metal particles (Ir, Pd, Pt, and Rh, used sequentially). The screen-printed carbon working electrodes used in this study are fabricated on a ceramic substrate and modified with rhodium, palladium, platinum, or iridium particles and were purchased from Metrohm DropSens (Llanera, Asturias, Spain). The K0265 Ag/AgCl Reference Electrode, Princeton Applied Research from AMETEK Scientific Instruments, was used as a reference electrode. K0266 Platinum Counter Electrode Assembly was used as a counter electrode (Princeton Applied Research from AMETEK Scientific Instruments, Oak Ridge, TN, USA).

The ultrapure water used was obtained using a Mili-Q Millipore water purification system (Bedford, MA, USA). Accurate weighing of substances was performed using an analytical scale, and an Elmasonic ultrasonic bath (Carl Roth GmbH, Karlsruhe, Germany) was used for dissolution of compounds and solution homogenization.

For the analysis of real samples, a quantity of cream containing phenylbutazone in identical concentrations (40 mg/g) was weighed. This was subjected to a sequential extraction in ethanol, using 1 mL of ethanol each time, repeating the extraction process five times. After extraction, the ethanol solution was dissolved in ultrapure water and then subjected to a sonication process for 30 min at 40 °C to facilitate dissolution. From the obtained stock solution, volumes of 50 µL were added to a volume of 50 mL of a 10⁻⁴ M catechol—10⁻¹ M KCl solution. This solution was used for the voltammetric determination of PBZ.

The electrochemical behaviour and analytical performance of four screen-printed carbon electrodes modified with iridium, palladium, platinum, and rhodium were studied: Ir/CSPE, Pd/CSPE, Pt/CSPE, and Rh/CSPE. All electrochemical measurements were performed using cyclic voltammetry as the detection technique, a versatile technique that allows the rapid detection of active compounds from various samples.

The pharmaceutical products analyzed for the validation of the method were creams from different producers. The composition of the pharmaceutical products is included in

Table 2. Excipients and active ingredients (the pharmaceutical products used, as specified by the manufacturer).

Pharmaceutical Product	Excipients According to the Leaflet
Antibiotic cream (PBZ 40 mg/g)	Camphor, menthol, emulsifying cetostearyl alcohol (type A), polysorbate 80, glycerol, white soft paraffin (white petrolatum), methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216), and purified water.
Fitterman cream (PBZ 40 mg/g)	White soft paraffin (white petrolatum), emulsifying cetostearyl alcohol (type A), glycerol, polysorbate 80, camphor, levomenthol, methyl parahydroxybenzoate (E218), n-propyl parahydroxybenzoate (E216), purified water, and ethyl alcohol 96% (ethanol).
Ducfarm cream (unspecified)	Carbopol, distilled water, diclofenac, menthol, camphor, benzocaine, and essential oils.

.

3. Results and Discussions

3.1. Preliminary Analyses

3.1.1. Preliminary Analysis in 10⁻¹ M KCl Solution

In the preliminary analysis, the behaviour of the sensors in 10⁻¹ M KCl solution was investigated. For each of the four screen-printed electrodes, Ir/CSPE, Pd/CSPE, Pt/CSPE, and Rh/CSPE, plus CSPE, cyclic voltammograms were recorded in the optimal potential range between −0.4 and 1.0 V at a scan rate of 0.05 V·s⁻¹. This study aimed to characterize the screen-printed electrodes by evaluating the stability and reproducibility of the signal in the supporting electrolyte solution. The analysis of the electrochemical response of each electrode allowed the identification of differences in the background current, thus providing a solid basis for further applications in the detection of electroactive organic compounds. Figure 2 presents the cyclic voltammograms of all sensors immersed in a 10⁻¹ M KCl solution.

The cyclic voltammograms of the sensors (Ir/CSPE, Pd/CSPE, and Rh/CSPE) recorded in 10⁻¹ M KCl solution did not show any redox peaks, as expected for an electrolyte lacking electroactive species. This behaviour confirms that the working electrode surfaces are free from impurities and that the materials used in the fabrication are of high purity and quality. The appearance of small anodic peaks in the case of Pt/CSPE could be related to the oxidation of oxygenated functional groups (i.e., carboxyl, hydroxyl) existent on the electrode surface, processes that are catalyzed by the Pt [38]. All tested electrodes showed relatively low background currents, indicating their suitability for voltammetric measurements.

3.1.2. Preliminary Study in Potassium Ferrocyanide/Ferricyanide 10⁻³ M–10⁻¹ M KCl Solution

Subsequently, measurements were performed in a 10⁻³ M potassium ferrocyanide/ferricyanide solution to evaluate the electrochemical response of the electrodes. In these preliminary studies, the experimental conditions were optimized to obtain stable signals with well-defined peaks and a low level of background current, thus ensuring the reproducibility and reliability of the measurements. The optimal parameters for cyclic voltammetry are the potential range between −0.4 and 1.0 V and the scan rate of 0.05 V·s⁻¹.

Figure 3 shows the cyclic voltammograms of all electrodes obtained in potassium ferrocyanide/ferricyanide solution under optimal conditions.

From the experimental data, information was obtained about the kinetics and reversibility of the redox process depending on the material used to modify the electrode. The results obtained are presented in

Table 3. Electrochemical parameters obtained from cyclic voltammograms of Ir/CSPE, Pd/CSPE, Pt/CSPE, Rh/CSPE, and CSPE immersed in 10⁻³ M potassium ferro-ferricyanide—10⁻¹ M KCl solution.

Electrode	Epa (V)	Epc (V)	E1/2 (V)	∆E (V)	Ipa (µA)	Ipc (µA)	Ipc/Ipa
Ir/CSPE	0.374	0.217	0.295	0.157	6.62	−6.63	1.00
Pd/CSPE	0.438	0.170	0.304	0.268	7.29	−6.97	0.96
Pt/CSPE	0.458	0.094	0.276	0.364	8.70	−5.22	0.60
Rh/CSPE	0.323	0.234	0.278	0.089	8.24	−7.77	0.94
CSPE	0.426	0.193	0.309	0.233	7.05	−6.15	0.87

E_pa, E_pc—anodic and cathodic peak potential, respectively; E_1/2—formal potential, calculated according to the equation E_1/2 = (E_pa + E_pc)/2; ∆E—potential difference between the anodic and cathodic peaks (E_pa − E_pc); I_pa, I_pc—currents corresponding to the anodic and cathodic peaks, respectively.

.

Relative standard deviations of potential values are 0.5%. Relative standard deviations of current values are 0.2%. Three electrodes were used for all the measurements to assess the variability of the measurements.

As shown in Table 3, all SPE have similar behaviour even when modified with metallic particles, with the I_pc/I_pa ratios close to the ideal value of 1, confirming the reversibility of the ferro-ferricyanide redox process at the sensor’s surfaces. The ∆E value is intermediate between the electrodes modified with Ir and Rh, with the lowest values, and the Pd and Pt electrodes, with higher ∆E values. The Rh/CSPE and Ir/CSPE sensors exhibited the lowest potential difference between the anodic peak and the cathodic peak (∆E) of 0.142 V and 0.162 V, respectively, indicating significantly faster electron transfer kinetics than the other electrodes. While E_1/2 values remained comparable across all sensors, suggesting similar thermodynamic behaviour, the superior ΔE and current ratios identify Rh and Ir as the most efficient modifiers, providing an optimal balance between kinetic rate and electrochemical reversibility.

To estimate the active area of the sensors, cyclic voltammograms were recorded in 10⁻³ M potassium ferro/ferricyanide—10⁻¹ M KCl solution at different scan rates from 0.05 to 0.5 V·s⁻¹ (Figure 4). For all four electrodes immersed in the solution containing an electroactive species, the anodic peak was found to increase with the scan rate. By analyzing the relationship between the anodic peak values and the square root of the scan rates, a linear correlation can be observed for all electrodes (Figure 4), demonstrating that the redox process of ferrocyanide ions at the sensor surface is controlled by diffusion.

To better highlight the differences between the electrodes, their active area was calculated using the Randles–Sevcik equation:

$$\mathrm{I}\mathrm{p}\mathrm{a}=\mathrm{268,600}\times n^{3/2}\times \mathrm{A}\times D^{1/2}\times C \times v^{1/2}$$

considering the diffusion coefficient of ferro-ferricyanide, D = 7.26 × 10⁻⁶ cm²·s⁻¹ [35]. This equation reflects the fact that the analyte moves freely by diffusion in the solution, and the electrochemical process is predominantly controlled by diffusion mechanisms [39].

Table 4. Active surfaces and roughness factors of the electrodes used in this study.

Electrode	Active Area (cm2)	Geometric Area (cm2)	RF
Ir/CSPE	1.0841 ± 0.0005	0.1256	8.6316 ± 0.0005
Pd/CSPE	1.0287 ± 0.0004		8.1903 ± 0.0004
Pt/CSPE	0.60082 ± 0.0002		4.7836 ± 0.0002
Rh/CSPE	1.0358 ± 0.0004		8.2469 ± 0.0004

R_F—roughness factor = active area/geometric area. Three electrodes were used for all the measurements to assess the variability of the measurements.

presents the obtained results, including the active surface of all electrodes examined in this study and the roughness factor, which represents the ratio between the electrochemically determined area and the geometric area of the electrodes.

Among all sensors used in this study, Ir/CSPE presented the largest active area (A = 1.0841 cm²), followed by Rh/CSPE (A = 1.0358 cm²) and Pd/CSPE (A = 1.0287 cm²). Pt/CSPE presented a much smaller active area compared to other electrodes. Additionally, the roughness factor (R_F) for Ir/CSPE demonstrated a value of 8.6316, higher than the other electrodes, indicating superior sensitivity and efficiency in the electrochemical process. These results emphasize that the highest active surface area and the roughness factors were obtained for Ir/CSPE and Rh/CSPE sensors, resulting in superior performance characteristics.

3.1.3. Preliminary Analysis in 10⁻⁴ M Catechol—10⁻¹ M KCl Solution

Subsequently, the results obtained using the sensors immersed in a 10⁻⁴ M catechol—10⁻¹ M KCl solution (Figure 5) are presented and discussed. This analysis highlights the differences in the electrochemical response of SPEs, demonstrating how the nature and properties of the noble metal particles influence the detection of electroactive organic compounds.

The cyclic voltammograms of Ir/CSPE, Pd/CSPE, Pt/CSPE, and Rh/CSPE recorded in the 10⁻⁴ M catechol—10⁻¹ M KCl solution showed one or two pairs of redox peaks (I and II) associated with the oxidation–reduction process of catechol at the active surface of the sensors. The summarized results are presented in

Table 5. Electrochemical parameters for the first peak pair (I) obtained from cyclic voltammograms of Ir/CSPE, Pd/CSPE, Pt/CSPE, and Rh/CSPE immersed in 10⁻⁴ M catechol—10⁻¹ M KCl solution.

Electrode	Epa (V)	Epc (V)	E1/2 (V)	∆E (V)	Ipa (μA)	Ipc (μA)	Ipc/Ipa
Ir/CSPE	0.133	-	-	-	4.71	-	-
Rh/CSPE	0.102	-	-	-	6.00	-	-
Pd/CSPE	0.073	0.02	0.046	0.053	15.59	−16.68	1.070
Pt/CSPE	0.058	−2.88	1.411	2.938	21.50	−18.26	0.850

and

Table 6. Electrochemical parameters for the second peak pair (II) obtained from cyclic voltammograms of Ir/CSPE, Pd/CSPE, Pt/CSPE, and Rh/CSPE immersed in 10⁻⁴ M catechol—10⁻¹ M KCl solution.

Electrode	Epa (V)	Epc (V)	E1/2 (V)	∆E (V)	Ipa (μA)	Ipc (μA)	Ipc/Ipa
Ir/CSPE	0.572	0.215	0.393	0.357	29.45	−22.89	0.777
Rh/CSPE	0.560	0.223	0.396	0.337	26.33	−22.89	0.869
Pd/CSPE	0.587	0.189	0.388	0.398	16.23	−16.11	0.993
Pt/CSPE	0.569	0.232	0.400	0.337	10.80	−10.47	0.969

.

The electrodes analyzed in catechol solution exhibit distinct electrochemical behaviours, influenced by their modification with metals such as iridium, rhodium, palladium, and platinum, which significantly improves the electrochemical performance.

The effect of the scan rate on the electrochemical response of the modified electrodes (Ir/CSPE, Pd/CSPE, Pt/CSPE, and Rh/CSPE) was evaluated in a solution of 10⁻⁴ M catechol—10⁻¹ M KCl (Figure 6).

For the Pd/CSPE and Pt/CSPE, the electrochemical process occurring at the electrode surface is diffusion-controlled, as shown by the linear dependence between the anodic peak current and v^1/2, according to the Randles–Sevcik equation. From the slope of I_ps vs. v^1/2 dependence, diffusion coefficients were calculated (

Table 7. Diffusion coefficients of catechol calculated for the electrochemical detection with Pd/CSPE and Pt/CSPE, respectively.

Electrode	Diffusion Coefficient (cm2·s−1)
Pd/CSPE	1.51 × 10−6
Pt/CSPE	1.43 × 10−5

Relative standard deviations of current values are 1.8%.

), obtaining values very close to those previously reported in the literature for catechol [40].

For Ir/CSPE and Rh/CSPE, the electrochemical process is controlled by an adsorption process, with linear dependence observed between the anodic peak current and the scan rate. Thus, to compare the performance of these sensors, the values of Γ (the degree of surface coverage with electroactive chemical species) were calculated using the Laviron equation:

$$I_{pa}={\displaystyle \frac{n^2{F}^2\Gamma Av}{4RT}}$$

where I_pa is the anodic current, n is the number of electrons transferred, F is the Faraday constant (96,485 C/mol), Γ is the degree of coverage of the electrode surface with the electroactive species (mol/cm²), A is the geometric area of the electrode (cm²), v is the scan rate (V·s⁻¹), R is the universal gas constant (8.314 J/mol·K), and T is the absolute temperature (K). For the redox process of catechol, the number of transferred electrons (n) is 2. The results obtained are presented in

Table 8. Data obtained from the study of the influence of scan rate on the response of Ir/CSPE and Rh/CSPE.

Sensor	Ipa vs. v	R2	Γ (mol/cm2)
Ir/CSPE	Ipa = (9.04 × 10−5)·v + 0.0264	0.9950	1.76 × 10−10
Rh/CSPE	Ipa = (7.49 × 10−5)·v + 0.0271	0.9911	1.46 × 10−10

Relative standard deviations of current values are 2.1%.

.

Comparing the calculated Γ values for both sensors, it is observed that the values are similar, which means similar sensitivity for catechol.

From these results, considering the highest sensitivity and the control of adsorption in the electrochemical detection for the next set of analysis, only the Ir/CSPE and Rh/CSPE were used.

3.2. Analysis in 10⁻¹ M KCl—10⁻⁴ M Catechol—10⁻⁴ Phenylbutazone Solution

Voltammetric measurements were also carried out in a 10⁻¹ M KCl—10⁻⁴ phenylbutazone solution using the Ir/CSPE and Rh/CSPE since no visible peaks were observed in the KCl-PBZ solution. The aim of the analysis in a 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁴ phenylbutazone solution was to achieve an electrochemical response related to the presence of PBZ in solution.

Among the four electrodes tested, Ir/CSPE and Rh/CSPE were selected, as they demonstrated the most favourable electrochemical responses both in ferrocyanide/ferricyanide and catechol, characterized by well-defined peaks, high sensitivity, and low background currents.

The results obtained when the sensors are immersed in 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁴ phenylbutazone solution towards the responses in 10⁻¹ M KCl—10⁻⁴ M catechol solution (scan rate 0.05 V·s⁻¹) are included in Figure 7.

As observed for both sensors, the presence of PBZ results in an increase in the first peak current and a decrease in the second peak current during the anodic sweep. Furthermore, the shift in both anodic peaks towards lower potential is observed. In the cathodic sweep, these changes are even more pronounced; two distinct peaks were observed, in contrast to the single peak obtained for catechol alone. Therefore, the sensors allow the detection of PBZ in the presence of catechol, which acts as a redox probe. The electrochemical detection mechanism of PBZ in the presence of catechol is shown in Figure 8.

According to the literature, it can be stated that in a first stage, a tautomerization reaction of PBZ occurs with the formation of a compound containing a hydroxyl group [27]. As can be observed in Figure 8, when the sensors are immersed in 10⁻¹ M KCl—10⁻⁴ M catechol solution, two anodic peaks (I and II) and one cathodic peak are observed. These peaks correspond to the oxidation of catechol to o-quinone and to the reduction of o-quinone to catechol [28,29]. When PBZ is added to the solution, a significant modification of the sensor signals is observed. For instance, the anodic peak II current decreases, and the anodic peak I current increases. In the cathodic sweep, the peak corresponding to o-quinone reduction (II) decreases in intensity. A second reduction peak (I) is also observed. The electrochemical oxidation of catechol in the presence of PBZ follows an electrochemical-chemical-electrochemical mechanism, where the o-benzoquinone electrochemically formed acts as a Michael acceptor for the nucleophilic compound PBZ [27,29]. A catechol-phenylbutazone adduct is formed (Figure 8). Therefore, the o-quinone formed by the oxidation of the catechol is consumed in the chemical reaction with phenylbutazone rather than being reduced to catechol. Furthermore, the catechol-phenylbutazone adduct is electroactive. The anodic peak I is related to the catechol-phenylbutazone adduct oxidation, which is oxidized at a lower potential than catechol, giving rise to an intense anodic peak. The anodic peak II decreases in intensity because of the catechol-phenylbutazone adduct formation. The cathodic peak II decreases in intensity and is shifted to lower potential because o-quinone is consumed in the chemical reaction with PBZ. The cathodic peak I corresponds to the electrochemical reduction in the catechol-phenylbutazone adduct (oxidized form), which is formed in the anodic scan (anodic peak I). Then, the o-quinone formed in the electrochemical oxidation of catechol reacts with the PBZ form containing one hydroxyl group, forming a new compound, one catechol-phenylbutazone adduct, containing two hydroxyl groups in the ortho- position. The catechol-phenylbutazone adduct is involved in a reversible redox process at the sensor surface, taking place at a lower potential compared to the catechol redox process [27,29].

After recording the stable response, the kinetics of electrochemical processes were performed at scan rates between 0.05 and 0.5 V·s⁻¹ for the two electrodes (Figure 9).

Figure 9 shows the cyclic voltammograms recorded for the Ir/CSPE and Rh/CSPE in 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁴ phenylbutazone solution at scan rates between 0.05 and 0.5 V·s⁻¹. Two anodic and cathodic peaks are observed, of which the first anodic peak was analyzed, being better defined and increasing linearly with the scan rate. The linear dependences obtained between the intensity of the anodic peak and the square root of the scan rate show that the electrochemical process is diffusion-controlled, according to the Randles–Sevcik equation. The linear dependence also indicated that the transport of electroactive species to the electrode surface is predominantly achieved by diffusion from the solution.

3.3. Spectrometric Analysis by the FT-IR Method

Pure PBZ and the pharmaceutical products were analyzed by the FT-IR method to identify the active compound and its relative concentration in these products.

Figure 10 shows the overlaid FT-IR spectra of the analyzed creams, which contain both the analyte of interest (PBZ) and various excipients, as listed in the corresponding pharmaceutical leaflet.

The FT-IR spectra were obtained in the 4000–500 cm⁻¹ range, in which several absorption bands characteristic of the functional groups in the PBZ structure can be observed. In the spectral region 3500–3200 cm^−1, the signals that appear can be attributed to stretching vibrations of the -OH groups, which can be influenced by the presence of excipients such as water, carbopol, or other hydrophilic components used in creams [41]. Peaks located in the region 3000–2800 cm⁻¹ are characteristic of the -CH stretching vibrations of the n-butyl residue of PBZ. At 1700–1500 cm⁻¹, low-intensity signals are characteristic of the stretching vibrations of the carbonyl group (>C=O). The peaks around 1500 cm⁻¹ correspond to the stretching vibrations of the C=C group in the phenyl residue, and those in the spectral region 1300–1100 cm⁻¹ correspond to the C-N stretching vibrations in the imidazole nucleus of PBZ [42]. The region 2800–3000 cm⁻¹ is the characteristic region for the stretching vibrations of aliphatic C-H bonds [43]. The Ducfarm cream (green line) has lower signals in the region 2800–3000 cm⁻¹ because the ingredients have fewer compounds containing aliphatic C-H bonds. For instance, Ducfarm cream contains as a major component carbopol, and Antibiotic cream and Fitterman cream contain white soft paraffin (Table 2). The Ducfarm cream has higher signals in the 3000–3500 cm⁻¹ region, which is related to the carbopol ingredient, which contains hydroxyl groups able to form inter- and intramolecular hydrogen bonds [41].

Analysis of the FT-IR spectra indicates that all three creams possess compounds with identical functional groups and consequently the same chemical constituents, present at varying concentrations, as reflected by the differences in band intensities. The spectra of Antibiotic (red line) and Fiterman (blue line) creams showed similar absorption bands, with small variations in intensity, whereas the spectrum of the Ducfarm cream (green line) shows wider and more intense bands that can be associated with much higher concentrations of hydrophilic compounds (excipients: menthol, carbopol), but also of PBZ, as well as the fact that this product has more bioactive compounds and not just PBZ.

3.4. Calibration Curve

Considering that so far, the iridium-modified electrode had the best results, to obtain the calibration curve, cyclic voltammograms were recorded with the Ir/CSPE in 10⁻¹ M KCl—10⁻⁴ M catechol solution containing different amounts of phenylbutazone. The concentration of PBZ ranged from 0.01 to 1.75 μM (Figure 11).

As can be observed in Figure 11a, the peak intensity (I_pa) increases proportionally with the increase in PBZ concentration. In Figure 11b, the dependence between the peak current and PBZ concentration is shown over the entire studied range. This dependence is typical for electrochemical sensors, a region where the current increases linearly with concentration, followed by a plateau where the current remains almost constant, despite further increases in concentration. From the linear dependence, the linear regression was obtained (equation of calibration).

The limits of detection (LOD) and limits of quantification (LOQ) were calculated using the linear regression equation.

$$LOD={\displaystyle \frac{3\mathsf{\sigma }}{\mathrm{m}}} \mathrm{and} LOQ={\displaystyle \frac{10\mathsf{\sigma }}{s}},$$

where σ is the standard deviation, and m represents the slope of the calibration equation [44]. The values obtained are shown in

Table 9. Data obtained from the cyclic voltammograms of Ir/CSPE immersed in 10⁻¹ M KCl—10⁻⁴ M catechol solution, adding PBZ in the concentration range from 0.01 to 1.00 μM.

Sensor	Linear Equation	R2	LOD (nM)	LOQ (nM)
Ir/CSPE	Ipa (μA) = 2.6543·c (μM) − 0.0088	0.9994	1.53	5.08

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3.5. Pharmaceutical Products Analysis

Voltammetric measurements were conducted using the Ir/CSPE sensor in 10⁻⁴ M catechol (10⁻¹ M KCl) solution on three pharmaceutical cream products. According to the manufacturers, the products from Antibiotic and Fitterman contained 40 mg/g of the active compound, while the concentration in the Ducfarm cream was not provided. Additionally, PBZ is not the sole active ingredient in this formulation, which also includes diclofenac, anaesthesin, and other components. The measured solutions were obtained according to the following protocol: 1 g of cream was extracted with 96% ethanol by adding 1 g of ethanol and mixing; the product was ultrasonicated for 10 min at 37 °C, filtered, and added to a 1 L volumetric flask of ultrapure water. For the analysis, 50 mL of the solution was employed. The electrochemical behaviour of the pharmaceutical products was evaluated using the addition method. For each product, three consecutive measurements were performed by adding volumes of 50 μL to the electrochemical cell containing 50 mL of 10⁻⁴ M catechol (10⁻¹ M KCl) solution. The analyses were carried out in triplicate, and the results are shown in Figure 12.

The cyclic voltammograms obtained for the analyzed creams reveal significant differences between manufacturers. The Antibiotic cream exhibits its own characteristic peak profile, Fiterman shows distinct variation in peak shape and intensity, and Ducfarm displays the most substantial deviations, indicating composition and concentration different from the other formulations.

Comparing with Figure 7, it is observed that the peak at 0.0 V maintains its position in all voltammograms, even though they may look different due to the different excipients found in each cream, which may introduce interference signals. The quantification of PBZ from the pharmaceutical products, considering the current corresponding to PBZ, dilution, and the calibration equation, is shown in Table 9.

The detection of PBZ by the UV spectrometric method was also carried out. The calibration curve was obtained by using a 10⁻⁵ M PBZ stock solution in ethanol and making subsequent dilutions in the 0.2–1.0 μM range. The UV spectrum is characterized by an absorption maximum at 264 nm. The calibration linear equation obtained is A = 1.832·c (μM), and the results obtained for the PBZ in pharmaceutical samples are included in

Table 10. PBZ content of pharmaceutical products under study.

Pharmaceutical Product	PBZ Content (mg/g)			Relative Error (%)
	Specified by Producer	Pharmacopeia Method	Obtained with Ir Sensor
Antibiotic cream	40	39.8 ± 0.8	38.6 ± 1.3	3.5
Fitterman cream	40	40.3 ± 0.9	41.5 ± 1.4	3.75
Ducfarm cream	-	52.1 ± 1.1	53.4 ± 1.6	-

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It can be pointed out that the method based on the Ir/CSPE sensor is useful in the detection of PBZ in pharmaceutical products, and the relative error of analysis is lower than 4%; therefore, it has reasonable accuracy.

3.6. Repeatability of the Ir/CSPE Signals

For the determination of the Ir/CSPE signal repeatability, 50 cyclic voltammograms were registered in 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M phenylbutazone solution (scan rate 0.05 V·s⁻¹). The variation in the current and potential of the cyclic voltammograms is lower than 4.5%. The overlaid 1st and 50th cyclic voltammograms are included in Figure 13.

3.7. The Reproducibility/Stability of the Ir/CSPE Signals

For the determination of the Ir/CSPE signal reproducibility, three cyclic voltammograms were registered in 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M phenylbutazone solution (scan rate 0.05 V·s⁻¹) every two days for 30 days. The peak from 0.0 V, related to PBZ, was monitored. The current of the peak is maintaining the value well for 18 days. After that, the current is slowly increasing. This fact is related to the adsorption of catechol and PBZ on the electrode surface. The evolution of the current corresponding to the peak from 0.0 V is presented in Figure 14.

3.8. Selectivity and Interference Studies

For the interference studies, the influence of some nonsteroidal anti-inflammatory compounds at a concentration level of 10⁻⁵ M on the Ir/CSPE response when it is immersed in 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M phenylbutazone solution. The influence on the potential and current of the peak related to PBZ (0.0 V) was quantified and included in

Table 11. Interference results in the voltammetric signal of Ir/CSPE in 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M phenylbutazone solution in the presence of interfering compounds.

Analyzed Solution	Interferent Compound	Measured Potential (V)	Potential Change (%)	Average Potential Change (%)	Measured Current (mA)	Current Change (%)	Average Current Change (%)
Solution 1	-	0.002	-	-	2.67	-	-
Solution 2	Diclofenac	0.009	3.5	2.67	2.96	0.11	0.14
Solution 3	Ibuprofen	−0.003	2.5		2.89	0.08
Solution 4	Paracetamol	0.006	2		2.06	0.23

Solution 1: 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M PBZ; Solution 2: 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M PBZ + 10⁻⁵ M diclofenac; Solution 3: 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M PBZ + 10⁻⁵ M ibuprofen; Solution 4: 10⁻¹ M KCl—10⁻⁴ M catechol—10⁻⁶ M PBZ + 10⁻⁵ M paracetamol.

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Experimental results (Table 11) show that anti-inflammatory compounds had almost no interference with the detection of PBZ. Consequently, it can be concluded that the Ir/CSPE is useful for PBZ detection in the presence of common interfering compounds. The average Ir/CSPE response change was found to be 2.67% for the peak potential and 0.14% for the peak current.

To detect the effects of the compounds from the pharmaceutical products in the form of creams, several measurements were carried out in real samples with the Ir/CSPE sensor using the standard addition method. To measure real samples, an amount of cream was added to a 10⁻¹ M KCl—10⁻⁴ M catechol solution and ultrasonicated for 10 min to prepare a solution with a concentration of 5 × 10⁻⁷ M PBZ. The response of the Ir/CSPE sensor in this solution was recorded by cyclic voltammetry. Before, 5 × 10⁻⁷ M pure PBZ was added to the real sample solution, and the Ir/CSPE sensor signal was recorded again by cyclic voltammetry. The analytical recovery was calculated. The procedure was applied for all three pharmaceutical products, and the analyses were carried out in triplicate. The results are summarized in

Table 12. PBZ data obtained from the analysis of pharmaceutical products by the standard addition method.

Sample	PBZ (M)	PBZ Added (M)	PBZ Found (M)	Recovery (%)
Antibiotic cream	5 × 10−7	-	4.94 × 10−7	98.8 ± 1.2
		5 × 10−7	9.94 × 10−7	99.4 ± 1.1
Fiterman cream	5 × 10−7	-	5.03 × 10−7	100.6 ± 1.3
		5 × 10−7	10.12 × 10−7	101.2 ± 1.4
Ducfarm cream	5 × 10−7	-	4.91 × 10−7	98.2 ± 1.3
		5 × 10−7	10.26 × 10−7	102.6 ± 1.5

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The quantification of PBZ in the cream samples yielded a mean recovery of 98.8 ± 1.2 for the Antibiotic cream, 101.2 ± 1.4 for the Fiterman cream, and 102.6 ± 1.5 for the Ducfarm cream. The Pharmacopeia limits for recovery for active substance quantitation are from 90% to 110% [45]. The recoveries obtained in this study for the quantification of PBZ in different brands of creams demonstrate the practical applicability of the sensor in the analysis of pharmaceutical products.

4. Conclusions

In summary, among the four electrodes analyzed in this study, the Ir/CSPE proved to be the most suitable for PBZ detection. Even in the preliminary analyses, both the Ir/CSPE and Rh/CSPE exhibited the lowest background currents, indicating their superior performance in voltammetric measurements. They also presented the smallest potential difference between the anodic and cathodic peaks, which indicates faster electron transfer kinetics compared to the other sensors analyzed. These observations highlight the advantageous properties of iridium- and rhodium-based electrodes for surface modification aimed at enhancing the kinetics and reversibility of redox processes.

Furthermore, iridium (Ir/CSPE) and rhodium (Rh/CSPE) proved to be the most efficient materials, providing an optimal balance between electron transfer kinetics and redox process reversibility. Overall, among all sensors tested, the Ir/CSPE demonstrated the largest electroactive surface area, and its roughness factor (R_F) was higher than that of the remaining electrodes, confirming its superior sensitivity and efficiency in the electrochemical detection. Based on the calculated surface coverage with electroactive species, as well as the preliminary findings, the Ir/CSPE yielded the most favourable results and was selected for the calibration process. The method displayed a linear range of 0.01 to 1.00 μM, with a detection limit of 1.53 nM and a quantification limit of 5.08 nM. Moreover, analyses carried out on real pharmaceutical samples confirmed that the peak observed at 0.0 V was consistent with that obtained for the pure compound, thereby validating the electrode’s applicability for reliable phenylbutazone detection and quantification.