key: cord-0977993-5ek31bwo
authors: Allahverdiyeva, Shabnam; Yunusoğlu, Oruc; Yardım, Yavuz; Şentürk, Zühre
title: First electrochemical evaluation of Favipiravir used as an antiviral option in the treatment of COVID-19: A study of its enhanced voltammetric determination in cationic surfactant media using a boron-doped diamond electrode()
date: 2021-03-18
journal: Anal Chim Acta
DOI: 10.1016/j.aca.2021.338418
sha: df2cf623d5dbaebfd2c7152e8de3a4b6ead5cf3b
doc_id: 977993
cord_uid: 5ek31bwo

Favipiravir, a promising antiviral agent, is undergoing clinical trials for the potential treatment of the novel coronavirus disease 2019 (COVID-19). This is the first report for the electrochemical activity of favipiravir and its electroanalytical sensing. For this purpose, the effect of cationic surfactant, CTAB was demonstrated on the enhanced accumulation of favipiravir at the surface of cathodically pretreated boron-doped diamond (CPT-BDD) electrode. At first, the electrochemical properties of favipiravir were investigated in the surfactant-free solutions by the means of cyclic voltammetry. The compound presented a single oxidation step which is irreversible and adsorption controlled. A systematic study of various operational conditions, such as electrode pretreatment, pH of the supporting electrolyte, concentration of CTAB, accumulation variables, and instrumental parameters on the adsorptive stripping response, was examined using square-wave voltammetry. An oxidation signal at around +1.21 V in Britton-Robinson buffer at pH 8.0 containing 6×10(-4) M CTAB allowed to the adsorptive stripping voltammetric determination of favipiravir (after 60 s accumulation step at open-circuit condition). The process could be used in the concentration range with two linear segments of 0.01-0.1 μg mL(-1) (6.4×10(-8)-6.4×10(-7) M) and 0.1-20.0 μg mL(-1) (6.4×10(-7) M-1.3×10(-4) M). The limit of detection values were found to be 0.0028 μg mL(-1) (1.8×10(-8) M), and 0.023 μg mL(-1) (1.5×10(-7) M) for the first and second segments of calibration graph, respectively. The feasibility of developed methodology was tested to the analysis of the commercial tablet formulations and model human urine samples.

Coronaviruses are enveloped, single-stranded RNA viruses that can infect a wide variety of hosts, including avian, wild, domesticated mammal species, and humans. Six of these wellknown viruses have been reported since the 1960s to cause disease in humans. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel type of coronavirus that gives rise to acute respiratory infections known as coronavirus disease 2019 . This respiratory disease is characterized with common symptoms including fever, dry cough, fatigue, body pain, nasal congestion, conjunctivitis, sore throat, loss of taste and smell, and headache. It was first identified in Wuhan (Central China) in December 2019, and was reported as a global pandemic by the World Health Organization (WHO) on March 11, 2020.

As of February 19, 2021 , the disease affected approximately 219 countries, infected more than 109 million people, and resulted in at least 2.4 million deaths worldwide. Currently, it appears that SARS-CoV-2 may pose a significant risk to the health system, due to its high contagiousness. However, to date, there is no specific effective approved drug for use in treating or preventing COVID 19, since there is not enough evidence. For this purpose, either a few recommended antiviral drugs approved for different infections or new alternatives that are still under trials, are being tested in various parts of the world [1] [2] [3] [4] [5] [6] [7] [8] . 4 for their phosphoribosylation (as will be explained below) and antiviral activity but does not require the presence of 6-fluoro substituent. After oral administration of favipiravir (formulation: tablet, 0.2 g), it is incorporated into the cells, and then ribosylated and phosphorylated by host cellular enzymes to form the active metabolite T-705-ribofuranosyl-5′-triphosphate (T-705-RTP). Possible routes for conversion of favipiravir into its active and inactive metabolites are presented in Fig.1 . This triphosphate form, T-705-RTP selectively inhibits the transcription and replication of the enzyme RNA dependent RNA polymerase (RdRp) of influenza and many other RNA viruses by incorporation into the virus RNA at low concentrations. However, it does not inhibit RNA and DNA synthesis in humans up to the half-maximal inhibitory concentration (IC 50 ) of 100 μg mL -1 . Following its oral administration, favipiravir reaches peak plasma concentrations in approximately 0.5 hours, and is eliminated significantly by the urinary system with a mean plasma elimination half-life (t½) of 1.5 hours. The inactive and major metabolite T-705M1 of favipiravir is generated as a result of liver oxidation, and is excreted in the hydroxylated form [9] [10] [11] [12] [13] [14] [15] .

Here Fig. 1 Given the facts mentioned above, the development of suitable analytical methodologies for the determination of promising antiviral agents used for COVID 19, such as favipiravir, is strongly required not only for routine quality control in pharmaceutical formulations but also to support pharmacokinetic and metabolic studies in biological samples of both human and animal origin. Apart from these requirements, such studies in biological matrices will assist in the development of new drugs [16] . Survey of the literature revealed that only two studies have been made on the quantification of favipiravir by using high performance liquid chromatography coupled with ultraviolet (UV) detection [17] , and spectrofluorimetric method [18] .

Among the most widely used instrumental techniques, electroanalytical approaches, especially voltammetric ones, may be suitable alternatives with simplicity of operation, low instrument cost, rapid response, portability, allowing the use of low toxicity reagents (usually aqueous buffer solutions), sufficient sensitivity accompanied by satisfactory selectivity, precision and accuracy. Undoubtedly, another remarkable feature of voltammetry is that it can provide useful information about the oxidation-reduction behavior of the analytes with redox active groups to design new strategies for their additional therapeutic effects.

It is very important to develop an outstanding electrode material to improve the performance of the voltammetric method. As reported in several extensive reviews over the past two decades, the boron-doped diamond (BDD), a relatively new form of carbon, received great attention as an eco-friendly electrode material in many fields, e.g., analytical chemistry, environmental science, biological science, materials science, and so on [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . In general, BDD provides exceptionally useful properties (without the need of any chemical modifications) among all metal (gold, platinum) and conventional sp 2 carbon (glassy carbon, carbon paste, pyrolytic graphite, etc.) electrode materials. This electrode is known to have the widest electrochemical potential window available without the interference of oxygen and hydrogen evolution, lower and stable voltammetric background current which gives it an advantage for potential scan analyses, good physical and chemical robustness which is very useful for maintaining the signal reproducibility, as well as high resistance to adsorption of most contaminants (due to the presence of sp 3 hybridized carbon atoms in diamond structure). strongly affects its physical, chemical and electronic surface properties. The as-prepared (untreated) surface of the BDD (both made in the lab and commercially available in polycrystalline form) is initially hydrophobic (free of C-O functionalities) with noticeable surface conductivity and negative electron affinity. It is mainly hydrogen-terminated as it is produced by hydrogen plasma process. Its surface can be altered to an oxygen-terminated surface using an anodic polarization process (anodic pretreatment -APT) by applying high positive potentials in the region of oxygen evolution reaction. This procedure introduces high oxygen-bonded carbon content such as ethers, ketones, alcoholic and/or carboxylic groups. Therefore BDD becomes hydrophilic with a relatively negative surface charge, lower conductivity and positive electron affinity depending on the polarization potential and period.

However, the hydrophobic nature of the BDD surface with noticeable surface conductivity can be restored by applying a cathodic polarization (cathodic pretreatment -CPT) in the region of hydrogen evolution reaction. In order to increase the sensitivity, provide the better selectivity, reduce the fouling effect and ensure the adequate repeatability, BDD properties should be tailored specifically for each electroanalytical application by selecting an appropriate electrochemical pretreatment step [28] .

In order to improve the analytical performance of voltammetric method, another simple and inexpensive way compared to the modified electrodes is the use of surfactant-containing solutions. As mentioned above, the bare BDD surface has assumed to be relatively inert to the adsorption of organic pollutants, which is considered one of its most important advantages [20] .

However, the authors of the present work have demonstrated the capability of the BDD electrode to adsorb a wider spectrum of organic compounds in the presence of surfactants (anionic and/or cationic) as pre-concentration agents, and thus to increase the sensitivity of the voltammetric method [29] [30] [31] [32] [33] [34] [35] [36] .

J o u r n a l P r e -p r o o f Taking these facts listed above into account, and considering the lack of electrochemical investigation on favipiravir, therefore, for the first time in the present study an effort will be made to examine the redox properties of this compound using voltammetric techniques and modification-free BDD electrode (only pretreated electrochemically) in the absence and the presence of cationic surfactant (cetyltrimethylammonium bromide, CTAB). For the following part, the practical applicability of the proposed voltammetric method will be tested in commercial pharmaceutical and model (spiked) human urine samples. Additionally, the results obtained for pharmaceutical formulation will be compared with those obtained by applying the UV-spectrophotometric method.

Electrochemical measurements were performed using a computer driven µAutolab Type III potentiostat/galvanostat (Metrohm Autolab B.V., The Netherlands) controlled by the GPES software (Version 4.9). For baseline correction, the raw signals of the square-wave (SW) voltammograms were mathematically processed by applying the moving average method (peak width: 0.01 V), and then the voltammograms were corrected using the Savicky and

The pH measurements were conducted using a WTW inoLab pH 720 meter with a combined glass-reference electrode (Xylem, New York, USA). Furthermore, to obtain comparative results, the quantitative assay of favipiravir in tablets by UV spectrophotometric method was carried out using an AE-S90 UV-Vis spectrophotometer with 1 cm quartz cell. Its absorption spectra in methanol, in the wavelength range from 200 to 800 nm, were recorded using methanol as a blank. Calibration graph was plotted using the absorbance value of favipravir at λ max 320 nm and its corresponding concentrations in µg mL -1 , and the regression equation was derived.

The reference standard of favipiravir was kindly provided by Atabay Pharmaceuticals and Fine Chemicals Inc. (Turkey) and used as received. Commercially available tablet samples containing the active compound were supplied from a local hospital in city of Van (Turkey).

Since favipiravir possesses poor aqueous solubility, its stock standard solution at 1 mg mL -1 was prepared by dissolving of the calculated amount in methanol and kept refrigerated when not in use. Analytical-grade reagents (acetic, boric and orthophosphoric acids) and highly pure deionized water from a Milli-Q water purifying system (Millipore, resistivity ≥ 18. 

Before starting any other electrochemical experiments, a cathodic or anodic (for comparative purposes) pretreatment of BDD electrode was carried out for its activation by applying either 

Cyclic voltammetry (CV) was carried out for preliminary studies to investigate the electrochemical behavior of favipiravir. Afterwards, it was followed by utilization of square- rest period (5 s), voltammograms were recorded by scanning the potential towards the positive direction over the range +0.50 to +1.60 V using SW waveform. The successive measurements were carried out by repeating the above assay protocol on the working electrode. Each voltammogram was repeated three times under laboratory conditions. It is important to mention that the methanol concentration in the working solutions was kept at the level of ≤ 2% (v/v). 

For the spectrophotometric assay applied to the analysis of tablet samples, the solution A (after filtration through paper) was diluted by a factor 1:4 (v/v) with methanol in order to fit into linear range of calibration curve previously determined, and analyzed by this technique.

In the next step, the human urine samples spiked with favipiravir were used in order to assess the feasibility of the voltammetric method for clinical trials. Drug-free urine samples were collected from a healthy laboratory co-worker (male, age 30 years) immediately before the experiments, and analyzed after resting for 30 min. Aliquot volume of fresh urine (500 

The described method was validated for the parameters such as linearity, limits of detection (LOD) and quantification (LOQ), precision and accuracy. LOD and LOQ values were calculated using the formulas 3 s/m and 10 s/m, respectively, where s represents standard deviation of the peak current (three runs) of the lowest level concentration (0.01 and 0.1 μg mL -1 for the 1st and 2nd linear segments of calibration graph, respectively) of the analytical curve, and m the slope of the related calibration equation [38] . To determine the precision, 0.01 or 0.1 μg mL -1 favipiravir was determined ten times within the same day (intra-day J o u r n a l P r e -p r o o f repeatability) and on three consecutive days (inter-day repeatability). Data were reported as the mean values and relative standard deviations (RSD %).

In order to understand the electrochemical response of favipiravir at the surface of BDD electrode, the experiments were performed in surfactant-free and surfactant-containing solutions by means of the CV and AdSV.

At first, the experiments were focused on the CV behavior of favipiravir using a cathodically It is also worth noting that when the electrode was scanned to a potential value of -0.8 V, no reduction peak was observed.

Here In order to determine the mass transport nature of favipiravir at the electrode/solution interface, the next step was an investigation of influence of scan rate on the electrooxidation response for 100 µg mL -1 (3.2×10 -4 M) favipiravir using CV technique in BR buffer at pH 8.0. Fig. 2B depicts the respective CVs for the potential scan rates from 10 to 150 mV s -1 (n=6). It should be noted at this point that worse current response was observed at a scan rate higher than 150 mV s -1 , making accurate measurement difficult.

As can be seen from the following equations, there is a linear relationship between the peak current (i p ) and the scan rate (ν):

It indicates an adsorption-controlled mechanism of the electrode reaction of favipiravir.

Meanwhile, i p linearly increased with ν 1/2 expressed as follow:

Such dependence indicated that the favipiravir oxidation process can be controlled by both adsorption and diffusion (less effective), i.e., there is a mixing mechanism. Similar findings have also been reported in the previous investigations for some compounds using not only the BDD electrode [33, 34, 36] but also any other carbon-based electrodes [39] [40] [41] .

On the other hand, there was a shift of the oxidation peak potential (E p ) towards more positive values as the scan rate gradually increased, confirming the above-mentioned irreversible behavior of the electrode reaction [42] . The plot of E p versus log v can be expressed by the following equation:

For an adsorption-controlled and irreversible electrode process [43] , the relationship between E p and v is defined according to the equation:

where α is charge transfer coefficient, k 0 the standard heterogeneous rate constant of the reaction, n the number of transferred electrons involved in favipiravir oxidation process, and E 0 the formal redox potential. Other symbols have their usual meanings. The slope obtained from the E p vs log v was 0.027; thus, by means of the above equation using T = 298 K, R= 8.314 J K -1 mol -1 , and F=96480 Cmol -1 , the value equals to about 2.33 was calculated for αn.

Generally α is assumed to be 0.5 for most organic molecules in total irreversible electrode process. Further, the value of n was calculated to be 4.3 (≈4). These results indicate that the oxidation of favipiravir involves four electrons per molecule.

Considering the above experimental data, it was examined whether favipiravir could be pre- This behavior could be indicative of the adsorptive accumulation of favipiravir on the electrode surface. Following this initial investigation, the measurements were performed by AdSV without applying a medium exchange step in the continuation of the study.

A preliminary study showed that suitable electroanalytical responses in terms of reproducibility and sensitivity of the measurements could not be obtained when using the unpretreated BDD electrode. In order to obtain the improved electrochemical response of the BDD electrode, two pretreatment procedures such as anodic polarization (APT, +1.8 V for 180 s) and cathodic polarization (CPT, -1.8 V for 180 s) were tested. The pretreatments were carried out as described in Experimental Section. Although a strong anodic polarization in the region of water decomposition reaction is required to complete oxygenation of the BDD surface, in the case of BDD electrode used in this study (with the diameter of 3 mm and boron content 1000 ppm), a distortion in the peak shape of favipiravir was observed at potential higher than +2.0 V. It has been demonstrated in some of our reports that anodic polarization potential between +1.6 and +1.8 V provides remarkable improvements for electroanalytical purposes [30, 33, 34] . On the other hand, a recently published study from our laboratory [33] showed that when negative potentials higher than -1.0 V are applied, cathodic pretreatment involves high value of current density, indicating that the magnitude of the applied potential polarization is sufficient for the surface of BDD to become completely hydrophobic.

Regarding studies on the pretreatment period (20-180 s), 180 s was found to be suitable for the highest current response of favipiravir compared to shorter pretreatment times. [44] [45] [46] . Although the ∆E p is quite different from the theoretical value of 59 mV for reversible systems, the findings indicate improved surface activity of BDD after cathodical pretreatment as reported in [47] . Furthermore, both pretreatment procedures were also tested for 25 μg mL -1 favipiravir in surfactant-free BR buffer, at pH 8.0 by SW-AdSV with an accumulation period of 30 s at an open-circuit condition. It is clearly seen from Fig. 3B that CPT-BDD offers higher peak intensity of the analyte than APT-BDD (the reason will be explained later). Therefore, the cathodic activation protocol was chosen in the continuation of this study and was conducted at the beginning of every experiment day. However, it should be underlined that prior to every voltammetric experiment in solutions containing CTAB, a simple mechanical polishing step (as described in the Experimental Section) is required prior to this activation protocol to remove favipiravir that has strongly adsorbed on the electrode surface by interacting with the surfactant.

Here Fig. 3 

With the aim of obtaining the best voltammetric results for analytical purposes, the next stage of experimental work was dedicated towards examining the effect of different pH values. In The slope of 67 mV/pH, which is close to the theoretical value of 59 mV suggests that the equal numbers of electrons and protons take part in the electrode reaction. Bearing the previous scan rate results of favipiravir in mind, this finding demonstrates that the electrode process for this compound is four-proton coupled four-electron transfer. Above pH 7.0, the position of the oxidation peak is not remarkably sensitive to the pH, indicating that a proton transfer step does not occur prior to the electron transfer rate-determination step at these pH values.

Until now there is no agreement on the assignment of pK a values of favipiravir available in literature. In the Drugbank database [48], its experimental pK a value has been reported to be 5. However in the same database, two predicted values such as pK a = 9.39 (strongest acidic) and pK a = -3.7 (strongest basic) are also mentioned. Moreover, very recently published paper reported eight different pK a values calculated using the direct, modified direct, and proton exchange thermodynamic cycles [49] . As seen from Fig. 1 , favipiravir (T-705, more stable enol form) and its analogues (T-705-MTP and T-705-RTP) (ketone form) belong to the potentially tautomeric pyrazinecarboxamide family. Thus, the conflicting results regarding pK a values can be due to the complexity of the proton transfer (ionization) process, which may involve several complicated steps to generate numerous products (keto and enol tautomers of favipiravir) after a series of tautomerization [49] . It should be emphasized that It is beyond the scope of the present study to describe the oxidation process of favipiravir in detail. On the other hand, more work needs to be carried out for collecting more information by using galvanostatic/potentiostatic coulometry with subsequent spectral analysis. However, a short comment can be made from the above results and considering the electrochemical behavior of the model compound pyrazinamide (pyrazinecarboxamide) used for tuberculosis treatment. Favipiravir differs from pyrazinamide by having a hydroxyl group in the position C3 of the pyrazine ring. As stated in the scientific literature, pyrazinamide does not undergo oxidation. In previously published reports, the electrochemical properties based on the reduction of this compound were investigated using several chemically modified electrodes [51] [52] [53] . With this knowledge in mind, we may assume that the hydroxyl group on this molecule also plays a role in the oxidation process of favipiravir.

As can be clearly verified from Fig. 4 , highest current response was obtained at pH 8.0, which was chosen for further studies and development of the methodology. Under the same experimental conditions, favipiravir signal recorded after applying an accumulation process was almost 2.0 times higher than that recorded without an accumulation step (see Fig. S1 ). 

Considering some studies from our laboratory on the significant role of CTAB towards the accumulation of the poorly water-soluble compounds at the hydrophobic surface of CPT-BDD [29, 31, 35, 36] , the attention was then turned to the effect of this surfactant in order to obtain more information for the adsorption process.

For this purpose, keeping the favipiravir concentration constant at 7.5 μg mL −1 (4.8×10 −5 M), the concentration of CTAB was increased from 2.5×10 −5 to 7×10 −4 M (note that CTAB was electrochemically inactive in the selected potential range). As can be seen in Fig. 5 , the peak potential remained practically constant when the electrolyte solution contains CTAB.

Regarding the peak currents, a remarkable enhancement was observed in the presence of CTAB. The peak current increased gradually with CTAB concentration up to 6.0×10 −4 M. At its higher concentration values, no significant change in the signal was observed (Fig. 5 inset) .

To sum up, the following experiments for the analytical application (see Section 3.2) were carried out by fixing the concentration of CTAB at 6.0×10 -4 M. It should be highlighted that in the case of the accumulation process after CTAB addition, favipiravir stripping signals were almost 5.5 times higher compared to the measurements in CTAB-free solutions. This finding demonstrates the possibility that CTAB can be used as a pre-concentration agent on the performance of the CPT-BDD electrode for the sensitive determination of favipiravir.

In solutions containing a certain concentration of CTAB (below critical micelle concentration in water, CMC = 0.92 to 1.0 mM) [54, 55] , its long hydrophobic tails are attracted to the hydrophobic surface of the CPT-BDD through the hydrophobic interaction. On the other hand, according to an accepted adsorption scenario [56] [57] [58] [59] [60] , small molecules with a low solubility like favipiravir may be interacted with adsorbed surfactant aggregates on the electrode surface, by an interaction mechanism very similar to the classical micellar J o u r n a l P r e -p r o o f solubilisation observed above the equilibrium CMC. As shown in the previous sections, favipiravir can be adsorbed on the surface of CPT-BDD when it is alone. However, in the presence of CTAB molecules, there is an enhancement of its surface concentration. This can be explained by the fact that a large part of the adsorption is the solubilisation of the favipiravir molecule inside surfactant aggregates. This phenomenon has been referred to as coadsorption. In some cases, the hydrophobic solute molecule normally is not adsorbed by the solid surface in the absence of surfactant but is adsorbed when a surfactant molecule is adsorbed, which is called adsolubilization. In general, the medium pH, the ionic strength of the aqueous phase, the surfactant and solute structures play an important role on the coadsorption/adsolubilization-oriented processes [58] . Reported studies have demonstrated that especially the hydrophobic tail length of surfactants (from C12 to C18) significantly affects the coadsorption/adsolubilization capacity of surfactant surface aggregates [61] . Both of these interaction mechanisms have recently found many applications in pharmacology (drug carrier targeting), engineering (chromatographic separation), materials science (surface modification), and environmental science (wastewater treatment).

Based on this fact, it is expected an adsorption of CTAB and subsequent coadsorption of favipiravir onto the surface of CPT-BDD, resulting in an increase in analytical signal of favipiravir.

Using the previously optimized experimental and operating conditions, the applicability of the CPT-BDD in combination with AdSV technique was tested by plotting the peak current against favipiravir concentration. Fig 6A and B Table 1 . The observation of the two linear segments may be a result of the adsorption effect that can occur in the presence of higher favipiravir concentration levels at the electrode surface. Therefore, the slope value and hence the sensitivity corresponding to the second linear segment was much lower than that recorded for the first linear segment. Moreover, it is clearly shown from Here Fig. 6 Here Table 1 LOD values achieved by the proposed method appears to be sufficient for favipiravir sensing in routine quality control analysis of commercial products and even in some biological samples such as urine (the principal route of excretion).

As it was mentioned earlier, a survey of the literature revealed that there are only two studies for the quantitative determination of favipiravir. In the first attempt, it was reported the use of high performance liquid chromatography with UV detection [17] . LOD value equaled to 1.20 mg mL -1 has been estimated in a mobile phase consisting potassium dihydrogen phosphate 50 mM (pH 2.3) and acetonitrile (90:10, v/v). The method was applied to the determination of favipiravir in pharmaceutical preparations. In the second published work [18] , spectrofluorimetric method has also been found to be applicable for its determination in pharmaceutical formulations and spiked human plasma with its LOD values J o u r n a l P r e -p r o o f of 9.44 ng mL -1 . From these data, it can be clearly seen that the proposed voltammetric method exhibits slightly better or even superior performance for the detection of favipiravir compared to other two techniques. In addition, the present electroanalytical methodology meets almost required characteristics such as simple and rapid treatment of the sample, minimum use of organic reagent, low cost, sufficient sensitivity and acceptable repeatability for application to real samples.

Before analyzing real samples, the effect of some possible interfering compounds, usually present in the pharmaceutical tablets and/or human urine, was investigated for 6.5×10 -6 M (~1.0 µg mL -1 ) favipiravir at the concentration ratios of 1:1, 1:10, and 1:50 (analyte:interfering compound) under the optimum experimental conditions. The maximum concentration of the selected interfering compounds, which caused an approximately ±5% relative error for the oxidation peak current of favipiravir was considered as the tolerance limit.

It was found that 50-fold excess of several excipients commonly co-formulated in tablet samples such as some metal ions (K + , Na + , Zn 2+ , Mg 2+ , Ca 2+ , Cu 2+ , Al 3+ , Fe 3+ , Ti 4+ ), some small biomolecules (lactose, sucrose, fructose, glucose), and some agents (microcrystalline cellulose, starch) did not show any interfering effects on the determination of favipiravir.

Although in case of some polyvalent cations, a precipitation may be observed in solutions at around pH 8.0, these ions are not or less extracted from methanol in real sample analysis.

Nevertheless, its detection in the presence of povidone (polyvinylpyrrolidone) and polyethylene glycol at a concentration ratio of 1:50 was somewhat complicated, which can be explained by the polymeric film formation on the surface of the BDD electrode. However, the interferences of these excipients are not expected since their contents in tablets are much lower than that here investigated. As a result, it can be concluded that the developed strategy presents sufficient selectivity and offers possibility for applying to the pharmaceutical formulations and biological matrices. Table 2 , satisfactory recovery values manifested the fact that the proposed protocol did not suffer from any considerable matrix effect.

Here Table 2 The results obtained by the proposed voltammetric procedure for commercial tablet formulations were also compared to those obtained by a simple UV spectrophotometric method which was developed in the present study. The representative absorption spectrum of favipiravir prepared in methanol is presented in Fig. S4 Table 3 . In the absence of favipiravir, there were no detectable peaks in the working potential range where its peak appeared. However, a broad peak at about +0.65 V was recorded, which might be probably due to the oxidation of common urinary compounds such as uric acid (see Fig. S2 ). This peak did not prevent the detection of favipiravir as it differs very well from that of analyte. In the presence of favipiravir, the distinct peak occurring at about +1.22 V could be attributed to its oxidation since its current increased proportionally after each addition of the stock favipiravir solutions. As can be seen in Table 3 , satisfactory recovery and RSD values indicate the potential applicability of the developed approach for analysis of these types of matrices.

Bearing the major urinary hydroxylated metabolite T-705M1 of favipiravir in mind (as explained above), the determination of this metabolite is necessary for closer examination and clarification of the analysis of human urine samples.

Here Fig. 7 Here Table 3 4

As mentioned in Introduction, this study is the first attempt describing the electrochemical investigation of favipiravir that is used as an antiviral option in the treatment of COVID-19. intra-day precision/RSD% 4.6 (peak current) 7.3 (peak current)

inter-day precision/RSD% 5.7 (peak current) 8.8 (peak current) J o u r n a l P r e -p r o o f Table 2 . The analysis of of the pharmaceutical tablets (Favimol ® ) fortified with favipiravir standard solutions using the proposed voltammetric method Added a (µg mL -1 ) Expected a (µg mL -1 ) 

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The authors thank the pharmaceutical company Atabay Pharmaceuticals and Fine Chemicals Inc., (Turkey) for the favipravir standard material. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.J o u r n a l P r e -p r o o f