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Adsorptive stripping voltammetric determination of zopiclone in tablet dosage forms and human urine
Colloids and Surfaces B: Biointerfaces 71 (2009) 79–83
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Adsorptive stripping voltammetric determination of zopiclone in tablet dosage forms and human urine Selehattin Yılmaz ∗ Canakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Analytical Chemistry, 17020 Canakkale, Turkey
a r t i c l e
i n f o
Article history: Received 14 October 2008 Received in revised form 2 January 2009 Accepted 7 January 2009 Available online 17 January 2009 Keywords: Zopiclone Adsorptive stripping voltammetry Determination Glassy carbon electrode Human urine Tablet dosage forms
a b s t r a c t Adsorptive stripping voltammetric (AdSV) techniques were proposed for the direct quantitative determination of zopiclone (ZP) in spiked human urine and tablet dosage forms for first time. The electrochemical oxidation and determination of ZP were easily carried out on glassy carbon electrode (CGE) using a variety of voltammetric techniques. Different conditions were investigated to optimize the analytical determination of ZP. The dependence of the intensities of currents and potentials on pH, concentration, scan rate, deposition time, deposition potential, and nature of the buffer were investigated. Oxidation of ZP was found to be adsorptive-controlled and irreversible. The best results for the determination of ZP were obtained by using differential pulse adsorptive stripping (DPAdSV) and osteryoung square wave voltammetric (OSWAdSV) techniques in Britton–Robinson buffer at pH 7.08 after a pre-concentration period of 120 s at 0.60 V. The peak current showed a linear dependence on the ZP concentration in the range of 6 × 10−7 to 2 × 10−5 mol L−1 for both techniques. The achieved limits of detection and quantitation were 2.78 × 10−7 and 5.28 × 10−7 mol L−1 for DPAdSV; 1.70 × 10−7 and 5.78 × 10−7 mol L−1 for OSWAdSV, respectively. The proposed techniques were successfully applied to direct determination of ZP in tablet dosage form and spiked human urine samples. Excipients did not interfere with the determination. Precision and accuracy of the developed method were checked by recovery studies in tablet dosage forms and spiked urine samples. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Zopiclone (ZP) [8-(5-chloropyridin-2-yl)-7-oxo-2,5,8-triazabicylo[4,3,0]nona-1,3,5-trien-9-yl]4-metylpiperazine-1-carboxylate (Scheme 1), is a non-benzodiazepine sedative hypnotic drug active material used for the short term treatment or management of insomnia. It also has anticonvulsant, antiaggressive and anticonflict actions in addition to its sedative and hypnotic effects. Overdose of zopiclone may be presented with excessive sedation, depressed respiratory function which may progress to coma and possibly death [1–3]. Therefore, the determination of ZP in biological media and pharmaceutical formulations is very important. The polarographic reduction of ZP was investigated by S¸entürk et al. [4] and Squella et al. [5]. Using a hanging mercury drop electrode (HMDE), but electrochemical oxidation on glassy carbon electrode (GCE) and adsorptive stripping voltammetric determination have not yet been published. Consequently, it would be of interest to investigate the adsorptive properties of oxidation process and determination of ZP on the GCE in real samples. The work presents a study of the factors that
∗ Tel.: +90 286 2180018/1857; fax: +90 286 2180533. E-mail address: [email protected]. 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.01.007
may influence both the accumulation process and the voltammetric response. In the proposed adsorptive stripping techniques, there are no complicated pretreatment or time-consuming extraction steps other than centrifugation when it is applied to determine of ZP in pharmaceutical formulations and biological fluids such as human urine. Moreover, the proposed technique was highly selective, sensitive, rapid and easy to perform and can be applied in routine clinical drug monitoring studies [6–8]. 2. Experimental 2.1. Apparatus A Model Metrohm 757 VA Trace Analyzer (Herisau, Switzerland) was used for the voltammetric measurements, with a threeelectrode system consisting of a glassy carbon working electrode, a platinum wire auxiliary electrode and Ag/AgCl (KCl 3 mol L−1 , Metrohm) reference electrode. All measurements were carried out after the deoxygenating of the solutions using argon for 5 min and for 30 s before each measurement. For each set of experiments, new fresh electrode surface was used. All pH measurements were made with Model Metrohm 744 pH meter (Herisau, Switzerland). All measurements were carried out at ambient temperature of the laboratory (25–30 ◦ C).
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S. Yılmaz / Colloids and Surfaces B: Biointerfaces 71 (2009) 79–83
Scheme 1. The structural formula of ZP.
For the analytical application, the following parameters were employed: pulse amplitude 50 mV; pulse time 0.04 s, voltage step 0.009 V, voltage step time 0.04, potential step 10 mV (DPV); pulse amplitude 50 mV; frequency 50 Hz, potential step 10 mV (OSWV); the scan rate in the range of 10–1000 mV s−1 (CV). 2.2. Reagents Zopiclone and its Imovane® tablets were kindly supplied by Eczacibasi Inc. (Istanbul, Turkey). A stock solution of 1.0 × 10−3 mol L−1 was prepared by dissolving an accurate mass of the drug in an appropriate volume of deionized water and kept in the dark in a refrigerator. The working solutions for the voltammetric investigations were prepared by dilution of the stock solution by deionized water containing 20 L HNO3 . All solutions were protected from light and were used within 24 h to avoid a possible decomposition. 0.5 mol L−1 sulphuric acid; pH 0.51 (Riedel, Germany, 95–97% (m/m)), 0.1 mol L−1 phosphate buffer (pH 4.53–7.52): sodium hydrogen phosphate (Na2 HPO4 , Riedel, Seelze, Germany) and sodium dihydrogen phosphate (NaH2 PO4 , Riedel, Seelze, Germany). 0.1 mol L−1 acetate buffer; pH: 3.51–5.51 (Acetic acid: Riedel, Seelze, Germany, 100* (m/m) and sodium hydroxide: Riedel, Seelze, Germany) and 0.04 mol L−1 Britton-Robinson buffer; pH 2.12–12.00 (acetic acid: Riedel, Seelze, Germany, 100% (m/m); boric acid; Merck, Darmstadt, Germany, and phosphoric acid, Carlo Erba, Rodeno, France, 85% (m/m)) were used for the supporting electrolytes. Deionized water obtained from Sartorius Arium model Ultra Pure Water Systems was used in order to prepare supporting electrolytes. Other chemicals were used at analytical-reagent grade (Merck). Imovane® tablets (Eczacibasi, Inc., Istanbul, Turkey) were labeled to contain 7.5 mg ZP per tablet. 2.3. Calibration graph for voltammetric determination ZP was dissolved in deionized water contained 20 L HNO3 to obtain 1 × 10−3 mol L−1 stock solution. This solution was diluted with deionized water to obtain serial ZP concentration. For optimum conditions described in the experimental section, a linear calibration curve for DPAdSV analysis was constructed in the ZP concentration range of 6 × 10−7 to 2 × 10−5 mol L−1 . The repeatability, accuracy and precision were all checked. 2.4. Procedure for the analysis of Imovane® tablets and recovery studies Ten tablets were weighed and ground to a fine powder. An adequate amount of this powder, corresponding to a stock solution of
concentration 1 × 10−3 mol L−1 , was weighed and transferred into a 10 mL calibrated flask and completed to the volume with deionized water. The contents of the flask were centrifuged for 15 min at 4000 rpm to affect complete dissolution and then diluted to the volume with the same solvent. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant liquor and diluting with selected supporting electrolyte solution. Each solution was transferred into the voltammetric cell and deoxygenated with argon (analytically pure with 99.99%) for 5 min and for 30 s before each measurement as for the pure ZP. The nominal content of the corresponding regression equations of previously plotted calibration plots [8]. To study the accuracy and repeatability of the applied DPAdSV technique, recovery experiments were carried out using the standard addition method. In order to know whether the excipients show any interference with the analysis, known amounts of the pure ZP were added to the pre-analyzed tablet formulations of ZP and mixtures were analyzed by the proposed DPAdSV method. The recovery results were calculated using the related calibration equations after five repeated experiments. 2.5. Working voltammetric procedure of spiked human urine Urine obtained daily from a volunteer was diluted 1:9 with deionize water. Firstly, 9.4 mL BR (pH 7.8) buffer put into voltammetric cell and its voltammogram was taken (blank). Then 600 L of the diluted urine solution was added to this solution and its voltammogram (urine + blank) was also taken. After that, 100 L urine sample (1 mL urine + 8 mL deionize water + 1 × 10−3 mol L−1 ZP solution) was put into this voltammetric cell and its voltammogram was recorded. Then 50 L of 8 × 10−6 mol L−1 ZP solution was added five times successively and their voltammograms were recorded individually after each addition. Calibration curve was plotted using obtained results [8,9]. 3. Results and discussion 3.1. Electrochemical oxidation of ZP The electrochemical oxidation process occurring on the GCE, cyclic and differential pulse and osteryoung square wave voltammetric techniques were carried out. Cyclic voltammetric measurements performed on 1 × 10−4 mol L−1 ZP at various scan rates at GCE in BR buffer (pH 7.08) are shown in Fig. 1. As can be seen from Fig. 1, ZP has one irreversible anodic peak appearing at about 1.00 V. The effects of the potential scan rate between 50 and 1000 mV s−1 on the peak potential and the peak current of ZP were evaluated. Scan rate studies were then performed to assess whether the processes on GCE were under diffusion- or adsorptioncontrol [6,8,9]. We employed two tests for this procedure. One of them is that the linear relationship existing between peak current and square root of the scan rate between 50 and 1000 mV s−1 (Ip (A) = 176.181/2 − 1276.8 (correlation coefficient 0.955) were observed and this may show that the oxidation process is diffusioncontrolled. However, the other test is that a plot of logarithm of peak current versus logarithm of scan rate gave a straight line (correlation coefficient 0.994) with a slope of 1.0093 (very close to 1.0), which is the expected value for an adsorption of surface species [9,10]. The second test is more validity for this aim. Therefore, an adsorptive component must be taken into account. In order to obtain the optimum experimental conditions, some variables affecting the peak current and peak potential with pH, supporting electrolyte, deposition potential and deposition time for a 4 × 10−6 mol L−1 ZP solution were studied at the GCE by proposed
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Fig. 1. The cyclic voltammograms of 1 × 10−4 mol L−1 ZP in BR buffer (pH 7.08) at GCE. Scan rates: (a) blank, (b) 50, (c) 75, (d) 100, (e) 150, (f) 200, (g) 250, (h) 300, (i) 400, (j) 500, (k) 750, (l) 1000 mV s−1 .
voltammetric techniques. The voltammetric response was strongly pH dependent. The peak potential of the oxidation peak shifted more negative values with increasing pH (Fig. 2a). The effect of pH on the peak current is maximum at pH 7.08 in BR buffer. Thus, this pH value was chosen for the electroanalytical studies (Fig. 2b). Error bars of five repeated experiment were also shown in both figures. DPAdSV technique and BR buffer pH 7.08 was also selected for further work due to the fact that it gave not only the highest peak current but also the best peak shape.
Fig. 3. Effect of deposition potential(Eacc ) (a) and deposition time (tacc ) (b) on the DPAdSV peak current of Zopiclone. Conditions are 4 × 10−6 M Zopiclone, BR buffer of pH 7.08 as the supporting electrolyte, and deposition potential (Eacc ) 0.60 V. (Each plots indicate average current and ±standard deviation of five repeated experiment).
The application of deposition potential in the range of 0.30–0.70 V indicates that the maximum value for the peak current occurred at 0.60 V (Fig. 3, curve a). Variation of the deposition time showed that the peak current increased with the deposition time and reached a plateau after a period longer than 120 s (Fig. 3, curve b). Error bars of five repeated experiment were also shown in both figures. According to these results, the deposition time, 120 s, was chosen in this study considering the good sensitivity and the relatively short analysis time. 3.2. Validation of the analytical procedure
Fig. 2. Effect of pH on (a) DPAdSV peak potential and (b) peak current of 4.0 × 10−6 mol L−1 Zopiclone; scan rate 100 mV s−1 (multiplication): sulphuric acid; (square): Britton–Robinson buffer; (triangle): acetate buffer; (guadrangle): phosphate buffer. (Each plots indicate average (a) potential and its ±standard deviation (b) current and its ±standard deviation of five repeated experiment).
On the basis of the electrochemical oxidation of ZP, DPADSV and OSWAdSV techniques were used to develop for quantitative determination of the pure drug active material in spiked pharmaceutical and urine. The optimum instrumental conditions were chosen from the studies of the variation of the peak current on pulse amplitude, and potential step. Using the optimum conditions described in experimental section, the voltammograms for various concentration of ZP were recorded in BR buffer pH 7.08 by both of the applied techniques (Figs. 4 and 5). Quantitative evaluation based on the linear correlation between the oxidation peak current and concentration was carried out. As a result of this, good correlation was obtained for ZP concentration between 6 × 10−7 and 2 × 10−5 mol L−1 for both techniques. The equation of the calibration plots is Ip (A) = 5.00 × 107 C (mol L−1 ) + 0.143 with a correlation coefficient, r = 0.995, which was obtained from five DPAdSV measurements. Standard deviations for intercept and slope of the calibration curve are in Table 1. Validation of the procedure for the quantitative determination of the ZP was examined via evaluation of the limit of detection (LOD), limit of quantification (LOQ), repeatability, accuracy and precision by both techniques (Table 1). LOD and LOQ were calculated on the oxidation peak current using the following equations: LOD = 3s/m, LOQ = 10 s/m (s is the
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S. Yılmaz / Colloids and Surfaces B: Biointerfaces 71 (2009) 79–83 Table 2 Analytical results for ZP in commercial tablets (Imovane® ) and mean recoveries in spiked tablets.
Nominal value (mg/per tablet) Amount found (mg)a R.S.D.% Bias (%) Added (mg) Found (mg)a Recovery (%) R.S.D.% of recovery Bias (%) a
Fig. 4. DPAdSV voltammograms of (a) blank (BR, pH 7.08); (b) 6 × 10−7 ; (c) 8.0 × 10−7 ; (d) 1 × 10−6 ; (e) 2.0 × 10−6 ; (f) 4 × 10−6 ; (g) 6.0 × 10−6 ; (h) 8 × 10−6 ; (i) 1.0 × 10−5 ; (j) 2.0 × 10−5 mol L−1 ZP.
DPAdSV
OSWAdSV
7.50 7.48 0.13 0.27 0.350 0.348 99.73 0.43 0.57
7.50 7.49 0.23 0.13 0.350 0.349 99.87 0.33 0.29
Each value is the mean of ten experiments.
Table 3 Application of the stripping voltammetric techniques to the quantitative determination of ZP in spiked human urine. Technique
DPAdSV
OSWAdSV
Medium ZP spiked (mol L−1 ) Number of measurements (n) ZP found (mol L−1 ) Average recovery (%) R.S.D.% Bias (%)
Human urine 8 × 10−6 5 7.91 × 10−6 98.88 0.27 1.13
Human urine 8 × 10−6 5 7.81 × 10−6 97.62 0.33 1.23
3.3. Pharmaceutical applications
Fig. 5. OSWAdSV voltammograms of (a) blank (BR, pH 7.08); (b) 6 × 10−7 ; (c) 8.0 × 10−7 ; (d) 1 × 10−6 ; (e) 2.0 × 10−6 ; (f) 4 × 10−6 ; (g) 6.0 × 10−6 ; (h) 8 × 10−6 mol L−1 .
standard deviation of the peak currents (six runs, m is the slope of the calibration curve) [8,9]. The achieved limits of detection and quantitation were 2.78 × 10−7 and 5.28 × 10−7 mol L−1 for DPAdSV; 1.70 × 10−7 and 5.78 × 10−7 mol L−1 for OSWAdSV, respectively. The repeatability of the current measurement was calculated for both techniques from five independent runs of 1 × 10−6 mol L−1 ZP solution. The relative standard deviations were calculated for peak current to be 1.10% for DPAdSV and 1.17% for OSWAdSV, respectively.
Table 1 Regression data of the calibration lines for quantitative determination of ZP calibration plots in BR buffer (pH 7.08) at GCE by both stripping voltammetric techniques. Parameters
DPAdSV
OSWAdSV
Measured potential (V) Linear concentration range (mol L−1 ) Slope (A mol−1 L) R.S.D. of slope Intercept (nA) R.S.D. of intercept Correlation coefficient, r Number of measurements (n) LOD (mol L−1 ) LOQ (mol L−1 ) Repeatability of peak current (R.S.D.%)
0.95 6 × 10−7 to 2.5 × 10−5
1.10 6 × 10−7 to 2.5 × 10−5
5.00 × 107 0.41 0.143 0.10 0.996
5.00 × 107 0.47 0.336 0.23 0.993
5
5
2.78 × 10−7 5.28 × 10−7 1.10 for 1.0 × 10−6
1.70 × 10−7 5.78 × 10−7 1.17 for 1.0 × 10−6
The amount of ZP in tablets was calculated by reference to the appropriate calibration plots. The results obtained are given in Table 2. The proposed techniques could be applied with great success to ZP assay in tablets without any interference. As far as we know, there is no official technique in the pharmacopoeias or other literature describing the determination of ZP in pharmaceutical dosage forms. For this reason, the proposed techniques were checked by performing recovery tests. To determine whether excipients in the tablets interfered with the analysis, the accuracy of the proposed methods were evaluated by recovery tests after addition of known amounts of pure drug to pre-analyzed formulations of ZP (Table 2). The results showed the validity of the proposed techniques for the quantitative determination of ZP in tablets. The proposed DPAdSV and OSWAdSV techniques proved to be sufficiently precise and accurate for reliable electro analytical analysis of ZP. 3.4. Application to biological samples The possibility of applying both voltammetric procedures to the quantitative determination of ZP in human urine was also tested successfully by the standard additions of pure drug as described in Section 2. The found amount of ZP in human urine was calculated from the related linear regression equations. The results of these analyses are summarized in Table 3. Good recovery of ZP was achieved from this type of matrix. Quantitative assay of urine samples by proposed techniques involved only dilution of urine samples, so it is time-saving and no other procedure steps are required. 4. Conclusion As far as we know, the electrochemical oxidation of ZP on GCE was studied for the first time in the literature. From the CV measurement, it is understood that electrode reaction process is irreversible and pH dependent. In BR (pH 7.08) medium DPAdSV and OSWAdSV technique were developed and successfully applied to the quantitative determination of ZP in tablet and human urine samples. The
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analysis was performed with good recoveries without any interference from the excipients in tablets and human urine. The principal advantage of these proposed techniques over the other techniques is that it may be applied directly to the analysis of pharmaceutical dosage forms and to the biological samples without the need for extensive sample preparation, since there was no interference from the excipients and endogenous substances. Another advantage is that the developed techniques are rapid, requiring about 5 min to run any sample and involves no sample preparing other than dissolving, diluting, precipitating, centrifuging and transferring an aliquot to the supporting electrolyte. This paper is not intended to be a study of the pharmacodynamic properties of ZP, because only healthy volunteers were used for sample collection and results might be of no significance. It only indicated that the possibility of monitoring this compound makes the technique useful for pharmacokinetic and pharmacodynamic purposes [6,8,9,11,12]. In addition all above, the proposed stripping techniques might be a rapid and simple alternative to more complicated LC or UV methods for routine analysis of ZP in any medium where considerably little interference takes place. Acknowledgements The author gratefully acknowledges to the Scientific and Technical Research Council of Turkey (TUBITAK, Grant No.: TBAG-2173;
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102T062). The author would also like to thank Eczacibasi, Inc. (Istanbul, Turkey) for supplying pure ZP and its commercial tablet forms for developing proposed voltammetric technique. We would like to thank to Kenan Dikilitas, the teaching staff at Language and Literature Department at Canakkale Onsekiz Mart University for providing help with the language used in the study. References [1] M. Partinen, C. Hublin. Epidemiology of sleep disorders in. Prindpies and practices of sleep medicine. Third edition, M.H. Kryger, T. Roth, W.C. Dement (eds.), 2000, p. 558. [2] British Pharmacopoeia: Published on the recommendation of Medicines Commission, U.K., London: Her Majesty’s Stationery Office; 1993 (Addendum 1996). p. 1848. [3] http://en.wikipedia.org/wiki/Zopiclone. [4] Z. S¸entürk, J.C. Vire, H. Zhang, G. Quarin, J. Patrarche, G.D. Christian, Talanta 40 (1993) 313. [5] J.A. Sequella, J.C. Sturm, A. Alvarez-Lufje, L.J. Nunez-Vergara, J. AOOC Int. 77 (1994) 768. [6] S. Özkan, B. Uslu, Anal. Bioanal. Chem. 372 (2002) 582. [7] M.O. Nan Sun, Hu. Weimin, S. Baoxiang, Zhenlu, Anal. Bioanal. Chem. 385 (2006) 161. [8] M. C¸ıtak, S. Yılmaz, Y. Dilgin, G. Türker, S. Yagmur, H. Erdugan, N. Erdugan, Curr. Pharm. Anal. 3 (2007) 141. [9] S. Skrzypek, W. Ciesielski, A. Sokolowski, S. Yilmaz, D. Kazmierczak, Talanta 66 (2005) 1146. [10] E. Laviron, J. Electroanal. Chem. 112 (1980) 11. [11] E. Suren, S. Yilmaz, M. Turkoglu, S. Kaya, Environ Monit. Assess. 125 (2007) 91. [12] B. Uslu, B. Dogan, S.A. Ozkan, Anal. Chim. Acta 537 (2005) 307.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Adsorptive stripping voltammetric determination of zopiclone in tablet dosage forms and human urine Selehattin Yılmaz ∗ Canakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Analytical Chemistry, 17020 Canakkale, Turkey
a r t i c l e
i n f o
Article history: Received 14 October 2008 Received in revised form 2 January 2009 Accepted 7 January 2009 Available online 17 January 2009 Keywords: Zopiclone Adsorptive stripping voltammetry Determination Glassy carbon electrode Human urine Tablet dosage forms
a b s t r a c t Adsorptive stripping voltammetric (AdSV) techniques were proposed for the direct quantitative determination of zopiclone (ZP) in spiked human urine and tablet dosage forms for first time. The electrochemical oxidation and determination of ZP were easily carried out on glassy carbon electrode (CGE) using a variety of voltammetric techniques. Different conditions were investigated to optimize the analytical determination of ZP. The dependence of the intensities of currents and potentials on pH, concentration, scan rate, deposition time, deposition potential, and nature of the buffer were investigated. Oxidation of ZP was found to be adsorptive-controlled and irreversible. The best results for the determination of ZP were obtained by using differential pulse adsorptive stripping (DPAdSV) and osteryoung square wave voltammetric (OSWAdSV) techniques in Britton–Robinson buffer at pH 7.08 after a pre-concentration period of 120 s at 0.60 V. The peak current showed a linear dependence on the ZP concentration in the range of 6 × 10−7 to 2 × 10−5 mol L−1 for both techniques. The achieved limits of detection and quantitation were 2.78 × 10−7 and 5.28 × 10−7 mol L−1 for DPAdSV; 1.70 × 10−7 and 5.78 × 10−7 mol L−1 for OSWAdSV, respectively. The proposed techniques were successfully applied to direct determination of ZP in tablet dosage form and spiked human urine samples. Excipients did not interfere with the determination. Precision and accuracy of the developed method were checked by recovery studies in tablet dosage forms and spiked urine samples. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Zopiclone (ZP) [8-(5-chloropyridin-2-yl)-7-oxo-2,5,8-triazabicylo[4,3,0]nona-1,3,5-trien-9-yl]4-metylpiperazine-1-carboxylate (Scheme 1), is a non-benzodiazepine sedative hypnotic drug active material used for the short term treatment or management of insomnia. It also has anticonvulsant, antiaggressive and anticonflict actions in addition to its sedative and hypnotic effects. Overdose of zopiclone may be presented with excessive sedation, depressed respiratory function which may progress to coma and possibly death [1–3]. Therefore, the determination of ZP in biological media and pharmaceutical formulations is very important. The polarographic reduction of ZP was investigated by S¸entürk et al. [4] and Squella et al. [5]. Using a hanging mercury drop electrode (HMDE), but electrochemical oxidation on glassy carbon electrode (GCE) and adsorptive stripping voltammetric determination have not yet been published. Consequently, it would be of interest to investigate the adsorptive properties of oxidation process and determination of ZP on the GCE in real samples. The work presents a study of the factors that
∗ Tel.: +90 286 2180018/1857; fax: +90 286 2180533. E-mail address: [email protected]. 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.01.007
may influence both the accumulation process and the voltammetric response. In the proposed adsorptive stripping techniques, there are no complicated pretreatment or time-consuming extraction steps other than centrifugation when it is applied to determine of ZP in pharmaceutical formulations and biological fluids such as human urine. Moreover, the proposed technique was highly selective, sensitive, rapid and easy to perform and can be applied in routine clinical drug monitoring studies [6–8]. 2. Experimental 2.1. Apparatus A Model Metrohm 757 VA Trace Analyzer (Herisau, Switzerland) was used for the voltammetric measurements, with a threeelectrode system consisting of a glassy carbon working electrode, a platinum wire auxiliary electrode and Ag/AgCl (KCl 3 mol L−1 , Metrohm) reference electrode. All measurements were carried out after the deoxygenating of the solutions using argon for 5 min and for 30 s before each measurement. For each set of experiments, new fresh electrode surface was used. All pH measurements were made with Model Metrohm 744 pH meter (Herisau, Switzerland). All measurements were carried out at ambient temperature of the laboratory (25–30 ◦ C).
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S. Yılmaz / Colloids and Surfaces B: Biointerfaces 71 (2009) 79–83
Scheme 1. The structural formula of ZP.
For the analytical application, the following parameters were employed: pulse amplitude 50 mV; pulse time 0.04 s, voltage step 0.009 V, voltage step time 0.04, potential step 10 mV (DPV); pulse amplitude 50 mV; frequency 50 Hz, potential step 10 mV (OSWV); the scan rate in the range of 10–1000 mV s−1 (CV). 2.2. Reagents Zopiclone and its Imovane® tablets were kindly supplied by Eczacibasi Inc. (Istanbul, Turkey). A stock solution of 1.0 × 10−3 mol L−1 was prepared by dissolving an accurate mass of the drug in an appropriate volume of deionized water and kept in the dark in a refrigerator. The working solutions for the voltammetric investigations were prepared by dilution of the stock solution by deionized water containing 20 L HNO3 . All solutions were protected from light and were used within 24 h to avoid a possible decomposition. 0.5 mol L−1 sulphuric acid; pH 0.51 (Riedel, Germany, 95–97% (m/m)), 0.1 mol L−1 phosphate buffer (pH 4.53–7.52): sodium hydrogen phosphate (Na2 HPO4 , Riedel, Seelze, Germany) and sodium dihydrogen phosphate (NaH2 PO4 , Riedel, Seelze, Germany). 0.1 mol L−1 acetate buffer; pH: 3.51–5.51 (Acetic acid: Riedel, Seelze, Germany, 100* (m/m) and sodium hydroxide: Riedel, Seelze, Germany) and 0.04 mol L−1 Britton-Robinson buffer; pH 2.12–12.00 (acetic acid: Riedel, Seelze, Germany, 100% (m/m); boric acid; Merck, Darmstadt, Germany, and phosphoric acid, Carlo Erba, Rodeno, France, 85% (m/m)) were used for the supporting electrolytes. Deionized water obtained from Sartorius Arium model Ultra Pure Water Systems was used in order to prepare supporting electrolytes. Other chemicals were used at analytical-reagent grade (Merck). Imovane® tablets (Eczacibasi, Inc., Istanbul, Turkey) were labeled to contain 7.5 mg ZP per tablet. 2.3. Calibration graph for voltammetric determination ZP was dissolved in deionized water contained 20 L HNO3 to obtain 1 × 10−3 mol L−1 stock solution. This solution was diluted with deionized water to obtain serial ZP concentration. For optimum conditions described in the experimental section, a linear calibration curve for DPAdSV analysis was constructed in the ZP concentration range of 6 × 10−7 to 2 × 10−5 mol L−1 . The repeatability, accuracy and precision were all checked. 2.4. Procedure for the analysis of Imovane® tablets and recovery studies Ten tablets were weighed and ground to a fine powder. An adequate amount of this powder, corresponding to a stock solution of
concentration 1 × 10−3 mol L−1 , was weighed and transferred into a 10 mL calibrated flask and completed to the volume with deionized water. The contents of the flask were centrifuged for 15 min at 4000 rpm to affect complete dissolution and then diluted to the volume with the same solvent. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant liquor and diluting with selected supporting electrolyte solution. Each solution was transferred into the voltammetric cell and deoxygenated with argon (analytically pure with 99.99%) for 5 min and for 30 s before each measurement as for the pure ZP. The nominal content of the corresponding regression equations of previously plotted calibration plots [8]. To study the accuracy and repeatability of the applied DPAdSV technique, recovery experiments were carried out using the standard addition method. In order to know whether the excipients show any interference with the analysis, known amounts of the pure ZP were added to the pre-analyzed tablet formulations of ZP and mixtures were analyzed by the proposed DPAdSV method. The recovery results were calculated using the related calibration equations after five repeated experiments. 2.5. Working voltammetric procedure of spiked human urine Urine obtained daily from a volunteer was diluted 1:9 with deionize water. Firstly, 9.4 mL BR (pH 7.8) buffer put into voltammetric cell and its voltammogram was taken (blank). Then 600 L of the diluted urine solution was added to this solution and its voltammogram (urine + blank) was also taken. After that, 100 L urine sample (1 mL urine + 8 mL deionize water + 1 × 10−3 mol L−1 ZP solution) was put into this voltammetric cell and its voltammogram was recorded. Then 50 L of 8 × 10−6 mol L−1 ZP solution was added five times successively and their voltammograms were recorded individually after each addition. Calibration curve was plotted using obtained results [8,9]. 3. Results and discussion 3.1. Electrochemical oxidation of ZP The electrochemical oxidation process occurring on the GCE, cyclic and differential pulse and osteryoung square wave voltammetric techniques were carried out. Cyclic voltammetric measurements performed on 1 × 10−4 mol L−1 ZP at various scan rates at GCE in BR buffer (pH 7.08) are shown in Fig. 1. As can be seen from Fig. 1, ZP has one irreversible anodic peak appearing at about 1.00 V. The effects of the potential scan rate between 50 and 1000 mV s−1 on the peak potential and the peak current of ZP were evaluated. Scan rate studies were then performed to assess whether the processes on GCE were under diffusion- or adsorptioncontrol [6,8,9]. We employed two tests for this procedure. One of them is that the linear relationship existing between peak current and square root of the scan rate between 50 and 1000 mV s−1 (Ip (A) = 176.181/2 − 1276.8 (correlation coefficient 0.955) were observed and this may show that the oxidation process is diffusioncontrolled. However, the other test is that a plot of logarithm of peak current versus logarithm of scan rate gave a straight line (correlation coefficient 0.994) with a slope of 1.0093 (very close to 1.0), which is the expected value for an adsorption of surface species [9,10]. The second test is more validity for this aim. Therefore, an adsorptive component must be taken into account. In order to obtain the optimum experimental conditions, some variables affecting the peak current and peak potential with pH, supporting electrolyte, deposition potential and deposition time for a 4 × 10−6 mol L−1 ZP solution were studied at the GCE by proposed
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Fig. 1. The cyclic voltammograms of 1 × 10−4 mol L−1 ZP in BR buffer (pH 7.08) at GCE. Scan rates: (a) blank, (b) 50, (c) 75, (d) 100, (e) 150, (f) 200, (g) 250, (h) 300, (i) 400, (j) 500, (k) 750, (l) 1000 mV s−1 .
voltammetric techniques. The voltammetric response was strongly pH dependent. The peak potential of the oxidation peak shifted more negative values with increasing pH (Fig. 2a). The effect of pH on the peak current is maximum at pH 7.08 in BR buffer. Thus, this pH value was chosen for the electroanalytical studies (Fig. 2b). Error bars of five repeated experiment were also shown in both figures. DPAdSV technique and BR buffer pH 7.08 was also selected for further work due to the fact that it gave not only the highest peak current but also the best peak shape.
Fig. 3. Effect of deposition potential(Eacc ) (a) and deposition time (tacc ) (b) on the DPAdSV peak current of Zopiclone. Conditions are 4 × 10−6 M Zopiclone, BR buffer of pH 7.08 as the supporting electrolyte, and deposition potential (Eacc ) 0.60 V. (Each plots indicate average current and ±standard deviation of five repeated experiment).
The application of deposition potential in the range of 0.30–0.70 V indicates that the maximum value for the peak current occurred at 0.60 V (Fig. 3, curve a). Variation of the deposition time showed that the peak current increased with the deposition time and reached a plateau after a period longer than 120 s (Fig. 3, curve b). Error bars of five repeated experiment were also shown in both figures. According to these results, the deposition time, 120 s, was chosen in this study considering the good sensitivity and the relatively short analysis time. 3.2. Validation of the analytical procedure
Fig. 2. Effect of pH on (a) DPAdSV peak potential and (b) peak current of 4.0 × 10−6 mol L−1 Zopiclone; scan rate 100 mV s−1 (multiplication): sulphuric acid; (square): Britton–Robinson buffer; (triangle): acetate buffer; (guadrangle): phosphate buffer. (Each plots indicate average (a) potential and its ±standard deviation (b) current and its ±standard deviation of five repeated experiment).
On the basis of the electrochemical oxidation of ZP, DPADSV and OSWAdSV techniques were used to develop for quantitative determination of the pure drug active material in spiked pharmaceutical and urine. The optimum instrumental conditions were chosen from the studies of the variation of the peak current on pulse amplitude, and potential step. Using the optimum conditions described in experimental section, the voltammograms for various concentration of ZP were recorded in BR buffer pH 7.08 by both of the applied techniques (Figs. 4 and 5). Quantitative evaluation based on the linear correlation between the oxidation peak current and concentration was carried out. As a result of this, good correlation was obtained for ZP concentration between 6 × 10−7 and 2 × 10−5 mol L−1 for both techniques. The equation of the calibration plots is Ip (A) = 5.00 × 107 C (mol L−1 ) + 0.143 with a correlation coefficient, r = 0.995, which was obtained from five DPAdSV measurements. Standard deviations for intercept and slope of the calibration curve are in Table 1. Validation of the procedure for the quantitative determination of the ZP was examined via evaluation of the limit of detection (LOD), limit of quantification (LOQ), repeatability, accuracy and precision by both techniques (Table 1). LOD and LOQ were calculated on the oxidation peak current using the following equations: LOD = 3s/m, LOQ = 10 s/m (s is the
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S. Yılmaz / Colloids and Surfaces B: Biointerfaces 71 (2009) 79–83 Table 2 Analytical results for ZP in commercial tablets (Imovane® ) and mean recoveries in spiked tablets.
Nominal value (mg/per tablet) Amount found (mg)a R.S.D.% Bias (%) Added (mg) Found (mg)a Recovery (%) R.S.D.% of recovery Bias (%) a
Fig. 4. DPAdSV voltammograms of (a) blank (BR, pH 7.08); (b) 6 × 10−7 ; (c) 8.0 × 10−7 ; (d) 1 × 10−6 ; (e) 2.0 × 10−6 ; (f) 4 × 10−6 ; (g) 6.0 × 10−6 ; (h) 8 × 10−6 ; (i) 1.0 × 10−5 ; (j) 2.0 × 10−5 mol L−1 ZP.
DPAdSV
OSWAdSV
7.50 7.48 0.13 0.27 0.350 0.348 99.73 0.43 0.57
7.50 7.49 0.23 0.13 0.350 0.349 99.87 0.33 0.29
Each value is the mean of ten experiments.
Table 3 Application of the stripping voltammetric techniques to the quantitative determination of ZP in spiked human urine. Technique
DPAdSV
OSWAdSV
Medium ZP spiked (mol L−1 ) Number of measurements (n) ZP found (mol L−1 ) Average recovery (%) R.S.D.% Bias (%)
Human urine 8 × 10−6 5 7.91 × 10−6 98.88 0.27 1.13
Human urine 8 × 10−6 5 7.81 × 10−6 97.62 0.33 1.23
3.3. Pharmaceutical applications
Fig. 5. OSWAdSV voltammograms of (a) blank (BR, pH 7.08); (b) 6 × 10−7 ; (c) 8.0 × 10−7 ; (d) 1 × 10−6 ; (e) 2.0 × 10−6 ; (f) 4 × 10−6 ; (g) 6.0 × 10−6 ; (h) 8 × 10−6 mol L−1 .
standard deviation of the peak currents (six runs, m is the slope of the calibration curve) [8,9]. The achieved limits of detection and quantitation were 2.78 × 10−7 and 5.28 × 10−7 mol L−1 for DPAdSV; 1.70 × 10−7 and 5.78 × 10−7 mol L−1 for OSWAdSV, respectively. The repeatability of the current measurement was calculated for both techniques from five independent runs of 1 × 10−6 mol L−1 ZP solution. The relative standard deviations were calculated for peak current to be 1.10% for DPAdSV and 1.17% for OSWAdSV, respectively.
Table 1 Regression data of the calibration lines for quantitative determination of ZP calibration plots in BR buffer (pH 7.08) at GCE by both stripping voltammetric techniques. Parameters
DPAdSV
OSWAdSV
Measured potential (V) Linear concentration range (mol L−1 ) Slope (A mol−1 L) R.S.D. of slope Intercept (nA) R.S.D. of intercept Correlation coefficient, r Number of measurements (n) LOD (mol L−1 ) LOQ (mol L−1 ) Repeatability of peak current (R.S.D.%)
0.95 6 × 10−7 to 2.5 × 10−5
1.10 6 × 10−7 to 2.5 × 10−5
5.00 × 107 0.41 0.143 0.10 0.996
5.00 × 107 0.47 0.336 0.23 0.993
5
5
2.78 × 10−7 5.28 × 10−7 1.10 for 1.0 × 10−6
1.70 × 10−7 5.78 × 10−7 1.17 for 1.0 × 10−6
The amount of ZP in tablets was calculated by reference to the appropriate calibration plots. The results obtained are given in Table 2. The proposed techniques could be applied with great success to ZP assay in tablets without any interference. As far as we know, there is no official technique in the pharmacopoeias or other literature describing the determination of ZP in pharmaceutical dosage forms. For this reason, the proposed techniques were checked by performing recovery tests. To determine whether excipients in the tablets interfered with the analysis, the accuracy of the proposed methods were evaluated by recovery tests after addition of known amounts of pure drug to pre-analyzed formulations of ZP (Table 2). The results showed the validity of the proposed techniques for the quantitative determination of ZP in tablets. The proposed DPAdSV and OSWAdSV techniques proved to be sufficiently precise and accurate for reliable electro analytical analysis of ZP. 3.4. Application to biological samples The possibility of applying both voltammetric procedures to the quantitative determination of ZP in human urine was also tested successfully by the standard additions of pure drug as described in Section 2. The found amount of ZP in human urine was calculated from the related linear regression equations. The results of these analyses are summarized in Table 3. Good recovery of ZP was achieved from this type of matrix. Quantitative assay of urine samples by proposed techniques involved only dilution of urine samples, so it is time-saving and no other procedure steps are required. 4. Conclusion As far as we know, the electrochemical oxidation of ZP on GCE was studied for the first time in the literature. From the CV measurement, it is understood that electrode reaction process is irreversible and pH dependent. In BR (pH 7.08) medium DPAdSV and OSWAdSV technique were developed and successfully applied to the quantitative determination of ZP in tablet and human urine samples. The
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analysis was performed with good recoveries without any interference from the excipients in tablets and human urine. The principal advantage of these proposed techniques over the other techniques is that it may be applied directly to the analysis of pharmaceutical dosage forms and to the biological samples without the need for extensive sample preparation, since there was no interference from the excipients and endogenous substances. Another advantage is that the developed techniques are rapid, requiring about 5 min to run any sample and involves no sample preparing other than dissolving, diluting, precipitating, centrifuging and transferring an aliquot to the supporting electrolyte. This paper is not intended to be a study of the pharmacodynamic properties of ZP, because only healthy volunteers were used for sample collection and results might be of no significance. It only indicated that the possibility of monitoring this compound makes the technique useful for pharmacokinetic and pharmacodynamic purposes [6,8,9,11,12]. In addition all above, the proposed stripping techniques might be a rapid and simple alternative to more complicated LC or UV methods for routine analysis of ZP in any medium where considerably little interference takes place. Acknowledgements The author gratefully acknowledges to the Scientific and Technical Research Council of Turkey (TUBITAK, Grant No.: TBAG-2173;
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102T062). The author would also like to thank Eczacibasi, Inc. (Istanbul, Turkey) for supplying pure ZP and its commercial tablet forms for developing proposed voltammetric technique. We would like to thank to Kenan Dikilitas, the teaching staff at Language and Literature Department at Canakkale Onsekiz Mart University for providing help with the language used in the study. References [1] M. Partinen, C. Hublin. Epidemiology of sleep disorders in. Prindpies and practices of sleep medicine. Third edition, M.H. Kryger, T. Roth, W.C. Dement (eds.), 2000, p. 558. [2] British Pharmacopoeia: Published on the recommendation of Medicines Commission, U.K., London: Her Majesty’s Stationery Office; 1993 (Addendum 1996). p. 1848. [3] http://en.wikipedia.org/wiki/Zopiclone. [4] Z. S¸entürk, J.C. Vire, H. Zhang, G. Quarin, J. Patrarche, G.D. Christian, Talanta 40 (1993) 313. [5] J.A. Sequella, J.C. Sturm, A. Alvarez-Lufje, L.J. Nunez-Vergara, J. AOOC Int. 77 (1994) 768. [6] S. Özkan, B. Uslu, Anal. Bioanal. Chem. 372 (2002) 582. [7] M.O. Nan Sun, Hu. Weimin, S. Baoxiang, Zhenlu, Anal. Bioanal. Chem. 385 (2006) 161. [8] M. C¸ıtak, S. Yılmaz, Y. Dilgin, G. Türker, S. Yagmur, H. Erdugan, N. Erdugan, Curr. Pharm. Anal. 3 (2007) 141. [9] S. Skrzypek, W. Ciesielski, A. Sokolowski, S. Yilmaz, D. Kazmierczak, Talanta 66 (2005) 1146. [10] E. Laviron, J. Electroanal. Chem. 112 (1980) 11. [11] E. Suren, S. Yilmaz, M. Turkoglu, S. Kaya, Environ Monit. Assess. 125 (2007) 91. [12] B. Uslu, B. Dogan, S.A. Ozkan, Anal. Chim. Acta 537 (2005) 307.