Adsorptive stripping voltammetry determination of methyldopa on the surface of a carboxylated multiwall carbon nanotubes modified glassy carbon electrode in biological and pharmaceutical samples

Accepted Manuscript Title: Adsorptive stripping voltammetry determination of methyldopa on the surface of a carboxylated multiwall carbon nanotubes modified glassy carbon electrode in biological and pharmaceutical samples Author: Behzad Rezaei Neda Askarpour Ali A. Ensafi PII: DOI: Reference:

S0927-7765(13)00247-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2013.04.004 COLSUB 5728

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

28-11-2012 6-3-2013 1-4-2013

Please cite this article as: B. Rezaei, N. Askarpour, A.A. Ensafi, Adsorptive stripping voltammetry determination of methyldopa on the surface of a carboxylated multiwall carbon nanotubes modified glassy carbon electrode in biological and pharmaceutical samples, Colloids and Surfaces B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.04.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Adsorptive stripping voltammetry determination of methyldopa on the surface of a carboxylated multiwall carbon nanotubes modified glassy carbon

ip t

electrode in biological and pharmaceutical samples

cr

Behzad Rezaei1*, Neda Askarpour, Ali A. Ensafi

us

Department of Chemistry, Isfahan University of Technology, Isfahan 8415683111, I.R. Iran Abstract

an

In the present work, a simple carboxylated multiwall carbon nanotubes (CMWCNTs) modified glassy carbon electrode was developed for sensitive determination of methyldopa

M

(MTD). The study of modified electrode and MTD electrochemical behavior at its surface were

d

investigated employing SEM, adsorptive stripping voltammetry, electrochemical impedance

te

spectroscopy and chronocoulometry. These studies show that the oxidation of MTD is facilitated at the surface of GCE which is casted with CMWCNTs and remarkably peak current enhanced

Ac ce p

comparing to the bare electrode due to its adsorption on the electrode surface. Also, because of the catalytic property of modified electrode onset potential decreased for oxidation of MTD. Under optimized conditions, the calibration curve was linear in two concentration ranges of 0.1 –30 and 30.0 – 300.0 µM with a detection limit of 0.08 µM and relative standard deviation (R.S.D.%) lower than 3.0% (n = 5). This modified electrode was used as a sensor for determination of MTD in pharmaceutical and human urine samples with satisfactory results. Keywords: Methyldopa, Adsorptive stripping voltammetry, Modified glassy carbon electrode, Carboxylated multiwall carbon nanotube, Preconcentration. 1

*Corresponding author. Tel: +98 311 3912351, Fax: +98 311 3912350 Email address: [email protected] or [email protected] (B. Rezaei)

Page 1 of 27

1. Introduction Methyldopa (MTD), known as (S)-2-amino-3-(3,4-dihydroxyphenyl)-2-methyl-propanoic

electrochemically

to

o-quinones.

MTD

is

converted

to

a

ip t

acid, is a catecholamine derivative. Catecholamines are aromatic vic-diols and can be oxidized methyldopamine

and

cr

α-methylnorepinephrine and widely used as an antihypertensive agent to reduce blood pressure in hypertensive patients [1,2]. Hypertension is a highly prevalent worldwide disease, constituting

us

one of the main risk factors for cardiovascular morbidity and mortality. Today, antihypertensive

an

drugs are widely used and with attention to side effects of these drugs, dosage control of them is very important. Side effects of a-methyldopa requiring drug discontinuation are relatively

M

infrequent. Sedation and fatigue may occur but are usually transient. Other side effects include positive Coombs test, drug-induced fever, and pancreatitis, hemolytic anemia and hepatic

te

important [3–6].

d

dysfunction. Thus, determination of MTD in pharmaceutical and biological samples is very

Ac ce p

Various methods like spectrophotometry [7–9], chromatography [10–12], mass spectrometry [13,14] and nuclear magnetic resonance spectroscopy [15] have been reported previously for the determination of MTD. Considering the complexity of the biological and pharmaceutical samples and low concentration of the analytes in such samples, new simple analytical methods have to be developed with high sensitivity and selectivity. Electrochemical methods have attracted more attention in recent years to determine drugs and metabolites in biological fluids due to their simplicity, accuracy and lower cost without requiring complex sample pretreatment. But ordinary electrochemical methods usually have not enough selectivity and sensitivity for analysis in complex matrix such as pharmaceutical and biological samples. Various methods are used to modify the surface of electrodes which can increase sensitivity and selectivity of the

Page 2 of 27

electrochemical sensors. Recently, electrodes have been modified with carbon nanotubes (CNTs) to increase sensitivity.

ip t

Carbon nanotubes opening a new and rapidly expanding research field on nanoscale materials because of their high surface area, good chemical stability and significant mechanical

cr

strength [16]. CNTs proved also to be excellent new electrode materials for a wide range of electrochemical applications such as energy, sensors and hydrogen storage [17–19]. The

us

extraordinary electrochemical features of CNTs make them suitable for applying in Faradaic

an

processes (e.g., the fast electrontransfer kinetics of CNTs, due to the presence of edge plane graphite sites within the walls and at the ends of CNTs) or in non-Faradaic processes (e.g., the

M

large changes in conductance when CNTs can accept or withdraw charges from or to molecules in their nearest chemical environment). Furthermore, CNT possess a hollow core suitable for

d

storing guest molecules and have the largest elastic modulus of any known materials. Also, their

te

surface can be activated with different chemical treatments to achieve special functional groups

Ac ce p

such as COOH and OH. These hydrophilic functional groups can be improved CNTs adsorption properties [20,21].

The objective of this research is to develop a MTD sensor using activated multiwall carbon nanotubes (CMWCNTs) based on its adsorption on the electrode surface that can serve as a preconcentration step for highly sensitive adsorptive stripping measurement. The electrochemical behavior of MTD on the modified glassy carbon electrode (CMWCNTs-GCE) was investigated employing cyclic voltammetry, chronocoulometry and electrochemical impedance spectroscopy (EIS). At the surface of modified electrode remarkably peak current for MTD oxidation enhanced and onset potential shifted to lower potential compare to bare GCE.

Page 3 of 27

2. Experimental 2.1. Apparatus and materials

ip t

Electrochemical measurements were carried out in a potentiostat-galvanostat Autolab connected to a conventional three electrode cell. The system was run on a PC by GPES and FRA

cr

4.9 software. A saturated Ag/AgCl reference electrode, platinum electrode and CMWCNTs

us

modified GCE was used as a reference, auxiliary, and working electrode, respectively. The EIS measurements were recorded within a frequency range of 100 kHz to 10 mHz. For stripping

an

voltammetric analysis a magnetic stirrer with stirring bar was used for the convective transportation of the analyte during its accumulation onto the surface of electrode.

M

The CMWCNTs were bought from Iran’s Research Institute of Petroleum Industry with a

d

length 50 µm and the purity of 95%. The modified glassy carbon electrode with CMWCNTs

te

layer was characterized using scanning electron microscopy (SEM).

Ac ce p

Reagent grade MTD was purchased from Aldrich and 10 mM of its solution was prepared and used as the stock solution. Other solutions were prepared freshly by sequential dilution of the stock solution. The pH of solution was adjusted with using 0.10 M phosphate buffer solutions in different pH values. All other chemicals used were reagent grade and doubly distilled water was used in preparation of all solutions. 2.2. Preparation of modified GCE

8.0 mg of the CMWCNTs was added to 4.0 mL of acetonitril, the mixture was sonicated to obtain a relative stable suspension. The GCE was carefully polished with 0.05 µm alumina slurry on a polishing cloth and rinsed with water to give a smooth electrode surface. Then the electrode

Page 4 of 27

was washed ultrasonically in ethanol-water (1:1, v:v) and water, respectively. Modified GCE was prepared by dropping 50 µL of the suspension of CMWCNTs on the surface of the cleaned

formed over the entire surface of the GCE after modification.

cr

2.3. Preparation of real samples

ip t

GCE and casting was done by evaporating of solvent at room temperature. A uniform film was

us

Tablet of methyldopa with the commercial name of Aldomet were prepared. Each tablet contains 250 mg methyldopa. Five methyldopa tablets were finely powdered using a mortar and

an

pestle and then, appropriate amount of this sample containing a known amount of the active material was weighed and dissolve with double distilled water. The prepared mixture was filtered

M

using paper filter (Whatman Filter Paper, No. 41) and diluted to appropriate amounts double distilled water. The urine samples were collected from healthy persons and were centrifuged (3

d

min with 3000 rpm) and then diluted 10 times with doubly distilled water without any additional

te

pretreatment. Before voltammetric determination, appropriate amount of prepared real samples

Ac ce p

were added to 10 mL of phosphate buffer solution with optimum pH (pH 8.0) and then was transferred to the electrochemical cell for electrochemical measurements. The standard addition method was used to determine methyldopa in the real and spiked samples.

3. Results and discussion

3.1. Characterization of the CMWCNT-modified GCE The structure and dispersing state of CMWCNTs were studied using scanning electron microscopy. Fig. 1 shows the SEM image of CMWCNT-GCE. This figure confirms that the

Page 5 of 27

surface of GCE was well covered by CMWCNTs. It is obvious that CMWCNTs were distributed uniformly on the surface of GCE. It can be seen that CMWCNTs formed a film with high surface

ip t

area. The microscopic areas of the CMWCNT-modified GCE and the bare GCE were obtained by

cr

CV method, using 1.0 mM K3Fe(CN)6 solution as a probe at different scan rates. For a reversible

us

process, the Randles-Sevcik equation has been used: Ipa = (2.69 × 105) n3/2CoDR1 /2 υ1/2A

(1)

an

Where Ipa (A) refers to the anodic peak current, n is the electron transfer number, DR (cm2 s-1) is the diffusion coefficient, Co (mol cm-3) is the concentration of K3Fe(CN)6, υ (V s-1) is the scan

M

rate and A (cm2) is the surface area of the electrode. For 1.0 mM K3Fe(CN)6 in the 0.1 M KCl

d

electrolyte: n=1, DR = 7.6×10−6 cm2 s−1 [22], then from the slope of the Ipa – υ1/2 relation

te

(I(µA) = 125.3υ1/2 – 16.107, R2 = 0.9967), the microscopic areas can be calculated. In the bare GCE, the electrode surface was 0.0314 cm2 and in MWCNT-modified GCE the microscopic

Ac ce p

surface was 0.1688 cm2 that was nearly 5.4 times greater. 3.2. Electrochemical behavior of methyldopa on GCE and CMWCNTs modified GCE The electrochemical oxidation of methyldopa was studied by cyclic voltammetry at the surface of the bare and CMWCNTs modified GCE (Fig. 1A). The oxidation of MTD shows a weak peak on the bare GCE but the experimental results for modified GCE show well-defined anodic peak at the peak potential of 0.214 V, respect to Ag/AgCl reference electrode. It could be observed that the oxidation peak current for modified electrode significantly increased and it was almost 50 times larger than unmodified electrode. This behavior is due to adsorption of MTD on the surface of CMWCNTs by interaction of MTD functional groups such as NH2, COOH and

Page 6 of 27

OH with carboxyl groups of activated MWCNTs on the surface of electrode. Thus sensitivity significantly enhanced due to preconcentration of MTD on the active surface of CMWCNTs. Also, as shown in the Fig.2A, the onset potential for MTD oxidation at CMWCNTs modified

ip t

GCE is lower than its oxidation at a bare GCE because of catalytic behavior of modified electrode. However, the potential peak at the bare GCE (0.133 V) is lower than the potential

cr

peak at the modified GCE (0.214 V) because the electrode response for the bare GCE is

us

controlled by diffusion while for the modified GCE the response is controlled by adsorption and diffusion. The oxidation peak of MTD in the pH of 8.0 is irreversible and thus with increase in

an

the peak height, the peak potential shifts to higher potential. Thus the peak potential of the modified GCE shifts to the higher potential compare to the bare GCE. But onset potential that

M

show the kinetic of the reaction, decreased for the modified GCE compare to the bare GCE and

d

thus sensitivity and selectivity increased because of these effects.

te

EIS is an electrochemical technique that allows for a wide variety of coating evaluations is

Ac ce p

effective in probing the interfacial properties of surface-modified electrodes. The Nyquist diagrams (the imaginary impedance (Zim) versus the real impedance (Zre)) and the appropriate equivalent circuit model are used to correlate the impedance with the capacitance and resistance of the electrodes. It has been known that the amount of Rct have reverse relationship with electrochemical activity of the species [23–25]. The Nyquist diagrams were recorded before and after modification of electrode with CMWCNTs (Fig. 2B). The results show, at the bare GCE, a semicircle with Rct about 315.5kΩ was obtained. However, the diameter of the semicircle was obviously reduced to 0.454 kΩ by modification of the electrode with CMWCNTs. Therefore, the charge transfer rate for oxidation

Page 7 of 27

of methyldopa on the CMWCNTs modified GCE is significantly higher than that of the bare GCE which is in agreement with CVs studies.

ip t

3.3. Effect of pH on the peak potential and peak current The peak current and potential are dependent on the pH of solution. To find the optimum pH,

cr

the influence of pH over the range of 3.0 – 10.0 on the performance of the sensor was

us

investigated. Experimental results for 100 µM methyldopa solution are shown in Fig.3A. It can be seen that the anodic peak current of MTD was increased by pH and reached to maximum

an

value at pH 8.0. Therefore, pH 8.0 was selected as the optimum pH for the determination of MTD. Increasing the peak current with the increase of the pH shows that the mechanism for

M

oxidation of MTD is a proton dependent reaction.

d

It was observed that as the pH of solution was increased, the oxidation peak potential was

te

shifted to negative potential values. The negative shift and the peak potential, showed a linear relationship with the slope of –53.1 mV pH–1 in the pH range of 5.0 – 8.0. This slope

Ac ce p

approximately revealed that the number of proton in the process is equal with the number of electron transfer in the oxidation reaction of MTD. 3.4. Effect of scan rate on the peak potential and peak current The dependence of the peak potential and the peak current on the scan rate at the modified GCE was examined using cyclic voltammetry in the presence of 100 µM methyldopa at pH 8.0. The anodic peak current increased linearly with the scan rate which indicates an adsorption controlled oxidation process occurring at the surface of modified GCE. Over the range of 10 – 150 mV s–1 the linear regression equation was Ipa (µA) = 0.8014 υ (mV s–1) + 18.56, with a correlation coefficient of 0.9970.In the higher scan rates, the oxidation peak of methyldopa was

Page 8 of 27

broad and unclear. Thus, the scan rate of 50 mV s–1 was selected for determination of methyldopa.

ip t

According to the relationship between the peak potential and ln (υ) and based on the slope of the fitted line, RT/(1− α)nαF = 0.0.093, the value of transfer coefficient (α) was calculated to be

cr

0.72. The Tofel plot (log (I) versus E) was obtained in the scan rate of 5 mV s–1. In this work, the slope of Tofel plot was found to be 4.0779 A V–1. Thus, transfer coefficient is found to be 0.76

us

for a one electron transfer process, which is the rate determining step [26].

an

From the obtained results and previous reports [27,28], a mechanism for the oxidation of methyldopa that can be proposed shown in scheme 1. Such as other catecholamines, oxidation of

M

methyldopa at the pH = 8.0 was irreversible and the oxidation process involved two electrons

d

and protons.

te

3.5. Effect of CMWCNTs amounts on the peak current

Ac ce p

The modifier amount has a significant effect on the response of electrode. So the electrochemical behavior of the MTD was studied by casting different amount of dispersed CNTs (10 µL to 70 µL of 2 mg/mL CMWCNTs/CH3CN suspension) onto the GCE surface. It was observed that the oxidation peak current for MTD increased when the volume of CNTs suspension deposited on the surface of the electrode was increased up to 50 µL (Fig. 3B). Beyond this point, the peak current decreased and the electrode became unstable. In all subsequent experiments 50 µL of CNTs suspension was selected as the optimum amount for the preparation of the modified GCE. 3.6. Effect of accumulation potential and time on the peak potential and peak current

Page 9 of 27

The influence of the accumulation potential on the oxidation peak current was examined by employing different potential from –0.4 to +0.1 V after accumulation for 5 min (Fig.3C). If the accumulation potential increases, the oxidation peak current of MTD will increase consequently.

ip t

Since under open circuit potential (OCP) peak current has maximum value, the accumulation

cr

was performed under this potential.

An increase in the accumulation time affects on the stripping voltammetric peak current

us

and thus improves the sensitivity. The effect of accumulation time on the peak current was

an

studied for different accumulation time in the presence of 10 and 100 µM methyldopa (Fig.3D). According to the results, the oxidation peak current amplifies at the first, and then reaches to a

M

steady amount after 6 minutes. This behavior indicates that adsorption saturation occurred at higher accumulation times. Thus, for all following experiments, 6 min and open circuit potential

te

3.7. Chronocoulometry (CC)

d

were used as accumulation time and potential, respectively.

Ac ce p

The determination of the amount of adsorbed reactant, Γ0, is possible by employing potential step chronocoulometry that the charge passed as a function of time, Q(t) is obtained. According to the Cottrell equation Q increases with time and a plot of its value vs. t1/2 is linear. The intercept of Q vs. t1/2 is Qdl + Qads. Where Qdl is the capacitive charge and Qads (=nFAΓ0) quantifies the faradic component given to the reduction of Γ0 mol/cm2 of adsorbed oxidant. An approximate value of the surface coverage (Γ0) can be estimated by comparing the intercept of the Q–t1/2 plot that obtained for a solution containing methyldopa, with the instantaneous charge passed in the same experiment performed with only supporting electrolyte [26,29,30].

Page 10 of 27

Chronocoulograms of MTD were obtained by setting the potential of the working electrode at 0.2 V for electrolyte and various concentration of MTD (50, 100 and 150 µM).The mean value of Γ0 was calculated to be 1.634ngcm–2. According to these results, due to the effect of

ip t

CMWCNTs, the electrode surface coverage increased which is in agreement with the results

cr

obtained from CV studies.

us

3.8. Determination of methyldopa

Dependence of the oxidation peak current of MTD to its concentration was investigated

an

using adsorptive stripping cyclic voltammetry under the optimum conditions selected as: pH = 8.0 with a scan rate of 50 mV s–1 , and the stripping time of 6 min in open circuit potential. The

M

calibration plot of the proposed sensor is linear in two concentration ranges of 0.1 – 30 and 30.0 – 300.0 µM. The regression equation over theses ranges are: I (µA) = 2.257 × CMTD (µM) –

d

0.645, R2 = 0.994 and I (µA) = 0.622 × CMTD (µM) + 51.36, R2 = 0.998, respectively (Fig.4).The

te

detection limit of the proposed sensor, that determined by S / N = 3, was 0.08 µM. The relative

Ac ce p

standard deviation (RSD) for MTD, 20 µM and 5 repeated measurements was less than 3%. Response characteristics of the proposed method are compared in Table 1 with previously reported electrochemical methods for determination of methyldopa [31–38]. It can be found that the modified CMWCNT-GCE offered a comparable detection limit for MTD determination in evaluation to other reported electrodes. 3.9. Interference studies To monitor the selectivity of the sensor, the effects of some various interferences in electrochemical determination of MTD which are common foreign species and present in real samples were evaluated. The tolerance limit for interfering species was defined as the maximum

Page 11 of 27

concentration that caused a relative error of 5.0 % for determination of 10 and 50 µM of MTD under the optimized conditions. The results showed that 1000-fold of Na+, K+, Cl–, NO3–, Br–, glucose, fructose, urea and uric acid, 500-fold of SO42–, thiourea and citric acid, 200 fold of F–

ip t

and sucrose and 50 fold of Mg2+ and Ca2+ and saturated starch solution did not affect the current response of methyldopa. This results show the peak current of methyldopa is not affected by

cr

conventional cations, anions, and organic substances. However, ascorbic acid interferes in the

us

analysis of MTD beyond 30 fold excess for 50 µM of MTD and 10 fold excess for 10 µM of

an

MTD and interference from it can be minimized using ascorbic acid oxidase if necessary. 3.10. Analytical application

M

To evaluate the applicability of the proposed method, CMWCNT/GCE was used to determine the concentrations of MTD in tow tablets and urine samples. The standard addition

d

method was used for the analysis of prepared samples. The results of triplicate measurements are

Ac ce p

102.9 %.

te

summarized in Table2. Satisfactory recoveries for all samples were obtained between 97.3 % and

4. Conclusion

The development of a simple and sensitive adsorptive stripping voltammetric sensor for determination of methyldopa using CMWCNTs modified GCE is described. The sensitivity not only significantly enhanced due to preconcentration of MTD on the active surface of CMWCNTs, but also increased because of its electrocatalytic effect. As well, electrochemical studies used to estimate MTD oxidation mechanism that shows the contribution of two electrons and two protons in the oxidation process. The proposed sensor, in comparison to other reported methods, has a good sensitivity with a low detection limit of 0.08 µM. After optimization of

Page 12 of 27

analytical conditions, the proposed modified electrode can be successfully applied as a sensor for

ip t

determination of trace amount of MTD in drug and urine samples.

Acknowledgements

cr

The authors wish to thank Isfahan University of Technology (IUT) Research Council and

an

us

Center of Excellence in Sensor and Green Chemistry for supporting of this work.

References

M

[1] N.J. Aradell, Phisical Desk References, 31st ed., Medical Economics Co., 1977, p. 1058.

d

[2] B.B. Hoffman, R.J. Lefkowitz, Catecholamines, Catecholamines, sympathomimetic drugs,

te

and adrenergic receptor antagonists, (Ed. A.G. Gilman) in: The Pharmacological Basis of

Ac ce p

Therapeutics, 9th ed., McGraw-Hill, New York, 1996, p. 211. [3] N.L. Benowitz, (Ed. B.G. Katzung), in: Basic and Clinical Pharmacology, 9th ed., McGrawHill, New York, 2004, p. 166.

[4] M. Dolezalova, M. Tkaczykova, J. Pharm. Biomed. Anal., 19 (1999) 555. [5] G. Moreira, J. Cipullo, J.V. Martin, L. Ciorlia, M. Godoy, C. Cesarino, J. Cordeiro, P. Lupino, G. Ciorlia and E. Burdmann, J. Hyperten., 27 (2009) 1900. [6] W. Vongpatanasin, K.Kario, S. A.Atlas and R. G.Victor,J. Clinic. Hyperten., 13 (2011) 658. [7] P.R.S. Ribeiro, J.A. Gomes Neto, L. Pezza and H.R. Pezza, Talanta, 67 (2005) 240.

Page 13 of 27

[8] I.C. Vieira and O. Fatibello-Filho, Talanta, 46 (1998) 559. [9] P.R. S.Ribeiro, L. Pezza and H.R. Pezza, J. Braz. Chem. Soc., 17 (2006) 674.

ip t

[10] S.F. Li, H.L. Wu, Y.J. Yu, Y.N. Li, J.F. Nie, H.Y. Fu and R.Q. Yu, Talanta, 81 (2010) 805.

cr

[11] G. Bahrami, A. Kiani and S. Mirzaeei, J. Chromatogr. B, 832 (2006) 197.

[12] M. Zečević, L. Živanović, S. Agatonovic-Kustrin and D, Minic, J. of Pharm. Biomed. Anal.,

us

24 ( 2001) 1019.

M

G.D. Nucci, J. Chromatogr. B, 768 (2002) 341.

an

[13] C.H. Oliveira, R.E. Barrientos-Astigarraga, M. Sucupira, G.S. Graudenz, M.N. Muscará and

[14] C.R. Freed, R.J. Weinkam, K.L. Melmon and N. Castagnoli, Anal. Biochem., 78 (1977)

d

319.

te

[15] Z. Talebpour, S. Haghgoo and M. Shamsipur, Anal. Chim. Acta, 506 (2004) 97.

Ac ce p

[16] S. Iijima, Nature, 354 (1991) 56.

[17] V. Lazarescu, Dekker Encyclopedia of Nanoscience and Nanotechnology, 2nd ed., Taylor and Francis, 2009, p. 586.

[18] K. Balasubramanian, M. Burghard and K. Kern, Dekker Encyclopedia of Nanoscience and Nanotechnology, second edition, Taylor and Francis, 2009, p. 620. [19] J. Wang, Electroanalysis, 17 (2005) 7. [20] P. Yáñez-Sedeño, J.M. Pingarrón, J. Riu and F.X. Rius, Trends in Anal. Chem., 29 (2010) 939.

Page 14 of 27

[21] G.A. Rivas, M.D. Rubianes, M.C. Rodriguez, N.F. Ferreyra, G.L. Luque , M.L. Pedano, S.A. Miscoria and C. Parrado, Talanta 74 (2007) 291.

ip t

[22] R.N. Adams, Electrochemistry at Solid Electrodes, Marcel-Dekker, New York, 1969. [23] A. Lasia, Electrochemical Impedance Spectroscopy and Its Applications, Modern Aspects

cr

of Electrochemistry, B. E. Conway, J. Bockris, and R.E. White, Edts., Vol. 32, Kluwer

an

[24] I.I. Suni, Trends in Anal. Chem., 27 (2008) 604.

us

Academic/Plenum Publishers, New York, 1999, p. 143.

[25] M. Rueda and F.P. Dapena, Collect. Czech. Chem. Commun., 76 (2011) 1825.

M

[26] A.J. Bard and L. R. Faulkner, Electrochemical Methods Fundamentals and Applications,

d

2nd ed. New York: Wiley, 2001.

te

[27] T.E. Young, B.W. Babbitt, and L.A. Wolfe, J. Org. Chem., 45 (1980) 2899.

5633.

Ac ce p

[28] A. Afkhami, D. Nematollahi, T. Madrakian and L. Khalafi, Electrochim. Acta, 50 (2005)

[29] A.W. Bott and W.R. Heineman, Curr. Sep., 20 (2004) 121. [30] F. Anson, Anal. Chem. 36 (1964) 932. [31] M.B. Gholivand and M. Amiri, Electroanalysis 21 (2009) 2461. [32] S. Shahrokhian, R.S. Saberi and Z. Kamalzadeh, Electroanalysis, 23 (2011) 2248. [33] A. Mohammadi, A. B.Moghaddam, R. Dinarvand, J. Badraghi, F. Atyabi and A.A. Saboury, Int. J. Electrochem. Sci., 3 (2008) 1248.

Page 15 of 27

[34] S.K. Moccelini, A.C. Franzoi, I.C. Vieira, J. Dupont and C.W. Scheeren, Biosens. and Bioelectronics 26 (2011) 3549.

ip t

[35] M. Fouladgar and H. Karimi-Maleh, Ionics, (2012) 1. [36] H. Karimi-Maleh, M.A. Khalilzadeh, Z. Ranjbarha, H. Beitollahi, A.A. Ensafi and D.

cr

Zareyee, Anal. Method., 4 (2012) 2088.

us

[37] A. Salmanipour, M.A. Taher and H. Beitollahi, Anal. Method., 4 (2012) 2982.

Ac ce p

te

d

M

an

[38] S. Shahrokhian and S. Rastgar, Electrochim. Acta, 58 (2011) 125.

Page 16 of 27

ip t

Table 1. Comparison of the characteristics of the proposed method with those of previously reported electrochemical methods. LDR (µM) 0.1 – 20 0.2 – 50 0.1 – 5 34.8 – 370.3 0.4–400.0 0.5–165.5 0.1–500 0.05–40 0.1 – 300

cr

LOD (µM) 0.05 0.06 0.02 5.5 0.1 0.2 0.08 0.01 0.08

us

Method DPV DPV DPV SWV SWV SWV SWV DPV ASV

an

Modifier polypyrrole/ nuclear fast red Polypyrrole/CNPs Tyrosinase/single-walled CNTs Cellulose acetate /BMI·N(Tf)2 MWCNTs and Ionic liquid MWCNTs and p-chloranil CNT and ferrocene MWCNT and Pt–RuNPs CMWCNTs

Ref. [31] [32] [33] [34] [35] [36] [37] [38] This work

M

Electrode Au Glassy carbon Glassy carbon Carbon paste Carbon paste Carbon paste Carbon paste Glassy carbon Glassy carbon

Ac ce p

te

d

LOD, limit of determination; LDR, linear dynamic range DPV, Differential Pulse Voltammetry; SWV, Square Wave Voltammetry; ASV, Adsorptive Stripping Voltammetry BMI·N(Tf)2, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

Page 17 of 27

Urine 1

Urine 2

5.0

4.71 ± 0.7

5.0

10.0

10.92 ± 0.5

15.0

20.0

20.11 ± 0.2

97.9

–

40.0

41.16 ± 0.3

102.9

20.0

60.0

60.04 ± 0.8

100.1

40.0

80.0

78.31 ± 0.5

97.9

5.0

5.0

10.0

10.0

20.0

20.0

30.0

30.0

40.0 a

us

94.2

109.2

5.32 ± 0.6

106.4

9.81 ± 0.9

98.1

20.67 ± 0.8

103.3

30.54 ± 0.1

101.8

40.0

40.72 ± 0.4

101.8

50.0

48.66 ± 0.4

97.3

Ac ce p

50.0

Recovery (%)

cr

–

an

c

Found (µM)

M

Tablet 2

b

Expected (µM)

d

Tablet 1

a

Added (µM)

te

Sample

ip t

Table 2. Determination of methydopa in drug and urine samples

Standard deviation (n=3) Zahravi Pharm. Co. (Tabriz, Iran) c Darou Pakhsh, Pharmaceutical Mfg. Co. (Tehran, Iran) b

Page 18 of 27

ip t cr

Figure and scheme captions

Fig. 1. SEM image of CMWCNTs modified GCE.

us

Scheme 1. Mechanism of methyldopa Oxidation with electron–proton transfer step.

an

Fig.2. (A) Background-subtraction stripping voltammograms of 100 µM of MTD at the CMWCNT-modified GCE (a) and at the bare GCE (b), in the scan rate of 50 mV s–1, pH=8.0 and

M

accumulation time of 5 min in OCP. Inset shows cyclic voltammogram of the bare GCE. (B) Nyquist plots of the impedance measurement of a 100 µM of MTD performed on CMWCNT-

te

d

modified GCE (a) and bare GCE (b). Inset shows Nyquist plots of the CMWCNT-modified

kHz.

Ac ce p

GCE. Bios was 0.12 V with 0.005 V ac voltage amplitude and frequency range of 0.01 Hz to 100

Fig.3. Effect of (A) the solution pH, (B) the amount of CMWCNTs, (C)the accumulation potential and (D) the accumulation time on the oxidation peak current and peak potential for 100 and 10 µM of MTD on the modified GCE in the scan rate of 50 mV s–1 and accumulation time of 5 min in OCP.

Fig.4. Calibration curves for determination of methyldopa at the optimum condition (pH 8.0,scan rate 50 mV s−1 and accumulation time of 6 min in OCP). Inset shows some raw voltammograms of methyldopa oxidation.

Page 19 of 27

ip t cr

Scheme 1.

HO

-2 H+, -2 e-

NH2 HO

O CH3

O

an

OH

us

O CH3

OH NH2

Ac ce p

te

d

M

O

Page 20 of 27

ip t

Ac ce p

te

d

M

an

us

cr

Fig. 1.

Page 21 of 27

ip t

100

I / mA

1.0

60

0

0.2

0.4

0.6

E/V

50

M

40 30 20

-1 0 -0 .2

-0 .1

te

B

0

a

d

10

0

Ac ce p

A  / I 

-1.0 -0.2

an

0.0

70

A

us

2.0

90 80

cr

Fig. 2.

0 .1

b

0 .2

0 .3

0 .4

0 .5

0 .6

E /V

B

Page 22 of 27

120

100

b

20

ip t cr

40

80 70 60 50 40 30 20 10 0

us

a 60 Z im / kΩ

Zim / kΩ

80

0

0 50

100

150

200

an

0

10

20

250

30 40 Z re / kΩ 300

50

60

350

M

Z re / kΩ

B

Ac ce p

A

te

d

Fig. 3.

Page 23 of 27

C

D

Ac ce p

te

d

M

an

us

cr

ip t

C

Fig. 4.

Page 24 of 27

Page 25 of 27

d

te

Ac ce p us

an

M

cr

ip t

Develop a methyldopa sensor using an activated carbon nanotubes modified electrode. Methyldopa determination is based on its strong adsorption on the surface of CNT.

ip t

Modified electrode used as a preconcentration tool for highly sensitive measurement.

cr

The proposed modified CMWCNT-GCE offered a very wide linear dynamic range.

Ac ce p

te

d

M

an

us

Low detection limit obtained for determination in comparison to other electrodes.

Page 26 of 27

an

us

cr

ip t

(A)

(B)

M

100 90 80

d

70

50

te

DI / mA

60

40

Ac ce p

30 20

a

10

b

0

-10

-0.2

-0.1 1

0

0.1

0.2

0.3

0.4

0.5

0.6

E /V

(A) SEM image of MWCNTs modified GCE.

(B) Stripping voltammograms of 100 µM of MTD at the bare GCE (a) and at the MWCNT MWCNT-modified modified GCE (b)

Page 27 of 27