ELECTROCHEMICAL TRANSFORMATIONS OF PARACETAMOL IN A WEAKLY ACIDIC ENVIRONMENT

ABSTRACT

Paracetamol (acetaminophen) is the most common non-steroidal drug among pharmaceutical preparations. It is used to relieve pain and reduce fever. However, consumption in high doses can lead to hepatotoxic effects, namely liver failure. Therefore, the cyclic voltammetry method using a platinum electrode was proposed for its detection. Paracetamol detection in the pH range of 3.5–4.0 using an acetate buffer resulted in the formation of oxidation and reduction peaks, based on these peaks, the formal potential value of paracetamol was determined to be 45 mV, while the anodic and cathodic diffusion potentials were calculated as Dox = 3.95 × 10⁻⁷ and Dred = 6.3× 10⁻⁸ using the Randles-Sevcik equation. The limit of detection (LOD) for paracetamol was found to be 4 nM, demonstrating the high sensitivity of the proposed method.

АННОТАЦИЯ

Парацетамол (ацетаминофен) является самым распространённым нестероидным препаратом среди лекарственных средств. Он используется для облегчения боли и снижения температуры. Однако употребление в высоких дозах может привести к гепатотоксическим эффектам, а именно к печёночной недостаточности. Поэтому для его определения был предложен метод циклической вольтамперометрии с использованием платинового электрода. Обнаружение парацетамола в диапазоне pH 3,5–4,0 с использованием ацетатного буфера привело к образованию пиков окисления и восстановления. На основе этих пиков формальный потенциал парацетамола был определён как 45 мВ, а анодный и катодный диффузионные потенциалы были рассчитаны с использованием уравнения Рэндлса-Севчика и составили Dox = 3,95 × 10⁻⁷ и Dred = 6,3 × 10⁻⁸ соответственно. Предел обнаружения (LOD) парацетамола составил 4 нМ, что свидетельствует о высокой чувствительности предлагаемого метода.

Keywords: cyclic voltammetry (CV), paracetamol, platinum wire electrode, supporting electrolyte, diffusion coefficient.

Ключевые слова: циклическая вольтамперометрия (ЦВА), парацетамол, платиновый проволочный электрод, поддерживающий электролит, коэффициент диффузии.

Introduction

Paracetamol or acetaminophen (N-acetyl-p-aminophenol, PAF) is a common non-steroidal drug among pharmaceutical preparations, widely used to reduce fever and relieve pain. High consumption of PAF can lead to hepatotoxicity, which may result in severe liver failure. Also, the widespread use of paracetamol has led to its distribution in the environment and has been observed to have harmful effects on crustaceans, fish, algae, and microorganisms, and its levels in environmental objects have been recorded at up to 10 μg/L. Therefore, it is necessary to develop precise, rapid, cost-effective, and selective methods for determining PAF. Various methods have been used for the determination of PAF, including titrimetric [1], spectrofluorimetric [2], chemiluminescence [3], spectrophotometry [4], ion chromatography [5], fluorimetric [6], and photoelectrochemical (PEC) [7]. These methods have high sensitivity; however, they have drawbacks such as the high cost of instruments, the complexity of sample preparation, and the time-consuming nature of the analysis. In contrast, electrochemical methods are particularly important due to their simplicity, cost-effectiveness, and rapid analysis. However, the sensitivity, selectivity, and stability of electrodes for PAF detection largely depend on the modifier materials used and the sensitivity of the applied electrochemical method. For this purpose, new modifiers with consistent electrochemical properties are continuously being developed [8]. For example, electrodes modified with silica gel [9], electrochemical sensors based on carbon nanomaterials (CNM) [10], and electrochemical sensors based on graphene [11] are available. A nickel oxide-modified electrode was used for the determination of paracetamol under neutral conditions in a phosphate buffer solution (pH = 7) using CV, DPV, and chronoamperometry [12]. A sensitive electroсchemical methods for the determination of PAF using adsorptive current voltammetry (AdsSV) on a modified electrode of multiwalled carbon nanotubes (MWCNT-BPPGE) is presented. Adsorption occurred at open-chain potential with an accumulation time of 1 min. The optimal scan rate was 100 MV/s and a pH=7.5, 0.05 M phosphate buffer solution was used. CV demonstrated a linear detection range for PAF from 0.1 to 25 µM, with a detection limit of 45 nM. Square-wave adsorptive stripping voltammetry, on the other hand, exhibited two detection ranges: 0.01 to 2 µM and 2 to 20 µM, with a detection limit of 10 nM in the first range. This method is currently considered the most sensitive for paracetamol detection [13]. Electrochemical methods for paracetamol detection using a platinum electrode are based on electron transfer and diffusion in the oxidation-reduction reaction, and they have been widely used previously [14].

Although the studies presented in these references demonstrate high sensitivity and reproducibility, the high cost of the instruments and the complexity of the analysis limit the applicability of these methods. The use of electrochemical methods in the quantification of pharmaceuticals, and the use of carbon electrodes in this field has been increasing significantly over the past decade. Unlike these traditional analytical methods, electroanalytical methods allow for high results with a large dynamic range, high sensitivity, accuracy, and low cost of the instrument.

The aim of the work was to develop a method for the electrochemical determination of paracetamol using a reusable, non-destructive, electrochemically inert platinum electrode and to perform the CV method.

Materials and Methods

All chemicals used in this study were of analytical reagent grade and used without further purification. Paracetamol, acetic acid (70.0%), sodium hydroxide (99.0%), nitric acid (99.95%), platinum (252), potassium chloride (99.0%).

CV was performed using a manual potentiostat CS350 device connected to an ASUS Core i3 personal computer. All experiments were conducted in a 30 ml quartz glass electrochemical cell using a working electrode (length 80 mm, diameter 6 mm, platinum wire diameter 0.2 mm), a reference electrode (saturated Ag/AgCl, length 50 mm, diameter 11 mm), and a counter electrode (Pt). The pH value was adjusted using a glass pH electrode. Before each measurement, free nitrogen gas was passed through the solutions in the cell for 10 minutes to eliminate the oxygen gas that wasn`t generated. A BIOBASE MS7-H550-S digital magnetic stirrer was used to ensure the uniform distribution of ions in the solution on the electrode surface. All solutions used in the experimental process were prepared with double-distilled water. Measurements were performed using an analytical balance.

Electrochemical measurement conditions using the CV method.

All electrochemical voltammograms in the CV experiment were obtained in three-electrode single-chamber glass cells. The experiment was conducted in an inert atmosphere (nitrogen) generated using a two-stage reducer system. A 0.0001 M solution of PAF was prepared by dissolving 0.0015 g of its powder in a 100 mL volumetric flask and diluting to the mark with bidi stilled water. The paracetamol solution was stored at room temperature in a dark place. Subsequent solutions used in the experiment were prepared by diluting this solution. A 0.1 M acetate buffer solution, used as the supporting electrolyte, was prepared by mixing 421.5 mL of 1 N acetic acid and 50 mL of 1 N sodium hydroxide in a 500 mL volumetric flask and diluting to the mark with bidi stilled water to obtain the desired pH solutions.

The CV curves were obtained by measuring solutions with a total volume of 25 ml, containing a 0.1 M sodium acetate supporting electrolyte at pH 3.8 and a diluted solution of paracetamol at room temperature. In this process, a potential scanning rate of 15–35 mV/s was applied to the electrode within a voltage range of 10 mV to 500 mV. The accumulation time of paracetamol on the electrode is 10 seconds. A Ag/AgCl electrode in 1 M KCl is used as the reference electrode. The surface of the platinum working electrode was cleaned by immersing it in concentrated nitric acid followed by rinsing with bidi stilled water. The solutions in the cell were refreshed after every 15 measurements, ensuring satisfactory repeatability of the results.

Results and Discussion

Effect of pH and buffer concentration.

Since the formation of distinct oxidation peaks for PAF in CV depends on the nature and environment of the buffer solution, the characteristics of the buffer solution were initially studied. Since the best signal was obtained in acetate buffer solution, it has been reported in the literature as the most suitable choice for the following experiments. The effect of acetate buffer on the increase in the anodic peak current of PAF was determined, and it was found to have the ability to maintain a stable pH value. The proper selection of the sample's pH level is a key factor in enhancing oxidation-reduction peaks. To study the effect of acetate buffer pH range on PAF detection in CV, buffer solutions with a pH range of 2.4–3.8 were prepared, and electrodes were immersed in the cell for analysis. The obtained results are presented in Figures 1 and 2.

Figure 1. The effect of pH on the determination of 0.004 µM PAF using the CV method in an acetate buffer solution (pH - 2.0, 2.5,3.0, 3.5, 3.8)

The pH value of the buffer solution directly influences the ionization state of the paracetamol molecule and its adsorption behavior on the electrode surface. These factors, in turn, indicate that the redox process possesses thermodynamically and kinetically reversible characteristics. This phenomenon can be explained by the reaction mechanism presented below.

1. Anodic process (oxidation):

In the anodic process, paracetamol (N-acetyl-para-aminophenol) (HOC₆H₄NHCOCH₃) is oxidized to N-acetyl-para-benzoquinone imine (CH₃–CO–NH=⟨C₆H₄⟩=O):

2. Cathodic process (reduction):

In the cathodic process, N-asetil-p-benzoquinonimin (CH₃CONH–C₆H₄=O) is reduced back to paracetamol (CH₃CONH–C₆H₄–OH), or depending on the number of electrons and protons, it is converted to a hydroxylamine derivative (N-acetyl-p-amino phenylhydroxylamine) (Ar–NHOH):

or

Figure 2. The effect of pH on the anodic current

The optimal pH value was in the range of 3.5 to 4.0, meaning that as the weakly acidic environment increased, the oxidation current of PAF reached a higher value, a decrease in the analytical signal was observed in the pH range from 3.5 to 2.0. This is because paracetamol is fully protonated in a weakly acidic environment, which promotes a higher electrooxidation reaction rate. At pH 3 and 2.0, the decrease in peak current is due to the decomposition of paracetamol. This led to a negative shift in the oxidation peak potential of PAF at higher pH values. Therefore, in subsequent analyses, the optimal pH was selected as 3.8.

Calculation of the diffusion coefficient of PAF.

To determine the diffusion coefficient of PAF using CV, a solution with a total volume of 25 mL was prepared in a 30 mL quartz glass cell. The solution consisted of 0.01 mL of 0.0001 M paracetamol, 2.0 mL of 0.1 M acetate buffer with pH 3.8, and 22.99 mL of bidi stilled water. Three electrodes were immersed into the solution. Then, to ensure a uniform distribution of ions in the solution, it was stirred using a magnetic stirrer at 400 TPM (turns per minute). To remove dissolved oxygen from the solution, nitrogen gas was bubbled through it for 10 minutes. After these procedures, the solution was allowed to settle, and the voltammograms during the electrolysis process were obtained by applying a potential of 15–35 mV/s to the electrode. Such an approach has also been successfully applied in the following studies (Karabayeva, G., et al. (2024). Development of a cyclic voltammetric method for the determination of cobalt (II) ions using nitrosophenol. Manuscript under review, International Journal of Analytical Chemistry; Karabayeva, G., et al. Determination of Ni (II) Ions by Cyclic Voltammetry via o-Nitrosophenol. Accepted manuscript, to be published in Univerisium Journal, August 2024). When a scan rate in the range of 40–60 mV/s was used, it was not possible to measure the anodic and cathodic peaks in the voltammograms because the analytical signal appeared as a broadened pattern. Therefore, the maximum current strength was found in the voltage range of 15-35 mV/s (Figure 2, Table 1).

Figure 3. The effect of scan rate on the analytical signal in the CV determination of paracetamol

(v = 15–35 mV/s, E = 10–500 mV, 1.0 μM PAF, pH 3.8 acetate buffer)

Table 1.

Voltametric parameters of paracetamol at different potential scan rates (15–35 mV/s, N=5)

The Randles–Sevcik equation (1) is one of the most useful equations for predicting the peak current of an electrochemical process based on certain analytical parameters. The square root of the diffusion coefficient (D) is directly proportional to the peak analytical signal and is affected by the solvent as well as the molecular weight. Additionally, the surface area of the electrode also increases the height of the analytical signal. Based on the obtained values, the diffusion coefficients of the anodic and cathodic peak currents of paracetamol were calculated using the Randles–Ševčík equation. The obtained results are presented in Tables 2, 3.

I𝑝= (2.69x10⁵) 𝑛^3/2 𝐴 C 𝐷^1/2𝑣^1/2  (1)

Table 2.

Diffusion coefficients of paracetamol

(T = 293.15 K, A = 0.277 cm², n = 2, C _PA = 1.0 μM)

The diffusion coefficients were calculated based on the Randles–Ševčík equation using the following program: https://www.calctool.org/physical-chemistry/randles-sevcik-equation

Based on the experimental data, the half-wave potential for the oxidation and reduction of paracetamol at the working electrode was 0.300 V. Within the potential scan range of 15–35 mV/s, the anodic peak appeared at 0.380–0.400 V and the cathodic peak at 0.130–0.134 V. The anodic current showed positive values ranging from 26.3 μA to 47.6 μA, while the cathodic current exhibited negative values ranging from –12.5 μA to –17.2 μA. The fact that the anodic diffusion coefficient is higher than the cathodic diffusion coefficient indicates that the oxidation of paracetamol at the working electrode occurs faster than its reduction. This suggests the oxidation of the hydroxyl (-OH) functional group to an enol (═O) group.

Effect of Paracetamol concentration on the analytical signal.

The effect of PAF concentration on the CV curve was investigated under previously optimized conditions: after an accumulation time of 10 seconds, within a potential range of 10 mV to 500 mV, at a scan rate of 30 mV/s, using an acetate buffer with pH 3.8. the analysis was performed in a 25 mL total solution containing 0.004, 0.008, 0.012, 00.16, 0.020 and 0.024 µM of PAF. The results are presented in Figures 4 and 5.

Figure 4. The effect of PAF concentration on the analytical signal

Figure 5. Linear relationship between analytical signal and PAF concentration

Based on the CV results shown in Figure 6, a linear relationship was observed between the analytical signal and PAF concentration in the range of 0.004–0.024 µM. At higher concentrations, deviations from linearity occurred. This is related to the correlation laws between molecular weight and diffusion coefficients (D) in specific solvents [15]. However, it has been emphasized that the relationship between D and molecular weight is very complex [16]. Initially, the main reason for the increase in current with increasing concentration is the presence of sufficient charge carriers in the solution, allowing charges to move more freely. Initially, the increase in current with rising concentration is mainly due to the presence of sufficient charge carriers in the solution, which allows charges to move more freely, leading to an enhancement of the analytical signal. after a certain concentration, an excessive increase in charge carriers may lead to crowding along the pathways through which the charges move, causing a slowdown in diffusion. As a result, the analytical signals decrease. Important factors affecting electrical conductivity include the mobility of ions or electrons and the internal structure of the substances.

In this study, for the first time, a method for the micro determination of paracetamol based on a reusable, electrochemically inert platinum electrode was developed. The potential scan range and buffer medium pH (3.8) were optimized using the cyclic voltammetry (CV) method.

• High accuracy and reproducibility were achieved by using a platinum wire electrode.
• Without adding any surface modifiers, a sufficiently high detection sensitivity was obtained, which simplifies the manufacturing process.
• Diffusion coefficients were calculated based on the oxidation and reduction currents of paracetamol using the Randles–Ševčík equation, aiding in the understanding of the reaction mechanisms.
• Based on the precise current responses in the voltammograms, the formal potential E₁/₂ was determined to be +45 mV, which shows a significant difference and advantage compared to many other methods.

The developed method was applied to a paracetamol-containing drug preparation, and the results were compared with the StSM method and presented in the table.

The results of the application of the developed method to the drug analysis of JSC URALBIOFARM

Table 3.

( C _PAF=12,0ng/25.0 ml; t_n =10 s; E _1/2 =45 MV, N = 5, R = 0.95 )

The Student's coefficient calculated based on the results of the table is equal to t_pcal=2.713, which indicates that t_ptab=2.776 is smaller than the value given in the table, i.e., t_ptab ˃ t_pcal. This value being smaller than the tabulated t_p-coefficient for 4 degrees of freedom confirms the accuracy of the developed method and indicates the absence of systematic error.

Differences from other scientific works:

• In most studies, modified carbon paste electrodes, graphene, nanocoating, metal oxides, MWCNTs, DPV, or photoelectrochemical (PEC) methods were used. Although they provide high sensitivity, the complexity of modification and preparation costs are significant.
• In this work, however, without any modification, a maximum detection current and high sensitivity (LOD up to 10⁻⁹ M) were achieved on a platinum electrode.
• Instrument and reagent costs were minimized, and experimental procedures were simplified, expanding the applicability of the method to practical use.

It becomes clear that the developed method enables the detection of paracetamol at the nanogram level using a platinum electrode. This also enables a comprehensive understanding of the oxidation-reduction process. The use of a platinum electrode offers additional advantages, such as eliminating the need for further procedures and highly qualified personnel, while allowing for the analysis of hundreds of paracetamol samples efficiently.

Conclusions. In this study, an electrochemical method for the determination of paracetamol was developed using cyclic voltammetry. The study provided an opportunity to investigate the oxidation and reduction reactions of PAF using a platinum electrode. The application of the CV method played a crucial role in enhancing the electroanalytical signal of paracetamol and determining the optimal conditions to lower its detection limit. During the study, the pH value, especially the optimal value of acetate buffer at pH 3.8, played a significant role in maximizing the oxidation peak of paracetamol. Under this pH value, the electrooxidation process of paracetamol occurred efficiently, improving the accuracy and repeatability of the signal. The limit of detection (LOD) for paracetamol was found to be 4 nM, demonstrating the high sensitivity of the proposed method. The diffusion coefficients of paracetamol were calculated using the Randles-Sevcik equation. The diffusion coefficients calculated based on the anodic and cathodic current intensities showed differences in the oxidation and reduction processes of paracetamol at the working electrode. The diffusion coefficient at the anode is higher than at the cathode, which confirms that the oxidation process of paracetamol occurs faster at the working electrode. The results indicate that the use of a platinum electrode and cyclic voltammetry provides high sensitivity for the accurate and efficient detection of paracetamol. The convenience and speed of this method ensure its widespread use in medicine, including for the determination of paracetamol in pharmaceuticals and the environment.

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