Electrochemical Sensing of Phenylbutazone using Multi-Walled Carbon Nanotube Paste Electrode in Pharmaceutical and Biological Fluids

The electrochemical performance of phenylbutazone (PBZ) was studied using a multi-walled carbon-nanotube-modified paste electrode (MWCNT/CPE) using a variety of voltammetric tools like cyclic voltammetry (CV), linear sweep voltammetry (LSV), and square wave voltammetry (SWV). The results showed that the MWCNT/CPE exhibited remarkable electro-catalytic action towards the electrochemical oxidation of PBZ in a phosphate buffer solution of physiological pH 7 compared to a bare carbon paste electrode. The electro-kinetic parameters like heterogeneous rate constant, transfer coefficient, scan rate, pH, and involvement of electrons in electro-oxidation of PBZ was investigated. For bare CPE, the peak current was noted to be 19.53 μA with peak potential of 0.6871 V. For MWCNT/CPE, the peak current was 30.53 μA with peak potential of 0.6792 V. The anodic peak was analyzed, and the process was diffusion controlled. For the estimation of PBZ, a SWV technique was developed with great precision and accuracy, with a detection limit of 5.2 nM and a limit of quantification of 17 nM, in the concentration range 1 × 10−7 to 10 × 10−6 M. The MWCNT/CPE has been used successfully for PBZ detection in injection, blood, and urine samples, with recovery rates of 98.9% to 101.5%, 96.3% to101.7% and 98.3% to 102.8%, respectively.

The advancement of a sensitive, simple, credible, and fast tool for estimating drug concentrations is critical.In comparison to other instrumental methods, electroanalytical techniques are strong alternatives.These methods have been proven to be effective in determining pharmaceutical, environmental, and biological compounds in a variety of matrices.Experimental electrochemical techniques have advanced in the field of drug analysis due to their simplicity, short analysis time and low cost when compared to other techniques.
Phenylbutazone (PBZ) named as 4-Butyl-1,2-diphenylpyrazolidine-3,5-dione (Scheme 1), often referred to as "bute," is a nonsteroidal anti-inflammatory 1 drug (NSAID) that can be used to treat fever, pain, and inflammation 2 that results from ankylosing spondylitis, 3 rheumatoid arthritis, 4,5 gout, and osteoarthritis. 6,7NSAIDs are nonnarcotic pain relievers, 8 used for a variety of reasons, including injury, menstrual cramps, 9 arthritis, and other musculoskeletal conditions. 10,11t is also widely used for the short-term treatment of fever and pain 12,13 in animals.However, PBZ is not a commonly used NSAID because of a unique potential for severe bone marrow toxicity, which results in dangerously low white blood cell counts.Therefore, it is often desirable to use the lowest effective dose to minimize side effects.The most common side effects of PBZ involves the gastrointestinal system.][24] However, the main issues with using such approaches, are the building up of detectors, time-consuming extraction, usage of costly instruments and separation practices.
In electrochemistry, multi walled carbon nanotubes (MWCNT) continue to draw a lot of attention.Due to their unique structural, mechanical, electrical, and chemical capabilities, MWCNT have been the focus of numerous studies in the chemical, physical, and material sciences. 25MWCNT may be able to facilitate charge transfer reactions when used as an electrode due to their subtle electronic properties.MWCNT have been used to modify electrode substrates for use in analytical sensing, and the results have been shown to produce low detection limits, high sensitivities, a decrease in overpotentials, and resistance to surface fouling.MWCNTs have been developed as electrocatalysts, and electrodes modified with MWCNT have been found to function exceptionally well in the investigation of a variety of biological species.This is the first report that we are aware of, that describes the investigation of PBZ and assessing PBZ in urine and blood plasma samples at multi walled carbon nanotube modified paste electrode (MWCNT/CPE) through voltammetric techniques. 26This research aims to find the best experimental conditions for investigating the voltammetric performance of PBZ at MWCNT/CPE using CV, LSV & SWV with remarkable accuracy and precision.The proposed sensor has several advantages, including a low limit of detection, high sensitivity, good reproducibility, and appropriate for both pharmacokinetic research and quality control laboratories for estimation of PBZ.

Experimental Segment
Chemical reagents and materials.-Thechemicals were used of purely analytical in nature.Phenylbutazone, MWCNT (>98%, O.D X L6-13nM X 2.5-20 μm) and graphite powder were purchased from Sigma-Aldrich, paraffin oil was procured from Molychem.The appropriate amount of PBZ was dissolved in carbinol to make the stock solution, a supporting electrolyte of phosphate buffer from pH 3.0 to 10.4 has been fabricated by mixing the appropriate proportion of H 3 PO 4 , NaH 2 PO 4 , and Na 2 HPO 4 in Millipore water. 27A native veterinary government facility provided PBZ content injection (Arigesic) for the analysis.0.1 mM Potassium ferrocyanide, 0.1 mM potassium chloride solutions were prepared & used to estimate the active surface area of the working electrode.Temperature 25 ± 1 °C was maintained throughout the experiment.
Instrument.-Electrochemical analysis has been conducted on the CH6156e electrochemical system procured from United States z E-mail: stnandibewoor@yahoo.com Preparation of MWCNT/CPE.-MWCNT/CPEworking electrode has been made by using a short hollow Teflon rod, which was provided with a cavity at one end.A heavy copper wire was inserted through the rod until it reaches the well's bottom for electrical contact.The paste was made by combining the graphite powder and multi walled carbon nanotube with paraffin oil (of 4:3:3 ratio respectively), in a mortar until the mass got evenly wetted.The paste was tapped into the well and polished to get a smooth surface area.At every interval, before inserting a new quantity into the electrode, the paste was carefully removed.
Preparation of real sample for analysis.-Theproposed methodology was evaluated to assess its efficacy in quantifying PBZ concentrations in biological matrices, specifically human urine and blood plasma.Blood plasma and urine samples were collected from healthy volunteers who were not under the influence of any drugs.These samples were obtained for the purpose of analysis.testing period.The collected drug-free urine sample were subjected to filtration using filter paper.Subsequently, the filtered samples were preserved in a refrigerator until the time of testing.Similarly, the Remi R-8C Centrifuge (REMI Sales & Engineering Ltd, India) was used to obtain blood plasma by centrifuging human blood at 7000 rpm for 20 min.Further, a phosphate buffer solution with a pH of 7.0 was utilized to carry out a 100-fold dilution of both the plasma and blood samples. 28Subsequently, the biological samples were supplemented with the standard PBZ solution as a spike.The calibration plot was generated utilizing the square wave voltammetry (SWV) method within the context of optimal experimental conditions.To evaluate the precision and accuracy of the proposed model, the samples were subjected to four replicates of analysis.Likewise, an examination was conducted to assess the accuracy of the proposed methodology by investigating the influence of excipients.
Analytical approach.-Forthe fine resolution of the voltammetric peak and high sensitivity, polishing of MWCNT/CPE was done to get a smooth, glossy surface area.Activation of the working electrode was done in 0.2 M PBS of pH 7.0 by CV sweep from 0.0 to +1.2 V till a steady voltammogram was recorded.Later on, electrodes were rinsed with distilled water and placed in a 10 ml solution containing phosphate buffer (0.2 M. pH7) and PBZ.Cyclic voltammograms were observed between 0 to +1.2 V, with a sensitivity: 1.0 × 10 -5 A/V and a sweep rate of 0.05 Vs −1 .Adequate voltammetric results were noted, after 10 s of accumulation in a closed circuit with stirring.

Results and Discussion
Area of the working electrode.-Atdifferent scan rates, the surface area of the working electrode was calculated using the CV technique with 1.0 mM K 4 Fe(CN) 6 as a probe.The following Randles-Sevcik's formula 1 29 was used for a reversible process.
in which I pa denotes the anodic peak current, A 0 indicates the electrode active surface area (cm 2 ), D 0 indicates diffusion coefficient, n denotes the total number of electrons transmitted, υ indicates the scan rate, C 0 is the concentration of K 4 Fe(CN) 6 , R, F refers to the universal gas constant, Faraday's constant respectively.For 1.0 mM K 4 Fe(CN) 6 in KCl 0.1 M electrolyte solution, T = 298 K, D 0 = 7.20 × 10 -6 cm 2 s −1 , F = 96,480 C mol −1 , n = 1, R = 8.314 J K −1 mol −1 , the electroactive area was calculated based on the slope of the plot I pa vs υ 1/2 .The electrode's active surface area was determined to be 0.021 cm 2 for bare CPE, while for MWCNT/CPE showed 1.5 times larger area than the nascent electrode in our experiment.
Cyclic voltammetric response of PBZ at MWCNT/CPE.-Voltammetriccharacteristics of PBZ at the MWCNT/CPE were recorded by means of cyclic voltammetry at PBS pH 7.0 (Fig. 1).The result obtained for 0.01 M PBZ solution at the MWCNT/CPE shows a well-defined oxidation peak, with a high current at 30.53 μA (Fig. 1 Inset), potential 0.6792 V as compared to bare CPE.With the rise in the number of successive sweeps, the anodic peak current decreased remarkably (Fig. 2).This clearly indicates the deposition of oxidation product on the surface of the electrode.Thus, the first cycle of the corresponding voltammogram was noted.On a reverse scan, no cathodic peak was observed, which ensures an irreversible electrochemical process.
Influence of supporting electrolyte.-ThepH of the medium may influence the electrode reaction.Electrochemical analysis of PBZ was studied in a different supporting electrolyte such as acetate buffer, Britton Robinson, phosphate buffer.Fine resolution of voltammetric peak and high sensitivity was noted in phosphate buffer.Hence the voltammetric behavior of 1.0 mM PBZ was studied in phosphate buffer of pH 3.0-10.4(Fig. 3A) by cyclic voltammetry.
A high peak current was observed with pH 7.0.As a result, pH 7.0 was chosen as a supporting electrolyte and used throughout the experiment.When we plot a graph of voltammetric peak potential E p vs pH (Fig. 3A Inset), as the pH of the solution increased, the oxidation peak potential shifted linearly to lesser positive values.As a result, we could see that E p and pH have a linear relationship.As in The slope of this equation was observed to be −27.2mV pH −1 , which is close to the theoretical value of −30 mV pH −1 , revealing that the rate-determining step includes unequal number of protons and electrons i.e., one proton and two electrons 30,31 transfer.It is obvious that the pH value has an impact on peak current.The best result in terms of sensitivity and the sharp response was obtained with pH 7 (Fig. 3B).
Influence of scan rate.-Theelectrochemical mechanism of the PBZ analyte was successfully proposed at the fabricated CPE by monitoring the scan rate variation.At 7.0 pH, the electron sensing of PBZ at the MWCNT/CPE electrode was studied at different sweep rates ranging from 05 to 300 mV s −1 using cyclic voltammetry (Fig. 4A), and linear sweep voltammetry (Fig. 5) to investigate its  A straight-line plot of the log of I p vs log of υ with slopes of 0.5639 for CV (Fig. 4C) and 0.5778 for LSV (Fig. 1S (B)), which is close to the theoretic value of 0.5, which once again proves that the electrode process is diffusion driven. 33Following are the respective With the rise in scan rate, the oxidation peak started shifting to positive potentials.In the range of 05 to 300 mV s −1 , a linear corelation was observed between E p and log v, which explains the physiochemical properties of the electrode reaction.The Eqs. 7 and 8 express a justified linear equation between E p and the log υ of for CV (Fig. 4D) and LSV (Fig. 1S (C)).The number of electrons were calculated using the Laviron equation, 34 as given in Eq. 9.  ECS Advances, 2024 3 026501 electrons migrated, υ (nu) represents the scan rate, and E 0 represents the formal redox potential.The meanings of the other symbols are as expected.Thus, the slope of E p vs log υ can easily be used to calculate the value of αn, which was found to be 0.0502 for CV and 0.0547 for LSV.Using standard gas constant R = 8.314462 J K −1 mol −1 and Faraday constant F = 96485 C mol −1 αn was calculated.According to Bard and Faulkner. 35Equation 10 α can be provided by: E pa/2 is the potential at which the current is half of its maximum value.As a result, we were able to calculate the α value for both CV and LSV methods, and the number of electrons (n) transferred during the electrooxidation of PBZ was determined to be 2.The proposed mechanism for the electrochemical oxidation of PBZ at MWCNT/CPE is as depicted in Scheme 2. The values of k 0 were obtained from the intercepts of the graph of E P vs log υ (Eq. 5. E 0 value in the equation was determined by extrapolating the intercept of the E P vs υ plot to the vertical axis at υ = 0.The results obtained from CV and LSV are given in Table I.
Calibration curve and limit of detection.-Toperform a quantitative analysis of PBZ, square wave voltammetric mode was adopted at pH 7, because of its detection limit even at very low concentrations, with significant sharper and defined peaks, as compared to a cyclic voltammetric method with low background current.
As shown in Fig. 6 Square wave voltammetry achieved with increasing concentrations of PBZ revealed that the peak current increases linearly with the increasing concentration of PBZ.A linear calibration curve was acquired for PBZ (Fig. 6 Inset) in the range of 1 × 10 -7 M to 10 × 10 -6 M as shown in Eq. 11.The limits of detection (LOD) and limits quantification (LOQ) were determined to be 5.2 nM and 17 nM, respectively.using the following Eq. 12 where slope procured from the calibration graph is denoted by "m" and standard deviation of the blank peak currents (four runs) is denoted by "s".The LOQ and LOD values of the present work is compared with the literature data given by the voltammetric and classical methods in the determination of PBZ, 23,36,37 as shown in Table II.From the table, we can see that the method proposed has significantly improved the limit of detection compared to the earlier reported methods that have been attested.Precision of the proposed method was investigated by intra-and inter-day determination of PBZ at two unlike concentrations for n = 4 (n = number of determinations).inside the linear range. 38Table 1S represents the accuracy of the methods.
Effect of excipients.-Impact of some commonly used excipients in pharmaceutical formulation was investigated as an analytical application for the recommended technique.The tolerance limit for PBZ determination was defined as the highest concentration of the interfering chemical substance that induce an error of less than 5%.Sample solutions with a predetermined amount of PBZ (10 μM) spiked with excess amounts of each interferent were analyzed under experimental conditions similar to the calibrations curve, to see how they affected the voltammetric response.The outcomes of the experiment (Table III) showed that hundredfold excess of Zn 2+ , Ca 2+ , Cu 2+ , Fe 3+ , Mg 2+ , citric acid, D-glucose, lactose, oxalic acid, ascorbic acid, sucrose, and tartaric acid did not affect the  ECS Advances, 2024 3 026501 voltammetric signal of PBZ (Fig. 7).As a result, the method was able to test PBZ in the existence of excipients, making it specific.
Determination of PBZ in urine sample.-Thehighly sensitive SWV technique was adopted to determine PBZ in urine samples, which was collected from a healthy individual, further phosphate buffer pH 7 was used to dilute urine samples hundred times before being analyzed.Quantitative determination was carried out by adding the known amount of PBZ solution to the collected urine sample.The calibration plot was used in order to determine the recovery data, and it was found to be 98.3% to 102.8%, with a R.S.D of 1.96%.The observed results (Table IV) show good PBZ recoveries at a 95% confidence level, indicating that the proposed methods are useful for assessing PBZ in blood plasma sample.Determination of PBZ in plasma sample.-Thedeveloped SWV procedure was also used to determine PBZ concentrations in spiked plasma samples.The plasma samples were prepared according to the procedures outlined in Instrument section.Further, the blood plasma sample was diluted hundred times in PBS pH 7.0 before analysis.The recoveries from blood plasma were recorded by spiking drugfree human plasma with known concentration of PBZ.For the quantitative analysis of PBZ spiked plasma samples, the calibration plot was used.Table V shows the amounts of PBZ spiked and the amounts detected.The recovery rate was determined to be between 96.3% to101.7%, with an RSD of 2.4%.PBZ recoveries remained good in these matrices with 95% confidence level.
Analytical application in pharmaceutical formulation.-TheMWCNT/CPE was used for the estimation of pharmaceutical injection containing phenylbutazone with a trading name Arigesic.The injection sample was provided by a native veterinary government hospital with specified content of PBZ of 200 mg and Sodium salicylate 20 mg.This sample was used to investigate PBZ in injection after proper dilution with PBS (0.2 M) pH 7.0 using the calibration curve method.The recovery and R.S.D were suitable, we can be 95% confidence for the detection of PBZ in injection sample (Table VI), indicating that the proposed technique could be effective in determining PBZ in injections with a good recovery.
Repeatability and reproducibility of the MWCNT/CPE.-Toevaluate the efficiency of the fabricated electrode, steadiness and reproducibility tests were performed by storing the prepared sensors in an airtight container for 15 d. and even after that the sensor retained 97.3% of its previous oxidation current response with RSD % of 2.46% for a 0.1 mM PBZ sample.This reveals the long shelflife of the MWCNT/CPE.

Conclusions
In the present work, the electrochemical oxidation of PBZ in physiological pH 7.0 at a MWCNT/CPE has been explored through the voltammetric techniques.The findings revealed that PBZ undergoes one proton, and two electrons transfer, and the sequence was controlled by diffusion.The square wave voltammetric method is effective, as evidenced by a high percentage recovery and an examination of the spiked urine, plasma, and injection.The procedure is free from the interferences of the generally used additives and excipients in the formulation of the drug.This technique could be the best option for determining PBZ analytically because it is simple, sensitive, quick, precise, and low-cost in comparison to other methods.Thus, the proposed technique can also be used in quality assurance labs and pharmacokinetic studies.

Declaration of Competing Interest
The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

ECS Advances, 2024 3
026501 by CH Instruments Inc.The voltammetric techniques such as CV and SWV for PBZ were performed in a 10 ml single conventional compartment of a three-electrode setup, reference electrode made of glass cell with Ag/AgCl (3 M potassium chloride), auxiliary electrode made of platinum wire, and working electrode made of MWCNT/CPE.All potentials are measured against a reference electrode of Ag/AgCl (3.0 M KCl).The pH was measured with an Elico LI 120 pH meter (Elico Ltd, India), which was previously calibrated with solutions of known pH 4 & 7.The Remi R-8C Centrifuge (REMI Sales & Engineering Ltd, India) was used to obtain blood plasma by centrifuging human blood.All the results were quoted for a CV, LSV and SWV are the average of five determinations.

Eqs. 5
and 6 for CV and LSV:

Figure 7 .
Figure 7. Effect of different types of excipients on the activity of PBZ.

Table I .
Results obtained by CV and LSV from the impact of scan rate.

Table II .
Comparison of detection limits and linear range for PBZ to different reported techniques.

Table III .
Impact of interferents on the voltammetric response of 10 × 10 -6 M PBZ.

Table IV .
Determination of PBZ in urine samples.
UCL-Upper Confidence Limit.a) Average of five measurements.

Table V .
Determination of PBZ in plasma samples.

Table VI .
Determination of PBZ in injection sample.