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Sensitive voltammetric sensor for determination of flumethasone pivalate, abused for doping by athletes
Sensors and Actuators B 137 (2009) 676–680
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Sensitive voltammetric sensor for determination of flumethasone pivalate, abused for doping by athletes Rajendra N. Goyal ∗ , Sanghamitra Chatterjee, Sunita Bishnoi Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, Uttaranchal, India
a r t i c l e
i n f o
Article history: Received 10 October 2008 Received in revised form 26 November 2008 Accepted 9 December 2008 Available online 24 December 2008 Keywords: Fullerene Pyrolytic graphite electrode Flumethasone pivalate Square wave voltammetry
a b s t r a c t The voltammetric reduction of flumethasone pivalate has been studied at fullerene-C60 -modified edge plane pyrolytic graphite electrode (PGE) and two well defined peaks are obtained with a peak potential of ∼−1220 mV and ∼−1351 mV respectively. The modified electrode showed a better response in comparison to a bare basal plane PGE and bare edge plane PGE. Linear calibration curves are obtained with sensitivity of 0.685 A M−1 and 0.570 A M−1 and the corresponding limits of detection at both the peaks have been found to be 13.81 × 10−8 M and 27.5 × 10−8 M. The recovery studies for flumethasone pivalate in biological samples were also investigated. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Corticosteroids affect the nervous system, cause euphoria, alleviate pain and improve athlete’s ability to concentrate in performance of endurance and power events and are thus considered as doping agents by World Anti-Doping Agency (WADA) [1–3]. Hence, their use is permitted only under strict medical supervision during competitive games as they are also included in the International Olympic Committee (IOC) list of restricted classes of substances [4,5]. Flumethasone pivalate (I), a topical corticosteroid of proven efficacy exhibits pronounced anti-allergic, antiproliferative, antipruritic and vasoconstrictive effects [6–9]. Its anti-inflammatory action is concentrated at the site of application which results in a prompt decrease in inflammation, exudation and itching [10–13]. It influences the skin diffusion profile of a ringing gel and exhibits an excellent chemical stability [9]. The use of corticosteroid agents in cosmetic products have been forbidden within the European Union, by the Directive 76/760 (Enclosure II) [14]. Corticosteroids can be illegally administered to cattle as growth promoting agents to improve meat production and flumethasone which is one of the active drugs of this class is registered in cattle for therapeutic use at a dosage of 1.25 mg/head per day [15,16].
∗ Corresponding author. Tel.: +91 1332 285794(O)/1332 274454(R). E-mail address: [email protected] (R.N. Goyal). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.12.022
The extensive use and misuse of flumethasone pivalate needs sensitive and selective analytical procedures to monitor clinical administration and to verify compliance with the law. In earlier times, the topical anti-inflammatory activity of the steroidal ointment was evaluated by croton oil ear edema method and homologous passive cutaneous anaphylaxis [17]. The determination by GC–MS provides good sensitivity and selectivity but most of the corticosteroids are thermally labile and their volatility is too low for direct GC analysis. The required derivatization of the analyte only complicates sample preparation. Several methods involving liquid chromatography–ionspray mass spectrometry [5], liquid chromatography–tandem mass spectrometry [15,16], immunoaffinity chromatography [18,19], high-resolution liquid chromatography/time-of-flight mass spectrometry [2] and
R.N. Goyal et al. / Sensors and Actuators B 137 (2009) 676–680
tandem thin-layer chromatography–high-performance liquid chromatography [14] have been reported for the determination of flumethasone pivalate. New techniques, mainly capillary electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography have also attracted wide interest in flumethasone analysis [20]. However, all these methods have the disadvantage of labour and time consuming steps, expensive instrumentation and running costs. To the best of our knowledge, no publications concerning the voltammetric determination of flumethasone pivalate is available in the literature. The electrochemical method presented in this work is a promising substitute to the frequently reported chromatographic methods owing to its simplicity, rapidity, reliability and low cost of analysis. In recent years, application of modified electrodes has been found to enhance the sensitivity of electrochemical determinations. In this paper, fullerene modified edge plane PGE is used for the voltammetric determination of flumethasone pivalate. 2. Experimental 2.1. Chemicals and reagents Flumethasone Pivalate was obtained from Sigma–Aldrich, Inc., USA and was used as received. C60 fullerene of purity >99.5% was purchased from Bucky USA, Houston, TX, USA. Phosphate buffer solutions were prepared using analytical grade chemicals by procedure described in the literature [21]. All other chemicals used were of analytical grade. Double distilled water was used to prepare the solutions. 2.2. Apparatus and procedure The voltammetric experiments were carried out using Bioanalytical System (BAS, West Lafayette, USA) CV-50 W voltammetric analyzer. The voltammetric cell used was a single compartment glass cell containing fullerene modified edge plane PGE (∼6 mm2 ) as working electrode, a platinum wire as counter electrode and Ag/AgCl (3 M NaCl) as reference electrode (BAS Model MF-2052 RB5B). The edge plane PGE was prepared as reported in literature [22]. All potentials are reported with respect to Ag/AgCl electrode at an ambient temperature of 27 ± 2 ◦ C. The stock solution of flumethasone pivalate (1 mM) was prepared by dissolving the required amount of the compound in methanol as it was insoluble in water. Required amount of stock
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solution was added to 3.0 ml phosphate buffer solution (1 M) and the total volume was made 6.0 ml with methanol, thus keeping the total methanolic content in the solutions as 50%. Oxygen might be a major interference at such a negative measurement potential and a complete exclusion of oxygen is important. Hence, before recording each voltammogram high-purity nitrogen was bubbled for 12–15 min to deoxygenate the solutions. The optimized parameters used for Osteryoung Square Wave Voltammetry (SWV) were: Initial E: 0 mV, Final E: −1800 mV, Square wave amplitude (Esw ): 25 mV, Potential Step (E): 4 mV, Square wave frequency (f): 15 Hz. The human blood samples from three healthy volunteers were obtained from the Institute hospital. The blood with EDTA as anticoagulant was ultra-centrifuged and the supernatant blood plasma obtained was taken for the analysis of flumethasone pivalate. Urine samples were received from laboratory personnel. The biological samples did not show any peak on scanning from 0.0 to −1.8 V due to the absence of other metabolites. The samples were therefore used for analysis without any dilution. 2.3. Preparation of fullerene-C60 -modified edge plane pyrolytic graphite electrode Prior to modification, the bare edge plane PGE was rubbed on the emery paper and then washed with double distilled water. It was then touched softly with tissue paper. Fullerene solution (150 M) was prepared by dissolving 0.54 mg of C60 in 5 ml dichloromethane solution using ultrasonic bath. A known volume (40 l) of this solution was coated onto the surface of the dried PGE using a microsyringe and dried using hair dryer. The electrode surface was then pretreated in 1 M KOH in potential range 0.0 to −1.5 V at the scan rate of 10 mV/s. Such a treatment caused partial reduction of fullerene layers to form conductive films. The partially reduced fullerene has been found to exhibit electrocatalytic activity, which leads to lowering of peak potential and enhancement of peak current in voltammetry [23]. Subsequently, the electrode is placed in phosphate buffer solution of pH 7.2, which was previously deoxygenated with high purity nitrogen for at least 10 min. Finally, the electrode was equilibrated by cyclic scanning in the potential range of 550 to −50 mV under a nitrogen atmosphere at a scan rate of 20 mV s−1 for 20 min. The fullerene-C60 -modified edge plane PGE was then ready for use. 3. Results and discussion 3.1. Voltammetric behavior of flumethasone pivalate
Fig. 1. A comparison of square-wave voltammograms of 60 M flumethasone pivalate at pH 7.2 at fullerene-C60 -modified edge plane PGE (—), bare basal plane PGE. (- - -), bare edge plane PGE (–·–·–), background phosphate buffer solution (pH 7.2) at fullerene-C60 -modified edge plane PGE (· · ·).
The electrocatalytic activity of the fullerene modified edge plane PGE is demonstrated by the comparison of the square wave voltammograms of 60 M flumethasone pivalate in 0.5 M phosphate buffer solution (pH 7.2) recorded at three different working electrodes (Fig. 1). On scanning from potential 0.0 to −1.8 V, two well-defined peaks are obtained at ∼−1220 mV and ∼−1351 mV at fullerene modified edge plane PGE. Under similar conditions, a small bump is noticed at potential ∼−1433 mV at bare basal plane PGE and two small peaks are observed at potential ∼−1324 mV and ∼−1558 mV at bare edge plane PGE. The significant improvement of peak current together with the sharpness of the peak clearly demonstrate that fullerene acts as an efficient promoter to enhance the kinetics of the electrochemical process. Further, a comparison of the voltammetric response of flumethasone pivalate was made between fullerene modified edge plane PGE and fullerene modified glassy carbon electrode (GCE). At fullerene modified GCE the reduction peaks were obtained at ∼−1280 mV and ∼−1460 mV indicating that edge plane PGE acts as a better substrate in comparison to GCE
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R.N. Goyal et al. / Sensors and Actuators B 137 (2009) 676–680
Fig. 2. Observed dependence of peak potential (−Ep ) of peak a () and peak b () on pH for 60 M flumethasone pivalate at fullerene-C60 -modified edge plane PGE.
for fullerene modification [22]. Hence, further studies were carried out at fullerene modified edge plane PGE. 3.2. Effect of pH and square wave frequency The pH of the supporting electrolyte has a significant influence on the reduction of flumethasone pivalate at the modified electrode. The pH effect was studied in the range 2.4–10.9 and the peak potential (Ep ) of both the peaks were found to shift towards more negative potentials with increase in pH (Fig. 2). The dependence of −Ep on pH for both the peaks can be expressed by the relations: −Ep (pH 2.4–10.9) = [967 + 34.58 pH] mV versus Ag/AgCl
[peak(a)]
−Ep (pH 2.4–10.9) = [923 + 63.37 pH] mV versus Ag/AgCl
Fig. 4. Osteryoung square wave voltammograms recorded for (a) phosphate buffer solution (background) at the modified electrode (· · ·) and (b) increasing concentration of flumethasone pivalate at the modified electrode (—) [curves were recorded at (i) 0.5; (ii) 5; (iii) 20; (iv) 40; (v) 60; (vi) 80; (vii) 100 M concentration in phosphate buffer solution of pH 7.2].
current and square wave frequency was found to be linear in the observed frequency range. The linear relation between ip and f can be represented by the following equations: ip (10−5 A) = 0.2557f − 0.8424
[peak(a)]
ip (10−5 A) = 0.2464f − 0.6013
[peak(b)]
[peak(b)]
having correlation coefficient 0.9972 and 0.9947 respectively. The slope of Ep versus pH corresponds to 34 mV/pH for the peak (a) which indicates that the reduction takes place by 2e− , H+ process. The slope for peak (b) was close to 60 mV/pH which suggested that equal number of protons and electrons are involved in the reduction. The square wave frequency also affects the peak potential values and the Ep is found to shift towards more negative potentials with increase in square wave frequency. The plot of −Ep versus log f was linear for both the peaks (Fig. 3) with a correlation coefficient of 0.9967 and 0.9962 respectively. Such a behavior indicated that the nature of redox reaction is reversible [24]. The variation of −Ep with log f can be expressed by the equations: −Ep (mV) = 60.555 logf + 1147.3
[peak(a)]
−Ep (mV) = 171.79 logf + 1158.0
[peak(b)]
The peak current (ip ) of both the peaks is found to increase with an increase in the square wave frequency. The effect of square wave frequency was studied in the range of 5–125 Hz as the peak merged with the background at higher frequencies. A plot between peak
having a correlation coefficient of 0.9970 and 0.9973 respectively. This indicates that the nature of electrode reaction is adsorption controlled [24–26]. 3.3. Concentration study Osteryoung square wave voltammograms at different concentrations of flumethasone pivalate at fullerene modified edge plane PGE were recorded. It was observed that at 0.1 M a single peak was obtained at potential ∼−1220 mV and its peak current increased with increase in concentration. At 0.3 M another peak started appearing at a higher negative potential (∼−1351 mV) whose peak current also increased linearly with concentration. The current values are obtained by subtracting the background current and are reported as an average of at least three replicate determinations. Fig. 4 depicts the systematic increase in the peak current values of both the peaks with an increase in the concentration in the range 0.5–100 M. The linear relation between the peak current and the concentration of flumethasone pivalate is expressed by the equation: ip (10−5 A) = 0.0685C (M) + 0.0757 ip (10−5 A) = 0.057C (M) + 0.3701
Fig. 3. Dependence of peak potential (−Ep ) of peak a () and peak b () on logarithm of square wave frequency for 60 M flumethasone pivalate at fullerene-C60 -modified edge plane PGE at pH 7.2.
[peak(a)]
[peak(b)]
where C is the concentration of flumethasone pivalate. The correlation coefficients for the equations are 0.9936 and 0.9969 respectively along with a sensitivity of 0.685 A M−1 and 0.570 A M−1 for both the peaks. The detection limit of the proposed method was calculated by using the formula 3/b, where is the standard deviation of the blank and b is the slope of the calibration curve. The detection limit for peak (a) and peak (b) was found to be 13.81 × 10−8 M and 27.5 × 10−8 M respectively.
R.N. Goyal et al. / Sensors and Actuators B 137 (2009) 676–680 Table 1 Recovery data of flumethasone pivalate added to human blood plasma. Spiked (M)
Detected (M)a
Recovery (%)
30.0 50.0 70.0
30.15 51.30 69.72
100.50 102.60 99.60
Sample 2
30.0 50.0 70.0
29.58 51.80 72.67
98.60 103.60 103.81
Sample 3
30.0 50.0 70.0
31.28 49.55 71.82
104.27 99.10 102.60
30.0 50.0 70.0
29.74 49.86 70.69
99.13 99.72 100.98
Sample 2
30.0 50.0 70.0
31.09 51.57 69.86
103.63 103.14 99.80
Sample 3
30.0 50.0 70.0
30.56 50.64 71.45
101.87 101.28 102.07
Peak a Sample 1
Peak b Sample 1
a
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plane PGE exhibits good stability and reproducibility for the determination of flumethasone pivalate. 3.5. Recovery test To study the accuracy of the proposed method, attempts were made to obtain samples of blood and urine from the patients undergoing treatment with flumethasone pivalate. However, due to major use of this steroid as an ointment, no samples could be obtained. Therefore, recovery experiments were carried out by standard addition method in biological samples obtained from healthy volunteers. The results observed are listed in Tables 1 and 2. The recoveries for peak (a) varied in the range from 98.60% to 104.27% in the case of human blood plasma and from 97.95% to 104.17% in case of urine samples. For peak (b) the recoveries lie in the range from 99.13% to 103.63% (human blood plasma) and from 98.97% to 102.58% (urine). Thus, it is quite evident that for both the peaks the recovery data lie in the acceptable range. The major metabolites present in human urine and blood plasma are uric acid and ascorbic acid. These metabolites did not interfere with the present investigation as they did not exhibit reduction peak in the range 0 to −1.8 V.
The R.S.D. value for determination was less than 2.1% for n = 3.
4. Conclusions 3.4. Stability and reproducibility of the modified electrode The reproducibility and stability of the fullerene modified edge plane PGE for the determination of flumethasone pivalate was investigated. The stability of the modified electrode was evaluated by measuring the current response at a fixed concentration of 60 M of flumethasone pivalate over a period of 10 days. The electrode was used daily and stored in the air. The experimental results indicated that the current responses deviated intraday by 1.8% (peak a) and 2.3% (peak b) and interday by 3.7% (peak a) and 4.2% (peak b), suggesting that the modified electrode possesses good stability for both the peaks. To characterize the reproducibility of the modified electrode, repetitive determinations of flumethasone pivalate were carried out at 60 M concentration at pH 7.2. The results of six replicate measurements showed a relative standard deviation of 1.8% for peak (a) and 2.2% for peak (b) indicating that the results are reproducible. To check the reproducibility of the results further, four different fullerene modified electrodes of almost the same area were prepared. They showed an acceptable reproducibility with a relative standard deviation of 1.2% for peak (a) and 1.6% for peak (b) for 60 M flumethasone pivalate. Thus, the fullerene modified edge Table 2 Analytical recovery of flumethasone pivalate added to urine samples.
Peak a Sample 1
Sample 2
Peak b Sample 1
Sample 2
a
Added (M)
Found (M)a
Recovery (%)
20.0 40.0 60.0
20.52 41.67 59.64
102.60 104.17 99.40
20.0 40.0 60.0
19.59 39.54 61.47
97.95 98.85 102.45
20.0 40.0 60.0
20.34 39.59 61.55
101.70 98.97 102.58
20.0 40.0 60.0
19.81 40.07 59.64
99.05 100.17 99.40
The R.S.D. value for determination was less than 3.5% for n = 3.
The fullerene modified electrode showed a marked increase in the sensitivity of flumethasone pivalate by accelerating the rate of electron transfer. Partially reduced fullerene-C60 -modified electrodes have been shown as an excellent working electrode owing to its properties such as electronic conductivity and good biocompatibility [27]. The reduction of fullerene is a multi-step transformation which involves three electrons per C60 molecule. KC60 and K2 C60 are the transient species showing ion-exchange properties and the conductive K3 C60 is the final product [28]. The partially reduced fullerene-C60 films have been found sufficiently conductive so that they could be used as electrodes for various electrochemical reactions. They have also been shown to have special electrocatalytic properties [27,28] e−
e−
e−
C60 −→KC60 −→K2 C60 −→K3 C60 Lowering of peak potential, improved reduction peak current with better peak shape and excellent reproducibility are the principal advantages of the partially reduced fullerene modified edge plane PGE. The available sites for reduction in flumethasone pivalate are the keto groups present at position 3 and 20 along with a carbonyl group in the pivalate part. The reduction peak (a) corresponds to the reduction of the carbonyl group in the ester part of the salt which undergoes reduction as stated in literature for esters by a 2e− , H+ process to give a carboxylate anion and an alkane [29]. The carbonyl group at position 20 does not undergo reduction as it is clearly stated in literature that in ketosteroids, a carbonyl group conjugated with a double bond undergoes reduction and not an isolated keto group [30]. The peak (b) is due to the reduction of the keto group present at position 3 by a 2e− , 2H+ process to give a hydroxyl group. Thus, the most probable sites for reduction are the carbonyl group present in the pivalate salt and at position 3 of the ketosteroid. The peak (a) with a lower value of detection limit and higher sensitivity is better in comparison to peak (b) for determination of flumethasone pivalate. The present studies clearly reveal that fullerene modified edge plane PGE gives a better response in comparison to a bare basal plane PGE and even bare edge plane PGE. The method described above is not only easy to perform, but also requires less time, financial input and sample amount than reported for other approaches.
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The method eliminates the need for derivitization prior to analysis. Hence, it is an attractive alternative to the chromatographic methods for the determination of flumethasone pivalate with good reproducibility and recovery along with a low detection limit. Acknowledgements One of the authors (S. Chatterjee) is thankful to the Council of Scientific and Industrial Research, New Delhi for the award of Junior Research Fellowship. Financial assistance for this work was provided by the Department of Science and Technology, New Delhi vide grant no. SR/S1/IC-21/2007. References [1] M.H. Spyridaki, P. Kiousi, A. Vonaparti, P. Valavani, V. Zonaras, M. Zahariou, E. Sianos, G. Tsoupras, C. Georgakopoulos, Anal. Chim. Acta 573 (2006) 242. [2] M.E. Touber, M.C. van Engelen, C. Georgakopoulos, J.A. van Rhijn, M.W.F. Nielen, Anal. Chim. Acta 586 (2007) 137. [3] K. Deventer, F.T. Delbeke, Rapid Commun. Mass Spectrom. 17 (2003) 2107. [4] K. Fluri, L. Rivier, A. Dienes-Nagy, C. You, A. Maitre, C. Schweizer, M. Saugy, P. Mangin, J. Chromatogr. A 926 (2001) 87. [5] V. Cirimele, P. Kintz, V. Dumestre, J.P. Goulle, B. Ludes, Forensic Sci. Int. 107 (2000) 381. [6] F. Gao, US Patent 6,541,263 (2003). [7] R.M. Loria, US Patent 5,387,583 (1995). [8] L. Bessone, Minerva Pediatr. 18 (1966) 2024.
[9] S. Holler, C. Valenta, Eur. J. Pharm. Biopharm. 66 (2007) 120. [10] R. Sackeyfio, J. Fong, N. Hurst, P. Manivasakam, E.R. Jost-Price, G. Zimmermann, C. Keith, A. Borisy, US Patent 6,897,206 (2005). [11] I. Villax, Z. Mendes, US Patent 6,528,666 (2003). [12] D. Bednarek, B. Zdzisinska, M. Kondracki, M. Kandefer-Szerszen, Vet. Microbiol. 96 (2003) 53. [13] V. Wong, L. Peng, US Patent 6,369,116 (1998). [14] L. Gagliardi, D. De Orsi, M.R.D. Giudice, F. Gatta, R. Porra, P. Chimenti, D. Tonelli, Anal. Chim. Acta 457 (2002) 187. [15] G. Brambilla, F. Buiarelli, G.P. Cartoni, F. Coccioli, C. Colamonici, A. Fagiolo, C. Giannini, B. Neri, J. Chromatogr. B 755 (2001) 265. [16] O.V.D. Hauwe, M. Schneider, A. Sahin, C.H.V. Peteghem, H. Naegeli, J. Agric. Food. Chem. 51 (2003) 326. [17] Y. Iizuka, Y. Endo, Y. Misawa, E. Misaka, Agents Actions 11 (1981) 254. [18] X. Gu, M. Meleka-Boules, C.L. Chen, J. Capillary, Electrophoresis 3 (1996) 43. [19] C.S. Creaser, S.J. Feely, E. Houghton, M. Seymour, J. Chromatogr. A 794 (1998) 37. [20] S. Gorog, Anal. Sci. 20 (2004) 767. [21] G.D. Christian, W.C. Purdy, J. Electroanal. Chem. 3 (1962) 363. [22] R.N. Goyal, V.K. Gupta, S. Chatterjee, Biosens. Bioelectron. (2009), doi:10.1016/j.bios.2008.08.024. [23] A. Szucs, M. Tolgyesi, M. Csiszar, J.B. Nagy, M. Novak, Electrochim. Acta 44 (1998) 613. [24] F. Quentel, C. Elleouet, Electroanalysis 13 (2001) 1030. [25] M.R.C. Massaroppi, S.A.S. Machado, L.A. Avaca, J. Braz. Chem. Soc. 14 (2003) 113. [26] S. Komorsky-Lovric, S. Gagic, R. Penovski, Anal. Chim. Acta 389 (1999) 219. [27] S. Fiorito, A. Serafino, F. Andreola, P. Bernier, Carbon 44 (2006) 1100. [28] R.N. Goyal, V.K. Gupta, N. Bachheti, Anal. Chim. Acta 597 (2007) 82. [29] H. Lund, M.M. Baizer, Organic Electrochemistry An Introduction and A Guide, third ed., Marcel Dekker, Inc., New York, 1991. [30] P. Zuman, M. Kolthoff, Progress in Polarography, vol. 2, Interscience Publishers, New York, 1962.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Sensitive voltammetric sensor for determination of flumethasone pivalate, abused for doping by athletes Rajendra N. Goyal ∗ , Sanghamitra Chatterjee, Sunita Bishnoi Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, Uttaranchal, India
a r t i c l e
i n f o
Article history: Received 10 October 2008 Received in revised form 26 November 2008 Accepted 9 December 2008 Available online 24 December 2008 Keywords: Fullerene Pyrolytic graphite electrode Flumethasone pivalate Square wave voltammetry
a b s t r a c t The voltammetric reduction of flumethasone pivalate has been studied at fullerene-C60 -modified edge plane pyrolytic graphite electrode (PGE) and two well defined peaks are obtained with a peak potential of ∼−1220 mV and ∼−1351 mV respectively. The modified electrode showed a better response in comparison to a bare basal plane PGE and bare edge plane PGE. Linear calibration curves are obtained with sensitivity of 0.685 A M−1 and 0.570 A M−1 and the corresponding limits of detection at both the peaks have been found to be 13.81 × 10−8 M and 27.5 × 10−8 M. The recovery studies for flumethasone pivalate in biological samples were also investigated. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Corticosteroids affect the nervous system, cause euphoria, alleviate pain and improve athlete’s ability to concentrate in performance of endurance and power events and are thus considered as doping agents by World Anti-Doping Agency (WADA) [1–3]. Hence, their use is permitted only under strict medical supervision during competitive games as they are also included in the International Olympic Committee (IOC) list of restricted classes of substances [4,5]. Flumethasone pivalate (I), a topical corticosteroid of proven efficacy exhibits pronounced anti-allergic, antiproliferative, antipruritic and vasoconstrictive effects [6–9]. Its anti-inflammatory action is concentrated at the site of application which results in a prompt decrease in inflammation, exudation and itching [10–13]. It influences the skin diffusion profile of a ringing gel and exhibits an excellent chemical stability [9]. The use of corticosteroid agents in cosmetic products have been forbidden within the European Union, by the Directive 76/760 (Enclosure II) [14]. Corticosteroids can be illegally administered to cattle as growth promoting agents to improve meat production and flumethasone which is one of the active drugs of this class is registered in cattle for therapeutic use at a dosage of 1.25 mg/head per day [15,16].
∗ Corresponding author. Tel.: +91 1332 285794(O)/1332 274454(R). E-mail address: [email protected] (R.N. Goyal). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.12.022
The extensive use and misuse of flumethasone pivalate needs sensitive and selective analytical procedures to monitor clinical administration and to verify compliance with the law. In earlier times, the topical anti-inflammatory activity of the steroidal ointment was evaluated by croton oil ear edema method and homologous passive cutaneous anaphylaxis [17]. The determination by GC–MS provides good sensitivity and selectivity but most of the corticosteroids are thermally labile and their volatility is too low for direct GC analysis. The required derivatization of the analyte only complicates sample preparation. Several methods involving liquid chromatography–ionspray mass spectrometry [5], liquid chromatography–tandem mass spectrometry [15,16], immunoaffinity chromatography [18,19], high-resolution liquid chromatography/time-of-flight mass spectrometry [2] and
R.N. Goyal et al. / Sensors and Actuators B 137 (2009) 676–680
tandem thin-layer chromatography–high-performance liquid chromatography [14] have been reported for the determination of flumethasone pivalate. New techniques, mainly capillary electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography have also attracted wide interest in flumethasone analysis [20]. However, all these methods have the disadvantage of labour and time consuming steps, expensive instrumentation and running costs. To the best of our knowledge, no publications concerning the voltammetric determination of flumethasone pivalate is available in the literature. The electrochemical method presented in this work is a promising substitute to the frequently reported chromatographic methods owing to its simplicity, rapidity, reliability and low cost of analysis. In recent years, application of modified electrodes has been found to enhance the sensitivity of electrochemical determinations. In this paper, fullerene modified edge plane PGE is used for the voltammetric determination of flumethasone pivalate. 2. Experimental 2.1. Chemicals and reagents Flumethasone Pivalate was obtained from Sigma–Aldrich, Inc., USA and was used as received. C60 fullerene of purity >99.5% was purchased from Bucky USA, Houston, TX, USA. Phosphate buffer solutions were prepared using analytical grade chemicals by procedure described in the literature [21]. All other chemicals used were of analytical grade. Double distilled water was used to prepare the solutions. 2.2. Apparatus and procedure The voltammetric experiments were carried out using Bioanalytical System (BAS, West Lafayette, USA) CV-50 W voltammetric analyzer. The voltammetric cell used was a single compartment glass cell containing fullerene modified edge plane PGE (∼6 mm2 ) as working electrode, a platinum wire as counter electrode and Ag/AgCl (3 M NaCl) as reference electrode (BAS Model MF-2052 RB5B). The edge plane PGE was prepared as reported in literature [22]. All potentials are reported with respect to Ag/AgCl electrode at an ambient temperature of 27 ± 2 ◦ C. The stock solution of flumethasone pivalate (1 mM) was prepared by dissolving the required amount of the compound in methanol as it was insoluble in water. Required amount of stock
677
solution was added to 3.0 ml phosphate buffer solution (1 M) and the total volume was made 6.0 ml with methanol, thus keeping the total methanolic content in the solutions as 50%. Oxygen might be a major interference at such a negative measurement potential and a complete exclusion of oxygen is important. Hence, before recording each voltammogram high-purity nitrogen was bubbled for 12–15 min to deoxygenate the solutions. The optimized parameters used for Osteryoung Square Wave Voltammetry (SWV) were: Initial E: 0 mV, Final E: −1800 mV, Square wave amplitude (Esw ): 25 mV, Potential Step (E): 4 mV, Square wave frequency (f): 15 Hz. The human blood samples from three healthy volunteers were obtained from the Institute hospital. The blood with EDTA as anticoagulant was ultra-centrifuged and the supernatant blood plasma obtained was taken for the analysis of flumethasone pivalate. Urine samples were received from laboratory personnel. The biological samples did not show any peak on scanning from 0.0 to −1.8 V due to the absence of other metabolites. The samples were therefore used for analysis without any dilution. 2.3. Preparation of fullerene-C60 -modified edge plane pyrolytic graphite electrode Prior to modification, the bare edge plane PGE was rubbed on the emery paper and then washed with double distilled water. It was then touched softly with tissue paper. Fullerene solution (150 M) was prepared by dissolving 0.54 mg of C60 in 5 ml dichloromethane solution using ultrasonic bath. A known volume (40 l) of this solution was coated onto the surface of the dried PGE using a microsyringe and dried using hair dryer. The electrode surface was then pretreated in 1 M KOH in potential range 0.0 to −1.5 V at the scan rate of 10 mV/s. Such a treatment caused partial reduction of fullerene layers to form conductive films. The partially reduced fullerene has been found to exhibit electrocatalytic activity, which leads to lowering of peak potential and enhancement of peak current in voltammetry [23]. Subsequently, the electrode is placed in phosphate buffer solution of pH 7.2, which was previously deoxygenated with high purity nitrogen for at least 10 min. Finally, the electrode was equilibrated by cyclic scanning in the potential range of 550 to −50 mV under a nitrogen atmosphere at a scan rate of 20 mV s−1 for 20 min. The fullerene-C60 -modified edge plane PGE was then ready for use. 3. Results and discussion 3.1. Voltammetric behavior of flumethasone pivalate
Fig. 1. A comparison of square-wave voltammograms of 60 M flumethasone pivalate at pH 7.2 at fullerene-C60 -modified edge plane PGE (—), bare basal plane PGE. (- - -), bare edge plane PGE (–·–·–), background phosphate buffer solution (pH 7.2) at fullerene-C60 -modified edge plane PGE (· · ·).
The electrocatalytic activity of the fullerene modified edge plane PGE is demonstrated by the comparison of the square wave voltammograms of 60 M flumethasone pivalate in 0.5 M phosphate buffer solution (pH 7.2) recorded at three different working electrodes (Fig. 1). On scanning from potential 0.0 to −1.8 V, two well-defined peaks are obtained at ∼−1220 mV and ∼−1351 mV at fullerene modified edge plane PGE. Under similar conditions, a small bump is noticed at potential ∼−1433 mV at bare basal plane PGE and two small peaks are observed at potential ∼−1324 mV and ∼−1558 mV at bare edge plane PGE. The significant improvement of peak current together with the sharpness of the peak clearly demonstrate that fullerene acts as an efficient promoter to enhance the kinetics of the electrochemical process. Further, a comparison of the voltammetric response of flumethasone pivalate was made between fullerene modified edge plane PGE and fullerene modified glassy carbon electrode (GCE). At fullerene modified GCE the reduction peaks were obtained at ∼−1280 mV and ∼−1460 mV indicating that edge plane PGE acts as a better substrate in comparison to GCE
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Fig. 2. Observed dependence of peak potential (−Ep ) of peak a () and peak b () on pH for 60 M flumethasone pivalate at fullerene-C60 -modified edge plane PGE.
for fullerene modification [22]. Hence, further studies were carried out at fullerene modified edge plane PGE. 3.2. Effect of pH and square wave frequency The pH of the supporting electrolyte has a significant influence on the reduction of flumethasone pivalate at the modified electrode. The pH effect was studied in the range 2.4–10.9 and the peak potential (Ep ) of both the peaks were found to shift towards more negative potentials with increase in pH (Fig. 2). The dependence of −Ep on pH for both the peaks can be expressed by the relations: −Ep (pH 2.4–10.9) = [967 + 34.58 pH] mV versus Ag/AgCl
[peak(a)]
−Ep (pH 2.4–10.9) = [923 + 63.37 pH] mV versus Ag/AgCl
Fig. 4. Osteryoung square wave voltammograms recorded for (a) phosphate buffer solution (background) at the modified electrode (· · ·) and (b) increasing concentration of flumethasone pivalate at the modified electrode (—) [curves were recorded at (i) 0.5; (ii) 5; (iii) 20; (iv) 40; (v) 60; (vi) 80; (vii) 100 M concentration in phosphate buffer solution of pH 7.2].
current and square wave frequency was found to be linear in the observed frequency range. The linear relation between ip and f can be represented by the following equations: ip (10−5 A) = 0.2557f − 0.8424
[peak(a)]
ip (10−5 A) = 0.2464f − 0.6013
[peak(b)]
[peak(b)]
having correlation coefficient 0.9972 and 0.9947 respectively. The slope of Ep versus pH corresponds to 34 mV/pH for the peak (a) which indicates that the reduction takes place by 2e− , H+ process. The slope for peak (b) was close to 60 mV/pH which suggested that equal number of protons and electrons are involved in the reduction. The square wave frequency also affects the peak potential values and the Ep is found to shift towards more negative potentials with increase in square wave frequency. The plot of −Ep versus log f was linear for both the peaks (Fig. 3) with a correlation coefficient of 0.9967 and 0.9962 respectively. Such a behavior indicated that the nature of redox reaction is reversible [24]. The variation of −Ep with log f can be expressed by the equations: −Ep (mV) = 60.555 logf + 1147.3
[peak(a)]
−Ep (mV) = 171.79 logf + 1158.0
[peak(b)]
The peak current (ip ) of both the peaks is found to increase with an increase in the square wave frequency. The effect of square wave frequency was studied in the range of 5–125 Hz as the peak merged with the background at higher frequencies. A plot between peak
having a correlation coefficient of 0.9970 and 0.9973 respectively. This indicates that the nature of electrode reaction is adsorption controlled [24–26]. 3.3. Concentration study Osteryoung square wave voltammograms at different concentrations of flumethasone pivalate at fullerene modified edge plane PGE were recorded. It was observed that at 0.1 M a single peak was obtained at potential ∼−1220 mV and its peak current increased with increase in concentration. At 0.3 M another peak started appearing at a higher negative potential (∼−1351 mV) whose peak current also increased linearly with concentration. The current values are obtained by subtracting the background current and are reported as an average of at least three replicate determinations. Fig. 4 depicts the systematic increase in the peak current values of both the peaks with an increase in the concentration in the range 0.5–100 M. The linear relation between the peak current and the concentration of flumethasone pivalate is expressed by the equation: ip (10−5 A) = 0.0685C (M) + 0.0757 ip (10−5 A) = 0.057C (M) + 0.3701
Fig. 3. Dependence of peak potential (−Ep ) of peak a () and peak b () on logarithm of square wave frequency for 60 M flumethasone pivalate at fullerene-C60 -modified edge plane PGE at pH 7.2.
[peak(a)]
[peak(b)]
where C is the concentration of flumethasone pivalate. The correlation coefficients for the equations are 0.9936 and 0.9969 respectively along with a sensitivity of 0.685 A M−1 and 0.570 A M−1 for both the peaks. The detection limit of the proposed method was calculated by using the formula 3/b, where is the standard deviation of the blank and b is the slope of the calibration curve. The detection limit for peak (a) and peak (b) was found to be 13.81 × 10−8 M and 27.5 × 10−8 M respectively.
R.N. Goyal et al. / Sensors and Actuators B 137 (2009) 676–680 Table 1 Recovery data of flumethasone pivalate added to human blood plasma. Spiked (M)
Detected (M)a
Recovery (%)
30.0 50.0 70.0
30.15 51.30 69.72
100.50 102.60 99.60
Sample 2
30.0 50.0 70.0
29.58 51.80 72.67
98.60 103.60 103.81
Sample 3
30.0 50.0 70.0
31.28 49.55 71.82
104.27 99.10 102.60
30.0 50.0 70.0
29.74 49.86 70.69
99.13 99.72 100.98
Sample 2
30.0 50.0 70.0
31.09 51.57 69.86
103.63 103.14 99.80
Sample 3
30.0 50.0 70.0
30.56 50.64 71.45
101.87 101.28 102.07
Peak a Sample 1
Peak b Sample 1
a
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plane PGE exhibits good stability and reproducibility for the determination of flumethasone pivalate. 3.5. Recovery test To study the accuracy of the proposed method, attempts were made to obtain samples of blood and urine from the patients undergoing treatment with flumethasone pivalate. However, due to major use of this steroid as an ointment, no samples could be obtained. Therefore, recovery experiments were carried out by standard addition method in biological samples obtained from healthy volunteers. The results observed are listed in Tables 1 and 2. The recoveries for peak (a) varied in the range from 98.60% to 104.27% in the case of human blood plasma and from 97.95% to 104.17% in case of urine samples. For peak (b) the recoveries lie in the range from 99.13% to 103.63% (human blood plasma) and from 98.97% to 102.58% (urine). Thus, it is quite evident that for both the peaks the recovery data lie in the acceptable range. The major metabolites present in human urine and blood plasma are uric acid and ascorbic acid. These metabolites did not interfere with the present investigation as they did not exhibit reduction peak in the range 0 to −1.8 V.
The R.S.D. value for determination was less than 2.1% for n = 3.
4. Conclusions 3.4. Stability and reproducibility of the modified electrode The reproducibility and stability of the fullerene modified edge plane PGE for the determination of flumethasone pivalate was investigated. The stability of the modified electrode was evaluated by measuring the current response at a fixed concentration of 60 M of flumethasone pivalate over a period of 10 days. The electrode was used daily and stored in the air. The experimental results indicated that the current responses deviated intraday by 1.8% (peak a) and 2.3% (peak b) and interday by 3.7% (peak a) and 4.2% (peak b), suggesting that the modified electrode possesses good stability for both the peaks. To characterize the reproducibility of the modified electrode, repetitive determinations of flumethasone pivalate were carried out at 60 M concentration at pH 7.2. The results of six replicate measurements showed a relative standard deviation of 1.8% for peak (a) and 2.2% for peak (b) indicating that the results are reproducible. To check the reproducibility of the results further, four different fullerene modified electrodes of almost the same area were prepared. They showed an acceptable reproducibility with a relative standard deviation of 1.2% for peak (a) and 1.6% for peak (b) for 60 M flumethasone pivalate. Thus, the fullerene modified edge Table 2 Analytical recovery of flumethasone pivalate added to urine samples.
Peak a Sample 1
Sample 2
Peak b Sample 1
Sample 2
a
Added (M)
Found (M)a
Recovery (%)
20.0 40.0 60.0
20.52 41.67 59.64
102.60 104.17 99.40
20.0 40.0 60.0
19.59 39.54 61.47
97.95 98.85 102.45
20.0 40.0 60.0
20.34 39.59 61.55
101.70 98.97 102.58
20.0 40.0 60.0
19.81 40.07 59.64
99.05 100.17 99.40
The R.S.D. value for determination was less than 3.5% for n = 3.
The fullerene modified electrode showed a marked increase in the sensitivity of flumethasone pivalate by accelerating the rate of electron transfer. Partially reduced fullerene-C60 -modified electrodes have been shown as an excellent working electrode owing to its properties such as electronic conductivity and good biocompatibility [27]. The reduction of fullerene is a multi-step transformation which involves three electrons per C60 molecule. KC60 and K2 C60 are the transient species showing ion-exchange properties and the conductive K3 C60 is the final product [28]. The partially reduced fullerene-C60 films have been found sufficiently conductive so that they could be used as electrodes for various electrochemical reactions. They have also been shown to have special electrocatalytic properties [27,28] e−
e−
e−
C60 −→KC60 −→K2 C60 −→K3 C60 Lowering of peak potential, improved reduction peak current with better peak shape and excellent reproducibility are the principal advantages of the partially reduced fullerene modified edge plane PGE. The available sites for reduction in flumethasone pivalate are the keto groups present at position 3 and 20 along with a carbonyl group in the pivalate part. The reduction peak (a) corresponds to the reduction of the carbonyl group in the ester part of the salt which undergoes reduction as stated in literature for esters by a 2e− , H+ process to give a carboxylate anion and an alkane [29]. The carbonyl group at position 20 does not undergo reduction as it is clearly stated in literature that in ketosteroids, a carbonyl group conjugated with a double bond undergoes reduction and not an isolated keto group [30]. The peak (b) is due to the reduction of the keto group present at position 3 by a 2e− , 2H+ process to give a hydroxyl group. Thus, the most probable sites for reduction are the carbonyl group present in the pivalate salt and at position 3 of the ketosteroid. The peak (a) with a lower value of detection limit and higher sensitivity is better in comparison to peak (b) for determination of flumethasone pivalate. The present studies clearly reveal that fullerene modified edge plane PGE gives a better response in comparison to a bare basal plane PGE and even bare edge plane PGE. The method described above is not only easy to perform, but also requires less time, financial input and sample amount than reported for other approaches.
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The method eliminates the need for derivitization prior to analysis. Hence, it is an attractive alternative to the chromatographic methods for the determination of flumethasone pivalate with good reproducibility and recovery along with a low detection limit. Acknowledgements One of the authors (S. Chatterjee) is thankful to the Council of Scientific and Industrial Research, New Delhi for the award of Junior Research Fellowship. Financial assistance for this work was provided by the Department of Science and Technology, New Delhi vide grant no. SR/S1/IC-21/2007. References [1] M.H. Spyridaki, P. Kiousi, A. Vonaparti, P. Valavani, V. Zonaras, M. Zahariou, E. Sianos, G. Tsoupras, C. Georgakopoulos, Anal. Chim. Acta 573 (2006) 242. [2] M.E. Touber, M.C. van Engelen, C. Georgakopoulos, J.A. van Rhijn, M.W.F. Nielen, Anal. Chim. Acta 586 (2007) 137. [3] K. Deventer, F.T. Delbeke, Rapid Commun. Mass Spectrom. 17 (2003) 2107. [4] K. Fluri, L. Rivier, A. Dienes-Nagy, C. You, A. Maitre, C. Schweizer, M. Saugy, P. Mangin, J. Chromatogr. A 926 (2001) 87. [5] V. Cirimele, P. Kintz, V. Dumestre, J.P. Goulle, B. Ludes, Forensic Sci. Int. 107 (2000) 381. [6] F. Gao, US Patent 6,541,263 (2003). [7] R.M. Loria, US Patent 5,387,583 (1995). [8] L. Bessone, Minerva Pediatr. 18 (1966) 2024.
[9] S. Holler, C. Valenta, Eur. J. Pharm. Biopharm. 66 (2007) 120. [10] R. Sackeyfio, J. Fong, N. Hurst, P. Manivasakam, E.R. Jost-Price, G. Zimmermann, C. Keith, A. Borisy, US Patent 6,897,206 (2005). [11] I. Villax, Z. Mendes, US Patent 6,528,666 (2003). [12] D. Bednarek, B. Zdzisinska, M. Kondracki, M. Kandefer-Szerszen, Vet. Microbiol. 96 (2003) 53. [13] V. Wong, L. Peng, US Patent 6,369,116 (1998). [14] L. Gagliardi, D. De Orsi, M.R.D. Giudice, F. Gatta, R. Porra, P. Chimenti, D. Tonelli, Anal. Chim. Acta 457 (2002) 187. [15] G. Brambilla, F. Buiarelli, G.P. Cartoni, F. Coccioli, C. Colamonici, A. Fagiolo, C. Giannini, B. Neri, J. Chromatogr. B 755 (2001) 265. [16] O.V.D. Hauwe, M. Schneider, A. Sahin, C.H.V. Peteghem, H. Naegeli, J. Agric. Food. Chem. 51 (2003) 326. [17] Y. Iizuka, Y. Endo, Y. Misawa, E. Misaka, Agents Actions 11 (1981) 254. [18] X. Gu, M. Meleka-Boules, C.L. Chen, J. Capillary, Electrophoresis 3 (1996) 43. [19] C.S. Creaser, S.J. Feely, E. Houghton, M. Seymour, J. Chromatogr. A 794 (1998) 37. [20] S. Gorog, Anal. Sci. 20 (2004) 767. [21] G.D. Christian, W.C. Purdy, J. Electroanal. Chem. 3 (1962) 363. [22] R.N. Goyal, V.K. Gupta, S. Chatterjee, Biosens. Bioelectron. (2009), doi:10.1016/j.bios.2008.08.024. [23] A. Szucs, M. Tolgyesi, M. Csiszar, J.B. Nagy, M. Novak, Electrochim. Acta 44 (1998) 613. [24] F. Quentel, C. Elleouet, Electroanalysis 13 (2001) 1030. [25] M.R.C. Massaroppi, S.A.S. Machado, L.A. Avaca, J. Braz. Chem. Soc. 14 (2003) 113. [26] S. Komorsky-Lovric, S. Gagic, R. Penovski, Anal. Chim. Acta 389 (1999) 219. [27] S. Fiorito, A. Serafino, F. Andreola, P. Bernier, Carbon 44 (2006) 1100. [28] R.N. Goyal, V.K. Gupta, N. Bachheti, Anal. Chim. Acta 597 (2007) 82. [29] H. Lund, M.M. Baizer, Organic Electrochemistry An Introduction and A Guide, third ed., Marcel Dekker, Inc., New York, 1991. [30] P. Zuman, M. Kolthoff, Progress in Polarography, vol. 2, Interscience Publishers, New York, 1962.