Simultaneous detection of ethambutol and pyrazinamide with IL@CoFe2O4NPs@MWCNTs fabricated glassy carbon electrode

For the first time, we report a novel electrochemical sensor for the simultaneous detection of ethambutol (ETB) and pyrazinamide (PZM) using 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF4]) ionic liquid (IL) assimilated with multiwalled carbon nanotubes (MWCNTs) decorated cobalt ferrite nanoparticles (CoFe2O4NPs) on the surface of glassy carbon electrode (GCE). The surface morphological and electrochemical properties of the IL@CoFe2O4NPs@MWCNTs was characterized with X-ray diffraction (XRD), transmission electron microscope (TEM), thermogravimetric analysis (TGA), fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry (CV), differential pulse voltammetry (DPV) respectively. Moreover, the obtained results of CV demonstrated that the 9-folds enhancement in the electrochemical signals was achieved with IL@CoFe2O4NPs@MWCNTs@GCE compared to that of a bare GCE. Additionally, the simultaneous electrochemical detection of ETB and PZM was successfully accomplished using IL@CoFe2O4NPs@MWCNTs over a wide-range of concentration with good limit of detection (3S/m) of 0.0201 and 0.010 μM respectively. The findings of this study identify IL@CoFe2O4NPs@MWCNTs@GCE has promising abilities of simultaneous detection of ETB and PZM in pharmaceutical formulations.

Scientific RepoRtS | (2020) 10:13563 | https://doi.org/10.1038/s41598-020-70263-z www.nature.com/scientificreports/ nanomaterial adopted in the fabrication of electrode surfaces due to their high flexibility, high tensile strength, elasticity and high conductivity of the electrons 14,15 . Thus, the MWCNTs was used as a fundamental electrode coating material in the present study.
Recently, metallic and transition metal oxide nanoparticles especially spinel ferrite MFe 2 O 4 has growing the interest of researchers in the development of ultrasensitive electrochemical biosensors with multifaceted applications ranging from environmental to point-of-care applications, whereas magnetic and electrical conducting nature of the MFe 2 O 4 nanocomposite depends on the M 2+ cation 16,17 . The cations such as Co, Mg, Cu, Ni, Fe, Zn and Mn were often used metals in the nanocomposites 18 . Generally, spinels ferrite substance CoFe 2 O 4 has significant features including magnetic, electronic, energy storage and analytical biochemical applications 19 . Nowadays ionic liquids (ILs) based electrochemical techniques with ion-selective sensors has attracted scientists in the fabrication of biosensor devices due to its improved lifetime, stability, promotes rapid electron transfer and sensitivity. The good catalytic ability together with the facile experimental methodologies significantly promotes the application of ILs-CNs based microelectrodes in the electrochemical sensing of different analytes [20][21][22] .
In this study, a selective and sensitive method was established to develop a robust electrochemical sensing platform with IL@CoFe 2 O 4 NPs@MWCNTs@GCE. To the best of our knowledge, IL@CoFe 2 O 4 NPs@MWCNTs@ GCE is the first report on its own for the simultaneous detection of ETB and PZM and successfully applied to the pharmaceutical formulations. instrumentation. All electrochemical studies were performed with 797VA computrace system (Metrohm Herisau, Switzerland) with conventional three-electrode system including Ag/AgCl (3 M KCl), GCE and platinum wire as reference, working and auxiliary electrodes respectively. Fourier transformation infrared (FTIR) characterization was performed using Varian 800 FTIR scimitar series (by SMM instruments). TGA/DSC analysis was conducted with STAR e system (model: 1 SF/1346) from METTLER TOLEDO. A CRISON digital micro pH meter with an accuracy of ± 0.1 was used for the pH adjustments. A TEM (model: JEM 2100 with a Lab 6 emitter) was used to evaluate the surface morphology of the nanocomposite. Furthermore, sonication was performed with a LABCON 5019 U model throughout the study. After adding of NaOH the pH of the solution was continuously observed. The solution mixture was continuously stirred using a magnetic stirrer until it reach pH 12. Additionally, a definite volume (2 mL) of oleic acid was added and subjected to heating for 60 min at 90 °C, resulting in a brown colour precipitate. The obtained precipitate was given several washings with deionized distilled water followed by the ethanol to eliminate free Na + , Cl − as well as other surfactant and dried for 15 h at 90 °C. Finally, the obtained solid NPs material was then grinded into a fine powder for further use.

Synthesis of cofe
Electrode modification. Before the fabrication process, the GCE was polished to a mirror like surface with 0.3 μm alumina slurry. Then after, the electrode was washed with deionized distilled water for five times. Thereafter, the GCE was sonicated with an aqueous solution of deionized distilled water and ethanol (1/1, v/v) for 15 min. The MWCNTs suspension was made by the previous described procedure with slight amendment 24    Real sample preparation. Five of each commercial brands were obtained from a local dispensary, exactly weighed, and then grounded to a fine residue with mortar and pestle. The obtained powder was dissolved in 10 mL deionized distilled water and sonicated for 20 min. The resulted mixture was filtered with a Whatman no: 1 paper and 10 mL of the successive solution was then transferred into a 15 mL standard flask and dilute with phosphate buffer solution.  422) and (533) 25 . The TEM analysis was performed to assess the exact particle size of CoFe 2 O 4 NPs. As shown in Fig. 3a, the measured particle size was found to be in the range of 8-25 nm, which is in the agreement with XRD data. The obtained TEM data shows that the most of the particles are spherical in shape with monodispersity (see Fig. 3a). Figure 3b shows the pure MWCNTs with a tubular network like structure. Figure 3c illustrates (Fig. 4a,b). The obtained results demonstrated that the IL@CoFe 2 O 4 NPs@MWCNTs@GCE exhibited the amplified electrochemical signals due to the rapid conductivity of the electrons compared to that of MWCNTs@GCE and bare GCE. Therefore, the active surface area was measured using Randles-Sevcik Equation 30,31 .    Fig. 5a,b). The response current of the ETB and PZM was increased with increasing pH from 3.0 to 7.0. Beyond pH 7.0, the response current was further decreased. Therefore, pH 7.0 was selected as an optimum throughout the study. As depicted in Fig. 5a,b the peak potentials of ETB and PZM shifted towards positive potential with the increase in pH, indicating the direct involvement of protons redox reaction procedures. The linear regression equations of ETB and PZM are stated as follows individually:

Results and discussion
The obtained slopes from the current vs pH in the pH range of 3.0-9.0 indicates equal number of protons and electrons are participating in the reduction of ETB and PZM as described previously 32 .
Deposition time. Figure 6 represents the effect of deposition time as a function of response current from 30-150 s. It was noted that the anodic and cathodic peak currents were increased proportionally with the deposition time between 30 and 150 s. It is well known that the sensitivity improves with a longer deposition time due to the availability of electrode surface to the analyte at the lower concentration, however upper detection limits also increases due to the attainment saturation of the electrode surface at higher concentration of the analyte. In this study, the maximum response current was observed at 60 s and the peak current decreases gradually with the increase in the deposition time due to the achievement of saturation of GCE. By considering the sensitivity factor in to account, the optimum deposition time of 60 s was selected throughout the experiment.   (Fig. 7a,b) and vice-versa. The obtained results were depicted in Figs. 7a and 8a,b. Figures 7a and 8a showed that the increase in the concentration of ETB, the current has increased linearly with the unaffected peak current of PZM. Moreover, the calibration curve for ETB and PZM were noted as I pa = 22.96ETB + 1.918 (R 2 = 0.9912) and I pa = 15.986PZM + 2.9451 (R 2 = 0.9913). Secondly, for the simultaneous detection, the concentration of the ETB and PZM were concurrently increased, resulting in the linear increase in the peak current ranging from 0.2 to 2.2 µM for ETB and 0.6 to 2.8 µM for PZM as illustrated in Fig. 9. The linear regression equations were shown below:   Fig. 9. Moreover, no significant interference effect was observed for the simultaneous detection of RTB and PZM due to the enhanced selectivity of developed IL@CoFe 2 O 4 NPs@MWCNTs@ GCE sensor. Additionally, the LOD's for ETB and PZM were individually and noted as 0.0201 and 0.010 μM respectively. The comparison of present sensor with the reported sensors were tabulated in Table 1.

Reproducibility, stability and interferences. The performance of the IL@CoFe 2 O 4 NPs@MWCNTs@
GCE was evaluated with DPV under the optimized conditions (pH: 7.0, accumulation time: 100 s and suspension volume: 5 μL). The reproducibility of the developed IL@CoFe 2 O 4 NPs@MWCNTs@GCE examined by using the same electrode modification for six individual measurements with 0.1 μM of ETB and PZM. The results obtained shows that the relative standard deviation (RSD) was 2.62%, indicating the acceptable reproducibility for the detection of ETB and PZM. To validate the stability, the IL@CoFe 2 O 4 NPs@MWCNTs@GCE was kept at ambient temperature in the laboratory for 15 days and then the electrochemical measurements were performed. Interestingly, it was found that 95% of the electrochemical signals were retained. The interference effect was studied by including organic compounds, common ions and electrochemical signals were noted with DPV at 0.1 μM ETB and PZM. The obtained results were presented in Table 2 demonstrated that the 50-fold increase in the concentration of ions such as Ca 2+ , K + , Mg 2+ , Na + , Cu 2+ , Cl − , NO 3− , Cd 2+ , and SO 4 2− does not interfere with the ETB and PZM signals. On the other hand, 100-fold increase in the concentration of ascorbic acid, glucose and uric acid also does not impact the detection of ETB and PZM.
Real sample analysis. The real applicability of IL@CoFe 2 O 4 NPs@MWCNTs@GCE was examined by considering the pharmaceutical formulations of ETB and PZM. The standard addition procedure was adopted for the detection of ETB and PZM and the obtained results were tabulated in Table 3. The acceptable recoveries were noted for the detection of ETB and PZM. The developed IL@CoFe 2 O 4 NPs@MWCNTs@GCE sensor might be competent in the detection of ETB and PZM in more commercial pharmaceuticals compared to that of other related techniques 16,33-44 . conclusions We report, for the first time, simultaneous electrochemical detection of ETB and PZM with IL@CoFe 2 O 4 NPs@ MWCNTs in pharmaceutical formulations more effectively. The investigated drugs are formulated together in a single pharmaceutical dosage procedures. In addition, these results confirmed that the IL@CoFe 2 O 4 NPs@ MWCNTs is highly selectivity, sensitivity and low detection limits for the simultaneous detection strategy due to the high surface area and rapid electron conductivity of CoFe 2 O 4 NPs. Finally, the capability of the developed (5) PZMI pa = 13.656PZM + 1.6796R 2 = 0.9904  Graphene oxide-poly arginine poly-l-methionine-glassy carbon electrode CV and DPV 3.28 Ref. 40 Poly-l-methionine-glassy carbon electrode CV and DPV 0.035 Ref. 41 Screen printed carbon electrode-poly-histidine prepared by histidine monomer electro polymerization DPV and SWV 68 Ref. 42 Graphene-zinc oxide nanoparticles-carbon paste electrode CV and DPV 0.0431 Ref. 43 Poly-l-methionine-reduced graphene oxide-glassy carbon electrode CV and DPV 0.  www.nature.com/scientificreports/ electrochemical sensor was tested on pharmaceutical formulations, yielding a good analytical performance and thus providing a promising alternative for sensing applications in the pharmaceutical and biochemical industries.