Determination of metronidazole in pharmaceutical dosage forms based on reduction at graphene and ionic liquid composite film modified electrode

Sensors and Actuators B 169 (2012) 81–87

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Determination of metronidazole in pharmaceutical dosage forms based on reduction at graphene and ionic liquid composite film modified electrode Jinyun Peng a,b , Chuantao Hou a , Xiaoya Hu a,∗ a b

College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China Department of Chemistry and Biological Science, Guangxi Normal University for Nationalities, Chongzuo 532200, China

a r t i c l e

i n f o

Article history: Received 6 January 2012 Received in revised form 10 March 2012 Accepted 13 March 2012 Available online 15 May 2012 Keywords: Graphene Ionic liquids Metronidazole Differential pulse voltammetric Determination

a b s t r a c t An electrochemical method has been successfully demonstrated for sensitive determination of Metronidazole (MTZ) with graphene (Gr)-room temperature ionic liquid (IL) of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6 ) composite modified glassy carbon electrode (GCE). The cyclic voltammetric results indicate that Gr-IL/GCE can remarkably enhance electrocatalytic activity towards the reduction of MTZ in neutral solutions. MTZ produce a cathodic peak at about −0.69 V at this electrode. The electrochemical parameters of MTZ in the composite film were carefully calculated with the results of the charge transfer coefficient (˛) as 0.625 and the number of electron transferred (n) as 4. The electrocatalytic behavior was further exploited as a sensitive detection scheme for the MTZ determination by differential-pulse voltammetry (DPV). Under optimized conditions, the concentration range and detection limit were 1.0 × 10−7 –2.5 × 10−5 mol L−1 and 4.7 × 10−8 mol L−1 (S/N = 3), respectively for MTZ. The method was successfully applied assay of the drug in the pharmaceutical dosage forms. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Metronidazole (2-methyl-5-nitroimidazole-1-ethanol, MTZ) is a nitroimidazole derivative that is used commonly to treat protozoal diseases including trichomoniasis and giardiasis [1]. MTZ is an effective drug against trichomonas, Vincent’s organisms, and anaerobic bacteria. Due to the critical roles of MTZ in the pharmaceutical industry, their determination is important in biological fluids and drug formulations. Several methods have been reported for determination of MTZ which include gas chromatography [2,3], TLC-Densitometric [4], high performance liquid chromatography (HPLC) [4–11], derivative spectrophotometry [12], fluorescence [13], and spectrophotometry [14–16]. These methods can be applied to the detection of MTZ in various samples, but they are generally time- consuming and/or complicated. So there still exists a need for improved an alternative method for MTZ determination with a high degree of selectivity and sensitivity. MTZ contains a nitro group which is the electrochemically active reducible center. MTZ can also be assayed and studied by using electrochemical methods. The reductive behaviour of MTZ at different electrode such as DNA modified glassy carbon electrode (GCE) [17,18], activated GCE [19], MWNT/GCE [20], carbon fiber microdisk electrode [21], SWCNTs/GCE [22], gold electrode using continuous pulsed-potential technique [23], hanging

∗ Corresponding author. Tel.: +86 514 87971818; fax: +86 514 87311374. E-mail address: [email protected] (X. Hu). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2012.03.040

mercury drop electrode (HMDE) [17,24], molecularly imprinted polymer–carbon paste electrode [25] was used for the determination of MTZ. Recently, a new kind of carbon material, graphene (Gr), has attracted tremendous attention from scientific communities based on its large specific surface area, extraordinary electrical and thermal conductivities [26,27], high mechanical stiffness [28], good biocompatibility [29], and low manufacturing cost [30]. Like carbon nanotubes (CNTs), which have been widely used for the study of electrode materials, Gr provides a new avenue for fabricating electrochemical devices due to its facilitative on electron transfer between electroactive species and electrodes. Moreover, they are more easily produced in mass quantities as compared with the CNTs. And ionic liquid (IL), another material, due to its wide electrochemical windows, high ionic conductivity and good solubility [31,32], has been widely applied in the fields of analytical chemistry and electrochemical sensors. Furthermore, the Gr-IL composite material has been used as a modifier for the chemically modified electrode based on the specific interaction of Gr with IL and their special properties. For example, based on functionalized Gr sheets/room temperature IL composite film, Zhu’s group determined nitrate at a low detection limit of 4.0 × 10−8 mol L−1 [33]. Niu and co-workers determined NADH using IL-functionalized Gr [34]. Dong and co-workers used IL-Gr as an enhanced material for electrochemical determination of trinitrotoluene [35]. Our group has recently reported a voltammetric method for the determination of azithromycin using GCE modified with Gr and IL composite film [36]. However, to the best of our knowledge, very little work has been performed on the determination of MTZ by the direct

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electrochemical measurement with the nanocomposites of Gr and IL electrode. The purpose of this work is to study the voltammetric behavior of MTZ at a Gr-IL modified GCE. This modified electrode strongly enhanced the reduction of MTZ and leads to considerable improvement of its cathodic peak current. This allows the development of a highly sensitive voltammetric sensor for determination of MTZ in pharmaceutical samples. Finally, this modified electrode was used for the analysis of MTZ in commercial tablets from different manufactures with satisfactory results. The method is simple, stable and sensitive. 2. Experiments 2.1. Reagents and apparatus MTZ reference standard was from Aladdin-Reagent Company (Shanghai, China), and its standard solution (2.30 × 10−3 mol L−1 ) was prepared with redistilled water and kept in the refrigerator at 4 ◦ C. Graphite powder (spectroscopically pure reagent) was obtained from Qingdao Hensen Graphite Co., Ltd. (China). Gr was synthesized according to previous report [37]. The room temperature IL, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ]), was purchased from Aladdin-Reagent Company (Shanghai, China). Other reagents were of analytical grade and used as received. Aqueous solutions were prepared with redistilled water. The image of scanning electron microscope (SEM) was obtained at Hitachi S-4800 (Japan). The Raman spectra were measured using Renishaw inVia (England). Electrochemical experiments were performed with a CHI660D electrochemical workstation (Shanghai Chenhua Co., China) with a conventional three-electrode cell. A bare or Gr modified GCE (ϕ = 3 mm) was used as working electrode. A saturated calomel electrode (SCE) and a platinum wire were used as reference electrode and auxiliary electrode, respectively.

Phosphate buffer solutions were carried out with a pHS-25 pH-meter (Shanghai Leici Instrument Plant, China) at room temperature. 2.2. Preparation of the Gr-modified electrode Before modification, the GCE surface (Alda Co. Ltd., China) was polished with 0.05 ␮m alumina slurry, and sonicated in redistilled water to give a clean surface. 1 mg Gr and 1 ␮L IL were added into 1 mL 1% chitosan (Chi) solution, followed by ultrasonication for 2 h to form homogenous Gr dispersion. 5 ␮L of Gr dispersion was deposited on the fresh prepared GCE surface (named as GrIL/GCE). After dried at room temperature for 30 min, the electrode surface was thoroughly rinsed with redistilled water. For comparison, Gr/GCE were also prepared with the similar process. 2.3. Procedure for tablets Five tablets were weighted accurately and crushed to a fine powder. The required amount of sample to prepare a solution of ca. 10−3 mol L−1 was transferred into a 100 mL standard flask containing 80 mL of PBS (pH 7.0). The contents of flask were stirred magnetically for 15 min and then diluted to volume with the same supporting electrolyte. The solution was filtered and the first 20 mL of the filtrate was removed. Appropriate solutions were prepared by taking suitable aliquots of the clear filtrate and diluting them with supporting electrolyte mentioned above. 2.4. Analytical procedure The pH 7.0 PBS was used as the supporting electrolyte for MTZ. After 30 s stirring, the differential pulse voltammetry curves were recorded from −1.1 to 0.6 V, and the reduction peak currents at −0.69 V was measured for MTZ. The pulse amplitude is 50 mV, the pulse width is 0.05 s, and the pulse period is 0.2 s.

Fig. 1. SEM image of Gr in DMF solvent (A), Gr/GCE (B), Gr-IL/GCE (C) and Raman spectroscopy of Gr (D).

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3. Results and discussion 3.1. Characterization of Gr dispersed in DMF Fig. 1A shows the SEM image of Gr (in DMF solvent) film on the surface of the GCE, revealing the typical crumpled and wrinkled Gr sheet structure on the rough surface of the film. However, for GrChi membrane (Fig. 1B) and Gr-Chi-IL membrane (Fig. 1C), a totally different morphology was obtained. It seemed that the Gr-Chi or Gr-Chi-IL film was characterized with uniform and smooth surface. Fig. 1D compares the Raman spectra of graphite, graphene oxide (GO), Gr measured at 532 nm excitation. G and D peaks lie at around 1580 and 1345 cm−1 respectively, which are the tow most intense features in the Raman spectra of carbons. Compared with graphite and GO, Gr samples had the highest D/G peak intensity ratio, which agrees with the results reported by other groups [38,39]. A stronger D peak in Gr compared to the GO may be attributed to a local disorder band induced by chemical reduction. 3.2. Electrochemical behaviors of MTZ The voltammograms of MTZ at a bare GCE, Gr/GCE and Gr-IL/GCE in PBS (pH 7.0) were shown in Fig. 2. It showed that no oxidation peak was observed in the reverse scan, suggesting that the electrochemical reaction was a totally irreversible process. As can be seen, the cathodic peak potential for the reduction of MTZ at Gr-IL/GCE and Gr/GCE are all −0.69 V. The reduction peak current value (Ip ) of MTZ at Gr-IL/GCE was 5.478 × 10−6 A, which is 5.7 times larger than the one at the Gr/GCE, and 9.0 times larger than that of Ip at the GCE. The data obtained clearly show that the combination of Gr and IL definitely improve the characteristics of MTZ reduction. So Gr-IL could be substituted for the reduction of MTZ. 3.3. Effect of pH value The effect of solution pH on the electrochemical response of 2.3 × 10−5 mol L−1 MTZ at Gr-IL/GCE was investigated with PBS solution in the pH range from 4.2 to 9.0 by cyclic voltammetry. As can be seen in Fig. 3, the peak current increased rapidly with the increase of pH when pH below 7.0, remained practically constant up to 9.0. The peak potential was closely dependent on the pH of the solution. It was found that the values of peak potential shifted to more negative values with the increase of pH below 9.0 (as shown in Fig. 3). Similar results have been reported by Hu and co-workers, they have reported that the peak potential shifted to more negative

Fig. 2. Voltammograms of Gr-IL/GCE (1 and 2), Gr/GCE (3 and 4) and bare GCE (5 and 6) in the presence (1, 3, 5) and absence (2, 4, 6) of MTZ. The concentration of MTZ is 2.3 × 10−5 mol L−1 . Supporting electrolyte: 0.10 mol L−1 phosphate buffer solution (pH 7.0); scan rate: 50 mV s−1 .

Fig. 3. Influence of pH on the reduction peak current and potential of 2.3 × 10−5 mol L−1 MTZ at the modified electrode in the sweep rate of 100 mV s−1 .

values with the increase of pH below 9.0 [20]. The peak potential (Ep ) moved in negative direction with pH rising in the range of 4.2–9.0, and they showed such relationship as: Ep (V) = −0.0465 pH −0.3614 (r = 0.9960). The slope of −46.5 mV pH−1 demonstrated that the numbers of electron and proton transferred in the electrochemical reaction of MTZ were equal.

3.4. Effect of scan rate Useful information involving electrochemical mechanism usually can be acquired from the relationship between peak current and scan rate. Therefore, the electrochemical behaviors of MTZ at different scan rates were investigated on the surface of the Gr-IL/GCE by cyclic voltammetry at pH 8.4 (Fig. 4). A linear relationship (Fig. 4, insert A) (Ip = 2.52 × 10−5 0.5 + 1.57 × 10−6 , n = 12, r = 0.9987) was observed between the peak current and the scan rate in the range of 0.01–0.3 V s−1 . This suggests that the process of electrode reaction is controlled by diffusion of MTZ, which is the ideal case for quantitative determinations. The peak potential shifted to more negative values with increasing the scan rates. The linear relation between peak potential and logarithm of scan rate can be expressed as Ep (V) = −0.0192 ln (V s−1 ) −0.7863 (n = 12, r = 9980) (Fig. 4 insert B). As for an

Fig. 4. Cyclic voltammograms of 2.3 × 10−5 mol L−1 MTZ in pH 8.4 PBS solution with different scan rates (from 1 to 12: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3 V s−1 ); insert A is the Ip vs  plot; insert B is the Ep vs ln  plot.

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Scheme 1. Proposed mechanism for the electrochemical behavior of MTZ on the surface of Gr-IL/GCE.

irreversible electrode process, according to Laviron [40], Ep is defined by the following equation: 

Ep = E  +

RT ln  (1 − ˛)nF

(1)

for the Gr/GCE and Gr-IL/GCE, the Rct nearly equal to zero, which indicated that the electrochemical probe to the substrate electrodes were accelerated. The result showed that the Gr and Gr-IL composite film can act as effective electron conduction pathway between the electrode and electrolyte.



where Ep is the peak potential (V vs. SCE), E␪ is the formal potential (V vs. SCE), R is the universal gas constant (8.314 J K−1 mol−1 ), T is the temperature (K), ˛ (alpha) is the charge transfer coefficient for the reduction step, n is the number of electrons involved in the rate determining step, F is the Faraday constant (96,485 C mol−1 ). According to Bard and Faulkner [41], for a totally irreversible wave, Ep is a function of scan rate, shifting (for a reduction) in a negative direction by an amount 1.15RT/˛F (or 30/˛ mV at 25 ◦ C) for each ten fold increase in v. So, from this we got the value of ˛ to be 0.625. Further, the number of electron (n) transferred in the electro-reduction of MTZ was calculated to be 3.57 (approximately equal to 4). The conclusions are well consistent with previous report on the electrochemical reduction of MTZ at MWNT/GCE [20]. The proposed mechanism on the Gr-IL/GCE may be expressed with the following equation, which involved four electrons and four protons reduction process (Scheme 1). 3.5. Electrochemical impedance spectroscopy (EIS) of different electrodes Electrochemical impedance spectroscopic measurement (EIS) was carried out in order to further understand the electron transfer on the modified electrode surface. The semicircle diameter of well conducting substrates equals to the electron transfer resistance (Rct ). The value of Ret varies when different substances are present on the electrode surface. By using 1.0 × 10−3 mol L−1 Fe(CN)6 3−/4− as redox probe, the EIS of different electrodes were recorded and the results were shown in Fig. 5. At the GCE, Rct can be estimated to be 2200 , indicating a big electron transfer resistance. Whereas,

Fig. 5. Measured (symbols) EIS of GCE, Chi/GCE, Gr/GCE and Gr-IL/GCE in a solution of 1.0 × 10−3 mol L−1 K3 [Fe(CN)6 ] + 1.0 × 10−3 mol L−1 K4 [Fe(CN)6 ] + 0.1 mol L−1 KCl attached by the corresponding fitted plots (lines). The applied perturbation amplitude was 0.005 V, init E was 0.236 V, the frequencies swept from 105 to 1 Hz, quiet time was 2 s.

3.6. Chronocoulumetry The electrochemically effective surface areas (A) of GCE Gr/GCE and Gr-IL/GCE were investigated by chronocoulometry using 1.0 × 10−4 mol L−1 K3 [Fe(CN)6 ] as model complex (the diffusion coefficient of K3 [Fe(CN)6 ] in 1 mol L−1 KCl is 7.6 × 10−6 cm2 s−1 [42]) based on Anson equation [43]: Q (t) =

2nFAcD1/2 t 1/2 1/2

+ Qdl + Qads

(2)

where c is substrate concentration, D is the diffusion coefficient, n is electron transfer number, Qdl is double layer charge which could be eliminated by background subtraction, Qads is Faradic charge. Other symbols have their usual meanings. Based on the slopes of the curves of Q versus t1/2 (Fig. 6A), A was calculated to be 0.097 cm2 (GCE), 0.172 cm2 (Gr/GCE) and 0.177 cm2 (Gr-IL/GCE). These results indicate Gr can significantly increase the reaction surface area of electrodes and enhance the current of the charge transfer reaction between the electrode and solution species for lower electrode polarization. 3.7. Calibration curve In order to develop a voltammetric method for determining the drug, we selected the differential-pulse voltammetric mode, because the peaks are sharper and better defined at lower concentration of MTZ than those obtained by cyclic voltammetry, with a lower background current, resulting in improved resolution. According to the obtained results, it was possible to apply this technique to the quantitative analysis of MTZ. The

Fig. 6. Plot of Q–t curve of GCE (3), Gr-IL/GCE (2) and Gr/GCE (1) in 1.0 × 10−4 mol L−1 K3 [Fe(CN)6 ] containing 1 mol L−1 KCl. Insert: plot of Q–t1/2 curve on GCE (3), Gr-IL/GCE (2) and Gr/GCE (1).

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Table 1 Performance comparison of the fabricated electrode for MTZ with other electrodes. Electrode Activated GCE MWNT-GCE Carbon fiber microdisk electrode SWCNTs-GCE Gold electrode HMDEc MIP-CPd Gr-IL/GCE a b c d

Linear range (mol L−1 )

Technique

−6

−4

2.0 × 10 –6.0 × 10 2.5 × 10−8 –1.0 × 10−5 5.0 × 10−6 –1.6 × 10−5 1.0 × 10−7 –2.0 × 10−4 5.0 × 10−7 –1.0 × 10−5 2.3 × 10−7 –1.8 × 10−6 3.3 × 10−10 –1.5 × 10−8 1.0 × 10−7 –2.5 × 10−5

a

LSV DPV SWASVb CV DPV DPV DPV

Correlation coefficient

LOD (mol L−1 )

RSD (%)

Ref.

0.9970 0.9970 0.9950 0.9971 0.9975 0.9998 0.9970 0.9990

1.1 × 10−6 6.0 × 10−9 5.0 × 10−7 6.3 × 10−8 1.5 × 10−7 3.6 × 10−8 1.5 × 10−10 4.7 × 10−8

1.7 4.8 3.7 – <5 – 4.5 2.1

[19] [20] [21] [22] [23] [24] [25] Our work

Linear sweep voltammetry. Square wave adsorptive stripping voltammetry. Hanging mercury drop electrode. Molecularly imprinted polymer–carbon paste electrode.

Table 2 Analysis of pharmaceutical formulations by proposed procedures. Pharmaceutical formulationa

Labeled values (mg/tablet)

Reference proceduresb (mg/tablet)

Proposed proceduresc (mg/tablet)

Add (mg/tablet)

Found (mg/tablet)

Tablet 1# Tablet 2#

100 750

102 745

99 ± 2 744 ± 3

98 492

199 1225

Recovery (%)

102 98

a Tablet 1#: batch no. 100603, expiry date: 01/2013, from Europharm laboratories Co., Ltd.; Tablet 2#: batch no. 20110404, expiry date: 03/2013, from Ventruepharm Pharmaceutical (Hainan) Co., Ltd. b Spectrophotometry method. c Average of five replicate measurements ± SD.

phosphate buffer solution of pH 7.0 was selected as the supporting electrolyte for the quantification of MTZ as it gave maximum peak current at pH 7.0. The peak at about −0.69 V was considered for the analysis. Differential-pulse voltammograms obtained with increasing amounts of MTZ showed that the peak current increased linearly with increasing concentration, as shown in Fig. 7. Using the optimum conditions described above, linear calibration curves were obtained for MTZ in the range of 1.0 × 10−7 –2.5 × 10−5 mol L−1 . The linear equation were Ip (A) = 0.0592 c (mol L−1 ) + 3.75 × 10−8 (r = 0.9990). The detection limit was estimated to be 4.7 × 10−8 mol L−1 (S/N = 3). The comparison of Gr-IL/GCE with other electrodes for MTZ determination was listed in Table 1. It can be seen that the Gr-IL/GCE offered reasonable linear range for MTZ detection and the detection limit was lower than some of previous reports. These results indicated that Gr-IL/GCE is an appropriate platform for the determination of MTZ.

3.8. Reproducibility, stability and interferences investigating the fabrication reproducibility, a For 5.0 × 10−6 mol L−1 MTZ solution was measured by six modified electrodes prepared independently and the RSD of the peak current was 2.1%, revealing excellent reproducibility. After the electrode was stored for 7 days at 4 ◦ C in humidity environment, it could retain 93.0% of its original response, suggesting acceptable storage stability. In addition, 300-fold concentration of lactose, glucose, sucrose, urea, NaCl, KNO3 , Ca(NO3 )2 ; 150-fold concentration of aminoacetic acid, thiamine hydrochloride, starch, dextrin, sodium dodecyl sulfate, ascorbic acid, MnSO4 ; 50-fold concentration of hydroxypropyl cellulose, FeCl3 , MgCl2 , ZnSO4 ; 20-fold concentration of Pb(NO3 )2 , Al(NO3 )3 do not interfere with the reduction signal of 5.0 × 10−6 mol L−1 MTZ (peak current change < 5%). These results indicated that Gr-IL/GCE has an excellent selectivity for MTZ, and it might be applied to determine MTZ in real samples. 3.9. Analytical application The applicability of the developed method was evaluated by analyzing two samples of commercial drugs containing MTZ commercialized in the market. The results obtained by the proposed and reference (spectrophotometry method [14] for the assay of MTZ was adopted) procedures for pharmaceutical formulations are compared in Table 2 and are in good agreement. 4. Conclusions

Fig. 7. Differential pulse voltammograms of Gr-IL/GCE in different concentrations of MTZ solutions. Line 1–12: 1.0 × 10−7 , 2.5 × 10−7 , 5.0 × 10−7 , 1.5 × 10−6 , 2.5 × 10−6 , 3.5 × 10−6 , 7.5 × 10−6 , 1.5 × 10−5 , 2.0 × 10−5 , 2.5 × 10−5 , 3.0 × 10−5 , 3.5 × 10−5 mol L−1 . Insert: calibration curve.

Gr-IL/GCE has been used successfully for electrocatalytic reduction of MTZ in neutral solutions. The results indicate that the modified electrode facilitates determination of MTZ with good sensitivity and reproducibility compared to similar based electrodes or other instrumental methods. This sensor can be used for amperometric determination of selected analytes as low as 4.7 × 10−8 mol L−1 with good reproducibility. In addition, the result obtained in the analysis of MTZ in medicines demonstrates the applicability of the method for real sample analysis.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 21075107).

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Biographies

Jinyun Peng received his M.Sc. in 2007 from Guilin University of Technology under the supervision of Prof. Linli Tang. He then spent (2007–2009) at Guangxi Normal University for Nationalities, China. Currently he is pursuing his Ph.D degree under the supervision of Prof. Xiaoya Hu in Yangzhou University, China. His current research interests are mainly focused on the synthesis and electrochemistry of graphene-based materials.

J. Peng et al. / Sensors and Actuators B 169 (2012) 81–87

Chuantao Hou received his B.Sc. in 2007 from Taishan University. Currently he is pursuing his PhD degree under the supervision of Prof. Xiaoya Hu in Yangzhou University, China. His current research interests are mainly focused on synthesis and electrochemistry with functionalized mesoporous materials.

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Xiaoya Hu is currently Professor in College of Chemistry and Chemical Engineering at Yangzhou University, China. He received his B.Sc. from Yangzhou Teachers’ College of China, and his PhD from Nanjing University, China. His current research interests include electroanalytical chemistry, bioelectrochemistry and sensors, physical electrochemistry and interfacial electrochemistry.