Voltammetric Determination of Isoniazid in the Presence of Acetaminophen Utilizing MoS2-Nanosheet-Modified Screen-Printed Electrode

We used MoS2 nanosheets (MoS2 NSs) for surface modification of screen-printed electrode (MoS2NSs-SPE) aimed at detecting isoniazid (INZ) in the presence of acetaminophen (AC). According to analysis, an impressive catalytic performance was found for INZ and AC electro-oxidation, resulting in an appreciable peak resolution (~320 mV) for both analytes. Chronoamperometry, differential pulse voltammetry (DPV), linear sweep voltammogram (LSV), and cyclic voltammetry (CV) were employed to characterize the electrochemical behaviors of the modified electrode for the INZ detection. Under the optimal circumstances, there was a linear relationship between the peak current of oxidation and the various levels of INZ (0.035–390.0 µM), with a narrow limit of detection (10.0 nM). The applicability of the as-developed sensor was confirmed by determining the INZ and AC in tablets and urine specimens, with acceptable recoveries.


Introduction
Drug analysis as one of the main branches of analytical chemistry is essential to control the quality of drugs. Drug analysis has used a variety of analytical techniques such as high-performance liquid chromatography (HPLC) [1,2], mass spectrometry [3], liquid chromatography-mass spectrometry/mass-spectrometry (LC-MS/MS) [4], and chemiluminescence [5][6][7]. Despite the many advantages of all of these methods, there are some drawbacks such as sophisticated analysis, high cost, low sensitivity, and long response times.
Screen-printing electrodes (SPEs) have been extensively employed for the massproduction of disposable electrochemical sensing systems [16]. The SPEs are affordable with the capability for mass production while maintaining sufficient reproducibility, with advantages of versatility and miniaturization [17][18][19][20][21]. The electrocatalytic activity of the bare electrode displays very substandard behavior [22]. Therefore, the electrode surface modification increases the sensitivity, reproducibility, and stability [23][24][25][26][27]. Detection of trace level of analytes can be increased by combining nanomaterials, significantly reinforcing the surface properties and electroconductivity of the electrodes, from such a function [28][29][30][31]. Nanomaterial-supported electrochemical sensors have recently attracted the attention of researchers [32][33][34]. three-electrode composition contained a 4 mm graphite as the working electrode, graphite as the auxiliary electrode, and a silver as pseudo-reference electrode. A Metrohm 713 pH meter (Metrohm, Herisau, Switzerland) equipped with a glass electrode was utilized to measure the pH values of all solutions. Deionized water from Direct-Q ®® 8 UV water purification system (Millipore, Darmstadt, Germany) was applied to freshly prepare all solutions. X-ray diffraction (XRD) spectra were obtained from Panalytical X'Pert Pro X-ray diffractometer (Etten Leur, The Netherlands) with Cu/Ká radiation at ë value of 1.5418 nm. Fourier-transform infrared (FTIR) patterns were obtained from a Tensor II spectrometer (Bruker, Mannheim, Germany). Energy dispersive X-ray (EDX) patterns and scanning electron microscopy (SEM) images were obtained by the MIRA3 scanning electron microscope (Tescan, Brno, Czech Republic).
All materials in this study were of analytical grade with no extra purification, sourced from Sigma-Aldrich. Phosphoric acid was utilized to prepare phosphate buffer solutions (PBSs) with various pH values adjusted by NaOH.

Fabrication of MoS 2 NSs
Based on a protocol, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (3 mmol) and thiourea (2.3 g) dispersed in deionized water (30 mL) were transferred to a Teflon autoclave (40-mL) at 200 • C for 24 h. The resultant product was adequately rinsed with ethanol and deionized water, followed by vacuum drying at 50 • C for six hours.

Preparation of MoS 2 NSs-SPE
For the preparation of MoS 2 NSs-SPE, 1 mg of synthesized MoS 2 NSs was poured into 1 mL of deionized water under ultrasonication, followed by drop casting of the prepared solution (4 µL) on SPE and subsequently drying at ambient temperature. The prepared MoS 2 NSs-SPE was used in electrochemical experiments.
The surface areas of the MoS 2 NSs-SPE and the bare SPE were obtained by CV using 1 mM K 3 Fe(CN) 6 at different scan rates. Using the Randles-Sevcik equation [52] for MoS 2 NSs-SPE, the electrode surface was found to be 0.16 cm 2 which was about 5.1 times greater than bare SPE.

Preparation of Real Specimens
Five AC tablets (containing 325 mg/tablet, Tehran Chemie Pharmaceutical Co., Tehran, Iran) were first powdered and then 325 mg of the powder was dissolved in water (25 mL) under ultrasonication. Next, various volumes of as-diluted solution were diluted to the mark of a 25 mL volumetric flask with PBS (pH = 7.0). The standard addition method was followed to determine the AC content.
Similarly, five INZ tablets (containing 300 mg/tablet, Tehran Chemie Pharmaceutical Co., Tehran, Iran) were first powdered and then 300 mg of the powder was dissolved in water (25 mL) under ultrasonication. Next, various volumes of as-diluted solution were diluted to the mark of a 25 mL volumetric flask with PBS (pH = 7.0). The standard addition method was followed to determine the AC content.
The instantly refrigerated urine specimens, at a certain volume (10 mL), were centrifuged at 2000 rpm for 15 min. Then, the supernatant was filtered by a 0.45 µm filter, and various volumes of it were diluted to the mark of a 25 mL volumetric flask with PBS (pH = 7.0). Next, the diluted specimens were spiked by various concentrations of INZ and AC.

Determination of MoS 2 NSs Characteristics
A scanning electron microscope was employed to capture images for the exploration of the morphology of MoS 2 nanostructures ( Figure 1). The MoS 2 nanostructure is composed of thin sheets and the sheets are slightly curved and look like clusters composed of randomly assembled NSs. Moreover, SEM images show that the as-prepared MoS 2 has a sheet-like morphology of about 12.8 nm thickness.

Determination of MoS2 NSs Characteristics
A scanning electron microscope was employed to capture images for the exploration of the morphology of MoS2 nanostructures ( Figure 1). The MoS2 nanostructure is composed of thin sheets and the sheets are slightly curved and look like clusters composed of randomly assembled NSs. Moreover, SEM images show that the as-prepared MoS2 has a sheet-like morphology of about 12.8 nm thickness. The XRD spectra was captured to determine the crystallographic structures of MoS2 NSs. Figure 2 shows the crystallite properties of MoS2 NSs based on the XRD spectra profiled at 57.8°, 35.3°, 32.22°, and 13.66° attributed to (110), (103), (100), and (002) crystal planes of the MoS2 structure, respectively, in line with the relevant standard card (JCPDS card No. . There were no peaks related to any impurity or other phases [53]. The XRD spectra was captured to determine the crystallographic structures of MoS 2 NSs. Figure 2 shows the crystallite properties of MoS 2 NSs based on the XRD spectra profiled at 57.

Electrochemical Evaluation of MoS 2 NSs-SPE towards INZ Detection
The electrochemical determinations of INZ are significantly influenced by the solution pH. Hence, we conducted the tests to determine the pH effect on electrocatalytic behavior of MoS 2 NSs-SPE towards INZ. The DPV was employed to study the effect of electrolyte solution pH (0.   Figure 5 shows the use of LSV to determine the scan rate influence on the INZ oxidation electrocatalytically on the MoS2 NSs-SPE. As seen in Figure 5, the peak potential of oxidation was towards more positive directions by elevating the scan rate, which means the kinetic restriction in electrochemical process. The peak height (Ip) plot versus the scan rate square root (ν 1/2 ) was linear ranging from 10 mV/s to 400 mV/s, which means the diffusion process is the main mechanism.  Figure 5 shows the use of LSV to determine the scan rate influence on the INZ oxidation electrocatalytically on the MoS 2 NSs-SPE. As seen in Figure 5, the peak potential of oxidation was towards more positive directions by elevating the scan rate, which means the kinetic restriction in electrochemical process. The peak height (Ip) plot versus the scan rate square root (ν 1/2 ) was linear ranging from 10 mV/s to 400 mV/s, which means the diffusion process is the main mechanism.   To study the rate-determining step as shown in Figure 6, the data related to the rising section of current vs. voltage curve obtained at 10 mV/s scan rate were applied to draw a Tafel plot for 100.0 µM of INZ. The linearity of E versus log I plot reveals the kinetics of the electrode process. The slope obtained from this plot was utilized to compute the electrons transfer number in the rate-determining step. Figure 6 illustrates the Tafel slope of 0.0989 V for a linear part of the plot, underlining the rate-limiting step of one-electron transfer having a transfer coefficient (α) of 0.4.

Chronoamperometric Measurement
Chronoamperometric determinations of INZ on the MoS 2 NSs-SPE surface were done by adjusting the potential of the working electrode at 810 mV (Figure 7). The findings from various INZ contents in PBS (at a pH value of 7.0) are depicted in Figure 8. The chronoamperometric measurement of electroactive moieties under the limited conditions of mass transfer was based on the Cottrell equation as follows: In this equation, D stands for the diffusion coefficient (cm 2 /s) and C b for the bulk concentration (mol/cm 3 ). The I plot against t −1/2 was on the basis of empirical data ( Figure 7A), with the optimal fits for various INZ contents. Then, the slopes from straight lines ( Figure 7A) were drawn against INZ content ( Figure 7B). At last, the slope from the plot in Figure 7B and the Cottrell equation were applied to calculate the mean D value, which was 1.0 × 10 −5 cm 2 /s. To study the rate-determining step as shown in Figure 6, the data related to the rising section of current vs. voltage curve obtained at 10 mV/s scan rate were applied to draw a Tafel plot for 100.0 µM of INZ. The linearity of E versus log I plot reveals the kinetics of the electrode process. The slope obtained from this plot was utilized to compute the electrons transfer number in the rate-determining step. Figure 6 illustrates the Tafel slope of 0.0989 V for a linear part of the plot, underlining the rate-limiting step of one-electron transfer having a transfer coefficient (α) of 0.4.

Chronoamperometric Measurement
Chronoamperometric determinations of INZ on the MoS2 NSs-SPE surface were done by adjusting the potential of the working electrode at 810 mV (Figure 7). The findings from various INZ contents in PBS (at a pH value of 7.0) are depicted in Figure 8. The chronoamperometric measurement of electroactive moieties under the limited conditions of mass transfer was based on the Cottrell equation as follows: In this equation, D stands for the diffusion coefficient (cm 2 /s) and Cb for the bulk

DPV Detection of INZ on the Developed Sensor Surface
DPV can increase sensitivity and better features for analytical purposes. There the voltammetric sensor of MoS2 NSs-SPE towards INZ detection was investigate DPV. Figure Table 1.

DPV Detection of INZ on the Developed Sensor Surface
DPV can increase sensitivity and better features for analytical purposes. Therefore, the voltammetric sensor of MoS 2 NSs-SPE towards INZ detection was investigated by DPV. Figure 8 shows  Table 1.

Determination of INZ in Combination with AC on MoS 2 NSs-SPE
The DPVs for the detection of INZ in combination with AC via MoS 2 NSs-SPE are presented in Figure 9. The peaks at 440 and 760 V were related to the AC and INZ oxidation, respectively. The peak current intensity was linearly elevated for both analytes by simultaneously elevating their concentrations.

Determination of INZ in Combination with AC on MoS2 NSs-SPE
The DPVs for the detection of INZ in combination with AC via MoS2 NSs-SPE are presented in Figure 9. The peaks at 440 and 760 V were related to the AC and INZ oxidation, respectively. The peak current intensity was linearly elevated for both analytes by simultaneously elevating their concentrations.

Stability
The DPV method was used to test the stability of MoS2 NSs-SPE in ambient conditions. Based on the observations, the peak current of the INZ (50.0 µM) on the modified electrode maintained 96.5% of its initial current after one week, 94.7% after two weeks, and 92.6% after four weeks, which demonstrates the exceptional long-term

Stability
The DPV method was used to test the stability of MoS 2 NSs-SPE in ambient conditions. Based on the observations, the peak current of the INZ (50.0 µM) on the modified electrode maintained 96.5% of its initial current after one week, 94.7% after two weeks, and 92.6% after four weeks, which demonstrates the exceptional long-term stability of the produced sensor.

Interference Studies
The possible interfering effect of some potentially coexisting species with INZ in real samples was investigated. The results showed that the presence of an 800-fold concentration of Na + , Mg 2+ , Cl − , and NO 3 − ; 500-fold concentration of glucose, Zn 2+ , Al 3+ , CO 3 2− , and SO 4 2− ; and a 150-fold concentration of dopamine, ascorbic acid, uric acid, and sodium citrate caused signal changes less than ±5% for 50.0 µM INZ. However, cysteine and tryptophan with two-folds excess showed interferences. The interference experiment showed that the MoS 2 NSs-SPE has good selectivity for determination of INZ.

Real Sample Analysis
The applicability of the as-developed MoS 2 NSs-SPE towards the detection of INZ and AC was tested for INZ tablets, AC tablets, and urine specimens using the standard addition method (Table 2). According to data, the proposed electrode could preserve its efficiency for sensing INZ and AC in real specimens. As seen, reasonable recovery of INZ and AC and also satisfactory reproducibility were confirmed based on the mean relative standard deviation (RSD).

Conclusions
A novel electrochemical sensor on the basis of MoS 2 -NSs-modified SPE was established for the determination of INZ in the presence of AC. According to the findings, the MoS 2 NSs exhibited a huge surface area and an admirable conductivity, thereby providing good electron transfer and unparalleled electrocatalytic performance in INZ and AC oxida-tion. There were distinct INZ and AC oxidation peaks that predisposed the detection of these two analytes concurrently on MoS 2 NSs-SPE. A low cost of production, impressive sensitivity, and narrow limit of detection make this sensor an appropriate candidate for selective determinations of target analytes in clinical and pharmaceutical preparations. The applicability of the as-developed sensor was confirmed by determining the INZ and AC in real tablets and urine specimens, with acceptable recoveries.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.