Journal of Electroanalytical Chemistry 698 (2013) 45–51

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Journ al of Electr oanalytica l Chemi stry

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Highly sensitive simultaneous determination of L-dopa and paracetamol using a glassy carbon electrode modified with a composite of nickel hydroxide nanoparticles/multi-walled carbon nanotubes

1572-6657/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jelechem.2013.01.021

⇑ Corresponding author at: Department of Chemistry, Faculty of Science, Arak University, Arak 38156-8-8349, Iran. Tel.: +98 861 4173401; fax: +98 861 4173406.

E-mail addresses: a-babaei@araku.ac.ir, ali.babaei08@gmail.com (A. Babaei).

Ali Babaei a,b,⇑, Masoud Sohrabi a, Ali Reza Taheri a

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

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 July 2012 Received in revised form 6 January 2013 Accepted 17 January 2013 Available online 26 March 2013

Keywords:L-DopaParacetamolMulti-walled carbon nanotubes Nickel hydroxide nanoparticles

The electrochemical oxidation of L-dopa (LD) and paracetamol (PAR) has been investigated by application of nickel hydroxide nanoparticles/multi-wall ed carbon nanotubes composite electrode (MWCNTs-NHNPs/GCE) using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperom- etry (CA) methods. The modified electrode showed electrochemical responses with high sensitivity for LDand PAR determina tion, which makes it a suitable sensor for simultaneou s sub- lmol L�1 detection of LDand PAR in aqueous solutions. Under the optimum conditions the electrode provides a linear response versus LD and PAR concentr ations in the range of 0.1–100 lM and 0.06–26 lM, respectively using the DPV method. Linear responses versus LD and PAR concentrations in the range of 1–672 lM and 1–960 lM, respective ly, were obtained using the CA method. The modified electrode was used for determi- nation of LD and PAR in human urine with satisfactory results.

� 2013 Elsevier B.V. All rights reserved.

1. Introductio n

The application of nanomaterials in various fields of science and technology has been extensively develope d due to the unique properties of these materials [1–3]. Metal nanoparticle has re- ceived considerable attention in recent years. It has unique chem- ical and electrical properties due to its size dependent properties.Hence there is currently an intense interest in the use of nanopar- ticles for the fabrication of modified electrodes and a wide range ofbioscience applications [4]. Many electrode s have been modifiedby Ni, NiO 2, Ni(OH)2 particles and nanoparticl es on traditional elec- trode surfaces such as gold [5], carbon or graphite [6,7]. In contrast to Ni nanomaterial s which are unstable and easily oxidized in air and solution, hydroxide (or oxide) of these materials are relatively stable [7,8]. Many precipita tion methods for preparing nickel hydroxide nanoparticl es (NHNPs) have been reported, however the method of coordina tion homogeneous precipitation (CHP) isnew and facile [9], needing no expensive raw materials or equip- ment, is also easy for mass production, and can be extended to syn- thesize other hydroxide or oxide nanocrystals. Therefore in this work, CHP method for synthesis of NHNPs was used.

Carbon nanotubes (CNTs), including single walled CNTs (SWNTs) and multi-walled CNTs (MWNTs), have been used in

various fields such as catalysis of redox reactions [10–12], nano- electroni cs [13], electrochemical sensors [14,15], etc., due to their unique structure , electrical and mechanical characteristics. Sensors based on CNTs have received a lot of attention and have largely im- proved the voltamm etric response (lower overvoltages and higher peak currents) of a variety of biological, clinical and environm ental compounds [16].

Paracetamol (PAR, N-acetyl- p-aminopheno l or acetaminop hen)is a long-establis hed substance being one of the most extensively employed drugs in the world. It is also found that overdoses ofPAR will damage liver and kidney. A great number of analytica lmethods such as: spectrophotom etry [17,18], high-perfor mance liquid chromatograp hy [19], near infrared transmittan ce spectros- copy [20], spectrofluorimetry [21,22] and capillary electrophoresis [23], have been develope d for the determination of PAR in pharma- ceutical formulations and biological fluids. However, these meth- ods suffer from some disadvantag es such as requirement for sample pretreatmen t, low sensitivity, high costs, the use of organic solvents and long analysis times. In contrast, electrochemi cal techniqu es are less time consuming, rapid, simple, without tedious procedures, inexpensive, and with high sensitivity. Several electro- chemical methods for the determinati on of PAR have been pro- posed [24–27].

Levodopa (LD, 3,4-dihydro xy-l-phenyl alanine or L-dopa) is one ofthe catechola mines and an essential precursor for the biosynth esis of dopamine. This drug can be principally metabolized by an enzy- matic reaction (dopa-descarboxilase) to dopamine compens ating

46 A. Babaei et al. / Journal of Electroanalytical Chemistry 698 (2013) 45–51

for the deficiency of dopamine in the brain [28]. Several methods for the determinati on of LD have been reported in literatures, such astitration [29], spectrophotom etry [30–32], high performanc e liquid chromatograp hy (HPLC) [33], photokinetic method [34] andcapillary zone electrophoresis [35]. Many electrochemi cal methods have been reported for LD determination [36–38].

It has been found that human absorption of PAR is very depen- dent on gastric emptying. Other drugs that alter gastric emptying can change the pharmacokine tics of PAR. It has been shown that LD can influence gastric emptying [39]. Therefore it would be use- ful to study simultaneous determination of PAR and LD. To the best of our knowledge, there is only one report on the electrochemical determination of PAR and LD using a glassy carbon electrode mod- ified with a single-walled carbon nanotube/ch itosan/1-ethy l-3- methyl-imid azoliumtetra-fluoroborat composite [40]. This work suffers from the low sensitivit y of the proposed electrode and low linear dynamic range. In this study, we report the preparati onand applicati on of nickel hydroxide nanoparticles/ multi-walled carbon nanotubes modified glassy carbon electrode (MWCNTs-NHNPs/GCE) as a new sensor for simultaneous determination ofPAR and LD. The modified electrode showed very high sensitivity,lower detection limit with wide linear dynamic range. The analyt- ical performanc e of the modified electrode in quantification of LDand PAR in human urine is evaluated with satisfactory results.

2. Experimen tal

2.1. Chemicals and reagents

Multi-walled carbon nanotubes (purity more than 95%) with number of walls 3–15, and tube length 1–10 lm were purchased from plasmaChem GmbH Company . The reagents were analytical grade and used without any further purification.

All solutions were freshly prepared with triply distilled water.Phosphate buffer solutions (PBS) were prepared from stock solu- tion of 0.1 M KH2PO4 and 0.1 M K2HPO4. pH was adjusted using concentrated H3PO4 or NaOH solutions. Electrochemi cal experi- ments on LD and PAR were carried out in 0.1 mol L�1 PBS atpH 7.0. Fresh human urine sample was purchased from Razi Insti- tute of Vaccine and Serum Company (Tehran, Iran). The urine sam- ple was filtered and diluted 40 times using a 0.1 M phosphate buffer solution of pH 7.0 and used for determination of spiked LDand PAR in the urine sample.

2.2. Synthesis of NHNPs

NHNPs were synthesized using a simple coordina tion precipita- tion procedure as previously reported [9]. Briefly, by adding con- centrated ammonia (28 wt.%) to nickel nitrate solution (1 M), adeep blue colored nickel hexamine complex solution was formed.The solution was added into a given amount of distilled water,the reaction was carried out under magnetic stirring for 1 h at70 �C. Finally, light green sediments were formed. The precipitate was separated by centrifuge and rinsed with distilled water and ethanol three times respectively to remove the adsorbed ions, then dried in a vacuum oven at 80 �C for 12 h to form a green powder ofNHNPs. The product obtained without the use of surfactant in the reaction process had a platelet-like shape.

2.3. Instrumentation

Electrochemical measureme nts were performed with an Auto- lab PGSTAT 30 Potentiostat Galvanostat (EcoChemie, The Nether- lands) running on GPES software coupled with a 663 VA stand (Metrohm, Switzerland ) with a conventional three electrode cell.

A circular 2 mm diameter glassy carbon electrode (Metrohm) and a platinum wire are used as the working electrode and counter electrode , respectively . All the cell potentials were measured with respect to that of an Ag/AgCl/3 M KCl reference electrode. Differen- tial pulse voltammetr y (DPV) experiments were carried out with pulse amplitud e of 50 mV, scan rate of 100 mV s�1 and a pulse interval of 0.2 s. All the measure ments were carried out at room temperat ure. pH measure ment were performed with a Metrohm 744 pH meter using a combination glass electrode . The morphol- ogy of the nano scale Ni(OH)2 was investigated by scanning elec- tron microscopy (SEM, Leica Cambridge, model S 360) and transmis sion electron microscopy (TEM, Philips CM10).

2.4. Preparation of MWCNTs-NHNPs /GCE modified electrode

The effect of composition of MWCNTs and NHNPs for modifica-tion of the GCE was tested using the cyclic voltammetr y method.The proportion of NHNPs influences the sensitivity of the biosensor.It was found that as the proportion by mass of NHNPs increased from 2% to 5%, the response of the electrode improved and when the proportion was more than 5%, the response decreased with lar- ger background current, which resulted in poor measure for PAR and LD (not shown). So 20 lL of the mixture of 5% NHNPs and 95% MWCNTs was chosen for the fabrication of the sensor.

Prior to Modification, the GCE was first polished with 0.3 and 0.05 lm aluminum oxide aqueous slurry and rinsed thoroughly with triply distilled water. It was then cleaned by sonication for 5 min, first in ethanol and then distilled water, and then dried un- der a nitrogen gas flow.

A stock solution of MWCNTs-N HNPs in DMF was prepared bydispersin g weighed amounts of MWCNTs and NHNPs (95:5% w/w) in 1 mL DMF using an ultrasonic bath and 20 lL of the prepared homogen eous suspension was cast on the electrode with a micro- syringe. After that, each MWCNTs-N HNPs/GCE was prepared bycoating the electrode surface with 20 lL of stock solution. The elec- trode was then dried at room temperature to obtain the modifiedelectrode . This fabricated MWCNTs- NHNPs/GCE was placed inthe electrochemi cal cell containing 0.1 mol L�1 PBS and several cy- cles in the potential windows of �0.1 to 0.7 V were performed using the CV method to obtain stable responses.

2.5. General procedure

The general procedure used to obtain voltammogram s was asfollows. Each sample solution (10 mL) containing 0.1 M phosphate buffer solution (pH 7.0) and appropriate amount of analytes was pipetted into a voltamm etric cell. The voltammogram s showed oxi- dation peak potentials about 0.12 and 0.33 V correspond ing to LDand PAR compounds . The amounts of LD and PAR were obtained using correspond ing peak heights. And also the density is corrected as shown in the modified electrode was regenerated by successive washing with triply distilled water and then 0.5% sodium hydroxide solution. The electrode was finally rinsed carefully with distilled water to remove all adsorbat e from the electrode surface and to pro- vide a fresh surface before running subsequent experime nts.

Electrochemi cal impedance spectroscopy (EIS) was performed in a solution containing 5 mM of each of FeðCNÞ3�6 and FeðCNÞ4�6

and 0.1 M KCl with the frequency swept from 105 to 0.01 Hz.

3. Results and discussion

3.1. Characterizati on of MWCNTs-NHNPs /GCE

Nanoscale NHNPs was characterized by means of SEM and TEM.Fig. 1A shows a typical image of the nanoscale NHNPs synthesized

Fig. 1. SEM image of the MWNTs and NHNPs nanoscale on glassy carbon (A) and TEM image of nickel hydroxide powders (B).

A. Babaei et al. / Journal of Electroanalytical Chemistry 698 (2013) 45–51 47

via coordinatio n precipitatio n method and MWNTs. It can be ob- served that it appears to have a platelet-like shape and with adimension of 50–80 nm, and weak agglomer ation can be seen.Fig. 1B displays TEM image of nanoscale NHNPs. The result shows the nanoparticles are in the same sizes as it is shown in the SEM image.

The CV from the MWCNTs- NHNPs/GCE in 0.1 M PBS (pH 7.0) isshown in Fig. 2C. The anodic and cathodic peaks in 0.1 M PBS solu- tion could be due to the Ni+2/Ni+3 redox couple. Similar behavior for NHNPs has been reported previousl y [8].

Electrochem ical impedance spectroscopy (EIS) can provide some informations on impedance changes of the electrode surface as a result of the modification process. Fig. 3 shows the Nyquist plots (�z0 0 vs. z0)for MWCNTs-N HNPs/GCE (Fig. A) and GCE (Fig. B) electrodes obtained when the electrodes were immersed in a 0.1 M KCl solution containing 5 mM in both K3[Fe(CN)6] and K4[Fe(CN)6]. The diameter of the semicircle for the MWCNT- NHNP/GCE is smaller than that of the GCE, which suggests the MWCNT-NHN P composite modification of the electrode provides lower electron transfer resistance.

The effect of modification of the electrode on active surface area was characterized by cyclic voltammogram s using MWCNTs- NHNPs/GCE, MWCNTs/G CE and GCE in 4 mM potassium ferricya- nide with phosphate buffer solution (pH 7.0) (not shown). K3-

Fe(CN)6 exhibited a pair of reversible redox peaks at a bare and modified GC electrode . However for the modified electrode s the re- dox peak currents are larger than for the GCE. On the other hand,under the same conditions, the anodic peak currents were linear

Fig. 2. Nyquist plots for MWCNTs-NHNPs/GCE (Fig. A) and GCE (Fig. B) electrodes obtained when the electrodes immersed into solutions of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1 M KCl solution. Cyclic voltammograms obtained in 0.1 M PBS solution at MWCNTs-NHNPs/GCE (Fig. C).

with the square root of scan rate (m1/2) on the GCE and other mod- ified GCE electrode s. The obtained regression equations for the three electrodes are as follows:

Ipa ðlAÞ ¼ 28:74m1=2 ðV s�1Þ1=2 þ 4:24 ðR2 ¼ 0:990Þ GCE

Ipa ðlAÞ ¼ 550:9m1=2 ðV s�1Þ1=2 þ 24:4 ðR2 ¼ 0:999ÞMWCNTs=GCE

Ipa ðlAÞ ¼ 651:9m1=2 ðV s�1Þ1=2 þ 21:83 ðR2 ¼ 0:999ÞMWCNTs� NHNPs=GCE

A reversible system should satisfy the Randles–Sevcik equation [41]:

Ip ¼ 2:69� 105 n3=2AD1=2m1=2C ð1Þ

The apparent area of the MWCNTs-NHN Ps/GCE and MWCNTs/ GCE were estimated about 22.7 and 19.2 times as large as that ofthe GC electrode , respectively . It can be concluded that the applica- tion of a MWCNTs-N HNPs composite leads to higher electrochem- ically active surface area than MWCNTs alone.

3.2. Electrochem ical studies of LD and PAR on MWCNTs-NHNPs /GCE

The electrochemi cal behaviors of LD and PAR at the MWCNTs- NHNPs/G CE have been investiga ted. The corresponding cyclic vol- tammogr am of LD and PAR in 0.1 M PBS (pH 7.0) is shown in Fig. 3.PAR, unlike LD showed a couple of redox peak.

The influence of scan rate on the oxidation peak potential (Epa)and current of LD and PAR at the MWCNTs- NHNPs/GCE in0.1 M PBS (pH 7.0) were studied by cyclic voltamm etry (not

Fig. 3. Cyclic voltammograms of 80 lM LD and 25 lM PAR at MWCNTs-NHNPs/GCE in 0.1 M phosphate buffer solution (pH 7.0) at scan rate of 50 mV s�1.

Fig. 4. Differential pulse voltammograms of 80 lM LD and 25 lM PAR at (a) GC, (b)MWCNTs/GCE and (c) MWCNTs-NHNPs/GCE in 0.1 M phosphate buffer solution (pH 7.0). Other conditions: Open circuit, tacc = 50 s, pulse amplitude = 50 mV, scan rate = 100 mV s�1, interval time = 0.5 s, modulation time = 0.2 s and step potential = 5 mV.

Fig. 5. (A) Differential pulse voltammograms of 80 lM LD and 25 lM PAR compounds at MWCNTs-NHNPs/GCE in 0.1 M phosphate buffer solutions atdifferent pHs: 4(a), 5(b), 6(c), 7(d), 8(e), 9(f), 10(g). Insets: (B) Plot of anodic peak currents (Ipa) of LD and PAR as a function of pH values. (C) Plot of potential values asa function of pH values.

48 A. Babaei et al. / Journal of Electroanalytical Chemistry 698 (2013) 45–51

shown). The Epa shifted to more positive potentials with increasing scan rate (v), confirming the kinetic limitation of the electrochem- ical reaction. The anodic peak current of 30 lM LD and 10 lM PAR was proportional to the scan rate over the range of 10–120 mV s�1

with linear regression equation s:

Ipa ðlAÞ ¼ 740:7mþ 29:75 ðmV s�1Þ ðR2 ¼ 0:976Þ LD

Ipa ðlAÞ ¼ 907:5mþ 23:37 ðmV s�1Þ ðR2 ¼ 0:993Þ PAR

These phenomena indicate oxidations of LD and PAR are adsorption- controlled processes at those scan rates. At sweep rates from 120 to 320 mV s�1 values, the plot of currents vs. scan rate deviate from linearity and the peak currents relate linearly with the square root of scan rate (m1/2). The results indicate diffusion- controlled mechanism s with linear regressio n equations:

Ipa ðlAÞ ¼ 200:1m1=2 ðmV s�1Þ1=2 þ 30:02 ðR2 ¼ 0:980Þ LD

Ipa ðlAÞ ¼ 296:6m1=2 ðmV s�1Þ1=2 þ 30:74 ðR2 ¼ 0:994Þ PAR

At scan rates higher than 200 mV s�1, peak separations (DEp) for PAR begins to increase, demonst rating the limitation due to charge transfer kinetics. Based on Laviron theory [42] the charge transfer coefficient (a) and electron transfer rate constant (ks) can be deter- mined by measuring the variation of DEp vs. log scan rate. The slopes of Epc vs. log (m) (not shown) are �0.099 and 0.153, respec- tively. Using the equations of:

Epc ¼ K � 2:303ðRT=acnFÞ log ðmÞ

Epa ¼ K þ 2:303ðRT=aanFÞ log ðmÞ

By considering two electrons transferred for PAR, cathodic (ac)and anodic (aa) charge transfer coefficients of 0.59 and 0.39 were obtained. Substituting the a value into the following equation, anapparent surface electron transfer rate constant, ks = 2.54 s�1,was obtained.

log ks ¼ a logð1� aÞ þ ð1� aÞ log a� logRTnFm

� �� a

For an irreversible anodic reaction, the relationship between Ep

and m describes by Laviron’s theory as follows:

Ep ¼ E0 � RTaanF

� �ln

RTKs

aanF

� �þ RT

aanF

� �ln m ð2Þ

According to the slope of the straight line of Ep against ln m, the value of aa of the LD was calculated to be 0.42. The value of E0 inEq. (1) can be obtained from the intercept of Ep vs. m curve byextrapolating to the vertical axis at m = 0 [43]. From E0 (0.173)and aa (0.42) values, the value of ks for the LD was calculated equal to 1.13 s�1.

The large value of the electron transfer rate constant shows the high ability of the modified electrode for promoting electron trans- fer between the PAR and the electrode surface. The surfaces of the MWCNTs-N HNPs composite contain a large number of defects and the MWCNTs-N HNPs nanostructu re may act as molecular wires,enhancing the direct electron transfer between PAR or LD and the electrode.

Electrochemical oxidation of 80 lM of LD and 25 lM of PAR were investigated by the different ial pulse voltamm etry method at bare GCE, MWCNTs/G CE and MWCNTs-N HNPs/GCE in the PBS (pH 7.0) (Fig. 4). Voltammogram a displays the LD and PAR data at the GCE. Voltammogram s b and c show results of LD and PAR under the same conditions at the MWCNTs/G CE and MWCNTs- NHNPs/GCE, respectively . It is obvious that the MWCNTs- NHNPs/ GCE exhibits enhanced electrocatalyti c oxidation with higher peak

current for the oxidation of LD and PAR in comparison to the bare GCE and MWCNTs/G CE.

Therefore, it was concluded that MWCNTs-N HNPs/GCE can beused for a highly sensitive simultaneou s electrochemi cal determi- nation of LD and PAR.

3.3. Effect of operational parameters

3.3.1. Buffer component and pH effects on the oxidation of LD and PAR The electrochemical responses of LD and PAR were studied in

different media, namely phosphate buffer solution, Britton–Robin-son buffer solution, and acetate buffer solutions at pH 7.0. The ano- dic peak potentials for these two species are well separated in the three media, but the maximal peak currents of LD and PAR were obtained in phosphat e buffer solution. The effect of the pH value on the voltamm etric behavior of LD and PAR at the MWCNTs- NHNPs/G CE was carefully investigated in the 4–10 pH range (Fig. 5A). Fig. 5B shows the changes with pH of the anodic peak currents (Ipa) of LD and PAR. The oxidation peak current of LDincreased gradually from pH 4.0–6.0 and then decrease d with pHchange from 6.0 to 10. In the case of PAR the peak current was

Fig. 6. Effect of accumulation time on the differential pulse voltammogram peak currents of 40 lM LD and 15 lM PAR in phosphate buffer (pH 7.0) solution atMWCNTs-NHNPs/GCE.

Fig. 7. Differential pulse voltammograms for different concentrations of LD and PAR mixture as (a) 0.1 + 0.06, (b) 1 + 1, (c) 3 + 3, (d) 5 + 6, (e) 10 + 9, (f) 25 + 14, (g)45 + 17, (h) 60 + 20, (i) 80 + 23, (j) 100 + 26, respectively, in which the first value isthe concentration of LD in lM and the second value is the concentration of PAR inlM. Insets: (A) plot of peak currents as a function of LD concentration. (B) Plot of the peak currents as a function of PAR concentration.

Fig. 9. Plots of peak currents obtained from amperometric experiments as afunction of (A) PAR and (B) LD concentrations.

A. Babaei et al. / Journal of Electroanalytical Chemistry 698 (2013) 45–51 49

increased from 4.0 to 7.0 and then decreased by raising pH from 7.0 to 10. So, the phosphate buffer with a pH of 7.0, which is close to biologica l pH value, was selected as the supportin g electrolyte for the simultaneou s determination of LD and PAR in the mixture.

Fig. 8. Amperometric response at rotating MWCNTs-NHNPs/GCE (rotating speed 2500 rpm) held at 0.45 V in PBS (pH 7) for simultaneous determination of PAR and LD by successive additions of (A) (a) 1 lM PAR and (b) 1 lM LD, (B) (c) 16 lM PAR ,(d) 16 lM LD and (C) (e) 96 lM PAR, (f) 96 lM LD.

The relationship s between the potentials and pH were linear (Fig. 5C), and the regression equation s were as follows:

Epa ðVÞ ¼ 0:474� 0:048 pH ðR2 ¼ 0:996Þ LD

Epa ðVÞ ¼ 0:663� 0:044 pH . . . ðR2 ¼ 0:991Þ PAR

The slopes are close to the Nernstian amounts which suggest anequal number of electron and proton transfers are involved in the electrochemi cal oxidations of LD and PAR.

3.3.2. Effects of accumula tion time Fig. 6. shows plots of the anodic peak currents, obtained from

DPV experiments , against accumulation time for solutions that in- clude 40 lM LD and 15 lM PAR in PBS (pH = 7.0) at the MWCNTs- NHNPs/G CE. As can be seen, the anodic peak currents of LD and PAR gently increased with increasing accumulation time and reached the maximum current response at 50 and 35 s respec- tively. Hence, an accumulation time of 50 s was chosen as an opti- mum time for the next experime nts.

3.4. Calibration plots and limits of detection

Fig. 7.exhibits the differential pulse voltammogram s (DPVs) ob- tained for LD and PAR mixture on MWCNTs-N HNPs modified GCE in the phosphate buffer solution by synchronous ly changing the concentr ations of LD and PAR. The peak currents of LD were pro- portional to the concentratio n in the range of 0.1–100 lM (Ip

(lA) = 2.099 c (lM) + 0.791) with a correlation coefficient of0.998. For PAR the oxidation peak currents increased linearly with concentr ation in the range of 0.06–26 lM (Ip (lA) = 8.914 c

Table 1Comparison of various electrochemical sensors for detection of LD and PAR.

Analyte Electrode pH LDR (lM) DL (lM) Ref.

LD Carbon paste electrode modified with trinuclear ruthenium ammine complex incorporated in NaY zeolite 4.8 120–1000 85 [36]Gold screen-printed electrode 3 99–1200 68 [37]Modified nanowire carbon paste electrode 7 0.01–1.0 0.004 [44]Tetraaminophthalocyanatonickel(II) (p-NiIITAPc) glassy carbon (GCE) 4 0.1–10 0.1 [45]Oxovanadium-salen thin film electrode 6 1.0–100 0.8 [46]Gold nanoelectrode ensembles 7 0.025–1.5 0.015 [47]Ppy-CNT-GC 7 1–100 0.1 [48]SWNT-modified electrode 5 0.5–20 0.3 [49]GC/SWCNTs-CHIT-IL 7 DPV: 2–450 DPV:0.155 [40]

CA: 10–560 CA: 0.85 MWCNTs-NHNPs/GCE 7 DPV:0.1–100 DPV:0.076 This work

CA: 1–672 CA: 0.21

PAR GC/nano-TiO 2/polymer 7 12–120 2 [50]GC/zirconium alcoxide porous gel 7.4 19.6–255 0.12 [51]BBNBH modified carbon paste electrode 8.0 15–1000 NR [52]Carbon ionic liquid 4.6 1–2000 0.3 [53]Carbon nanotubes modified screen-printed 10 2.5–1000 0.1 [54]Boron-doped diamond 4.5 0.5–83 0.49 [55]Carbon-coated nickel magnetic nanoparticles/GCE 3.0 7.8–110 0.6 [56]GC/SWCNTs-CHIT-IL 7.0 DPV:1–420 DPV:0.092 [40]

CA: 10–420 CA: 0.62 MWCNTs-NHNPs/GCE 7.0 DPV:0.06–26 DPV:0.017 This work

CA: 1–960 CA: 0.25

Table 2Maximum tolerable conc entration of interfering species.

Interfering species LD Cint (lM)a PAR Cint (lM)a

Ascorbic acid 450 500

L-Glutamic acid 750 600

L-Alanin 1000 900

Aspartic acid 550 500 Tryptophane 450 450 Tyrosine 850 700

a Cint refers to interfering compound concentration.

Table 3Estimation of LD and PAR diluted (40-fold) urine.

Spiked (lM) Found (lM) R.S.D. (%)a Recovery

LD PAR LD PAR LD PAR LD PAR

10 10 9.7 10.3 1.6 1.8 97.0 103.0 20 20 21 20.7 0.9 1.2 105.0 103.5

a Average of five determinations at optimum conditions.

50 A. Babaei et al. / Journal of Electroanalytical Chemistry 698 (2013) 45–51

(lM) + 2.8) with correlation coefficient of 0.997. With a signal-to- noise ratio (S/N) of 3, the detection limit were 0.076 lM and 0.017 lM for LD and PAR, respectively.

The chronoamperom etry as well as the other electrochemical methods was employed for investigatio n of the electro-oxidati onof LD and PAR at MWCNTs-N HNPs/GCE (Fig. 8). For PAR the peak currents were proportional to concentr ation (Fig. 9A) between 1and 960 lM with regressio n equation of Ip (lA) = 0.271 c(lM) + 2.086 (R2 = 0.996). The calibration plot (Fig. 9B) of LD is lin- ear between 1 and 672 lM with regression equation of Ip

(lA) = 0.33 c (lM) + 1.559 (R2 = 0.994). The correspond ing detec- tion limits were 0.21 lM and 0.25 lM for LD and PAR, respectively.

The electrochemi cal parameters for determination of LD and PAR were compare d with the reports and the results were listed in Table 1. It can be seen that the present method can provide high sensitivity and wide linear range and comparable detection limit.

3.5. Stability and repeatabili ty of the MWCNTs-NHNPs /GCE

The repeatability of the method was checked by consecut ive determinati on (n = 8) for 80 lM of LD and 20 lM of PAR for anaccumulati on time of 50 s. The relative standard deviations (RSDs)were 0.52% and 0.67%, respectively indicating that the modifiedelectrode is not subject to surface fouling by oxidation products during the voltamm etric measure ments. Electrode-to -electrode reproduci bility was also satisfacto ry. A series of eight modifiedelectrode s which were prepared individually gave an RSD of 5.4%and 5.8% for determination of 50 lM LD and 20 lM PAR,respectivel y.

The stability of the modified electrode was measured by deter- mining the decrease in peak currents during repetitive DPV mea- suremen ts of LD and PAR after storing the electrode in 0.1 M PBS (pH 7.0). When the modified electrode was subjected to an exper- iment for ten times, after 12 h it gave no more than 7.6% and 8.3%decrease in the current response for LD and PAR, respectively .However , storing the modified electrode in air for 10 days gave only about 4.2% and 6.4% current decrease for LD and PAR, respec- tively. The results showed that the MWCNTs-N HNPs/GCE has very good stability to use for detection of these compounds .

3.6. Effect of interferences

The effects of common interfering species in solutions of 80 lMLD and 20 lM PAR were investigated in the optimum measure -ment conditions. Table 2 lists the tolerance limit for each potential interfere nt, which is defined as the concentratio n of the interferent that gives an error of 610% in the determination of LD or PAR. The data show that interfere nces are only significant at relatively high concentr ations, confirming that the proposed method is likely to befree from interferences from common components of biologica lsamples.

3.7. Analytical application s

The proposed method was successfully applied to the simulta- neous determination of LD and PAR in human urine at optimum

A. Babaei et al. / Journal of Electroanalytical Chemistry 698 (2013) 45–51 51

conditions by differential pulse voltammetry method. The samples were diluted 40 times before analysis and spiked with appropriate amounts of LD and PAR. The results are summarized in Table 3 inour experiments , the concentratio n of LD and PAR was calculated using standard additions method in order to prevent of any matrix effect. Good recoveries were obtained for spiked samples providing further evidence that this is a reliable method for the direct deter- mination of LD and PAR in urine samples. These confirm that the proposed method can be used for the consistent simultaneou sdetermination of these compounds in biological fluids.

4. Conclusion

This work demonstrat es that MWCNTs-NHN Ps modified glassy carbon electrode is suitable for a sensitive simultaneous determi- nation of trace amounts of LD and PAR. The very high sensitivity of the modified electrode can be related to high surface area, low resistance and electrocatalyti c effect of the MWCNTs- NHNPs nano- composite. In addition the fabricated sensor showed the lowest detection limit with excellent repeatabi lity in determination ofLD and PAR. The high sensitivit y of the MWCNTs-N HNPs/GCE for the detection of LD and PAR proves its potential for sensing appli- cations in real samples. The interfering study of some species showed no significant interfere nce with determinati on of LD and PAR. The sensor was successfully utilized for the analysis of LDand PAR levels in human urine.

Acknowled gements

The authors gratefully appreciate the financial support from re- search council of Arak University. Special thanks to Professor A.J.McQuillan from Otago Universit y in New Zealand for his valuable comments.

References

[1] N.F. Atta, M.F. El-Kady, A. Galal, Sens. Actuat. B. 141 (2009) 566–574.[2] J. Heo, Y.W. Lee, M. Kim, W.S. Yun, S.W. Han, Chem. Commun. 15 (2009) 1981–

1983.[3] H. Parham, N. Rahbar, J. Hazard. Mater. 177 (2010) 1077–1084.[4] S. Shahrokhian, M. Ghalkhani, M. Adeli, M.K. Amini, Biosens. Bioelectron. 24

(2009) 3235–3241.[5] I.G. Casella, M. Gatta, Anal. Chem. 72 (2000) 2969–2975.[6] Q. Li, L.S. Wang, B.Y. Hu, C. Yang, L. Zhou, L. Zhang, Mater. Lett. 61 (2007) 1615–

1618.[7] G.r. Fu, Z.a. Hu, L.j. Xie, X.q. Jin, Y.l. Xie, Y.x. Wang, Z.y. Zhang, Y.y. Yang, H.y.

Wu, Int. J. Electrochem. Sci. 4 (2009) 1052–1062.[8] A. Safavi, N. Maleki, E. Farjami, Biosens. Bioelectron. 24 (2009) 1655–1660.[9] G. Xiao-yan, D. Jian-cheng, Mater. Lett. 61 (2007) 621–625.

[10] J.J. Davis, R.J. Coles, H.A.O. Hill, J. Electroanal. Chem. 440 (1997) 279–282.[11] H. Luo, Z. Shi, N. Li, Z. Gu, Q. Zhuang, Anal. Chem. 73 (2001) 915–920.[12] J.M. Nugent, K.S.V. Santhanam, A. Rubio, P.M. Ajayan, Nano Lett. 1 (2001) 87–

91.[13] S.J. Tans, A.R.M. Verschueren, C. Dekker, Nature 393 (1998) 49–52.[14] J.J. Gooding, Electrochim. Acta 50 (2005) 3049–3060.

[15] J. Kong, N.R. Franklin, C. Zhou, M.G. Chapline, S. Peng, K. Cho, et al., Science 287 (2000) 622–625.

[16] A. Babaei, M. Afrasiabi, S. Mirzakhani, A.R. Taheri, J. Braz. Chem. Soc. 22 (2011)334–341.

[17] H.N. Dogan, A. Duran, Pharmazie 53 (1998) 781–784.[18] R. Sandulescu, S. Mirel, R. Oprean, J. Pharm. Biomed. Anal. 23 (2000) 77–87.[19] C. Nebot, S.W. Gibb, K.G. Boyd, Anal. Chim. Acta 598 (2007) 87–94.[20] A. Eustaquio, M. Blanco, R.D. Jee, A.C. Moffat, Anal. Chim. Acta 383 (1999) 283–

290.[21] J. Vilchez, R. Blanc, R. Avidad, A. Navalon, J. Pharm. Biomed. Anal. 13 (1995)

1119–1125.[22] J.A. Murillo Pulgarin, L.F. Garcia Bermejo, Anal. Chim. Acta 333 (1996) 59–69.[23] T. Pérez-Ruiz, C. Martínez-Lozano, V. Tomás, R. Galera, J. Pharm. Biomed. Anal.

38 (2005) 87–93.[24] W. Peng, T. Li, H. Li, E. Wang, Anal. Chim. Acta 298 (1994) 415–421.[25] A. Babaei, M. Afrasiabi, M. Babazadeh, Electroanalysis 22 (2010) 1743–1749.[26] A. Babaei, B. Khalilzadeh, M. Afrasiabi, J. Appl. Electrochem. 40 (2010) 1537–

1543.[27] H.R. Zare, N. Nasirizadeh, Int. J. Electrochem. Sci. 4 (2009) 1691–1705.[28] A.S. Fauci, E. Braunwald, D.L. Kasper, S.L. Hauser, D.L. Longo, J.L. Jameson, J.

Loscalzo, Harrison0s Principles Internal Medicine, seventeeth ed., Mc-Graw Hills companies, New York, 2008 (Chapter 366).

[29] E.J. Greenhow, L.E. Spencer, Analyst 98 (1973) 485–492.[30] A. Afkhami, D. Nematollahi, H.A. Khatami, Asian J. Chem. 14 (2002) 333–338.[31] J. Coello, S. Maspoch, N. Villegas, Talanta 53 (2000) 627–637.[32] P. Nagaraja, K.C. Srinivasa Murthy, K.S. Rangappa, N.M. Made Gowda, Talanta

46 (1998) 39–44.[33] G. Cannazza, A.D. Stefano, B. Mosciatti, D. Braghiroli, M. Baraldi, F. Pinnen, P.

Sozio, C. Benatti, C. Parenti, J. Pharmaceut. Biomed. Anal. 36 (2005) 1079–1084.

[34] C. Martinez-Lozano, T. Perez-Ruiz, V. Tomas, O. Val, Analyst 116 (1991) 857–859.

[35] S. Zhao, W. Bai, B. Wang, M. He, Talanta 73 (2007) 142–146.[36] M.F.S. Teixeira, M.F. Bergamini, C.M.P. Marques, N. Bocchi, Talanta 63 (2004)

1083–1088.[37] M.F. Bergamini, A.L. Santos, N.R. Stradiotto, M.V.B. Zanoni, J. Pharm. Biomed.

Anal. 39 (2005) 54–59.[38] G. Cannazza, A. Di Stefano, B. Mosciatti, D. Braghiroli, M. Beraldi, F. Pinnen, P.

Sozio, P. Benatti, C. Parenti, J. Pharm. Biomed. Anal. 36 (2005) 1079–1084.[39] D.R.C. Robertson, A.G. Renwick, B. Macklin, S. Jones, D.G. Waller, C.F. George,

J.S. Fleming, Eur. J. Clin. Pharmacol. 42 (1992) 409–412.[40] A. Babaei, M. Babazadeh, M. Afrasiabi, Sensor Lett. 10 (2012) 992–998.[41] A.J. Bard, L.R. Faulkner, Electrochemical Methods, Fundamentals and

Applications, second ed., Wiley, New York, 2001 .[42] E. Laviron, J. Electroanal. Chem. 101 (1979) 19–28.[43] Y. Wu, X. Ji, S. Hu, Bioelectrochemistry 64 (2004) 91–97.[44] P. Daneshgar, P. Norouzi, M.R. Ganjali, A. Ordikhani-Seyedlar, H. Eshraghi,

Colloids Surf. B 68 (2009) 27–32.[45] A. Sivanesan, S. Abraham John, Biosens. Bioelectron. 23 (2007) 708–713.[46] M.F.S. Teixeira, L.H. Marcolino-Junior, O. Fatibello-Filho, E.R. Dockal, M.F.

Bergamini, Sens. Actuat. B 122 (2007) 549–555.[47] S. Viswanathan, W. Liao, C. Huang, W.L. Hsu, J.A. Ho, Talanta 74 (2007) 229–

234.[48] S. Shahrokhian, E. Asadian, J. Electroanal. Chem. 636 (2009) 40–46.[49] X. Yan, D. Pang, Z. Lu, J. Lu, H. Tong, J. Electroanal. Chem. 569 (2004) 47–52.[50] S.A. Kumar, C.F. Tang, S.M. Chen, Talanta 76 (2008) 997–1005.[51] V. Sima, C. Cristea, F. Lapadus, I.O. Marian, A. Marian, R. Sandulescu, J. Pharm.

Biomed. Anal. 48 (2008) 1195–1200.[52] M. Mazloum-Ardakani, H. Beitollahi, M.A. Sheikh Mohseni, A. Benvidi, H.

Naeimi, M. Nejati-Barzoki, N. Taghavinia, Colloids Surf. B 76 (2010) 82–87.[53] X. ShangGuan, H. Zhang, J. Zheng, Anal. Bioanal. Chem. 391 (2008) 1049–1055.[54] P. Fanjul-Bolado, P.J. Lamas-Ardisana, D. Hernandez-Santos, A. Costa-Garcia,

Anal. Chim. Acta 638 (2009) 133–138.[55] B.C. Lourencao, R.A. Medeiros, R.C. Rocha-Filho, L.H. Mazo, O. Fatibello-Filho,

Talanta 78 (2009) 748–752.[56] S.F. Wang, F. Xie, R.F. Hu, Sens. Actuat.: B 123 (2007) 495–500.