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Determination of lamotrigine by adsorptive stripping voltammetry using silver nanoparticle-modified carbon screen-printed electrodes
Available online at www.sciencedirect.com
Talanta 74 (2007) 59–64
Determination of lamotrigine by adsorptive stripping voltammetry using silver nanoparticle-modified carbon screen-printed electrodes M. Encarnaci´on Burgoa Calvo, Olga Dom´ınguez Renedo, M. Julia Arcos Mart´ınez ∗ ´ Departamento de Qu´ımica, Area de Qu´ımica Anal´ıtica, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Ba˜nuelos s/n, E-09001 Burgos, Spain Received 22 February 2007; received in revised form 9 May 2007; accepted 18 May 2007 Available online 24 May 2007
Abstract Carbon screen-printed electrodes (CSPE) modified with silver nanoparticles present an interesting alternative in the determination of lamotrigine (LTG) using differential pulse adsorptive stripping voltammetry. Metallic silver nanoparticle deposits have been obtained by electrochemical deposition. Scanning electron microscopy measurements show that the electrochemically synthesized silver nanoparticles are deposited in aggregated form. The detection limit for this analytical procedure was 3.72 × 10−7 M. In terms of reproducibility, the precision of the above mentioned method in %R.S.D. values was calculated at 2.58%. The method was applied satisfactorily to the determination of LTG in pharmaceutical preparations. © 2007 Published by Elsevier B.V. Keywords: Silver nanoparticles; Screen-printed electrodes; Lamotrigine; Adsorptive stripping voltammetry
1. Introduction Lamotrigine (LTG), 3,5-diamino-6-(2,3-dichlorophenyl)1,2,4-triazine (Fig. 1) is a new-generation antiepileptic drug registered for treatment of patients with refractory partial seizures with or without secondary generalization [1,2]. Lamotrigine is thought to act at voltage-sensitive neuronal membranes and inhibit the release of excitatory amino acid neurotransmitters, in particular glutamate and aspartate, which play an important role in the generation and spread of epileptic seizures. HPLC [3–7] and capillary electrophoresis [8] are among the different techniques generally used for the measurement of LTG concentrations in pharmaceutical products and biological fluids. Electrochemical techniques applied to the determination of LTG provide an interesting alternative to the chromatographic methods that are widely used at present. Together with the recognized advantage of the relatively low cost of electrochemical ∗
Corresponding author. E-mail addresses: [email protected] (M.E. Burgoa Calvo), [email protected] (O. Dom´ınguez Renedo), [email protected] (M.J. Arcos Mart´ınez). 0039-9140/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.talanta.2007.05.026
instrumentation, one should bear in mind the high sensitivity of methods such as stripping voltammetry, based on the adsorption exhibited by numerous organic compounds on some electrodes. Despite the presence of redox groups in this molecule, only one work can be found in the literature describing the electrochemical analysis of LTG by means of adsorptive stripping voltammetry using a HMDE electrode [9]. Screen-printed electrodes are planar devices with plastic substrates that are coated with layers of electroconductive and insulating inks at controlled thickness. The advent of screen-printed (thick-film) technology has made it possible to mass-produce inexpensive disposable electrodes for use with electrochemical instruments [10–14]. Their use in potentiometric, amperometric and voltammetric devices have been reported for the detection of different compounds [15–27] although the bibliography shows very few examples of determination of drugs such as lamotrigine [28,29]. The great versatility of screen-printed electrodes resides in their wide range of possible modifications. In fact, the composition of the inks used in the printing process can be modified by the addition of substances of a very different nature, such as metals, enzymes, polymers, complexing agents, etc. Moreover, the possibility also exists of modifying the electrodes once they
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M.E. Burgoa Calvo et al. / Talanta 74 (2007) 59–64
Fig. 1. Chemical structure of lamotrigine.
have been printed through the deposition of films containing those substances. The design of new nanoscale materials has acquired evergreater importance in recent years due to their wide-ranging applications in various fields. Among these materials, metallic nanoparticles are of great interest due to their important properties and their numerous possible applications [30,31]. The bibliography lists numerous methods describing the synthesis of metallic silver nanoparticles in solution as well as by deposition on solid surfaces. These include chemical synthesis by means of reduction with different reagents [32], UV light or electronbeam irradiation [33] and electrochemical methods [34–39]. The latter provides an easy and rapid alternative for the preparation of metallic nanoparticle electrodes in a short period of time. The aim of this work is to determine LTG by differential pulse adsorptive stripping voltammetry (DPAdSV) using screen-printed electrodes modified with silver nanoparticles and, to the best of the author’s knowledge, presents the first ever electrochemical detection of this molecule with these types of electrodes. 2. Experimental
Silver nitrate was from VWR International and hydrochloric acid was purchased from Merck (analytical-reagent grade, Merck, Darmstadt, Germany). Commercial capsules of LAMICTAL® were obtained from GlaxoSmithKline. The excipients of the tablet are: blackcurrant flavour, calcium carbonate, low-substituted hydroxypropylcellulose, magnesium aluminium silicate, magnesium steareate, povidone, saccharin sodium and starch glycolate. 2.2. Apparatus Screen-printed electrodes were produced on a DEK 248 printing machine (DEK, Weymouth, UK) using polyester screens with appropriate stencil designs mounted at 45◦ to the printer stroke. Voltammetric measurements were taken using a Autolab electrochemical system with GPES software (Eco Chemie, Utrecht, The Netherlands). Centrifugation of samples was carried out in an Angular 6 centrifuge (Selecta, Barcelona, Spain). Scanning electron microscopy (SEM) images were obtained using a JEOL JSM-6460LV with an INCA elemental X-ray analysis system. 2.3. Screen printed electrodes preparation Hand-made screen-printed electrodes were used in the determination of LTG. For the construction of the screen-printed electrodes successive layers of different inks were printed onto a PVC strip substrate (30 mm × 15 mm, 0.5 mm thick) using four different screens with appropriate stencils to transfer the required design (Fig. 2). The printing procedure was as follows:
2.1. Reagents All solutions were prepared with deionized water obtained with a Barnstead NANO Pure II system. Electrodag PF-407 A (carbon ink), Electrodag 418 SS (silver ink), Electrodag 6037 SS (silver/silver chloride ink) and Electrodag 452 SS (dielectric ink) were supplied by Achenson Colloiden (Scheemda, The Netherlands). Britton-Robinson solutions were used as buffers. A 0.04 M Britton-Robinson buffer solution for the o-boric, o-phosphoric and acetic acids was prepared using Merck analytical grade reagents. Solutions of different pH values were prepared from this by the addition of 0.2 M sodium hydroxide (analyticalreagent grade, Merck, Darmstadt, Germany).
(1) Firstly, three parallel conducting base-patterns were screenprinted on the PVC strip substrate with commercial silver ink, giving them an effective conductive surface, and they were then cured for 15 min at 90 ◦ C. The base-pattern at the left was used as the counter electrode. (2) A silver/silver chloride reference electrode was screenprinted using silver/silver chloride ink on the silver base-pattern at the right, as can be seen in Fig. 2, and then cured for 15 min at 90 ◦ C. (3) The working electrode was formed by screen printing a graphite layer over the silver base pattern at the centre using commercial graphite ink and was then cured for 15 min at 90 ◦ C.
Fig. 2. Schematic diagram of the sensor preparation procedure.
M.E. Burgoa Calvo et al. / Talanta 74 (2007) 59–64
(4) An insulator layer was finally printed over all of the sensor strip except for the three electrode surfaces and the electrical connection at the reverse end of the sensor strip, which was subsequently cured under UV radiation. 2.4. Preparation of nanoparticle-modified electrodes Despite the potential advantages of using silver nanoparticlemodified carbon screen-printed electrodes as working electrodes in electrochemical techniques, no works have been found in the reference literature on the applications of such electrodes for drug analysis. In comparison to other electrochemical methods described for the generation of silver nanoparticles [38,39], this work describes a new and easy procedure to obtain silver nanoparticlemodified CSPE. The method consists in the electrochemical deposition of silver on the working-electrode surface, using a solution of AgNO3 in Britton-Robinson (pH 2). The deposition was performed by applying an accumulation potential during a time under stirring. 3. Results and discussion 3.1. Optimization of the experimental parameters for silver nanoparticle-modified CSPE formation No analytical signal for LTG was observed when Ag solid electrodes were used. However, modification of the graphite electrode by the deposition of silver nanoparticles produced signals of a sufficiently high quality for analytical purposes, as may be observed from Fig. 3. This figure shows a peak due to
Fig. 3. Differential pulse voltammograms obtained for LTG in BrittonRobinson buffer, using silver nanoparticle-modified CSPE. pH 5.5, tdep = 200 s, Edep = −0.9 V and [LTG] = 1.56 × 10−5 M. Optimum silver concentration was used for the preparation of the electrode.
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reduction of LTG at a potential about −1.06 V, which is notably influenced by the features of the silver coating. Previous experiments showed that very acid pH values are needed to deposit silver in the carbon surface of the electrode. For this reason pH 2 Britton-Robinson solution was used in the deposition stage. In the same way, to deposit silver in the carbon surface of the electrodes, long deposition times or very negative potentials were needed so, we decided to use −1.20 V and 120 s as conditions for the deposition. Finally, silver concentration was optimized by addition of different volumes of 5.0 × 10−3 M AgNO3 solution into a 10 mL cell containing Britton-Robinson pH 2. The silver nanoparticle modified CSPE was introduced into a solution containing 1.56 × 10−5 M of LTG and a DPAdSV scan was carried out under the following conditions, pH 5.5, Edep = −0.90 V and tdep = 200 s. As shown in Fig. 4 the highest peak intensity was reached for a concentration of AgNO3 of 0.83 mM. It has been observed that a poor response for LTG is obtained for high concentrations of AgNO3 . This behaviour can be explained by the formation of a partial silver film on the CSPE electrode as can be seen in Fig. 5, which shows the SEM images of the silver nanoparticles electrodeposited onto the CSPE under different operating conditions. 3.2. Characterization of silver deposition SEM analysis shows the formation of silver nanoparticles on the CSPE surface which are deposited in aggregated form. The effect of the silver concentration in the solution is shown in Fig. 5, which depicts different images taken, as well as the image of a clean CSPE. It can be observed that the higher silver concentration, the greater the nanoparticles size obtained (Fig. 5b–d).
Fig. 4. Effect of silver concentration in the LTG reduction peak using a silver nanoparticle-modified CSPE. pH 5.5, tdep = 200 s, Edep = −0.9 V and [LTG] = 1.56 × 10−5 M.
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Fig. 5. SEM images of no modified CSPE and silver nanoparticle-modified CSPE: (a) no modified CSPE; (b–d) silver nanoparticle-modified CSPE prepared by electrodeposition at a potential of −1.2 V during 120 s from a pH 2 Britton-Robinson solution containing various concentrations of AgNO3 ; (b) 0.45 mM AgNO3 ; (c) 0.83 mM AgNO3 ; (d) 1.15 mM AgNO3 .
Moreover, energy-dispersive X-ray analysis (EDX) of CSPE confirmed the presence of deposited silver. 3.3. Optimization of adsorptive stripping parameters for LTG determination Once silver coated has been optimized the next step is to optimize the parameters which influence on DPAdSV. It is well known that deposition potential (Edep ), deposition time (tdep ) and pH value have a great influence in DPAdSV. Previous experiments have shown that there is no analytical signal for pH values very different of 5.5. For this reason a BrittonRobinson buffer solution of pH 5.5 was chosen as the supporting electrolyte. In order to obtain a sensitive analytical signal a 22 central composite design was carried out with the remaining factors. The values which correspond to the high (+) and low (−) levels and to the central point (0) for each factor are as follows: Edep (+)=−0.70 V Edep (−)=−1.10 V Edep (0)= − 0.90 V tdep (+) = 250 s tdep (−) = 50 s tdep (0) = 150 s The response to be optimized was the intensity (−iP ), at a potential of −1.06 V, of a LTG sample at a concentration of 2.0 × 10−6 M. From analysis of the variance (ANOVA) in Table 1, it can be seen that a second order function is adequate to model the data because the lack of fit is not significant at the 95% confidence level. A maximum can be observed in Fig. 6 which corresponds to a potential of −0.90 V and an accumulation time of 147 s.
3.4. Calibration and detection limit Once the optimal experimental conditions were found for the analysis of LTG by means of silver nanoparticle-modified CSPE, a calibration was performed using a least-median-squares regression (LMS) to detect the existence of anomalous points [40], which might have led to incorrect adjustments altering the sensitivity and the detection limit. The calibration equation obtained by DPAdSV for standard solutions containing LTG concentrations of between 3.30 × 10−7 and 1.50 × 10−6 M was I = −33.98 + 1.25 × 108 C (R2 = 0.99, syx = 4.99).
Table 1 ANOVA of the data obtained with the 22 central composite design for optimization of experimental variables in LTG determination with silver nanoparticle-modified CSPE Effect
SS
d.f.
MS
Fratio
Plevel
A: Edep B: tdep AA AB BB Lack of fit Pure error Total R2 = 0.933
888.06 173.83 52050.70 190.44 32833.70 3493.91 1730.34 72781.70
1 1 1 1 1 3 2 10
888.06 173.83 52050.70 190.44 32833.70 1165.64 865.17
1.03 0.20 60.16 0.22 37.95 1.35
0.418 0.698 0.016a 0.685 0.025 0.453
[LTG] = 2.0 × 10−6 M. SS, sum of squares; d.f., degrees of freedom; MS, mean squares; Fratio : MSfactor /MSerror . Plevel , probability level. a Significant factor at α = 0.05.
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3.6. Analytical application The concentration of LTG in commercial capsules of LAMICTAL® (GlaxoSmithKline) with a known concentration of analyte, was determined using the DPAdSV method, so as to evaluate the accuracy of the proposed method. In order to determine the concentration of LTG in LAMICTAL® 10 different capsules of the pharmaceutical preparation were analyzed. The following procedure was carried out for each capsule:
Fig. 6. Response surface for the 22 central composite design for optimization of experimental variables in LTG determination by DPAdSV.
(1) The tablet was pulverized with a pestle and dissolve in HCl 0.1 M. (2) The resulting solution was centrifuged at 5000 rpm for 1 h. (3) After centrifugation the supernatant was collected and used as a stock solution of the sample.
A key feature of any analytical method is its detection limit; the smallest concentration of the analyte that can be detected to a specified degree of certainty. Calculation of the detection limit, based on the variability of ten samples with a very low analyte concentration, was calculated according to [41] and ISO 11843–2 [42]. At the chosen probability level of 5% (α = β = 0.05), the detection limit was 3.72 × 10−7 M. This limit of detection value is one order less than the values obtained when LTG was measured with carbon and mercuryfilm carbon screen-printed electrodes. Moreover, as can be seen in Fig. 7, the analytical signal is higher when LTG is analyze with nanoparticle-modified electrodes
Finally, the determination of the LTG content was made in an aliquot of the stock solution described above by means of the AdSV using the silver nanoparticle-modified CSPE developed in this work. Good agreement was obtained between the amount of LTG found by the standard addition 26 ± 1 mg with n = 10 and α = 0.05 and the value supplied by the manufacturer 25 ± 1 mg. These results were also checked using the HPLC method described in Ref. [4] as a reference technique obtaining 25 ± 1 mg (n = 3, α = 0.05).
3.5. Precision
The silver nanoparticle-modified carbon screen printed electrodes developed in this work present an environmentallyfriendly method for the analysis of LTG. Their most important advantage is the high sensitivity in the determination LTG in real samples. In fact, the detection limit obtained was less than the values found when LTG was measured with carbon and mercuryfilm carbon screen-printed electrodes. For this reason we can assure that this is the best way, known at the present time, to analyze LTG using screen printed electrodes.
This parameter was calculated in terms of reproducibility. A series of measurements of three different samples containing 2.0 × 10−7 M of LTG were carried out obtaining a %R.S.D. value of 2.58%.
4. Conclusions
Acknowledgements The financial support made available by the Junta de Castilla y Le´on (Burgos 08/04) and the Ministerio de Ciencia y Tecnolog´ıa (BQU2001-1126) is gratefully acknowledged. The ´ authors also thank Dr. Alvaro Colina from Universidad de Burgos for the help with SEM images. M. Encarnaci´on Burgoa Calvo also thanks Ministerio de Educaci´on y Ciencia for the F.P.U. grant. References Fig. 7. DPAdSV voltammograms of 5 × 10−6 M of LTG in: (1) Hg-film-CSPE (pH 5.53, tdep = 105 s and Edep = 0.40 V); (2) CSPE (pH 5.50, tdep = 28 s and Edep = 0.05 V); (3) silver nanoparticle-modified CSPE (pH 5.5, tdep = 147 s and Edep = −0.90 V). Measurements were carried out in the optimal conditions for each electrode.
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Talanta 74 (2007) 59–64
Determination of lamotrigine by adsorptive stripping voltammetry using silver nanoparticle-modified carbon screen-printed electrodes M. Encarnaci´on Burgoa Calvo, Olga Dom´ınguez Renedo, M. Julia Arcos Mart´ınez ∗ ´ Departamento de Qu´ımica, Area de Qu´ımica Anal´ıtica, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Ba˜nuelos s/n, E-09001 Burgos, Spain Received 22 February 2007; received in revised form 9 May 2007; accepted 18 May 2007 Available online 24 May 2007
Abstract Carbon screen-printed electrodes (CSPE) modified with silver nanoparticles present an interesting alternative in the determination of lamotrigine (LTG) using differential pulse adsorptive stripping voltammetry. Metallic silver nanoparticle deposits have been obtained by electrochemical deposition. Scanning electron microscopy measurements show that the electrochemically synthesized silver nanoparticles are deposited in aggregated form. The detection limit for this analytical procedure was 3.72 × 10−7 M. In terms of reproducibility, the precision of the above mentioned method in %R.S.D. values was calculated at 2.58%. The method was applied satisfactorily to the determination of LTG in pharmaceutical preparations. © 2007 Published by Elsevier B.V. Keywords: Silver nanoparticles; Screen-printed electrodes; Lamotrigine; Adsorptive stripping voltammetry
1. Introduction Lamotrigine (LTG), 3,5-diamino-6-(2,3-dichlorophenyl)1,2,4-triazine (Fig. 1) is a new-generation antiepileptic drug registered for treatment of patients with refractory partial seizures with or without secondary generalization [1,2]. Lamotrigine is thought to act at voltage-sensitive neuronal membranes and inhibit the release of excitatory amino acid neurotransmitters, in particular glutamate and aspartate, which play an important role in the generation and spread of epileptic seizures. HPLC [3–7] and capillary electrophoresis [8] are among the different techniques generally used for the measurement of LTG concentrations in pharmaceutical products and biological fluids. Electrochemical techniques applied to the determination of LTG provide an interesting alternative to the chromatographic methods that are widely used at present. Together with the recognized advantage of the relatively low cost of electrochemical ∗
Corresponding author. E-mail addresses: [email protected] (M.E. Burgoa Calvo), [email protected] (O. Dom´ınguez Renedo), [email protected] (M.J. Arcos Mart´ınez). 0039-9140/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.talanta.2007.05.026
instrumentation, one should bear in mind the high sensitivity of methods such as stripping voltammetry, based on the adsorption exhibited by numerous organic compounds on some electrodes. Despite the presence of redox groups in this molecule, only one work can be found in the literature describing the electrochemical analysis of LTG by means of adsorptive stripping voltammetry using a HMDE electrode [9]. Screen-printed electrodes are planar devices with plastic substrates that are coated with layers of electroconductive and insulating inks at controlled thickness. The advent of screen-printed (thick-film) technology has made it possible to mass-produce inexpensive disposable electrodes for use with electrochemical instruments [10–14]. Their use in potentiometric, amperometric and voltammetric devices have been reported for the detection of different compounds [15–27] although the bibliography shows very few examples of determination of drugs such as lamotrigine [28,29]. The great versatility of screen-printed electrodes resides in their wide range of possible modifications. In fact, the composition of the inks used in the printing process can be modified by the addition of substances of a very different nature, such as metals, enzymes, polymers, complexing agents, etc. Moreover, the possibility also exists of modifying the electrodes once they
60
M.E. Burgoa Calvo et al. / Talanta 74 (2007) 59–64
Fig. 1. Chemical structure of lamotrigine.
have been printed through the deposition of films containing those substances. The design of new nanoscale materials has acquired evergreater importance in recent years due to their wide-ranging applications in various fields. Among these materials, metallic nanoparticles are of great interest due to their important properties and their numerous possible applications [30,31]. The bibliography lists numerous methods describing the synthesis of metallic silver nanoparticles in solution as well as by deposition on solid surfaces. These include chemical synthesis by means of reduction with different reagents [32], UV light or electronbeam irradiation [33] and electrochemical methods [34–39]. The latter provides an easy and rapid alternative for the preparation of metallic nanoparticle electrodes in a short period of time. The aim of this work is to determine LTG by differential pulse adsorptive stripping voltammetry (DPAdSV) using screen-printed electrodes modified with silver nanoparticles and, to the best of the author’s knowledge, presents the first ever electrochemical detection of this molecule with these types of electrodes. 2. Experimental
Silver nitrate was from VWR International and hydrochloric acid was purchased from Merck (analytical-reagent grade, Merck, Darmstadt, Germany). Commercial capsules of LAMICTAL® were obtained from GlaxoSmithKline. The excipients of the tablet are: blackcurrant flavour, calcium carbonate, low-substituted hydroxypropylcellulose, magnesium aluminium silicate, magnesium steareate, povidone, saccharin sodium and starch glycolate. 2.2. Apparatus Screen-printed electrodes were produced on a DEK 248 printing machine (DEK, Weymouth, UK) using polyester screens with appropriate stencil designs mounted at 45◦ to the printer stroke. Voltammetric measurements were taken using a Autolab electrochemical system with GPES software (Eco Chemie, Utrecht, The Netherlands). Centrifugation of samples was carried out in an Angular 6 centrifuge (Selecta, Barcelona, Spain). Scanning electron microscopy (SEM) images were obtained using a JEOL JSM-6460LV with an INCA elemental X-ray analysis system. 2.3. Screen printed electrodes preparation Hand-made screen-printed electrodes were used in the determination of LTG. For the construction of the screen-printed electrodes successive layers of different inks were printed onto a PVC strip substrate (30 mm × 15 mm, 0.5 mm thick) using four different screens with appropriate stencils to transfer the required design (Fig. 2). The printing procedure was as follows:
2.1. Reagents All solutions were prepared with deionized water obtained with a Barnstead NANO Pure II system. Electrodag PF-407 A (carbon ink), Electrodag 418 SS (silver ink), Electrodag 6037 SS (silver/silver chloride ink) and Electrodag 452 SS (dielectric ink) were supplied by Achenson Colloiden (Scheemda, The Netherlands). Britton-Robinson solutions were used as buffers. A 0.04 M Britton-Robinson buffer solution for the o-boric, o-phosphoric and acetic acids was prepared using Merck analytical grade reagents. Solutions of different pH values were prepared from this by the addition of 0.2 M sodium hydroxide (analyticalreagent grade, Merck, Darmstadt, Germany).
(1) Firstly, three parallel conducting base-patterns were screenprinted on the PVC strip substrate with commercial silver ink, giving them an effective conductive surface, and they were then cured for 15 min at 90 ◦ C. The base-pattern at the left was used as the counter electrode. (2) A silver/silver chloride reference electrode was screenprinted using silver/silver chloride ink on the silver base-pattern at the right, as can be seen in Fig. 2, and then cured for 15 min at 90 ◦ C. (3) The working electrode was formed by screen printing a graphite layer over the silver base pattern at the centre using commercial graphite ink and was then cured for 15 min at 90 ◦ C.
Fig. 2. Schematic diagram of the sensor preparation procedure.
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(4) An insulator layer was finally printed over all of the sensor strip except for the three electrode surfaces and the electrical connection at the reverse end of the sensor strip, which was subsequently cured under UV radiation. 2.4. Preparation of nanoparticle-modified electrodes Despite the potential advantages of using silver nanoparticlemodified carbon screen-printed electrodes as working electrodes in electrochemical techniques, no works have been found in the reference literature on the applications of such electrodes for drug analysis. In comparison to other electrochemical methods described for the generation of silver nanoparticles [38,39], this work describes a new and easy procedure to obtain silver nanoparticlemodified CSPE. The method consists in the electrochemical deposition of silver on the working-electrode surface, using a solution of AgNO3 in Britton-Robinson (pH 2). The deposition was performed by applying an accumulation potential during a time under stirring. 3. Results and discussion 3.1. Optimization of the experimental parameters for silver nanoparticle-modified CSPE formation No analytical signal for LTG was observed when Ag solid electrodes were used. However, modification of the graphite electrode by the deposition of silver nanoparticles produced signals of a sufficiently high quality for analytical purposes, as may be observed from Fig. 3. This figure shows a peak due to
Fig. 3. Differential pulse voltammograms obtained for LTG in BrittonRobinson buffer, using silver nanoparticle-modified CSPE. pH 5.5, tdep = 200 s, Edep = −0.9 V and [LTG] = 1.56 × 10−5 M. Optimum silver concentration was used for the preparation of the electrode.
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reduction of LTG at a potential about −1.06 V, which is notably influenced by the features of the silver coating. Previous experiments showed that very acid pH values are needed to deposit silver in the carbon surface of the electrode. For this reason pH 2 Britton-Robinson solution was used in the deposition stage. In the same way, to deposit silver in the carbon surface of the electrodes, long deposition times or very negative potentials were needed so, we decided to use −1.20 V and 120 s as conditions for the deposition. Finally, silver concentration was optimized by addition of different volumes of 5.0 × 10−3 M AgNO3 solution into a 10 mL cell containing Britton-Robinson pH 2. The silver nanoparticle modified CSPE was introduced into a solution containing 1.56 × 10−5 M of LTG and a DPAdSV scan was carried out under the following conditions, pH 5.5, Edep = −0.90 V and tdep = 200 s. As shown in Fig. 4 the highest peak intensity was reached for a concentration of AgNO3 of 0.83 mM. It has been observed that a poor response for LTG is obtained for high concentrations of AgNO3 . This behaviour can be explained by the formation of a partial silver film on the CSPE electrode as can be seen in Fig. 5, which shows the SEM images of the silver nanoparticles electrodeposited onto the CSPE under different operating conditions. 3.2. Characterization of silver deposition SEM analysis shows the formation of silver nanoparticles on the CSPE surface which are deposited in aggregated form. The effect of the silver concentration in the solution is shown in Fig. 5, which depicts different images taken, as well as the image of a clean CSPE. It can be observed that the higher silver concentration, the greater the nanoparticles size obtained (Fig. 5b–d).
Fig. 4. Effect of silver concentration in the LTG reduction peak using a silver nanoparticle-modified CSPE. pH 5.5, tdep = 200 s, Edep = −0.9 V and [LTG] = 1.56 × 10−5 M.
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Fig. 5. SEM images of no modified CSPE and silver nanoparticle-modified CSPE: (a) no modified CSPE; (b–d) silver nanoparticle-modified CSPE prepared by electrodeposition at a potential of −1.2 V during 120 s from a pH 2 Britton-Robinson solution containing various concentrations of AgNO3 ; (b) 0.45 mM AgNO3 ; (c) 0.83 mM AgNO3 ; (d) 1.15 mM AgNO3 .
Moreover, energy-dispersive X-ray analysis (EDX) of CSPE confirmed the presence of deposited silver. 3.3. Optimization of adsorptive stripping parameters for LTG determination Once silver coated has been optimized the next step is to optimize the parameters which influence on DPAdSV. It is well known that deposition potential (Edep ), deposition time (tdep ) and pH value have a great influence in DPAdSV. Previous experiments have shown that there is no analytical signal for pH values very different of 5.5. For this reason a BrittonRobinson buffer solution of pH 5.5 was chosen as the supporting electrolyte. In order to obtain a sensitive analytical signal a 22 central composite design was carried out with the remaining factors. The values which correspond to the high (+) and low (−) levels and to the central point (0) for each factor are as follows: Edep (+)=−0.70 V Edep (−)=−1.10 V Edep (0)= − 0.90 V tdep (+) = 250 s tdep (−) = 50 s tdep (0) = 150 s The response to be optimized was the intensity (−iP ), at a potential of −1.06 V, of a LTG sample at a concentration of 2.0 × 10−6 M. From analysis of the variance (ANOVA) in Table 1, it can be seen that a second order function is adequate to model the data because the lack of fit is not significant at the 95% confidence level. A maximum can be observed in Fig. 6 which corresponds to a potential of −0.90 V and an accumulation time of 147 s.
3.4. Calibration and detection limit Once the optimal experimental conditions were found for the analysis of LTG by means of silver nanoparticle-modified CSPE, a calibration was performed using a least-median-squares regression (LMS) to detect the existence of anomalous points [40], which might have led to incorrect adjustments altering the sensitivity and the detection limit. The calibration equation obtained by DPAdSV for standard solutions containing LTG concentrations of between 3.30 × 10−7 and 1.50 × 10−6 M was I = −33.98 + 1.25 × 108 C (R2 = 0.99, syx = 4.99).
Table 1 ANOVA of the data obtained with the 22 central composite design for optimization of experimental variables in LTG determination with silver nanoparticle-modified CSPE Effect
SS
d.f.
MS
Fratio
Plevel
A: Edep B: tdep AA AB BB Lack of fit Pure error Total R2 = 0.933
888.06 173.83 52050.70 190.44 32833.70 3493.91 1730.34 72781.70
1 1 1 1 1 3 2 10
888.06 173.83 52050.70 190.44 32833.70 1165.64 865.17
1.03 0.20 60.16 0.22 37.95 1.35
0.418 0.698 0.016a 0.685 0.025 0.453
[LTG] = 2.0 × 10−6 M. SS, sum of squares; d.f., degrees of freedom; MS, mean squares; Fratio : MSfactor /MSerror . Plevel , probability level. a Significant factor at α = 0.05.
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3.6. Analytical application The concentration of LTG in commercial capsules of LAMICTAL® (GlaxoSmithKline) with a known concentration of analyte, was determined using the DPAdSV method, so as to evaluate the accuracy of the proposed method. In order to determine the concentration of LTG in LAMICTAL® 10 different capsules of the pharmaceutical preparation were analyzed. The following procedure was carried out for each capsule:
Fig. 6. Response surface for the 22 central composite design for optimization of experimental variables in LTG determination by DPAdSV.
(1) The tablet was pulverized with a pestle and dissolve in HCl 0.1 M. (2) The resulting solution was centrifuged at 5000 rpm for 1 h. (3) After centrifugation the supernatant was collected and used as a stock solution of the sample.
A key feature of any analytical method is its detection limit; the smallest concentration of the analyte that can be detected to a specified degree of certainty. Calculation of the detection limit, based on the variability of ten samples with a very low analyte concentration, was calculated according to [41] and ISO 11843–2 [42]. At the chosen probability level of 5% (α = β = 0.05), the detection limit was 3.72 × 10−7 M. This limit of detection value is one order less than the values obtained when LTG was measured with carbon and mercuryfilm carbon screen-printed electrodes. Moreover, as can be seen in Fig. 7, the analytical signal is higher when LTG is analyze with nanoparticle-modified electrodes
Finally, the determination of the LTG content was made in an aliquot of the stock solution described above by means of the AdSV using the silver nanoparticle-modified CSPE developed in this work. Good agreement was obtained between the amount of LTG found by the standard addition 26 ± 1 mg with n = 10 and α = 0.05 and the value supplied by the manufacturer 25 ± 1 mg. These results were also checked using the HPLC method described in Ref. [4] as a reference technique obtaining 25 ± 1 mg (n = 3, α = 0.05).
3.5. Precision
The silver nanoparticle-modified carbon screen printed electrodes developed in this work present an environmentallyfriendly method for the analysis of LTG. Their most important advantage is the high sensitivity in the determination LTG in real samples. In fact, the detection limit obtained was less than the values found when LTG was measured with carbon and mercuryfilm carbon screen-printed electrodes. For this reason we can assure that this is the best way, known at the present time, to analyze LTG using screen printed electrodes.
This parameter was calculated in terms of reproducibility. A series of measurements of three different samples containing 2.0 × 10−7 M of LTG were carried out obtaining a %R.S.D. value of 2.58%.
4. Conclusions
Acknowledgements The financial support made available by the Junta de Castilla y Le´on (Burgos 08/04) and the Ministerio de Ciencia y Tecnolog´ıa (BQU2001-1126) is gratefully acknowledged. The ´ authors also thank Dr. Alvaro Colina from Universidad de Burgos for the help with SEM images. M. Encarnaci´on Burgoa Calvo also thanks Ministerio de Educaci´on y Ciencia for the F.P.U. grant. References Fig. 7. DPAdSV voltammograms of 5 × 10−6 M of LTG in: (1) Hg-film-CSPE (pH 5.53, tdep = 105 s and Edep = 0.40 V); (2) CSPE (pH 5.50, tdep = 28 s and Edep = 0.05 V); (3) silver nanoparticle-modified CSPE (pH 5.5, tdep = 147 s and Edep = −0.90 V). Measurements were carried out in the optimal conditions for each electrode.
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