Highly selective differential pulse voltammetric determination of phenazopyridine using MgCr2O4 nanoparticles decorated MWCNTs-modified glassy carbon electrode

Colloids and Surfaces B: Biointerfaces 111 (2013) 270–276

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Highly selective differential pulse voltammetric determination of phenazopyridine using MgCr2 O4 nanoparticles decorated MWCNTs-modified glassy carbon electrode Ali A. Ensafi a,∗ , B. Arashpour a , B. Rezaei a , Ali R. Allafchian b a b

Department of Chemistry, Isfahan University of Technology, Isfahan, 84156-83111, Iran Nanotechnology and Advanced Materials Institute, Isfahan University of Technology, Isfahan, 84156-83111, Iran

a r t i c l e

i n f o

Article history: Received 18 February 2013 Received in revised form 2 June 2013 Accepted 7 June 2013 Available online 19 June 2013 Keywords: MgCr2 O4 nanoparticles Modified multiwall carbon nanotubes Phenazopyridine Voltammetry

a b s t r a c t A selective modified glassy carbon electrode based on multiwall carbon nanotubes decorated with MgCr2 O4 nanoparticles was fabricated and used for the determination of phenazopyridine using differential pulse voltammetry. The electrochemical response of the modified electrode toward phenazopyridine was characterized by different electrochemical methods including differential pulse voltammetry (DPV), cyclic voltammetry (CV), and impedance spectroscopy. The prepared electrode showed an efficient synergic effect on the oxidation of phenazopyridine at pH 6.0. The oxidation peak current was proportional to the concentration of phenazopyridine from 0.05 to 7.5 ␮mol L−1 . The detection limit was 0.025 ␮mol L−1 . The applicability of the method was confirmed with satisfactory results obtained through the assay of phenazopyridine in human plasma, urine samples, and pharmaceuticals. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Phenazopyridine is a type of azo dye and a very important organic compound for its optical and electrochemical properties as well as its medicinal relevance [1,2]. Other azo dyes, formerly used in textiles, printing, and plastic manufacturing, have been implicated as carcinogenic agents that can cause bladder cancer [3]. Evidence from animal models suggests that it is potentially carcinogenic [4], while phenazopyridine has never been shown to cause cancer in humans. Phenazopyridine is prescribed for its local analgesic effects on the urinary tract [5]. Moreover, it is sometimes used in conjunction with an antibiotic or other anti-infective medication at the inception of treatment to help provide immediate symptomatic relief [6–8]. Phenazopyridine frequently causes a distinct color change in the urine, typically to a dark orange to reddish color [9]. This effect is common and harmless, and indeed a key indicator of the presence of the drug in the body. Less frequently, it can cause a pigment change in the skin or eyes to a noticeable yellowish color. Other such side effects include fever, confusion, shortness of breath, skin rash, and swelling of the face, fingers, feet, or legs. Long-term use may cause yellowing of nails [10]. It is, therefore, necessary to develop a rapid and sensitive method with simple

∗ Corresponding author. Tel.: +98 311 3913269; fax: +98 311 3912350. E-mail addresses: Ensafi@cc.iut.ac.ir, aaensafi@gmail.com (A.A. Ensafi). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.06.017

sample preparation and determination steps for phenazopyridine analysis in biological fluids. Several analytical techniques are available for the assay of phenazopyridine in biological fluids. The methods commonly used for the determination of phenazopyridine include amperometry [11], gravimetry [12], polarography [13], voltammetry [14], UV spectrophotometry [15], and potentiometric sensor [16]. High performance liquid chromatography has been used with UV detection to determine phenazopyridine in a compound dosage form [17–19]. Some researchers have used liquid chromatography–mass spectroscopy (LC–MS) [20] and gas chromatography–mass spectrometry (GC–MS) [21,22] for the determination of this drug. The facile and intense voltammetric response due to the reduction of N N into NH NH and NH2 has provided a great deal of information about their optical, structural, electrochemical, and thermodynamic properties [1]. In recent years, substantial efforts have been devoted to the field of electrochemical methods based on modified electrodes, for their enhanced selectivity and sensitivity [23]. Carbon nanotubes are molecular-scale tubes with high electrical conductivity, high chemical stability, and very high tensile strength and modulus [24]. There has been much interest in diverse applications of carbon nanotubes such as in scanning probes [25,26], electron field emission sources [27], actuators [28], nanoelectronic devices [29], batteries [30], potential hydrogen storage material [31], and chemical sensors [32]. MNPs modified electrodes have also received serious consideration due to their high surface

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Table 1 Comparison of the proposed electrochemical sensor with the other reported method for the determination of PAP. Technique

Limit of detection (␮mol L−1 )

Linear dynamic range (␮mol L−1 )

Reference

Amperometry Differential pulse polarography Potentiometric Square wave voltammetry Differential pulse voltammetry

0.8 0.3 8.0 0.07 0.025

4.0–120.0 0.32–4.0 10–10000 0.2–2.5 0.05–40.0

[11] [13] [16] [35] This method

area, effective mass transport, catalysis, and control over the local microenvironment [33,34]. In the present work, we have used a magnetic nanocomposite of MWCNTs decorated with spinel MgCr2 O4 as a working electrode using the citrate sol–gel method for the study of the voltammetric behavior and assay of phenazopyridine with differential pulse voltammetry. The results show that the proposed method is highly selective and sensitive in the determination of phenazopyridine. Its applicability to the determination of phenazopyridine levels in pharmaceuticals, urine samples, and human blood serum was also evaluated. The detection limit, linear dynamic range, and sensitivity for phenazopyridine determination using this method were found to be appreciably better than those of other electrochemical methods reported in the literature (Table 1). 2. Experimental 2.1. Apparatus The voltammograms were obtained with a Metrohm instrument, Model 797 VA processor. A three-electrode electrochemical cell that contained of a Pt-wire as an auxiliary electrode, an Ag/AgCl (3.0 mol L−1 KCl) reference electrode and the unmodified or the modified-GCE as a working electrode were used throughout the experiment. Electrochemical impedance spectroscopy (EIS) was performed in solutions with 0.1 mol L−1 KCl as a supporting electrolyte, using a current voltage of 5 mV, within the frequency range of 100 kHz to 1.0 kHz by Autolab instrument. (PGSTAT 12 and FRA2 boards, run on a pc using GPES and FRA 4.9 software). Fourier transform-IR spectra were recorded using a JASCO FT-IR (680 plus). The spectra of solids were obtained using KBr pellets. Atomic force microscopy (AFM) was done with a Bruker Nano instrument (Germany). Tunneling electron microscopy (TEM) was obtained using a Philips CH 200, LaB6-Cathode160 kV. Scanning electron microscope (SEM) was performed with a Philips XLC. A coring pH-meter, Model 140, with a glass electrode (conjugated with an Ag/AgCl reference, Model 140) was used to indicate of pH solution. 2.2. Chemicals Phenazopyridine was supplied by Sigma–Aldrich and phenazopyridine tablet (100 mg per tablet) was purchased from Shahre Daru Company (Tehran, Iran). Other chemicals were obtained from Merck and used without further treatment. Diluted solutions were prepared from the chemicals of analytical grade using double distilled water. A stock solution of 0.01 mol L−1 phenazopyridine was prepared by dissolving 0.0624 g of pure phenazopyridine in an appropriate volume of double distilled water. Phosphate buffer solution (0.1 mol L−1 ) with different pH values were used to adjusting solutions pH. Multiwall carbon nanotubes (>90% MWCNT basis, with a diameter of 20–30 nm and a length of 5–15 ␮m) were prepared from Aldrich.

2.3. Preparation of MgCr2 O4 -MWCNTs modified-GCE Nitric acid was used to purify MWCNTs before its modification [35]. For this purpose, 20 mL of 3.0 mol L−1 nitric acid was added to 1.00 g of MWCNTs. The mixture was refluxed for 15 h. After cooling the reaction mixture to room temperature, it was diluted with deionized water, vacuum-filtered through a 3 ␮m porosity filter paper, and allowed to dry at room temperature. The suspension of activated MWCNTs was sonicated in dimethyl formamide (0.10 mg MWCNTs per 5 mL). These conditions lead to the removal of impurities from the carbon nanotubes to open the tube caps [36]. To modify the MWCNTs, first 0.750 g of the MWCNTs was mixed with 10 mL of 1.0 mol L−1 citric acid and placed in an ultrasonic bath for 10 min. Into the suspension solution were added 10 mL of 0.5 mol L−1 Mg(NO3 )2 ·6H2 O of analytical grade and 1.0 mol L−1 Cr(NO3 )3 ·9H2 O. Next, the pH level of the solution was raised to 9.0 using 0.10 mol L−1 ammonium hydroxide (NH4 OH) solution. To complete the process, the reaction mixture was stirred at 30 ◦ C for 48 h, and the resulting product was dried in an oven at 100 ◦ C for 12 h. Finally, the product mixture was calcinated at 630 ◦ C for 2 h to remove impurities in the furnace under argon atmosphere [37]. This operation led to the production of MgCr2 O4 -MWCNTs. Afterward; a glassy carbon electrode was polished with aqueous slurry of 0.05 mm alumina powder on a polishing micro-cloth for 3 min until a mirror-like finish was obtained. The GCE thus prepared was sonicated in a mixture of ethanol/water solution (50% v/v). Finally, 10 ␮L of the stable suspension was dropped onto the surface of the electrode and dried in hot air flow at 50 ◦ C. Due to the magnetic properties of this mediator, the magnet was used for equalizing the surface of the electrode. 2.4. Real samples preparation For tablet analysis, 5 tablets of phenazopyridine (labeled 100 mg of phenazopyridine per tablet) were completely ground and homogenized. Then, 0.0111 g of the powder was accurately weighed and dissolved in water by sonication for 5 min (equal to 0.001 mol L−1 phenazopyridine). Finally, 250 and 350 ␮L of the solution plus 1.0 mL of phosphate buffer (pH 6.0) were transferred in 5.0-mL volumetric flasks and diluted with water. The result solutions were transferred into the electrochemical cell to be analyzed without any further treatment. Standard addition method was used for the determination phenazopyridine. Urine and blood plasma samples were stored in a refrigerator after collection. 4.0 mL of the centrifuged (for 5 min at 1500 rpm) urine sample was diluted with 1.0 mL of 0.10 mol L−1 buffer (pH 6.0). The result solution was transferred into the electrochemical cell to be analyzed without any further treatment. Standard addition method was used for the determination phenazopyridine. 3. Result and discussion 3.1. Morphology and structure of the modified electrodes The structures of the obtained MWCNTs-GCE and MgCr2 O4 MWCNTs were studied by AFM, XRD, FT-IR, TEM, SEM and EIS.

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Fig. 1. (A) (a) SEM image of MWCNTs modified-GCE; (b) TEM image of the surface of MWCNTs modified-GCE. (B) (a) SEM image of MgCr2 O4 -MWCNTs modified-GCE; b) 2D, and (c) 3D AFM topology of MgCr2 O4 -MWCNTs-GCE; and (d) TEM image of the surface of MgCr2 O4 -MWCNTs modified-GCE.

SEM image of the surfaces of the MWCNTs modified-GCE and MgCr2 O4 -MWCNTs modified-GCE is shown in Fig. 1A(a) and Fig. 1B(a), respectively. The SEM images of the MWCNTs modifiedGCE and MgCr2 O4 -MWCNTs modified-GCE shows that the surface of GCE was completely covered with MWCNTs and MgCr2 O4 MWCNTs nanoparticles, respectively. AFM was also used to obtain further details of the surface structure such as roughness and thickness. Fig. 1B(b) and (c) show AFM topology of the surface of MgCr2 O4 -MWCNTs modified-GCE corresponding to 2D (Figs. 1B(b)) and 3D (Fig. 1B(c)) images recorded over an area of 1.00 × 1.00 ␮m. The existence of particles with less than 20 nm size at MgCr2 O4 -MWCNTs modified electrode surface is clearly reflected in 2D and 3D AFM images. Fig. 1A(b) and B(d) shows TEM images (sample morphology) of MWCNTs-GCE and MgCr2 O4 -MWCNTs-GCE, respectively. These figures confirm that MWCNTs and MgCr2 O4 -MWCNTs were distributed on the surface of the GCE and they did not change the morphology of MWCNTs. The spaghetti-like MgCr2 O4 -MWCNTs and MWCNTs formed a porous structure. The entangled crosslinked fibrils offered a good accessible surface area. XRD spectra of the MgCr2 O4 -MWCNTs magnetic nanocomposite shows fifteen characteristic peaks occur at 2 of 30.32◦ , 35.72◦ , 37.35◦ , 43.41◦ , 47.52◦ , 57.44◦ , 63.06◦ , 66.29◦ , 74.60◦ , 75.64◦ , 79.65◦ , 82.65◦ , 87.53◦ , 90.47 and 95.37◦ which are marked by their corresponding index (2 2 0), (3 1 1), (2 2 2), (4 0 0), (3 3 1), (5 1 1),

(4 4 0), (5 3 1), (5 3 3), (6 2 2), (4 4 4), (5 5 1), (6 4 2), (7 3 1) and (8 0 0), respectively. The diffraction peaks at 2 of 26.35◦ , 42.61◦ , 44.83◦ and 54.23◦ are the typical Bragg peaks of pristine CNTs and can be indexed to (0 0 2), (1 0 0), (1 0 1) and (0 0 4) reflection of MWCNTs. This reveals that the particles are pure MgCr2 O4 -MWCNTs with a cubic structure. No diffraction peaks of other impurities such as MgO or Cr2 O3 were observed. The diffractive peaks of MgCr2 O4 -MWCNTs are broadened, implying that the crystalline size of the MgCr2 O4 -MWCNTs particles is quite small. The mean particle size of MgCr2 O4 -MWCNTs calculated by Scherer equation is about 25 nm. FT-IR spectra (in the range of 4000-400 cm−1 ) of the MWCNTs decorated with MgCr2 O4 clearly shows an absorption bands around 3437 cm−1 , which are characteristic stretching vibration of hydroxyl functional group (O H) on the surface of MWCNTs or adsorbed water in the sample. The absorption band at 1733 cm−1 corresponds to stretching vibration of carbonyl group (C O). The stretching vibration of the carboxylate group (C O) is observed around 1380 cm−1 . Stretching vibrations of C C group also localized at 1626 cm−1 . The absorption band around 1113 cm−1 is assigned the stretching vibration of C C C group. Also three at absorption bands at 633, 497 and 426 cm−1 are corresponding to the vibration of tetrahedral and octahedral complexes, respectively, which are indicative of formation of spinel chromite structure [38]. As it is seen from the spectra, the normal mode of vibration

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Fig. 2. (a): Differential pulse voltammograms of the blank solution at GCE electrode; (b): The blank solution at MWCNTs modified-GCE; (c): The blank solution at MgCr2 O4 -MWCNTs modified-GCE; (d): Phenazopyridine (10.0 ␮mol L−1 ) at unmodified-GCE; (e): Phenazopyridine (10.0 ␮mol L−1 ) at MWCNTs modified-GCE; (f): Phenazopyridine (10.0 ␮mol L−1 ) at MgCr2 O4 -MWCNTs modified-GCE. Conditions: Pulse amplitude of 100 mV, Pulse time of 20 ms, Sweep rate of 80 mV s−1 ; Phosphate buffer (0.1 mol L−1 , pH 6.0).

of tetrahedral cluster (633 cm−1 ) is higher than that of octahedral cluster (426 and 497 cm−1 ). This can be due to the shorter bond length of tetrahedral cluster than the octahedral cluster. 3.2. Study of the electrochemical behavior of phenazopyridine at MgCr2 O4 -MWCNTs film-GCE In order to investigate the role of the mediator’s synergic effect, the oxidation process of phenazopyridine on GCE, on the electrode modified with MWCNTs, and on MgCr2 O4 -MWCNTs was studied by cyclic voltammetry. Voltammograms of the phosphate buffer (pH 7.0) as an electrolyte were recorded on the bare glassy carbon electrode, modified electrode with MWCNTs, and MgCr2 O4 MWCNTs modified electrode (curves a, b, and c, respectively, Fig. 2). The curves d, e, and f in Fig. 2 show the cyclic voltammograms of 50.0 ␮mol L−1 phenazopyridine at pH 7.0 on the surface of GCE, MWCNTs-GCE and MgCr2 O4 -MWCNTs-GCE, respectively. The experimental results revealed a weak oxidation peak current for phenazopyridine on the surface of GCE, whereas it was seen to significantly increase in the presence of the modifier (MgCr2 O4 MWCNTs) at the surface of the GCE. In other words, the data show that the combination of MWCNTs and MgCr2 O4 -MWCNTs improved the characteristics of the electrode for the oxidation of phenazopyridine. MWCNTs decorated with MgCr2 O4 increase the electrode surface area, which is the reason why the oxidation current of phenazopyridine increases on the surface of the modified electrode. In order to determine the active surface area of the modified electrode, voltammograms of 1.0 mmol L−1 K3 Fe(CN)6 as a probe were recorded at different scan rates. For a reversible process, the Randles-Sevcik formula was used (at 25 ◦ C) as follows: Ipa = 2.69 n3/2 AC0 D1/2 1/2

(1)

where, Ipa (A) designates the anodic peak current, n is the electron transfer number, A (cm2 ) is the surface area of the electrode, D (cm2 s−1 ) is the diffusion coefficient, C0 (mol cm−3 ) is the concentration of K3 Fe(CN)6 , and  (V s−1 ) represents scan rate. In this case, 1.0 mmol L−1 K3 Fe(CN)6 was used in the presence of 0.10 mol L−1 KCl electrolyte with n = 1 and D = (7.6 ± 0.2) × 10−6 cm2 s−1 . In this equation, all the parameters for potassium hexacyanoferrate are

Fig. 3. Effect of pH on the (A): peak current; and (B): on the peak potentials of 6.0 ␮mol L−1 phenazopyridine in phosphate buffer (0.1 mol L−1 ) at MgCr2 O4 MWCNTs modified-GCE. Conditions: Pulse amplitude, 100 mV; Pulse time, 20 ms; and sweep rate, 80 mV s−1 (the error bars are standard deviations for n = 3).

given except the surface area of electrode. The microscopic areas of the electrodes were calculated from the slope of the Ipa vs. 1/2 . The results showed that the electrode surface area was 0.0391 cm2 for the bare GCE, 0.1564 cm2 for the MWCNTs-GCE, and about 0.2307 cm2 for the MgCr2 O4 -MWCNTs modified-GCE. This means that the surfaces area for the MWCNTs-modified-GCE and MgCr2 O4 -MWCNTs modified-GCE are 4.0 and 5.9 times, respectively, greater than that for the bare GCE. However, the increase in the peak current of the analyte at the surface of the MgCr2 O4 MWCNTs modified-GCE is not only due to the surface area, but also due to the synergic effect of MgCr2 O4 -MWCNTs nanoparticles on the oxidation of phenazopyridine at pH 7.0 3.3. Optimization of measurement conditions In order to optimize the response of the sensor to phenazopyridine oxidation, we characterized the effect of pH on the oxidation of phenazopyridine in 0.1 mol L−1 phosphate buffer solution with various pH values (3.0 < pH < 9.0) at the surface of the MgCr2 O4 MWCNTs modified-GCE using differential pulse voltammetry. As seen in Fig. 3, the oxidation peak current and the peak potential were closely related to the pH of the solution. The best shape and highest current were observed with pH 6.0. The anodic peak potential shifted linearly toward less positive values when the solution pH increased with a slope of −0.0565 V pH−1 . These results confirm that the ratio of the participating protons to the transferred electrons through the MgCr2 O4 -MWCNTs is 1:1. Based on discussions presented elsewhere [1], it may be suggested that the electrode reaction is a redox process of the azo group. These compounds are known for their facile reduction of the azo moiety in aqueous media. The effect of potential scan rate () (between 10 and 100 mV s−1 ) on the peak current was investigated by cyclic voltammetry. When the potential scan rate was increased from 10 to 100 mV s−1 in the presence of 100.0 ␮mol L−1 phenazopyridine at pH 7.0, a linear relationship was observed between the peaks current and scan rate ()

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Fig. 4. Nyquist plot of 1.0 mmol L−1 Fe(CN)6 3−/4− in 0.10 mol L−1 KNO3 (a): at unmodified GCE; (b) at MWCNTs-GCE; and (c) at MgCr2 O4 -MWCNTs modified-GCE. Inset: Fitted circuit for the electrochemical impedance spectroscopic data.

with a regression equation of Ip (␮A) = 0.455 + 2.349 (r = 0.9959). This equation indicates that the process is adsorption-controlled process. To learn more about the adsorption of phenazopyridine at MgCr2 O4 -MWCNTs coated GCE, the influence of accumulation potential and time on peak current was evaluated. The oxidation peak current of phenazopyridine at different potentials from 0.45 V to 0.70 V was examined. Increasing potential from 0.45 V to 0.60 V led to an increase in the adsorption of the analyte and, thus, increased the oxidation peak current. However, when the potential reached near the oxidation potential of phenazopyridine, the intensity reduced. This suggests that phenazopyridine oxidizes at this potential and that it is not collected at the electrode surface. Therefore, 0.60 V was selected as a suitable accumulation potential. Accumulation time also strongly influenced peak current. The oxidation peak current of 100.0 ␮mol L−1 phenazopyridine increased within an accumulation time of 60 s before it leveled off. This was caused by the saturation of phenazopyridine adsorbed on the surface of the MgCr2 O4 -MWCNTs coated GCE. Therefore, 60 s was selected as the proper accumulation time for further study. The optimum differential pulse voltammetry (DPV) parameters were chosen from a study of the variation oxidation peak current of 100.0 ␮mol L−1 phenazopyridine in the ranges of 10-100 mV, 10400 ms, and 10-100 mV s−1 for pulse amplitude, pulse width, and scan rate, respectively. The results obtained showed that maximum oxidation peak current of the analyte was achieved for a pulse amplitude of 80 mV, a pulse width of 200 ms, and at a scan rate of 50 mV s−1 .

3.4. Electrochemical impedance spectroscopy To investigate the effect of nano-composite synergic effect, the electrochemical impedance spectroscopy was used. This technique contains useful information about changes in resistance of the electrode surface during the oxidation process. Fig. 4 shows the Nyquist curves of imaginary impedance (Zim ) vs. the real impedance (Zre ) of the EIS at different electrode surfaces in a solution containing 1.0 mmol L−1 Fe(CN)6 4−/3− in 0.10 mol L−1 KNO3 . Based on the impedance spectrograms, the electrochemical impedance spectroscopic data of the three electrodes are comparable with the equivalent circuit shown in Fig. 4B. The circuit consists of Rs (solution resistance), CPE (a constant phase element corresponding to the double layer capacitance), Rct (charge transfer

Fig. 5. Calibration plot for 0.05–7.5 ␮mol L−1 phenazopyridine; Inset: Differential pulse voltammograms of phenazopyridine at various concentrations at the surface of MgCr2 O4 -MWCNTs modified-GCE (the error bars are standard deviations for n = 3).

resistance), and Zw (Warburg impedance) coupled to Rct , which is related to the Nernstain diffusion. The Nyquist diagram includes both a semicircle at high frequencies, whose diameter depends on the electron transfer resistance (Rct ), and a straight line with a slope of nearly 45, which is due to the mass transport process via diffusion. The results show that for the MgCr2 O4 -MWCNTs modified-GCE (Fig. 4a, c), the diameter of the semicircle is smaller than those in the cases of the MWCNTs modified-GCE (Fig. 4A, b) and the unmodified GCE (Fig. 4A, a). This is due to the presence of a high conductive mediator and the synergic effect of MgCr2 O4 -MWCNTs nanoparticles on the electron transfer. All this demonstrates that phenazopyridine could be successfully oxidized at the surface of the MgCr2 O4 -MWCNTs modified-GCE. It may, therefore, be concluded that MWCNTs decorated with MgCr2 O4 make nanoparticles that have a synergic effect on the oxidation of phenazopyridine. 4. Dynamic range and detection limit In this study, differential pulse voltammetry was used to generate the calibration curve due to its higher sensitivity compared to other voltammetric techniques. Under the optimized conditions, the electrode response was linearly related to phenazopyridine concentration over the range of 0.057.5 ␮mol L−1 phenazopyridine with a regression equation of Ip (␮A) = (0.1166 ± 0.0031)CPhenazopyridine + (0.3211 ± 0.0123), (r2 = 0.9964, n = 6). The detection limit was obtained to be 0.025 ␮mol L−1 phenazopyridine based on S/N = 3. Fig. 5 shows a typical DPV plus the associated calibration curve for the oxidation of phenazopyridine at the surface of the modified electrode. The modified electrode was studied for its reproducibility and stability by five replicate measurements of phenazopyridine using DPV under the optimum conditions. The relative standard deviation (RSD%) for ten successive measurements of 100.0 ␮mol L−1 phenazopyridine was found to be 1.1%. When using five different electrodes, the RSD% for five measurements was 1.6%. These results confirm the excellent stability and reproducibility of the modified electrode for the determination of phenazopyridine. 5. Effect of chemical interferences In order to assay the selectivity of the modified electrode for determination of phenazopyridine, the influence of different compounds as potential interference substances on the determination

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Table 2 Interference study under the optimum conditions. Species

Tolerance limit (molSubstance /molPhenaopyridine )

Glucose, Sucrose, Lactose, Fructose, Ascorbic acid, Citric acid, Urea, Co2+ , Cu2+ , Mg2+ , Ca2+ , K+ , NH4 + , SO4 2− , NO3− , F− , Br− Salicylic acid, Tartaric acid, Glycine, Histidine Methionine, Uric acid

1000a

a

800 500

Maximum concentration of the tested ions.

Table 3 Determination of PAP in real sample at pH 6.0 (n = 3). Sample

PAP added (␮mol L−1 )

PAP found (␮mol L−1 )

Recovery (%)

Differential pulse polarography [13] (␮mol L−1 )

Tablet Tablet Urine Urine Urine Plasma Plasma Plasma

50.0 70.0 – 40.0 50.0 – 50.0 70.0

48.9 ± 1.2 68.7 ± 0.7
99.8 98.1 – 97.3 99.0 – 97.0 100.1

48.2 ± 1.5 –
of phenazopyridine were investigated under the optimum conditions in the presence of 30.0 ␮mol L−1 of phenazopyridine. The tolerance limit was taken as the maximum concentration of the interfering substances, which caused a relative error of approximately ±5% in the determination of phenazopyridine. The results are presented in Table 2. All of the checked potential interfering substances except uric acid interfered at their concentrations higher than their tolerance limits because of their adsorption at the electrode surface. Moreover, uric acid at more than 500-fold interfered because it oxidized at the electrode surface, near the phenazopyridine peak potential. These results confirm that no interference could be observed for the tested compounds such as different cations, anions and organic compounds. 6. Analysis of real samples The high sensitivity of the method allows for phenazopyridine being determined in real samples. To assess the applicability of this method to real samples, an attempt was made to determine phenazopyridine in tablet, urine, and plasma samples. The standard addition method was used for measuring phenazopyridine concentrations in the samples. Based on the results obtained, a good agreement is seen to hold between the results obtained from the proposed method and those from the standard method (Table 3). The results also confirm the capability of the modified-GCE for the voltammetric determination of phenazopyridine in real samples with good recoveries. In addition, comparison of the analytical results with those from an established method [13] (Table 3) confirms the accuracy of the proposed method. 7. Conclusion In this study, MgCr2 O4 -MWCNTs modified-GCE was prepared and the application of MgCr2 O4 -MWCNTs for the determination of phenazopyridine in phosphate buffer solution (pH 6.0) was described. The magnetic nano-composite of multiwall carbon nanotubes decorated with MgCr2 O4 nanoparticles was satisfactorily used as a suitable mediator in the electrochemical method for the detection of phenazopyridine. The synergic effect of the bare GCE toward phenazopyridine oxidation was improved by the formation of a uniform MWCNTs film, which was incorporated with MgCr2 O4 nanoparticles. The electrochemical renewal of the electrode surface in the phosphate buffer was found to be efficient, while it also ensured the reproducibility of measurements. In the differential pulse voltammetric determination, the detection limit

of phenazopyridine was estimated to be as low as 0.025 ␮mol L−1 . The MgCr2 O4 -MWCNTs modified-GCE showed excellent sensitivity, selectivity, and anti-fouling properties for the voltammetric determination of phenazopyridine. Although the sensitivity of the proposed method is comparable with those reported for electrochemical methods [35], the proposed method has the additional advantage that it is free from compounds potentially interfering in the detection of phenazopyridine. Finally, the capability of the proposed method was demonstrated for the determination of phenazopyridine in real samples with satisfactory results. Acknowledgements The authors wish to thank Isfahan University of Technology (IUT) Research Council and Center of Excellence in Sensor and Green Chemistry for their support. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]

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