Journal of Electroanalytical Chemistry 692 (2013) 9–16

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Journal of Electroanalytical Chemistry

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Determination of lamotrigine by using molecularly imprinted polymer–carbonpaste electrode

Mohammad Bagher Gholivand ⇑, Ghodratollah Malekzadeh, Maryam TorkashvandDepartment of Analytical Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran

a r t i c l e i n f o

Article history:Received 19 July 2012Received in revised form 18 December 2012Accepted 21 December 2012Available online 2 January 2013

Keywords:LamotrigineMolecularly imprinted polymerCarbon paste electrodeDPV

1572-6657/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jelechem.2012.12.016

⇑ Corresponding author. Tel.: +98 831 4274557; faxE-mail address: mbgholivand@yahoo.com (M.B. Gh

a b s t r a c t

By using a molecularly imprinted polymer (MIP) as a recognition element, construction of a high selectivevoltammetric sensor for lamotrigine (LTG) was performed. A LTG selective MIP and a non-imprinted poly-mer (NIP) were synthesized and then incorporated in the carbon paste (CP) electrodes. The sensor wasapplied for LTG determination using differential pulse voltammetric (DPV) method. The MIP–CP electrodeshowed very high recognition ability in comparison to NIP–CPE. Some parameters affecting the sensorresponse were optimized and then the calibration curve was plotted. Two dynamic linear ranges of0.8–25 and 25–400 nM were obtained. The detection limit of the sensor was calculated as 0.21 nM. Thissensor was used successfully for LTG determination in pharmaceutical preparations.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

Lamotrigine (LTG), 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine is a new-generation antiepileptic drug registered for treat-ment of patients with refractory partial seizures with or withoutsecondary generalization [1,2]. Lamotrigine is thought to act atvoltage-sensitive neuronal membranes and inhibit the release ofexcitatory amino acid neurotransmitters, in particular glutamateand aspartate, which play an important role in the generationand spread of epileptic seizures. In the case of overdoses, the mostfamous side effect of LTG is life-threatening skin rashes including aform called Stevens–Johnson syndrome, which is characterizedby painful blistering of the skin and mucous membranes and isoften fatal [3]. Owing to the dangerous side effect of LTG, thepharmaceutical quality control of LTG is vital. So, development ofa sensitive and versatile analytical method is needed for itsdetermination.

Lamotrigine and its metabolites in pharmaceutical products andbiological fluids typically have been monitored by high perfor-mance liquid chromatography (HPLC) [4–12], gas chromatographywith nitrogen phosphorus detector [13], capillary electrophoresis[14,15], chromatography-thermospray mass spectrometry [16],immuno fluorometric assay [17] and radioimmuno assay [18].

Due to high efficiency, accuracy, sensitivity, simplicity and lowcost, use of electrochemical techniques in pharmaceutical analysisattracted more attention. Despite the presence of redox groups in

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: +98 831 4274559.olivand).

this drug, only a few works can be found in the literature describ-ing the electrochemical analysis of LTG [19–23]. Molecularimprinting technology [24,25] gets increasingly interesting forthe preparation of useful materials with predetermined selectivityfor application in several areas of analytical chemistry [26]. MIPsare crosslinked synthetic polymers obtained by copolymerizing amonomer with a crosslinker in the presence of a template mole-cule (print molecule). The polymer, with its template being washedaway, contains recognition sites that are complementary in size,shape and chemical functionality to the template molecules. Theproduced imprinted polymer is able to rebind selectively withthe template (analyte) and its analogous structures. The highlyselective recognition characteristics of the molecular imprintedpolymers are comparable to those of the natural biological speciessuch as receptors and antibodies. However, MIPs possess severaladvantages over their biological counterparts, including low cost,ease of preparation, and good physical and chemical stability overa wide range of experimental conditions and solvents. Many pub-lications have dealt with the use of MIPs for specific purposes, e.g.,stationary phases for chromatography [27] and capillary electro-chromatography [28], electrochemical sensors [29], quartz crystalmicrobalance [30], biomimetic sensors [31], solid phase extraction[32], and membrane separation [33]. MIPs are promising materialscontinually being used in sensor fields as recognition elements ormodifying agents. The application of MIPs in electrochemistry israther recent and was directed to combine their intrinsic proper-ties to selected electrochemical reactions, in order to improve theresponse of the electrode. Literature survey showed that Mohajeriet al. have synthesized the LTG–MIP which has been applied fordrug assay in human serum [34].

10 M.B. Gholivand et al. / Journal of Electroanalytical Chemistry 692 (2013) 9–16

The carbon paste electrode (CPE) is one of the convenient con-ductive matrixes to prepare chemically modified electrodes(CMEs), by simply mixing the graphite/binder paste and the mod-ifier. CPEs are inexpensive and possess many advantages, such aslow background current, easy fabrication and rapid renewal [35].Newly, CPEs have been used as suitable conductive matrixes byour group for the construction of herbicide voltammetric sensors[36,37].

In the present work the LTG–MIP was synthesized as sensingmaterials to develop a modified carbon paste electrode for selectivemonitoring the low level of LTG. The procedure is based on thereduction of LTG after its selective extraction in the carbon pasteelectrode. The measurements with MIP modified sensors were car-ried out based on a three-step methodology, namely analyte extrac-tion in the electrode, electrode washing, and electrochemicalmeasurement. The developed sensor has been successfully appliedto determine LTG in pharmaceutical preparations.

2. Experimental

2.1. Instruments and reagents

Electrochemical data were obtained with a three-electrode sys-tem using a potentiostat/galvanostat model PGSTAT30, AUTOLAB.The differently prepared MIP or NIP involved sensors were usedas a working electrode. A platinum wire and an Ag/AgCl electrode(Saturated KCl) were used as the counter and reference electrodes,respectively. Metrohm pH-meter (model 827 pH Lab) was also ap-plied for pH measurements. A Hewlett–Packard 8453 diode-arrayspectrophotometer controlled by a computer and equipped witha 1 cm path length quartz cell was used for spectra acquisition.Absorbance at 266 nm was performed with UV–Visible ChemSta-tion program (Agilent Technologies), running under Windows XP.

Commercial tablets of Lamtrigine� were obtained from BakhtarBioshimi Pharmaceutical Co. (Kermanshah, Iran). Methacrylic acid(MAA), obtained from Sigma–Aldrich (Munich, Germany), waspurified by passing it through a short column of neutral alumina,followed by distillation under reduced pressure. Ethylene glycoldimethacrylate (EGDMA), obtained from Fluka (Buchs, Switzer-land), was distilled under reduced pressure in the presence of ahydroquinone inhibitor, and stored at 4 �C until used. 2, 2-Azobis-isobutyronitrile (AIBN) and Lamotrigine were supplied by Sigma–Aldrich (Munich, Germany), and used as received. Graphite powderwas purchased from Fluka (Buchs, Switzerland). All other chemi-cals were of analytical grade and were purchased from Merck(Darmstadt, Germany).

Scheme 1. The electrochemical reduction mechanism of LTG.

2.2. Molecularly imprinted polymers preparation

To prepare the MIP, a noncovalent molecular imprintingapproach was used. The procedure adopted for preparation of theLTG imprinted polymer was based on that conventional bulk poly-merization according to previous report [34]. LTG (0.4 mmol) asthe template, MAA (2 mmol) as the functional monomer, EGDMA(8 mmol) as the cross-linker, and AIBN (0.06 mmol) as the initiatorwere dissolved in 7 mL THF/ACN (4:3, v/v) in a thick walled glasstube. It was purged with N2 for 5 min and the glass tube was sealedunder this atmosphere and heated at 60 �C for 17 h. The polymerobtained was ground using a mortar and pestle. A steel sieve wasemployed to select particles with sizes <200 lm and the templatewas removed by soxhlet extraction with THF for 14 h. The com-plete removal of template from the polymer was traced by the dif-ferential pulse voltammetric method. Non imprinted polymer(NIP) was prepared similar to MIP except that the template wasnot present in the polymerization media.

2.3. Preparation of the sensors

The bare carbon paste was prepared by thoroughly mixing ana-lytical grade graphite and paraffin oil, in a 65:35 (w/w%) ratio. TheLTG modified carbon paste was also prepared by mixing differentpercentages of graphite powder, paraffin oil, and MIP (or NIP). Thismixture was mixed in a mortar for at least 10 min to becomehomogeneous. The paste was packed into an end of a Teflon holderin which electrical contact was made with a copper rod that runsthrough the center of the electrode body. The electrode surfacewas polished using a butter paper to produce reproducible workingsurface. Electrochemical behavior of LTG at these different elec-trodes was investigated using cyclic voltammetric technique. Bestresults were obtained at 12:58:30 (w/w%) MIP (or NIP), paraffin oiland ratio of graphite powder. This optimized electrode composi-tion was then used for the voltammetric determination of LTG.

2.4. General method for electrochemical measurements

The electrochemical measurement of LTG and its selected com-petitors were carried out according to the following sequentiallyprocedure:

Extraction step: each prepared electrode (CPE, NIP–CPE and orMIP–CPE) was inserted into the solutions containing the knownor unknown concentrations of LTG and or other interfering com-pounds, which their pH were fixed at 5.5 by acetate buffer. All solu-tions were stirred at fixed stirring rate (500 rpm) during extractiontime (7 min).

Washing step: The electrode was removed from the first solu-tion and then immersed into water/methanol (95:5 w/w%) as awashing solution, remaining in this solution for 60 s.

Analyzing step: after washing, the electrode was placed in theelectrochemical cell containing 10 mL acetate buffer (0.025 M,PH = 5.5). For recording the cyclic and DP voltammograms, thepotential was scanned from the �0.3 to �1.4 and �0.5 to �1.5 Vrespectively using the sweep rate of 100 mV s�1.

3. Result and discussion

3.1. Electrochemical behavior of lamotrigine (LTG)

The electrochemical behavior of LTG at the pyrolytic graphiteelectrode (PGE) using cyclic voltammetry technique has beeninvestigated by Saberi and Shahrokhian [22]. They have reportedthat, the electrochemical reduction of LTG at pyrolytic graphiteelectrode (PGE) is an irreversible reaction according to the mecha-nism presented in Scheme 1.

The same results have also been reported by other researchers[19,21]. To elucidate the electrode reaction of LTG at the surfaceof the carbon paste electrode, the cyclic voltammetric techniquewas used. The obtained cyclic voltammogram at the surface ofCPE exhibited a well-defined cathodic peak at about �1.1 V, withno peak on the reversed scan (Fig. S1) which indicated an irrevers-ible reduction process, which confirmed the previous report [19].

M.B. Gholivand et al. / Journal of Electroanalytical Chemistry 692 (2013) 9–16 11

The effect of potential scan rate, t, on the peak current of LTG atthe CPE was evaluated (Fig. S1A). A linear relationship was ob-served between log I and log t over the scan range 10–500 mV s�1

(Fig. S1B). The slope of the log I versus log t plot gave a value of0.658, suggesting a mixture of diffusion-and surface-controlledprocesses. (Slopes of 0.50 and 1.00 are expected for ideal reactionsof solutions and surface processes, respectively) [35]. On the otherhand, as scan rate increased, the potential shifted to more negativevalues as expected for an irreversible reduction process [38]. Fur-thermore, according to the reduction mechanism of LTG, the peakpotential of the drug would be strongly pH dependent. The effect ofpH on the electrochemical responses of LTG was investigated overthe pH range 2.0–9.0 and the results are summarized in the Fig. 1.As it is seen from Fig. 1A the maximum peak current was appearedat pH = 5.5. The same pH was reported for LTG determination byother workers [19–21]. Furthermore, as can be found in Fig. 1Bthe reduction peak potential (Epc) was almost pH independent atpH lower than 5 which is close to its pKa (5.7) of protonated formof LTG [39,40], and then Epc shifted negatively with pH rising from5 to 9 based on the following equation:

EpcðVÞ ¼ �0:061pH� 0:821ðR2 ¼ 0:997Þ ð1Þ

The slope of this plot indicated participation of the equal num-bers of electrons and protons in the electro-reduction of LTG.

A

B

Fig. 1. Effect of pH on the cyclic voltammograms of 1.0 � 10�4 M LTG at CPE at scanrate 100 mV s�1, (A) cyclic voltammograms at different pH; (B) plot of Ep vs. pH.

To study the LTG recognition ability of MIP, the MIP–CP, NIP–CPand CP electrodes were prepared and inserted into the LTG con-taining solutions. After 7 min the electrodes were removed fromthe LTG solution and their CVs were recorded in the acetate buffer(pH = 5.5) solution. As it is seen from Fig. 2 the CV signal of MIP–CPelectrode is higher than those for NIP–CP and CP electrodes. Thisindicates that the MIP in the carbon paste electrode can uptakeLTG intensively from the acetate buffer (pH = 5.5) in comparisonto NIP–CP and CP electrodes.

3.2. Estimation of the active surface area of electrodes

The area of the CPE, NIP–CPE and MIP–CPE were calculatedusing 1.0 � 10�3 M K3Fe(CN)6 as a probe at different scan rates[41] by cyclic voltammetric technique. For a reversible process,the following Randles–Sevcik formula can be used:

Ipa ¼ 2:69� 105n3=2 � A � D1=2R � C � m1=2 ð2Þ

where n is the number of electrons participating in the redox reac-tion, A is the area of the electrode (cm2), DR is the diffusion coeffi-cient of the molecule in solution (cm2 s�1), C is the concentrationof the probe molecule in the bulk solution (mol cm�3), and t isthe scan rate of the potential perturbation (V s�1). Fig. 3 representssteady-state CVs (second cycle recorded) and their correspondingplot of Ip vs. ˆ1/2 for the MIP–CPE. For 1.0 � 10�3 K3Fe(CN)6 in0.1 M KCl electrolyte, n = 1, DR = 7.6 � 10�6 cm2 s�1, then fromthe slope of the plot of Ipa vs. ˆ1/2, the surface area of the electrodecan be calculated. The average value of the electroactive surfacearea were (4.5 ± 0.1) � 10�2, (6.3 ± 0.3) � 10�2 and (8.7 ± 0.2) �10�2 cm2 for CPE, NIP–CPE and MIP–CPE respectively. With respectto Fig. 2, these results indicate that MIP–CPE not only can uptakeLTG intensively from the buffer solution in comparison to NIP–CPE and CPE, but also have higher electroactive surface area thanNIP–CPE and CPE.

3.3. Evaluation of selectivity

The selectivity of designed MIP–CP sensor was evaluated bypreparing the MIP–CP, and CP electrodes and then inserting theminto the aqueous solutions of LTG and or some LTG similar com-pounds such as 2,4,6-triamino- pyrimidine, 2,6-diamine pyridine,

Fig. 2. Cyclic voltammograms of different electrodes immersed in the 1.0 � 10�6 MLTG solutions after 7 min preconcentration. Determination conditions: acetatebuffer pH = 5.5 and scan rate 100 mV s�1.

A

B

Fig. 3. (A) Cyclic voltammograms for the MIP–CP electrode in 1.0 � 10�3 MK3Fe(CN)6 over the scan range 10–500 mV s�1 in 0.1 M KCl electrolyte. (B)Corresponding plot of Ip vs. ˆ1/2.

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2,4,6-Tris(chloroamino)-1,3,5-triazine, 3-amino-5,6-dimethyl-1,2,4-triazine and 3-amino- 1,2,4-triazine. The chemical structures ofthese compounds are shown in Scheme 2. As can be seen all repre-sented compounds contain azo group similar to LTG which isresponsible for electroactivity of these compounds. Fig. 4A showsthe recorded DP voltammetric responses of the CP electrode in-serted in the solutions of LTG and other tested compounds. Ascan be seen, the current magnitude of most compounds are as highas that of LTG and all of them have electrochemical signal in theLTG reduction potential range. This indicates that these com-pounds can easily interfere in the LTG determination. Therefore,

(a) (b

(d) (eScheme 2. Structure of compounds which were examined for MIP–CP electrode selectTris(chloroamino)-1,3,5-triazine; (e) 3-amino-5,6-dimethyl-1,2,4-triazine; (f) 3-amino-1

the carbon paste electrode was modified with synthesized MIPand NIP and the same experiments were carried out. The obtainedresults are presented in Fig. 4B and C respectively. It is evident that,the presence of MIP in the carbon paste electrode makes the elec-trode very selective for LTG whereas the responses of all othertested compounds except LTG reach to small amount. The responseon the NIP–CP and CP electrodes are compared with that of MIP–CPelectrode in Table 1. These result approved the powerful ability ofMIP–CP electrode for LTG determination purpose.

3.4. Optimization of parameters for LTG detection

Optimization of analytical parameters for the designed sensorwas divided into three sections including: optimization of carbonpaste composition, LTG extraction and electrochemical determina-tion conditions.

3.4.1. MIP–CP composition optimizationTo find the best composition for MIP–CP electrodes, the amount

of different ingredients of the electrode including MIP, carbon andparaffin oil were changed in the fixed extraction and voltammetricdetection conditions. For initial optimization purposes, the MIP–CPelectrodes were prepared with fixed amounts of carbon and paraf-fin oil and different amounts of MIP. The resulted electrode at eachcase was used for LTG extraction and determination. The maxi-mum response for the prepared sensor appeared (Fig. S2) in theMIP amount of 0.01 g in presence of 0.05 g carbon and 0.026 g ofparaffin oil (12:58:30 (w/w%) of MIP, graphite and paraffin oil).Higher amounts ratio of graphite powder of MIP in the MIP–CPelectrode can increase the sensor response due to providing morerecognition sites on the electrode surface. However, enhancementthe MIP amount more than a threshold level, leads to a decrease inthe sensor response, probably because of decreasing electroactivesurface area. Similar experiments were also carried out in orderto investigate the effect of carbon and paraffin oil amounts onthe prepared electrode response. From the obtained results, theoptimum amounts of carbon and paraffin oil were found to be0.05 g and 0.026 g, respectively. Increasing the carbon content ofMIP–CP electrode leads to an increase in the corresponding elec-trode response because of electron transferring capabilityenhancement of the electrode in the presence of higher carboncontent. However, after a certain point, further increase in carboncontent results in lowering the corresponding signal that may beattributed to this fact that, larger carbon amount on the electrode

) (c)

) (f)ivity. (a) LTG; (b) 2,4,6-triamino- pyrimidine; (c) 2,6-diamine pyridine; (d) 2,4,6-,2,4-triazine.

A B C

Fig. 4. The DPV response of sensors based on and CPE (A), MIP–CPE (B) and NIP–CPE (C) for different compound immersed into the solutions containing 1.0 � 10�7 M of (a)LTG; (b) 2,4,6-triamino- pyrimidine; (c) 2,6-diamine pyridine; (d) 2,4,6-Tris(chloroamino)-1,3,5-triazine; (e) 3-amino-5,6-dimethyl-1,2,4-triazine; (f) 3-amino-1,2,4-triazine.Extraction conditions: 0.025 M acetate buffer (pH 5.5), extraction time = 7 min and stirring rate = 500 rpm. Measurement conditions: pH = 5.5, pulse amplitude = 100 mV,pulse width = 20 ms and scan rate = 100 mV s�1.

Table 1Comparison of the DPV signals of the LTG similar compounds at CPE, NIP–CPE and MIP–CPE relative to its LTG response.

Compound MIP–CP electrode NIP–CP electrode CP electrode

I (lA) % of LTG response I (lA) % of LTG response I (lA) % of LTG response

a �9.25 100.0 �3.22 34.8 �0.47 5.0b �2.97 32.1 �1.13 12.3 �0.39 4.2c �1.98 21.4 �0.70 7.6 �0.11 1.1d �1.48 16.0 �0.55 6.0 �0.18 1.9e �3.74 40.5 �1.45 15.7 �0.64 6.9f �2.97 32.1 �1.15 12.4 �0.42 4.6

(a) LTG; (b) 2,4,6-triamino- pyrimidine; (c) 2,6-diamine pyridine; (d) 2,4,6-Tris(chloroamino)-1,3,5-triazine; (e) 3-amino-5,6-dimethyl-1,2,4-triazine;(f) 3-amino-1,2,4-triazine.

M.B. Gholivand et al. / Journal of Electroanalytical Chemistry 692 (2013) 9–16 13

surface leads to decrease MIP content of the electrode surface. Alsothe amounts of binder (paraffin oil) higher than 0.026 g in the MIP–CP electrode leads to a decrease in electrode response that may bedue to the insulating effect of the binder and its results is decreas-ing the electroactive surface area.

3.4.2. LTG extraction conditions optimizationThe extraction time, stirring rate in the extraction period, the

pH of the test solution and electrode washing after extraction ascommonly considering parameters were noticed and their effecton the LTG extraction were studied. In order to investigate theeffect of pH, the prepared electrode was inserted into the LTG solu-tion with various pHs (2–9), and then was incubated for 7 min at aconstant stirring rate. After removal electrode from the solutionand washing with 5% methanol–water mixture, it was immersedinto the solution of the electrochemical cell. The amount of LTGextracted in the electrode was increased up to pH = 5.5 and thenleveled off at higher pH values (Fig. S3). Thus, the pH of 5.5 whichfixed by acetate buffer, was selected for the extraction step in thedeveloped method.

The extraction time is the other main parameter that must beinvestigated. In order to optimize the extraction time, LTG wasextracted in the prepared MIP–CP electrodes at various extractiontime, whereas the other extraction parameters were kept constant.The electrode was removed from the solution and after washing,followed by electrochemical determination. Increasing of theextraction time leads to intensive increase in the LTG extractionamount in the electrode till about 7 min (Fig. S4) and afterwardsthe increasing extraction time has no considerable effect on thevoltammetric response (the response at 7 min is about 96% of12 min extraction time). 7 min was selected as optimum extraction

time which it is accompanied with high sensitivity and short ana-lyzing time.

Since the electrode contacting area with the LTG containingsolution was partially small therefore the analyte extraction inthe electrode was carried out with stirring. Thus, the effect of thestirring rate on the LTG extraction in the MIP–CP electrode at fixedextraction period and optimum pH value was investigated. Thegrowth in LTG voltammetric response with the stirring rateincreasing was seen up to 500 rpm. However, further enhancementin stirring rate does not affect considerably on the LTG extraction.Therefore, the stirring rate of 500 rpm was chosen for subsequentuses.

The constructed MIP particles, during the noncovalentapproach, usually contain selective sites with various affinitiesfor template. Some of them are cavities with matchable sizes tothe template molecule. These are template recognition sites, con-structed with regular and perfect shape in the polymerization per-iod and thus have more affinity for LTG. The washing of electrodemodified with MIP, does not noticeably disrupt the correspondinginteractions, [25]. The cavities with incomplete or irregular shape,as well as the nonselective binding sites cannot absorb LTG mole-cules so tightly. The portion of template molecules absorbed bysuch mentioned binding sites can be removed from MIP–CP elec-trode during the washing process. Therefore, to improve the sensorselectivity and omitting the interference effects, washing solutionwith different nature including water and water containing smallamount of organic solvents such as ethanol, methanol, acetoneand acetonitrile on the response MIP–CPE, NIP–CPE and bare CPEwere investigated. The maximum difference between the responseof MIP–CP and NIP–CP electrodes obtained when 5% methanol–water mixture was used as washing solution. Thus, this mixture

14 M.B. Gholivand et al. / Journal of Electroanalytical Chemistry 692 (2013) 9–16

solvent was selected for further uses. Fig. 5 shows the response ofLTG extracted by MIP–CP, NIP–CP and CP electrodes under opti-mum extraction conditions before and after washing with 5%methanol–water mixture as selected washing solution. In the caseof NIP–CPE, and CPE washing the electrode removed the adsorbedLTG from the electrode easily because the binding site in the NIPand CPE are nonselective and acts almost similar to nonselectivesites of MIP. The importance of the application of the washing stepin our proposed method is explicit because of the presence of non-selective and poorly selective sites in the MIP and the non-selectiveabsorption property of carbon particles in the MIP–CPE.

The effect of washing time on the response of electrode was alsostudied. It was found that the difference between response of MIP–CPE and NIP–CPE increase till 60 s and afterwards the aimed signalreached to partly steady state. Thus 60 s was chosen as optimalwashing time.

3.4.3. Electrochemical conditions optimizationIn order to achieve a highly sensitive sensor, the selection

of a proper electrochemical technique is of great importance.

A

B

Fig. 5. The DPV responses of different electrodes immersed in solution containing1 � 10�7 M of LTG before (A) and after (B) washing. (a) MIP–CPE, (b) NIP–CPE and(c) CPE, washing time = 60 s, other conditions are same as Fig. 4.

Therefore, differential pulse stripping voltammetry (DPSV) as asensitive method was selected and used for further investigation.

As mentioned before the reduction of LTG is pH dependent,thus, the effect of solution pH on the electrochemical signal of1.0 � 10�7 M LTG was investigated in pH ranging from 2 to 9.The results (Fig. S5) showed that the signal was increased byincreasing the pH up to 5.5 and after that, the response of the elec-trode was decreased. The same pH was reported for voltammetricdetermination of LTG [19–21].

Therefore, different buffer solutions such as acetate, maleate,phosphate and Britton–Robinson with pH = 5.5 were tested as sup-porting electrolyte. The best result was obtained when acetate buf-fer was used. Therefore, acetate buffer with pH = 5.5 was selectedand used as the supporting electrolyte in all voltammetricdeterminations.

In the case of differential pulse measurement the main impor-tant parameters which could be optimized were: pulse amplitude,pulse width and scan rate. During the study, each variable waschanged while the other two were kept constant. The variablesof interest were studied over the ranges 25–100 mV, 30–100 msand 10–100 mV s�1 for pulse amplitude, pulse width and scan raterespectively. The best sensitivity and well-shaped wave with rela-tively narrow peak width were obtained when the values of100 mV, 20 ms and 100 mV s�1 were chosen for pulse amplitude,pulse width and scan rate, respectively.

3.5. Analytical application

In order to study the LTG recognition ability of the MIP–CP elec-trode, it was inserted into the different LTG concentration contain-ing solutions. After 7 min the electrodes were removed from theLTG solution and after washing, their differential pulse voltammo-grams were recorded. The DPV signals obtained for tested differentconcentrations of LTG besides the plotted calibration curves areshown in Fig. 6. It is worth noticing that the values of currentresponse used for the calibration curve are actually the absolutevalues of the reductive peak current observed after electrodeincubating in different concentration of LTG solution. Two linearrelationships are obtained over LTG concentration in the range of0.8 to 25 and 25 to 400 nM respectively. The linear regressionequations are:

IpðlAÞ ¼ 0:1234CLTG ðnMÞ þ 0:2282 ðr2 ¼ 0:995Þ ð3Þ

IpðlAÞ ¼ 0:0624CLTG ðnMÞ þ 2:6882 ðr2 ¼ 0:993Þ ð4Þ

The limits of detection (LOD) and quantitation (LOQ) were cal-culated using the relation kSb/m [42], where k = 3 for LOD and 10for LOQ, Sb representing the standard deviation of the peaks cur-rent of the blank (n = 12) and m representing the slope of the firstcalibration curve for LTG. Both LOD and LOQ values are found to be0.21 and 0.69 nM respectively, which these values indicate the sen-sitivity of the proposed method. Three freshly packed electrodeswere prepared on three consecutive days and the peak current val-ues of a solution containing 1.0 � 10�8 M of LTG was measured foreach electrode. The obtained results show a relative standard devi-ation (RSD) of DP voltammogarm’s currents for five replicate lessthan 4.2%. This result indicates the acceptable reproducibility forthe proposed electrode.

3.6. Determination of lamotrigine in real samples

To evaluate the accuracy of the proposed method, the concen-tration of LTG in commercial tablets of lamotrigine (25 mg fromBakhtar Bioshimi Co.) was determined using the DPV method.Ten tablets of LTG were accurately weighed in order to find the

A B

C

Fig. 6. DPV of different concentration of LTG (A) and its calibration curves (B and C), conditions are same as Fig. 4.

Table 2Comparison of characteristic of the proposed sensor with other reported sensors.

Method Electrode Linear range (M) LOD (M) References

DPASVa HMDEb 4.0 � 10�9–1.2 � 10�7 4.68 � 10�9 [19]SWASVc HMDE 3.0 � 10�9–1.9 � 10�8 5.02 � 10�9

DPASV AgNP-CSPEd 3.3 � 10�7–1.5 � 10�6 3.72 � 10�7 [20]DPASV CSPEe 5.0 � 10�6–2.1 � 10�5 5.00 � 10�6 [21]DPASV Hg-CSPEf 2.0 � 10�6–5.0 � 10�6 2.00 � 10�6

CASVg PGEh 1.0 � 10�7–1.0 � 10�5 8.00 � 10�8 [22]1.0 � 10�5–1.0 � 10�4

Potentiometry MIP sensor 1 � 10�6–1 � 10�3 8.00 � 10�7 [23]DPV MIP–CP 8.0 � 10�10–2.5 � 10�8 2.10 � 10�10 This work

2.5 � 10�8–4.0 � 10�7

a DPASV: differential pulse adsorptive stripping voltammetry.b HMDE: hanging mercury drop electrode.c SWAdSV: square wave adsorptive stripping voltammetry.d AgNP-SCPE: silver nanoparticles modified carbon screen printed electrode.e CSPE: screen printed electrodes.f Hg-CSPE Hg modified carbon screen printed electrode.g CASV: cyclic adsorptive striping voltammetry.h PGE: pyrolytic graphite electrode.

M.B. Gholivand et al. / Journal of Electroanalytical Chemistry 692 (2013) 9–16 15

average weight of each tablet. Then, the tablets were powderedand mixed carefully. Then a portion of tablet powder containing25 mg of LTG was transferred to a backer and dissolved in HCl0.1 M. The resulting solution was centrifuged at 5000 rpm forand then, the supernatant was collected and diluted to 100 mland used as a stock solution of the sample. Before determination,the sample solution was diluted appropriately to ensure the con-centration was within the linear range. Finally, the determinationof the LTG content was made in an aliquot of the stock solutionby means of the DP voltammetric technique using the standardaddition method. The amount of LTG was found to be 25.0 ±1.2 mg with n = 3 and a = 0.05 (RSD = 1.93%). This result was alsocompared with that obtained by UV detection at k = 266 nm(25.0 ± 1.05 mg, n = 3 and a = 0.05). As it is seen, there is a goodagreement between these two methods.

Repeatability was examined by performing seven replicatemeasurements for 1.0 � 10�8 M LTG. The recovery of analyzeddrug was calculated to be above 97%, and the relative standard

deviation (RSD) was lower than 3%. This level of precision is suit-able for the routine quality control analysis of the drug in pharma-ceutical dosage form.

4. Conclusion

In summary, in this paper, the application of a MIP-modifiedcarbon paste electrode for determination of LTG is demonstrated.A three-step procedure including extraction, washing andelectrochemical measurement was developed for selective LTGdetermination using the MIP-based LTG sensor. The MIP func-tioned as both preconcentrator and high selective recognition ele-ment in the carbon paste structure. The extraction method is costeffective and does not consume large amounts of organic and toxicsolvents. The proposed sensor was used successfully for LTG deter-mination in the pharmaceutical preparations. In comparison withpreviously reported LTG sensors it provided a better detection limit(better sensitivity) and a wider linear range (Table 2).

16 M.B. Gholivand et al. / Journal of Electroanalytical Chemistry 692 (2013) 9–16

Appendix A. Supplementary material

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jelechem.2012.12.016.

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