- Email: [email protected]
Adsorptive stripping square-wave voltammetric behavior of rofecoxib
Microchemical Journal 72 (2002) 35–41
Adsorptive stripping square-wave voltammetric behavior of rofecoxib A. Radi* Department of Chemistry, Faculty of Science, Mansoura University, 34517 Dumyat, Egypt Received 26 March 2001; received in revised form 16 October 2001; accepted 21 October 2001
Abstract Adsorption and reduction of rofecoxib were investigated by cyclic and square-wave voltammetry on a hanging mercury drop electrode in electrolytes of various pH values. The reduction process on hanging mercury drop electrodes gave rise to a single peak within the entire pH range (2.0–11.5). In alkaline solutions, rofecoxib gave a sensitive adsorptive reductive peak; approximately 10 times larger than those obtained by applying a square-wave scan without prior accumulation. Application of the method to the determination of rofecoxib in two pharmaceutical products (Vioxx 12.5 and 25 mg), without sample pretreatment, resulted in acceptable deviation from the stated concentrations. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Stripping voltammetry; Square-wave; Rofecoxib; Vioxx
1. Introduction Rofecoxib (Vioxx) is a potent and selective inhibitor of the COX-2 isoform of cyclooxygenase, which is used as a non-steroidal anti-inflammatory drug (NSAID). It is indicated in the symptomatic relief of pain due to osteoarthritis. The initial oral dosage of rofecoxib is 12.5 mg once daily in adults, and this dose may be increased up to a maximal dosage of 25 mg once daily if necessary w1x. Its clinical efficacy seems similar to that of other NSAIDs at maximal recommended dosages, but its safety profile, especially gastrointestinal tolerance, is much better because of the COX-2 *Tel.: q20-5034-7054; fax: q20-57-403868. E-mail address: [email protected] (A. Radi).
selectivity w2,3x. Rofecoxib is designated chemically as 4-(4-methanesulfonylphenyl)-3-phenyl5H-furan-2-one (structural formula in Scheme 1). Analytical procedures for rofecoxib determination are few. Currently the most commonly employed techniques for the determination of the rofecoxib drug have been based on HPLC w4,5x. Adsorptive stripping voltammetry (AdSV) has been demonstrated to be a sensitive analytical method for a wide range of organic compounds that adsorb, in a reproducible way, on electrode surfaces w6x. This paper shows that rofecoxib is adsorbed onto a mercury electrode. By using this phenomenon and by accumulation of this compound at the hanging mercury-dropping electrode (HMDE) prior to square-wave voltammetric measurement, higher sensitivities can be readily
0026-265X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 1 . 0 0 1 5 3 - 9
A. Radi / Microchemical Journal 72 (2002) 35–41
36
metry, a frequency f , 100 Hz; scan increment dE, 10 mV and amplitude Esw, 25 mV. A Knick pH meter was used for pH measurements. 2.3. Procedure
2. Experimental
Ten milliliters of the supporting electrolyte solution were pipetted into the cell and deoxygenated with nitrogen for 8 min. The accumulation potential (usually –1.0 V) was applied to a fresh mercury drop while the solution was stirred. Following the accumulation period, the stirring was stopped and after 5 s the voltammogram was recorded by applying a negative-biased squarewave scan. After background stripping voltammograms were obtained, the entire cycle was repeated in the presence of the analyte, using a new mercury drop. All data were obtained at room temperature.
2.1. Materials
2.4. Pharmaceutical preparations
Rofecoxib and Vioxx tablets were obtained from Global Nabi Pharm. Egypt. Stock solutions (1.0=10y3 M) of rofecoxib were prepared in methanol each week and stored under refrigeration. Britton–Robinson buffers (0.04 M in each of acetic, o-phosphoric, and boric acids, adjusted to the required pH with 0.2 M sodium hydroxide) were used as supporting electrolytes. All other reagents were of analytical-reagent grade and purified water was obtained by passing distilled water through a Milli-Q water purification system.
Ten tablets of each pharmaceutical product (Vioxx 12.5 or 25 mg) were powdered and an amount corresponding to 15 mg of rofecoxib were weighed and dissolved in methanol by sonication for 5 min, transferred to a 100-ml volumetric flask and diluted to the mark with water. After the nondissolved excipients settled at the bottom of the flask, 1 ml of the clear supernatant was transferred to a 10-ml volumetric flask and diluted with water. A 1.0-ml portion added to 9.0 ml supporting electrolyte solution in the cell was used for the voltammetric analysis.
Scheme 1. Structure of rofecoxib.
achieved. The possibility of determining rofecoxib at ultratrace levels and the applicability of the method in determining rofecoxib in two different formulations of the same product Vioxx (25, 12.5 mg) has also been demonstrated.
2.2. Apparatus 3. Results and discussion Voltammograms were obtained with a 394 Electrochemical Trace Analyzer combined with a PAR Model 303A hanging mercury drop electrode (HMDE) incorporating an AgyAgCl reference electrode and a platinum wire auxiliary electrode. A magnetic stirrer (PAR 305) and stirring bar provided the convective transport during accumulation. The whole procedure was automated and controlled through the programming capacity of the apparatus. The following parameters were used throughout unless otherwise stated: cyclic voltammetry, scan rate 100 mV sy1; square-wave voltam-
3.1. Cyclic voltammetry The nature of the electrochemical process was studied by cyclic voltammetry (CV). Fig. 1 shows two different cyclic voltammograms for a 5.0=10y6 M solution of rofecoxib without accumulation and after a 30-s accumulation time step (scan rate 100 mV sy1). The reduction process was not accompanied by an anodic wave, which indicates that the redox reaction is totally irreversible. The dependence of the peak intensity of the
A. Radi / Microchemical Journal 72 (2002) 35–41
37
the value of the charge transfer coefficient is as 0.87. 3.2. Effect of pH
Fig. 1. Cyclic voltammograms for 5.0=10y6 M rofecoxib in Britton–Robinson buffer (pH 9.0) with scan rate ns100 mV sy1 following accumulation, tacc: (a) 0 and (b) 30 s at accumulation potential Eaccsy1.0 V.
reduction process (ip) at the HMDE on the scan rate (n) was examined. A linear plot of ip vs. n1y2 should be obtained when the electrode process is diffusion-controlled, whereas the adsorptioncontrolled process should result in linear plot of ip vs. n w7x. When the potential was scanned at increasing rates from 10 to 200 mV sy1, under the same experimental conditions, a linear relationship was observed between the peak intensity ip and the scan rate n (ip (mA)s0.046q5.108 n), suggesting the adsorption of rofecoxib on the electrode surface. For a totally irreversible interfacial reaction the relationship between the peak potential (Ep) and scan rate (n) can be expressed as w8x:
The influence of pH on the square-wave voltammetric response of 2.0=10y6 M rofecoxib was examined between pH 1.0 and 11.5 (Fig. 2.). From this study, the current value of ip without adsorption (Fig. 2a) is not practically sensitive to the pH of solutions. The peak potentials vary very little and change from –1.52 V at pH 2.0 to –1.58 V at pH 12.0. One hypothesis to explain this behavior could be that the protonation is probably preceded by the rate determining charge transfer step w9x. From Fig. 2b one can see that the response preceded by adsorption (taccs30 s and Eaccsy 1.0 V) increases dramatically in alkaline solutions. This behavior suggests a higher rate of adsorption when the uncharged neutral form of rofecoxib predominates in alkaline solution. A value of pH 9.0 was considered suitable and the methods were developed at this pH value. 3.3. Effect of accumulation potential and accumulation time The effect of the accumulation potential (Eacc) on peak intensity (ip) was evaluated for 5.0=10y8 M rofecoxib solution following tacc of
EpsŽ2.303RTyanaF.logŽRTkfoyanaF. yŽ2.303RTyanaF.logn The dependence of the peak potential on the decimal logarithm of the scan rate is a straight line following the equation: Ep (V)sy1.533y 0.034 log n. Using the value of the slope, a value of ans1.74 is obtained. Considering the molecular structure of rofecoxib the possibility of obtaining a reduction signal may be attributed to the hydrogenation of C_C double bond in the heterocyclic moiety. The number of electrons exchanged by the molecule was assumed to be ns2. Hence,
Fig. 2. Influence of pH on the accumulation of a c solution of rofecoxib using square-wave voltammetry (frequency f s100 Hz, scan increment dEs10 mV and pulse amplitude Esws25 mV) following accumulation, tacc: (a) 0 and (b) 30 s at Eaccs y1.0 V and equilibration time 5 s.
38
A. Radi / Microchemical Journal 72 (2002) 35–41
Fig. 3. Effect of accumulation potential (Eacc) on the squarewave stripping signal (ip) for 5.0=10y8 M rofecoxib for taccs 30 s. Other conditions as in Fig. 2.
30 s for accumulation potentials varying from y 0.2 to y1.4 V (Fig. 3). The ip reaches the maximum values over the Eacc range from –0.6 to –1.0 V. The observed decrease in ip value is probably a consequence of desorption of the substance at much more negative or positive potentials than the zero charge potential, where maximum adsorption of uncharged organic molecules can be expected. The application of an accumulation potential of y1.0 V was chosen as optimal accumulation potential. Different square-wave voltammograms with increasing accumulation times were recorded for a solution containing rofecoxib at two concentration levels (5.0=10y8 M and 1.0=10y7 M) using the selected conditions (Fig. 4). The resulting peaks showed a linear relationship between peak intensity and accumulation time up to 90 and 120 s for the studied solutions, respectively. Thus, when saturation of the electrode area is reached, interactions among the molecules in the adsorbed state become noticeable and the peak intensity started to decrease but with a smaller slope.
Fig. 4. Stripping peak current vs. accumulation time plots for (a) 5.0=10y8 M and (b) 1.0=10y7 M rofecoxib in Britton– Robinson buffer (pH 9.0) following accumulation at Eaccsy 1.0 V. Square-wave with f s100 Hz, dEs10 mV and Esws 25 mV.
to Ads-DPV and Ad-LSV, respectively. Although the peak corresponding to the DP waveform is more distant from the background discharge, the SW waveform is preferable because of its highest sensitivity and its high scan rate, which is time efficient. 3.5. Square-wave (SW) parameters With the study of the influence of SW parameters on the electrochemical response for
3.4. Choice of electroanalytical technique Fig. 5 shows a comparison of square-wave with differential-pulse and linear sweep adsorptive stripping voltammograms for 2.0=10y6 M (taccs30 s; Eaccsy1.0 V). The reduction peak of Ad-SWV is 265 and 25 times larger than that corresponding
Fig. 5. Square-wave, differential-pulse and linear sweep adsorptive stripping voltammograms for 2.0=10y6 M rofecoxib (taccs30 s; Eaccsy1.0 V). Square wave with f s100 Hz, dEs10 mV and Esws25 mV; differential-pulse with ns10 mV sy1 and Edps25 mV and linear sweep with ns100 mV sy1.
A. Radi / Microchemical Journal 72 (2002) 35–41
39
1.0=10y7 M rofecoxib solution in pH 9.0 following tacc of 60 s and Eacc of y1.0 V, the optimization of these parameters for analytical applications can be deduced. For a constant scan increment of 10 mV and pulse amplitude of 25 mV, the current intensity is a linear function of the logarithm of square-wave frequency for the whole range studied (10–120 Hz), which conformed to the following equation: ipwmAxs0.411q0.028fwHzx rs0.998 From the slope of this straight line a coating w10x index for the electrode of (7.05"0.45)=10y11 mol cmy1 was calculated. The area of the mercury drop electrode surface area covered by one molecule was calculated to be f0.43 nm2. Moreover, the peak potential (Ep) shifted to more negative values with increases in frequency according to the equation: EpŽV.sy1.498y0.036logf rs0.991 The slope of this straight was used to calculate the charge transfer coefficient for the electrochemical reaction since the variation of the peak potential with that of square-wave frequency conforms to the following equation: DEp yDlog f syRTy Fna w11x. Assuming ns2, a resulting charge transfer coefficient as0.82 can be evaluated, which is very close to the value obtained by cyclic voltammetry. Up to a square-wave amplitude of 50 mV, the peak current increases with the amplitude, and for higher values the peak current remains nearly constant. An amplitude of 25 mV was adopted as optimum. The influence of the scan increment on the analytical signal was studied varying Ds from 2 to 10 mV for a frequency of 120 Hz at amplitude 25 mV. A linear relationship between peak intensity, ip, and Ds was found. A scan increment at 10 mV was adopted for the remainder of the study. Once all the parameters that can affect the determination of rofecoxib were studied, the variation of the peak current with concentration of rofecoxib was performed. Fig. 6a shows stripping square-wave voltammograms obtained for solutions of increasing rofecoxib concentration (1.0=10y8 to 3.0=10y8 M) after a 30-s accu-
Fig. 6. (a) Stripping square-wave voltammograms obtained for solutions of increasing rofecoxib concentration over the range 1.0=10y8 to 3.0=10y8 M (a–e); taccs30 s; Eaccsy1.0 V. (b) Calibration plots for different accumulation times, tacc: (a) 0 (b) 30 and (c) 60 s.
mulation time. The very favorable signal-to-background characteristics permit convenient measurements of nanomolar concentrations. Calibration plots over a wide concentration range (5.0=10y9 to 5.0=10y8), obtained by using different accumulation times, are also shown in Fig. 6b. For 0- and 30-s accumulation, the response is linear over the entire range (slopes of 1.5=10y4 and 3.1=10y2 mA nMy1, respectively). With 60s accumulation, linearity prevails up to 3.5=10y8 M (slope of 0.055 mA nMy1). Such curvature is expected for a process that is limited by adsorption. The detection limit calculated from the calibration curves as 3Syx yb w12x; was estimated as 1.0=10y9 M following an accumulation of 60 s. The precision was estimated from 10 repetitive
A. Radi / Microchemical Journal 72 (2002) 35–41
40
Table 1 Recovery values and determination of rofecoxib in analyses of synthetic preparations and commercial tablets using SWV and AdSWV methods (ns5) SWV Synthetic preparations Nominal (mg) Found (mg) R.S.D.% Commercial tablets Nominal (mg) Found (mg) R.S.D.%
Ad-SWV (taccs30)
Ad-SWV (taccs60)
12.5 12.61 1.8
25.0 24.99 2.1
12.5 12.41 1.7
25.0 24.91 1.8
12.5 12.45 2.2
25.0 24.98 2.3
12.5 12.43 1.9
25.0 25.1 2.2
12.5 12.36 2.2
25.0 24.98 2.7
12.5 12.37 2.0
25.0 24.89 2.9
measurements of 1.0=10y8 M rofecoxib (30 s accumulation) using optimal conditions. This yielded a relative standard deviation of 2.8% (mean peak current of 290 nA, with a range of 281–300 nA). Hence, high precision is maintained despite the remarkably low concentration. 3.6. Analytical applications The determination of rofecoxib content in two different pharmaceutical formulations, Vioxx tablets (12.5 and 25 mg) was achieved by both SW and Ad-SWV. The composition of these pharmaceutical products are: rofecoxib, microcrystalline cellulose, lactose monohydrate, hydoxypropyl cellulose, croscarmellose sodium, yellow ferric oxide, red ferric oxide and magnesium stearate. The amounts of rofecoxib in two different pharmaceutical formulations of rofecoxib were calculated by reference to the regression equation of the peak current vs. the rofecoxib concentration. The accuracy of the method was carried out by spiking a placebo (mixture of the tablet excipients) with rofecoxib at various concentrations of the commercial tablets. From this preparation five aliquots were taken and analyzed by the two techniques. The results of these experiments, as well as the results of analyses of commercial tablets, are given in Table 1. As can be seen, the quality control assay of rofecoxib in Vioxx tablets, expressed as the percentage of the label claim, gave result which were near to 100% with a relative standard deviation (R.S.D.) under 3% for both formulations of rofecoxib, Vioxx (12.5 and 25 mg). A t-test was carried out on the data to statistically examine the
validity of the obtained results. At the 95% level the values of t (experimental) were less than that of t (theoretical) showing that the method has no systematic error. 4. Conclusions This work shows that rofecoxib can be determined using voltammetric techniques on the basis of its reduction process over the hanging mercury drop electrode. The method is sensitive, precise and fast enough to be used in routine control analysis. Moreover, application of the method to pharmaceutical preparations was possible after suitable dilution of the sample without interference from the excipients present in the tablets. Based on the Ad-SWV procedure, we plan to develop a very sensitive method for the determination of rofecoxib in biological samples. References w1x J. Scheen, Rev. Med. Liege 55 (2000) 751. w2x N. Futaki, K. Yoshikawa, Y. Hamasaka, et al., Gen. Pharmacol. 24 (1993) 105. w3x K. Seibert, Y. Zhang, K. Leahys, et al., Adv. Immunol. 62 (1994) 167. w4x C.M. Chavez-Eng, M.L. Constanzer, B.K. Matuszewski, J. Chromatogr. B 748 (2000) 31. w5x A.J. Woolf, I. Fu, B.K. Matuszewski, J. Chromatogr. B 730 (2000) 221. w6x J. Wang, in: A.J. Bard (Ed.), Electroanalytical Chemistry, 16, Marcell Dekker, New York, 1989, p. 39. w7x A.J. Bard, L.R. Faulkner, Electrochemical Methods Fundamentals and Applications, Wiley, New York, 1980, p. 522.
A. Radi / Microchemical Journal 72 (2002) 35–41 w8x E. Laviron, J. Electroanal. Chem. 52 (1974) 255. w9x M. Heyrovsky, S. Vavieka, J. Electroanal. Chem. 36 (1972) 203. w10x M. Lovric, S. Komorsky-Lovric, R.W. Murray, Electrochem. Acta 33 (1988) 739.
41
w11x J.G. Osteryoung, R.A. Osteryoung, Anal. Chem. 57 (1985) 101A. w12x J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry; Ellis Horwood Series, PTR Prentice Hall, NY, London, 1993, p. 119.
Adsorptive stripping square-wave voltammetric behavior of rofecoxib A. Radi* Department of Chemistry, Faculty of Science, Mansoura University, 34517 Dumyat, Egypt Received 26 March 2001; received in revised form 16 October 2001; accepted 21 October 2001
Abstract Adsorption and reduction of rofecoxib were investigated by cyclic and square-wave voltammetry on a hanging mercury drop electrode in electrolytes of various pH values. The reduction process on hanging mercury drop electrodes gave rise to a single peak within the entire pH range (2.0–11.5). In alkaline solutions, rofecoxib gave a sensitive adsorptive reductive peak; approximately 10 times larger than those obtained by applying a square-wave scan without prior accumulation. Application of the method to the determination of rofecoxib in two pharmaceutical products (Vioxx 12.5 and 25 mg), without sample pretreatment, resulted in acceptable deviation from the stated concentrations. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Stripping voltammetry; Square-wave; Rofecoxib; Vioxx
1. Introduction Rofecoxib (Vioxx) is a potent and selective inhibitor of the COX-2 isoform of cyclooxygenase, which is used as a non-steroidal anti-inflammatory drug (NSAID). It is indicated in the symptomatic relief of pain due to osteoarthritis. The initial oral dosage of rofecoxib is 12.5 mg once daily in adults, and this dose may be increased up to a maximal dosage of 25 mg once daily if necessary w1x. Its clinical efficacy seems similar to that of other NSAIDs at maximal recommended dosages, but its safety profile, especially gastrointestinal tolerance, is much better because of the COX-2 *Tel.: q20-5034-7054; fax: q20-57-403868. E-mail address: [email protected] (A. Radi).
selectivity w2,3x. Rofecoxib is designated chemically as 4-(4-methanesulfonylphenyl)-3-phenyl5H-furan-2-one (structural formula in Scheme 1). Analytical procedures for rofecoxib determination are few. Currently the most commonly employed techniques for the determination of the rofecoxib drug have been based on HPLC w4,5x. Adsorptive stripping voltammetry (AdSV) has been demonstrated to be a sensitive analytical method for a wide range of organic compounds that adsorb, in a reproducible way, on electrode surfaces w6x. This paper shows that rofecoxib is adsorbed onto a mercury electrode. By using this phenomenon and by accumulation of this compound at the hanging mercury-dropping electrode (HMDE) prior to square-wave voltammetric measurement, higher sensitivities can be readily
0026-265X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 1 . 0 0 1 5 3 - 9
A. Radi / Microchemical Journal 72 (2002) 35–41
36
metry, a frequency f , 100 Hz; scan increment dE, 10 mV and amplitude Esw, 25 mV. A Knick pH meter was used for pH measurements. 2.3. Procedure
2. Experimental
Ten milliliters of the supporting electrolyte solution were pipetted into the cell and deoxygenated with nitrogen for 8 min. The accumulation potential (usually –1.0 V) was applied to a fresh mercury drop while the solution was stirred. Following the accumulation period, the stirring was stopped and after 5 s the voltammogram was recorded by applying a negative-biased squarewave scan. After background stripping voltammograms were obtained, the entire cycle was repeated in the presence of the analyte, using a new mercury drop. All data were obtained at room temperature.
2.1. Materials
2.4. Pharmaceutical preparations
Rofecoxib and Vioxx tablets were obtained from Global Nabi Pharm. Egypt. Stock solutions (1.0=10y3 M) of rofecoxib were prepared in methanol each week and stored under refrigeration. Britton–Robinson buffers (0.04 M in each of acetic, o-phosphoric, and boric acids, adjusted to the required pH with 0.2 M sodium hydroxide) were used as supporting electrolytes. All other reagents were of analytical-reagent grade and purified water was obtained by passing distilled water through a Milli-Q water purification system.
Ten tablets of each pharmaceutical product (Vioxx 12.5 or 25 mg) were powdered and an amount corresponding to 15 mg of rofecoxib were weighed and dissolved in methanol by sonication for 5 min, transferred to a 100-ml volumetric flask and diluted to the mark with water. After the nondissolved excipients settled at the bottom of the flask, 1 ml of the clear supernatant was transferred to a 10-ml volumetric flask and diluted with water. A 1.0-ml portion added to 9.0 ml supporting electrolyte solution in the cell was used for the voltammetric analysis.
Scheme 1. Structure of rofecoxib.
achieved. The possibility of determining rofecoxib at ultratrace levels and the applicability of the method in determining rofecoxib in two different formulations of the same product Vioxx (25, 12.5 mg) has also been demonstrated.
2.2. Apparatus 3. Results and discussion Voltammograms were obtained with a 394 Electrochemical Trace Analyzer combined with a PAR Model 303A hanging mercury drop electrode (HMDE) incorporating an AgyAgCl reference electrode and a platinum wire auxiliary electrode. A magnetic stirrer (PAR 305) and stirring bar provided the convective transport during accumulation. The whole procedure was automated and controlled through the programming capacity of the apparatus. The following parameters were used throughout unless otherwise stated: cyclic voltammetry, scan rate 100 mV sy1; square-wave voltam-
3.1. Cyclic voltammetry The nature of the electrochemical process was studied by cyclic voltammetry (CV). Fig. 1 shows two different cyclic voltammograms for a 5.0=10y6 M solution of rofecoxib without accumulation and after a 30-s accumulation time step (scan rate 100 mV sy1). The reduction process was not accompanied by an anodic wave, which indicates that the redox reaction is totally irreversible. The dependence of the peak intensity of the
A. Radi / Microchemical Journal 72 (2002) 35–41
37
the value of the charge transfer coefficient is as 0.87. 3.2. Effect of pH
Fig. 1. Cyclic voltammograms for 5.0=10y6 M rofecoxib in Britton–Robinson buffer (pH 9.0) with scan rate ns100 mV sy1 following accumulation, tacc: (a) 0 and (b) 30 s at accumulation potential Eaccsy1.0 V.
reduction process (ip) at the HMDE on the scan rate (n) was examined. A linear plot of ip vs. n1y2 should be obtained when the electrode process is diffusion-controlled, whereas the adsorptioncontrolled process should result in linear plot of ip vs. n w7x. When the potential was scanned at increasing rates from 10 to 200 mV sy1, under the same experimental conditions, a linear relationship was observed between the peak intensity ip and the scan rate n (ip (mA)s0.046q5.108 n), suggesting the adsorption of rofecoxib on the electrode surface. For a totally irreversible interfacial reaction the relationship between the peak potential (Ep) and scan rate (n) can be expressed as w8x:
The influence of pH on the square-wave voltammetric response of 2.0=10y6 M rofecoxib was examined between pH 1.0 and 11.5 (Fig. 2.). From this study, the current value of ip without adsorption (Fig. 2a) is not practically sensitive to the pH of solutions. The peak potentials vary very little and change from –1.52 V at pH 2.0 to –1.58 V at pH 12.0. One hypothesis to explain this behavior could be that the protonation is probably preceded by the rate determining charge transfer step w9x. From Fig. 2b one can see that the response preceded by adsorption (taccs30 s and Eaccsy 1.0 V) increases dramatically in alkaline solutions. This behavior suggests a higher rate of adsorption when the uncharged neutral form of rofecoxib predominates in alkaline solution. A value of pH 9.0 was considered suitable and the methods were developed at this pH value. 3.3. Effect of accumulation potential and accumulation time The effect of the accumulation potential (Eacc) on peak intensity (ip) was evaluated for 5.0=10y8 M rofecoxib solution following tacc of
EpsŽ2.303RTyanaF.logŽRTkfoyanaF. yŽ2.303RTyanaF.logn The dependence of the peak potential on the decimal logarithm of the scan rate is a straight line following the equation: Ep (V)sy1.533y 0.034 log n. Using the value of the slope, a value of ans1.74 is obtained. Considering the molecular structure of rofecoxib the possibility of obtaining a reduction signal may be attributed to the hydrogenation of C_C double bond in the heterocyclic moiety. The number of electrons exchanged by the molecule was assumed to be ns2. Hence,
Fig. 2. Influence of pH on the accumulation of a c solution of rofecoxib using square-wave voltammetry (frequency f s100 Hz, scan increment dEs10 mV and pulse amplitude Esws25 mV) following accumulation, tacc: (a) 0 and (b) 30 s at Eaccs y1.0 V and equilibration time 5 s.
38
A. Radi / Microchemical Journal 72 (2002) 35–41
Fig. 3. Effect of accumulation potential (Eacc) on the squarewave stripping signal (ip) for 5.0=10y8 M rofecoxib for taccs 30 s. Other conditions as in Fig. 2.
30 s for accumulation potentials varying from y 0.2 to y1.4 V (Fig. 3). The ip reaches the maximum values over the Eacc range from –0.6 to –1.0 V. The observed decrease in ip value is probably a consequence of desorption of the substance at much more negative or positive potentials than the zero charge potential, where maximum adsorption of uncharged organic molecules can be expected. The application of an accumulation potential of y1.0 V was chosen as optimal accumulation potential. Different square-wave voltammograms with increasing accumulation times were recorded for a solution containing rofecoxib at two concentration levels (5.0=10y8 M and 1.0=10y7 M) using the selected conditions (Fig. 4). The resulting peaks showed a linear relationship between peak intensity and accumulation time up to 90 and 120 s for the studied solutions, respectively. Thus, when saturation of the electrode area is reached, interactions among the molecules in the adsorbed state become noticeable and the peak intensity started to decrease but with a smaller slope.
Fig. 4. Stripping peak current vs. accumulation time plots for (a) 5.0=10y8 M and (b) 1.0=10y7 M rofecoxib in Britton– Robinson buffer (pH 9.0) following accumulation at Eaccsy 1.0 V. Square-wave with f s100 Hz, dEs10 mV and Esws 25 mV.
to Ads-DPV and Ad-LSV, respectively. Although the peak corresponding to the DP waveform is more distant from the background discharge, the SW waveform is preferable because of its highest sensitivity and its high scan rate, which is time efficient. 3.5. Square-wave (SW) parameters With the study of the influence of SW parameters on the electrochemical response for
3.4. Choice of electroanalytical technique Fig. 5 shows a comparison of square-wave with differential-pulse and linear sweep adsorptive stripping voltammograms for 2.0=10y6 M (taccs30 s; Eaccsy1.0 V). The reduction peak of Ad-SWV is 265 and 25 times larger than that corresponding
Fig. 5. Square-wave, differential-pulse and linear sweep adsorptive stripping voltammograms for 2.0=10y6 M rofecoxib (taccs30 s; Eaccsy1.0 V). Square wave with f s100 Hz, dEs10 mV and Esws25 mV; differential-pulse with ns10 mV sy1 and Edps25 mV and linear sweep with ns100 mV sy1.
A. Radi / Microchemical Journal 72 (2002) 35–41
39
1.0=10y7 M rofecoxib solution in pH 9.0 following tacc of 60 s and Eacc of y1.0 V, the optimization of these parameters for analytical applications can be deduced. For a constant scan increment of 10 mV and pulse amplitude of 25 mV, the current intensity is a linear function of the logarithm of square-wave frequency for the whole range studied (10–120 Hz), which conformed to the following equation: ipwmAxs0.411q0.028fwHzx rs0.998 From the slope of this straight line a coating w10x index for the electrode of (7.05"0.45)=10y11 mol cmy1 was calculated. The area of the mercury drop electrode surface area covered by one molecule was calculated to be f0.43 nm2. Moreover, the peak potential (Ep) shifted to more negative values with increases in frequency according to the equation: EpŽV.sy1.498y0.036logf rs0.991 The slope of this straight was used to calculate the charge transfer coefficient for the electrochemical reaction since the variation of the peak potential with that of square-wave frequency conforms to the following equation: DEp yDlog f syRTy Fna w11x. Assuming ns2, a resulting charge transfer coefficient as0.82 can be evaluated, which is very close to the value obtained by cyclic voltammetry. Up to a square-wave amplitude of 50 mV, the peak current increases with the amplitude, and for higher values the peak current remains nearly constant. An amplitude of 25 mV was adopted as optimum. The influence of the scan increment on the analytical signal was studied varying Ds from 2 to 10 mV for a frequency of 120 Hz at amplitude 25 mV. A linear relationship between peak intensity, ip, and Ds was found. A scan increment at 10 mV was adopted for the remainder of the study. Once all the parameters that can affect the determination of rofecoxib were studied, the variation of the peak current with concentration of rofecoxib was performed. Fig. 6a shows stripping square-wave voltammograms obtained for solutions of increasing rofecoxib concentration (1.0=10y8 to 3.0=10y8 M) after a 30-s accu-
Fig. 6. (a) Stripping square-wave voltammograms obtained for solutions of increasing rofecoxib concentration over the range 1.0=10y8 to 3.0=10y8 M (a–e); taccs30 s; Eaccsy1.0 V. (b) Calibration plots for different accumulation times, tacc: (a) 0 (b) 30 and (c) 60 s.
mulation time. The very favorable signal-to-background characteristics permit convenient measurements of nanomolar concentrations. Calibration plots over a wide concentration range (5.0=10y9 to 5.0=10y8), obtained by using different accumulation times, are also shown in Fig. 6b. For 0- and 30-s accumulation, the response is linear over the entire range (slopes of 1.5=10y4 and 3.1=10y2 mA nMy1, respectively). With 60s accumulation, linearity prevails up to 3.5=10y8 M (slope of 0.055 mA nMy1). Such curvature is expected for a process that is limited by adsorption. The detection limit calculated from the calibration curves as 3Syx yb w12x; was estimated as 1.0=10y9 M following an accumulation of 60 s. The precision was estimated from 10 repetitive
A. Radi / Microchemical Journal 72 (2002) 35–41
40
Table 1 Recovery values and determination of rofecoxib in analyses of synthetic preparations and commercial tablets using SWV and AdSWV methods (ns5) SWV Synthetic preparations Nominal (mg) Found (mg) R.S.D.% Commercial tablets Nominal (mg) Found (mg) R.S.D.%
Ad-SWV (taccs30)
Ad-SWV (taccs60)
12.5 12.61 1.8
25.0 24.99 2.1
12.5 12.41 1.7
25.0 24.91 1.8
12.5 12.45 2.2
25.0 24.98 2.3
12.5 12.43 1.9
25.0 25.1 2.2
12.5 12.36 2.2
25.0 24.98 2.7
12.5 12.37 2.0
25.0 24.89 2.9
measurements of 1.0=10y8 M rofecoxib (30 s accumulation) using optimal conditions. This yielded a relative standard deviation of 2.8% (mean peak current of 290 nA, with a range of 281–300 nA). Hence, high precision is maintained despite the remarkably low concentration. 3.6. Analytical applications The determination of rofecoxib content in two different pharmaceutical formulations, Vioxx tablets (12.5 and 25 mg) was achieved by both SW and Ad-SWV. The composition of these pharmaceutical products are: rofecoxib, microcrystalline cellulose, lactose monohydrate, hydoxypropyl cellulose, croscarmellose sodium, yellow ferric oxide, red ferric oxide and magnesium stearate. The amounts of rofecoxib in two different pharmaceutical formulations of rofecoxib were calculated by reference to the regression equation of the peak current vs. the rofecoxib concentration. The accuracy of the method was carried out by spiking a placebo (mixture of the tablet excipients) with rofecoxib at various concentrations of the commercial tablets. From this preparation five aliquots were taken and analyzed by the two techniques. The results of these experiments, as well as the results of analyses of commercial tablets, are given in Table 1. As can be seen, the quality control assay of rofecoxib in Vioxx tablets, expressed as the percentage of the label claim, gave result which were near to 100% with a relative standard deviation (R.S.D.) under 3% for both formulations of rofecoxib, Vioxx (12.5 and 25 mg). A t-test was carried out on the data to statistically examine the
validity of the obtained results. At the 95% level the values of t (experimental) were less than that of t (theoretical) showing that the method has no systematic error. 4. Conclusions This work shows that rofecoxib can be determined using voltammetric techniques on the basis of its reduction process over the hanging mercury drop electrode. The method is sensitive, precise and fast enough to be used in routine control analysis. Moreover, application of the method to pharmaceutical preparations was possible after suitable dilution of the sample without interference from the excipients present in the tablets. Based on the Ad-SWV procedure, we plan to develop a very sensitive method for the determination of rofecoxib in biological samples. References w1x J. Scheen, Rev. Med. Liege 55 (2000) 751. w2x N. Futaki, K. Yoshikawa, Y. Hamasaka, et al., Gen. Pharmacol. 24 (1993) 105. w3x K. Seibert, Y. Zhang, K. Leahys, et al., Adv. Immunol. 62 (1994) 167. w4x C.M. Chavez-Eng, M.L. Constanzer, B.K. Matuszewski, J. Chromatogr. B 748 (2000) 31. w5x A.J. Woolf, I. Fu, B.K. Matuszewski, J. Chromatogr. B 730 (2000) 221. w6x J. Wang, in: A.J. Bard (Ed.), Electroanalytical Chemistry, 16, Marcell Dekker, New York, 1989, p. 39. w7x A.J. Bard, L.R. Faulkner, Electrochemical Methods Fundamentals and Applications, Wiley, New York, 1980, p. 522.
A. Radi / Microchemical Journal 72 (2002) 35–41 w8x E. Laviron, J. Electroanal. Chem. 52 (1974) 255. w9x M. Heyrovsky, S. Vavieka, J. Electroanal. Chem. 36 (1972) 203. w10x M. Lovric, S. Komorsky-Lovric, R.W. Murray, Electrochem. Acta 33 (1988) 739.
41
w11x J.G. Osteryoung, R.A. Osteryoung, Anal. Chem. 57 (1985) 101A. w12x J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry; Ellis Horwood Series, PTR Prentice Hall, NY, London, 1993, p. 119.