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Voltammetric Oxidation and Determination of Atenolol Using a Carbon Paste Electrode Roopa H. Patil, Rajesh N. Hegde, and S. T. Nandibewoor* P.G. Department of Studies in Chemistry, Karnatak UniVersity, Dharwad 580003, India
The electrochemical oxidation of atenolol, an antihypertensive drug, was studied in a phosphate medium at a carbon paste electrode. Cyclic voltammetric results showed one well-defined oxidation peak in the potential range from 0.2 to 1.2 V under different pH conditions, but the best results were obtained at pH 10.4. The oxidation was irreversible and exhibited diffusion-controlled behavior. The response was evaluated with respect to pH and scan rate. The number of electrons transferred in the oxidation process was calculated, and the probable oxidation mechanism was proposed. The proposed differential pulse voltammetric method was successfully applied to atenolol determination in pharmaceutical samples. The analytical performance of this method has been evaluated for detection of analyte in urine as a real sample. Introduction Hypertension is a growing disease of medical concern. Tremendous increase in the use of antihypertensive medications such as β-blockers has pointed toward an increasing number of hypertension cases in last few decades. The β-blocker drug atenolol (ATN) is used as a antihypertensive drug.1 ATN, designated chemically as 4-(2-hydroxy-3-isopropylaminopropoxy) phenylacetamide, is a hydrophilic β1-selective (cardioselective) adrenoceptor antagonist.2 It has the following structure:
determination of ATN by CPE and applying it to the pharmaceutical and urine samples. There are no sample preparation and time-consuming extraction steps other than sonication for the determination of ATN in tablet form by the proposed voltammetric method. The obtained results by the proposed methods have been compared with the labeled values of tablets, and the proposed method yields accurate, fast, and reproducible results. The present work has shown a very good detection limit as compared to C60-modified glassy carbon electrode.11 Experimental Section
ATN is used for antiangina treatment to relieve symptoms and improve tolerance and as an antiarrhythmic to help to regulate heartbeat and infections. It is also used in management of alcohol withdrawal, in anxiety state migraine prophylaxis, hyperthyroidism, and tremors.3,4 The derivative of the oxidation product of ATN finds its importance in biological systems such as plant growth hormones, herbicides, and so forth. β-blockers are exceptionally toxic and have a narrow therapeutic range. The overdose of ATN will lead to disorders of the respiratory drive, lethargy, wheezing, sinus, pause, bradycardia, congestive heart failure, hypotension, bronchospasm, and hypoglycemia.5 A review of the literature reveals that several chromatographic methods have been developed for the determination of ATN in previous reports.6-9 However, these methods are generally complicated and tedious. In recent reports, various voltammetric techniques have been employed for the investigation and determination of ATN using different electrodes such as nanogold modified indium tin oxide electrode,10 C60-modified glassy carbon electrode,11 and glassy carbon electrode.12 The main objective of this work is the development of a simple, rapid, and sensitive voltammetric method for the * To whom correspondence should be addressed. Tel.: +91 836 2770524. Fax: +91 836 2747884. E-mail:
[email protected].
Chemicals. Pure ATN in powdered form was generously provided by S. S. Antibiotics Pvt. Ltd., Aurangabad, India, and was used without further purification. A 0.01 M stock solution was made in doubly distilled water. The Britton Robinson, acetate, KCl + NaOH, and phosphate buffers were prepared in doubly distilled water by using standard procedure. To check the effect of tetraalkylammonium salt, tetramethyl ammonium chloride and ATN were dissolved in methanol. Graphite powder (particle size < 50 µm) and paraffin oil (IR grade) were obtained from s. d. fine chem. India. Other reagents used were of analytical or chemical grade, and their solutions were prepared with doubly distilled water. ATN containing tablets were purchased from a local pharmacy. Instrumentation. All voltammetric measurements were carried out with a model EA-201 Electro Analyzer coupled with a single compartment glass cell of conventional three-electrode system consisting of a saturated calomel as reference electrode, self-made CPE as working electrode, and a platinum wire as counter electrode. All scans were taken at a rate of 50 mV s-1. All the potentials given in this paper are referenced to the calomel electrode. The pH measurements were made with Elico pH meter model LI120. Preparation of CPE. The CPE was prepared by mixing 1.0 g of graphite powder and 0.5 mL of paraffin oil in a small agate mortar, and this mixture was then homogenized. After that, the paste was pressed manually into the cavity of the electrode body, and the surface was smoothed against weighing paper. Unless otherwise stated, the paste was carefully removed prior to pressing a new portion into the electrode after every measurement. The area of the electrode was obtained by the cyclic voltammetric method using 1.0 × 10-3 M K3Fe(CN)6 as a probe
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at different scan rates. For a reversible process, the following Randles-Sevcik eq 1 can be used.13 Ip ) (2.69 × 105)n3/2AD01/2Co*υ1/2
(1)
where Ip refers to the anodic peak current, n is the number of electrons transferred, A is the surface area of the electrode, D0 is diffusion coefficient, υ is the scan rate, and Co* is the concentration of K3Fe(CN)6. For 1.0 × 10-3 M K3Fe(CN)6 in 0.1 M KCl electrolyte, n ) 1 and D0 ) 7.6 × 10-6 cm2 s-1, and then from the slope of the plot of Ipa vs υ 1/2, the surface area of the electrode can be calculated. In the CPE the surface area was found to be 0.0135 cm2. Analytical Procedure. The CPE was first activated in phosphate buffer (0.2 M, pH 10.4) by cyclic voltammetric sweeps between 0.2 to 1.2 V until a stable cyclic voltammogram was obtained. Then electrodes were transferred into another cell containing aliquots of the stock solution of ATN which was diluted with an appropriate amount of phosphate buffer of desired pH to a total volume of 20 mL. The potential scan was initiated, and cyclic voltammograms were recorded between 0.2 to 1.2 V with a scan rate of 50 mV s-1. All measurements were carried out at room temperature of 25 ( 0.1 °C. Sample Preparation. ATEN-50 tablets (Zydus Cadila, Healthcare, India) labeled as containing 50 mg of ATN per tablet were weighed and ground to a homogeneous fine powder in a mortar. A portion equivalent to a stock solution of a concentration of about 0.01 M was accurately weighed and transferred into a 100 mL calibrated flask and completed to the volume with doubly distilled water. The contents of the flask were sonicated for 15 min to affect complete dissolution. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant liquor and diluting them with the phosphate buffer solution. Each solution was transferred to the voltammetric cell and analyzed by standard addition method. The differential pulse voltammograms were recorded between 0.5 and 1.1 V. The oxidation peak current of ATN was measured at scan rate of 50 mV s-1. To study the accuracy of the proposed method and to check the interferences from excipients used in the dosage form, recovery experiments were carried out. The concentration of ATN was calculated using the standard addition method. Results and Discussion Electrooxidation of ATN. Cyclic Voltammetric Behavior of ATN. The electrochemical behavior of ATN at CPE was studied by cyclic voltammetry at pH 10.4. The cyclic voltammogram obtained for 1.0 × 10-3 M ATN solution at a scan rate of 50 mV s-1 exhibits an anodic peak at about 0.9 V at CPE. The results are as shown in the Figure 1. On the reverse scan, no corresponding reduction peak was observed, indicating that the electrode process of ATN is an irreversible process. Nevertheless, it was found that the oxidation peak current of ATN showed a remarkable decrease during the successive cyclic voltammetric sweeps (Figure 2). A decrease of the oxidation peak current occurs with the number of successive sweeps. This phenomenon may be due to the fact that the adsorption of its oxidative product occurs at the electrode surface. Therefore, the voltammograms corresponding to the first cycle was generally recorded. Effect of Supporting Electrolyte. To check the effect of supporting electrolytes, cyclic voltammograms were recorded in Britton Robinson, acetate, KCl + NaOH, phosphate buffer, and tetramethyl ammonium chloride. However, the best results with respect to sensitivity accompanied with sharper
Figure 1. Cyclic voltammogram at CPE (a) without ATN (b) and with 1.0 × 10-3 M ATN at scan rate of 50 mV s-1 in phosphate buffer with pH 10.4.
Figure 2. Successive cyclic voltammograms of 1.0 × 10-3 M ATN on CPE at scan rate of 50 mV s-1 in phosphate buffer with pH 10.4.
response were obtained with phosphate buffer. Below pH 8.0 there was no oxidation peak observed. The electro-oxidation of 1.0 × 10-3 M ATN was studied over the pH range 8.0-11.2 in phosphate buffer solution by cyclic voltammetry, which is as shown in Figure 3. With the rise in pH, the peak potential linearly shifts to less positive values, and the linear relation between Ep and pH (Figure 4) can be expressed as Ep ) 1.5911 - 0.0651 pH; r ) 0.9835. A slope of 0.0651 per pH unit suggests that the number of electrons transfered is equal with that of hydrogen ions taking part in the electrode reaction. The solution pH influences the peak current considerably. From the plot of Ip vs pH (Figure 5) it is clear that peak current is affected by the pH value. The peak current reached the highest value at around pH 10.4-11.2 in phosphate buffer. Phosphate buffer at pH 10.4 was selected for further work because height of the peak reaches a maximum at pH 10.4, and after that it decreases. Influence of Scan Rate. The effect of scan rate on the electrooxidation of ATN was examined by cyclic voltammetry (Figure 6). The influence of the square root of scan rate on the peak current showed a linear relationship in the range of 0.025 to
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Ep ) E 0 +
Figure 3. Influence of pH on the shape of anodic peak of 1.0 × 10-3 M ATN on CPE at scan rate of 50 mV s-1 in phosphate buffer.
(
) ( ) (
2.303RT RTko 2.303RT log log υ + RnF RnF RnF
)
(2)
where R is the transfer coefficient, k0 is the standard rate constant of the reaction, n is the number of electrons transferred, υ is the scan rate, and E0 is the formal redox potential. The other symbols have their usual meanings. Thus the value of Rn can be easily calculated from the slope of Ep versus log υ. In this system, the slope is 0.063, taking T ) 298, R ) 8.314, and F ) 96480, and the Rn was calculated to be 0.9387. Generally R is assumed to be 0.5 in the total irreversible electrode process.16 So the number of electrons (n) transferred in the electrooxidation of ATN was calculated to be 2. The value of k0 can be determined from the intercept on Figure 7 if the value of E0 is known. The value of E0 in eq 2 can be obtained from the intercept of the Ep vs υ curve by extrapolating to the vertical axis at υ ) 0.17 In our system the intercept for Ep vs log υ plot was 0.9626, and E0 was obtained to be 0.8671; k0 was calculated to be 1195.91 s-1. In the proposed method the number of electrons transferred in the electro-oxidation was 2, and the mechanism is as shown in Scheme 1. This observation was in accordance with our previous work on the oxidation of ATN, where the number of electrons transferred is 2 and the product was identified as 2-[4(3-isopropylamino-2-oxo-propoxy)-phenyl]-acetamide.12 Calibration Curve and Detection Limit. To develop a voltammetric method for determining the drug, we selected the differential pulse voltammetry mode, because the peaks are sharper and better defined at lower concentration of ATN, with
Figure 4. Influence of pH on the potential of 1.0 × 10-3 M ATN on CPE at scan rate of 50 mV s-1 in phosphate buffer.
Figure 5. Variation of current with pH of 1.0 × 10-3 M ATN on CPE at scan rate of 50 mV s-1 in phosphate buffer.
0.3 V s-1, which is typical of a diffusion controlled process,14 and the equation can be expressed as Ip ) 26.3υ1/2 - 0.5866; r ) 0.9844. A linear relationship was observed between log Ip and log υ, and the corresponding equation can be expressed as log Ip ) 0.5169 log υ + 1.4033; r ) 0.9924. The slope of 0.5169 was close to the theoretically expected value of 0.5 for a purely diffusion controlled process,14 which in turn further confirms that the electro-oxidation of ATN was diffusion controlled. With an increase in scan rate, the peak potential shifted to a more positive value, and a linear relationship was observed in the range of 0.025 to 0.3 V s-1 as shown in Figure 7. The equation can be expressed as Ep ) 0.063 log υ + 0.9626; r ) 0.9964. As for an irreversible electrode process, according to Laviron, Ep is defined by the following equation:15
Figure 6. Cyclic voltammograms of 1.0 × 10-3 M ATN on CPE with different scan rates from 25 (a), 50 (b), 100 (c), 200 (d), and 300 (e) mV s-1 respectively, in phosphate buffer with pH 10.4.
Figure 7. Relation between potential and logarithm of scan rates of 1.0 × 10-3 M ATN on CPE in phosphate buffer with pH 10.4.
Ind. Eng. Chem. Res., Vol. 48, No. 23, 2009 Scheme 1. Proposed Mechanism for Electro-Oxidation of ATN at CPE
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Table 1. Influence of Potential Interferents on the Voltammetric Response of 0.5 × 10-4 M ATN interferent
concentration (10-4 M)
signal change (%)
glucose starch sucrose dextrose citric acid oxalic acid
1.0 1.0 1.0 1.0 1.0 1.0
+2.0 +1.6 +1.8 -2.8 +0.6 +1.4
Table 2. Determination of ATN in Urine Samples urine
a lower background current, in improved resolution. According to the obtained results, it was possible to apply this technique to the quantitative analysis of ATN. The phosphate buffer solution of pH 10.4 was selected as the supporting electrolyte for the quantification of ATN as it gave maximum peak current at pH 10.4. Differential pulse voltammograms obtained with increasing amounts of ATN showed that the peak current increased linearly with increasing concentration, as shown in Figure 8. Using the working conditions described above, linear calibration curves were obtained for ATN in the range of 2.0 × 10-5 to 1.0 × 10-4 M. The linear equation was Ip (µA) ) 5.78C (mM) + 0.136; r ) 0.994. Deviation from linearity was observed for more concentrated solutions, due to the adsorption of its oxidation product on the electrode surface. Related statistical data of the calibration curves were obtained from four different calibration curves. The limit of detection (LOD) and quantification (LOQ) were 0.587 and 1.96 µM, respectively. The LOD and LOQ were calculated using the following equations: LOD ) 3s/m; LOQ ) 10s/m, where s is the standard deviation of the peak currents of the blank (four runs) and m is the slope of the calibration curve. Sample solutions recorded after 48 h did not show any appreciable change in the assay values. This method was better as compared with other reported electrochemical methods. The detection limit for nanogold modified indium tin oxide was reported as 0.13 µM; for the C60-modified glassy carbon electrode, it was 0.16 mM; and for CPE it was found to be 0.58 µM in the present work. For 0.1 mM ATN the oxidation signal for nanogold modified indium tin oxide was reported as 0.81 nA; for the C60-modified glassy carbon electrode it was 0.85 µA; for the glassy carbon electrode it was 0.11 µA; and for CPE it was found to be 0.71 µA in the present work.
Figure 8. Differential pulse voltammograms of ATN at CPE at different concentrations 0.2 (a), 0.4 (b), 0.6 (c), 0.8 (d), and 1.0 (e) × 10-4 M, respectively, at pulse amplitude, 50 mV; sample width, 20 ms; pulse width, 50 ms; pulse period, 200 ms; in phosphate buffer with pH 10.4. Inset: Plot of current against the concentration of ATN.
sample sample sample sample sample a
1 2 3 4 5
spiked (10-4 M)
found (10-4 M)a
recovery (%)
0.2 0.4 0.6 0.8 1.0
0.1946 0.3924 0.5952 0.8096 0.9822
97.30 98.10 99.20 101.20 98.22
Average of four determinations.
Carbon paste electrodes (CPEs), due to unique characteristics such as versatility of chemical modification, renew ability of the electrode surface, and compatibility with various electron mediators has extensively been used in these studies. Tablet Analysis and Recovery Test. To evaluate the applicability of the proposed method in the real sample analysis, it was used to detect ATN in tablets (ATEN-50 (50 mg per tablet)). The procedures for the tablet analysis were followed as described in the sample preparation section. The results were in good agreement with the content marked in the label. The detected content was 48.5 mg per tablet with 97% recovery. The recovery test of ATN ranging from 2.0 × 10-5 to 1.0 × 10-4 M was performed using differential pulse voltammetry. The recoveries in different samples were found to lie in the range from 95.86% to 98.18%, with RSD of 0.58%. Interference. Under the working conditions, the effects of potential interferents on the voltammetric response of 0.5 × 10-4 M ATN were evaluated. The experimental results (Table 1) showed that twofold of glucose, starch, sucrose, dextrose, citric acid, and oxalic acid did not interfere with the voltammetric signal of ATN. Detection of ATN in Urine Samples. The developed differential pulse voltammetric method for the ATN determination was applied to urine samples. The recoveries from urine were measured by spiking drug free urine with known amounts of ATN. The urine samples were diluted 100 times with the phosphate buffer solution before analysis without further pretreatments. A quantitative analysis can be carried out by adding the standard solution of ATN into the detected system of urine sample. The calibration graph was used for the determination of spiked ATN in urine samples. The obtained detection results of five urine samples are listed in Table 2. The recovery determined was in the range from 97.30% to 101.20%, and the RSD was 2.28%. Reproducibility of the Carbon Paste Electrode. To study the reproducibility of the electrode preparation procedure, a 0.1 mM ATN solution was measured with the same CPE (renewed every time) for several hours within the day, and the RSD of the peak current was 1.62% (number of measurements ) 6). As to the reproducibility between days, it was similar to that within a day if the temperature was kept almost unchanged. Owing to the adsorption of ATN or its oxidative products on to the electrode surface, the current response of the electrode
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would decrease after successive use. In this case, the electrode should be prepared again. Conclusions In this work, ATN undergoes oxidation at CPE. When the potential was made to move, ATN produced one anodic peak at about 0.9 V in phosphate buffer (0.2 M, pH 10.4). The oxidation process was irreversible and diffusion-controlled. A suitable oxidation mechanism was proposed. This method has been successfully used to determine ATN in the pharmaceutical sample. The proposed method offered the advantages of accuracy and time savings as well as simplicity of reagents and apparatus. In addition, the results obtained in the analysis of ATN in spiked urine samples demonstrated the applicability of the method for real sample analysis. Furthermore, the present method could possibly be adopted for pharmacokinetic studies as well as clinical and quality control laboratories. Acknowledgment R.H.P. thanks UGC, New Delhi, for the award of Research Fellowship in Science for Meritorious Students (RFSMS). Nomenclature and Abbreviations ATN ) atenolol CPE ) carbon paste electrode n ) number of electrons transferred in the reaction A ) surface area of the electrode D0 ) diffusion coefficient υ ) scan rate Co* ) concentration Ep ) peak potential Ip ) peak current R ) transfer coefficient k0 ) standard rate constant of the reaction F ) Faraday constant E0 ) formal redox potential R ) Gas constant T ) temperature LOD ) limit of detection LOQ ) limit of quantification s ) standard deviation of the peak currents m ) slope of the calibration curve RSD ) relative standard deviation
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ReceiVed for reView July 21, 2009 ReVised manuscript receiVed September 17, 2009 Accepted October 16, 2009 IE901163K
