Accepted Manuscript

Title: Computational study and multivariate optimization ofhydrochlorothiazide analysis using molecularly imprintedpolymer electrochemical sensor based on carbonnanotube/polypyrrole film

Author: <ce:author id="aut0005" biographyid="vt0005">Azizollah Nezhadali<ce:author id="aut0010"biographyid="vt0010"> Maliheh Mojarrab

PII: S0925-4005(13)01022-8DOI: http://dx.doi.org/doi:10.1016/j.snb.2013.08.086Reference: SNB 15896

To appear in: Sensors and Actuators B

Received date: 19-6-2013Revised date: 13-8-2013Accepted date: 26-8-2013

Please cite this article as: A. Nezhadali, M. Mojarrab, Computational study andmultivariate optimization of hydrochlorothiazide analysis using molecularly imprintedpolymer electrochemical sensor based on carbon nanotube/polypyrrole film, Sensorsand Actuators B: Chemical (2013), http://dx.doi.org/10.1016/j.snb.2013.08.086

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Computational study and multivariate optimization of hydrochlorothiazide

analysis using molecularly imprinted polymer electrochemical sensor based on

carbon nanotube/polypyrrole film

Azizollah Nezhadali a, b,*, Maliheh Mojarrab a

a Department of Chemistry, Payame Noor University (PNU), Mashhad, Iranb Department of Chemistry, Payame Noor University, PO.B.19395-4697, Tehran 19569, Iran

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Abstract

An electrochemical sensor was developed by modifying multi-walled carbon nanotubes-molecularly imprinted polymer onto a pencil graphite electrode (PGE). A computational approach was used to screening functional monomers and polymerization solvent for rational design of molecularly imprinted polymer (MIP). Based on computational results, pyrrole and ethanol were selected as functional monomer and polymerization solvent, respectively. The MIP film was fabricated by electropolymerization of pyrrole in the presence of hydrochlorothiazide (HCT) as template molecule after electrodepositing of carboxyl functionalized multi-walled carbon nanotubes onto the surface of pencil graphite electrode. A Plackett–Burmanexperimental design was used to evaluate the influence of several variables on theanalytical response (current). Then, the significant parameters were optimized using acentral composite design, simultaneously. Under the optimized conditions, thedetection limit (3sb, n=7) was found to be 1.0×10−10 M. The calibration curve showed two dynamic linear ranges including 9×10−10-1×10−5 M and 1×10−5-1×10−2M with correlation coefficients (r2) of 0.9986 and 0.9921, respectively. The prepared sensorshowed a suitable reproducibility (RSD % of 3.36, n=3) and regeneration capacity.The sensor showed good results for determination of HCT in serum and pharmaceutical samples.

Keywords:Molecularly imprinted polymer, Experimental design, Hydrochlorothiazide, Multi-walled carbon nanotubes, Density functional theory.

______________________________________________________________________________*Corresponding author at: Department of Chemistry, Payame Noor University(PNU), Mashhad, Iran. Tel.: +98 5118683968; Fax: +98 5118683001.E-mail address: aziz_nezhadali@yahoo.com; aziz_ nezhadali@pnu.ac.ir ( A. Nezhadali)

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1. Introduction

HCT, or 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide

(Scheme I), is a diuretic of the class of benzothiadiazines that widely used in

antihypertensive pharmaceutical formulations, alone or in combination with other

drugs, which decreases active sodium reabsorption and reduces peripheral vascular

resistance [1]. HCT is official in the European pharmacopoeia 4 [2]; the drug and its

tablets are official in the United States Pharmacopeia (USP) 26 which describes high-

performance liquid chromatographic (HPLC) procedures for their quantitations [3].

Due to the frequency in which HCT is prescribed and its abuse for reduction of body

weight by some athletes (who are categorized in several body weight classes), its

analysis is of great importance. Several analytical procedures have been described for

the individual determination of HCT and jointly with other pharmaceutical substances,

including spectrophotometric [4, 5], HPLC [6, 7], chemometric methods [8, 9],

conventional and differential pulse polarography [10], capillary electrophoresis [11],

chemiluminescence [12], differential pulse anodic voltammetry [13] and adsorptive

stripping voltammetry [14] procedures.

Molecularly imprinted polymers (MIPs) are synthesized using template (target)

molecules, which are cross-linked into monomers. The target-monomer complex is

then polymerized and the template molecules are removed to leave the polymer matrix

with ‘‘holes’’ specific to the target molecules [15-17]. The holes capture the target

molecules from a sample even if they are present in small amounts. MIP materials

have high recognition affinity to the target molecules. The holes are so specific that

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they allow only the target molecules to enter them and reject all others. MIPs are

robust, cost effective, and easy to design [18]. MIPs have been developed in many

fields, such as liquid chromatography [19], capillary electrochromatography [20], drug

delivery [21], solid-phase extraction [22], antibody and receptor mimics [23], artificial

enzymes [24], determination of drugs [25], cancer biomarkers and viruses [26], and

sensing devices [27]. Electrochemical [27], piezoelectric [28], and optical [29]

methods could achieve the detection of adsorbed molecules in the imprinted polymers.

Among these methods, electrochemical method is probably the easiest and low cost

method for fabricating a commercial sensor.

Multi-walled carbon nanotubes (MWCNTs), owing to their unique structures, high

stabilities, low resistivities, and high surface-to-volume ratios, are extremely attractive

in the field of electrochemical sensors because they increase the surface areas of the

electrodes, enhance conductivity and facilitate the electron transfers [30].

The most important task in synthesizing MIP is finding the best monomer, which can

evolve the highest interactions with the template at ground state in order to achieve

high selectivity and rebinding capacity. To facilitate the selection of imprinting

conditions such as template, functional monomer, and the best suitable solvent,

various processes such as computer simulation and molecular mechanics [31, 32] have

come enforce towards the development of MIP motifs. Over the past few years, a

number of studies have been reported describing the application of ab initio and

density functional theory (DFT) computational methods to the rational design of

molecularly imprinted polymers [33]. DFT, which has become very popular in recent

years, enables novel molecules of theoretical interest to be accurately studied at a

lower computational cost as compared to ab initio methods [34].

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Multivariate techniques are cost-effective, powerful and efficient statistical

approaches, widely applied for optimization of analytical procedures. Compared with

the traditional univariate methods, they provide several advantages such as the

possibility of evaluating the effects of parameters and their mutual interactions by a

reduced number of experiments [35, 36]. Among the most relevant multivariate

techniques used in analytical optimization is response surface methodology (RSM).

Firstly, a large number of continuous factors are screened and insignificant ones are

eliminated in order to obtain a smaller, more manageable set of factors. The remaining

factors could be optimized by a response surface modeling methodology. Finally, after

model building and optimization, the predicted optimum is verified experimentally [37].

In this study, a DFT-based computational approach was used to the selection of

functional monomer and suitable solvent to design of MIPs for HCT as template

molecule. Then a novel kind of MIP-based electrochemical sensor was fabricated

successively by electrodepositing of carboxyl functionalized MWCNTs (MWCNTs-

COOH) and electropolymerizing of pyrrole in the presence of HCT onto a PGE

surface. The prepared sensor was characterized by scanning electron microscopy

(SEM).The influences of several parameters on the efficiency of sensor response were

investigated using multivariate methods. Plackett–Burman design (PBD) for screening

and central composite design (CCD) for optimization were carried out and optimum

conditions were determined. Under the optimized conditions, the prepared sensor

showed very high recognition ability toward HCT.

Insert Scheme I

2. Experimental

2.1. Reagents

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Multi-walled carbon nanotubes (MWCNTs) with diameters of 10-40 nm and lengths

of 1-25 µm were purchased from Iran's Research Institute of Petroleom Industry.

Sodium perchlorate (99-102%), boric acid (99.9999 Suprapur), acetic acid (99.5%),

pyrrole (≥ 97%), and ethanol (99.9%), were purchased from Merck (Darmstadt,

Germany). Benzimidazole (98%), methimazole (≥ 99%), acetaminophen (≥ 99%) and

aspirin (≥ 99%) were obtained from Aldrich (St. Louis, MI, United States). HCT (Iran

Darou Company, Iran), 2-aminobenzimidazole (97%, Fluka, Steinheim, Germany),

diphenoxylate (Temad Company, Iran), ascorbic acid (> 99.5%, Fluka, Steinheim,

Germany), sodium hydroxide (98%, Lobachemie, Mumbai, India), and other reagents

were commercially available as analytical grade and used without further purification.

Stock solutions of HCT and buffer solutions were prepared in ethanol and distilled

water, respectively.

2.2. Apparatus

The electrochemical studies were performed using Autolab PGSTAT 12 potentiostat-

galvanostat controlled by GPES 4.9 software (Ecochemie, The Netherlands). A three-

electrode system was used for all measurements; a modified pencil graphite electrode

(Owner, HB, 0.7 mm diameter, Korea) as the working electrode a platinum auxiliary

electrode. All measurements were carried out with a silver/silver chloride reference

electrode. A Metrohm (Model 827 pH-lab, Switzerland) was employed for pH

measurements. Surface evaluations were performed by scanning electron microscopy

(SEM) in an Oxford S360 SEM (Britain) microscope. The sonication process was

performed using Hielscher ultrasonic processor (UP400S, Germany).

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2.3. Hardware and softwares

All computations were carried out on a computer with 8 GB memory and an Intel

Pentium 7 3.07 GHz CPU. Quantum calculations were carried out using Gaussian 03

[38] software. GaussView 3.07 (Gaussian, Inc.) produced the optimized 3-dimensional

molecular structures. The softwares of Minitab 16 and Matlab 07 were used for

experimental designs, statistical evaluation and model fitting in this work.

2.4. Computational approach

In order to understand the properties of MIP at molecular level, the model of the

template-monomer model complexes were set up. Then all calculations were carried

out using the Gaussian 03 [38] program. The electronic energies were calculated

through DFT using the popular hybrid functional B3LYP. The geometry optimization

was performed at the B3LYP/6-311G (d) level. Finally, the solvent effect in different

solvents was studied using the polarizable continuum model (PCM), developed by

Tomasi and coworkers [39-41], in order to select the most appropriate one.

2.5. Fabrications of the MWCNT- and MIP-modified PGEs

MWCNTs (0.5 g) were oxidized with 60 mL of concentrated nitric acid solution at

100 ◦C for 12 h [42]. After cooling to room temperature, the mixture was filtered and

washed thoroughly with distilled water for several times until the pH of final wash

(filtrate) came down to neutral. The filtered solid was dried under vacuum to obtain

carboxylic acid functionalized MWCNTs (MWCNTs-COOH). After sonication, a

chronoamperometry method was employed to deposit the MWCNTs-COOH onto the

bare PGE surface using +1.7 V for 400 s in a solution containing 0.37 gL-1 of

MWCNTs-COOH; this process gave MWCNTs-COOH-modified PGE

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(MWCNTs/PGE) [43]. The resulting MWCNTs/PGE was immersed into a solution

containing supporting electrolyte (NaClO4, 0.1 M), functional monomer (pyrrole, 0.02

M) and template (HCT, 0.01 M). The cyclic voltammetry (CV) technique was

performed from +0.60 V to +2.00 V for 10 cycles at a scan rate of 100 mVs-1 to obtain

the polymer-modified MWCNTs/PGE. Subsequently, the embedded HCT were

extracted by scanning between +0.75 and +1.75 V at a scan rate of 16 mVs-1 in

Britton–Robinson buffer solution (pH 2.7) for several cycles until no obvious

oxidation peak for HCT could be observed; this process gave MIP-modified

MWCNTs/PGE (MIPs/MWCNTs/PGE). A stock Britton–Robinson buffer solution

containing a mixture of boric, acetic and phosphoric acids (each 0.04 M) were

prepared, and its pH value was adjusted to 2.7 by addition of NaOH solution (0.2 M).

As a control, a non-molecularly imprinted polymer (NIP) modified MWCNTs/PGE

(NIPs/MWCNTs/PGE) was prepared and treated in exactly the same manner, except

for the omission of HCT in the electropolymerization process.

2.6. Electroanalytical measurements

Differential pulse voltammetric measurements were carried out in a three-electrode

cell, in Britton–Robinson buffer at pH 2.7. The current measurements were performed

using differential pulse voltammetry (DPV) in a potential range between +0.75 and

+1.75 V. To record differential pulse voltammograms, the following conditions were

used: step potential 8 mV, modulation amplitude 50 mV and scan rate 16 mVs-1. All

electroanalytical measurements were made at room temperature.

2.7. Statistical treatment of data

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The experimental design and statistical treatment of results were performed using

Minitab 16 software (Minitab Inc., USA).

2.8. The interferences

The selectivity of MIP electrode was evaluated in the presence of ascorbic acid,

methimazole, 2-aminobenzimidazole, benzimidazole, acetaminophen, aspirin and

diphenoxylate as the interfering molecules.

2.9. Pharmaceutical and serum sample analysis

The pharmaceutical samples were chosen from three HCT brand tablets. Four HCT

tablets of each brand were precisely weighed in order to get the average weight of

each tablet. Then, the tablets were lightly powdered. An equivalent quantity of the

powder including a known amount of active material was weighed and placed into a

glass vial containing ethanol to sonication for 15 min. Finally, it was filtered to make

a sample solution of HCT.

A 0.5 mL human serum sample was spiked with analyte to give a working

concentration of HCT (2, 4 and 6 mM). This sample was placed into a glass vial

including 4.0 mL of ethanol, vortexes for 10 s and centrifuged at 2500 rpm for 25

min. A 1.0 mL aliquot of ethanol layer was placed into another glass vial and 3.0 mL

methanol was added and centrifuged again at 2500 rpm for 10 min. A 0.50 mL of the

supernatant was diluted to 10 mL with ethanol solution [44]. After 5 min adsorption,

the MIP/MWCNTs film was washed by distilled water to wash out unwanted

materials, which were chemically close to the analyte or the analyte molecules

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adsorbed to nonspecific binding point of MIP. Eventually, voltametric detection was

applied to record analyte current at 1.35 V.

3. Results and discussion

3.1. Theoretical selection of functional monomers

The selection of suitable functional monomers is a crucial factor in the study of MIP.

In this work, four functional monomers, pyrrole (PY), thiophene (TH), furan (FU) and

3-methyle pyrrole (MPY) were theoretically selected as possible functional

monomers. The conformation optimization was performed using DFT computations at

B3LYP level with 6-311G (d) basis set. The binding energies of template-monomer

complexes, ∆E, were calculated through the template and monomer energies as

follows:

∆E = E (template–monomer) − E (template) − ∑E (monomer) (1)

The optimized conformations of HCT and the four functional monomers are shown in

Fig. 1. The suggested structures of the most stable complexes between HCT–PY,

HCT–TH, HCT–FU and HCT–MPY are shown in Fig. 2. The results of calculated

binding energies are listed in Table 1. Based on theory, the monomer with the highest

binding energy in the monomer-template interaction would be more suitable for

monomer to prepare MIP [33]. Table 1 shows the ∆E order is PY-HCT ~ MPY-HCT

> FU-HCT > TH-HCT. This is indicating that HCT interacts most strongly and most

weakly with PY and TH, respectively. In addition, Table 1 shows the MIP synthesized

with PY is expected to give good adsorption and a high recognition capacity for HCT.

Insert Fig. 1

Insert Fig. 2

Insert Table 1

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3.2. Theoretical selection of the solvent

The more realistic modeling of the imprinting molecular systems should not include

the effects of solvent, but most of the reaction occurred in the solvent, which lead to

have different results. Solvation model based on polarizable continuum dielectrics

have proved flexible and accurate, in particular, when the solute is accommodated in a

cavity of realistic molecular shape. In this section, PCM model was carried out for

monomer and polymer molecular systems containing solvent in order to predict the

interaction energies between HCT and functional monomers dissolved in a solvent.

Five solvents; water (H2O), acetonitrile (AN), dimethyl sulfoxide (DMSO), methanol

(MeOH) and ethanol (EtOH); were considered for the study. PY was used as

monomer for the study. The calculated interaction energies (∆E, kJ mol-1) for HCT-

PY in H2O, AN, DMSO, MeOH and EtOH were -101.8239378, -103.0712878, -

103.7146578, -104.4761978 and -112.1231098, respectively. As the results show, the

combination of PY and EtOH as functional monomer and polymerization solvent

leads to the most stable complex. Therefore, the electropolymerization of PY was

done in EtOH as solvent.

3.3. Electropolymerization of molecularly imprinted polypyrrole

The PGE was washed by water and dried at room temperature before experiments.

Then, PGE was immersed in a sonicated solution containing 0.37 gL-1 of MWCNTs-

COOH and a chronoamperometry method was employed to deposit the MWCNTs-

COOH onto the bare PGE surface by using +1.7 V for 400 s. The resulting

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MWCNTs/PGE was immersed in the polymerization solution. The electrochemical

behavior of PY was investigated in ethanolic solution of 0.1 M NaClO4 using potential

cycling between +0.6 and +2.0 V (versus Ag/AgCl). Electro oxidation of PY

monomer occurs at the anode and the resulting polymer deposits onto the surface of

MWCNTs/PGE [16, 17].

Fig. 3a shows the cyclic voltammograms taken during the electropolymerization of

PY (0.02 M) onto a MWCNTs/PGE. The formation and growth of the polymer film

can be easily seen in Fig. 3a. A broad oxidation peak was observed at the peak

potential of +1.25 V. For imprinted electropolymerizations, HCT was added to the

electrochemical cell at a concentration of 0.01M. During the electrodeposition of the

conductive polymer, HCT template molecules are trapped in the polymer matrix

because of ability of these molecules to interact with the PY monomers. As seen in

Fig. 3b, a new oxidation peak at +1.5 V indicates that the template is becoming part of

the polymeric chain. Fig. 3a and b shows 10 cycles obtained by electropolymerization

of PY in NIP and MIP process on the surface of MWCNTs/PGE, respectively. The

interaction of PY and HCT could be evaluated by comparing the cyclic

voltammograms obtained by NIP and MIP in Fig. 3a and b, respectively.

Insert Fig. 3

3.4. Optimization of experimental parameters

3.4.1. Screening of significant factors using Plackett-Burman design

A large number of factors could potentially affect the MIP technique and therefore a

Plackett-Burman design was used as a screening method to select the most statistically

significant parameters for further optimization. The Plackett-Burman (PB) factorial

design can identify main factors affecting the electrode preparation, HCT extraction in

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the prepared electrode and electrochemical measurement processes by a relatively few

experiments. A Plackett–Burman type (III) resolution design was carried out for eight

factors, consisted of 36 non-randomized runs.

The evaluated factors can be divided into three different sections. Concentration of

MWCNT ([MWCNT]), deposition time of MWCNTs onto the bare PGE surface

(Deposition time), concentration of pyrrole ([PY]) and number of cycles in

electropolymerization (Cycles) which are related to the electrode preparation. On the

other hand, pH of buffer solution (pH), concentration of hydrochlorothiazide ([HCT]),

stirring rate of solution (S.R.) and electrode loading time (Loading time) related to the

HCT extraction in the prepared electrode and electrochemical measurement processes.

Based on the preliminary experiments, variations in the parameters considered might

affect the analytical signal. The amounts and levels of the factors as low (–) and high

(+), are listed in Table 2. Table 3 shows the results of PB experimental design matrix

together with the analytical response (expressed as peak height) for each run.

Insert Table 2

Insert Table 3

The statistical evaluation of results (Table 3) produced the standardized main effect

Pareto chart as shown in Fig. 4 and offered a minimum t-value of 2.052 at a

confidence level of 95.0%. In Fig. 4, lengths are proportional to the absolute values of

estimated effects and t-value is included as a vertical reference line. The variables

were considered as statistically significant factors. Furthermore, the positive and

negative signs (corresponding to a white and grey bar filling, respectively) showed

that whether the response would be improved from the low to high level or not.

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Insert Fig. 4

As shown in Fig. 4, pH of buffer solution, the stirring rate of solution, the

concentration of HCT and concentration of PY are statistically significant and the

effects of these variables are negative leading to enhanced analytical signals at their

lower values. Thus, the lower values of these factors were selected for subsequent

experiments. For the studied range of parameters, the deposition time of MWCNTs

onto the bare PGE surface and the electrode loading time were not significant showing

negative effects. However, the concentration of MWCNT and the number of cycles in

electropolymerization were the next insignificant variables that were having positive

statistical effects. Thus, the higher values of concentration of MWCNT and the

number of cycles in electropolymerization and lower deposition time of MWCNTs

onto the bare PGE surface and electrode loading time were selected for further

experiments.

3.4.2. Optimization using central composite design

Based on the results of the screening design, a new optimization procedure was

performed. Four variables including pH of buffer solution (pH), the stirring rate of

solution (S.R.), the concentration of hydrochlorothiazide ([HCT]) and the

concentration of pyrrole ([PY]) which all significantly influenced the analytical

response were simultaneously optimized using a central composite design (CCD) and

the effects as well as their mutual interactions were studied. This three-level fractional

factorial design allows estimation of a second order (quadratic) model with linear,

quadratic and interaction terms. The CCD with twenty seven experiments was carried

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out, consisting 24 full factorial design, augmented with 2×4 axial (or star) points

(α=1.00) and 3 replicates of the center point. The complete design matrix and the

corresponding analytical response of each run are presented in Table 4.

Insert Table 4

To find the most suitable fitting with the experimental data, a response surface model

was developed using the regression analysis by considering different combinations of

the linear, squared and interaction terms in polynomial equations. The adequacy of

each model was checked using the analysis of variance (ANOVA) and a maximum p-

value for lack-of-fit (LOF) of 0.081 was obtained for the following equation

suggesting that the quadratic model was significant. By applying the regression

analysis, the following second-order polynomial equation was established to express a

semi empirical model for the electrolytic technique:

I = 18.1740 -18.4788 × pH - 0.000216042 × S.R. - 1389.36 × [HCT] + 1713.74 ×

[PY] + 5.37039 × (pH)2 – 0.000286911 × (S.R.)2 + 493662 × ([HCT])2 - 28964.4 ×

([PY])2 + 0.0176231 × (pH)(S.R.) - 1525.62 × (pH)([HCT]) - 393.338 × (pH)([PY]) -

0.361250 × (S.R.)([HCT]) + 0.856625 × (S.R.)([PY]) + 134815 × ([HCT])([PY])

Where "I" is the response of sensor to HCT values and pH, S.R., [HCT] and [PY] are

the actual values of the significant parameters.

The R2 and R2-(adj) for the model were obtained 99.99% and 99.98%, respectively.

The adequacy of fit of calculated models was evaluated statistically. According to

Table 5, regression statistics information (p-value<0.05 and F-ratio>19.42) and those

for lack-of-fit (p-value>0.05, F-ratio<19.40) showed the regression validity and

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presence no lack of fit in the model. It is also evident that the coefficients for linear,

square and interaction parameters are meaningful statistically.

Insert Table 5

In order to achieve the maximum response, the obtained equation was solved and the

parameters of pH, S.R., [HCT] and [PY] were calculated to be 2.67, 110.14 r.p.m.,

0.00288 M and 0.0198 M, respectively.

3.5. Scanning electron microscopy (SEM) of NIPs/MWCNTs/PGE and

MIPs/MWCNTs/PGE

The structures of both polymeric electrodes were studied by SEM. There are

appreciable differences in morphology of SEM of NIP and MIP ( data was not

shown). The surface of MIP was exhibited a more porous and rough structure than

NIP. The cavities in the MIP were probably caused by the structure of target molecule,

HCT. The results were shown that MIP has more adsorption capacity to HCT than

NIP.

3.6. Calibration curve of MIPs/MWCNTs/PGE

The calibration curve was plotted by drawing oxidation peak current (A) Vs. HCT

concentration for MIP electrode at optimal conditions. The results show linearity over

two concentration ranges of 9×10−10 to 1×10−5 M and 1×10−5 to 1×10−2 (data was not

shown). The calibration linear equations of Ip, a = 0.040Ca + 6×10-7 (r2 = 0.9986) and

Ip, b = 0.021Cb + 2×10-6 (r2 = 0.9921) were obtained at optimal conditions. The

detection limit (based on S/N = 3) of HCT was obtained 1.0×10−10 M.

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3.7. Reproducibility of MIPs/MWCNTs/PGE

The reproducibility of MIPs/MWCNTs/PGE was investigated using a HCT

concentration of 3 × 10-3 M. The peak current response of HCT was determined using

three different electrodes, which were produced under the same conditions. The peak

current intensity showed a relative standard deviation of 3.36%, confirming that the

prepared sensor was reproducible. Furthermore, the MIPs/MWCNTs/PGE could be

used more than 10 times after subsequent cycles of washing and measuring operations.

For investigation of inter day stability of electrode, current response was measured,

the current was unaltered and a decrease of 4.7% in current response occurred after

90th day.

3.8. Effect of interferences

The selectivity of MIP electrode in this work was evaluated in the presence of

different interfering molecules. The voltammetric responses of HCT imprinted

polypyrrole films were examined in the presence of some interfering substances like

ascorbic acid, methimazole, 2-aminobenzimidazole, benzimidazole, acetaminophen,

aspirin and diphenoxylate. The differential pulse voltammograms were taken for the

oxidation of HCT (2.5 mM) after addition of different concentration (2.5, 5.0 and 7.5

mM) of each interference compound. These results are graphically shown in Fig. 5. As

can be seen (Fig. 5), these changes in current would correspond to a change in

concentration of less than 0.15 mM analyte when the concentration of interference

components is 2.5 mM. As can be found, however, there is no serious interfering

effect for the method in the most cases; the selectivity appears to decrease as the mole

ratio of interference to analyte increases.

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Insert Fig. 5

3.9. Real sample analysis

In order to establish the ability of the MIP/MWCNT electrode, the sensor was applied

to determine HCT in tablets according to the recommended method. To investigating

the capability of proposed sensor for determination of HCT in complex matrix of

clinical samples, a spike method was chosen. Three measurements were performed for

each concentration (2, 4 and 6 mM of HCT). As can be seen from Table 6, good

recoveries and RSD were obtained revealing that the recommended method has

capability in determination of HCT serum and pharmaceutical samples.

Insert Table 6

4. Conclusions

A HCT-imprinted electrode successfully fabricated by the cyclic voltammetry

electropolymerization of a polypyrrole film on MWCNT-functionalized PGE.

The DFT calculations predict that PY/EtOH is the best combination of functional

monomer/solvent, which leads to the most stable prepolymerization complexes with

HCT as template. Experimental designs such as PBD and CCD were applied to find a

model for optimizing the technique. The imprinted sensor was easily constructed at

low cost and was showed good repeatability, selectivity, and accuracy. The

recommended method capability was successfully applied to determination of HCT in

pharmaceutical and serum samples.

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determination of losartan and hydrochlorothiazide in tablets by high-performance

liquid chromatography, Journal of Pharmaceutical and Biomedical Analysis 23

(2000) 185-189.

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Biographies

Azizollah Nezhadali is an associate Professor of Department of

Chemistry Payame Noor University in Mashhad, Iran. He received his

PhD in Department of Chemistry (2000) from Ferdowsi University of

Mashhad, Iran and the University of Sydney, Australia (guest PhD student

for six months). Now he is a Faculty member of Department of Chemistry,

Payame Noor University, Mashhad, in Iran. His research interests cover

separation (micro and bulk), electroanalytical chemistry and GC/MS. He has

written a chapter of macrocylic chemistry book, Nova Publisher,NY,

2010.

Maliheh Mojarrab is a Ph.D. candidate in department of chemistry, Payame

Noor University of Mashhad. She received her master of science degree in

analytical chemistry ( 2012) from Payame Noor University ( Dr. A Nezhadali

research group) and bachelor degree in chemistry (2010) from Ferdowsi

University, Iran. Her research interest is mainly in molecularly imprinted

polymers and works with research group of Dr. A. Nezhadali.

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Fig. 1. The optimized conformations of HCT, PY, TH, FU and MPY.

Fig. 2. The optimized geometries for the most stable complexes between HCT and PY, TH, FU and MPY.

Fig. 3. The voltammetric cycles for the preparation of (a) NIPs/MWCNTs/PGE and (b) MIPs/MWCNTs/PGE in an ethanolic solution of 0.1 M NaClO4 and 0.02 M pyrrole.

Fig. 4. The standardized main effect Pareto chart for Plackett–Burman design.

Fig. 5. The evaluate selectivity of HCT (2.5 mM) in the presence of different interfering molecules a: 0; b: 2.5; c: 5.0 and d: 7.5mM.

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Table 1

The calculated binding energies (∆E) of HCT with PY, TH, FU and MPY.

Molecule E(au) ∆E(au) ∆E(kJmol-1)HCT -1993.13247580 PY -210.16589071 TH -553.00262846 FU -230.02058141 MPY -249.4840153 HCT-PY -2203.31081722 -0.0124502 -32.6942252HCT-TH -2546.14144396 -0.0063392 -16.6467392HCT-FU -2223.1614265 -0.0083692 -21.9775192HCT-MPY -2242.6289288 -0.0124372 -32.6600872

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Table 2

The experimental field definition for PBD.

Variable Symbol Low(-) High(+)

Concentration of MWCNT (mg)

[MWCNT] 0.3 5.5

Deposition time of MWCNTs onto the bare PGE surface (s)

Deposition time 400 1600

Concentration of pyrrole (M)

[PY] 0.01 0.04

Number of cycles in electropolymerization

Cycles 5 10

pH of buffer solution pH 2 5

Concentration of hydrochlorothiazide (M)

[HCT] 0.005 0.01

Stirring rate of solution (r.p.m)

S.R 100 400

Electrode loading time (min)

Loading time 5 15

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Table 3

The results of Plackett–Burman experimental design matrix.

Run order

[MWCNT]

(mg)

Deposition time (s)

[PY] (M)

Cycles pH [HCT] (M)

S.R (r.p.m)

Loading time (min)

Current (µA)

1 5.5 400 0.04 5 2 0.005 4 15 159.00

2 5.5 1600 0.01 10 2 0.005 1 15 200.00

3 0.3 1600 0.04 5 5 0.005 1 5 99.50

4 5.5 400 0.04 10 2 0.01 1 5 80.20

5 5.5 1600 0.01 10 5 0.005 4 5 17.10

6 5.5 1600 0.04 5 5 0.01 1 15 4.92

7 0.3 1600 0.04 10 2 0.01 4 5 89.30

8 0.3 400 0.04 10 5 0.005 4 15 21.50

9 0.3 400 0.01 10 5 0.01 1 15 79.70

10 5.5 400 0.01 5 5 0.01 4 5 86.00

11 0.3 1600 0.01 5 2 0.01 4 15 22.00

12 0.3 400 0.01 5 2 0.005 1 5 207.00

13 5.5 400 0.04 5 2 0.005 4 15 162.00

14 5.5 1600 0.01 10 2 0.005 1 15 286.00

15 0.3 1600 0.04 5 5 0.005 1 5 109.00

16 5.5 400 0.04 10 2 0.01 1 5 102.00

17 5.5 1600 0.01 10 5 0.005 4 5 22.00

18 5.5 1600 0.04 5 5 0.01 1 15 2.87

19 0.3 1600 0.04 10 2 0.01 4 5 72.60

20 0.3 400 0.04 10 5 0.005 4 15 23.00

21 0.3 400 0.01 10 5 0.01 1 15 154.00

22 5.5 400 0.01 5 5 0.01 4 5 108.00

23 0.3 1600 0.01 5 2 0.01 4 15 39.50

24 0.3 400 0.01 5 2 0.005 1 5 278.00

25 5.5 400 0.04 5 2 0.005 4 15 160.50

26 5.5 1600 0.01 10 2 0.005 1 15 268.00

27 0.3 1600 0.04 5 5 0.005 1 5 164.00

28 5.5 400 0.04 10 2 0.01 1 5 125.00

29 5.5 1600 0.01 10 5 0.005 4 5 85.80

30 5.5 1600 0.04 5 5 0.01 1 15 3.89

31 0.3 1600 0.04 10 2 0.01 4 5 106.00

32 0.3 400 0.04 10 5 0.005 4 15 45.90

33 0.3 400 0.01 10 5 0.01 1 15 157.00

34 5.5 400 0.01 5 5 0.01 4 5 97.00

35 0.3 1600 0.01 5 2 0.01 4 15 19.00

36 0.3 400 0.01 5 2 0.005 1 5 163.00

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Table 4

The central composite design matrix and the experimental results.

Stdorder Runorder pH S.R (r.p.m)

[HCT] (M)

[PY] (M)

Current (µA)

2 1 3 0 0.0025 0.010 51.727 2 1 200 0.0075 0.010 247.3015 3 1 200 0.0075 0.020 407.0017 4 1 100 0.0050 0.015 261.0014 5 3 0 0.0075 0.020 133.7721 6 2 100 0.0025 0.015 87.0027 7 2 100 0.0050 0.015 121.3025 8 2 100 0.0050 0.015 121.4213 9 1 0 0.0075 0.020 460.7322 10 2 100 0.0075 0.015 215.301 11 1 0 0.0025 0.010 145.825 12 1 0 0.0075 0.010 313.2516 13 3 200 0.0075 0.020 154.1020 14 2 200 0.0050 0.015 79.9511 15 1 200 0.0025 0.020 178.5026 16 2 100 0.0050 0.015 122.229 17 1 0 0.0025 0.020 224.314 18 3 200 0.0025 0.010 56.9012 19 3 200 0.0025 0.020 73.5023 20 2 100 0.0050 0.010 71.6519 21 2 0 0.0050 0.015 103.2610 22 3 0 0.0025 0.020 50.153 23 1 200 0.0025 0.010 80.208 24 3 200 0.0075 0.010 67.806 25 3 0 0.0075 0.010 65.8024 26 2 100 0.0050 0.020 154.4618 27 3 100 0.0050 0.015 87.00

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Table 5

ANOVA results for evaluation of mathematical models of sensitivity in relation to

electrode preparation factors, obtained by response surface design.

Source DF Seq SS Adj SS Adj MS F-ratio P-valueRegression 14 2981.05 2981.05 212.93 8555.00 0.000Linear 4 2399.40 2399.40 599.85 24100.29 0.000Square 4 188.81 188.81 47.20 1896.48 0.000Interaction 6 392.83 392.83 65.47 2630.49 0.000Residual error 12 0.30 0.30 0.02Lack-of-fit 10 0.29 0.29 0.03 11.74 0.081Pure error 2 0.01 0.01 0.00Total 26 2981.35

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Table 6

The results of HCT determination in real samples (n = 3).

Sample HCT added (mM) Average of HCTfound (mM) ± RSDa

Recovery (%)

Serum sample

0 Not detected -

2 2.06±0.0096 103.004 3.96±0.0143 99.006 6.1±0.0108 101.67

Tablet b 0 2.58±0.0120 -2 4.67±0.0169 104.504 6.47±0.0114 97.25

Tablet c 0 2.63±0.0104 -2 4.62±0.0113 99.504 6.84±0.0140 105.25

Tablet d 0 2.54±0.0082 -2 4.57±0.0123 101.504 6.77±0.0096 105.75

aRelative standard deviation (n=3)

bDaroupakhsh Co.

cSobhandarou Co.

dIrandarou Co.

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Graphical abstract

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Scheme I

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Figure 5