First report adsorptive differential pulse voltammetric sensor for sensitive determination of dronedarone in dosage form and biological samples based on tungsten oxide nanostructure: DFT assisted mechanistic insight

Abstract

The present study illustrated the construction, characterization, and application of a novel tungsten oxide nanoparticle (WO₂NPs) based sensors for sensitive adsorptive differential pulse voltammetric quantification of dronedarone (DRO) in dosage forms and biological samples. The content of the WO₂NPs was optimized with full characterization of the surface morphology and the electroanalytical features of the fabricated sensors. At pH 2, DRO molecule recorded an irreversible oxidation peak at 1.08 V following adsorption-controlled mechanism. The molecular orbital calculations and electroanalytical studies sustained the electrooxidation of the nitrogen atom (N5) through the transfer of two-electron/two-proton reaction. Calibration graphs showed linear dynamic range covering the DRO concentration ranged from 0.010 to 2.772 µgmL^− 1, with LOD value 0.0031 µgmL^− 1. The developed WO₂NPs based voltammetric sensor has been successfully applied for the quantification of DRO in biological and pharmaceutical samples. The outcomes were contrasted with the previously reported analytical methods, with the advantages of acceptable performance, wide linear range, and low LOD value. The green assessment of the method acknowledged the environmental friendliness, with the minimal waste generation.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13065-026-01890-2.

Introduction

Improving the quality of life and clinical outcomes for patients with atrial fibrillation (AF) is a primary goal of therapy, specifically to reduce the risk of complications and hospital readmissions [1]. Dronedarone (DRO, Multaq) is a multichannel-blocking antiarrhythmic drug (AAD) indicated for patients with a history of atrial fibrillation (AF) or atrial flutter [2]. DRO inhibits various cardiac ion channels, and stabilizes the heart’s electrical activity to maintain sinus rhythm following successful pharmacological or electrical cardioversion [3]. DRO was approved by the FDA in 2009 as a safer alternative to the popular antiarrhythmic agent, amiodarone (AMI) [4]. While AMI is effective, it causes organ toxicity due to its iodine content. To mitigate these toxic effects, DRO features a modified chemical structure (N-(2-Butyl-3-(p-(3- (dibutylamino)propoxy)benzoyl)-5-benzofuranyl)methane sulfonamide) in which the iodine moieties of AMI molecule were replaced with a methyl sulfonamide group [5]. Furthermore, DRO is less lipophilic than AMI, resulting in reduced accumulation in the thyroid, lungs, and other peripheral tissues.

High-performance liquid chromatographic (HPLC) methods accompanied with UV and mass spectrometric detectors are the primary analytical standards for the quantification of DRO. Dronedarone and its active metabolite, debutyldronedarone (DBD), are frequently quantified in human plasma using HPLC-MS/MS [4]. For comparative studies in animal models, UPLC-MS/MS separation technique has been validated for the simultaneous determination of both DRO and AMI [6]. For metabolic profiling in both plasma and myocardial tissue, DRO, AMI, and their respective metabolites are commonly assessed using HPLC with UV detection [7]. For quality assessment of pharmaceutical formulations, DRO and its degradation products in tablets are assessed by stability-indicating reverse-phase high-performance liquid chromatography RP-HPLC [8, 9]. A stability-indicating micellar electrokinetic chromatography (MEKC) method was successfully applied for the quality control of DRO in commercial film-coated tablets with high accuracy and intermediate precision [10]. Nevertheless, UV spectrometric methods [2, 11, 12], and colorimetric method [13] were also reported.

Generally, the spectrometric measurements are more applicable for quality control of the bulk and marketed pharmaceutical formulations, but they are unsuitable in complex biological matrices. Chromatographic separation techniques are typically instrument- and solvent-dependent, with skilled operators and tedious analysis protocol. Therefore, novel precise and cost-effective analytical procedures are encouraged to ensure rapid, economical approach for monitoring of the pharmaceutically active compounds in various matrices. Electroanalytical techniques afforded adequate sensitivity with minimal consumptions of reagents, namely the analyte solution and supporting buffer solution [14–16]. Electrochemical sensors offer rapid analysis time, with excellent sensitivity, detection limits, and compatibility with portable devices [17]. Voltammetric measurements stand out based rapid analysis time, high selectivity, and capability to detect multi analytes in the sub-nanomolar range [18].

Transition metal oxides are increasingly utilized to enhance the performance of electrochemical sensors. Their effectiveness stems from a unique capacity for valence cycling, which facilitates rapid electron transfer process at the electrode surface [19–21]. Transitions between valence states on the electrode surface facilitate direct detection, while the presence of oxygen-containing functional groups increases adsorptive capacity and electron supply; together, these features significantly enhance both selectivity and sensitivity [21]. Gao et al. synthesized porous, rod-like tungsten oxide (WO₃) for the electrochemical detection of cupric ions in real samples. The W^{+ 5} and W^{+ 6} redox couple inherent to WO₃ facilitates valence transitions that significantly catalyze the reduction process on the electrode surface [22]. Tungsten oxide-based nanomaterials (WO₃, WO_3−x) exhibited exceptional optoelectronic qualities, including a wide bandgap, high electron-hole mobility, and tunable oxygen vacancies. Furthermore, their high specific surface, rapid, reversible redox-driven color changes and high coloration efficiency enhance optical and electrochromic performance [23–25].

A review of the literature reveals that the analytical determination of dronedarone has so far relied exclusively on chromatographic and optical techniques, including HPLC, UPLC-MS/MS, and photometric methods. To the best of our knowledge, no electrochemical (voltammetric, amperometric, potentiometric, or impedimetric) method has yet been reported for the determination of dronedarone itself. This gap is notable given that several electrochemical sensors have been developed for amiodarone [26]. Furthermore, although tungsten oxide nanostructures have been successfully employed as electrocatalytic electrode modifiers for the voltammetric determination of several biomolecules, they have not, to our knowledge, previously been applied to the electroanalysis of any antiarrhythmic agent. The present work therefore reports, for the first time, a tungsten oxide nanostructure-based differential pulse voltammetric sensor for the sensitive determination of dronedarone in pharmaceutical dosage forms and biological samples, supported by density functional theory (DFT) calculations that provide mechanistic insight into the electrode–analyte interaction and the oxidation pathway of the drug. Such analytical capabilities are essential for maintaining the therapeutic integrity of dronedarone in managing atrial fibrillation. Moreover, their environmental impact was assessed using the Green Analytical Procedure Index (GAPI) and the Analytical Greenness (AGREE) measure.

Experimental

Materials and reagents

Highly pure dronedarone hydrochloride standard sample (99.40 ± 0.70%, C₃₁ClH₄₅N₂O₅, molecular weight 556.76 gmol^− 1) was kindly supplied by Al Andalous for Pharmaceutical Industries (Cairo, Egypt). The stock DRO solution was prepared by dissolving an appropriate amount of the standard powder in 25mL water. Synthetic graphite powder (GR), sodium dodecyl sulphate (SDS), cetyltrimethylammonium bromide (CTAB), Triton X-100, and WO₃ (CAS 1314-35-8) were purchased from Sigma-Aldrich (Sigma-Aldrich Louis, USA). The pH value of the Britton-Robinson buffer (4 × 10^− 2molL^− 1) was adjusted using NaOH (2 × 10^− 2molL^− 1) solution. Potassium ferricyanide (FCN, Sigma-Aldrich) was used for the cyclic voltammetric (CV) and electrochemical impedance spectroscopic (EIS) measurements. The drug free biological samples were obtained from VACSERA (Giza, Egypt).

Instrumentation

All experimental studies were carried out at 25 ± 0.1 °C. Voltammetric measurements were carried out by using Metrohm 797 VA Computrace electroanalyzers (Switzerland) equipped with VA Computrace software (version 1.3.1), which enables precise control of experimental parameters. WO₂NPs-modified electrode was used as a working electrode, an Ag/AgCl reference electrode, and a platinum auxiliary electrode. Electrochemical impedimetric spectroscopic measurements (EIS) were performed on a CHI 660D electrochemical analyzer (CH Instrument Company, China). The morphological characterization of the electrode surface was carried out on a field-emission scanning electron microscope (FE-SEM, Quanta FEG 250, Thermo Fisher Scientific/FEI).

Working electrode

The working electrodes were fabricated using a sophisticated powder metallurgy approach in which the partial substitution of graphite was performed with different percentages of the tungsten dioxide particles, namely 0, 2, 5, 7, 10, and 12%. The nanostructured electrode modifier was prepared through the method of solid-state carbothermic reduction, where various amounts of WO₃ were carefully mixed with an excessive amount of graphite powder as the reducing agent and the conducting matrix, respectively. The mixture was calcined in a sealed crucible under anaerobic conditions and an optimum temperature of 600 °C for 2 h, which enabled the phase transformation from WO₃ to sub-stoichiometric tungsten oxides (WO₃–x) and/or nano-WO₂. After calcination, the materials were cooled at a rate of 5 °C min^− 1 to avoid thermal stress and preserve the microstructure. The calcined powders were then reground for an additional 30 min to further refine particle size and promote homogeneity, after which they were sieved through a 50 μm mesh to obtain a uniform particle distribution. Finally, the processed powders were compacted using a hydraulic press under a pressure of 5 tons cm^− 2 for 5 min, producing standardized pellet-type electrodes with approximate dimensions of 3.0 mm in diameter and 10 mm in length. Each pellet was encased in a PVC plastic tube, which was custom-cut from a standard glucose intravenous (IV) set. The pellet was positioned such that one end remained exposed to the measurement solution, while the opposite end was connected to a copper wire to ensure a secure electrical connection to the measuring device. Prior to electrochemical measurements, the surface of the working electrode was meticulously polished with emery tissue paper (grade 4000, Struers, Denmark) to remove residual contaminant layers and manufacturing artifacts, thereby ensuring a clean, uniform, and reproducible electrode surface. Polishing was performed gently in a unidirectional manner for 30 s under moderate pressure, followed by thorough rinsing with deionized water (resistivity 15 MΩ cm, Milli-Q system) and drying under a gentle stream of high-purity nitrogen gas to prevent aerial oxidation or impurity adsorption.

The electroactive surface area (EASA) of the fabricated sensors enriched with different WO₂NPs ratios were measured by recording successive cyclic voltammograms were in FCN/KCl solution at different scan rates. The anodic peak current linearly correlated against the square root of the scan rate (ν^1/2), confirming a diffusion-controlled reaction. EASA values were subsequently calculated using the Randles–Ševčík equation [27].

Analytical procedures

To assess the optimal conditions and construct the calibration curve, the voltammetric behavior of DRO at the WO₂NPs-based sensor was evaluated using BR buffer at pH 2.0, containing 2.0 mL of 10^{− 3} molL^{− 1} SDS solution. Specified aliquots were added to the measuring cell and the differential pulse voltammograms (DPV) were recorded at the optimized electroanalytical parameters: deposition potential 0.2 V; deposition time 100 s, scan rate of 0.050 Vs^{− 1}, pulse amplitude 0.050 V, and pulse time of 0.04 s. The average of three successive replicates at each concentration was estimated to create the calibration curve by plotting the peak current (µA) against the DRO concentration (µgmL^{− 1}). To validate the method, specificity, precision, accuracy, and linearity were performed as per the ICH guidelines [28]. The standard deviation of the blank was used to compute the limit of detection (LOD), and the limit of quantification (LOQ) values.

Analysis of DRO samples

Two tablets of the DRO marketed pharmaceutical sample (Vasodarone, 400 mg DRO/tablet, Al Andalous for Pharmaceutical Industries, Cairo, Egypt) were ground to obtain a fine powder. The DRO sample solution was prepared by dissolving an appropriate amount of the tablet powder in water, sonication for 30 min to ensure complete dissolution and filtration through a PTFE syringe filter (0.45 μm, Agilent Technologies). Suitable aliquots of the sample solution were added to the measuring cell, and the standard addition method was employed by spiking the pharmaceutical formulation with different amounts of the standard DRO standard solution. Differential pulse voltammograms were recorded, and recoveries were calculated compared with the RP-HPLC procedure [8]. DRO free serum sample solution was mixed with suitable volumes of the DRO standard solution, shaking well for 2 mints, adding 2.0 mL of ethanol and 0.5 mL of 5% ZnSO4. After centrifugation for 5 min at 13,000 rpm, a suitable volume of the clear supernatant was transferred to the measuring vessel and underwent differential voltammetric measurements under the previously optimized parameters, and the recovery was evaluated using the standard addition method to account for potential matrix effects.

Design of forced degradation studies

Forced degradation studies were performed on the stock DRO solution to provide an indication of the specificity and the stability-indicating property of the proposed method [8]. Stress studies included heat (105 °C), UV light exposure, acid/base hydrolysis (HCl/NaOH refluxed for 12 h), neutral hydrolysis, and oxidative degradation with H₂O₂ (5% at 90 °C and refluxing for 12 h). The residual, underrated DRO was assayed spectrophotometrically at 290 nm and voltammetrically using the proposed sensor.

Result and discussion

Morphological investigation and electrical behavior of WO₂NPs-based sensors

The surface morphology of the developed electrodes was characterized to assess the modification process. Figure S1a shows the surface topography of the unmodified carbon-based electrode, which appears relatively smooth and uniform, consisting of closely packed layers of carbon due to the high pressure applied during fabrication process. This surface shows a relatively low level of porosity and a smaller effective surface area, which correlates with the lower electrochemical response observed in initial studies. Conversely, the surface topography of the WO₂NPs-modified electrode, as shown in Figure S1b, displays a significantly altered structure, indicating successful integration of WO₂NPs throughout the electrode matrix. This surface features a high degree of roughness, with a porous, three-dimensional structure and a uniform distribution of WO₂NPs. This enhance the effective electroactive surface area of the electrode and provides more catalytically active sites, thus enabling faster electron transfer kinetics, which aligns with the high sensitivity seen in the detection of DRO.

As indicated (Figure S2, Table S3), the EDX spectrum confirms the chemical purity of the material as well as the modification of the carbon electrode’s surface with WO₂NPs, as indicated by three main peaks, which define the composition of the nanocomposite material. The peak at 0.277 keV indicates the carbonaceous matrix of the electrode, while the sharp peaks at 1.77 keV, corresponding to the tungsten M lines, as well as the L lines at 8.39 keV, confirm the incorporation of the metal into the composite, as indicated by the 9.15% weight%. This shows excellent correspondence with the theoretical values of 7.0%. The oxygen peak at 0.525 keV confirms the incorporation of the metal as WO₂, while the stoichiometric ratio of oxygen to tungsten indicates the formation of a sub-oxide, which has better electrocatalytic properties than the metal itself. The absence of impurity peaks, such as Cu, Fe, Cl, etc., confirms the purity of the synthesized material, while the sharp, well-defined peaks indicate a uniform distribution of active sites over the surface, thereby enhancing the oxidation of DRO.

To explore the electrochemical behavior of the fabricated sensors enriched with different WO₂NPs ratios, cyclic voltammograms (CVs) and EIS were performed in FCN solution as a common redox probe. As illustrated in Figure S4a, the value of the peak current increased upon loading of the WO₂NPs into the electrode matrix. Incorporation of 2.0% WO₂NPs duplicated the peak current; where the maximum peak current was recorded at 12.0% WO₂NPs ratio (about 6 folds compared to the unmodified one). Similarly, the EASA values were enhanced based on the WO₂NPs ratio. Sensors containing 12.0% WO₂NPs showed EASA 0.265cm², compared to 0.082 cm^− 2 for the blank matrix. The surface roughness factor (R_f, which represents the ratio between the electroactive and geometric surface areas), was about 3.75 and can be attributed to increased surface roughness, porosity, or surface activation effects.

As an effective electroanalytical tool, EIS can explore the alterations of the electrode surface that takes place upon modification of the electrode matrix. As presented in Nyquist plots (Table 1, Figure S4b), the 12.0% ratio showed the lowest Rct value (1145 Ω) compared with 3189 Ω for the bare electrode indicating the high electron transfer process at the electrode surface. In general, the collective results from SEM, EDX, CVs, and EIS studies demonstrate that the modification of the electrode with WO₂NPs nanoparticles has a profound effect on improving the electrochemical sensitivity and efficiency of charge transfer for the determination of DRO and the success of the modification process.

Table 1.

Peak characteristics, EASA, and Rct values of the WO₂NPs-based sensors

WO2NPs%	Ep.a. (V)	Epc (V)	ΔE (V)	Ip.a. (µA)	Ipc (µA)	Ipc/Ip.a.	EASA (cm2) ± SE*	Rct
0.0%	0.400	0.271	0.129	2.6500	−2.7488	−1.038	0.082 ± 0.015	3189
2.0%	0.405	0.262	0.143	5.4891	−5.5907	−1.019	0.120 ± 0.017	2896
5.0%	0.410	0.253	0.157	8.8986	−9.1149	−1.024	0.130 ± 0.021	2657
7.0%	0.42	0.242	0.178	11.7299	−13.2415	−1.129	0.180 ± 0.025	2193
10.0%	0.437	0.225	0.212	13.3394	−14.5743	−1.093	0.240 ± 0.015	1935
12.0%	0.44	0.222	0.218	14.7500	−16.2477	−1.099	0.265 ± 0.018	1145

*Average of three measurements

Electrochemical behavior of DRO at the WO₂NPs-based electrode

To the best of our knowledge, no previous investigations were performed electrochemical behavior of DRO, therefore, the present study explored for the first time the electrochemical oxidation of DRO at carbon-based electrodes loaded with different WO₂NPs ratios. As illustrated in Fig. 1a, cyclic voltammograms recorded for all of modified electrodes showed well-defined single anodic peaks around 1.08 V, without a corresponding cathodic peak in the reverse direction. This is indicative of a purely irreversible electrochemical oxidation of DRO at the modified electrode surface. Out of all of the modified electrodes, the electrode with 7.0% WO₂NPs showed the highest electrocatalytic activity for DRO oxidation, which is indicated by a high peak current and sensitivity compared to the bare and lower-percentage modified electrodes (Fig. 2b).

Fig. 1.

a The recorded cyclic voltammograms at carbon based sensors loaded with different WO₂NPs ratios; and b peak current values against the modifier content. Scan rate 50mVs^–1 at pH 2, and 2.0µgmL^− 1 DRO. The potential was recorded against Ag/AgCl electrode

Fig. 2.

a DPVs recorded at different pH values; and b peak potential and peak current as a function of pH value. DRO concentration: 2.0µgmL^-1. The potential was recorded against Ag/AgCl electrode

The enhanced electrochemical response observed via incorporation of WO₂NPs may be attributed to a dual-interaction mechanism operating at the electrode–analyte interface. First, the oxygen-rich, polar WO₂NPs surface anchors onto the carbon substrate via electrostatic and van der Waals interactions, creating a conductive nanocomposite that increases the density of catalytically active sites while preserving efficient electron transport through the underlying carbon framework; this is reflected in the progressive rise in EASA and the corresponding drop in Rct with increasing WO₂NPs loading (Table 1, Figure S4). Second, the protonated tertiary amine group within the DRO moiety (under the working acidic conditions at pH 2) is electrostatically attracted to the electronegative WO₂NPs surface, promoting adsorptive pre-concentration of the analyte. At the same time, the redox-active tungsten centers facilitate electron abstraction from the electron-rich benzofuran/methanesulfonamide moiety, lowering the oxidation overpotential and amplifying the peak current relative to the bare electrode (Fig. 1). This synergistic carbon–WO₂NPs–DRO interaction is further supported by our DFT calculations, which indicate a favorable adsorption geometry and a reduced HOMO–LUMO gap upon analyte binding, consistent with the experimentally observed electrocatalytic enhancement.

Effect of surfactants

Successive cyclic voltammetric measurements showed a gradual diminishing of the peak performance with noticeable shifting of the peak potential to the anodic direction. This behavior may be explained based on the possible adsorption of the DRO molecule and/or its possible oxidation products onto the electrode surface, which may oppose the electron transfer process [29, 30]. To preserve the analytical signal performance, a suitable surface-active agent was added to the supporting electrolyte solution. The impact of various surfactants (anionic, cationic, and non-ionic) on the voltammetric response was investigated using a 7.0% WO₂-modified sensor (Figure S5). The presented data indicating the superiority of SDS (with the final concentration 2 × 10^{− 4}molL^{− 1}), which significantly enhances the anodic peak currents. More significantly, the addition of the SDS surfactant made the electrochemical reaction more efficient by shifting the peak potential to more negative values, thereby demonstrating the electrode’s catalytic activity. The significant enhancement in the voltammetric response upon the addition of SDS can be attributed to the synergistic interaction between the drug’s chemical state and the modified electrode surface. At pH 2.0, the DRO molecules exist predominantly in their protonated cationic form and the presence of the anionic surfactant at the electrode interface facilitates a strong electrostatic attraction between its negatively charged headgroups and the positively charged drug cations. This interaction promotes the pre-concentration of the analyte within the electrical double layer of the WO₂NPs-modified, thereby increasing the mass transfer rate and the resulting anodic peak current. Furthermore, the observed shift in the peak potential toward less positive values suggests that SDS effectively reduces the activation energy required for the electrochemical process, enhancing the electron transfer kinetics. In contrast, the use of CTAB introduced electrostatic repulsion with cationic DRO molecule, while TXT likely created a steric hindrance effect. Both mechanisms resulted in the blockage of active redox sites on the electrode surface, which accounts for the inhibited peak currents and decreased sensitivity observed with these surfactants.

Voltammetric behavior at different pH values

The influence of the pH value on the electrochemical behavior of DRO was explored by recording DPVs at different pH values ranged from 2 to 7 (Fig. 2a). A gradual decrease of the peak current upon increasing the pH value was recorded, with a maximum value at pH 2. At higher pH values (6, 7), complete diminishing of the DPV was observed.

The oxidation potential was shifted to more negative values against the pH value indicating the possible participation of the hydrogen protons in the electrooxidation of DRO molecule at the WO₂NPs-based sensors. Linear relationship was illustrated between the peak potential versus the pH value [E_p=(1.1771 ± 0.0046) – (0.0464 ± 0.001) [pH], r²= – 0.99916, Fig. 2b] with Nernstian slope value suggesting the involvement of equal numbers of protons/electrons in the electrooxidation reaction, while the lower intercept indicates the absence of side reaction at the electrode surface [31–33].

Voltammetric behavior at different scan rate values

To explain the electrode reaction mechanism and estimate the number of electrons that participate in the electrode reaction, successive cyclic voltammograms were recorded in the presence of DRO at different scan rates ranged from 0.020 to 0.200Vs^-1 (Fig. 3a). DRO molecule showed a single irreversible anodic oxidation peak with improved current values at higher scan rates. In addition, the peak potentials were shifted to more positive values, indicating the irreversibility of the oxidation process. A robust linear correlation (r²= 0.9884) was observed between the anodic peak current (Ip) and the scan rate values (Fig. 3b). This linearity initially indicates the irreversibility of the electrode reaction and the oxidation process is governed by an adsorption-controlled process rather than a purely semi-infinite diffusion mechanism.

Fig. 3.

a The recorded cyclic voltammograms at different scan rate values; b plot of peak current versus the scan rate value; c log–log plot of peak current against scan rate; and d peak potential versus log ν. Measurements were performed at pH 2 in the presence of 2.0 µg mL^-1DRO solution, and data represent the average of three independent experiments. The potential was recorded against Ag/AgCl electrode

To further validate the adsorptive nature of the electrode reaction, the double-logarithmic relationship between the peak current and scan rate was analyzed. The resulting regression equation, [log I_(µA)= (0.7516 ± 0.0289) + (0.7435 ± 0.0269) log [ʋ], r² = 0.9909; Fig. 3c] yielded a slope of 0.7435 confirms that the electroactive species are pre-concentrated on the electrode surface via adsorption [31–33]. Therefore, the influence of the accumulation parameters was tested (Figure S6). The peak current increased gradually via the deposition potential to reach a maximum value at 0.2 V. Next, the deposition time was prolonged from 0 to 200 s, and 100 s was the most suitable.

The surface coverage (Γ) of the electroactive species at the WO₂NPs-based sensors can be estimated from the integrated charge () under the oxidation peak using the following relation:

where: n) is the number of electrons transferred, F is the Faraday constant (96,485Cmol⁻¹), and A is the geometric area of the working electrode. The charge was obtained by numerical integration of the anodic peak area after baseline correction. The calculated Γ value (2.31 × 10^− 10 molcm^− 2 ≈ 1.39 × 10¹⁴ molecules cm⁻²) was typical for monolayer adsorption of electroactive complexes. Therefore, the surface coverage data and the log-log slope value (0.7435) consistently supported the adsorption-controlled reaction.

Furthermore, as the scan rate increased, a systematic positive shift in the peak potential (Ep) was observed (Fig. 3d), accompanied by a concomitant increase in current magnitude. This diagnostic shift confirms the irreversible nature of the heterogeneous electron transfer process. A linear relationship between Ep against logν was established [E_p=(1.1924 ± 0.0016) + (0.06662 ± 0.0015) log [ʋ], r² = 0.9980], which is characteristic of irreversible electrochemical systems where the rate-determining step is kinetically hindered. The number of electrons transferred in the electrooxidation of DRO molecule can be calculated based on Laviron equation [34]. Based on the slope value of the previously mentioned linear equation, the value of αn equal to 1.4449 and the estimated number of electrons is equal to 1.806 (two electrons).

Kinetic studies of the DRO molecule

The Tafel region is controlled by the electron transfer kinetics between the electrode surface and DRO molecule. If the DRO protonation process is sufficiently fast step, and, assuming the number of electrons transferred in the rate-determining step of αn = 1, the ionic exchanging current density (j) and charge transfer coefficient (α) can be estimated from the slopes and intercepts of the Tafel plot (Figure S7) [35].

where I₀ is the exchange current density.

The peak potential (E_p) was linearly correlated against the logarithmic value of the peak current [E_p=1.8504 + 0.1380 Log (I_µA); r² = 0.9978]. As is evident in Figure S7, kinetic analysis within the Tafel region yielded a slope of 138 mVdec^− 1, confirming an irreversible electron-transfer process governed by a single electron in the rate-determining step (n = 1).

According to the Laviron framework, the calculated charge transfer coefficient ((1 − α) n) was 0.0666, providing robust evidence that the total number of electrons involved in the anodic peak is n = 2. The linear dependence of peak potential on pH suggested a 2e⁻/2H⁺ mechanistic pathway, primarily localized at the sulfamide moiety. Furthermore, the calculated heterogeneous rate constant (ks ≈ 4.2 × 10^− 4 s^− 1) demonstrates the electrocatalytic efficiency of the tungsten oxide matrix in reducing the activation overpotential. The kinetic parameters data are listed in Table S7.

Quantum chemical calculations

Full geometry optimization of the dronedarone molecule (C₃₁H₄₄N₂O₅S, 83 atoms, Figure S9) was carried out using the PM7 semi-empirical Hamiltonian as implemented in MOPAC2016 (Version 18.184 W, 64-bit). The COSMO (Conductor-like Screening Model) implicit solvation model was applied with EPS = 999 to approximate an aqueous polar environment. Additional keywords employed were: ALLVEC (all eigenvectors printed), MULLIK (Mulliken population analysis), DISP (dispersion and hydrogen bond energy contributions), GRAPH (graphical output), POLAR (polarizability calculation), and SHIFT = 80 (convergence damping). The optimization converged with a gradient norm of 0.874 (0.096 per atom), completing after 454 SCF cycles. All global reactivity descriptors were derived from the frontier orbital energies using Koopmans’ theorem.

The following global reactivity descriptors were computed according to standard conceptual DFT relationships:

• Ionization Potential: IP = − E(HOMO).

• Electron Affinity: EA = − E(LUMO).

• Electronegativity: χ = (IP + EA) / 2.

• Chemical Hardness: η = (IP − EA) / 2.

• Chemical Softness: σ = 1 / (2η).

• Chemical Potential: µ = −χ = −(IP + EA) / 2.

• Electrophilicity Index: ω = µ² / (2η).

The chemical potential (µ=−4.549 eV) is the negative of the electronegativity and represents the escaping tendency of electrons from the equilibrium state. The negative value confirms that dronedarone acts as an electron donor in its interaction with the electrode surface, consistent with the observed oxidation. The heat of formation ΔHf = − 189.122 kcal mol^− 1 indicates a thermodynamically stable ground-state structure.

Mulliken charge distribution and reactive site identification

Mulliken population analysis provides atomic-level charge distributions that are critical for identifying the preferred sites for electrochemical oxidation. The key Mulliken charges (Table S10, S11) reveal the following:

The tertiary amine nitrogen N5 (atom 5 in the numbering scheme of Figure S9) carries a Mulliken charge of − 0.443 e. This moderate negative charge, combined with its structural accessibility (aliphatic, non-conjugated lone pair), identifies N5 as the primary electroactive center. At pH 2.0, the Ip–ν^0.5 linear relationship and the 2e⁻/2H⁺ stoichiometry strongly support that N5 undergoes the stepwise oxidation sequence: N5 → [N5]•⁺ (radical cation) → iminium C = N⁺, as illustrated in the reaction scheme.

The sulfonamide nitrogen N33 bears a higher negative Mulliken charge (− 0.852 e), attributable to the electron-withdrawing resonance effect of the adjacent sulfonyl group (S34 = O36 and S34 = O37). This lone pair delocalization drastically reduces the nucleophilicity and electrochemical accessibility of N33, consistent with its inertness in the observed CV experiment.

Among the oxygen atoms, O13 (ether oxygen of the propoxy chain, − 0.339 e) and O21 (carbonyl oxygen of the benzoyl group, − 0.446 e) carry negative charges but are constrained by conjugation (O21) or by the absence of an adjacent ionizable proton (O13), precluding their direct participation in the primary oxidation step. The COSMO solvation descriptors—surface area A = 603.63 Ų and volume V = 703.06 ų—are consistent with the large, amphiphilic molecular structure of dronedarone and confirm its effective solvation in the electrolyte medium.

Dipole moment and solvation

The computed total dipole moment of 6.962 Debye (components: x = 3.188 D, y = − 6.186 D, z = 0.190 D) is a relatively large value for an organic drug molecule, reflecting the highly polar character imparted by the sulfonamide group, the carbonyl, and the amine functionality. A large dipole moment facilitates orientation of the molecule at the electrode–solution interface in a specific geometry favorable for electron transfer, potentially contributing to the well-defined, sharp CV peak observed experimentally. The molecular mass of DRO (556.76 gmol^− 1) combined with the large COSMO surface area (603.63 Ų) and the relatively compact COSMO volume (703.06 ų) yields a reasonable density-like parameter, consistent with the molecule’s amphiphilic, partially planar benzofuran core and flexible aliphatic chains.

Suggested reaction mechanism

Based on the cyclic voltammetric evidence and the PM7 Mulliken atomic charge analysis, the mechanistic scheme for the anodic oxidation of dronedarone at pH 2.0 can be described as follows. The compound contains two nitrogen centers: the tertiary aliphatic amine N5 (dibutylamino group, Mulliken charge = − 0.443 e) and the sulfonamide nitrogen N33 (charge = − 0.852 e). Despite the higher formal negative charge on N33, its lone pair is strongly delocalized into the adjacent sulfonyl group and conjugated benzofuran ring, rendering it essentially non-nucleophilic toward the electrode. Conversely, N5 bears an accessible lone pair that, at pH 2.0 (below the pKa of the amine, approximately 9.2), is present in a protonated ammonium equilibrium; however, the free base form in equilibrium at the electrode surface participates in the oxidative electron transfer. The electrochemical oxidation proceeds as a net 2-electron, 2-proton process (2e⁻−2 H⁺) at E = 1.05 V vs. reference electrode, converting the tertiary amine N–C bond to an iminium C = N⁺ species. This is fully consistent with the known mechanism of electrochemical oxidation of tertiary aliphatic amines, which typically proceeds in two sequential one-electron steps: (1) formation of a nitrogen-centered radical cation (aminyl radical cation, [N•]⁺) in the first oxidation step, followed by rapid deprotonation from the α-carbon to yield an α-amino radical, and (2) a second one-electron oxidation to produce the stable iminium cation (C = N⁺R₂). Scheme 1).

Scheme 1.

Electrooxidation mechanism of DRO molecule at WO2NPs-based sensor surface at pH 2

Analytical performance of the WO₂NPs-based sensors

For improved sensitivity and better peak morphology, differential pulse voltammograms were recorded at different DRO concentrations (from 0.01 to 2.772 µgmL^− 1) to illustrate the calibration graphs (Fig. 4a, b). Linearity parameters including the linear dynamic range (LDR), limit of quantification (LOQ), limit of detection (LOD), precision and accuracy were tabulated in Table 2. Integration of the electrode matrix with WO₂NPs significantly enhanced the sensitivity, LOD and LOQ values (3.1 ngmL^− 1, and 10 ngmL^− 1, respectively).

Fig. 4.

a Typical output of the differential pulse voltammograms recorded at different DRO concentrations applying WO₂NPs-based sensors at pH 2, and b peak current against DRO concentration. The potential was recorded against Ag/AgCl electrode

Table 2.

Validation and linearity parameters of the fabricated WO₂NPs-based sensors against dronedarone at pH 2

Parameter		Value
Peak potential		1.05 (V)
pH value		2
Linearity
LDR (µgmL− 1)		0.01–2.772 (µgmL− 1)
Slope		0.5984
Intercept		0.4138
Correlation coefficient (r)		0.9994
LOD a		0.0031(µgmL− 1)
LOQa		0.0100 (µgmL− 1)
Accuracy b (mean ± SD)		100.22 ± 0.11
Precision (RSD%)		1.17
Repeatability c		0.98
Intermediate precision d
Operational lifetime (weeks)		16

^aLOD and LOQ were estimated based on the signal-to-noise ratio

^bAverage of three independent determinations

^cIntraday precision was evaluated at three concentration levels, each analyzed in triplicate within a single day

^dInterday precision was evaluated at the same three concentration levels, each analyzed in triplicate across three consecutive days

The reproducibility of measurement was evaluated through successive measurements of 2.5 µgmL^− 1 DRO at the same WO₂NPs-based electrode surface (Figure S12). The recorded DPVs were highly consistent, recording reproducible peak current values with RSD value 2.65%. Moreover, the operational lifetime of the WO₂NPs-based sensors was monitored during an extended storage period. Sable voltammetric responses were maintained within the first 2 months, retaining 95.5 ± 2.4% of the original peak current for the fresh electrodes (Figure S13). The response declined moderately to 92.4 ± 3.7% after 3 months of continuous measurements and diminished to 87.4 ± 4.2% at the end of the storage period (4 months).

The performance characteristics of the developed DRO sensor based onWO₂NPs were compared with the reported DRO spectrometric and chromatographic measurements (Table S14). The presented data indicated adequate sensitivity in terms of the linear dynamic range and LOD. These outcomes indicated that the presented method particularly well-suited the precise detection of DRO in biomedical and pharmaceutical analysis.

To objectively assess the degree of environmental sustainability of the proposed electrochemical approach, its comparison with the previously reported HPLC technique [8] based on twelve key criteria was performed [36, 37]. As shown in Table S15, the WO₂NPs-based technique possesses substantially greater potential in terms of being eco-friendly than its predecessor. While the HPLC technique involves the continuous flow of highly toxic solvents (Acetonitrile and Methanol), resulting in the generation of large volumes of chemical waste and exposing operators to health risks, the new DPV technique employs an exclusively aqueous solvent matrix (BR buffer). The reduced size of the sensor implies an extremely low quantity of samples needed for analysis, whereas the absence of HPLC pumps and column heating makes this method energy effective. Moreover, the electrochemical technique does not require any complicated sample pretreatment or derivatization.

Environmental performance of the developed DPV approach was assessed via two validated metrics – the Green Analytical Procedure Index (GAPI) and the Analytical Greenness (AGREE) measure [38, 39]. As follows from the GAPI chart (Figure S16), the prevalence of the green areas, especially those connected with sample preparation, reagents, and wastes, testifies to the environmental friendliness of the proposed approach. For instance, the E-factor was kept at a very low level – only 0.1 (1.0E-01). Such an impressive result can be associated with numerous benefits offered by the Screen-Printed Electrodes (SPE) technology, allowing miniaturization and thus requiring microliter amounts of the samples and electrolytes without any organic solvents. The AGREE score achieved its high value of 0.78, because the majority of the twelve GAC principles were complied with. Dark green sections in the AGREE chart, such as segments 10, 11, and 12, testify to low energy consumption, safety of the reagents used, and increased safety of operators, respectively. Absence of complicated sample preparation and derivatization procedures makes the approach more environmentally friendly compared to traditional methods.

Method selectivity

Dronedarone is extensively metabolized (CYP3A4), to debutyldronedarone (DBD, Figure S17) which is one-tenth to one-third as potent as the parent DRO compound [40, 41]. The parent compound has a well-defined anodic peak at 1.08 V due to the oxidation of the tertiary aliphatic amine. The major metabolite [4], resulting from N-dealkylation as confirmed by MS fragmentation, lacks this electroactive center. The metabolite is expected to show a significantly reduced or shifted anodic response due to the loss of the easily oxidizable tertiary amine, but residual electroactive moieties may still contribute to a weaker oxidation signal at higher potential.

Dronedarone showed significant stability when subjected to heat, light, basic, and neutral hydrolysis conditions. On the other hand, significant degradation of DRO was detected under acidic and oxidative degradation conditions (12.0 and 20%, respectively), leading to the formation of two major n degradation products, namely debutyldronedarone (DBD) and N-oxide DRO. The N-oxide DRO degradation product is expected to show no oxidation peak due to the absence of the lone pair of electrons at the active tertiary aliphatic amine.

Excipients and additives that are present in the marketed pharmaceutical samples require the development of a robust analytical approach that is capable to selective quantifying of the parent compound in the presence of excipients and additives. Therefore, the selectivity of the presented voltammetric method for quantification of 2.0 µgmL^− 1 was critically tested in the presence of various interfering species, i.e., pharmaceutical excipients, co-administered drugs, biological substances, and inorganic ions. As depicted in Table S18, the proposed method was highly selective for most of the tablet additives, as all the calculated percentage relative errors of the anodic peak current were found to be below ± 5%. Among different pharmaceutical excipients, Magnesium Stearate (1:10 ratio) was found to cause maximum percent relative error, i.e., 1.24%, which indicates that DRO oxidation is not hindered by tablet fillers. Although endogenous biological interferents, i.e., Bovine Serum Albumin (BSA), were found to cause maximum interference, i.e., 2.24% relative error at a 1:18 ratio, which might be due to adsorption effects, the results still confirm the proposed method’s high selectivity. Moreover, physiological concentrations of inorganic ions, i.e., Na⁺, K⁺, Ca²⁺, and Cl⁻, were found not to cause any interference, as their percentage relative errors were.

Sample analysis

With improved sensitivity and selectivity of the developed WO₂NPs-based sensors, the proposed differential pulse voltammetric approach was effectively utilized for the quantification of DRO residues in Vasodarone tablets and biological samples (Tables S19-S120). The calculated F-values were lower than the critical values at the 95% confidence level with three degrees of freedom (df = 3), concluding the absence of significant difference in precision between the proposed and the corresponding chromatographic methods.

Conclusion

Heine, a novel sensor based on WO₂NPs platform was introduced for sensitive differential pulse voltammetric determination of dronedarone. With a prolonged operational lifetime and high measurement reproducibility, and presented sensors showed linear dynamic range from 0.01 to 2.772 µgmL⁻¹and successful applicability for analysis of DRO residues in pharmaceutical and biological samples. The proposed electrooxidation of DRO molecule at the electrode surface was established based on the DFT and electroanalytical studies. The green analytical assessment highlighted the method’s environmental friendliness compared with chromatographic approach with minimal release of hazardous wastes.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Rehab Alatawi1, Jawza Sh. Alnawmasi2,: Conceptualization, Methodology, Software ; Data curation, Writing- Original draft preparation. Deemah M. Alenazy3, Ahmed H. Jawhari4,: Formal analysis, Investigation; Razan M. Snari5, Omer A. Azher6,: Formal analysis, Investigation. Software, Validation. Nasser A. Alamrani1, Nashwa M. El-Metwaly5: Writing- Reviewing and Editing, Supervision; Revision.

Funding

This research work was funded by Umm Al-Qura University, Saudi Arbia Under grant number: (26UQU4350527GSSR12).

Data availability

The corresponding author will provide the data supporting the study’s conclusions upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Human participants

Not applicable.

Competing interests

The authors declare no competing interests.