Elsevier Editorial System(tm) for Sensors & Actuators: B. Chemical Manuscript Draft Manuscript Number: Title: Nanomolar simultaneous determination of levodopa and melatonin at a new cobalt hydroxide nanoparticles and multi-walled carbon nanotubes composite modified carbon ionic liquid electrode Article Type: Research Paper Keywords: Levodopa; Melatonin; Carbon Ionic Liquid Electrode; Electrochemical Sensor; Cobalt Hydroxide nanoparticles Corresponding Author: Dr Ali Babaei, PhD Corresponding Author's Institution: Arak University First Author: Ali Babaei, PhD Order of Authors: Ali Babaei, PhD; Ali Reza Taheri; Iman Khani Farahani Abstract: A novel modified carbon ionic liquid electrode (CILE) is prepared as an electrochemical sensor for simultaneous determination of Levodopa (L-dopa) and Melatonin (Mel). The experimental results suggest that a carbon ionic liquid electrode modified with multi-walled carbon nanotubes and cobalt hydroxide nanoparticles accelerates the electron transfer reactions of L-Dopa and Mel. The fabricated sensor revealed some advantages such as convenient preparation, good stability and high sensitivity. The DPV data in 0.1 M phosphate buffer soloution (PBS) (pH 7.5) allowed a method to be developed for the determination of L-Dopa and Mel concentrations in the ranges 0.1 to 300 and 0.01 to 50 μM, with the detection limits of 0.075 and 0.004 μM, respectively. The proposed method was successfully applied to determinations of these compounds in some pharmaceutical and human urine samples. Suggested Reviewers: Sheng Shui Hu sshu@whu.edu.cn Yuzhong Z. Zhang zhangyz65@hotmail.com Hamid Reza Zare hrzare@yazduni.ac.ir Codrura Cofan cofancodruta@umft.ro Jiannong Ye jnye@ecnu.edu.cn

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Nanomolar simultaneous determination of levodopa and melatonin at

a new cobalt hydroxide nanoparticles and multi-walled carbon

nanotubes composite modified carbon ionic liquid electrode

Ali Babaeia,b,

, Ali Reza Taheria, Iman Khani Farahani

c

a Department of Chemistry, Faculty of Science, Arak University, Arak, 38156-8-8349,

Iran

b Research Center for Nanotechnology, Arak University, Arak, 38156-8-8349, Iran

c Department of Biology, Faculty of Science, Yasouj University, Yasouj, 75918-74831,

Iran

Corresponding author. Tel.: +98 861 4173401; Fax: +98 861 4173406.

E-mail address: a-babaei@araku.ac.ir, ali.babaei08@gmail.com

*ManuscriptClick here to view linked References

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Abstract

A novel modified carbon ionic liquid electrode (CILE) is prepared as an electrochemical

sensor for simultaneous determination of Levodopa (L-dopa) and Melatonin (Mel). The

experimental results suggest that a carbon ionic liquid electrode modified with multi-

walled carbon nanotubes and cobalt hydroxide nanoparticles accelerates the electron

transfer reactions of L-Dopa and Mel. The fabricated sensor revealed some advantages

such as convenient preparation, good stability and high sensitivity. The DPV data in 0.1

M phosphate buffer soloution (PBS) (pH 7.5) allowed a method to be developed for the

determination of L-Dopa and Mel concentrations in the ranges 0.1 to 300 and 0.01 to

50 μM, with the detection limits of 0.075 and 0.004 μM, respectively. The proposed

method was successfully applied to determinations of these compounds in some

pharmaceutical and human urine samples.

Keywords: Levodopa; Melatonin; Carbon Ionic Liquid Electrode; Electrochemical

Sensor; Cobalt Hydroxide nanoparticles.

1. Introduction

Melatonin (Mel, N-acetyl-5-methoxytryptamine) is a lipophilic hormone, mainly

produced and secreted at night by the pineal gland. The mechanisms that control its

synthesis within the pineal gland have been well characterized [1] and the retinal and

biological clock processes that modulate the circadian production of Mel in the pineal

gland are rapidly being unraveled [2]. Main and best-known effect of Mel is restoring

the natural cycle of organism functions[3]. It is safe and non-addictive sleep-inducing

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drug, which can eliminate disruptions in our circadian rhythm, in such situations as shift

working, changing of time zones (during intercontinental air travelling) or insomnia.

However, researchers also have shown that Mel has chronobiotic activities to

resynchronize sleep and circadian rhythms disturbances and it is also involved in the

regulation of seasonal reproduction, body weight and energy balance [4]. Mel can be

detected in biological samples by several methods, such as HPLC [5],

spectrofluorimetric and colorimetric methods[6, 7], electrochemical methods [8, 9], GC-

MS methods [10], radioimmunoassay (RIA) [11], flow injection analysis system

(FIAED)[12] and capillary electrophoresis (CE) [13]. Furthermore, Mel content in

pharmaceutical products has also been determined by capillary electrophoretic methods

with UV [14] or electrochemical detection[15]. However, some drawbacks of these

methods include complicated pretreatment of sample, high cost or low sensitivity.

Levodopa (L-dopa) is a naturally occurring dietary supplement and psychoactive

drug found in certain kinds of herbs and food and is synthesized from the essential

amino acids L-phenylalanine and L-tyrosine in the brain and mammalian body. L-Dopa

is currently the therapeutic drug in the treatment of Parkinson’s disease and required by

the brain to produce dopamine which compensates the deficiency of dopamine in the

organism and decreases the symptoms of Parkinson’s disease[16]. Various analytical

methods have been developed for L-Dopa determination, such as

spectrophotometry[17], liquid chromatography[18], and capillary zone

electrophoresis[19].

Carbon nanotubes (CNTs) are carbon materials that have a new kind of porous

nanostructure, have been found to possess properties such as high surface area, high

electrical conductivity, significant mechanical strength and chemical stability [20].

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Multiwalled carbon nanotubes (MWCNTs) can be used to promote electron transfer

reactions when used as electrode materials in electrochemical Sensors [21].

Room temperature ionic liquids (RTILs) such as 1-butyl-3-methylimidazolium

hexafluorophosphate, (BMIM)(PF6), have been proposed to be very interesting and

efficient pasting binder in place of non conductive organic binders such as Nojul or

paraffin oil for the preparation of carbon ionic liquid electrodes (CILEs)[22]. This new

composite has several advantages compared to other traditional carbon paste (CPEs)

such as high electrical conductivity, high stability and very low vapor pressure [23],

where it offers improved sensitivities toward a number of compounds, and at the same

time lower detection potentials using a very small amount of MWCNTs[24].

Transition-metal nanoparticles, in different forms, have emerged as a novel

family of catalysts able to promote more efficiently a variety of organic transformations

because of their extremely large surface-to-volume ratio and small size [25, 26]. Many

nanoparticles have been successfully introduced onto CNTs, such as TiO2[27],

CdTe[28], Au [29], Cu[30] and Ag [31]. Some electrodes such as platinum gauze[32] ,

glassy carbon electrode [33], and carbon paste electrode [34, 35] have been modified by

Co and Co(OH)2 particles and nanoparticles. Cobalt hydroxide nanoparticles (CHNPs)

with a low crystallinity and nano-flake network structure show a high proton diffusion

coefficient, giving excellent electrochemical performance. Various methods of

preparation of cobalt hydroxide nanoparticles, ranging from spray pyrolysis [36],

sonication[37], sputtering [38] and electrodeposition[33] to precipitate them at various

pH values, have been considered. The method of precipitation is new and facile,

needing no expensive raw materials or equipment. It is also easy for mass production

and can be extended to synthesize other hydroxide or oxide nanocrystals[32].

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Simultaneous determination of L-Dopa and Mel is important, since numerous

reports demonstrated that L-Dopa and Mel influence each other in their respective

releasing [39-43] and also they coexist in a biological system. Lynch et al. reported L-

Dopa administration also causes profound increases in the pineal Mel content and its

biosynthesis [39]. This response is also potentiated by sympathetic denervation of the

pineal. Srinivasan et al. studied Mel secretion patterns in patients suffering from

Parkinson disease [40]. A phase advance of the nocturnal Mel maximum was noted in

L-Dopa-treated but not in untreated patients. Under medication with L-Dopa, daytime

Mel was additionally increased, a finding discussed in terms of an adaptive mechanism

in response to the neurodegenerative process and possibly reflecting a neuroprotective

property of Mel. However, to the best of our knowledge no study has reported yet about

the simultaneous determination of L-Dopa and Mel.

In the present work an RTIL, (BMIM)(PF6), is used as the binder for fabrication

of a CILE and modified with a nanocomposite film which contains MWCNTs and

CHNPs, based on the idea that the MWCNTs with CHNPs could enhance the electron

transfer rate for L-Dopa and Mel, due to synergistic electrocatalysis which leads to

increasing the sensitivity. The fabricated electrode was used as a new sensor for

simultaneous determination of Mel and L-Dopa in some real samples.

2. Experimental

2.1 Reagents and solutions

L-Dopa and Mel were obtained from Acros and Sigma chemical companies,

respectively. (BMIM)(PF6) was obtained from Hangzhou Kemer Chemical Limited

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Company. Spectrally pure graphite powder (average particle size 50 μm) from Merck

was used as received. Multiwalled carbon nanotubes (MWCNTs) (>95 wt%, 5-20 nm)

were purchased from PlasmaChem GmbH company. Phosphate buffer 0.1 M solution

(PBS) was prepared by dissolving appropriate amounts of sodium hydrogen phosphate

and sodium dihydrogen phosphate in a 250 mL volumetric flask. The solution pH was

adjusted to appropriate value by addition of 7.5 M sodium hydroxide solution. All

electrochemical experiments were carried out in 0.1 M PBS at pH 7.5. The other

chemicals were of analytical reagent grade purchased from Merck and used without

further purification.

2.2 Synthesis of nanoscale Co (OH)2

CHNPs were synthesized according to a literature method[32]. Briefly, Co(OH)2

nanoparticles were prepared by a simple precipitation method. The first step was the

dissolving of cobalt chloride as aqueous solution (1 M, 25 ml) in a glass beaker, using a

magnetic stir bar. The cobalt chloride solution was slowly adjusted to pH 9 by addition

of 5 wt. % NH3·H2O (30 ml) at a temperature around 10 C. The NH3·H2O was added

drop wise with a constant time interval of 5 s. The resulting suspension was stirred at

this temperature for an additional 3 h. Then the solid was filtered, washed with a

copious amount of distilled water several times. The obtained CHNPs product was dried

at 100C.

2.3 Instrumentation

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All the voltammetric measurements were carried out using the MWCNTs-

CHNPs/CILE electrode as the working electrode, Ag/AgCl 3 M KCl as the reference

electrode and platinum wire as an auxiliary electrode at room temperature. A magnetic

stirrer was used for the convective transport of the analyte. Cyclic voltammetry was

scanned between -0.2 and 1 V at the scan rate of 0.1 V s-1

. Amperometric measurement

was conducted under forced convection (stirring) by applying the appropriate potentials

and allowing the transient currents to decay to a steady-state value. All experiments

were done under a nitrogen atmosphere at room temperature by using an Autolab

PGSTAT 30 Potentiostat Galvanostat (EcoChemie, The Netherlands) coupled with a

663 VA stand (Metrohm Switzerland). The pH measurements were performed with a

Metrohm 744 pH meter using a combination glass electrode. X-ray diffraction (XRD)

measurements were performed at a speed of 0.01 s

−1 by a Bruker Axs diffractometer

(Germany) with Cu Kα (λ=1.5418 nm) operating at 40 kV, 30 mA. The morphology of

the nanoscale CHNPs was investigated by scanning electron microscopy (SEM, Leica

Cambridge, model S 360) and transmission electron microscopy (TEM, Philips CM10).

2.4. Electrode modification

The carbon ionic liquid electrode (CILE) was prepared by mixing graphite

powder and (BMIM)(PF6) (w/w 4:1) thoroughly in a mortar to form a carbon paste. A

portion of the carbon paste was firmly filled into one end of a glass tube (ca. 1.8 mm i.d.

and 10 cm long) and a copper wire was inserted through the opposite end to establish an

electrical contact. The surface of the CILE was smoothened on a piece of weighing

paper. The fabricated CILE was used as the basic solid electrode. A stock solution of

8

MWCNTs–CHNPs in DMF was prepared by dispersing weighed amounts of MWCNTs

and CHNPs (94/6% : w/w) in 1 mL DMF using ultrasonic bath until a homogeneous

solution resulted, and 20µL of the prepared suspension was casted on the CILE surface

with a microsyring and dried at room temperature. During these procedures a small

bottle was fitted tightly over the electrode so that the solvent could evaporate slowly

and a uniform film was formed. The fabricated electrode was stored at 4 C when not in

use. For comparison, MWCNTs/CILE and CHNPs/CILE were prepared with similar

procedures and used for further investigation.

2.5 General procedure

The electrode was first activated in PBS by cyclic voltammetric sweeps between

-0.1 and 1.1V until stable cyclic voltammograms were obtained. Each sample solution

(10 mL) containing 0.1 M PBS (pH 7.5) and appropriate amounts of L-Dopa and Mel

were pipetted into a voltammetric cell. The open-circuit accumulation time was 90 s.

Upon using the differential pulse voltammetric technique the oxidation peaks for L-

Dopa and Mel appeared at 0.075 and 0.70 V respectively. After every measurement, the

electrode was regenerated by soaking then rinsing thoroughly with triply distilled water

and then 0.5% sodium hydroxide solution for few seconds to remove adsorbed

substances. The electrode was finally rinsed carefully with distilled water to remove all

adsorbate from the electrode surface and to provide a fresh surface before running the

next experiments. All sample solutions were deoxygenated by purging with N2 gas

before each experiment.

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3. Results and Discussion

3.1. Characterization of the CHNPs

The response of a sensor is related to physical morphologyof its surface.

Scanning electron microscopy (SEM) was performed to the CHNPs synthesized through

the precipitation method (Fig. 1a). The morphology of CHNPs shows a network-like

structure which consists of interconnected nano-flakes. The SEM image shows the

agglomerated Co(OH)2 particles with an average size of less than 100 nm. It can be seen

that the CHNPs are very homogeneous in size. Fig. 1b displays TEM image of

Nanoscale CHNPs. The result shows the nanoparticles are in the same sizes as it is

shown in the SEM image.

Figure 1

It is known that cobalt hydroxide can be crystallized into a hexagonal layered structure

with two polymorphs: α and β [44].The α-hydroxides thus have a larger interlayer

spacing (usually >7 Å, dependent on intercalated anions) than that of the β-form (4.6 Å)

and are theoretically expected to exhibit superior electrochemical activity as compared

to the β–form [44-46].Therefore, the α-hydroxides of CHNPs may be more promising

electrode materials. The crystal structure of CHNPs is shown in Fig. 2 by powder X-ray

diffraction (XRD). No obvious peaks of β-Co(OH)2 have been observed in the XRD

pattern of the CHNPs material, and it correspond to the layered α-Co(OH)2 structure

(PDF, card no. 46-0605) with low crystallinity [47].

Figure 2

3.2. Electrochemical behavior of L-Dopa and Mel at MWCNTs-CHNPs/CILE

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The electrochemical behavior of L-Dopa and Mel at the different modified

electrode in phosphate buffer at pH 7.5 was examined using cyclic voltammetry (CV).

Fig. 3 shows the cyclic voltammograms for 20 µM L-Dopa and 5 µM Mel in

deoxygenated 0.1 M phosphate buffer solution. As can be seen at the CILE (curve a in

Fig. 3), a broad and small irreversible oxidation peak for Mel around 0.7 V and ill-

defined redox peak for L-Dopa were observed around 0.1 V. Fig. 3b demonstrates that

MWCNTs can effectively catalyze the electro-oxidation of L-Dopa and Mel and greatly

improve the peak shapes. This can mainly be attributed to the large surface area, subtle

electronic properties of MWCNTs; meanwhile, the oxidation peak potential (Epa) shifts

negatively, and the reduction peak potential (Epc) shifts positively. The modified

MWCNTs/CILE not only improves the redox peak currents but also makes the redox

reaction of L-Dopa more reversible. It can also be seen that the peak current of L-Dopa

and Mel increases further at the CHNPs modified CILE (curve c in Fig. 3). The

enhancement in peak currents and the lowering of overpotentials are clear evidences of

catalytic effects of CHNPs toward the L-Dopa and Mel redox reactions. Fig. 3d strongly

suggests that the hybrid film of MWCNTs and CHNPs on the carbon ionic liquid

electrode can combine the advantages of all of them and accelerate electron transfer

significantly; therefore, resulting in a remarkably increased response towards the redox

reactions of L-Dopa and Mel in contrast to the behavior from other electrode

modifications.

Figure 3

3.3 Optimization of Experimental Variables

3.3.1. Effects of the modified electrode composition materials

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Modification of the CILE with different amounts of MWCNTs and CHNPs was

tested. The amount ratio of CHNPs to MWCNTs nanoparticles influences the sensitivity

of the sensor. It was found that as the amount ratio of CHNPs was increased from 0 to

6% (w/w), the response of the electrode improved and when the ratio was more than

6%, the response decreased with larger background current, which resulted in poor

determinations for L-Dopa and Mel (Fig. 4a). Therefore 20µL of 6% CHNPs (94%

MWCNTs) in DMF solution was chosen for the fabrication of the modified electrode.

Fig. 4

3.3.2. Effect of accumulation time

To study the adsorption of L-Dopa and Mel at the MWCNTs-CHNPs film–

coated CILE, the influences of accumulation time was investigated. Fig.4b shows the

variation of differential pulse anodic peak currents with respect to accumulation times.

The anodic peak currents of L-Dopa and Mel improve with accumulation time. After 60

and 90 seconds of accumulation time for L-Dopa and Mel respectively, they remained

almost constant. This may be due to saturation of the amount of L-Dopa and Mel

adsorbed on the modified electrode surface.

3.3.3 Effect of solution pH

The effect of pH of supporting electrolyte on differential pulse voltammograms

of L-Dopa and Mel are shown in Fig. 5a. Fig 5b shows that at pH 7.5, higher anodic

peak currents for both compounds were obtained. Variations of anodic peak potentials

for L-Dopa and Mel with pH were fitted with following equations:

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L-Dopa: Epa (V) = −0.057 pH + 0.490 (R2 = 0.996) (1)

Mel: Epa (V) = −0.036 pH + 0.971 (R2 = 0.997) (2)

The results showed that the oxidation potentials of L-Dopa and Mel shift to less

positive potential with increasing solution pH which is a consequence of the

deprotonation involved in the oxidation process that is facilitated at higher pH values.

The slope of 0.057 and 0.036 VpH-1

for L-Dopa and Mel respectively, suggests that the

oxidations of L-Dopa involve the same number of transferred electrons and protons and

the number of electrons transferred in the oxidation of Mel is twice of protons, which is

in agreement with previous reports. [9, 48, 49]

Fig. 5

3.4 Effect of the scan rate

In order to investigate the effect of scan rate, cyclic voltammetry of solutions of

20 µM L-Dopa and 5 µM Mel were obtained in the range 0.01–0.8 Vs−1

. The effect of

scan rate on the oxidative peak potential (Epa) and peak current (Ipa) of L-Dopa and Mel

are shown in Fig. 6. The results showed that the peak currents vary linearly with the

scan rate over the range 0.01-0.15 Vs-1

(Figure 6b,d) for both compounds which

confirm the adsorption-controlled process for electro-oxidation of L-Dopa and Mel on

the surface of the electrode as following equations:

L-Dopa : Ipa = 0.251ν + 5.20 (R2 = 0.997) (3)

Mel : Ipa = 0.269ν + 7.59 (R2 = 0.998) (4)

13

At sweep rates between 0.2 V s−1

and 0.4 V s−1

, the plot of peak currents vs.

scan rate plot deviates from linearity and the peak current becomes proportional to the

square root of the scan rate which confirm diffusion controls of the systems(Figure

6c,e).

Fig. 6

The plot of Ep versus the logarithm of scan rate (log(ν)) was not linear for L-

Dopa, but showed a linear behavior for Mel according to Laviron theory [50]. The

charge transfer coefficient (α) can be determined by measuring the variation of Ep vs.

log(v). The slope of the Ep vs. log(ν), was about, 0.056 V. Using the equation of:

Ep = K − 2.3030 (RT/αnF) log(ν)

By considering two electrons transferred for Mel, charge transfer coefficient (α) of

0.530 was obtained which is in agreement with the results explained for the oxidation

process of Mel at the other electrodes[9, 49, 51]

3.5 Linear dynamic range and detection limit of the method

To obtain the linear dynamic range of the modified electrode under optimum

conditions for simultaneous determination of L-Dopa and Mel, the differential pulse

voltammetric behaviors of the mixed analytes were obtained (Fig 7 and 8). The

electrochemical response of additions of L-Dopa from 0.1 to 300 µM in the co-

existence of 5 µM Mel under the optimized conditions is depicted in Fig.7. By

application of the DPV method two linear ranges were obtained. The first linear

dynamic range was from 0.1 to 7.5 μM, with a calibration equation of Ip(µA) = 1.59c

14

(M) + 0.21 (R2=0.9993) and the second linear dynamic range was between 10 to 300

μM with a calibration equation of Ip(µA) = 1.55c (M) + 0.52 (R2=0.9996). A detection

limit of 0.075 µM (S/N = 3) was obtained.

Fig. 7

Figure 8 shows differential pulse voltammograms and the corresponding calibration

curves obtained from 0.01 to 50 µM of Mel in presence of 20 µM L-Dopa. The first

linear dynamic range was from 0.01 to 0.5 μM, with a calibration equation of Ip(µA) =

7.491 (M) + 0.329 (R2=0.9998) and the second linear dynamic range was between 1

μM to 50 μM with a calibration equation of Ip(µA) = 6.71c (M) + 2.44 (R2=0.9995). A

detection limit of 0.004 µM (S/N = 3) was obtained.

Fig. 8

Figure 9 displays a hydrodynamic chronoamperogram of oxidations of various

concentrations of L-Dopa and Mel at applied potential of 0.85 V in PBS (pH 7.5) using

the rotated modified electrode (2500 rpm). For Mel the linear dynamic range was from

0.3 to 49.2 µM, with a calibration equation of Ip(µA) = 3.45c (mol L-1

) + 1.21

(R2=0.9998) (Fig 9B) . A detection limit of 0.042 µM (S/N = 3) was obtained. For L-

Dopa the linear relationship was in the range of 0.8 to 98.2 µM with a calibration

equation of Ip(µA) = 1.49c (mol L-1

) – 0.22 (R2=0.9995) (Fig 9C). In addition a

detection limit of 0.10 µM was obtained.

Fig. 9

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3.6 Repeatability and stability of the modified electrode

To evaluate the repeatability of the MWCNTs-CHNPs/CILE, the peak currents

of 20 successive measurements by DPV in a mixture solution of 20 µM L-Dopa and 5

µM Mel were determined. The relative standard deviation (R.S.D.) of 2.60% and 2.75%

were obtained for L-Dopa and Mel, respectively; indicating that the MWCNTs-

CHNPs/CILE is not subject to surface fouling by the oxidation products. The stability

of the proposed sensor was investigated. After 100 cyclic runs, the voltammetric

response to 20 µM L-Dopa and 5 µM Mel almost remained 88% and 87% of the initial

response, respectively (data not shown). The storage stability of the proposed sensor

was also studied. When not in use, the electrode was suspended in PBS at 4 C in a

refrigerator. The response to determination of 20 µM L-Dopa and 5 µM Mel were tested

intermittently. After 7 and 15 days of storage, the sensor retained 93% and 91% of its

initial response current in 20 µM L-Dopa, respectively. In addition the response to 5 µM

Mel was also tested and the sensor retained 94% and 92% of its initial response current

after 7 and 15 days respectively. The results indicate that the modified electrode has a

good stability.

3.7 Effects of interferences on the behaviors of L-Dopa and Mel

The effects of the common interfering species in solutions of 10 µM L-Dopa and

Mel were investigated in the optimum measurement conditions using differential pulse

voltammetric method. The tolerance limit was defined as the maximum concentration of

the interfering substance that causes an error less than 5% for determination of Mel and

L-Dopa. It was found that a 450-fold excess NaCl and KCl, 350-fold excess Ca(NO3)2,

16

300-fold excess MgCl2, and Cu(NO3)2, 220-fold excess of folic acid and oxalic acid,

200-fold excess of Ascorbic acid and Uric acid, 100-fold excess citric acid and

glutamic acid, 50-fold excess of glucose and L-histidine did not interfere with the

measurement of L-Dopa and Mel.

3.8 Determination of L-Dopa and Mel in real samples

The proposed method was applied to simultaneously determination of L-Dopa

and Mel in several commercially available pharmaceutical formulations and human

urine samples in order to demonstrate the capability of the modified electrode. Ten

tablets of each sample were weighed and pulverized by gentle grinding. An accurate

weight of the powder was dissolved in PBS (pH=7.4) in a water bath (450C) and

sonicated for 10 min. Then all sample solutions and running buffer were filtered

through a filter paper (Whatman No. 1) prior their use. Solutions obtained by dissolving

of L-Dopa and Mel tablets were subsequently diluted by PBS so that concentration lies

in the linear ranges. The prepared solutions were analyzed and the results obtained with

the MWCNTs-CHNPs/CILE were compared to the spectrophotometric method, as the

official method (Table 1). The analysis of L-Dopa and Mel for each sample was realized

in triplicate (n = 3). According to the t-test [52], there were no significant differences

between the calculated and comparative values at the 95% confidence level and within

an acceptable range of error, indicating that the modified electrode can be used for

voltammetric determinations of L-Dopa and Mel in the real samples.

Table 1

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4. Conclusion

In the current study, a CHNPs and MWCNTs modified carbon ionic liquid

electrode (CILE) was fabricated. The combination of CHNPs nanoparticles and

MWCNTs show the characteristics of large surface area, good dispersing properties and

fast electron transfer. Due to the co-contribution of CILE and modifiers on the electrode

surface, the resulting electrode exhibited a good electrocatalytic performance to

simultaneous trace determination of L-Dopa and Mel. The analytical parameters of the

proposed electrode for determination of L-Dopa or Mel were compared with the earlier

reports and the results were listed in Table 2. A wide linear range, low detection limit,

high stability and good reproducibility suggest that this electrode will be an attractive

candidate for practical applications.

Table 2

Acknowledgments

The authors gratefully acknowledge the research council of Arak University for

providing financial support (grant number 89.13326) for this work.

18

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22

Biographies

Ali Babaei received his BS degree in 1989 from Shahid Beheshti University, Tehran,

Iran; MS degree in 1991 from Mazandarn University, Babolsar, Iran and PhD degree

from Otago University, Dunedin, New Zealand. At present, he is associate professor of

chemistry at Arak University , Arak, Iran. His main area of interest at present is

electroanalytical chemistry.

Ali Reza Taheri received his BS degree in 2003 from Isfahan University, Isfahan, Iran;

MS degree in 2007 from Arak University, Arak, Iran. At present, he is a PhD student in

chemistry department of Arak University, Arak, Iran.

Iman Khani Farahani received his BS degree in 2008 from Arak University, Arak,

Iran;. At present, he is a MS student in biology department of Yasouj University,

Yasouj, Iran.

Caption for Tables

Table 1. Determination of L-Dopa and Mel in real samples

Table 2. Comparison of the proposed electrode for L-Dopa and Mel with other types of

nanocomposite material modified electrode

Table(s)

Table 1.

Sample L-Dopa added

(µM)

Mel added

(µM)

L-Dopa added (µM) Mel added (µM)

proposed methodb Recovery (%)

Official method

[53]

proposed methodb Recovery (%)

Official method

[14]

Tableta 10.0 5.0 10.1 ±0.2 101.0 10.2 ±0.2 4.9 ±0.3 98.0 5.1 ±0.3

20.0 10.0 20.2 ±0.3 101.0 19.9 ±0.4 10.2 ±0.4 102.0 10.2 ±0.3

30.0 15.0 29.8 ±0.2 99.3 29.5 ±0.3 14.8 ±0.3 98.7 14.7 ±0.4

Urine - - <LOD

- <LOD <LOD

- <LOD

20.0 10.0 19.7 ±0.4 98.5 19.5 ±0.4 9.7 ±0.5 97.0 9.8 ±0.4

a: L-Dopa,100 mg ( Ramofarmin Co. IRAN) and Mel, 3 mg (Daru pakhsh Co. IRAN)

b: Values reported are the average of three independent analysis of each spiked sample.

Table 2.

Analyte Electrode pH LDR (µM) LOD (µM) References

L-Dopa

Ru-red/NaY/CPE 4.8 120-10000 85 [54]

Gold screen-printed electrode 3.0 99–1200 68 [55]

FCMCNPE 7 2-50 1.2 [56]

PbO2- modified electrode (MCPE) 4.0 260-1200 25 [57]

oxovanadium-salen thin film electrode 6.0 10-100 0.8 [48]

MWCNTs/CHT/GCE 7.0 2-220 0.6 [58]

MWCNTs-CHNPs/CILE 7.5 0.1-300 0.075 This work

Mel

castor oil–graphite paste electrode 2.0 0.05-0.1 0.001 [59]

MWNTs-DHP-GCE 7.5 0.08-10 0.02 [49]

GC 1.0 20-80 5.86 [8]

AGCE 6.7 0.8-10 0.05 [51]

CPE 1.69 3-550 2.3 [9]

MWCNTs-CHNPs/CILE 7.5 0.01-50 0.004 This work

Ru-red/NaY/CPE :Carbon paste electrode modified with trinuclear ruthenium ammine complex incorporated in NaY zeolite,

FCMCNPE:ferrocene modified carbon nanotubes paste electrode, AGCE:activated glassy carbon electrode by pretreatment in

sodium hydroxide solution, MWNTs-DHP-GCE : Multi-wall carbon nanotubes- Dihexadecyl hydrogen phosphate film coated

glassy carbon electrode

Legends for Figures:

Figure 1. SEM (a) and TEM (b) image of the CHNPs.

Figure 2. Typical X-ray diffraction patterns ofCHNPs.

Figure 3.CVs of 20 µM L-Dopa and 5 µM Mel in 0.1 M PBS at CILE(a), MWCNTs/CILE (b),

CHNPs/CILE(c) and MWCNTs-CHNPs/CILE(e).

Figure 4.Effect of the amount ratio of CHNPs (a) and accumulation time (b), on themodified

electrodes using differential pulse anodic peak currents of 20 μM L-Dopa and 5 μM Mel in 0.1

M PBS (pH 7.5) atscan rate of 100mVs−1

.

Figure 5. DPVs of MWCNTs-CHNPs/CILE in 0.1 M PBs at different pHs.(curves A–F: 4.5, 5.5,

6.5, 7.5, 8.5, 9.5)in the presence of 20 µM of L-Dopa and 5 µM of Mel(a) and plot of peak

current vs. pH values (b).

Figure 6.CVs of 20 µM of L-Dopa and 5 µM of Mel at different scan rates (from A to L) 0.01,

0.025, 0.075, 0.1, 0.125, 0.150, 0.200, 0.250, 0.300, 0.350 and 0.4V s-1

(a).Plot of peak currents

vs. scan rate for L-Dopa (b), plot of peak currents vs. square root of scan rate for L-Dopa (c) ,(d)

as (b) for Mel, (e) as (c) for Mel.

Figure 7.DPVs of L-Dopa at MWCNTs-CHPs/CILE in the presence of 5 μM Mel. L-Dopa

concentrations (from A to L) are: 0.1, 0.5, 1, 3, 7.5, 12.5, 20, 45, 75, 130, 200, 300 μM. Insets:

Figure(s)

Plot of peak currents as a function of L-dopa concentration at low concentrations (a) and at high

concentrations (b).

Figure 8.DPVs of Mel at MWCNTs-CHNPs/CILE in the presence of 20 μM L-Dopa. Mel

concentrations (from A to L) are: 0.01, 0.05, 0.1, 0.15, 0.2, 0.5, 1.5, 5, 10, 15, 25, 50 μM. Insets:

Plot of peak currents as a function of Mel concentration at low concentrations (a)and at high

concentrations (b).Other conditions are the same as in Fig. 4.

Figure 9. Hydrodynamic amperometric response (rotating speed 2500 rpm) held at 0.85 V in

PBS (pH 7.5) for simultaneous determination of L-Dopa and Mel by successive additions of (a)

10 µM L-Dopa and (b) 5 µM Mel. Insets: (A) successive additions of (c) 0.8 µM L-Dopa and (d)

0.3 µM Mel; (B) Plot of currents as a function of Mel concentration and (C) Plot of the currents

as a function of L-Dopa concentration.

Figure 1.

Figure 2.

Figure 3.

-35

-15

5

25

45

65

85

-0.3 -0.1 0.1 0.3 0.5 0.7 0.9

I / µ

A

E / V

d

c

ba

Figure 4.

0

10

20

30

40

0 30 60 90 120

I / µ

A

Time / S

Levodopa

Melatonin

b

0

10

20

30

40

0 2 4 6 8 10 12

I /µ

A

% Co(OH)2 ratio

Levodopa

Melatonin

a

Figure 5..

0

10

20

30

40

50

60

70

80

90

100

-0.15 0.05 0.25 0.45 0.65 0.85

I/ µ

A

E / V

a

AF

0

10

20

30

40

50

3.54.55.56.57.58.59.5

I/µ

A

pH

Melatonin

Levodopa

b

Figure 6.

-150

-100

-50

0

50

100

150

200

-0.3 -0.1 0.1 0.3 0.5 0.7 0.9

I / µ

A

E / V

A

La

-50

0

50

0 50 100 150 200

I / µ

A

ν / (mV/s)

0

20

40

0 50 100 150 200

I / µ

Aν / (mV/s)

b

d

-200

-50

100

10 15 20 25 30

I/µ

A

ν1/2 / (mv/s)1/2

25

50

75

100

10 15 20 25 30

I/µ

A

ν1/2 / (mv/s)1/2

e

c

Figure 7.

0

100

200

300

400

500

600

-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

I / µ

A

E / V

L

A

0

5

10

15

0 2 4 6 8

I / µ

A

C / µM

a

0

150

300

450

0 100 200 300 400

I / µ

A

C / µM

b

Figure 8.

0

50

100

150

200

250

300

350

400

-0.15 0.25 0.65 1.05 1.45

I / µ

A

E / V

L

A

0

2

4

6

0 0.2 0.4

I / µ

A

C / µM

a

0

150

300

450

0 20 40 60

I / µ

A

C / µM

b

Figure 9.

0

100

200

300

400

500

600

700

0 300 600 900 1200

I / µ

A

t/s

a

ab

ba

ab

ba

a

aa

a aa

bb

b b

b b

0

10

20

30

0 200 400 600 800

I / µ

A

t/s

cc

cccc

cccc

dd

dddd

dddd

A

y = 3.54x + 1.21R² = 0.99980

50100150200

0 20 40 60

I / µ

A

C / µM

B

y = 1.49x - 0.22R² = 0.9995

050

100150200

0 40 80 120 160

I / µ

A

C / µM

C