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Electro-oxidation and determination of antihistamine drug, cetirizine dihydrochloride at glassy carbon electrode modified with multi-walled carbon nanotubes
Colloids and Surfaces B: Biointerfaces 83 (2011) 133–138
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Electro-oxidation and determination of antihistamine drug, cetirizine dihydrochloride at glassy carbon electrode modified with multi-walled carbon nanotubes Roopa H. Patil, Rajesh N. Hegde, Sharanappa T. Nandibewoor ∗ P. G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad-580 003, Karnataka, India
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 29 October 2010 Accepted 9 November 2010 Available online 16 November 2010 Keywords: Cetirizine dihydrochloride Multi-walled carbon nanotubes Voltammetric determination Modified electrode
a b s t r a c t A multi-walled carbon nanotube (MWCNT) film-modified glassy carbon electrode (GCE) was constructed for the determination of an antihistamine drug, cetirizine dihydrochloride (CTZH) using cyclic voltammetry (CV). Owing to the unique structure and extraordinary properties of MWCNT, the MWCNT film has shown an obvious electrocatalytic activity towards oxidation of CTZH, since it facilitates the electron transfer and significantly enhances the oxidation peak current of CTZH. All experimental parameters have been optimized. Under the optimum conditions, the oxidation peak current was linearly proportional to the concentration of CTZH in the range from 5.0 × 10−7 to 1.0 × 10−5 M. The detection limit was 7.07 × 10−8 M with 180 s accumulation. Finally, the proposed sensitive and simple electrochemical method was successfully applied to CTZH determination in pharmaceutical and urine samples. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Drug analysis is one of the important tools for drug quality control. Therefore, the development of simple, sensitive, rapid and reliable method for the determination of drug is of great importance. Cetirizine dihydrochloride ([2-[4-[(4chlorophenyl)phenylmethyl]-piperazin-1-yl]ethoxy] acetic acid, dihydrochloride) (CTZH) (Fig. 1) is an active metabolite of hydroxyzine, a first generation H1-receptor antagonist [1]. Marked affinity of cetirizine for peripheral histamine H1 receptors results in antiallergic properties, but has the advantage that it lacks the CNS depressant effects often encountered in anti-histamines [2]. The pKa values of CTZH as a basic compound with three ionizable groups, the carboxylic group, the tertiary amine group and the nitrogen heterocyclic group are 2.2, 2.9 and 8.0 respectively [3]. It is used for the treatment of seasonal and perennial allergetic rhinitis and chronic urticaria [4]. Literature survey reveals that various analytical methods have been reported in literature for determination of CTZH in its pharmaceutical preparations. These include gas chromatography [5], ion-selective electrode [6], fluorimetry [7], high-performance thinlayer chromatography [8], liquid chromatography [9], titration [10], calorimetry [11], high-performance liquid chromatography [12], spectrophotometry [13] and potentiometric methods [14]. Most of
∗ Corresponding author. Tel.: +91 836 2770524; fax: +91 836 2747884. E-mail address: [email protected] (S.T. Nandibewoor). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.11.008
the reported methods require sample pretreatment and extraction of the drug prior to the analysis. These methods are time consuming, intensive solvent-usage and require expensive devices and maintenance. Apparently, there is a need for the development of highly selective, low-cost, stable, and facile CTZH sensors for complex matrixes of pharmaceuticals and industrial fields. Electrochemical detection of analyte is a very elegant method in analytical chemistry [15]. The interest in developing electrochemical-sensing devices for use in environmental monitoring, clinical assays or process control is growing rapidly. Electrochemical sensors satisfy many of the requirements for such tasks particularly owing to their inherent specificity, rapid response, sensitivity and simplicity of preparation for the determination of organic molecules, including drugs and related molecules in pharmaceutical dosage forms and biological fluids [16,17]. Carbon electrodes, especially glassy and paste electrodes are widely used in electrochemical investigations. Till date there is only one report on electro-oxidation of CTZH with glassy carbon electrode [18]. Electrochemical sensors based on carbon nanotubes (CNTs) represent a new and interesting alternative for quantification of different analytes. There are reports on the synthesis of multi-walled carbon nanotubes (MWCNTs) [19] and single-walled carbon nanotubes (SWCNTs) [20]. These materials have attracted enormous interest because of their unique structural, mechanical, electronic and chemical properties [21]. Some of these properties include high chemical and thermal stability, high elasticity, high tensile strength and in some instances, metallic conductivity. The subtle
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OH N
H
Cl
H
Cl
O N
walled carbon nanotubes were purchased from Sigma–Aldrich (>90%, O.D.: 10–15 nm, I.D.: 2–6 nm, length: 0.1–10 M). Phosphate buffer solutions (Ionic strength = 0.2 M) were prepared according to the reported method [27]. The tablets containing CTZH (Zyncet, Unichem Lab. Ltd, India) were purchased from a local pharmacy. All other reagents used were of analytical grade and their solutions were prepared with doubly distilled water. 2.3. Preparation of MWCNT modified electrode
O
Fig. 1. Chemical structure of cetirizine dihydrochloride.
electronic properties suggest that CNTs have capability to promote electron transfer reactions and improve sensitivity in electrochemistry and thus they are widely used as electrodes [22]. CNT modified electrodes have been proved to have excellent electroanalytical properties, such as wide potential window, low background current, low detection limits, high sensitivities, reduction of over potentials and resistance to surface fouling. There are reports which reveal that CNT modified electrodes have shown electrocatalytic behavior [23–25] with excellent performance in the study of a number of biological species [26]. To the best of our knowledge, there is no report on the electrooxidation and determination of CTZH at glassy carbon electrode (GCE) modified with MWCNTs. The objective of the present work is to develop a convenient and sensitive electroanalytical method for the determination of CTZH based on the unique properties of MWCNT modified electrode. Here we report the electro-oxidation of CTZH by cyclic voltammetric method at GCE modified with MWCNTs. The ability of the modified electrode for voltammetric response of CTZH was evaluated. The experimental results showed that the oxidation peak current of CTZH was found to increase to a greater extent for GCE modified with MWCNTs than that of bare GCE. We optimized all the experimental parameters for the determination of CTZH and developed an electroanalytical method for its determination. The modified electrode was also tested for the analysis of CTZH in pharmaceutical and urine samples. The resultant biosensor exhibits high sensitivity, rapid response, good reproducibility and it is independent from other potentially interfering species.
MWCNTs were refluxed in the mixture of concentrated H2 SO4 and HNO3 for 4–5 h, then washed with doubly distilled water and dried in vacuum under ambient conditions. The MWCNT suspension was prepared by dispersing 10 mg of MWCNTs in 10 ml acetonitrile using ultrasonic agitation to obtain a relatively stable suspension. The GCE was carefully polished with 0.30 and 0.05 M ␣-alumina slurry on a polishing cloth, and then washed in an ultrasonic bath of methanol and water, respectively. The cleaned GCE was coated by casting 40 l of the black suspension of MWCNTs and dried in air. The surface areas of the MWCNT-modified GCE and the bare GCE were obtained by cyclic voltammetry using 1.0 × 10−3 M K3 Fe(CN)6 as a probe at different scan rates. For a reversible process, the Randles–Sevcik formula [28] has been used, which is as given below, 1/2
Ipa = (2.69 × 105 )n3/2 ADo Co∗ 1/2
(1)
where Ipa refers to the anodic peak current, n is the number of electrons transferred, A is the surface area of the electrode, Do is diffusion coefficient, is the scan rate and Co∗ is the concentration of K3 Fe(CN)6 . For 1.0 × 10−3 M K3 Fe(CN)6 in 0.1 M KCl electrolyte, n = 1, Do = 7.6 × 10−6 cm2 s−1 , then from the plot of Ipa vs 1/2 , the electroactive area was calculated. In bare GCE, the electrode surface area was found to be 0.04822 cm2 and for MWCNT-modified GCE, the surface was nearly 3.0 times greater than that of bare GCE. 2.4. Analytical procedure The MWCNT-modified GCE was activated in the potential range 0–1.60 V in presence of phosphate buffer (pH 3.0, Ionic strength = 0.2 M) until stable cyclic voltammograms were obtained. Then electrodes were transferred into another cell of phosphate buffer (pH 3.0, Ionic strength = 0.2 M) containing proper amount of CTZH. After accumulating for 180 s at open circuit under stirring and following quiet for 10 s, potential scan was initiated and cyclic voltammograms were recorded between 0.60 and 1.40 V, with a scan rate of 50 mV s−1 . All measurements were carried out at room temperature of 25 ± 0.1 ◦ C.
2. Experimental 2.5. Sample preparation 2.1. Apparatus Electrochemical measurements were carried out on a CHI1110A electrochemical analyzer coupled with a conventional threeelectrode cell (CH Instrument Company, USA). A three-electrode cell was used with a Ag/AgCl as reference electrode, Pt wire as counter electrode and a bare GCE of diameter 3 mm as working electrode, respectively. pH measurements were performed with Elico LI120 pH meter (Elico Ltd., India). All the potentials are given against the Ag/AgCl (3 M KCl). 2.2. Reagents CTZH was obtained from Dr Reddy’s Laboratory, India, and used without further purification. A stock solution of CTZH (1.0 × 10−3 M) was prepared in doubly distilled water. Multi-
Ten pieces of CTZH tablets were powdered in a mortar. A portion equivalent to a stock solution of a concentration of about 1.0 × 10−3 M was accurately weighed and transferred into a 100 ml calibrated flask and diluted with water and was followed by sonication for 10 min for complete dissolution. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant liquid and diluting them with the phosphate buffer solutions. Each solution was transferred to the voltammetric cell and analyzed by standard addition method. The parameters for cyclic voltammetry were set at sample interval 0.001 V and scan rate of 50 mV s−1 . The cyclic voltammograms were recorded between 0.60 and 1.40 V after open-circuit accumulation for 180 s under stirring. The oxidation peak current of CTZH was measured. To study the accuracy of the proposed method and to check the interferences from excipients used in the dosage form, recovery experiments were carried
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Fig. 2. Cyclic voltammograms of 5.0 × 10−5 M CTZH on MWCNT-modified GCE (a) and bare GCE (c). Blank CVs of MWCNT-modified GCE (b) and bare GCE (d). Supporting electrolyte: phosphate buffer with pH 3.0, scan rate: 0.05 V s−1 , accumulation time: 180 s (at open circuit), volume of MWCNT suspension: 40 l.
out. The concentration of CTZH was calculated using standard addition method. 3. Results and discussion 3.1. Cyclic voltammetric behavior of CTZH Fig. 2 shows the cyclic voltammograms at modified and unmodified GCE in phosphate buffer (pH = 3.0, Ionic strength = 0.2 M) at a scan rate of 50 mV s−1 . As seen Fig. 2 the oxidation process of CTZH for both bare and modified GCE was irreversible, but the anodic peak current for the modified GCE (Fig. 2a) is greatly enhanced with the shift in the peak potential for bare GCE from 1.17 V to 1.08 V in case of modified GCE. This result clearly shows the electrocatalytic effect caused by MWCNTs. The reason for the better performance of the MWCNT-modified GCE may be due to the increase in the effective area of the electrode, nanometer dimensions of the MWCNTs, the electronic structure and the topological defects present on the MWCNT surfaces [29]. The modified electrode has no electrochemical activity in phosphate buffer solution (Fig. 2b), but the background current has increased which implies that the electrode surface area has increased after the modification.
Fig. 3. Cyclic voltammograms for the oxidation of CTZH at various suspension (1.0 mg ml−1 ) volumes of MWCNTs. Inset: Influence of MWCNT suspension (1.0 mg ml−1 ) volume used on the anodic peak current. Other conditions are as in Fig. 2.
was adopted. The peak current increases greatly as the accumulation time is increased from 30 to 180 s, which might be due to rapid adsorption of CTZH on the surface of modified electrode. For accumulation time greater than 180 s, the peak current was almost constant (Fig. 4) presumably indicates the saturation accumulation. As too long accumulation time might reduce the stability of MWCNT film, 180 s was generally chosen as accumulation time. The relative standard deviation (RSD) values for accumulation time 30–180 s were in the range from 1.04% to 1.21% (number of measurements = 4). 3.4. Influence of pH The electrode reaction might be affected by the buffer solution and pH of the medium. For controlling pH, buffers such as Britton Robinson, acetate and phosphate buffers were used. The best results with respect to sensitivity accompanied with sharper response were obtained with phosphate buffer (0.2 M). Within the range of pH 3.0–11.2 for phosphate buffer (Fig. 5), the peak potential shifted to less positive value with increasing the pH of the buffer solution. However by increasing the pH, the peak potential
3.2. Influence of amount of MWCNTs
365
Fig. 3 shows the influence of amount of MWCNTs on the peak current. At 40 l of MWCNTs, the peak current was highest. And for further higher concentrations it decreased, which was greatly influenced by the thickness of the film. If the film was too thin, the CTZH amount adsorbed was small, resulting in the small peak current. For thicker films, the film conductivity reduces and the film just peels off from the electrode surface, with decrease in peak current. Therefore, 40 l MWCNT suspension solution was used in the remaining studies.
315
3.3. Influence of accumulation potential and time The influences of accumulation potential and accumulation time have been studied by cyclic voltammetric method as these could affect the amount of adsorption of CTZH at the electrode. The concentration of CTZH used was 5.0 × 10−5 M. When accumulation potential was varied from +0.4 to −0.4 V, there was no considerable change in the peak current. Hence, accumulation at open-circuit
135
Current / µA
265 215 165 115 65 15
30
80
130
180
230
280
330
Accumualtion time / s Fig. 4. Variation of the anodic peak current with accumulation time. Other conditions are as in Fig. 2.
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900
Current / µA
750 600 450 300 150 0 0.00
0.04
0.08
0.12
υ Fig. 5. Variation of peak currents of CTZH with pH. Other conditions are as in Fig. 2. Inset: (A) Influence of pH on peak potential. Other conditions are as in Fig. 2. (B) Influence of pH on peak current. Other conditions are as in Fig. 2.
is shifted to less positive value till pH 8.0, then becomes almost pH independent (Fig. 5A). Basically, two linear regions were obtained, one between pH 3.0 and 8.0 with a slope of 59 mV/pH and another between pH 8.0 and 11.2 with a slope of 9 mV/pH. The intersection of the curve was located around pH 8.0, which is pKa of the nitrogen heterocyclic moiety [3]. From the plot of Ipa vs pH (Fig. 5B) it is clear that, peak current is affected by the pH value. However, the best result with respect to sensitivity accompanied with sharper response was obtained with pH 3.0. So pH 3.0 was selected for further experiment. 3.5. Influence of scan rate Useful information can be acquired from the relationship between peak current and scan rate. Therefore, the electrochemical behavior of CTZH at different scan rates from 5 to 150 mV s−1 was also studied (Fig. 6). There was a good linear relationship between peak current and scan rate and it can be expressed as Ip = 4873.0 + 51.74; r = 0.970 as shown in the Fig. 7. This indicates that the electrode process was controlled by adsorption rather than diffusion. The RSD for Ip vs was 1.36% (number of measurements = 4).
0.16
0.20
/ V s -1
Fig. 7. Dependence of the oxidation peak current on the scan rate. Other conditions are as in Fig. 2.
In addition, there was a linear relation between log Ip and log corresponding to the equation: log Ip = 0.833 log + 3.588; r = 0.993. The slope of 0.833 was close to the theoretically expected value of 1.0 for an adsorption controlled process [30]. With an increase in scan rate, the peak potential shifted to more positive value. The linear relation between peak potential and logarithm of scan rate can be expressed as Ep = 0.130 log + 1.271; r = 0.958 as shown in the Fig. 8. The RSD for Ep vs log was 1.17% (number of measurements = 4). As for an irreversible electrode process, according to Laviron [31], Ep is defined by the following equation: Ep = E0 +
2.303RT ˛nF
log
RTk0 ˛nF
2.303RT +
˛nF
log
(2)
where ˛ is the transfer coefficient, k0 is the standard heterogeneous rate constant of the reaction, n is the number of electrons transferred, is the scan rate and E0 is the formal redox potential. Other symbols have their usual meanings. Thus, the value of ˛n can be easily calculated from the slope of Ep vs log plot. In this system, the slope was 0.130, taking T = 298 K, R = 8.314 J K−1 mol−1 and F = 96,480C, the ˛n was calculated to be 0.4549. Generally ˛ is assumed to be 0.5 in total irreversible electrode process [32]. So the 1.4
1.0
Ep / V
1.2
0.8
-2.6
-2.0
-1.4
-0.8
0.6
log υ / V s -1 Fig. 6. Cyclic voltammograms for the oxidation of CTZH at different scan rates: (a) 5, (b) 10, (c) 25, (d) 50, (e) 75, (f) 100, and (g) 150 mV s−1 . Other conditions are as in Fig. 2.
Fig. 8. Relationship between peak potential and logarithm of scan rate. Other conditions are as in Fig. 2.
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Table 1 Recovery test of CTZH. Added (M)
Found (M)
Recovery (%)
2.0 4.0 6.0 8.0
2.0294 3.9944 6.0546 7.9120
101.47 99.86 100.91 98.90
Table 2 Influence of potential interferents on the voltammetric response of 1.0 × 10−5 M CTZH.
Fig. 9. Cyclic voltammograms of MWCNT-modified GCE in CTZH solution at different concentrations: 0.5 (1), 2.0 (2), 5.0 (3), 6.0 (4) and 10.0 (5) M. Inset: Plot of the peak current against the concentration of CTZH.
number of electrons (n) transferred in the electro-oxidation of CTZH was calculated to be 0.9098–1. The value of k0 can be determined from the intercept of the above plot if the value of E0 is known. The value of E0 in Eq. (2) can be obtained from the intercept of Ep vs curve by extrapolating to the vertical axis at = 0 [33]. In our system the intercept for Ep vs log plot was 1.271 and E0 was obtained to be 0.998, the k0 was calculated to be 3.951 × 103 s−1 . 3.6. Calibration curve According to the obtained results, it was possible to apply this technique to the quantitative analysis of CTZH. The phosphate buffer solution of pH 3.0 was selected as the supporting electrolyte for the quantification of CTZH as it gave maximum peak current. Cyclic voltammograms obtained with increasing amounts of CTZH showed that the peak current increased linearly with increasing concentration, as shown in Fig. 9. Using the optimum conditions described above, linear calibration curves were obtained for CTZH in the range of 5.0 × 10−7 –1.0 × 10−5 M. The linear equation was Ipa (A) = 13.32C (M) + 66.64; r = 0.994. Deviation from linearity was observed for more concentrated solutions, due to the adsorption of CTZH or its oxidation product on the electrode surface. Related statistical data of the calibration curves were obtained from five different calibration curves. The limit of detection (LOD) and quantification (LOQ) were 7.07 × 10−8 M and 2.36 × 10−7 M, respectively. The LOD and LOQ were calculated using the following equations: s LOD = 3 ; m
s LOQ = 10 m
where s is the standard deviation of the peak currents of the blank (five runs), and m is the slope of the calibration curve. The LOD and LOQ carried out by this method were better as compared to the reported LOD and LOQ for electro-oxidation of CTZH with GCE [18]. In order to study the reproducibility of the electrode preparation procedure, a 5.0 × 10−6 M CTZH solution was measured with the same electrode (renewed every time) for several hours within a day, the RSD of the peak current was 2.48% (number of measurements = 6). Reproducibility of the electrode on different days was similar to that of within a day reproducibility if the temperature was kept unchanged. Owing to the adsorption of oxidative product of CTZH on to the electrode surface, the current response of the modified electrode would decrease after successive use. In this case, the electrode should be modified again.
Interferent
Concentration (10−4 M)
Signal change (%)
Glucose Starch Sucrose Citric acid Oxalic acid Gum acacia
1.0 1.0 1.0 1.0 1.0 1.0
+8.83 −0.55 +1.33 −6.94 −7.00 +4.80
3.7. Tablet analysis and recovery test In order to evaluate the applicability of the proposed method in the real sample analysis, it was used to detect CTZH in tablets (Zyncet (10 mg per tablet)). The procedure for the tablet analysis was followed as described in Section 2.5. The results are in good agreement with the content marked in the label. The detected content was 9.85 mg per tablet with 98.5% recovery. Recovery studies were carried out after the addition of known amounts of the drug to various pre-analyzed formulations of CTZH. The results are listed in Table 1. The recovery test of CTZH ranging from 2.0 × 10−6 M to 8.0 × 10−6 M was performed using cyclic voltammetry. The recoveries in different samples were found to lie in the range from 98.90% to 101.47%, with RSD of 0.68%. 3.8. Interference The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error less than ±5% for determination of CTZH. Under the optimum experimental conditions, the effects of potential interferents on the voltammetric response of 1.0 × 10−5 M CTZH as a standard were evaluated. The experimental results (Table 2) show that ten-fold excess concentration of starch, sucrose and gum acacia did not interfere; however, glucose, citric acid and oxalic acid interfered with the voltammetric signal of CTZH. 3.9. Detection of CTZH in urine samples The developed cyclic voltammetric method for the CTZH determination was applied to urine samples. The recoveries from urine were measured by spiking drug free urine with known amounts of CTZH. The urine samples were diluted 100 times with the phosphate buffer solution before analysis without further pretreatments. A quantitative analysis was carried out by adding the standard solution of CTZH into the detect system of urine sample. The calibration graph was used for the determination of spiked Table 3 Determination of CTZH in urine samples. Urine
Spiked (M)
Detected (M)a
Recovery (%)
SD ± R.S.D. (%)
Sample 1 Sample 2 Sample 3 Sample 4
1.0 3.0 5.0 8.0
1.0012 2.9925 4.9750 8.0672
100.12 99.75 99.50 100.84
0.0039 0.0090 0.0127 0.0826
a
Average of five determinations.
± ± ± ±
0.39 0.30 0.25 1.02
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CTZH in urine samples. The detection results of four urine samples obtained are listed in Table 3. The recovery determined was in the range from 99.50% to 100.84% and the standard deviation and RSD are listed in Table 3. 4. Conclusion In this work, a multi-walled carbon nanotube modified glassy carbon electrode has been successfully developed for electrocatalytic oxidation of CTZH in phosphate buffer solution. MWCNTs showed electrocatalytic action for the oxidation of CTZH, characterizing by the enhancement of the peak current, which was probably due to the larger surface area of MWCNTs. The electrochemical oxidation of CTZH was an irreversible and adsorption controlled process. The oxidation mechanism involves transfer of one electron. The peak current was linear to CTZH concentrations over a certain range under the selected conditions. This sensor can be used for voltammetric determination of selected analyte as low as 7.07 × 10−8 M with good reproducibility. The proposed method has distinct advantages over other existing methods regarding sensitivity, accuracy, time saving and minimum detectability. In addition, no sophisticated instrumentation is required. The modified electrode has been used to determine CTZH in pharmaceutical samples. In addition, the results obtained in the analysis of CTZH in spiked urine samples demonstrated the applicability of the method for real sample analysis. References [1] [2] [3] [4]
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Electro-oxidation and determination of antihistamine drug, cetirizine dihydrochloride at glassy carbon electrode modified with multi-walled carbon nanotubes Roopa H. Patil, Rajesh N. Hegde, Sharanappa T. Nandibewoor ∗ P. G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad-580 003, Karnataka, India
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 29 October 2010 Accepted 9 November 2010 Available online 16 November 2010 Keywords: Cetirizine dihydrochloride Multi-walled carbon nanotubes Voltammetric determination Modified electrode
a b s t r a c t A multi-walled carbon nanotube (MWCNT) film-modified glassy carbon electrode (GCE) was constructed for the determination of an antihistamine drug, cetirizine dihydrochloride (CTZH) using cyclic voltammetry (CV). Owing to the unique structure and extraordinary properties of MWCNT, the MWCNT film has shown an obvious electrocatalytic activity towards oxidation of CTZH, since it facilitates the electron transfer and significantly enhances the oxidation peak current of CTZH. All experimental parameters have been optimized. Under the optimum conditions, the oxidation peak current was linearly proportional to the concentration of CTZH in the range from 5.0 × 10−7 to 1.0 × 10−5 M. The detection limit was 7.07 × 10−8 M with 180 s accumulation. Finally, the proposed sensitive and simple electrochemical method was successfully applied to CTZH determination in pharmaceutical and urine samples. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Drug analysis is one of the important tools for drug quality control. Therefore, the development of simple, sensitive, rapid and reliable method for the determination of drug is of great importance. Cetirizine dihydrochloride ([2-[4-[(4chlorophenyl)phenylmethyl]-piperazin-1-yl]ethoxy] acetic acid, dihydrochloride) (CTZH) (Fig. 1) is an active metabolite of hydroxyzine, a first generation H1-receptor antagonist [1]. Marked affinity of cetirizine for peripheral histamine H1 receptors results in antiallergic properties, but has the advantage that it lacks the CNS depressant effects often encountered in anti-histamines [2]. The pKa values of CTZH as a basic compound with three ionizable groups, the carboxylic group, the tertiary amine group and the nitrogen heterocyclic group are 2.2, 2.9 and 8.0 respectively [3]. It is used for the treatment of seasonal and perennial allergetic rhinitis and chronic urticaria [4]. Literature survey reveals that various analytical methods have been reported in literature for determination of CTZH in its pharmaceutical preparations. These include gas chromatography [5], ion-selective electrode [6], fluorimetry [7], high-performance thinlayer chromatography [8], liquid chromatography [9], titration [10], calorimetry [11], high-performance liquid chromatography [12], spectrophotometry [13] and potentiometric methods [14]. Most of
∗ Corresponding author. Tel.: +91 836 2770524; fax: +91 836 2747884. E-mail address: [email protected] (S.T. Nandibewoor). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.11.008
the reported methods require sample pretreatment and extraction of the drug prior to the analysis. These methods are time consuming, intensive solvent-usage and require expensive devices and maintenance. Apparently, there is a need for the development of highly selective, low-cost, stable, and facile CTZH sensors for complex matrixes of pharmaceuticals and industrial fields. Electrochemical detection of analyte is a very elegant method in analytical chemistry [15]. The interest in developing electrochemical-sensing devices for use in environmental monitoring, clinical assays or process control is growing rapidly. Electrochemical sensors satisfy many of the requirements for such tasks particularly owing to their inherent specificity, rapid response, sensitivity and simplicity of preparation for the determination of organic molecules, including drugs and related molecules in pharmaceutical dosage forms and biological fluids [16,17]. Carbon electrodes, especially glassy and paste electrodes are widely used in electrochemical investigations. Till date there is only one report on electro-oxidation of CTZH with glassy carbon electrode [18]. Electrochemical sensors based on carbon nanotubes (CNTs) represent a new and interesting alternative for quantification of different analytes. There are reports on the synthesis of multi-walled carbon nanotubes (MWCNTs) [19] and single-walled carbon nanotubes (SWCNTs) [20]. These materials have attracted enormous interest because of their unique structural, mechanical, electronic and chemical properties [21]. Some of these properties include high chemical and thermal stability, high elasticity, high tensile strength and in some instances, metallic conductivity. The subtle
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OH N
H
Cl
H
Cl
O N
walled carbon nanotubes were purchased from Sigma–Aldrich (>90%, O.D.: 10–15 nm, I.D.: 2–6 nm, length: 0.1–10 M). Phosphate buffer solutions (Ionic strength = 0.2 M) were prepared according to the reported method [27]. The tablets containing CTZH (Zyncet, Unichem Lab. Ltd, India) were purchased from a local pharmacy. All other reagents used were of analytical grade and their solutions were prepared with doubly distilled water. 2.3. Preparation of MWCNT modified electrode
O
Fig. 1. Chemical structure of cetirizine dihydrochloride.
electronic properties suggest that CNTs have capability to promote electron transfer reactions and improve sensitivity in electrochemistry and thus they are widely used as electrodes [22]. CNT modified electrodes have been proved to have excellent electroanalytical properties, such as wide potential window, low background current, low detection limits, high sensitivities, reduction of over potentials and resistance to surface fouling. There are reports which reveal that CNT modified electrodes have shown electrocatalytic behavior [23–25] with excellent performance in the study of a number of biological species [26]. To the best of our knowledge, there is no report on the electrooxidation and determination of CTZH at glassy carbon electrode (GCE) modified with MWCNTs. The objective of the present work is to develop a convenient and sensitive electroanalytical method for the determination of CTZH based on the unique properties of MWCNT modified electrode. Here we report the electro-oxidation of CTZH by cyclic voltammetric method at GCE modified with MWCNTs. The ability of the modified electrode for voltammetric response of CTZH was evaluated. The experimental results showed that the oxidation peak current of CTZH was found to increase to a greater extent for GCE modified with MWCNTs than that of bare GCE. We optimized all the experimental parameters for the determination of CTZH and developed an electroanalytical method for its determination. The modified electrode was also tested for the analysis of CTZH in pharmaceutical and urine samples. The resultant biosensor exhibits high sensitivity, rapid response, good reproducibility and it is independent from other potentially interfering species.
MWCNTs were refluxed in the mixture of concentrated H2 SO4 and HNO3 for 4–5 h, then washed with doubly distilled water and dried in vacuum under ambient conditions. The MWCNT suspension was prepared by dispersing 10 mg of MWCNTs in 10 ml acetonitrile using ultrasonic agitation to obtain a relatively stable suspension. The GCE was carefully polished with 0.30 and 0.05 M ␣-alumina slurry on a polishing cloth, and then washed in an ultrasonic bath of methanol and water, respectively. The cleaned GCE was coated by casting 40 l of the black suspension of MWCNTs and dried in air. The surface areas of the MWCNT-modified GCE and the bare GCE were obtained by cyclic voltammetry using 1.0 × 10−3 M K3 Fe(CN)6 as a probe at different scan rates. For a reversible process, the Randles–Sevcik formula [28] has been used, which is as given below, 1/2
Ipa = (2.69 × 105 )n3/2 ADo Co∗ 1/2
(1)
where Ipa refers to the anodic peak current, n is the number of electrons transferred, A is the surface area of the electrode, Do is diffusion coefficient, is the scan rate and Co∗ is the concentration of K3 Fe(CN)6 . For 1.0 × 10−3 M K3 Fe(CN)6 in 0.1 M KCl electrolyte, n = 1, Do = 7.6 × 10−6 cm2 s−1 , then from the plot of Ipa vs 1/2 , the electroactive area was calculated. In bare GCE, the electrode surface area was found to be 0.04822 cm2 and for MWCNT-modified GCE, the surface was nearly 3.0 times greater than that of bare GCE. 2.4. Analytical procedure The MWCNT-modified GCE was activated in the potential range 0–1.60 V in presence of phosphate buffer (pH 3.0, Ionic strength = 0.2 M) until stable cyclic voltammograms were obtained. Then electrodes were transferred into another cell of phosphate buffer (pH 3.0, Ionic strength = 0.2 M) containing proper amount of CTZH. After accumulating for 180 s at open circuit under stirring and following quiet for 10 s, potential scan was initiated and cyclic voltammograms were recorded between 0.60 and 1.40 V, with a scan rate of 50 mV s−1 . All measurements were carried out at room temperature of 25 ± 0.1 ◦ C.
2. Experimental 2.5. Sample preparation 2.1. Apparatus Electrochemical measurements were carried out on a CHI1110A electrochemical analyzer coupled with a conventional threeelectrode cell (CH Instrument Company, USA). A three-electrode cell was used with a Ag/AgCl as reference electrode, Pt wire as counter electrode and a bare GCE of diameter 3 mm as working electrode, respectively. pH measurements were performed with Elico LI120 pH meter (Elico Ltd., India). All the potentials are given against the Ag/AgCl (3 M KCl). 2.2. Reagents CTZH was obtained from Dr Reddy’s Laboratory, India, and used without further purification. A stock solution of CTZH (1.0 × 10−3 M) was prepared in doubly distilled water. Multi-
Ten pieces of CTZH tablets were powdered in a mortar. A portion equivalent to a stock solution of a concentration of about 1.0 × 10−3 M was accurately weighed and transferred into a 100 ml calibrated flask and diluted with water and was followed by sonication for 10 min for complete dissolution. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant liquid and diluting them with the phosphate buffer solutions. Each solution was transferred to the voltammetric cell and analyzed by standard addition method. The parameters for cyclic voltammetry were set at sample interval 0.001 V and scan rate of 50 mV s−1 . The cyclic voltammograms were recorded between 0.60 and 1.40 V after open-circuit accumulation for 180 s under stirring. The oxidation peak current of CTZH was measured. To study the accuracy of the proposed method and to check the interferences from excipients used in the dosage form, recovery experiments were carried
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Fig. 2. Cyclic voltammograms of 5.0 × 10−5 M CTZH on MWCNT-modified GCE (a) and bare GCE (c). Blank CVs of MWCNT-modified GCE (b) and bare GCE (d). Supporting electrolyte: phosphate buffer with pH 3.0, scan rate: 0.05 V s−1 , accumulation time: 180 s (at open circuit), volume of MWCNT suspension: 40 l.
out. The concentration of CTZH was calculated using standard addition method. 3. Results and discussion 3.1. Cyclic voltammetric behavior of CTZH Fig. 2 shows the cyclic voltammograms at modified and unmodified GCE in phosphate buffer (pH = 3.0, Ionic strength = 0.2 M) at a scan rate of 50 mV s−1 . As seen Fig. 2 the oxidation process of CTZH for both bare and modified GCE was irreversible, but the anodic peak current for the modified GCE (Fig. 2a) is greatly enhanced with the shift in the peak potential for bare GCE from 1.17 V to 1.08 V in case of modified GCE. This result clearly shows the electrocatalytic effect caused by MWCNTs. The reason for the better performance of the MWCNT-modified GCE may be due to the increase in the effective area of the electrode, nanometer dimensions of the MWCNTs, the electronic structure and the topological defects present on the MWCNT surfaces [29]. The modified electrode has no electrochemical activity in phosphate buffer solution (Fig. 2b), but the background current has increased which implies that the electrode surface area has increased after the modification.
Fig. 3. Cyclic voltammograms for the oxidation of CTZH at various suspension (1.0 mg ml−1 ) volumes of MWCNTs. Inset: Influence of MWCNT suspension (1.0 mg ml−1 ) volume used on the anodic peak current. Other conditions are as in Fig. 2.
was adopted. The peak current increases greatly as the accumulation time is increased from 30 to 180 s, which might be due to rapid adsorption of CTZH on the surface of modified electrode. For accumulation time greater than 180 s, the peak current was almost constant (Fig. 4) presumably indicates the saturation accumulation. As too long accumulation time might reduce the stability of MWCNT film, 180 s was generally chosen as accumulation time. The relative standard deviation (RSD) values for accumulation time 30–180 s were in the range from 1.04% to 1.21% (number of measurements = 4). 3.4. Influence of pH The electrode reaction might be affected by the buffer solution and pH of the medium. For controlling pH, buffers such as Britton Robinson, acetate and phosphate buffers were used. The best results with respect to sensitivity accompanied with sharper response were obtained with phosphate buffer (0.2 M). Within the range of pH 3.0–11.2 for phosphate buffer (Fig. 5), the peak potential shifted to less positive value with increasing the pH of the buffer solution. However by increasing the pH, the peak potential
3.2. Influence of amount of MWCNTs
365
Fig. 3 shows the influence of amount of MWCNTs on the peak current. At 40 l of MWCNTs, the peak current was highest. And for further higher concentrations it decreased, which was greatly influenced by the thickness of the film. If the film was too thin, the CTZH amount adsorbed was small, resulting in the small peak current. For thicker films, the film conductivity reduces and the film just peels off from the electrode surface, with decrease in peak current. Therefore, 40 l MWCNT suspension solution was used in the remaining studies.
315
3.3. Influence of accumulation potential and time The influences of accumulation potential and accumulation time have been studied by cyclic voltammetric method as these could affect the amount of adsorption of CTZH at the electrode. The concentration of CTZH used was 5.0 × 10−5 M. When accumulation potential was varied from +0.4 to −0.4 V, there was no considerable change in the peak current. Hence, accumulation at open-circuit
135
Current / µA
265 215 165 115 65 15
30
80
130
180
230
280
330
Accumualtion time / s Fig. 4. Variation of the anodic peak current with accumulation time. Other conditions are as in Fig. 2.
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900
Current / µA
750 600 450 300 150 0 0.00
0.04
0.08
0.12
υ Fig. 5. Variation of peak currents of CTZH with pH. Other conditions are as in Fig. 2. Inset: (A) Influence of pH on peak potential. Other conditions are as in Fig. 2. (B) Influence of pH on peak current. Other conditions are as in Fig. 2.
is shifted to less positive value till pH 8.0, then becomes almost pH independent (Fig. 5A). Basically, two linear regions were obtained, one between pH 3.0 and 8.0 with a slope of 59 mV/pH and another between pH 8.0 and 11.2 with a slope of 9 mV/pH. The intersection of the curve was located around pH 8.0, which is pKa of the nitrogen heterocyclic moiety [3]. From the plot of Ipa vs pH (Fig. 5B) it is clear that, peak current is affected by the pH value. However, the best result with respect to sensitivity accompanied with sharper response was obtained with pH 3.0. So pH 3.0 was selected for further experiment. 3.5. Influence of scan rate Useful information can be acquired from the relationship between peak current and scan rate. Therefore, the electrochemical behavior of CTZH at different scan rates from 5 to 150 mV s−1 was also studied (Fig. 6). There was a good linear relationship between peak current and scan rate and it can be expressed as Ip = 4873.0 + 51.74; r = 0.970 as shown in the Fig. 7. This indicates that the electrode process was controlled by adsorption rather than diffusion. The RSD for Ip vs was 1.36% (number of measurements = 4).
0.16
0.20
/ V s -1
Fig. 7. Dependence of the oxidation peak current on the scan rate. Other conditions are as in Fig. 2.
In addition, there was a linear relation between log Ip and log corresponding to the equation: log Ip = 0.833 log + 3.588; r = 0.993. The slope of 0.833 was close to the theoretically expected value of 1.0 for an adsorption controlled process [30]. With an increase in scan rate, the peak potential shifted to more positive value. The linear relation between peak potential and logarithm of scan rate can be expressed as Ep = 0.130 log + 1.271; r = 0.958 as shown in the Fig. 8. The RSD for Ep vs log was 1.17% (number of measurements = 4). As for an irreversible electrode process, according to Laviron [31], Ep is defined by the following equation: Ep = E0 +
2.303RT ˛nF
log
RTk0 ˛nF
2.303RT +
˛nF
log
(2)
where ˛ is the transfer coefficient, k0 is the standard heterogeneous rate constant of the reaction, n is the number of electrons transferred, is the scan rate and E0 is the formal redox potential. Other symbols have their usual meanings. Thus, the value of ˛n can be easily calculated from the slope of Ep vs log plot. In this system, the slope was 0.130, taking T = 298 K, R = 8.314 J K−1 mol−1 and F = 96,480C, the ˛n was calculated to be 0.4549. Generally ˛ is assumed to be 0.5 in total irreversible electrode process [32]. So the 1.4
1.0
Ep / V
1.2
0.8
-2.6
-2.0
-1.4
-0.8
0.6
log υ / V s -1 Fig. 6. Cyclic voltammograms for the oxidation of CTZH at different scan rates: (a) 5, (b) 10, (c) 25, (d) 50, (e) 75, (f) 100, and (g) 150 mV s−1 . Other conditions are as in Fig. 2.
Fig. 8. Relationship between peak potential and logarithm of scan rate. Other conditions are as in Fig. 2.
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Table 1 Recovery test of CTZH. Added (M)
Found (M)
Recovery (%)
2.0 4.0 6.0 8.0
2.0294 3.9944 6.0546 7.9120
101.47 99.86 100.91 98.90
Table 2 Influence of potential interferents on the voltammetric response of 1.0 × 10−5 M CTZH.
Fig. 9. Cyclic voltammograms of MWCNT-modified GCE in CTZH solution at different concentrations: 0.5 (1), 2.0 (2), 5.0 (3), 6.0 (4) and 10.0 (5) M. Inset: Plot of the peak current against the concentration of CTZH.
number of electrons (n) transferred in the electro-oxidation of CTZH was calculated to be 0.9098–1. The value of k0 can be determined from the intercept of the above plot if the value of E0 is known. The value of E0 in Eq. (2) can be obtained from the intercept of Ep vs curve by extrapolating to the vertical axis at = 0 [33]. In our system the intercept for Ep vs log plot was 1.271 and E0 was obtained to be 0.998, the k0 was calculated to be 3.951 × 103 s−1 . 3.6. Calibration curve According to the obtained results, it was possible to apply this technique to the quantitative analysis of CTZH. The phosphate buffer solution of pH 3.0 was selected as the supporting electrolyte for the quantification of CTZH as it gave maximum peak current. Cyclic voltammograms obtained with increasing amounts of CTZH showed that the peak current increased linearly with increasing concentration, as shown in Fig. 9. Using the optimum conditions described above, linear calibration curves were obtained for CTZH in the range of 5.0 × 10−7 –1.0 × 10−5 M. The linear equation was Ipa (A) = 13.32C (M) + 66.64; r = 0.994. Deviation from linearity was observed for more concentrated solutions, due to the adsorption of CTZH or its oxidation product on the electrode surface. Related statistical data of the calibration curves were obtained from five different calibration curves. The limit of detection (LOD) and quantification (LOQ) were 7.07 × 10−8 M and 2.36 × 10−7 M, respectively. The LOD and LOQ were calculated using the following equations: s LOD = 3 ; m
s LOQ = 10 m
where s is the standard deviation of the peak currents of the blank (five runs), and m is the slope of the calibration curve. The LOD and LOQ carried out by this method were better as compared to the reported LOD and LOQ for electro-oxidation of CTZH with GCE [18]. In order to study the reproducibility of the electrode preparation procedure, a 5.0 × 10−6 M CTZH solution was measured with the same electrode (renewed every time) for several hours within a day, the RSD of the peak current was 2.48% (number of measurements = 6). Reproducibility of the electrode on different days was similar to that of within a day reproducibility if the temperature was kept unchanged. Owing to the adsorption of oxidative product of CTZH on to the electrode surface, the current response of the modified electrode would decrease after successive use. In this case, the electrode should be modified again.
Interferent
Concentration (10−4 M)
Signal change (%)
Glucose Starch Sucrose Citric acid Oxalic acid Gum acacia
1.0 1.0 1.0 1.0 1.0 1.0
+8.83 −0.55 +1.33 −6.94 −7.00 +4.80
3.7. Tablet analysis and recovery test In order to evaluate the applicability of the proposed method in the real sample analysis, it was used to detect CTZH in tablets (Zyncet (10 mg per tablet)). The procedure for the tablet analysis was followed as described in Section 2.5. The results are in good agreement with the content marked in the label. The detected content was 9.85 mg per tablet with 98.5% recovery. Recovery studies were carried out after the addition of known amounts of the drug to various pre-analyzed formulations of CTZH. The results are listed in Table 1. The recovery test of CTZH ranging from 2.0 × 10−6 M to 8.0 × 10−6 M was performed using cyclic voltammetry. The recoveries in different samples were found to lie in the range from 98.90% to 101.47%, with RSD of 0.68%. 3.8. Interference The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error less than ±5% for determination of CTZH. Under the optimum experimental conditions, the effects of potential interferents on the voltammetric response of 1.0 × 10−5 M CTZH as a standard were evaluated. The experimental results (Table 2) show that ten-fold excess concentration of starch, sucrose and gum acacia did not interfere; however, glucose, citric acid and oxalic acid interfered with the voltammetric signal of CTZH. 3.9. Detection of CTZH in urine samples The developed cyclic voltammetric method for the CTZH determination was applied to urine samples. The recoveries from urine were measured by spiking drug free urine with known amounts of CTZH. The urine samples were diluted 100 times with the phosphate buffer solution before analysis without further pretreatments. A quantitative analysis was carried out by adding the standard solution of CTZH into the detect system of urine sample. The calibration graph was used for the determination of spiked Table 3 Determination of CTZH in urine samples. Urine
Spiked (M)
Detected (M)a
Recovery (%)
SD ± R.S.D. (%)
Sample 1 Sample 2 Sample 3 Sample 4
1.0 3.0 5.0 8.0
1.0012 2.9925 4.9750 8.0672
100.12 99.75 99.50 100.84
0.0039 0.0090 0.0127 0.0826
a
Average of five determinations.
± ± ± ±
0.39 0.30 0.25 1.02
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CTZH in urine samples. The detection results of four urine samples obtained are listed in Table 3. The recovery determined was in the range from 99.50% to 100.84% and the standard deviation and RSD are listed in Table 3. 4. Conclusion In this work, a multi-walled carbon nanotube modified glassy carbon electrode has been successfully developed for electrocatalytic oxidation of CTZH in phosphate buffer solution. MWCNTs showed electrocatalytic action for the oxidation of CTZH, characterizing by the enhancement of the peak current, which was probably due to the larger surface area of MWCNTs. The electrochemical oxidation of CTZH was an irreversible and adsorption controlled process. The oxidation mechanism involves transfer of one electron. The peak current was linear to CTZH concentrations over a certain range under the selected conditions. This sensor can be used for voltammetric determination of selected analyte as low as 7.07 × 10−8 M with good reproducibility. The proposed method has distinct advantages over other existing methods regarding sensitivity, accuracy, time saving and minimum detectability. In addition, no sophisticated instrumentation is required. The modified electrode has been used to determine CTZH in pharmaceutical samples. In addition, the results obtained in the analysis of CTZH in spiked urine samples demonstrated the applicability of the method for real sample analysis. References [1] [2] [3] [4]
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