Electro-sensing base for mefenamic acid on a 5% barium-doped zinc oxide nanoparticle modified electrode and its analytical application

Abstract

In the present work, surface enhanced electro-oxidation of mefenamic acid (MFA) at a glassy carbon electrode modified with 5% barium doped ZnO nanoparticles was studied. Cyclic voltammograms with a maximum peak current at pH 5 in the pH range of 3.0–11.0 were recorded at a scan rate of 50 mV s⁻¹, with a distinct oxidation peak in the range of 0.2–1.0 V (versus Ag/AgCl). Two protons and electrons accompanied the electrode reaction. Based on the experimental results, a possible electro-oxidation mechanism for MFA was proposed. Quantitative analysis of MFA was carried out using a differential-pulse voltammetric method (DPV). Linearity was observed in the range of 500 nM to 10.0 × 10³ nM. LOD and LOQ were calculated to be 6.02 nM and 20 nM, respectively. In addition, the developed method was used in the in vitro analysis of MFA in pharmaceutical formulations and spiked human urine samples.

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Introduction

Electrochemical methods with comparatively low cost, high sensitivity and selectivity, more accuracy and wide linear dynamic range have been used as flexible measurement tools in biological,^1,2 food^3,4 and environmental^5,6 investigations. The use of electrochemistry in pharmaceutical⁷ analysis including drug discovery, particularly for investigation of the physiological behaviour of their dosage form, has emerged as an important approach. These techniques can provide useful information on the electro-oxidation mechanism of pharmaceutical molecules, which can give an insight into their drug activity or transformation during metabolism. Nevertheless, the use of bare electrodes in electro-analytical methods has limitations such as slow electron transfer and fouling problems.⁸ These limitations may be solved by the use of highly sensitive and selective electrodes with different modifications. Nanoparticles, surfactants, carbohydrates, dyes, etc. can be used as modifiers to increase the sensitivity and selectivity of the electrodes.

Nanocrystalline materials have attracted broad interest owing to their diverse character and huge potential applications in nanodevice fabrication.^9–11 Advantages such as specific surface area, non-toxicity, optical transparency, chemical and photochemical stability, ease of fabrication, and their electrochemical activity have allowed ZnO nanoparticles to attain a unique position among the different nanomaterials.¹² ZnO nanostructures grown with the use of single crystal substrates have provided a suitable platform for the development of electronic applications. These nanostructures possess a high surface area to volume ratio and exceptional mechanical steadiness, which is suitable for sensor design based on ZnO nanomaterials.¹³ Recently, a number of researchers have completely focused on improving ZnO nanoparticle properties by using a dopant. At present, there are a few reported methods for ZnO nanoparticles which include the usage of Ag, Co, Sn, Cu, Cd, Pd and Cr as a dopant.^14–20

Mefenamic acid (MFA), a fenamate class of non-steroidal anti-inflammatory drug, is a well studied and effective pain mitigating non-steroidal anti-inflammatory drug, which is used for the treatment of many pathological conditions such as osteoarthritis, non-articular rheumatism, sports injuries and other musculoskeletal illnesses including menstrual pain.^21,22 Consumption of MFA has lead to unusual cases of nephrotoxicity and hepatotoxicity.²³ Hepatotoxicity induced by drug toxicity may be explained due to the formation of metabolites which are able to interact and covalently bind with tissue proteins leading to dysfunction or immunotoxicity.^24,25 MFA mediated toxicity is also due to the formation of chemically reactive metabolites which lead to adverse immunological responses by covalently binding to proteins in liver and kidney tissues.^26,27 Further, carboxylic acid-containing drug–protein adducts are assumed to form antigens, which lead to idiosyncratic allergic reactions.²⁸ Recent investigations have revealed that MFA is metabolized to mefenamyl-1-O-acyl glucuronide leading to MFA-mediated idiosyncratic toxicity.^26,29 In addition, MFA is third placed in the European Union list of priority contaminants.³⁰ Several studies on MFA removal have reported that MFA cannot be eliminated from conventional wastewater treatment plants and it has been detected in the effluent water of treatment plants.^31–34 In view of this, there is a need for development of more reliable and sensitive techniques for trace determination of MFA and understanding the oxidation pathway of MFA. In the past few years, a few reported methods for MFA determination have become available which include spectrophotometry,^35,36 fluorimetry,^37,38 potentiometry,^39,40 chromatography,^41,42 chemiluminescence,^43,44 and capillary electromigration.^45,46 In addition, reported electrochemical methods include the use of a cobalt hydroxide modified glassy carbon electrode,⁴⁷ a multiwalled carbon nanotube–polymer composite electrode⁴⁸ and a carbon nanotube–gold nanoparticle composite film modified electrode.⁴⁹

In the present work, Ba doped ZnO nanoparticles were synthesized and characterized using XRD, EDX, SEM and TEM. Further, the electrochemical behaviour and utility of a Ba doped ZnO nanoparticle modified electrode as a sensor for the determination of MFA were investigated. To the best of our knowledge, until now, the synthesis, characterization and electro-catalytic activity of Ba doped ZnO nanoparticles has not been explored. It is of interest to combine barium doped ZnO nanoparticles on an electrode surface to study the electrochemical behaviour and the determination of MFA. Hence, these studies were undertaken. Compared to a bare electrode and a ZnO nanoparticle modified electrode, an enhanced peak current with high selectivity and sensitivity was observed from a Ba doped ZnO nanoparticle modified electrode. Additionally, low detection and quantification limits compared to the earlier reported methods fulfil the need for trace determination of MFA. Good recoveries from urine and pharmaceutical samples enabled us to develop a method for determination of MFA in real samples, which can be adopted for pharmacokinetic studies along with applications in clinical and quality control purposes. The good percentage of RSD values obtained indicate the superiority in terms of reproducibility and repeatability of the proposed method.

Experimental

Reagents and chemicals

A pure form of MFA (Sigma-Aldrich) was used to prepare a 1.0 mM stock solution by dissolving an appropriate amount of MFA in ethanol stored at low temperature in the dark. Phosphate buffer was used as a supporting electrolyte (I = 0.2 M) and to sustain the solution pH between 3.0–11.0. Double distilled water and analytical grade chemicals and reagents were employed right through the experiment.

Apparatus

With a three electrode system and a 10 ml single compartment, electrochemical experiments were carried out in a CHI Company, USA (Model D630) electrochemical analyzer at an ambient temperature of 25 ± 0.1 °C. The oxidation of MFA was carried out on the surface of a 5% Ba doped ZnO nanoparticle modified glassy carbon electrode (working electrode) and the potential was measured against Ag/AgCl (reference electrode) and platinum wire as a counter electrode. The pH of the buffer solutions was recorded using an Elico pH meter (Elico Ltd., India). For the characterization of Ba doped ZnO nanoparticles, a Siemens X-ray diffractometer (XRD) (Cu source) AXS D5005 was used. Surface morphology was examined using a scanning electron microscope (SEM and EDX) JEOL, JSM-6360, and the topography and particle size were measured using a JEOL, JEM-2010.

Synthesis of 5% barium doped ZnO nanoparticles

5% Ba doped ZnO nanoparticles were prepared using a co-precipitation method. 100 ml of 0.1 M zinc nitrate (Zn(NO₃)₂·4H₂O) solution was mixed with a 5% molar ratio of barium nitrate and 10 mg L⁻¹ of sodium dodecyl sulfate and kept under constant stirring for one hour using a magnetic stirrer to completely dissolve the zinc nitrate. Sodium dodecyl sulfate is a surfactant which serves as a capping agent to control the particle size.⁵⁰ After complete dissolution of zinc nitrate, 100 ml of 0.2 M NaOH aqueous solution was added to the zinc nitrate solution with vigorous stirring (2000 rpm), drop by drop (slowly for 45 minutes). The reaction was allowed to proceed for two hours after the complete addition of sodium hydroxide. The beaker was sealed under these conditions and kept for two hours. After completion of the reaction, the solution was allowed to settle overnight and further, the supernatant solution was separated carefully. The remaining solution was centrifuged for ten minutes, and the precipitate was removed. Then, the precipitated ZnO nanoparticles were washed repeatedly with deionized water and ethanol to remove the by-products which were bound with the nanoparticles and then air dried at 60 °C for four hours. The obtained product was calcined at 500 °C for three hours using a muffle furnace. Calcination at 500 °C will decrease the volume resistivity of nanoparticles.⁵¹ Hence, a low resistivity indicates high moment of electric charge. The mechanism by which the barium dopes the ZnO nanoparticles is by entering into the crystal lattice of ZnO. It occupies an interstitial position in the ZnO lattice. Similar results were reported by W. Water et al.⁵²

Preparation of a 5% barium doped zinc oxide nanoparticle modified glassy carbon electrode (5% Ba doped ZnO nanoparticles/GCE)

1 mg of 5% Ba doped zinc oxide nanoparticles was dispersed in 10 ml double distilled water using an ultrasonicator to give a white suspension. Alumina particles of 3.0 μm size were used to polish the surface of the glassy carbon electrode. Alumina particles that settled on the electrode surface were removed by rinsing with ethanol and then with distilled water. The cleaned GCE was coated with 25 μL of the suspension of 5% Ba doped ZnO nanoparticles and dried in air. To activate the electrode surface, cyclic voltammograms were recorded in phosphate buffer solution (pH = 5) between 0.2 V to 1.0 V until a steady voltammogram was obtained.

Electro-active surface area of the electrode

The Randles–Sevcik formula was used to calculate the electro-active area of the electrode using 1.0 mM K₃Fe(CN)₆ as a probe at different scan rates and 0.1 M KCl as a supporting electrolyte in a cyclic voltammetric method. At T = 298 K and for a reversible process, the equation is as follows:⁵³

I_p = (2.69 × 10⁵)n^3/2A₀D_R^1/2ν^1/2C₀ (1)

In eqn (1) for 1.0 mM K₃Fe(CN)₆ and 0.1 M KCl as supporting electrolyte; I_p: anodic peak current, n: number of electrons transferred during the electrode reaction = 1, A₀: surface area of the electrode, D_R: diffusion coefficient = 7.6 × 10⁻⁶ cm² s⁻¹, ν: scan rate and C₀: concentration of K₃Fe(CN)₆. Hence, A₀ was calculated using the slope of the plot, I_p vs. ν^1/2. The A₀ of the bare GCE was calculated to be 0.0423 cm² and the surface area of the 5% Ba doped ZnO nanoparticle modified GCE shows an increment in surface area of about 3–4 times and was calculated to be 0.165 cm².

Sample preparation

Ten pieces of MFA tablets were ground in a mortar. A weight corresponding to a 1.0 × 10⁻³ M stock solution was taken in a 100 ml calibrated flask and made to volume with double distilled water. After sonication for ten minutes to attain complete dissolution, suitable aliquots of clear supernatant liquid were diluted with phosphate buffer solution (pH = 5.0) to prepare appropriate solutions. Each solution was transferred to a voltammetric cell and analyzed by a standard addition method. Differential pulse voltammograms were recorded between 0.2 to 0.8 V after open circuit accumulation of 60 seconds with stirring, and the oxidation peak current of MFA was measured. The parameters for differential pulse voltammetry were a pulse increment of 0.004 V, pulse width of 0.06 s, pulse amplitude of 0.05 V and pulse period of 0.2 s. The accuracy of the proposed method was tested using recovery experiments. Further, the concentration of MFA was calculated using a standard addition method (a known amount of standard solution of a different concentration was added to several solutions containing the same amount of solution of unknown concentration).

Results and discussion

Characterization of 5% Ba doped ZnO nanoparticles

Characterization of 5% Ba doped ZnO nanoparticles was done using XRD, EDX, SEM and TEM. Fig. 1 shows XRD patterns with distinct diffraction peaks, corresponding to the (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) lattice planes, revealing that the prepared nanoparticles have a wurtzite (hexagonal) structure. The XRD pattern of 5% Ba doped ZnO nanoparticles shows that the peaks of ZnO shift towards lower angles in relation to pure ZnO nanoparticles. Such shifts of the XRD peaks occur due to the difference in the ionic radii of the elements (Zn²⁺ = 0.088 nm, Ba²⁺ = 0.135 nm), revealing a lattice distortion due to the substitution of Ba for Zn in the ZnO crystal lattice. Similar results have been reported in the literature.^54,55 No segregation of pure Ba and BaO was detected in the XRD pattern which confirms the single phase of Ba doped ZnO. The average sizes of the crystallite, D, were calculated from the full widths at half maximum (FWHM), β, of the peaks using Scherrer’s formula⁵⁶ (ignoring the effect of stress):where θ is the Bragg angle, λ is the radiation wavelength (0.15406 nm), and k is a constant which depends on the peak shape, crystallite habit, and particle shape. It is found that the average crystallite size of pure ZnO is in the range of 26 ± 2 nm and the average crystallite size of 5% Ba doped ZnO is 36 ± 3 nm.

Fig. 1 X-ray diffraction patterns of undoped and Ba doped ZnO nanoparticles.

Fig. 2 shows the EDX spectrum of 5% barium doped ZnO nanoparticles. EDX analysis indicates that the atomic and weight percentages respectively of elements present were, Zn = 71.26 and 41.58, O = 23.95 and 57.10, and Ba = 4.78 and 1.33. From the SEM image of the 5% Ba doped ZnO nanoparticles (Fig. 3), it can be observed that all the films were uniform and homogenous with closely packed spherical grains. The doped films are compact relative to the doping level. This could be due to the fact that the incorporation of Ba in the starting solution improves the nucleation process. From the TEM image (Fig. 4) it can be concluded that the average crystal size of the nanoparticles was 20 nm, which is closely related to the data obtained from XRD.

Fig. 2 EDX spectrum of 5% Ba doped ZnO nanoparticles.

Fig. 3 SEM image of 5% Ba doped ZnO nanoparticles.

Fig. 4 TEM image of 5% Ba doped ZnO nanoparticles.

Cyclic voltammetric behaviour of MFA

Cyclic voltammograms of MFA at the GCE, ZnO/GCE and 5% Ba doped ZnO nanoparticles/GCE were recorded in a phosphate buffer of pH 5 (Fig. 5).

Fig. 5 Cyclic voltammograms of 1.0 × 10⁻⁴ M MFA in phosphate buffer solution of pH 5 (I = 0.2 M); scan rate: 50 mV s⁻¹, accumulation time 60 s, for the (a) blank bare GCE, (b) blank with 5% Ba doped ZnO nanoparticle modified GCE, (c) MFA run on GCE, (d) MFA run on ZnO nanoparticle modified GCE, and (e) MFA run on 5% Ba doped ZnO nanoparticle modified GCE.

As a result of sluggish electron transfer the peak current at the bare GCE and ZnO nanoparticle modified electrode was weak, whereas the response was significantly enhanced with high current when the 5% Ba doped ZnO nanoparticles/GCE was employed. The enhancement in the peak for the Ba doped ZnO nanoparticles/GCE was attributed to the better catalytic activity of barium.⁵⁷ Further, it may also be presumed that, at pH 5.0, MFA is almost in a negatively charged form which increases the interaction between MFA and the 5% Ba doped ZnO nanoparticles/GCE.⁵⁸ MFA exhibits one anodic peak at 0.66 V with an anodic current of 9.0 μA. Irreversibility of the process was revealed due to the absence of the peak when a reverse scan was carried out. During consecutive cyclic voltammetric sweeps, a decrease in the peak current was observed due to surface adsorption of the oxidized product or MFA.

Effect of barium percentage on oxidation behaviour of MFA

Cyclic voltammetric behaviour of MFA upon varying the percentage of barium as a dopant was investigated to optimize the barium concentration. From the plot of percentage of barium versus peak current, it was observed that the maximum peak current was observed at 5 percent doping. Hence, 5% Ba doped ZnO nanoparticles were considered for further investigations (Fig. 6).

Fig. 6 Plot of percentage of barium versus current/μA, 1.0 × 10⁻⁴ M MFA; in phosphate buffer solution of pH 5 (I = 0.2 M) at 50 mV s⁻¹.

Effect of accumulation time

Fixing the accumulation time for which adsorption studies were undertaken, gives insight into the response of the peak current towards the adsorption of MFA on an electrode surface. A cyclic voltammetric technique for MFA in a range of 0–120 seconds was undertaken for the investigation. An optimal accumulation time of 60 seconds was selected for further experiments, since the maximum peak current was observed at 60 seconds (Fig. 7).

Fig. 7 Variation of the anodic peak current with time for 1.0 × 10⁻⁴ M MFA in phosphate buffer solution of pH 5 (I = 0.2 M) at a scan rate 50 mV s⁻¹.

Effect of the amount of 5% Ba doped ZnO nanoparticles

The influence of immobilized 5% Ba doped ZnO nanoparticle quantities on the peak current response of MFA was investigated using a cyclic voltammetric technique. An electrode surface was casted with 5% Ba doped ZnO nanoparticle solutions of disparate volumes. 5, 10, 15, 20, 25, 30 and 50 μL of 5% Ba doped ZnO nanoparticle solutions were used. An increase in volume, from 5–25 μL of 5% Ba doped ZnO nanoparticle solution, engendered an increment in peak current. A constant peak current was observed for more than 25 μL of 5% Ba doped ZnO nanoparticle solutions. Hence, 25 μL was established as the optimised amount of 5% Ba doped ZnO nanoparticle solution required to catalyze the oxidation of MFA, and was used further.

Effect of pH

Fig. 8 represents the voltammograms of MFA recorded in the range of pH from 3.0–11.0 at 50 mV s⁻¹. From the I_p versus pH plot (Fig. 8A), the maximum peak current was obtained at pH 5 and it shows that the variation of solution pH affected the peak current. Hence, pH 5 was selected as optimal for all measurements. The linear relationship between E_p and pH is expressed in Fig. 8B; and the equation is E_p (V) = 0.910 − 0.040pH; R² = 0.999. From the plot of E_p versus pH the peak potential was observed to shift towards a less positive value as the pH of the solution increased, and this indicates the participation of protons in the electrode process. Involvement of an equal number of protons and electrons was evidenced by the slope of E_p versus pH, which was 40.0 mV per pH unit. The involvement of an equal number of protons and electrons was in good agreement with the previously reported methods.⁵⁹ In one of the earlier reports,⁷⁴ the authors reported the electrochemical behaviour of MFA at pH 7. Although that pH is optimum for biological fluids, past reported work has revealed that MFA is in its negatively charged form at pH 5, which may increase electrode surface and MFA interactions to give an enhanced peak current at pH 5 rather than at pH 7. Further, there are several studies which have reported the electrochemical studies of MFA at pH 5.^58,60,70 It also confirms that the determination of MFA at pH 7 has a low detection limit as compared to that in an acid medium.

Fig. 8 Influence of pH on the shape of the anodic peak; pH: (a) 3.0, (b) 4.0, (c) 5.0, (d) 6.0, (e) 7.0, (f) 8.0, (g) 9.0, (h) 10.0 and (i) 11.0. Accumulation time: 60 s; supporting electrolyte: 0.2 M phosphate buffer. (A) Variation of peak current I_p/μA of MFA with pH. (B) Influence of pH on the peak potential E_p/V of MFA.

Effect of scan rate

Investigation of the cyclic voltammograms of MFA at different scan rates, rendered information regarding the dependence of peak current on scan rate. Hence, using a cyclic voltammetric method, the effect of scan rate on peak current was investigated in detail for MFA on the modified electrode surface. The irreversibility of the electrode process of MFA was indicated by the appearance of only an anodic peak (Fig. 9). An increase in peak current was observed when scan rate was increased. Further information regarding the adsorption controlled process on the surface of the electrode was obtained from the linear relationship between peak current and scan rate (Fig. 10A) with the linear regression equation: I_p (μA) = 7.904ν (V s⁻¹) + 0.360; R² = 0.994. Also there is a linear relationship between log I_p and log ν (Fig. 10B) corresponding to the equation: log I_p (μA) = 0.811 log ν (V s⁻¹) + 0.869; R² = 0.999. A value close to the theoretical value of 1.0 for an adsorption controlled process, from the slope of log I_p and log ν (0.811 V s⁻¹) was obtained which confirms the electrode process to be adsorption rather than diffusion controlled.⁶¹ Further, with an increase in scan rate, peak potential shifted positively (Fig. 10C). A good linear relationship with the regression equation E_p (V) = 0.052 log ν (V s⁻¹) + 0.713, R² = 0.999, was obtained for the linear relationship between peak potential and logarithm of scan rate. The relationship between peak potential and scan rate for an irreversible electrode process can be expressed by Laviron’s theory:⁶²where ‘α’ is the transfer coefficient, ‘k⁰’ is the standard rate constant of the reaction in s⁻¹, ‘n’ is the number of electrons transferred, ‘ν’ is the scan rate and ‘E⁰’ is the formal redox potential. From the slope of E_p versus log ν (0.052), ‘αn’ was calculated to be 1.12. The Bard and Faulkner⁶³ equation can be used to calculate α:

Fig. 9 Cyclic voltammograms for the oxidation of 0.1 mM MFA at different scan rates, (a) blank, (b) 50, (c) 100, (d) 150, (e) 250, (f) 350, (g) 400 and (h) 500 mV s⁻¹.

Fig. 10 (A) Dependence of peak current I_p/μA on scan rate ν/V s⁻¹. (B) Plot of logarithm of peak current (log I_p/μA) versus logarithm of scan rate (log ν/V s⁻¹). (C) Plot of variation of peak potential E_p/V with logarithm of scan rate.

Hence, α was calculated to be 0.56 from the above equation and electrons transferred was taken to be 2. Using dE_p/dpH = 0.059X/αn, where dE_p/dpH is the slope of the plot of E_p vs. pH, i.e. 0.040, X is the number of protons transferred and is calculated to be 1.6 ≈ 2. Further from eqn (3),^64,65 E⁰ can be calculated from the intercept of the E_p versus ν curve by extrapolating to the vertical axis at ν = 0. The E⁰ value was found to be 0.653 and the k⁰ value was calculated to be 575.43 s⁻¹.

Reaction mechanism

Based on the above results, the electrode reaction mechanism for oxidation of MFA was elucidated and is shown in Scheme 1. The results suggest that the transfer of two electrons and two protons is involved in this process.

Scheme 1 Reaction mechanism of MFA.

Calibration curve

A series of MFA solutions of different concentrations were measured at optimized experimental conditions (Fig. 11).

Fig. 11 Differential pulse voltammograms for the determination of MFA at the 5% Ba doped ZnO nanoparticle electrode as a function of concentration of the drug; (a) blank, (b) 0.001 μM, (c) 0.002 μM, (d) 0.01 μM, (e) 0.02 μM, (f) 0.04 μM, (g) 0.08 μM, (h) 0.1 μM, (i) 0.2 μM, (j) 0.4 μM, and (k) 0.6 μM; pH = 5 (I = 0.2 M); accumulation time 60 s.

In the range of 500 nM to 10.0 × 10³ nM, linearity of the peak current towards concentration of MFA was observed with the following equation: I_p (μA) = 6.650C (μM) + 0.359; R² = 0.997. LOD (limit of detection) and LOQ (limit of quantification) were calculated^66,67 from the equation

LOD = 3S/m; LOQ = 10S/m (5)

where ‘S’ is the standard deviation of the peak currents of the blank (six replicates) and ‘m’ is the slope of the calibration curve. LOD and LOQ were calculated to be 6.02 nM and 20 nM, respectively. Analytical characteristics of the calibration plot are summarized in Table 1. The LODs reported at different electrodes are tabulated in Table 2 which presents the superiority of the present method. The percentage of RSD values indicate good repeatability and reproducibility.

Table 1 Characteristics of MFA calibration plot using differential pulse voltammetry at 5% Ba doped ZnO nanoparticles/GCE

Linearity range (M)	5.0 × 10^−7 to 1.0 × 10^−5
Slope of the calibration plot (μA M^−1)	6.58
Intercept (μA)	0.37
Correlation coefficient (r)	0.9954
RSD of slope (%)	1.95
RSD of intercept (%)	3.59
Number of data points	8.00
LOD (nM)	6.02
LOQ (nM)	20.0
Repeatability (RSD%)	2.07
Reproducibility (RSD%)	10.90

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Table 2 Comparison of detection limits of MFA by different methods^a

Method	LOD		Reference
a GCE: glassy carbon electrode, RTIL: room temperature ionic liquid, MWCNT: multiwalled carbon nanotubes, CHIT: chitosan, CNT: carbon nanotubes.
Spectrophotometry	11 × 10^5 nM		34
Capillary zone electrophoresis	3 × 10^3 nM		43
Chemiluminescence detection	50 × 10^3 nM		69
HPLC	13.79 × 10^3 nM		70
Electrochemical based sensors
GCE		15 × 10^3 nM	71
MWCNT–graphite/Ag electrode		16 nM	72
RTIL–MWCNT–CHIT/GC electrode		1235 nM	73
MWCNT–CHIT/GCE electrode		660 nM	74
CNT/gold nanoparticles composite film		10 nM	49
Ba doped ZnO nanoparticles modified GCE		6.02 nM	This work

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Tablet analysis and recovery test

A commercially available medicinal sample containing MFA, i.e. Meftal (100 mg per tablet) was used to appraise the applicability of the proposed method in pharmaceutical sample analysis. Concentrations of MFA were prepared so as to fall in the range of the calibration plot. Identical conditions used for calibration plot construction were maintained during tablet analysis. The results obtained were in good conformity with the content marked in the label. All these results are listed in Table 3. The recoveries were found to be in the range of 96.2–100.5% with an RSD of 1.8%.⁶⁸

Table 3 Analysis of MFA in tablets by DPV and recovery studies

	Meftal-p
Labelled claim (mg)	100
Amount found (mg)*	98.4
RSD (%)	1.05
t-Test of significant	0.49
F-Test of significant	1.01
Bias (%)	1.50
Added (mg)	1.00
Found (mg)*	0.97
Recovered (%)	97.2
RSD (%)	0.86
Bias (%)	−2.80

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Effect of excipients

Since common excipients used in pharmaceutical dose preparations can interfere with analytical applications, the effects of some commonly used excipients were examined. Effects were studied using 0.1 mM MFA in the presence of 1.0 mM excipients using a differential-pulse voltammetric technique. The results (Table 4) suggest that a 100-fold excess of different excipients did not impede the voltammetric signal of MFA. Hence, an MFA assay can be carried out in the presence of excipients and it can be considered specific.

Table 4 Influence of potential interferents on the voltammetric response of 1.0 × 10⁻⁴ M MFA

Interferent	Concentration (mM)	Signal change (%)
Oxalic acid	1.0	2.60
Citric acid	1.0	1.56
Sucrose	1.0	−0.02
Starch	1.0	−0.01
Gum Acacia	1.0	1.44
Dextrose	1.0	0.27

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Detection of MFA in urine samples

The applicability of the proposed method for the determination of MFA in spiked human urine was investigated. Urine samples from three healthy volunteers free from drugs were filtered and stored until usage. Sample analysis was carried out using the developed differential-pulse voltammetric technique.

Drug free urine was spiked with known amounts of MFA and used for a recovery study. The results obtained denoted that the recovery lay between 94.2–101.1% with an RSD of 2.6%. From the calibration graph determination of spiked MFA in urine samples was carried out. The detection results of four urine samples (Table 5) showed good recovery suggesting the applicability of the method for MFA analysis in biological fluid.

Table 5 Application of DPV for the determination of MFA in spiked human urine samples

Urine sample	Spiked (10^−6 M)	Detected (10^−6 M)	Recovery (%)	RSD (%)
1	0.4	0.398	99.5	2.62
2	0.5	0.491	94.2	2.77
3	0.8	0.900	101.1	2.58
4	1.0	0.981	97.1	2.69
5	1.3	1.278	98.3	2.65

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Reproducibility of the 5% Ba doped ZnO nanoparticle modified GCE

Reproducibility of the prepared electrode was studied with 0.1 mM MFA solution and the same 5% Ba doped ZnO nanoparticle modified GCE at every few hours within a day. We found that the % of RSD was 2.23% (number of measurements = 5), which suggests good reproducibility for the determination of MFA. If the temperature was kept constant, reproducibility between days can be obtained. Owing to the adsorption of the oxidative product on the surface of the electrode, current response would decrease after successive use. In this case, the electrode should be prepared again.

Conclusion

5% Ba doped ZnO nanoparticles were synthesized and characterized using XRD, EDX, SEM and TEM. A cyclic voltammetric technique was used to investigate the electrochemical behavior of MFA on the surface of a 5% Ba doped ZnO nanoparticle modified glassy carbon electrode. It shows that, the process was adsorption-controlled and involves a two electron and two proton process. Based on the results obtained a probable reaction mechanism was proposed. A DPV technique was adopted to determine MFA for analytical applications. The present work is superior in terms of limit of detection (LOD) compared with the earlier reported methods. And it is proved that it can be a good alternative for determination of MFA. Further, commercially available MFA, i.e. Meftal, was analyzed using the DPV technique and also some of the common excipients present in Meftal were studied. The analysis of pharmaceutical samples showed good recovery values and none of the excipients interfered with the pharmaceutical dose. Also it can be observed from the analysis of MFA in spiked human urine samples that the proposed method is applicable to real samples.

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