Dfc

HOa

b

c

d

a

ARRAA

KFMPD

1

plTnlbb

immddtm

Sf

0d

Sensors and Actuators B 161 (2012) 755– 760

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h o mepage: www.elsev ier .com/ locate /snb

ifferential pulse voltammetric determination of ciprofibrate in pharmaceuticalormulations using a glassy carbon electrode modified with functionalizedarbon nanotubes within a poly(allylamine hydrochloride) film

umberto H. Takedaa,b,c, Bruno C. Janegitza, Roberta A. Medeirosa, Luiz H.C. Mattosob,rlando Fatibello-Filhoa,d,∗

Departamento de Química, Universidade Federal de São Carlos, P.O. Box 676, 13.560-970, São Carlos, SP, BrazilEmbrapa Instrumentac ão Agropecuária, Laboratório Nacional de Nanotecnologia para o Agronegócio 13560-970, São Carlos, SP, BrazilDepartamento de Engenharia de Alimentos, Fundac ão Universidade Federal de Rondônia, 76872-862, Ariquemes, RO, BrazilInstituto Nacional de Ciências e Tecnologia em Bioanalítica, 13083-970, Campinas, SP, Brazil

r t i c l e i n f o

rticle history:eceived 11 August 2011eceived in revised form 31 October 2011ccepted 14 November 2011

a b s t r a c t

A glassy carbon electrode (GCE) modified with functionalized multiwalled carbon nanotubes withina poly(allylamine hydrochloride) film (CNTs–PAH) to determine ciprofibrate using differential pulsevoltammetry (DPV) is proposed. The CNTs/PAH film was previously valued and characterized with thezeta potential technique and scanning electronic microscopy (SEM). The DP voltammograms presented a

vailable online 22 November 2011

eywords:ibric acid determinationultiwalled carbon nanotubes

AH – poly(allylamine hydrochloride) filmifferential pulse voltammetry

ciprofibrate oxidation peak potential at 0.98 V in a 0.1 mol L−1 phosphate buffer solution (pH 7.0). The ana-lytical curve was linear in the ciprofibrate concentration range from 1.33 × 10−5 to 1.32 × 10−4 mol L−1,with a detection limit of 8.34 × 10−6 mol L−1. Precise and accurate results are in agreement with thoseobtained by the chromatographic method at a 95% confidence level.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Fibric acids are drugs with antihyperlipidemic agents, whichromote the reduction of high lipid (cholesterol and triglycerides)

evels in the blood [1]. The use of such acids began in 1962 whenhope and Waring observed that the fibric acid ethyl chlorophe-oxiisobutirate (clofibrate) derivative caused the reduction of lipid

evels in rats. Since then, many other fibric acid derivatives haveeen developed, such as clofibrate, genfibrozile, fenofibrate, bezafi-rate and ciprofibrate (Fig. 1) [2].

Fibrates are indicated for patients for whom tests confirmed anncrease in endogenous triglycerides due to poor nutrition. Com-

only prescribed, such fibrates are generally well tolerated. Theost frequent side effects include upper gastrointestinal system

iscomfort, nausea, headaches, anxiety, and skin rashes. Rhab-

omyolysis is an unusual but serious adverse effect in fibratereatment. Rhabdomyolysis can injure the skeletal muscles or

yocardium and lead to renal failure, infections, disseminated

∗ Corresponding author at: Departamento de Química, Universidade Federal deão Carlos, P.O. Box 676, 13.560-970, São Carlos, SP, Brazil. Tel.: +55 16 33518098;ax: +55 16 33518350.

E-mail address: bello@ufscar.br (O. Fatibello-Filho).

925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2011.11.028

intravascular coagulation, or even death [2]. Thus, the determi-nation of ciprofibrate in pharmaceutical formulations is necessaryto ensure the fulfillment of legal requirements as well as quality-control procedures in the pharmaceutical industry.

Only a few methods for determining ciprofibrate have beenreported, based on chromatography [3–5], capillary electrophore-sis [6] and spectrophotometry [7]. However, these methods areprone to many drawbacks, such as high expense, and complicatedand lengthy procedures.

Polyelectrolytes are long-chain macromolecules (polymers),obtained by a combination of simple monomers, and whendissolved in polar solvents, especially water, they have posi-tive or negative charges. They are commonly used to constructhybrid, nanostructured and layer-by-layer (LBL) materials [8–10].Poly(allylamine hydrochloride) (PAH) and other gel polyelec-trolytes are used in various applications, such as in the formationof thin films used in sensors for displaying small variations in thevolume of transition at different temperatures, pH values and indifferent solvents [8,10]. As such, this molecule seems suited todeveloping films and electrodes.

Carbon nanotubes (CNTs), since their discovery by Iijima [11],have been attracted considerable attention due to their fascinatingand unique structural, mechanical, electrical and electrochemi-cal properties. They have been widely used for the development

756 H.H. Takeda et al. / Sensors and Actu

oicetaatasoo

oarftctdo

2

2

cafs(t(t

2

epacpmZ2

tIb

aA(m

Fig. 1. Ciprofibrate molecular structure.

f chemically modified electrodes providing special electrochem-cal properties, such as good conductivity and enhanced sensingapabilities in sensors and biosensors [12–19]. Moreover, extensivefforts have been devoted to designing novel CNTs-modified elec-rodes to improve voltammetric determination of organic [20–22]nd inorganic [23,24] compounds. Pretreatment of CNTs is usu-lly necessary to eliminate metallic impurities, and/or to improvehe electron transfer properties and/or to allow further function-lization. Pretreatment consists of exposing the CNTs to an acidicolution of sulfuric [25], nitric [26–29] or hydrochloric [30,31] acid,r a mixture of these acids at room temperature, under refluxingr sonication for different times [29].

Electrochemical techniques, such as voltammetric techniques,ffer promising alternatives to classical approaches due to their rel-tively low operational cost, good miniaturization potential, andapid and sensitive detection procedures, which are suitable foraster analyses. In this paper we offer a report on the coupling ofhe differential pulse voltammetric (DPV) technique with a glassyarbon electrode (GCE) modified with multiwalled carbon nano-ubes and poly(allylamine hydrochloride) film (CNTs–PAH) for theevelopment and optimization of a method for the determinationf ciprofibrate in several pharmaceutical formulation samples.

. Experimental

.1. Reagents and standards

Ciprofibrate and PAH were acquired from Sigma. Multiwalledarbon nanotubes (20–30 nm in diameter, 1–2 nm wall thickness,nd 0.5–2 nm in length with a 95% purity level) were purchasedrom Aldrich. All other chemicals were of analytical grade. Allolutions were prepared with Millipore Milli-Q nanopure waterresistivity > 18 M� cm). The 0.1 mol L−1 phosphate buffer solu-ions at pH 7.0, which were made from Na2HPO4 and NaH2PO4both Aldrich), were consistently employed as supporting elec-rolyte.

.2. Apparatus

Voltammetric measurements were performed with a three-lectrode system, including CNTs–PAH/GCE as working electrode, alatinum plate as counter electrode, and Ag/AgCl (3.0 mol L−1 KCl)s reference electrode, at 25 ◦C. Voltammetric measurements werearried out using a PalmSens (Palm Instruments BV – Netherlands)otentiostat/galvanostat controlled by PalmSens software. Theorphologies were verified using FEG-SEM (Supra 35-VP, Carl

eiss, Germany) equipment with an electron beam energy of5 keV.

The stability of the CNTs–PAH film was verified with zeta poten-ial measurements in Zeta Series Nano equipment from Malvernnstruments coupled with a MPT-2 Multipurpose Titrator managedy Dispersion Technology Software 5.02.

The HPLC ciprofibrate determination was carried out using

n LC-10AT Shimadzu system, with a UV/vis detector (SPD-M10-VP) set at a wavelength of 220 nm. A Shim-Pack CLC-ODS

4.6 mm × 150 mm, 5 �m) chromatographic column was used. Theobile phase was an acetonitrile/0.02 mol L−1 H3PO4 (55:45, v/v)

ators B 161 (2012) 755– 760

at a flow-rate of 1.0 mL min−1, and the injection volume was 20 �L[4].

2.3. Functionalization of the CNTs

The acid treatment of carbon nanotubes is very important toeliminate metallic impurities presents in the synthesis and appearcarboxylic groups, which can increasing the analytical signal, canimprove the electron transfer properties and/or to allow furtherfunctionalization [24,32–34]. The carbon nanotubes were submit-ted to a chemical treatment using a mixture of concentrated sulfuricand nitric acids 3:1 (v/v) for 12 h at room temperature (using a ratio1 g of CNTs: 500 mL of acid solution) under stirring. Then, the sus-pension was separated by centrifugation and the solid was washedwith ultrapure water until pH 6.5–7.0 and then it was dried at 120 ◦Cfor 5 h.

2.4. CNTs–PAH/GCE electrode preparation

The GC electrode (5 mm diameter) was polished sequentiallywith metallographic abrasive paper (No. 6) and slurries of 0.3and 0.05 �m alumina microparticles to a mirror finish. After beingrinsed with Milli-Q water, the electrode was sonicated in absoluteethanol and then with Milli-Q water for about 1 min, respectively.

The two CNTs suspensions used in the stability study were pre-pared by dispersing 1.0 mg of functionalized CNTs in 30 mL of a1.0 × 10−4 mol L−1 PAH (1) and in 30 mL of ultrapure water (2). Bothdispersions, 1 and 2, were subjected to ultrasonication for 30 min.

Film formation was obtained by the dispersion of 1.0 mg of func-tionalized CNTs in 1.0 mL of 1.0 × 10−4 mol L−1 PAH solution thatwas subjected to ultrasonication for 30 min. Following this, 10 �L ofthe CNTs–PAH suspension was cast on the surface of a GC electrodeand the solvent was allowed to evaporate at room temperature for2 h. This electrode was designated as PAH–CNTs/GCE. When notused immediately, the electrode was stored in a desiccator at 25 ◦C.

2.5. Sample preparation

Pharmaceutical formulations containing ciprofibrate were pur-chased from a local drugstore, which were analyzed by theproposed method and by an HPLC comparative method. To pre-pare commercial sample solutions, a representative number oftablets (5 or 10) of each pharmaceutical sample were reduced toa fine homogeneous powder in a mortar. Then, a quantity of onetablet (100 mg) was weighed, dissolved and transferred to a 10 mLvolumetric flask, the volume being made up with a 0.1 mol L−1

phosphate buffer solution at pH 7.0. All insoluble pharmaceuticalexcipients of samples were removed by filtration using filter paper.To prepare sample solutions in concentration ranges of the ana-lytical curve, an appropriate aliquot was diluted with 0.1 mol L−1

phosphate buffer solution (pH 7.0) solution in a 50.0 mL calibratedflask.

3. Results and discussion

3.1. CNTs–PAH film characterization

Fig. 2 shows the study of stability of CNTs–PAH film and CNTs inwater (CNTs–H2O). Using the zeta (�) potential as a function of thepH, the behavior and stability of both dispersions were observed.According to the literature, suspension stability may be affected by

changes in the pH and/or ionic strength, and the higher value ofzeta potential indicates greater dispersion stability [30].

CNTs–PAH dispersion presented higher values of zeta (�) poten-tial, between 40.2 and 50.2 mV, in a pH range from 2.7 to 8.6, and at

H.H. Takeda et al. / Sensors and Actuators B 161 (2012) 755– 760 757

Fs

ppwtastsaaiahsct

intbee

taa

ig. 2. Dependence of zeta potential of CNTs–PAH and CNTs–H2O dispersions witholution pH.

H values higher than 8.6 the � potential decreased considerably. Arobable explanation for these results is related to the pKa of PAH,hich is equal to 8.5 [28]; therefore, at pH values lower than 8.5,

he PAH solution presents a cationic character (polycation) gener-ted by the protonated amino group and there is consequently atrong electrostatic attraction with the CNTs. At pH values higherhan 8.5–8.6 the PAH molecules were neutral and the repulsivetrength increased gradually between PAH molecules and CNTs,nd the stability of CNTs–PAH dispersion decreased. These resultsre in agreement with the work proposed by Iamsamai et al. [31],n which the stability of chitosan dispersion in CNTs was studiednd it was observed that the stability of this dispersion occurs atigh values of zeta (�) potential (between 34 and 42 mV) due to thetrong electrostatic attraction between protonated amino groups ofhitosan with the carboxyl group of functionalized CNTs. As such,he results indicate the stability of the proposed CNT–PAH film.

The electroactive area of GCE and CNTs–PAH/GCE was estimatedn 0.1 mol L−1 KCl in the presence of 1.0 mmol L−1 [Fe(CN)6]4− (dataot shown) according to the Randles-Sevcik equation [35]. The elec-roactive areas of CNTs–PAH/GCE and bare GCE were calculated toe 0.10 cm2 and 0.050 cm2, respectively. The CNTs increased thelectroactive surface area by a factor of 2 compared to the barelectrode.

Fig. 3 shows SEM images of PAH and PAH–CNTs. It can be seen

hat the PAH film has a reasonably uniform polymeric structure,nd the PAH–CNTs presented CNTs distributed in the polymer films expected.

Fig. 3. SEM images of PAH (

Fig. 4. DP voltammograms for 4.0 × 10−5 mol L−1 ciprofibrate in a 0.1 mol L−1 phos-phate buffer solution at pH 7.0 on PAH-CNT/GC (solid line) and GC (dashed line)electrodes; � = 10 mV s−1, a = 40 mV and t = 100 ms.

3.2. Investigation of the electrochemical behavior

Initially, measurements were performed by DPV to investi-gate the electroactivity of ciprofibrate using CNTs–PAH/GCE andGCE. DPV measurements were performed with a scan rate (�) of10 mV s−1, a pulse amplitude (a) of 40 mV and a pulse time (t) of100 ms for a 4.0 × 10−5 mol L−1 ciprofibrate in 0.1 mol L−1 phos-phate buffer solution (pH 7.0) (Fig. 4). As can be observed, thevoltammogram obtained employing the PAH–CNTs/GCE presenteda higher anodic peak current and a lower peak potential whencompared with the DP voltammogram obtained using the bareGCE. On the reverse scan, no corresponding reduction peak wasobserved. These results are in agreement with those presented inthe literature for carbon nanotube electrodes [36,37] and suggestelectrocatalysis.

The proposed electrode, PAH–CNTs/GCE, presents an oxidationpeak current for 4.0 × 10−5 mol L−1 ciprofibrate that increases lin-early with the scan rate � in the range from 50 to 500 mV s−1,using linear sweep voltammetry. The linear regression equationobtained was Ip/�A = −0.81 + 0.23�1/2 (mV s−1), (r = 0.992). Theseresults indicate that the electrode process is mainly controlled bymass transport. As for a totally irreversible electrode, the rela-tionship between the peak potential Ep and the scan rate � was

followed by the Laviron equation [38]. The oxidation peak poten-tial Ep of ciprofibrate shifted linearly with log � in the range ofscan rate from 10 to 200 mV s−1 with the linear regression equa-tion of Ep/V = 0.127 + 0.046log � (mV s−1), (r = 0.993). If the value of

A) and CNTs/PAH (B).

758 H.H. Takeda et al. / Sensors and Actuators B 161 (2012) 755– 760

oxidation mechanism of ciprofibrate.

˛totc

ae6aoba

3p

d(irfofs

ipnsst

3

octpc[odc

5caeoa

Fig. 6. Differential pulse voltammograms obtained using the PAH–CNTs/GCE forciprofibrate solution in the concentrations of: (1) 1.3 × 10−5, (2) 2.6 × 10−5, (3)

−5 −5 −5 −5 −5 −4

The proposed method was applied to ciprofibrate determinationin pharmaceutical samples and the results were compared withthose results obtained using the HPLC method. Table 1 presents

Table 1Results obtained in the determination of ciprofibrate in pharmaceutical formula-tions (tablets) using the comparative HPLC and proposed DPV methods.

Ciprofibrate (mg/tablet)

Sample Label value HPLC methoda DPV methoda Relative error1c (%)

A 100 98 ± 5 100 ± 3 2B 100 100 ± 7 99 ± 4 −1

Fig. 5. Proposal for the possible

is assumed to be 0.5, a common value for organic molecules,hese results indicate that the oxidation of ciprofibrate involvesne electron per molecule, according to the Laviron equation. Fromhese results, a proposal for the possible oxidation mechanism ofiprofibrate is show in Fig. 5.

The effect of pH on the DP voltammograms was examined for 9.0 × 10−5 mol L−1 ciprofibrate solution using the PAH–CNTs/GClectrode in 0.1 mol L−1 phosphate buffer solutions at pH values of.1, 6.4, 7.0, 7.4 and 7.8. The highest analytical signal was observedt pH 7.0–7.4. The great advantage in using a solution that is neutralr close to neutral is the possibility to determine ciprofibrate iniological samples. Thus, the 0.1 mol L−1 phosphate buffer solutiont pH 7.0 was selected for further studies.

.3. Optimization of the differential pulse voltammetryarameters

The peak current obtained in differential pulse voltammetryepends on various instrumental parameters such as the scan rate�), amplitude (a) and modulation time (t). These parameters arenterrelated, having a combined influence on the peak currentesponse. Hence, in order to establish the optimum conditionsor the determination of ciprofibrate in this study, the influencef these instrumental parameters on the peak current responseor 2.6 × 10−5 mol L−1 ciprofibrate in 0.1 mol L−1 phosphate bufferolution (pH 7.0) was investigated.

For the scan rate (�), it was observed that the peak currentncreases to 12 mV s−1, which was selected for further studies. Theulse amplitude (a), which also strongly influences the DPV sig-al, increased linearly to 60 mV, and this value was selected for theubsequent analytical application. The modulation time increasedignificantly for values up to 100 ms, and then leveled off; therefore,his value was chosen for further studies.

.4. Analytical curve and applications

Under the optimized parameters a calibration curve wasbtained for ciprofibrate (Fig. 6). The anodic peak current foriprofibrate was linearly dependent on its concentration (c) inhe range from 1.33 × 10−5 to 1.32 × 10−4 mol L−1 in 0.1 mol L−1

hosphate buffer solution at pH 7.0 using the PAH–CNTs/GCE. Theorresponding calibration equation is Ipa/�A = −0.701 + 4.75 × 104

ciprofibrate/mol L−1] with r = 0.996 and the detection limitbtained was 8.34 × 10−6 mol L−1, based on three times the stan-ard deviation for the blank solution by the slope of the analyticalurve.

The inter-day repeatability of CNTs–PAH/GCE was realized with determinations in five different days for a 8.0 × 10−5 mol L−1

iprofibrate solution using the same electrode, which presented

relative standard deviation of 0.2%. In addition, using 3 differ-nt electrodes (CNTs–PAH/GCE) prepared in the same way it wasbtained a relative standard deviation of 1.0% for 5 measurementsnd 8.0 × 10−5 mol L−1 ciprofibrate solution (reproducibility). The

4.0 × 10 , (4) 5.3 × 10 , (5) 6.6 × 10 , (6) 7.9 × 10 , (7) 9.2 × 10 , (8) 1.0 × 10 ,(9) 1.1 × 10−4 and (10) 1.3 × 10−4 mol L−1, in a 0.1 mol L−1 phosphate buffer solutionat pH 7.0; � = 12 mV s−1, a = 60 mV and t = 100 ms.

CNTs–PAH/GCE was stored in a desiccator at 25 ◦C, which main-tained 90% of voltammetric response after using uninterrupted 15days (ca. 120 determinations per each film).

Possible interference of excipient substances frequently foundwith ciprofibrate in pharmaceutical formulations, such as sodiumlauryl sulfate, lactose and starch, was evaluated using the proposedelectrode. DPV measurements containing only 5.0 × 10−5 mol L−1

ciprofibrate were compared with DPV measurements contain-ing ciprofibrate and excipients (10-fold excess). All excipientspresented an interference level lower than 5%. Therefore, the excip-ients commonly present in this pharmaceutical formulation do notaffect ciprofibrate determination using the PAH–CNTs/GCE.

Recovery studies were also realized to evaluate the matrixeffect after standard-solution additions. The results obtained showrecoveries ranging from 98.8% to 101% of ciprofibrate for thesepharmaceutical formulations, indicating an absence of matrixeffects.

C 100 100 ± 4 99 ± 6 −1D 100 104 ± 2 100 ± 6 −4

Relative error1c = 100 × (voltammetric value − HPLC value)/HPLC value).

a Average of three measurements.

H.H. Takeda et al. / Sensors and Actu

Table 2Comparison of the analytical data between present work and some recently reportedpapers for ciprofibrate determination.

Method Linear range (mol L−1) LOD (mol L−1) Reference

−5 −4 −5

t[(o

sHtcaeltttd

4

Cp(qlm

sfdafbottMc

A

I

R

[

[[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

Spectrophotometric 4.84 × 10 to 2.08 × 10 3.46 × 10 [39]HPLC 8.65 × 10−7 to 1.73 × 10−4 2.42 × 10−7 [4]CNTs–PAH/GCE 1.33 × 10−5 to 1.32 × 10−4 8.34 × 10−6 This work

he results obtained using both methods. Applying paired t-tests39], the calculated t value (2.84) is smaller than the critical value3.18, = 0.05) indicating that there is no difference between thebtained results at a confidence level of 95%.

There are few papers for ciprofibrate determination. Table 2 pre-ented the linear range and LOD of a spectrophotometric [7] andPLC [4] methods for ciprofibrate quantification. A capillary elec-

rophoresis achiral separation [6] for racemic hypolipidaemic agentiprofibrate and its main metabolite (ciprofibrate glucuronide) islso presented, nevertheless the linear range and LOD were notvaluated. The proposed voltammetric method presented a similarinear range and a LOD smaller than that obtained using the spec-rophotometric method and a higher linear range and LOD thanhose obtained using an HPLC method. Nevertheless, the concen-ration of the analyte in the pharmaceutical product is high and ailution step is required previous to voltammetric determination.

. Conclusions

The results reported here allow the conclusion that theNTs–PAH dispersion presented good stability, according to zetaotential study, which is probably due to the cationic characterpolycation) generated by protonated amino groups, and conse-uently a strong electrostatic attraction with the CNTs, at pH values

ower than 8.5. The casting technique proved to be adequate for theodification of the GC electrode with CNTs–PAH film.The polymer used to immobilize the CNTs in the CG electrode

urface increasing the stability, repeatability, the analytical per-ormance and lifetime of electrode. The electroanalytical methodeveloped with the CNTs–PAH/GCE, as working electrode, is a goodlternative for the determination of ciprofibrate in pharmaceuticalormulations, and this methodology is most likely readily applica-le to other matrix samples. Indeed, very low detection limits werebtained in the determination of ciprofibrate (8.34 �mol L−1). Fur-hermore, addition and recovery studies allowed the conclusionhat the matrix effect did not present any significant interference.

oreover, the proposed method is simple, inexpensive, quick, andan be carried out with good precision and accuracy.

cknowledgements

The authors acknowledge financial support from CAPES, FAPESP,NCT of Bioanalytical and CNPq (Brazil).

eferences

[1] A.M. Hierlihy, M. Cooke, I.H.A. Curran, R. Mehta, L. Karamanos, C.A. Price, Effectsof ciprofibrate on testicular and adrenal steroidogenic enzymes in the rat,Reprod. Toxicol. 22 (2006) 37–43.

[2] G. Paragh, I. Seres, M. Harangi, A. Erdei, M. Audikovszky, L. Debreczeni, A. Kovác-say, L. Illyés, G. Pados, Ciprofibrate increases paraoxonase activity in patientswith metabolic syndrome, Br. J. Clin. Pharmacol. 61 (2006) 694–701.

[3] G. Misztal, L. Komista, Determination of bezafibrate and ciprofibrate in pharma-ceutical formulations by densitometric and videodensitometric TLC, J. PlanarChromatogr.: Mod. TLC 18 (2005) 188–193.

[4] L.D. Masnatta, L.A. Cuniberti, R.H.J. Rey, Determination of bezafibrate, ciprofi-

brate and fenofibric acid in human plasma by high-performance liquidchromatography, J. Chromatogr. B 687 (1996) 437–442.

[5] G.B. Park, C.E. Biddlecome, C. Koblantz, J. Edelson, Determination of ciprofibratein human-plasma by high-performance liquid-chromatography, J. Chromatogr.227 (1982) 534–539.

[

ators B 161 (2012) 755– 760 759

[6] H. Huttemann, G. Blaschke, Achiral and chiral determination of ciprofibrate andits glucuronide in human urine by capillary electrophoresis, J. Chromatogr. B729 (1999) 33–41.

[7] G.N.L. Nascimento, D.L. Rosa, H. Nishijo, T.A. Aversi-Ferreira, Validation of aspectrophotometric method to determine ciprofibrate content in tablets, Braz.J. Pharm. Sci. 47 (2011) 23–29.

[8] H. Kong, P. Luo, C. Gao, D. Yan, Polyelectrolyte-functionalized multiwalledcarbon nanotubes: preparation, characterization and layer-by-layer self-assembly, Polymer 46 (2005) 2472–2485.

[9] V. Jain, H.M. Yochum, R. Montazami, J.R. Heflin, L.B. Hu, G. Gruner, Modificationof single-walled carbon nanotube electrodes by layer-by-layer assembly forelectrochromic devices, J. Appl. Phys. 103 (2008) 074504–74511.

10] G.A. Rivas, S.A. Miscoria, J. Desbrieres, G.D. Barrera, New biosensing plat-forms based on the layer-by-layer self-assembling of polyelectrolytes onNafion/carbon nanotubes-coated glassy carbon electrodes, Talanta 71 (2007)270–275.

11] S. Iijima, Helical microtubules of graphitic carbon, Nature 354 (1991) 56.12] E.W. Wong, P.E. Sheehan, C.M. Lieber, Nanobeam mechanics: elasticity,

strength, and toughness of nanorods and nanotubes, Science 277 (1997)1971–1975.

13] J. Riu, A. Maroto, F.X. Rius, Nanosensors in environmental analysis, Talanta 69(2006) 288–301.

14] J.W. Mintmire, B.I. Dunlap, C.T. White, Are fullerene tubules metallic, Phys. Rev.Lett. 68 (1992) 631–634.

15] J. Liu, A.G. Rinzler, H.J. Dai, J.H. Hafner, R.K. Bradley, P.J. Boul, A. Lu,T. Iverson, K. Shelimov, C.B. Huffman, F. Rodriguez-Macias, Y.S. Shon,T.R. Lee, D.T. Colbert, R.E. Smalley, Fullerene pipes, Science 280 (1998)1253–1256.

16] R.H. Baughman, C.X. Cui, A.A. Zakhidov, Z. Iqbal, J.N. Barisci, G.M. Spinks, G.G.Wallace, A. Mazzoldi, D. De Rossi, A.G. Rinzler, O. Jaschinski, S. Roth, M. Kertesz,Carbon nanotube actuators, Science 284 (1999) 1340–1344.

17] L. Kavan, L. Dunsch, H. Kataura, Electrochemical tuning of electronic structureof carbon nanotubes and fullerene peapods, Carbon 42 (2004) 1011–1019.

18] M.E. Ghica, R. Pauliukaite, O. Fatibello-Filho, C.M.A. Brett, Applicationof functionalized carbon nanotubes immobilized into chitosan films inamperometric enzyme biosensors, Sens. Actuators B: Chem. 142 (2009)308–315.

19] R. Pauliukaite, M.E. Ghica, O. Fatibello-Filho, C.M.A. Brett, Comparative study ofdifferent cross-linking agents for the immobilization of functionalized carbonnanotubes within a chitosan film supported on a graphite-epoxy compositeelectrode, Anal. Chem. 81 (2009) 5364–5372.

20] W.S. Huang, W.B. Hu, J.C. Song, Adsorptive stripping voltammetric determina-tion of 4-aminophenol at a single-wall carbon nanotubes film coated electrode,Talanta 61 (2003) 411–416.

21] J. Wang, S.B. Hocevar, B. Ogorevc, Carbon nanotube-modified glassy carbonelectrode for adsorptive stripping voltammetric detection of ultratrace levelsof 2,4,6-trinitrotoluene, Electrochem. Commun. 6 (2004) 176–179.

22] R. Pauliukaite, M.E. Ghica, O. Fatibello-Filho, C.M.A. Brett, Graphite-epoxy elec-trodes modified with functionalised carbon nanotubes and chitosan for therapid electrochemical determination of dipyrone, Comb. Chem. High Through-put Screen 13 (2010) 590–598.

23] K.B. Wu, S.S. Hu, J.J. Fei, W. Bai, Mercury-free simultaneous determination ofcadmium and lead at a glassy carbon electrode modified with multi-wall carbonnanotubes, Anal. Chim. Acta 489 (2003) 215–221.

24] B.C. Janegitz, L.H. Marcolino-Junior, S.P. Campana-Filho, R.C. Faria, O. Fatibello-Filho, Anodic stripping voltammetric determination of copper (II) using afunctionalized carbon nanotubes paste electrode modified with crosslinkedchitosan, Sens. Actuators B: Chem. 142 (2009) 260–266.

25] M. Musameh, J. Wang, A. Merkoci, Y.H. Lin, Low-potential stable NADHdetection at carbon-nanotube-modified glassy carbon electrodes, Electrochem.Commun. 4 (2002) 743–746.

26] A. Salimi, C.E. Banks, R.G. Compton, Abrasive immobilization of carbon nano-tubes on a basal plane pyrolytic graphite electrode: application to the detectionof epinephrine, Analyst 129 (2004) 225–228.

27] K.P. Gong, Y. Dong, S.X. Xiong, Y. Chen, L.Q. Mao, Novel electrochemical methodfor sensitive determination of homocysteine with carbon nanotube-based elec-trodes, Biosens. Bioelectron. 20 (2004) 253–259.

28] M.L. Guo, J.H. Chen, D.Y. Liu, L.H. Nie, S.Z. Yao, Electrochemical characteristicsof the immobilization of calf thymus DNA molecules on multi-walled carbonnanotubes, Bioelectrochemistry 62 (2004) 29–35.

29] C.G. Hu, W.L. Wang, K.J. Liao, G.B. Liu, Y.T. Wang, Systematic investigation on theproperties of carbon nanotube electrodes with different chemical treatments,J. Phys. Chem. Solids 65 (2004) 1731–1736.

30] S.R. Bhatia, S.F. Khattak, S.C. Roberts, Polyelectrolytes for cell encapsulation,Curr. Opin. Colloid Interface Sci. 10 (2005) 45–51.

31] C. Iamsamai, S. Hannongbua, U. Ruktanonchai, S. Soottitantawat, S.T. Dubas,The effect of the degree of deacetylation of chitosan on its dispersion of carbonnanotubes, Carbon 48 (2010) 25–30.

32] B.C. Janegitz, R. Pauliukaite, M.E. Ghica, C.M.A. Brett, O. Fatibello-Filho, Directelectron transfer of glucose oxidase at glassy carbon electrode modified withfunctionalized carbon nanotubes within a dihexadecylphosphate film, Sens.

Actuators B: Chem. 158 (2011) 411–417.

33] B.C. Janegitz, L.C.S. Figueiredo-Filho, H. Luiz, S.P.N. Marcolino-Junior, E.R. Souza,O. Pereira-Filho, Fatibello-Filho, Development of a carbon nanotubes pasteelectrode modified with crosslinked chitosan for cadmium(II) and mercury(II)determination, J. Electroanal. Chem. 660 (2011) 209–216.

7 d Actu

[

[

[

[

[

[

B

HeoRne

60 H.H. Takeda et al. / Sensors an

34] F.C. Vicentini, L.C.S. Figueiredo-Filho, B.C. Janegitz, A. Santiago, E.R.Pereira-Filho, O. Fatibello-Filho, Factorial design and response sur-face: voltammetric method optimization for the determination of Ag(I)employing a carbon nanotubes paste electrode, Quim. Nova 34 (2011)825–830.

35] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applica-tions, 2nd ed., John Wiley & Sons, Inc., New York, 2001.

36] Q. Zhao, Z.H. Gan, Q.K. Zhuang, Electrochemical sensors based on carbon nano-tubes, Electroanalysis 14 (2002) 1609–1613.

37] J. Li, J.E. Koehne, A.M. Cassel, H. Chen, H.T. Ng, Q. Ye, W. Fan, J. Han, M.Meyeyyappan, Inlaid multi-walled carbon nanotube nanoelectrode arrays forelectroanalysis, Electroanalysis 17 (2005) 15–27.

38] E. Laviron, General expression of the linear potential sweep voltammogramin the case of diffusionless electrochemical systems, J. Electroanal. Chem. 101(1979) 19–28.

39] R.L. Anderson, Practical Statistics for Analytical Chemists, Van Nostrand Rein-hold Company, New York, 1987.

iographies

umberto H. Takeda received his PhD degree in Chemistry from São Carlos Fed-

ral University, São Carlos, SP, Brazil in 2010. Currently, he is an active professorf chemistry in the Department of Food Engineering at the Federal University ofondônia, Brazil. His research interest consists in de electroanalytical chemistry,anostructured materials, modified electrodes surfaces, conducting polymers andlectrochemical sensors and biosensors.

ators B 161 (2012) 755– 760

Bruno C. Janegitz received the MS degree from the Federal University of São Carlos,São Carlos, SP, Brazil in 2009, and actually is PhD student in the same University.His research interests include electroanalytical chemistry, nanostructured electrodematerials and modified electrode surfaces, electrochemical sensors and biosensors.

Roberta A. Medeiros received her PhD degree in analytical chemistry from SãoCarlos Federal University, São Carlos, SP, Brazil in 2011, and currently conductsher posdoctorade in the same University. Her research interests consist in thedevelopment of electroanalytical procedures for identification and determinationof differently analytes mainly in food samples and pharmaceutical formulations,employed electrochemical sensors, modified electrode surfaces, voltammetric,amperometric and multiple pulse amperometric techniques and flow injection anal-ysis.

Luiz H.C. Mattoso received his PhD in materials science and engineering in1993 from Materials Engineering Department at Federal University of São Carlos(DEMa-UFSCar), Brazil. He is currently a full researcher at Embrapa AgriculturalInstrumentation Center in Brazil. His field of interests includes conducting poly-mers, chemical and biological sensors, nanostructured materials, polymeric blendsand nanocomposites.

Orlando Fatibello-Filho received his PhD degree in Analytical Chemistry from SãoPaulo University and postdoctorate in 1989 from the University of New Orleans, USA.Currently, he is a full professor of analytical chemistry in the Department of Chem-

istry at the Federal University of São Carlos, Brazil. His research interests consistin the development of analytical procedures employing chemical sensors, biosen-sors, solid-phase reactors and flow-injection systems and their application to thedetermination of analytes in pharmaceutical formulations, environmental and foodsamples.