Talanta 46 (1998) 83 89

Differential pulse polarographic determination of ooxacin in pharmaceuticals and biological uids

M. Rizk, F. Belal *, F.A. Aly, N.M. El-Enany
Department of Analytical Chemistry, Faculty of Pharmacy, Uni6ersity of Mansoura, Mansoura 35516, Egypt Received 8 October 1996; received in revised form 23 July 1997; accepted 4 August 1997

Abstract A sensitive method is described for the determination of ooxacin in its pure form, dosage forms and biological uids. The proposed method depends upon the polarographic activity of ooxacin in Britton Robinson buffers, whereby a well-dened cathodic wave is produced over the pH range 4.1 10.3. The wave was characterized as being irreversible, diffusion-controlled with limited adsorption properties. The current-concentration relationship was found to be rectilinear over the range 5 10 5 5 10 4 M and 1 10 5 5 10 4 M using the DCt and DPP modes respectively, with a minimum detectability (S/N = 3) of 3 10 7 M. The proposed method was successfully applied to the determination of ooxacin in tablets and biological uids. The results obtained were found to be in agreement with those obtained by a reference method. 1998 Elsevier Science B.V. All rights reserved. Keywords: Ooxacin; Polarography; Pharmaceutical analysis; Biological uids

1. Introduction

Ooxacin is a synthetic uorinated quinolone

* Corresponding author.

derivative having activity against both gram negative and gram positive bacteria through inhibition of their DNA gyrase [1]. It is widely used in the treatment of respiratory tract and urinary tract infections [2]. Various techniques have been utilized for the determination of ooxacin either per SE or in dosage forms. These techniques include: potentiometric titration [3], spectrophotometry [47], spectrouorometry [5,6], HPLC [817], microbiology [18] and adsorptive stripping voltammetry [19]. During the preparation of this manuscript, a paper dealing with the polarographic determination of ooxacin appeared [20]. Astonishingly, the authors used BRb of pH 4 for their determination, at that medium, the wave was very poorly

0039-9140/98/$19.00 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 2 4 9 - X

84

M. Rizk et al. / Talanta 46 (1998) 8389

dened. The wave was found to be substantially improved as the pH value of the medium was increased up to pH 8.36 (the one we used) whereby well-dened wave was obtained. Meanwhile the authors [20] had limited their method to the use of DCt mode, therefore we attempted the Differential Pulse Polarography (DPP) mode which allowed better working range and lower detection limit. 2. Experimental

The effect of mercury height was studied using Sarget-Welch Polarograph Voltametric Analyzer equipped with Ag0/AgCl reference electrode and a platinum wire as the auxiliary electrode. The cyclic voltametry apparatus consisted of a Wenking LB 75 potentiostat, a Wenking VSG 72 function generator, a Goldstar DM 6135 digital multimeter and a Philips PM 8271 xyt recorder, a three-electrode system composed of a graphite working electrode, Ag0/AgCl reference electrode, and a platinum auxiliary electrode was used.

2.1. Materials
Ooxacin was kindly provided by Hoechst Oriented Pharmaceutical Company (Cairo, Egypt) and was used as received. Tablets (each containing 200 mg of ooxacin) were obtained from commercial sources. Reagents Britton Robinson buffers (0.08 M) [21], covering the pH range 2.7 10.3. Methanol: AR grade (Aldrich). A stock solution (2.5 10 3 M) of ooxacin was prepared in methanol, and was further diluted with the same solvent to give the appropriate concentrations. The methanol concentration in the polarographic cell was kept always at 20%. The solutions were purged with pure nitrogen for 5 min, then polarographed at ambient temperature.

2.2. Apparatus
The polarographic study and DPP measurements were carried out using the Polarecord E 506 Metrohm (Herisau, Switzerland). The drop time (1 s) was electronically controlled using a 663 VA Stand from the same company. The polarograms were recorded using a potential scan of 10 mV s 1. A three-electrode system composed of a Dropping Mercury Electrode (DME), Ag0/AgCl reference electrode, and a graphite rod as the auxiliary electrode, was used. Phase-selective ACt polarograms were recorded using the same instrument; the superimposed alternating voltage being 15 mV at a frequency of 75 Hz and a phase angle of 90.

Fig. 1. Typical polarogram of ooxacin (1 10 4 M) in BRb pH 8.36 containing 20% methanol.

M. Rizk et al. / Talanta 46 (1998) 8389

85

Fig. 2. Effect of pH on the development of the polarographic waves of ooxacin (5 10 4 M).

The voltammogram of 5 10 4 M solution were recorded at different scan rates of 20, 50, 100, 200 and 500 mV s 1.

2.3. Procedure for tablets

Weigh and pulverize 10 tablets. Transfer a weighed quantity of the powder equivalent to 20 mg of ooxacin. Dissolve in 100 ml of methanol then lter. Transfer 2 ml of the ltrate into 25 ml volumetric ask and complete to the mark with BRb of pH 8.36. Pour the contents of the ask into the polarographic cell. Record the DCt and DPP polarograms. Calculate the nominal content of the tablet using previously plotted calibration graph or the corresponding regression equation.

(90:10 V/V). Allow the extract to dry under nitrogen gas at room temperature. Dissolve the residue in methanol and lter into 50 ml volumetric ask. Complete to the mark with methanol, proceed as described above.

3. Results and discussion Fig. 1 shows a typical (DCt and DPP) polarogram of ooxacin in BRb of pH 8.36 containing 20% methanol. Methanol was added as a solubilizer for ooxacin, meanwhile it decreased adsorption interferences. Reduction of ooxacin at the DME was found to be pH dependent. The E1/2 was shifted to more negative values upon increasing the pH as shown in Fig. 2. A plot of E1/2 versus pH gave two straight lines with a break at pH 6.1 (Fig. 3) which corresponds to the second pka value of ooxacin (the rst one at 0.9 did not appear on the curve because the pH range over which reduction occurs began from 4.1) by analogy to the second pka value of nalidixic acid which has almost the same function group [26].

2.4. Procedure for spiked urine

Add a quantity of ooxacin to the urine to obtain a nal concentration of 0.5 mg ml 1 then add 2 ml of phosphate buffer (pH = 7.0). Stir the mixture, extract with 3 10 ml of dichloromethane isopropyl alcohol mixture

86

M. Rizk et al. / Talanta 46 (1998) 8389

E1/2 = 0.4994 0.1089 pH

(R = 0.99959) (2)

Fig. 3. Effect of pH on the E1/2 (mV) values of ooxacin (5 10 4 M) in BRb containing 20% methanol.

Over the pH range of 7.510.3. Logarithmic analysis of the reduction waves obtained in BRb of different pH values resulted in straight lines. Assuming that the rate determining step involves the transfer of two electrons (a free radical, one electron-transfer is not likely to occur), the values of the slopes suggest that the reduction process is irreversible in nature. The 8 na values were calculated using the treatment of Meites and Israel [22] and were listed in Table 1. It is noticed that, the degree of reversibility increased as the pH is raised up to pH 8.36 then it began to decrease. This character was further conrmed by CV measurements. The cyclic voltamograms of ooxacin (5 10 4 M solution) in BRb of pH values 4.0, 7.0 and 10.0 show one cathodic peak using different scan rates (10500 mV s 1). The peak potentials of the wave display a cathodic shift on increasing the scan rate, thus revealing the irreversible nature of the reduction process [23]. The number of protons, Z, consumed in the electrode reaction is given by the following equation [24]: E1/2/pH = 0.059Z 8 na

The relation between E1/2 and pH for the wave is expressed by the following equations: E1/2 = 0.9622 0.0577 pH (R = 0.99560) (1) Over the pH range of 2.6 7.0 and

where is the transfer coefcient. The value of 8 na was calculated from the following equation: E = E1/2 (0.0559/na)log[(i /id i )]

Table 1 Effect of pH on the development of the polarographic waves of ooxacin pH E1/2 (V) 4.10--1.250 5.00--1.270 6.10--1.310 7.00--1.360 7.50--1.390 8.36--1.450 9.10--1.490 9.90--1.575 10.30--1.620 E1/2/pH (mV) 28 36 51 60 68 57 106 112 Half-peak width (W1/2) (mV) 13 12 12 12 14 11 14 Number of protons (Z ) 0.34 0.63 1.01 1.12 1.27 1.13 1.94 1.99 8 na 0.9 1.03 1.08 1.10 1.10 1.17 1.08 1.05 0.93

M. Rizk et al. / Talanta 46 (1998) 8389

87

Fig. 4. Alternating current behavior of ooxacin (5 10 4 M) in BRb of different pH values. Superimposed alternating voltage: 15 mV; frequency: 75 Hz; phase angle: 90 (SE: supporting electrolyte).

Where id is the limiting current. At pH 8.36 Z was found to be 1.13, i.e. two protons are probably consumed in the electrode reaction.

3.1. Study of the wa6e characteristics

Increasing the mercury height (h ) resulted in a corresponding increase in the waveheight (w ); a plot of
h vs. the waveheight gave a straight line. A plot of log w gave a straight line, the slope of which was 0.49. Changing the buffer concentration over the range 0.006 to 0.06 M resulted in a negligible decrease in the waveheight. These two characters point out to the diffusion-controlled nature of the wave. The alternating current behavior (ACt) of ooxacin was studied using a phase-selective angle of 90. In BRb of pH 5.0, 7.0 and 8.36, the summit potentials (Es) were shifted to more negative values of 119, 99.27 and 90.625 mV respectively. Fig. 4 demonstrates that a pH values of 7.0 and 8.36, adsorption of the reduction product of the depolarizer may occur. The diffusion coefcient (D ) was calculated using the Ilkovic equation [25] and was found to be 2.72 10 7 cm2. S 1 in BRb of pH 8.36 (Table 2).

Solutions of ooxacin in BRb of pH 8.36 containing 20% methanol were found to be stable for about 2 h, after which the waveheight began to decrease slowly. The relation between the limiting current id (mA) and the concentration, C (mM), was found to be rectilinear over the concentration range 5 10 5 5 10 4 M and 1 10 5 5 10 4 M in the DCt and DPP modes, respectively. C = 0.0002 + 0.838id using DCt mode, and C = 0.0002 + 0.774id (r = 0.99999) (r = 0.99998)

using DPP mode respectively, with a minimum detectability of 3 10 7 M (S/N = 3). The diffusion current constant [Id = id/C m2/3 1/6 t ] was calculated at 25 and found to be 0.63 9 0.01.

3.2. Number of electrons in6ol6ed in the electrode reaction

The number of electrons consumed during the reduction was accomplished through comparison of the waveheight of ooxacin with that obtained from an equimolar solution of a closely related compound, i.e. nalidixic acid [26]. In BRb of pH

88

M. Rizk et al. / Talanta 46 (1998) 8389

Table 2 Correlation between concentration and limiting current in the DCt mode No. 1 2 3 4 5 6 x 9SD Concentration (mM) 5102 0.10 0.20 0.30 0.40 0.50 Current (mA) 0.060 0.119 0.237 0.359 0.475 0.597 id/C (mA mM1) 1.20 1.190 1.185 1.197 1.188 1.194 1.192 0.005 Id id/cm2/3 t1/6 0.65 0.856 0.853 0.861 0.855 0.859 0.858 0.004

NB: The results are the average of 18 separate determinations.

8.36 both compounds gave one peak of the same height pointing out to a two electron transfer process. Based on these facts, the following mechanism is proposed. O 3 R C + 2H + + 2e R CH OH

3.3. Analysical application

Polarogram of ooxacin in BRb of pH 8.36 exhibits cathodic wave. The current is diffusioncontrolled and proportional to the concentration over a convenient range of concentration. Both DCt and DPP modes were successfully applied to the assay of ooxacin in commercial tablets (200 mgtablet) and the results obtained are shown in Table 3. The percentage recoveries based on the average of nine separate determinations were 98.5 9 0.4 and 99.01 9 0.3, respectively. The results obtained were favourably compared with a

reference [5] was 98 9 0.5 based on measuring the native uorescence of ooxacin in 1 M sulphuric acid at 512 nm after excitation at 298 nm. Statistical analysis [27] of the results obtained by both methods using the Student t -test and Variance Ratio F -test, shows no signicant difference between the performance of the two methods regards the accuracy and precision, respectively (Table 3). Ooxacin is given by mouth in doses of 200 400 mg daily. It is metabolized to desmethyl and N -oxide metabolites. It is eliminated mainly by the kidneys. Excretion is affected by tubular secretion and glomerular ltration, and 7580% of the dose is excreted unchanged in the urine over 2448 h, resulting in high urinary concentration [28] (Table 4). The DPP mode could be successfully applied to the determination of ooxacin in urine over the concentration range of 4180 mg ml 1. Thus the proposed study could be applied to the determination of ooxacin in spiked urine. The results are

Table 3 Statistical analysis of the results obtained by the proposed and reference method for pure sample of ooxacin Method (1) (2) (3) (4) (5) Number of experiments Mean found (%) Variance Students t -value Variance ratio F -test DCt 6 100.09 0.32 Reference method [29] 3 100.03 0.08 0.17 (2.365) 4.0 (5.79) DPP mode 8 99.90 0.30 0.38 (2.262) 3.75 (4.74)

NB: Figures in parentheses are the tabulated t - and F -values, respectively, at P = 0.05 [27].

M. Rizk et al. / Talanta 46 (1998) 8389 Table 4 Assay of ooxacin in commercial tablets using the proposed and a reference method No. 1 2 3 x 9SD DCt 98.89 98.15 98.50 98.5 0.40 t = 0.28 F = 1.14 DPP 99.28 99.0 98.75 99.01 0.30 t = 1.48 F = 2.29 Reference method [5]

89

References
[1] K. Sato, U. Matsura, M. Inone, T. Ueno, Y. Osada, H. Ogawa, M. Mitshuhashi, Antimicrob. Agents Chemother. 22 (1982) 548. [2] N. Ichihara, H. Tachizawa, M. Tusumura, T. Une, K. Sato, Chemotherapy, Tokyo 32 (1984) 118. [3] D.S. Lee, H.J. Han, K. Kim, W.B. Park, J.K. Cho, J.H. Kim, J. Pharm. Biomed. Anal. 12 (1994) 157. [4] G. Garlucci, P. Mazzeo, T. Fantozzi, Anal. Lett. 26 (1993) 2193. [5] F.A. Elyazbi, Spectros. Lett. 25 (1992) 279. [6] Y. Fujitta, I. Mori, K. Fujitta, Y. Nakahashi, T. Tanaka, Chem. Pharm. Bull. 35 (1987) 5004. [7] S.C. Mathur, Y. Kumar, N. Murugesau, Y.K.S. Rathore, P.D. Sethi, Indian Drugs 29 (1992) 376. [8] A. Mizuno, T. Uematsu, M. Nakashima, J. Chromatogr. 653 (1994) 187. [9] R. Jain, C.L. Jain, Liq. Chromatogr. Gas Chromatogr. 10 (1992) 707. [10] D.K. Xu, A.Z. Ding, Y.S. Yuan, Y. Dioa, Yaoxue. Xuebao 27 (1992) 462; A.A. Thro 55 8G (1993) 112. [11] A. Le Cogenic, R. Bidualt, R. Farinotti, A. Dauphin, J. Chromatogr. 434 (1988) 320. [12] K. Matsu Bayashi, T. Une, Y. Osada, J. Chromatogr. 495 (1989) 354. [13] D.J. Griggs, R. Wise, J. Antimicrob. Chemother. 24 (1989) 437. [14] T. Ohkubo, T. Nambara, J. Chromatogr. 527 (1990) 441. [15] O. Oka Zaki, H. Aoki, H. Hakus, J. Chromatogr. 563 (1991) 31. [16] T. Ohkubo, M. Kudo, K. Sugawara, Anal. Sci. 7 (1991) 741. [17] T. Ohkubo, M. Kudo, K. Sugawara, J. Chromatogr. 573 (1992) 289. [18] M.R. Lockley, R. Wise, J. Dent, J. Antimicrob. Chemother. 14 (1984) 647. [19] A. Tamer, Anal. Chim. Acta. 231 (1990) 129. [20] G. Zhou, J. Pan, Anal. Chim. Acta. 307 (1995) 49. [21] J. Heyrovsky, P. Zuman, in: Practical Polarography, Academic Press, New York, 1968, pp. 163, 179. [22] L. Meites, Y. Israel, J. Am. Chem. Soc. 83 (1961) 4903. [23] P. Delahy, in: New Instrumental Methods in Electrochemistry, Interscience, New York, 1953. [24] J. Proszt, V. Cielezky, K. Gyorbiro, in: Polarographie Text Book, Akademia Kiado, Budapest, 1987, p. 385. [25] J. Heyrovsky, J. Kuta, in: Principles of Polarography, Czechoslovak Academy of Science, Prague, 1965, p. 82. [26] W.J. Vaoort, R.H.A. Sorel, D. Brusse, S.G. Schulman, P. Zuman, J. Denhartigh, Anal. Chem. Acta. 149 (1983) 175. [27] J.D. Hichen, Practical Statistics for Research, Methuen, London, 1969. [28] J.G.F. Reynolds (Ed), Martindale, The Extra-Pharmacopeia, 30th ed., The Pharmaceutical Press, London, 1993. [29] The United States Pharmacopeia XXII, National Formulary XVII, Rockville, USA Convention, 1990, pp. 912, 963.

98.60 0.40 t = (2.78) F = (19.00)

*Tarivid tablets (ooxacin, 200 mg/tablets), product of Hoechst Orient Pharm., Cairo, Egypt. NB: Figures in parentheses are the tabulated t and F values, respectively, at P = 0.05 [27].

abridged in Table 5. It can also be adopted for the pharmacokinetic studies of ooxacin. The within-day precision was evaluated through replicate analysis of urine samples spiked with ooxacin at certain concentration level. The percentage recoveries based on the average of four separate determinations were 97.36 9 0.73, the corresponding RSD% was 0.75 indicating high precision of the method. The between-day precision was also evaluated as proceeding above. The percentage recoveries based on the average of four separate determinations were 96.25 9 1.62, the corresponding RSD% was 1.68 indicating also the high precision of the method.

Table 5 Polarographic determination of ooxacin in spiked urine using DPP mode Concentration (mM)* mM (taken) mM (found) 0.05 0.08 0.10 0.20 x 9SD 0.0484 0.0769 0.0966 0.1943 % found Proposed method 96.80 96.13 96.60 97.15 96.67 90.43

*Average of at least three triplicate determinations.