Gliclazide voltammetric sensor based on electropolymerized molecularly imprinted polypyrrole film onto glassy carbon electrode

Sensors and Actuators B 204 (2014) 42–49

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Gliclazide voltammetric sensor based on electropolymerized molecularly imprinted polypyrrole film onto glassy carbon electrode Hajer Hrichi a , Mohamed Radhouan Louhaichi b , Lotfi Monser a , Nafaâ Adhoum a,c,∗ a b c

National Institute of Applied Sciences and Technology, Carthage University, Centre Urbain Nord, B.P.N 676, 1080 Tunis Cedex, Tunisia National Laboratory for Pharmaceutics Control, Tunis, Tunisia Preparatory School for Engineering Studies, Kairouan University, Tunisia

a r t i c l e

i n f o

Article history: Received 12 May 2014 Received in revised form 6 July 2014 Accepted 14 July 2014 Available online 24 July 2014 Keywords: Molecularly imprinted polymer Polypyrrole Glassy carbon electrode Electropolymerization Gliclazide

a b s t r a c t Gliclazide sensing was investigated based on differential pulse voltammetry measurements of an electropolymerized molecularly imprinted polymer (E-MIP) film. The E-MIP polymer was prepared via anodic electropolymerization of pyrrole in the presence of GLZ onto glassy carbon electrodes using cyclic voltammetry (CV). GLZ molecules are successfully trapped into the polypyrrole matrix creating, after their subsequent removal, shape-complementary artificial recognition sites. The effect of several significant operational parameters (monomer and template concentrations, number of CV scans, pH and incubation time) on film analytical performances were investigated and optimized. Under optimized conditions, the sensor response exhibited high sensitivity toward the target template and was linearly proportional to GLZ concentration (R2 = 0.998) over the range 5 × 10−11 –4 × 10−10 M, with a detection limit (3 /m) of 1.2 × 10−11 M. The precision of the method (R.S.D., n = 6) for within and between-days is better than 1.4% and 2.48%, respectively at 10−10 M. Moreover, the selectivity of E-MIP sensor, against potentially competing molecules (Imp. B, Imp. E, glipizide, glibenclamide, glimepiride), was demonstrated. The developed E-MIP sensor was successfully applied to the determination of GLZ in three pharmaceutical products and gave results in close agreement with the reference HPLC method with mean recoveries between 95.4 and 98.8%, showing promising potential in practical applications. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Gliclazide (GLZ) is a second-generation sulfonylurea oral antidiabetic drug, effective in controlling blood glucose in type 2 diabetes mellitus. It acts mainly on pancreatic sulphonylurea receptors (SURs), at the surface of ␤-cells, by increasing the secretion of insulin [1]. In contrast with the first-generation drugs, the gliclazide preparations are of very high efficiency, therefore treatment doses are two to three times lower [2]. Furthermore, gliclazide is sparingly soluble in water, making the development of simple new analytical methods of better specificity and higher sensitivity of significant importance [3]. During the past years, several analytical methods including UV and visible spectrophotometry [4–6], high performance liquid chromatography (HPLC) [7–11], thin-layer chromatography

∗ Corresponding author at: National Institute of Applied Sciences and Technology, Carthage University, Centre Urbain Nord, B.P.N 676, 1080 Tunis Cedex, Tunisia. Tel.: +216 1 703627; fax: +216 1 704329. E-mail address: [email protected] (N. Adhoum). http://dx.doi.org/10.1016/j.snb.2014.07.056 0925-4005/© 2014 Elsevier B.V. All rights reserved.

(TLC) often in combination with densitometry [12–15] and gas chromatography coupled with flame ionization detection (GC-FID) [16,17] have been commonly used for quantitative determination of gliclazide in blood, plasma and pharmaceutical samples. Comparatively, only few reports dealing with the electrochemical behavior of gliclazide and its electroanalytical determination using voltammetry [18–20] and coulometry [21] were published. Over the past two decades, molecular imprinting emerged as a powerful technique for preparation of polymeric materials with high recognition ability and has attracted tremendous research interest from scientists engaged in sensor development due to their long-term stability, low cost and easy preparation [22]. The general principal of molecular imprinting is based on polymerizing a monomer with a cross-linker in the presence of a template molecule (print molecule), which is subsequently removed from the cross-linked matrix, yielding host cavities that can rebind the template molecules reversibly and selectively in the presence of their interferents. Most commonly used preparation strategies, including bulk polymerization, sol–gels and surface grafting polymerization suffer from some inherent drawbacks, such us slow mass transfer, heterogeneous distribution of the binding sites and poor assembly ability on the transducer’s surface [23]. The

H. Hrichi et al. / Sensors and Actuators B 204 (2014) 42–49

electrochemical polymerization offers a straightforward alternative to overcome these disadvantages by enabling the direct growth of thin molecularly imprinted polymer (MIP) films onto electrode surface. Moreover, electropolymerization procedure allows easy preparation of multilayer thin films of accurately controlled thickness with improved sensitivity and good adherence to a transducer of any size and shape. Many conjugated polymers such as polypyrrole, polyaniline, polythiophenes and others were described as promising materials suited for MIP formation [24,25]. Among various polymers, polypyrrole (PPy) has received particular interest owing to its convenience of preparation, high stability and wide range of applications [26]. Besides, PPy has good electric conductivity and electrochemical redox activity even in pH-neutral solutions allowing successful entrapment of a wide range of templates [27–34]. A detailed survey of the literature reveals that a large number of MIPbased chemo-sensors have already been reported [35,36], whereas the development of electrochemical sensors has been significantly lower [37]. Indeed, the electrochemical approach in tandem with MIP protocol is rather recent and intended to combine the intrinsic high selectivity of the molecular recognition technique with the high sensitivity of electrochemical determination [30,37,38]. In this paper, we report on the development of a gliclazide electrochemical sensor based on the molecular recognition of the analyte by a molecularly imprinted polymer film electrochemically deposited on glassy carbon electrode. The sensitivity and selectivity of electropolymerized MIP (E-MIP) film were evaluated using differential pulse voltammetry and its successful application for GLZ determination in pharmaceutical samples has been demonstrated. To the best of our knowledge, the proposed method is the first application of E-MIP electrode for GLZ determination.

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The glassy carbon electrode was polished at the beginning of the study, using an aqueous slurry of 0.01 ␮m alumina powder on a smooth polishing cloth until a mirror-like finish was obtained. Every day, the electrode was sonicated in distilled water and in acetone for 5 min before its use in voltammetric measurements. Fourier-transform infrared spectra were recorded on a (PerkinElmer, Model 2000) spectrometer to characterize the imprinted film. The pH of all sample solutions, were adjusted using a MettlerToledo 340 pH-meter with accuracy of 0.01 pH unit. 2.3. Preparation of E-MIP and NIP electrodes The gliclazide molecularly imprinted film was prepared by electrochemical polymerization of pyrrole on the surface of glassy carbon electrode, using cyclic voltammetry in the potential range between −600 and 1000 mV (versus SCE), for 3 cycles at a scan rate of 100 mV s−1 . The polymerization mixture consisted of an aqueous solution containing 0.1 M pyrrole, 0.4 mM gliclazide and 0.1 M NaClO4 , used as supporting electrolyte. After electropolymerization of pyrrole, the working electrodes were thoroughly rinsed with distilled water to remove any loosely bound gliclazide molecules. Then, embedded gliclazide molecules were removed from the polymeric film by immersing the E-MIP electrode into a stirred 2 M phosphate buffer solution of pH 5, until the signal corresponding to the oxidation of GLZ disappeared. The template removal from MIP matrix during washing treatment was monitored using a previously published HPLC method [11]. A non-imprinted electrode (NIP) has been prepared under similar conditions without the addition of template and used as a control electrode. 2.4. Electroanalytical measurements

2. Materials and methods 2.1. Chemical and reagents Unless otherwise stated, all chemicals used in this work were of analytical-reagent grade and used without further purification. Gliclazide, Imp. B, Imp. E, glipizide, glibenclamide and glimepiride powders were kindly supplied by the National Laboratory of Drugs Control (Tunisia). Three brands of tablets Diabenorm® , Diamicron® and Diamivance® were procured from local pharmacies. Pyrrole and NaClO4 were obtained from Sigma–Aldrich (Steinheim, Germany). Methanol, KH2 PO4 and K2 HPO4 were all purchased from Prolabo (France). Phosphate buffer solutions (0.2 M), used as supporting electrolyte, were prepared by dissolving the appropriate amounts of KH2 PO4 and K2 HPO4 in double distilled ultrapure water. The pH of the solution was adjusted to the required value by adding few drops of concentrated phosphoric acid or sodium hydroxide solution. Stock standard solution of gliclazide (10 mM) was prepared by dissolving appropriate amount of drug in methanol and kept in the freezer at −18 ◦ C. Working standards of gliclazide were freshly prepared just before assay, by adding appropriate amounts of stock solution directly to the voltammetric cell. 2.2. Instrumentation All experiments of cyclic and differential pulse voltammetry were performed using a Radiometer potentiostat–galvanostat model PGZ 402 Voltalab 80 equipped with a conventional threeelectrode system consisting of a glassy carbon working electrode (area of 0.196 cm2 ), a saturated calomel (SCE) reference electrode and a platinum wire as auxiliary electrode. The system was monitored with a personal computer using Voltamaster 4 software package for data acquisition and subsequent analysis.

The electrochemical performance of E-MIP sensor was evaluated by differential pulse voltammetry (DPV), carried out in a 0.2 M phosphate buffer solution (PBS) of pH 5.0 In a typical run, a 5 mL of the supporting electrolyte was transferred into a clean, dry cell and the required volume of the standard stock solution of GLZ was added by micropipette (Gilson, France). After a stirring period of 5 min, a differential pulse voltammogram was recorded. Differential pulse voltammograms were recorded in the potential range between 0.6 and 1 V (versus SCE), using a scan rate of 10 mV s−1 with pulse amplitude of 50 mV and a pulse width of 20 ms. All electroanalytical measurements were made at room temperature (20 ± 2). The calibration curve was constructed by plotting the intensity of the anodic (DPV) current peak against the corresponding GLZ concentration. 2.5. Analysis of pharmaceutical dosage forms For the determination of gliclazide in pharmaceuticals, 10 tablets were weighed, powdered and an accurately weighted portion corresponding to one tablet was transferred to a 100 mL volumetric flask and dissolved in methanol. The mixture was sonicated for 5 min in an ultrasonic bath. A suitable volume of the clear supernatant liquor was then added to a voltammetric cell containing 5 mL of phosphate buffer, to make a final concentration in the working range. The differential pulse voltammogram was then recorded and the content of the drug in tablet was quantified by referring to the calibration graph. It should be noticed that we checked that the presence of methanol (added to pharmaceuticals) does not produce any significant change on the background current (<0.5% when 50 ␮l methanol was added to 5 mL of the supporting electrolyte).

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Fig. 1. The structures of gliclazide, glipizide, glibenclamide and glimepiride.

3. Results and discussion 3.1. Electropolymerization of molecularly imprinted polypyrrole Electropolymerization of the pyrrole monomer occurs at the anode via radical cation formation and their subsequent coupling to form a conductive polymer onto the surface of GCE. Fig. 2 shows typical cyclic voltammograms obtained during the synthesis of EMIP and NIP films, carried out by cycling the potential between −0.6 and 1 V (versus SCE). The film growth was monitored through changes in current over cycles. The oxidation current attributed to pyrrole monomer oxidation, increased progressively upon cycling, suggesting a stepwise growth of the polymer film on the electrode surface. After the second cycle, a broad peak ascribed to PPy oxidation appeared at about 40 mV for both E-MIP and NIP electrodes, whereas a second peak at 590 mV was exclusively recorded with E-MIP films. Furthermore, the deposition of imprinted polymer resulted into a relatively higher increase in anodic and cathodic current compared to non-imprinted film, pointing out the differences between MIP and NIP growth. This behavior, evidently attributed to the presence of GLZ, provides supporting evidence on its effective incorporation into the polymer matrix [34]. Indeed, the oxygen atoms in the O S O and C O groups of GLZ molecules are able to interact with the hydrogen atom in the N H group of pyrrole units via H-bonding and other possible non-covalent intermolecular interactions (Fig. 3). Such causes the entrapment of GLZ molecules which are in the vicinity of the electrode into the polymer matrix and enhances the cross-linking of monomers resulting in higher current density.

The entrapped template molecules were removed by immersing the MIPs film-coated electrode into a stirred phosphate buffer solution of pH 5. The time evolution of released GLZ during washing was determined using a previously published HPLC procedure. The recorded HPLC profile (Fig. 4), showed rapid release of GLZ during the first 15 min which then slowed down to reach a plateau, probably indicating a complete removal of all trapped template molecules. This behavior ascertained for successful incorporation of the template molecules into the polymer matrix and evidenced its efficient removal during the washing step. The optimum time of washing, beyond which there was no significant further release of GLZ, was fixed at about 1 h. 3.5

MIP

3

NIP

2.5

Current/mA cm-2

The selectivity of imprinted film toward GLZ was assessed by performing DPV analysis in the presence of variable amounts of five related molecules: impurities B and E (Imp. B, Imp. E), glipizide, glibenclamide and glimepiride, presenting structural similarity with GLZ (Fig. 1).

2 1.5 1 0.5 0 -0.5 -1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

E/V (vs. SCE) Fig. 2. Cyclic voltammograms obtained during the preparation of the imprinted and non-imprinted PPy films at glassy carbon electrode. Experimental conditions: [pyrrole] = 0.1 M; [gliclazide] = 0.4 mM; scan rate = 100 mV s−1 ; [NaClO4 ] = 0.1 M; number of cyclic scans = 4.

H. Hrichi et al. / Sensors and Actuators B 204 (2014) 42–49

Fig. 3. Schematic representation of possible interactions between gliclazide and polypyrrole film deposited onto glassy carbon electrode.

3.2. Optimization of operational parameters The effects of main operational parameters such as the monomer and template concentrations and the number of CV scans were investigated and optimized, as they may significantly affect the structural integrity of the resulting film and sensor performance [39]. The optimization was carried out using the univariate method by comparing imprinted and non-imprinted DPV responses. In order to assess the effect of monomer concentration, a series of experiments were carried out in which the concentration of pyrrole was changed between 0.05 and 0.2 M and all other operational parameters were kept constant. Each resulting film was compared to NIP films prepared under similar conditions. Fig. 5A depicts the distinct analytical response of the imprinted film against the non-imprinted in sensing gliclazide, denoting successful imprinting. These results provide further support to the assumption that template molecules were able to penetrate and rebind with the recognition sites present within the imprinted polymer network, whereas NIP film was nearly impermeable to gliclazide. It was also observed that the E-MIP film sensitivity was highly affected by monomer concentration present in polymerization mixture. Indeed, the sensitivity tends to increase upon increasing pyrrole concentration up to 0.1 M. Above this concentration, 0.1

45

a dramatic decrease of sensor sensitivity was observed. These findings are in agreement with results published by other groups [29,34,39] reporting similar trends in sensor response variation with monomer concentration. This behavior was ascribed to a substantial increase of film thickness with monomer concentration. It is assumed that increasing monomer concentration resulted in concomitant increase of polymer loading at the electrode surface, which is coincident with a corresponding increase in film thickness alongside with an increase in the number of template molecules trapped within the polymer matrix [39]. This is regarded to be responsible for the generation of more binding cavities, which enhances the sensing capability of the polymer toward GLZ. However, too high thickness gives rise to poor recognition sites accessibility implying a drastic reduction in the efficiency of template release and rebinding. To further illustrate this point, film thickness were estimated based on the charge passed through the electropolymerization process [40] (inset of Fig. 5A), and were found to support the above proposed explanation. Therefore, the optimum monomer concentration was fixed at 0.1 M for all subsequent measurements, as it gave the highest sensitivity. The influence of template on the film sensitivity was investigated by varying its concentration in the range between 0.05–0.6 mM. As shown in Fig. 5B, imprinted film sensitivity was found to increase upon increasing the template concentration up to 0.4 mM, and stayed fairly constant afterward. This trend is ascribed to the higher number of specific cavities generated within the polymer network as the template concentration was increased. Nevertheless, higher loading of entrapped GLZ molecules may results in relatively thicker films with lower recognition sites accessibility. Thus it may be speculated that simultaneous increase of thickness and recognition sites number have produced two opposite effects on sensing capability which led to the observed plateau. Therefore, it could be concluded that the optimum template concentration was approximately 0.4 mM. Furthermore, it has been reported [29,30] that sensor response depends to a great extent on the number of cyclic scans. This is due to the well-known fact that sensing performance depends on film thickness which in turn is related to the number of electropolymerization cycles. As expected, the MIP coated-electrode sensitivity was found to increase upon increasing cycle number up to 3 (Fig. 5C). More cycles led to thicker PPy film with less accessible imprinted sites, consistent with the recorded response decrease. Besides, NIP film response was found to be very low and fairly constant. Therefore, the optimum number of cyclic scans was fixed at 3 cycles.

3.3. FTIR characterization of MIP-modified electrodes

Gliclazide concentration/nM

0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0

0

10

20

30

40

50

60

70

80

90

Time/min. Fig. 4. Time evolution of gliclazide released during its removal from the imprinted electrode.

The IR spectroscopy was employed in this study to further ascertain the successful incorporation of GLZ into PPy network. Fig. 6 displays typical infrared spectra of NIP and MIP electrodes, before and after removing the template molecules. Major vibrational bands attributed to polypyrrole and associated with C N and C C ring stretching can be seen at 1420 and 1524 cm−1 , respectively on all recorded spectra. Compared with the non-imprinted spectrum, the imprinted film (Fig. 6A) displayed the characteristic peaks of sulfonamide groups from GLZ molecules at 1565 cm−1 (N H deformation band), 1327 cm−1 (S O asymmetrical vibrational band) and 1182 cm−1 (S O symmetrical vibrational band) [41]. These data provide supporting evidence on the effective incorporation of gliclazide into PPy matrix. Moreover, GLZ characteristic peaks disappeared completely after washing, leading to IR spectrum (Fig. 6B) identical to that of the non-imprinted sample (Fig. 6C). This leads to assumption of efficient and complete removal of the templates.

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Fig. 5. The effects of the monomer concentration (A), template concentration (B) and number of cyclic scans (C) on MIP and NIP modified GCE electrodes sensitivity. The response was measured through differential pulse voltammetric response to 10−10 M GLZ in phosphate buffer medium at pH 5.0.

3.4. Evaluation of the MIP analytical performance Initially, the optimal instrumental parameters for the quantitation of gliclazide were studied and the effect of scan rate, pulse height and width were investigated. With regard to peak current intensity and symmetry, the following parameters: 10 mV s−1 , 50 mV and 20 ms were chosen as the most convenient values. It was also noteworthy to discuss the effect of pH and incubation time on the sensor response, in order to achieve the highest sensitivity. As demonstrated in Fig. 7, DPV oxidation peak height increased with increasing pH between 2 and 5, to reach its maximum value at pH 5.0. Above this value, a dramatic decrease was recorded. This behavior could tentatively be attributed to the deprotonation of sulfonamide moiety (pKa = 5.8) [40] at pH ≥6 and thus weakening template molecules interaction with binding sites. However, the observed sensitivity decrease at pH lower than 5, is likely due to loose of GLZ electrochemical activity at acidic pH [19]. On the other hand, the recorded analytical signal is directly related to the number of rebinded recognition sites at the moment of measurement which evidently depends on rebinding kinetic. Therefore, the effect of sensor immersion time was investigated. The obtained results showed a progressive increase of analytical signal up to 5 min, after which a steady state sensor response was reached. This relatively short response time is probably due to the thin structure of the E-MIP film, allowing for fast rebinding kinetic.

The E-MIP sensor prepared under optimized conditions, was used to evaluate its analytical performances under the above mentioned optimized detection conditions (incubation time 5 min, pH 5). Fig. 8 displays differential pulse voltammograms for solutions containing increasing quantities of GLZ. The analytical response of the E-MIP film, measured as the peak current, was found to increase linearly with GLZ concentration in the range from 5 × 10−11 to 4 × 10−10 M. Very good linearity with an excellent linear regression coefficient (R2 = 0.998) and high sensitivity (3.1 ␮A/nM) were obtained (see inset of Fig. 8). The intra- and inter-days precision of the proposed method was evaluated as the relative standard deviation (RSD) of 6 repeated determinations of 10−10 mol L−1 standard solution of gliclazide. The precision was found to be satisfactory with an average of intra- and inter-days RSD values of 1.4% and 2.48%, respectively. The E-MIP electrodes exhibited high reproducibility and were very stable for at least 15 days with subsequent cycles of washing and measuring operations, since its response decreased only by 5.7%. The limit of detection (LOD) and quantification (LOQ) were calculated as (3 /m) and (10 /m), respectively, where  is the standard deviation of the intercept and m is the slope of the calibration plot. The calculated LOD and LOQ were found to be 1.2 × 10−11 M and 4 × 10−11 , respectively. This excellent detection limit could probably be explained by the very thin structure (∼75 nm) of the sensing layer, allowing for faster and easier recognition sites accessibility.

H. Hrichi et al. / Sensors and Actuators B 204 (2014) 42–49

47

1

Transmittance

0.95

0.9

1327

0.85

0.8

3188

1182

A

1580 500

1000

1500

2000 2500 Wavenumber/cm-1

3000

3500

4000

Transmittance

1

0.95

0.9

B 500

1000

1500

2000 2500 Wavenumber/cm-1

3000

3500

4000

structures similarly related to that of GLZ). The sensor responses shown in Table 1 proved that no significant interference could be measured even when an anlyte–interferent ratio of 1:3 was used. This reflects on the highly specific recognition between the binding cavities and GLZ molecules and provides support to the assumption that E-MIP film was nearly impermeable to these potentially interfering molecules. That is, the recognition sites formed within the polymer network were spatially oriented to GLZ and almost complementary to this molecule in terms of size and shape.

1

Transmittance

Fig. 8. Differential pulse voltammograms of E-MIP sensor for varying gliclazide concentrations. The inset shows the corresponding calibration plot. Experimental conditions: scan rate, 10 mV s−1 ; pulse amplitude, 50 mV; pulse width, 20 ms. Gliclazide concentrations: (1) 0.05 nM; (2) 0.1 nM; (3) 0.15 nM; (4) 0.2 nM; (5) 0.3 nM; (6) 0.4 nM.

0.95

0.9

C 0.85

1000

500

1500

2000 2500 Wavenumber /cm-1

3000

3500

4000

Fig. 6. FTIR spectra of: (A) MIP film, (B) MIP after GLZ removal and (C) NIP film.

The selectivity of GLZ molecularly imprinted film was investigated by exposing the sensor to 10−10 M gliclazide standard solution in the presence of increasing concentrations of five potentially interfering molecules: impurities B and E, glipizide, glibenclamide and glimepiride (sulfonylurea molecules, having 0.35 MIP

The potential applicability of the developed E-MIP sensor for gliclazide analysis in real samples was evaluated by analyzing three brands of commercial tablets (Diabenorm, Diamicron and Diamivance). The obtained results were compared with those obtained with a reference HPLC method. As can be seen (Table 2), the values obtained by both methods were in close agreement and compared Table 1 Effect of interferents on the differential pulse voltammetric response at the MIP electrode. Interferent molecule

Concentration (nM)a

Signal increase (%)b

RSD (%) (n = 3)

Imp. B

0.1 0.2 0.3

0.65 0.87 0.85

1.22 0.75 2.12

Imp. E

0.1 0.2 0.3

0.96 1.23 1.35

1.75 1.10 0.85

Glipizide

0.1 0.2 0.3

1.10 1.29 1.76

1.64 1.19 1

Glibenclamide

0.1 0.2 0.3

1.68 1.93 2.40

1.2 0.63 0.43

Glimepiride

0.1 0.2 0.3

1.33 1.55 1.64

0.38 0.50 1.03

NIP

0.3 0.25

Current/µ µA

3.5. Application to analysis of real samples

0.2 0.15 0.1 0.05 0

1

2

3

4

5

6

7

8

9

10

11

pH

Spiked concentration to 5 mL of 10−10 M gliclazide solution. Percent increase of analytical signal following the addition of interferent molecule. a

Fig. 7. Effect of pH on MIP and NIP differential pulse responses to 10−10 M gliclazide in a phosphate buffer solution at pH 5.0.

b

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Table 2 Determination of gliclazide in pharmaceutical formulations using the developed E-MIP sensor compared statistically with the reference HPLC method. Tablets

Reported content (mg per tablet)

Diabenorm Diamicron Diamivance

80 30 30

a b

Mean ± SD (n = 6) E-MIP sensor

HPLC method

77.84 ± 1.20 28.26 ± 0.40 29.61 ± 0.46

79.58 ± 0.49 29.86 ± 0.44 29.70 ± 0.42

t-testa

F-testb

1.84 1.13 0.86

1.41 0.32 0.27

Tabulated t-value for significance level p = 0.05 and n = 6 is 2.228. Tabulated F-value for significance level p = 0.05 and f1 = f2 = 5 is 5.0.

Table 3 Recovery of the E-MIP method determined by analyzing spiked pharmaceuticals and urine samples. Samples

Initial content (nM)

Amount spiked (nM)

Amount found (nM)

Recovery (%)

R.S.D. (%)

Diabenorm

0.241 0.241 0.088 0.088 0.093 0.093 – –

0.050 0.100 0.050 0.100 0.050 0.100 0.050 0.100

0.285 0.338 0.133 0.184 0.139 0.191 0.049 0.991

97.9 99.1 96.3 97.9 97.2 98.9 98.0 99.1

0.95 1.23 2.15 1.72 1.46 1.12 0.39 0.68

Diamicron Diamivance Urine

reasonably well with the label claim of all pharmaceutical formulations. Furthermore, the obtained data were compared statistically using student’s t-test and the variance ratio F-test (Table 2). The experimental values were below the theoretical values in either test, indicating that there was no significant difference between the compared methods (confidence limit 95%). Finally, the sensor accuracy was further assessed by performing the recovery test which consists in spiking real samples (drug and urine) with known amounts of gliclazide standard (Table 3). The calculated mean recoveries were ranged from 95.4% to 99%, indicating that excipients are electrochemically inactive and have no interference effect on the analysis of gliclazide. Therefore the proposed method is precise and accurate and could be applied to gliclazide determination in final pharmaceutical products.

4. Conclusion A highly sensitive molecularly imprinted polypyrrole sensor has been developed and applied to the analysis of gliclazide in pharmaceutical formulations. The electrochemical preparation procedure allowed for an accurate control of the film thickness which ensured a fast and efficient recognition sites accessibility. After optimizing fabrication and determination operational parameters, the developed E-MIP sensor showed high analytical performances in term of precision, selectivity and sensitivity. In addition, acceptable longterm stability was observed, since only a slight decrease (∼5.7%) of the analytical response was recorded after 15 days of utilization. In view of the foregoing discussion, the developed E-MIP sensor proved to be a suitable and advantageous alternative for GLZ determination, as it offers interesting assets such as simplicity, low cost and high sensitivity.

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Biographies Hajer Hrichi received her master degree in Industrial Chemistry from The National Institute of Applied Sciences and Technologies (Tunisia) in 2010. She is now a PhD student at Carthage University. She is currently working on the development of molecularly imprinted sensor for environmental and pharmaceutics control. Mohamed Radhouan Louhaichi obtained his PhD degree in Chemistry and Physical Chemistry of Macromolecules from the University Louis Pasteur (Strasbourg, France) in 2001. He is currently assistant professor at The National Laboratory of Drug Control (Laboratoire National de Contrôle des Médicaments, Tunisia). His current research interests are centred on analytical chemistry and drug control. Lotfi Monser obtained his PhD degree in analytical chemistry from the University of Hull, United Kingdom, in 1996. He is currently professor at The National Institute of Applied Sciences and Technologies (Tunisia). His current research interests are centered on analytical and environmental chemistry. Nafaâ Adhoum received his PhD degree in electrochemistry from the Polytechnic National Institute (Grenoble, France) in 1996. He is presently Professor of Chemistry at the Preparatory Institute for Engineering Studies (Kairouan University, Tunisia). His research interests are centered on electroanalytical and environmental chemistry with particular recent emphasis on molecularly imprinted sensors.