Voltammetric quantitation at the mercury electrode of the anticholinergic drug flavoxate hydrochloride in bulk and in a pharmaceutical formulation

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1 DOI: /s Research article CEJC 5(2) Voltammetric quantitation at the mercury electrode of the anticholinergic drug flavoxate hydrochloride in bulk and in a pharmaceutical formulation M.M. Ghoneim 1, M.A. El-Attar 1, S.A. Razeq 2 1 Analytical Chemistry Research Unit, Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt 2 Department of Analytical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Nacer City - Cairo, Egypt Received 16 November 2006; accepted 2 January 2007 Abstract: Flavoxate hydrochloride, 2-piperidinoethyl 3-methyl-4-oxo-2-phenyl-4-H-chromene-8- carboxylate, is a smooth muscle antispasmodic. Its electrochemical behavior was studied at the mercury electrode in buffered solutions containing 30% (v/v) methanol using dc-polarography, differential-pulse polarography, cyclic voltammetry, and linear sweep- and square-wave adsorptive stripping voltammetry. Sensitive and precise procedures were developed for determination of bulk flavoxate hydrochloride and in the pharmaceutical formulation Genurin R S.F, without sample pretreatment or extraction. Limits of quantitation (LOQ) of ,5 10 6, and M flavoxate hydrochloride were achieved by dc-polarography, differential-pulse polarography, linear sweep and square-wave adsorptive stripping voltammetric, respectively. c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved. Keywords: Flavoxate hydrochloride, Genurin R stripping voltammetry, quantitation tablets, polarography, cyclic voltammetry, adsorptive 1 Introduction Flavoxate hydrochloride (Scheme 1), 2-piperidinoethyl 3-methyl-4-oxo-2-phenyl-4-H-chromene- 8-carboxylate), belongs to a series of flavone derivatives which exhibit strong smoothmuscle activity, especially on the urogenital tract [1, 2]. mmghoneim@usa.net

2 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) N 7 6 O 5 8 O 1 O O CH 3. HCl Scheme 1 Flavoxate hydrochloride. Few analytical methods have been reported for the determination of flavoxate in pharmaceutical formulations and human biological fluids. These include spectrophotometry [3], capillary electrophoresis [4] and high performance liquid chromatography [5]. No information is available concerning the electrochemical behavior or quantitation of flavoxate hydrochloride. In this work we throw light on the electrochemical reduction of flavoxate hydrochloride at the mercury electrode in buffered solutions and describe simple and precise electroanalytical procedures for its quantitation in bulk form and in a pharmaceutical formulation without sample pretreatment or extraction. 2 Experimental 2.1 Apparatus A Model 4001 (Sargent Welch, USA) pen recording Polarograph and the cell described by Mietes [6] were used for the polarographic measurements. The characteristics of the dropping mercury electrode (DME) were: m = 1.1 mg / s, t = 3.3 s (in 0.1 M KCl at open circuit) at a mercury height of 60 cm. A saturated calomel electrode (SCE) was the reference. PAR computer-controlled Electrochemical Analyzer Models 273A and 263A (Princeton Applied Research, Oak Ridge, TN, USA) were used for differential-pulse polarography as well as cyclic, linear-sweep, and square-wave voltammetry. The electrode assembly was a PAR Model 303A with a micro-electrolysis cell incorporating a three electrode system comprised of a hanging mercury drop working electrode (area of HMDE: cm 2 ), an Ag/AgCl/KCl s reference electrode and a platinum wire auxiliary electrode. A magnetic stirrer (PAR 305) was used to provide transport during the accumulation step of the stripping voltammetric measurements. All measurements were automated and controlled through programming.

3 498 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) Materials and solutions Solutions of bulk flavoxate hydrochloride A stock solution of M flavoxate hydrochloride was prepared by weight in methanol and stored in a dark glass bottle at 4 C. More dilute solutions ( M) were prepared by accurate dilution with methanol just before use. Flavoxate hydrochloride solutions are stable at room temperature and their concentrations did not change over five days. This was demonstrated by the reproducibility of the differential-pulse polarography peak current of dilutions of the stock solution of flavoxate hydrochloride measured at room temperature in the ph 4 acetate buffer Solutions of Genurin R S.F. tablets Genurin R S.F. tablets (Medical Union Pharmaceuticals Co. Abu- Sultan-Ismailia, Egypt, under licence from Recordati Milano, Italy) labeled as containing 200 mg flavoxate hydrochloride was used. Ten tablets were powdered and mixed. A quantity of the homogeneous powder equivalent to 400 mg of flavoxate hydrochloride was diluted to volume with methanol in a 100 ml volumetric flask, then sonicated for 15 min. The desired concentrations were obtained by accurate dilution with methanol. These solutions were then analyzed Supporting electrolyte Britton-Robinson universal buffers (ph 2-11) and acetate buffers (ph ) were prepared [7] from analytical-grade chemicals and used as supporting electrolytes. The ph-meter (Crison, Barcelona, Spain) and the glass electrode were calibrated in pure aqueous buffer. The ph values of the electrolysis solutions in 30% (v/v) methanol are apparent values. A Mettler balance (Toledo-AB104, Switzerland) was used for weighing the solid materials. Deionized water was supplied from a Purite Still Plus Deionizer connected to a Hamilton Aquamatic double distillation water system. 3 Results and discussion 3.1 DC polarography DC polarograms of M flavoxate hydrochloride in ph 2-11 B-R universal buffers containing 30% (v/v) methanol exhibited two irreversible cathodic waves (Fig. 1). The limiting current of the second wave was almost double that of the first at ph 9. The total limiting current was practically ph-independent up to ph 9. At higher ph values the limiting current of the second wave decreased and the shape of the wave distorted. The observed reduction of flavoxate hydrochloride at the mercury electrode may be attributed to the reduction of the 2-phenyl-4-H-chromene moiety (Scheme 1, positions 1 8). This suggestion was supported by molecular orbital energy calculations (Gaussion 98) for

4 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) a b c d e f g h 2.0 i j i ( A) E (V) vs. SCE Fig. 1 DC polarograms of 2.5x10 4 M flavoxate hydrochloride in Britton-Robinson buffers containing 30% (v/v) methanol: ph (a) 2, (b) 3, (c) 4, (d) 5, (e) 6, (f) 7, (g) 8, (h) 9, (i) 10 and (j) 11. flavoxate, since the 2-phenyl-4-H-chromene moiety was found to have an electron deficiency (i.e. a higher orbital energy than that of the other centers), which facilitates the consumption of electrons. These calculations also showed that the LUMO level is delocalized over the 2-phenyl-4-H-chromene moiety. Analysis of the polarographic waves of flavoxate hydrochloride using the fundamental equation for irreversible polarographic waves [6] exhibited linear E d.e. vs. log (i/i d i) plots over the ph range 2 9 with slope values S 1 (S 1 =59mv/αn a ) of mv, from which values of αn a ( ) and the symmetry coefficient α ( ) were estimated. These values confirmed the irreversible nature of the reduction of flavoxate hydrochloride at the mercury electrode. The half-wave potential (E 1/2 ) shifted to more negative values with increased ph, indicating the involvement of protons in the electrode reaction, and suggesting that proton transfer precedes electron transfer [8]. The E 1/2 -phplotsforthe1 st and 2 nd waves over the ph range 2 9 are straight lines with a slope S 2 (S 2 =P59mv/αn a )of40mv.thenumberofprotonsp participating in the rate-determining step of the reduction was calculated from the relation [6, 9]: P =(δe 1/2 /δph)/(59/αn a )=S 2 /S 1 =1 (1) In addition, the previously estimated symmetry coefficient α values ( ) were obtained when the number of electrons involved in the rate-determining step (n a )equals two (i.e. the ratio P/n a =0.5). DC polarograms of to M flavoxate hydrochloride were recorded in ph 4 B-R universal buffer and in ph 4 acetate buffer. The polarograms exhibited two waves; the total limiting current was better developed in the acetate buffer. In this buffer the total limiting current (i l ) vs. the flavoxate hydrochloride concentration (C) was linear: i l (μa) =0.006 C (μm)+0.1 (r =0.999; n =7) (2)

5 500 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) Three replicate calibration curves were obtained over the range M. Limits of detection (LOD) and quantification (LOQ) of and M, respectively, were estimated using the relation k SD / b [10], where k =3forLODand 10 for LOQ, SD is the standard deviation of the intercept (or the blank) and b is the slope of the calibration curve. For 5 5 M flavoxate hydrochloride the mean recovery was ± 0.52% (n = 4) by dc-polarography. 3.2 Differential-pulse polarography Differential-pulse polarograms (DPP) of to M flavoxate hydrochloride in ph 4 acetate buffer at a scan rate of 2 mv s 1 exhibited a main irreversible cathodic peak (E p = V). A linear variation of the peak current (i p ) with concentration (C) was obtained: i p (μa) =0.009C(μM)+0.07 (r =0.999 and n =7) (3) LOD and LOQ of and M, respectively, were achieved by DPP. For M flavoxate hydrochloride the mean recovery was ± 0.53% (n = 4). 3.3 Cyclic voltammetry Cyclic voltammograms of M flavoxate hydrochloride at the HMDE in ph 2-11 B-R buffers containing 30% (v/v) methanol exhibited a main irreversible cathodic peak. The peak potentials (E p ) shifted to more negative values with increased ph and scan rate v ( mv s 1 ). These shifts confirm the involvement of protons in the electrode reaction [8] and the irreversibility of the reduction [11]. The interfacial adsorption of flavoxate hydrochloride was studied in ph 4 acetate buffer by cyclic voltammetry of M flavoxate hydrochloride following accumulation onto the HMDE at open circuit (Fig. 2a) and at -0.9 V (2b) for 60 s. The behavior shown in Figure 2 indicates interfacial adsorption of flavoxate hydrochloride onto the mercury surface. A substantial decrease in cathodic peak current was observed in a subsequent scan (2c) at the same mercury drop without stirring, indicating rapid loss of flavoxate hydrochloride from the electrode surface. The adsorption of flavoxate hydrochloride was also identified by recording its cyclic voltammograms for M at different scan rates (v) mv s 1, following preconcentration onto the HMDE by adsorptive accumulation at 0.9 V for 30 s. A linear log (i p )versuslog(v) plot followed the relation: log(i p )=0.94 log(v)+0.44 (r =0.999 and n =7) (4) was obtained. The slope (0.94) is very close to the theoretical value (1.0) for the ideal reaction of a surface species [12], indicating interfacial adsorption of flavoxate hydrochloride onto the mercury surface.

6 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) b 0.4 i ( A) c a E (V) vs. Ag / AgCl / KCl s Fig. 2 Cyclic voltammograms of M flavoxate hydrochloride in ph 4 acetate buffer at v = 300 mv s 1 following its accumulation onto the HMDE: (a) at open circuit, (b) at E acc = 0.9 V for 60 s, and (c) a replicate scan at the same mercury drop. The electrode surface coverage (Γ o mol cm 2 ) was evaluated using the relation Γ = Q/nFA,where Q is the amount of charge consumed by the surface process (calculated by integration of the area under the cyclic voltammogram peak corrected for residual current [13]), n is the number of electrons transferred per reactant molecule (n =4),F is Faraday s constant ( C) and A is the mercury electrode surface area (0.026 cm 2 ). On dividing the number of coulombs transferred (0.362 μc) at ph 4 by (nfa) a monolayer surface coverage of mol cm 2 was obtained. Each adsorbed molecule therefore occupied an area of 4.6 nm Stripping voltammetry A single well-defined irreversible cathodic peak was exhibited by both linear sweep and square-wave voltammograms of M flavoxate hydrochloride in ph 2 11 B-R universal buffers and ph acetate buffers, following accumulation onto the HMDE for 30 s. The peak was well developed and sharper in the ph 4 acetate buffer; therefore it was the supporting electrolyte in the rest of the study Linear-sweep (LS) voltammetry The linear-sweep adsorptive cathodic stripping (LSAdCS) voltammograms of M flavoxate hydrochloride at the HMDE in ph 4 acetate buffer at various scan rates ( mv s 1 ) exhibited a single well developed irreversible cathodic peak, which was

7 502 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) sharpest at 100 mvs 1. The LSAdCS peak current ( M flavoxate hydrochloride over the potential range 0.0 to 1.1 V following accumulation onto the HMDE for 30 s) was practically independent of the accumulation potential (E acc ) within the range -0.2 V to 1.1 V. An accumulation potential of 0.4 V was chosen for the rest of the study. A linear relationship between peak current (i p ) and accumulation time (t acc )upto60swas obtained for M flavoxate hydrochloride Square-wave (SW) voltammetry The square-wave adsorptive cathodic stripping (SWAdCS) voltammetry peak current ( M flavoxate hydrochloride in ph 4 acetate buffer following accumulation onto the HMDE at 0.4 V for 30 s) was optimized by changing the pulse-amplitude (a), frequency (f) and scan increment (Δs) within the ranges mv, Hz and 2-10 mv, respectively. Although the peak current was almost directly proportional to each of a, f and Δs, the best peak current and shape were obtained at a =20mV,f = 140 Hz and Δs =10 mv. The peak current intensity was practically independent of the accumulation potential (E acc. ) from 0.0 to 1.0 V ( M flavoxate hydrochloride in ph 4 acetate buffer following accumulation onto the HMDE for 30 s (Fig. 3)). Therefore, an accumulation potential of -0.3 V was chosen for the rest of the measurements. Under these optimized conditions the peak current gave a linear relationship with the accumulation time up to 150 s and 60 s for M(Figure4a) and M (4b), respectively. Each i p t acc curve showed a plateau which may indicate saturation of the HMDE with flavoxate. This means that the accumulation time should be chosen according to the concentration of flavoxate i p ( A) E acc. (V) vs. Ag / AgCl / KCl s Fig. 3 Effect of accumulation potential (E acc ) on the SWAdCS voltammetric peak current (i p )of M flavoxate hydrochloride in ph 4 acetate buffer following accumulation onto the HMDE for 30 s; f= 140 Hz, Δs =10mVanda =20mV.

8 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) b i p ( A) a t acc. (s -1 ) Fig. 4 Effect of accumulation time (t acc ) on the SWAdCS voltammetric peak current (i p ) of flavoxate hydrochloride in ph 4 acetate buffer: (a) Mand(b) M; f = 140 Hz, Δs = 10 mv, a =20mVandE acc =-0.3V. SWAdCS voltammograms for various concentrations of flavoxote hydrochloride are shown in Figure f e d i p ( A) a c b E (V) vs. Ag / AgCl / KCl s Fig. 5 SWAdCS voltammograms for flavoxate hydrochloride: (a) Background, (b) M, (c) M, (d) M, (e) M, (f) M; E acc = 0.3 V and t acc = 150 s. Other operational parameters were as in Figure 4.

9 504 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) Method validation Voltammograms of various concentrations of flavoxate hydrochloride were recorded under the optimized conditions for each procedure. The linear ranges, regression equations, limits of detection (LOD) and quantitation (LOQ) reported in Table 1 indicate their reliability for the assay of bulk flavoxate hydrochloride within the sensitivity range. Table 1 Calibration curve characteristics for determination of flavoxate hydrochloride in ph 4 acetate buffer. Procedure/ Linearity Least square equation (r) LOD LOQ t acc. range Intercept Slope (M) (M) (M) (µa) (µa /µm) DCP DPP LSV 60 s SWV 60 s s Reproducibility [14] was examined by performingfive replicateswadcs voltammetric measurements ( M flavoxate hydrochloride accumulated for 60 s under the optimal conditions) on the same day using the same standard solution, and on three successive days using different standard solutions. The results (Table 2) confirm both the good precision of the proposed procedure and the stability of the drug solutions. The robustness [14] of the SWAdCS procedure is demonstrated by noting that small variations in ph (4 ± 0.5), accumulation potential E acc. (-0.3 V ± 0.05) and accumulation time t acc (60 ± 5s) have no significant effect on recovery for M flavoxate hydrochloride (Table 2). The results from the LS, DPP and DCP procedures are similar. The interlaboratory reproducibility of the LSAdCS and SWAdCS procedures was examined by assay of flavoxate hydrochloride using two potentiostats in separate laboratories, PAR 273A (lab. 1) and PAR 263A (lab. 2). The achieved recoveries and standard deviations from laboratories 1 and 2 show no significant difference (Table 2). 3.6 Analysis of Genurin R SF tablets The dc-polarography (DCP), differential-pulse polarography (DPP), linear-sweep adsorptive cathodic stripping voltammetry (LS-AdCSV) and square-wave adsorptive cathodic stripping voltammetry (SWAdCSV) procedures described were successfully applied to the determination of flavoxate hydrochloride in Genurin R SF tablets (200 mg / tablet). Re-

10 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) Table 2 Effects of small variations in the SWAdCS procedure on the recovery and standard deviation: M flavoxate hydrochloride; f = 140 Hz, Δs = 10 mv and a =20 mv. Variable Conditions %R±SD (n=5) ph of the medium 3.5 E acc = -0.3 V ± t acc = 60 s ± ± 0.80 Accumulation potential (E acc ) ph = ± t acc = 60 s ± ± 0.20 Accumulation time (t acc ) 55 ph = ± E acc = -0.3 V ± ± 0.56 Elapsed time (Days) 1 ph = ± E acc = -0.3 V ± t acc = 60 s ± 0.33 Potentiostat Lab (1): PAR Model 273 A ph = ± 0.26 Lab (2): PAR Model 263 A E acc = -0.3 V ± 0.52 t acc =60s coveries of flavoxate hydrochloride based on the average of four replicate measurements, using both the calibration curve and standard addition methods [15] are reported in Table 3. The results were compared with those obtained spectrophotometrically [3]. Values of F -calculated, F-theoretical, t-calculated and t-theoretical are also included in Table 3. Since the calculated value of F did not exceed the theoretical value there was no significant difference in reproducibility between the electrochemical and spectrophotometric methods [16]. Also, the t-value shows no significant difference in accuracy and precision between the methods [16]. 4 Conclusion The electrochemical behavior of flavoxate hydrochloride at the mercury electrode was studied and discussed. Four electroanalytical procedures (DCP, DPP, LSAdCSV and SWAdCSV) for quantitation of flavoxate hydrochloride in bulk and in its pharmaceutical formulation (Genurin R SF tablets) were described. The sensitivity increases in the order:

11 506 M.M. Ghoneim et al. / Central European Journal of Chemistry 5(2) Table 3 Assay of flavoxate hydrochloride in Genurin R SF tablets (200 mg / tablet) by the electroanalytical procedures and spectrophotometry [3]. Procedure (% R ± S.D*) (Calculated**) Calibration curve Standard addition F-value t-test method method DCP ± ± DPP ± ± LS-AdCSV ± ± SW-AdCSV ± ± Reported [3] ± * Average of four determinations. ** Calculated from data of the calibration curve method Theoretical F-value =6.6andt-test = 2.45 at 95% confidence limit for n 1 =4andn 2 =4. DCP < DPP < LS-AdCSV < SW-AdCSV. The proposed electroanalytical procedures could be applied to the analysis of this and other similar formulation products containing flavoxate hydrochloride without sample pretreatment or extraction. Acknowledgment The authors express their deep gratitude to the Alexander von Humboldt Foundation (Bonn, Germany) for donating the Electrochemical Analyzer (PAR 263A) to the first author. We are also grateful to Prof. Dr. M.K. Awad at our Institute for carrying out the molecular orbital energy calculations. References [1] E. Pedersen: Studies on effect and mode of action of flavoxate in human urinary bladder and sphincter, Urol. Int., Vol. 32, (1977), pp [2] D.V Bradley and R.J. Cazort: Relief of bladder spasm by flavoxate: A comparative study, J. Clin. Pharm. & New Drugs, Vol. 10, (1970), pp [3] A. Gova and I. Setnikar: Flavoxate and 3-methylflavone-8-carboxylic acid. Assay methods in blood plasma red cells repartition and stability, Drug Research, Vol. 25, (1975), pp [4] C.X. Zhang, Z.P. Sun and D.K. Ling: Determination of 3- methylflavoxate in human urine by capillary electrophoresis with direct injection, J. Chromatogr., Vol. 612 (1993), pp [5] M.T. Sheu, G.C. Yeh, W.T. Ke and H.O. Ho: Development of a high-performance liquid chromatographic method for bioequivalence study of flavonate tablets, Chromatogr. B, Vol. 751, (2001), pp

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