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. 2022 Nov 8;7(46):42537–42544. doi: 10.1021/acsomega.2c05904

N-Methylfulleropyrrolidine-Based Multimode Sensor for Determination of Butoconazole Nitrate

Bianca-Maria Ţuchiu †,‡, Raluca-Ioana Stefan-van Staden †,‡,*, Jacobus (Koos) Frederick van Staden †, Hassan Y Aboul-Enein §
PMCID: PMC9685749  PMID: 36440171

Abstract

graphic file with name ao2c05904_0006.jpg

A multimode sensor (a sensor responding simultaneously to more than one mode, e.g., stochastic mode, amperometric mode, voltammetric mode) based on graphite paste modified with N-methylfulleropyrrolidine was proposed for the determination of butoconazole nitrate in its pharmaceutical formulation. The stochastic mode and square wave voltammetry mode were applied for the determinations. Both the stochastic mode and square wave voltammetry mode were applied for a qualitative and quantitative assay of butoconazole nitrate. The sensor can be used between 1.68 × 10–6 and 1.68 × 104 μmol L–1 when the stochastic mode is used and between 0.168 and 16.80 μmol L–1 when the square wave voltammetry mode is used. The multimode sensor was reliably used for the determination of butoconazole nitrate in its pharmaceutical formulation, Gynofort cream, the recorded recoveries being higher than 99.00%, with RSD (%) values of lower than 2.00%.

Introduction

Butoconazole (BTC, (±)1-[4-(4-chlorophenyl)-2-(2,6-dichlorophenyl)sulfanylbutyl]imidazole) is an imidazole derivative, part of the azole antifungal drugs class. Its nitrate salt (Figure 1) is used in pharmaceutical formulations such as creams for the local treatment of vulvovaginal fungal infection caused by Candida species. BTC is usually well tolerated, with mild adverse effects occurring at the administration site.1,2 Studies have revealed minimal absorption from the vagina into the systemic circulation.3 However, one report showed a possible link between BTC administration and severe reversible thrombocytopenia, caused either by BTC cream treatment or by an interaction among BTC, ibuprofen, and methotrexate.4

Figure 1.

Figure 1

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Chemical structure of butoconazole nitrate.

The compendial assay method provided in the United States Pharmacopoeia endorses the determination of butoconazole nitrate in its pure state and its pharmaceutical formulation (vaginal cream) using high-performance liquid chromatography (HPLC) with UV detection.5 A literature survey revealed several other methods proposed for the determination of BTC from various matrices such as pure form, human plasma and urine, rat plasma and tissue, cream, and water samples. These methods include ultraperformance liquid chromatography (UPLC) with UV detection,6 reversed-phase liquid chromatography (RPLC) with UV detection,7 high-performance thin-layer chromatography (HPTLC)-densitometry,8 fabric phase sorptive extraction-HPLC with photodiode array detection (FPSE-HPLC-PDA),9 microextraction by packed sorbent-HPLC (MEPS-HPLC-DAD),10 liquid chromatography-tandem mass spectrometry (LC-MS/MS),11,12 capillary electrophoresis (CE),13 and spectrophotometry.14 Since BTC is a chiral molecule, liquid chromatography-electrospray ionization coupled with tandem mass spectrometry (LC-ESI-MS/MS) was proposed to detect its enantiomers.15,16 Although these methods are reliable and generate accurate results, they have several limitations such as high equipment costs, the consumption of large amounts of solvents, and laborious processes of extraction and separation of target analytes. Therefore, the attention of the scientific community has turned toward electrochemical methods due to their low cost, the simplicity of the principle, and the construction of the sensors as well as the fast, sensitive, and selective response.

The development of electrochemical methods is needed to provide an alternative to standard methods of analysis presented in different pharmacopeias. They could help improve drug quality analysis, expanding to a larger scale, toward the pharmaceutical industry, drug analysis laboratories, and even clinical trials. To our knowledge, no electrochemical method for BTC analysis has been reported to date. Therefore, this paper proposes a multimode sensor based on stochastic and square-wave voltammetry (SWV) modes. An N-methylfulleropyrrolidine (MFP)-modified graphite paste electrode (MFP/GPE) was successfully applied in pharmaceutical formulation samples for the determination of BTC. N-Methylfulleropyrrolidine was selected due to its properties of being a good electrocatalyst (needed for the voltammetric mode) and also its capacity of having the channels needed for stochastic detection.

A multimode electrochemical sensor is an electrode that responds simultaneously to more than one mode (e.g., stochastic mode, amperometric mode, voltammetric mode).17 To date, several multimode sensors have been developed to detect and quantify hormones, neurotransmitters, biomarkers, endocrine disruptors, and enantiomers in various matrices.18−22

Carbon-paste electrodes (CPEs) have been successfully employed as working electrodes in electrochemical analysis due to their stable response, robustness, chemical inertness, low non-Faradaic current, low Ohmic resistance, quickly renewable surface, low cost, and ease of fabrication. Moreover, they can be easily modified by either chemical or physical processes with a wide array of materials and molecules to improve their response and sensitivity to electroactive molecules.23−26 Graphite is the most common carbon-based conductive material used in CPE construction. Under normal temperature and pressure conditions, it is the most stable allotrope of carbon and has important characteristics in terms of electric conductivity, thermal stability, and chemical inertness.27 Electrochemical sensors have proved to be powerful tools when they are used for pharmaceutical analysis.28−31

Fullerenes are a class of stable and hydrophobic molecules made up entirely of carbon atoms arranged in pentagonal, hexagonal, or heptagonal rings. Fullerenes can take spherical, ellipsoidal, or tubular shapes depending on the number of rings. The most used are those comprised of 60 π carbon atoms (C60 fullerenes). C60 consists of 12 pentagonal and 20 hexagonal rings in which carbon atoms are linked by single and conjugated double bonds, thus forming a symmetrical truncated-icosahedral closed-cage shape. Due to the mixed sp2 and sp3 bonds, C60 has a high electron affinity, promoting its reduction while hindering oxidation. Fullerene materials are suitable for electrochemical analysis due to their good physical (size, hydrophobicity), electrochemical (electronic configuration), and optical properties.32 Fulleropyrrolidines are a class of chemically functionalized C60 molecules that exhibit higher conductivity and are water-soluble. They are obtained through the Prato reaction, in which azomethine ylides are added to a [6,6] junction of the C60 in a 1,3-dipolar cycloaddition. This addition of the pyrrolidine ring transforms the C60 into an n-type semiconductor.33,34

Experimental Section

Materials and Reagents

Butoconazole nitrate, N-methylfulleropyrrolidine, graphite powder (<20 μm, synthetic), methanol, monosodium phosphate, disodium phosphate, sodium nitrate, sodium chloride, potassium nitrate, potassium chloride, d-sorbitol, glycerol, EDTA disodium salt, dl-histidine, sodium benzoate, lidocaine, and fusidic acid were purchased from Sigma-Aldrich (Milwaukee, USA); paraffin oil (d4,20 0.86 g cm–1) was obtained from Fluka (Buchs, Sweden).

A phosphate buffer solution (PBS, 0.1 mol L–1) was prepared using monosodium phosphate and disodium phosphate. Using different quantities of 0.1 mol L–1 NaOH or HCl solutions, the pH of the buffer solution was adjusted to obtain various pHs (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5). The stock solution of 4.21 × 104 μmol L–1 butoconazole nitrate was prepared in methanol.

Apparatus and Methods

Electrochemical measurements (cyclic voltammetry (CV), square wave voltammetry (SWV), electrochemical impedance spectroscopy (EIS), and stochastic) were carried out using an EmStat Pico mini potentiostat connected to a laptop with PSTrace software version 5.8 (PalmSens BV, Houten, The Netherlands) for data acquisition. A regular three-electrode system was constructed using MFP/GPE, an Ag/AgCl wire (1 mol L–1 KCl), and a Pt wire as the working electrode, reference electrode, and counter electrode, respectively. A Mettler Toledo pH meter was used for the pH adjustment.

All measurements were performed at room temperature.

Design of the MFP/GPE Sensor

The MFP/GPE electrochemical sensor was prepared by physically mixing 100 mg of graphite powder with 20 mg of MFP and an adequate quantity of paraffin oil for 15–20 min until a homogeneous paste was obtained. The paste was inserted into a plastic tube, and an Ag wire served as an electrical contact. Before each measurement, the electrode was cleaned with deionized water. The MFP/GPE surface was renewed by polishing on aluminum foil until a smooth surface was achieved. The sensor was stored at room temperature, in a dry place away from light when not in use.

Procedures

Stochastic Mode

The chronoamperometry technique was employed to determine ton and toff values. All measurements were performed at a constant potential of 1.36 V vs Ag/AgCl, which was found to be optimal. Standard solutions of various BTC concentrations were used to calibrate the sensor. The toff value (BTC signature) read in the diagrams served for the qualitative analysis, whereas the ton value was found to be related to the BTC concentration through the calibration equation of the sensor35

graphic file with name ao2c05904_m001.jpg 1

where a is the intercept and b is the slope/sensitivity.

The calibration equation was obtained by applying the linear regression method. The unknown BTC concentration in the sample was calculated by introducing the ton values in the calibration equation.

Square-Wave Voltammetry

All SWV measurements were carried out at a potential domain ranging from −500 to +800 mV, a step potential of 25.0 mV s–1, a 100 mV modulation amplitude, and a frequency of 5.0 Hz. The calibration curve was obtained by plotting BTC concentrations ranging from 0.168 to 16.80 μmol L–1 against their corresponding peak heights with a correlation coefficient of above 0.997. Baseline correction was applied to the SWV peaks.

Samples

The proposed electrochemical sensor was used for the BTC determination in its pharmaceutical formulation, Gynofort cream, using stochastic and square-wave voltammetric methods. Besides BTC as the active pharmaceutical ingredient, Gynofort contains the following excipients: sorbitol, propylene glycol, disodium edetate, liquid paraffin, polyglyceryl-3-oleate, glyceryl monoisostearate, microcrystalline wax, hydrophobic colloidal silicon dioxide, methyl p-hydroxybenzoate (E 218), propyl p-hydroxybenzoate (E 216), and purified water.

For the stochastic mode, the sample was analyzed without any pretreatment in 10 different aliquots taken from the Gynofort. For the voltammetric mode, a weighed amount of 50 mg of the cream was transferred to a 100 mL volumetric flask and treated with ethanol for complete BTC dissolution; 0.1 mol L–1 KNO3 (supporting electrolyte) and PBS pH 2.5 were added. Ten solutions were prepared in this way using different aliquots taken from Gynofort.

Results and Discussion

Electrochemical Characterization of the Sensor

The bare (GPE) and modified (MFP/GPE) electrodes were electrochemically characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The electrochemical response of the MFP/GPE was studied using the CV method. The CVs (Figure S1A) were recorded in a 5.0 × 10–3 mol L–1 K3[Fe(CN)6] (0.1 mol L–1 KCl) solution in a potential range of −0.6 to +1.0 V, at a scan rate of 0.1 V s–1, employing the GPE and MFP/GPE as working electrodes. The modified sensor showed an increased conductivity in comparison with the bare sensor, indicating that the modification with MFP improved the electrochemical response.

The interface of the bare and modified electrodes was investigated through the EIS technique, in the frequency range between 105 and 10–1 Hz. The measurements were performed in a 5.0 × 10–3 mol L–1 K3[Fe(CN)6] (0.1 mol L–1 KCl) solution. In Figure S1B,C are shown the Nyquist and Bode plots, respectively, recorded for GPE (red line) and MFP/GPE (blue line). The Randles equivalent circuit was chosen to fit the EIS data. This circuit is among the simplest and most commonly used circuits to describe simple electrochemical systems associated with interface/electrolyte processes. As can be observed in the Nyquist plot, GPE presented a large and well-defined semicircle in a low-frequency range, corresponding to high electrical resistance (Rct = 1.055 × 106 Ω). After the modification of GPE with MFP, a semicircle with a smaller diameter (Rct = 2.522 × 104 Ω) was recorded. The decrease in the semicircle diameter and Rct value indicates that MFP/GPE yields a faster electron transfer rate. The EIS results are in good agreement with CV measurements.

The electrochemical behavior of the GPE and MFP/GPE was also studied by employing the SWV technique in pH 2.5 PBS containing 0.1 mol L–1 KNO3 as the supporting electrolyte and 6.72 μmol L–1 BTC. As shown in Figure S1D, MFP/GPE gave better BTC oxidation results than GPE.

Using the Randles–Sevcik equation36 for quasi-reversible redox processes controlled by diffusion, the electrocatalytic activity of the sensors was studied by calculating the electroactive surface area. The anodic and cathodic peak current intensities are directly proportional to the square root of the scan rate as

graphic file with name ao2c05904_m002.jpg 2

where Ip is the peak current (μA), n is the number of transferred electrons (in this case n = 1), A is the electrode active surface area (cm2), C0 is the concentration of K3[Fe(CN)6] (mol cm–3), DR is the diffusion coefficient (7.60 × 10–6 cm2 s–1), and ν is the scan rate (V s–1). The “+” notation corresponds to Ipa,while “–” corresponds to Ipc. The measurements (Figure S2A) were performed in a 5 × 10–3 mol L–1 K3[Fe(CN)6] (0.1 mol L–1 KCl) solution. For the bare and modified electrodes, both the anodic and cathodic peaks (Ipa and Ipc) showed a linear dependence on the square root of the scan rate, indicating a diffusion-controlled redox process. The linear dependence of Ipa and Ipc vs ν1/2 is presented in Figure S2B. By applying the Randles–Sevcik equation, the MFP/GPE presented a larger electroactive area, 0.0015 cm2, compared to 0.0010 cm2 recorded for the GPE.

Optimization of the pH Values and the Supporting Electrolyte

The influence of pH and supporting electrolytes on BTC oxidation were investigated to optimize the working conditions. For the pH influence study, solutions of PBS at various pH values ranging from 2.0 to 5.5 containing 0.168 μmol L–1 BTC were analyzed. As can be observed in Figure S3A, the highest oxidation peak of BTC was obtained in an acidic medium (pH 2.5). Accordingly, pH 2.5 PBS was used for all further measurements. As presented in Figure S3B, the peak potential decreases linearly from pH 2.0 to 5.5 according to the linear relation Epa = 0.18 – 0.05 × pH, with a good regression coefficient (R2 = 0.9999). The value of the slope is close to the Nernstian theoretical value (0.059 V pH–1), which indicates that the number of protons involved in the electrochemical reaction is equal to the number of electrons.

The supporting electrolyte influence on the electrooxidation of BTC was studied by separately adding 0.1 mol L–1 NaCl, KCl, NaNO3, and KNO3 in pH 2.5 PBS containing 0.168 μmol L–1 BTC. As can be observed in Figure S3C, the highest peak current was recorded when 0.1 mol L–1 KNO3 was used. Thus, all further BTC working solutions were prepared using 0.1 mol L–1 KNO3 as supporting electrolyte.

Response Characteristics of the Sensor in the Stochastic Mode

Stochastic sensing is a two-step process based on channel conductivity. In the first step, the analyte flows through the channel and blocks it (the amount of time while the channel is blocked is called toff and represents the signature of the analyte) and the current drops to 0. In the second step, the analyte binds to the channel wall (a process characterized by the ton parameter) and an equilibrium equation takes place

graphic file with name ao2c05904_m003.jpg 3

where Ch is the channel and I is the interface.37

Table 1 presents the response characteristics of the MFP/GPE sensor. A linear concentration range from 1.68 × 10–6 to 1.68 × 104 μmol L–1 BTC and a limit of quantification (LOQ) of 1.68 × 104 μmol L–1 BTC were obtained when the MFP/GPE sensor was employed in the stochastic mode. The calibration curve is presented in Figure S4. When these results were correlated with the high sensitivity obtained, it could be concluded that the sensor can be used for the BTC analysis in pharmaceutical samples without any pretreatment. This method could prove useful when purity tests are performed on the active pharmaceutical ingredient and also for quality control of its pharmaceutical formulation, Gynofort, and can be applied for uniformity content tests of BTC pharmaceutical formulations.

Table 1. Response Characteristics of the MFP/GPE Sensor in Stochastic Mode.

calibration eq and correlation coeff (r) linear concentration range(μmol L–1) toff (s) sensitivity (s–1 mol L–1) LOQ (μmol L–1)
1/ton = 0.13 + 221.64 × CBTC, r = 0.9999 1.68 × 10–6 to 1.68 × 104 1.2 221.64 1.68 × 10–6
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Response Characteristics of the Sensor in Square-Wave Voltammetry Mode

To determine the response characteristics of the sensor in square wave voltammetry the influence of the scan rate on the electrooxidation of butoconazole nitrate had to be determined. The electrochemical behavior of BTC was studied by the CV technique, using the MFP/GPE at various scan rates ranging from 0.005 to 0.07 V s–1. The CVs were recorded in pH 2.5 PBS containing 0.1 mol L–1 KNO3 as supporting electrolyte and 0.168 μmol L–1 BTC. The anodic and cathodic peak currents increased with an increase in the scan rate, as presented in Figure S5A.

A linear correlation between the peak current and the square root of the scan rate (ν1/2) (Figure S5B) was observed, as given by the regression equation:

graphic file with name ao2c05904_m004.jpg 4

A linear dependence was observed when the logarithm of current intensity was plotted against the logarithm of the scan rate (Figure S5C). Their relationship is characterized by the regression equation log Ipa = −2.01 + 0.73 × log ν (R2 = 0.9973). A slope of 0.50 indicates a diffusion-controlled process, while a slope of 1.00 represents a process controlled by adsorption. Furthermore, obtaining an intermediate value suggests a mixed diffusion–adsorption process.38 Therefore, the calculated slope value of 0.73 indicates a mixed diffusion–adsorption process of BTC with the MFP/GPE surface.

To determine the heterogeneous rate constant, the Laviron equation was used:39

graphic file with name ao2c05904_m005.jpg 5

where E° is the formal potential (V), R is the universal gas constant (8.314 J mol–1 K–1), T is the absolute temperature (298.15 K), α is the electron transfer coefficient of the BTC oxidation, n is the number of electrons transferred, F is Faraday’s constant (96485 C mol–1), ks is the heterogeneous rate constant of the electrochemical reaction (s–1), and ν is the scan rate (V s–1). The formal potential of BTC (E° = 0.17) is the intercept value of the regression equation obtained by plotting the peak potential against the scan rate:

graphic file with name ao2c05904_m006.jpg 6

The oxidation peak potential shifts positively with the increase of the scan rate, as depicted in Figure S5D.

Figure S5E presents the linear dependence of the anodic peak potential on the natural logarithm of the scan rate given by the regression equation

graphic file with name ao2c05904_m007.jpg 7

By using the intercept and the slope values from this equation, the heterogeneous rate constant of the electrochemical reaction was calculated to be 0.07 s–1. The kinetic parameters α and n were calculated to be 0.60 and 0.80 (∼1 electron), respectively. Thus, the electrooxidation of BTC involves one electron and one proton, as described in Scheme 1.

Scheme 1. Proposed Mechanism for Butoconazole Oxidation.

Scheme 1

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The response characteristics of the MFP/GPE toward the BTC oxidation were investigated. SWVs were recorded in pH 2.5 PBS containing 0.1 mol L–1 KNO3 as supporting electrolyte and various concentrations of BTC (Figure 2A). By plotting the peak current response against the concentration of BTC (Figure 2B), a linear dependence is observable in the range between 0.168 and 16.80 μmol L–1, characterized by the following regression equation:

graphic file with name ao2c05904_m008.jpg 8

According to the equation, the sensitivity (m) was 1.26 × 10–4 μA μmol L–1. The calculated LOQ and limit of detection (LOD) were determined as per ICH guidelines, by applying the following equations: LOQ = 10s/m and LOD = 3s/m, where s is the standard deviation of the peak current of the blank (five measurements) and m is the slope of the calibration curve.40,41 Accordingly, the LOQ and LOD were 0.168 and 5 × 10–2 μmol L–1, respectively. The proposed sensor was further assessed using validation parameters such as repeatability, reproducibility, and precision. Table 2 presents the parameters of the calibration curve.

Figure 2.

Figure 2

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(A) SWVs recorded with MFP/GPE in pH 2.5 PBS containing 0.1 mol L–1 KNO3 as the supporting electrolyte and different concentrations of BTC ranging between 0.168 and 16.80 μmol L–1. (B) Calibration curve obtained with MFP/GPE in the linear concentration range from 0.168 to 16.80 μmol L–1 BTC. The blank curve was obtained in pH 2.5 PBS.

Table 2. Parameters of the Calibration Curve.

parameter value
linear range (μmol L–1) 0.168–16.80
intercept (μA) 2.65 × 10–3
sensitivity (μA μmol L–1) 1.26 × 10–4
correlation coeff 0.9981
LOQ (μmol L–1) 0.168
LOD (μmol L–1) 5 × 10–2
no. of data points 7
RSD of the slope (%) 2.84 × 10–4
RSD of the intercept (%) 2.68 × 10–3
reproducibility of the peak current (RSD %) 3.87
reproducibility of the peak potential (RSD %) 6.10
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A wider linear concentration range as well as a lower limit of quantification were obtained in the stochastic mode. In comparison with classical methods (FPSE-HPLC-PDA),9 the proposed methods showed wider linear concentration ranges (0.168 and 16.80 μmol L–1 in SWV mode and 1.68 × 10–6 to 1.68 × 104 μmol L–1 in stochastic mode vs 0.21–16.84 μmol L–1 for FPSE-HPLC-PDA) and lower LOQs (0.168 μmol L–1 in SWV mode and 1.68 × 10–6 μmol L–1 in stochastic mode vs 0.21 μmol L–1 for FPSE-HPLC-PDA).

Interference Studies

The effect of seven possible interfering molecules was studied under optimum conditions using the MFP/GPE. The following organic species were selected as possible interfering species in the determination of BTC: d-sorbitol, glycerol, dl-histidine, fusidic acid, EDTA disodium salt, lidocaine, and sodium benzoate.

Interference Studies in Stochastic Mode

In stochastic mode, the selectivity of MFP/GPE toward BTC is determined by the toff parameter values (signature) recorded for the possible interfering molecule. Table 3 presents the individual signatures obtained for the selected molecules when MFP/GPE is usedin stochastic mode. The values obtained proved a high selectivity of the sensor and enabled its use in pharmaceutical samples.

Table 3. Selectivity of the Multimode Sensor MFP/GPE in the Stochastic Mode.
  interfering species
  d-sorbitol glycerol EDTA disodium salt dl-histidine sodium benzoate lidocaine fusidic acid
toff (s) 0.3 0.8 2.2 1.6 0.5 1.0 2.0
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Interference Studies Using SWV Mode

The study was performed byemploying the SWV technique in pH 2.5 PBS containing 0.1 mol L–1 KNO3 as supporting electrolyte, 6.72 μmol L–1 BTC, and various concentrations of one of the selected interfering species. The experimental results revealed no major changes in the current intensity of BTC for 100-fold excesses of d-sorbitol, glycerol, histidine, and fusidic acid, 50-fold excesses of EDTA disodium salt and lidocaine, and a 10-fold excess of sodium benzoate. Moreover, there was no notable shift of the peak potential when the possible interfering species were analyzed together with a constant concentration of BTC. Figure 3 presents the ratio (I/I0) between the peak current of BTC in the presence of one of the interfering molecules and the peak current of BTC. As can be seen, the ratio value is close to 1, meaning that the selected molecules did not interfere with BTC detection.

Figure 3.

Figure 3

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Interference study results of the MFP/GPE sensor used in SWV mode.

The investigation proved that the proposed sensor presents a high selectivity toward BTC detection when it is employed in the SWV mode. The high sensitivity at low concentrations as well as the high selectivity of the proposed sensor is due to the electrocatalytic activity of MFP; MFP electrocatalyst selectivity is very high for BTC, meaning that it favors the redox processes of BTC, while no electrocatalytic activity was recorded for d-sorbitol, glycerol, dl-histidine, fusidic acid, EDTA disodium salt, lidocaine, and sodium benzoate.

No interference from the substances that may be found in the pharmaceutical formulations was recorded in any of the modes used for BTC determination.

Reproducibility, Repeatability, and Stability

The reproducibility, repeatability, and stability of the MFP/GPE sensor were tested using a solution of BTC (6.72 μmol L–1) under the optimum working conditions, in the SWV mode. To assess the reproducibility, three modified sensors were prepared in a like manner and tested. The relative standard deviation (RSD%) obtained was 1.34% (n = 5). For one of the sensors, the measurement repeatability was studied, giving an RSD value of 1.14% (n = 6). The stability of the MFP/GPE was evaluated after it was stored at room temperature, in a dry place. After 2 weeks, the current intensity of BTC was retained at around 90.27 ± 3.98% compared with the initial response. The supporting voltammograms for reproducibility, repeatability, and stability are represented in Figure S6.

The multimode sensor stability was also investigated when it was used in the stochastic mode for 3 months. The responses obtained were consistent during this period with no major changes concerning the sensitivity of the sensor (RSD% was lower than 1.00%).

Determination of Butoconazole Nitrate in Gynofort Vaginal Cream

The applicability of the proposed multimode sensor was investigated in Gynofort, a pharmaceutical vaginal cream sample containing BTC, using both SWV and stochastic modes.

For the SWV mode, the 10 samples prepared according to the principle previously described were introduced one by one into the electrochemical cell and the peak current was recorded (Figure 4A). By replacing the value of the peak current into the calibration equation, the BTC concentrations in samples were determined. Table 4 shows the results of the BTC assay in pharmaceutical samples. The calculated recoveries range from 95.39% to 105.35% with the RSD (%) and bias (%) ranging from 0.03% to 1.98% and from −5.35% to 4.83 %, respectively.

Figure 4.

Figure 4

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Diagrams obtained using MFP/GPE for the determination of BTC in Gynofort using (A) SWV mode and (B) stochastic mode.

Table 4. Determination of BTC in Gynofort Vaginal Cream (20 mg/g Butoconazole Nitrate) Using MFP/GPE in SWV Modea.

  BTC (μmol L–1)
      
sample declared amount found amount BTC recovery (%) RSD (%) bias (%)
Gynofort 1 2.10 2.11 100.68 0.09 –0.68
Gynofort 2 2.10 2.19 104.33 0.85 –4.15
Gynofort 3 2.10 2.17 103.37 1.27 –3.26
Gynofort 4 2.10 2.10 99.83 1.98 0.17
Gynofort 5 2.10 2.21 105.35 0.04 –5.35
Gynofort 6 2.10 2.14 101.96 1.17 –1.92
Gynofort 7 2.10 2.14 102.07 1.35 –2.03
Gynofort 8 2.10 2.00 95.39 1.14 4.83
Gynofort 9 2.10 2.11 100.68 0.05 –0.68
Gynofort 10 2.10 2.08 99.08 0.03 0.93
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a

All measurements are the average of five determinations.

For the stochastic mode, the sample was analyzed without any pretreatments or dilutions, as the concentration of the active substance BTC is within the linear concentration range of MFP/GPE. Thus, the proposed sensor was used for the qualitative and quantitative analysis of BTC in Gynofort. First, the BTC signature was identified in the diagram (Figure 4B), subsequently, the ton value was read and used, as described above in the stochastic mode paragraph, for the determination of BTC concentration. The results obtained are presented in Table 5.

Table 5. Determination of BTC in Gynofort Vaginal Cream (20 mg/g Butoconazole Nitrate) Using MFP/GPE in Stochastic Modea.

  BTC (mg/g)
      
sample declared amount found amount BTC recovery (%) RSD (%) bias (%)
Gynofort 20 19.82 ± 0.03 99.10 0.01 0.90
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a

All measurements are the average of ten determinations.

The results obtained using both modes indicate that the proposed sensor can be reliably used for a qualitative and quantitative reliable analysis of BTC in real samples with minimum interference from the excipients contained by the cream. Additionally, the stochastic mode enables a uniformity content test of the cream without any processing of the sample.

Conclusions

A multimode sensor based on a functionalized C60 fullerene and graphite paste was proposed for a butoconazole nitrate assay in Gynofort. Low and very low limits of determinations were obtained using the stochastic mode and square-wave voltammetry mode, respectively. The sensor presented high sensitivities in both modes, and it was reliable when it was employed for the determination of butoconazole nitrate in real samples. The features of the sensor are its utilization in the pharmaceutical industry for the quality control of butoconazole nitrate, as well as for a uniformity content assay of its pharmaceutical formulations.

Acknowledgments

This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI-UEFISCDI, project number PN-III-P4-ID-PCE-2020-0059, within PNCDI III.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c05904.

  • Additional figures as described in the text obtained during characterization of the proposed multimode sensor (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao2c05904_si_003.pdf (754.1KB, pdf)

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