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Cathodic Stripping Voltammetric Determination of Ceftazidime in Urine at a Hanging Mercury Drop Electrode
MICROCHEMICAL JOURNAL ARTICLE NO.
57, 115–122 (1997)
MJ971516
Cathodic Stripping Voltammetric Determination of Ceftazidime in Urine at a Hanging Mercury Drop Electrode Valdir S. Ferreira,* M. Valnice B. Zanoni,* and Arnold G. Fogg† *Departamento de Quimica Analitica, Instituto de Quimica, Universidade Estadual Paulista, Caixa Postal 355, 14800-900, Araraquara, Sa˜o Paulo, Brazil; and †Chemistry Department, Loughborough University, Loughborough, Leicestershire LE11 3TU, United Kingdom Received May 2, 1997; accepted May 12, 1997 A method was developed for the differential-pulse cathodic stripping voltammetric determination of ceftazidime with a hanging mercury drop electrode using its reduction peak at 00.43 V in Britton–Robinson buffer pH 4.0. The optimum accumulation potential and time were 00.15 V and up to 60 s, respectively. Linear calibration graphs were obtained from 1 1 1008 M and 1.5 1 1007 M. The limit of determination was calculated to be 5 1 1009 M. The coefficient of variation was 4% (n Å 7) at 1 1 1007 M ceftazidime. The effect of various components of urine on the voltammetric response was studied, and creatinine, uric acid, urea, and glucose were shown to interfere in the method. Ceftazidime bound to human albumin gives a unique stripping peak at 00.48 V. Recoveries of 87% { 2% of the ceftazidime (n Å 5) were obtained from urine spiked with 1.27 mg ml01 using C-18 solid phase extraction cartridges. q 1997 Academic Press
INTRODUCTION
Ceftazidime [7-[2-(2-aminothiazol-4-yl)-2-(1-carboxy-1-methylethoxyimino) acetamido]-3(1-pyridiniomethyl)-3-cephem-4-carboxylate pentahydrate] is a b-lactam antibiotic of the third-generation cephalosporin family, active against a wide variety of bacterial infections (1). Although ceftazidime has shown an excellent record of clinical success, rigorous control of dosage is required since high dosage can cause renal tubular necrosis in humans (2). Its therapeutic and nephrotoxic effects need rapid and sensitive methods for determination of trace levels. Various analytical methods have been employed for this purpose. Fluorescence ELISA assay and liquid chromatographic methods with spectrophotometric and electrochemical detection for the determination of ceftazidime in human serum, urine, and bile are reported (3–8). Stripping voltammetry has been used successfully for the determination of other cephalosporin drugs (10 – 12). A differential pulse polarographic method of determining ceftazidime was reported previously by the present authors (9). Indications of the adsorption of ceftazidime on the mercury electrode, particularly in acidic conditions, were observed. The aim of the present study was to establish suitable experimental conditions for the determination of ceftazidime by adsorptive stripping voltammetry at the hanging mercury drop electrode, and, particularly, its determination in human urine. EXPERIMENTAL
Adsorptive stripping voltammetry was carried out using a Metrohm (Herisau, Switzerland) E-506 Polarecord and Voltammetric Scanner E612 and a Houston Instruments 115 0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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2000 X–Y recorder. A Metrohm 663 VA Stand was used in the hanging mercury drop electrode (HMDE) mode. The three electrode system was completed by means of a glassy carbon auxiliary electrode and an Ag/AgCl (3 M KCl) reference electrode: all potentials are quoted relative to this electrode. Supporting electrolytes and buffers were prepared using Suprapur grade reagents supplied by Merck and demineralized water from a Milli-Q system (Millipore, Milford, MA). Ceftazidime stock standard solutions (1 1 1004 –1 1 1005 M) were prepared from the pure compound (after drying) supplied by Glaxo Pharmaceuticals in demineralized water. Britton–Robinson (B–R) buffer (pH 2.0–8.0) was prepared by mixing 0.004 M orthophosphoric acid, 0.004 M acetic acid, and 0.004 M boric acid with the appropriate amount of 0.02 M sodium hydroxide solution. Hydrochloric acid solution (0.01– 2.5 M) was used for voltammetric measurements at pH õ 2.0. Solutions were deoxygenated for 12 min initially. Adsorptive accumulation was carried out while the solution was stirred, and cathodic scans were carried out after 15 s to allow the solution to become quiescent. Scan rates of 50 and 5 mV s01 were used with linear sweep stripping voltammetry and differential pulse stripping voltammetry, respectively. A pulse amplitude of 50 mV s01 and a pulse interval of 1 s were used with the differential pulse mode. Extraction Procedure for the Determination of Ceftazidime in Human Urine A Waters Associates Sep-Pak C18 extraction cartridge was prewashed once with 10 ml of methanol and then with 10 ml of water. For the extraction, 50 ml of human urine containing 63.7 ng of ceftazidime diluted in 5 ml of water was passed through the Sep-Pak cartridge, the ceftazidime being adsorbed on the Sep-Pak column. The column was then rinsed with 4 ml of water. For elution of the drug, 4-ml portions of water/ethanol 50% (v/v) were passed through the matrix. The total eluent was dried under a stream of nitrogen. The residue was reconstituted with 10 ml of B – R buffer at pH 4.0. The voltammograms were recorded under the optimum instrumental conditions. RESULTS AND DISCUSSION
Ceftazidime in acidic solution is polarographically reduced in two reduction processes (9). The first cathodic step is attributed to the reduction of the methylethoxyimino group and the second cathodic step to the reductive elimination of the pyridine group. At slightly less acidic pH values both reduction peaks show the existence of adsorption processes at the mercury electrode (9), which indicates that adsorption could be used as an effective preconcentration step before voltammetric measurements. Typical cyclic voltammograms of a 5 1 1007 M solution of ceftazidime in 0.004 M Britton – Robinson (B – R) buffer at pH 4.0 after 30 s accumulation at 00.15 V are shown in Fig. 1. A single well-defined peak at 00.43 V, corresponding to an irreversible process, can be seen. The peak obtained in the second scan, without further accumulation, is much smaller, indicating the adsorptive nature of the process. The second reduction peak previously observed polarographically is masked by the electrolyte discharge. Ceftazidime was shown to be stable in these solutions for at least 6 h. Comparison of the voltammetric responses obtained with
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FIG. 1. Cyclic voltammograms for ceftazidime (5 1 1007 M) in Britton–Robinson buffer at pH 4.0. Accumulation potential Å 00.15 V. (I) First scan after accumulation time 30 s. (II) Second scan without further accumulation.
FIG. 2. Effect of pH on differential-pulse adsorptive stripping peak current for 5 1 1007 M ceftazidime in Britton–Robinson buffer with 30 s accumulation at 00.15 V.
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FIG. 3. Effect of accumulation time on peak current for ceftazidime. (A) 5 1 1008 M, (B) 1 1 1007 M at 00.15 V.
linear scan and differential-pulse waveforms showed that the use of the pulse technique improves the sensitivity. The effect of pH on the differential pulse adsorptive stripping voltammograms of ceftazidime (5 1 1007 M ) is shown in Fig. 2. Maximum signal is observed between pH 3.0 and 5.5. Outside this pH range the signal is very small. This is in broad agreement with the polarographic results, as the polarographic wave for the first process becomes increasingly small at pH ú 4. For analytical purposes pH 4.0 was therefore chosen for the adsorptive stripping voltammetric determination of ceftazidime. The effect of accumulation potential on the stripping current at potentials from 0 to 00.30 V was minimal and an accumulation potential of 00.15 V was chosen as satisfactory. The peak current increased with the drop size on the voltammetric stand and the larger drop size (nominally 0.4 mm2) was chosen for use. Increased stirring rate (positions 0 to 6 on the voltammetric stand) produced a linear increase of the signal up to stirring speed 4; stirring speed 3 (approximately 1500 rev min01) was chosen as giving the best results. Variations of pulse amplitude (5–100 mV) and scan rate (5–20 mV s01) established that for 1 1 1007 M ceftazidime solutions with an accumulation time of 30 s the peak current increased with an increase of either parameter. A pulse amplitude of 50 mV/s and a scan rate of 5 mV/s produced the best signal in intensity and resolution. A rest time between 0 and 30 s after the stirring ended and the potential scan began had no effect on the size of the peak current. A rest time of 15 s was chosen as the measurements provided good reproducibility. The influence of accumulation time on the peak heights observed with 5 1 1008 M and 1 1 1007 M solutions of ceftazidime at pH 4.0 is shown in Fig. 3. A rectilinear
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FIG. 4. Effect of various urine components on the stripping peak current of 1 1 1007 M ceftazidime in Britton–Robinson buffer pH 4.0, with an accumulation time of 60 s. (a) creatinine; (b) glucose; (c) urea; (d) uric acid.
relationship is observed up to 200 and 90 s, respectively. Above these times marked decreases in the peak current are seen: these may be caused by multilayer adsorption or some reorientation of the molecules on the surface (12). Hence, care has to be taken in choosing accumulation times and shorter accumulation times are recommended for determining ceftazidime. Linear calibration graphs were obtained from voltammograms recorded at accumulation times of 60 s from 1.0 1 1008 to 1.5 1 1007 M ceftazidime in B – R buffer at pH 4.0 with an accumulation potential of 00.15 V. With an accumulation time of 60 s, limits of determination around 5 1 1009 M could be obtained. Mean standard deviations of 4% were obtained by measuring seven ceftazidime solutions (1 1 1007 M ). In order to investigate the possibility of applying cathodic stripping voltammetry to the determination of ceftazidime in urine samples, a study was carried out to determine the influence of various urine constituents on the peak current. Certain urine
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FIG. 5. Differential pulse adsorptive stripping voltammograms of 1 1 1007 M ceftazidime in Britton– Robinson buffer at pH 4.0 for 30 s at 00.15 V (Voltammogram I) and after addition of albumin 2.4 mg ml01 (Voltammogram III); 3.2 mg ml01 (Voltammogram IV) and 10 mg ml01 (Voltammogram V). Albumin (4.0 mg ml01) in Britton–Robinson buffer pH 4.0 (Voltammogram II).
components, viz., uric acid, urea, glucose, and creatinine, were found to lower the voltammetric response, as shown in Fig. 4. The presence of human albumin has a very interesting effect on the stripping voltammograms of ceftazidime in Britton–Robinson buffer at pH 4.0 after 30 s accumulation at 0 V. Stripping voltammograms for human albumin and ceftazidime separately are shown in Fig. 5, voltammograms I and II, respectively: those for mixtures of the two are shown in voltammograms III–V. The stripping voltammetric behavior of human albumin is well known (13). Human albumin gives a stripping peak at 00.34 V (marked in Fig. 5 as peak C), compared with the stripping peak of ceftazidime at 00.40 V (marked in Fig. 5 as peak A). The addition of 2.4 mg ml01 of albumin to the ceftazidime solution (voltammogram III) causes a slight decrease in the ceftazidime peak and the appearance of a new peak at 00.48 V (marked in Fig. 5 as peak B). The addition of 3.2 mg ml01 of albumin again decreases peak A and enhances peak B, and a shoulder corresponding to peak C becomes apparent. With 10 mg ml01 of albumin peak C becomes predominant with only a slight sign of peak B, and none of peak A, remaining. The heights of peaks A, B, and C are plotted in Fig. 6 against albumin concentration. Consideration of the results suggests that the appearance of peak B probably can be attributed to the interaction of ceftazidime with albumin, i.e. the protein binding of ceftazidime. The addition of excess albumin swamps the signals of the bound ceftazidime. These results with albumin are in agreement with information in the literature (8, 14): the extent of protein binding of ceftazidime in serum was previously determined
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FIG. 6. Effect of albumin concentration on the height of the stripping peaks of ceftazidime (1 1 1007 M) (peak A), free albumin (peak C), and ceftazidime bound with albumin (peak B).
by HPLC, and associated with the pharmacological efficacy of ceftazidime (3). In human plasma, at least 21 { 6% of ceftazidime has been shown to be bound to proteins. From the results given above it is apparent that the direct determination of the drug in urine by cathodic stripping voltammetry is not possible. For that reason a prior extraction of ceftazidime using the solid-phase extraction procedure described in the experimental section was applied. Sep-Pak C18 cartridges were used successfully for the extraction of the drug. Urine samples with differing ceftazidime contents were analysed. The stripping peaks obtained after extraction were of the same form as those obtained for aqueous solutions prepared directly from the pure drug. A standard addition method was used for the determination in urine. At 1.27 mg ml01 of ceftazidime in urine, the recovery was 87% { 2% (5 determinations) using a more rapid accumulation time of 15 s and an accumulation potential of 00.25 V. In conclusion, ceftazidime is adsorbed spontaneously onto a hanging mercury drop
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electrode, and the method of cathodic stripping voltammetry can be applied to the determination of ceftazidime in urine samples in conjunction with a Sep-Pak C18 cartridge. ACKNOWLEDGMENTS The authors thank CAPES/The British Council and CNPq for financial support for this collaboration.
REFERENCES 1. Gilman, A. G.; Rall, T.; Nies, A.; Taylor, P. Goodman and Gilman’s The Pharmacological Basis of Therapeuticals, 8th ed., p - 1065. Pergamon, Elmsford, NY, 1990. 2. Barza, M. J. Infect. Dis., 1978, 137, 560. 3. Farrel, C. D.; Rowell, F. J.; Cumming, R. H. Anal. Proc. Anal. Commun., 1995, 32, 205. 4. Airton, J. J. Antimicrob. Chemother. B, 1981, 8, 227. 5. Myers, T. S.; Blumer, T. L. Antimicrob. Agents Chemother., 1983, 24, 343. 6. Ogtrop, M. L.; Mattie, H.; Guiot, H. F. L.; Van Striten, E.; Dokkuman, A. M. H.; Furth, R. Antimicrob. Agents Chemother., 1990, 34, 1932. 7. Jenl, F.; Brickel, P.; Monteil, H. J. Chromatogr., 1987, 413, 109. 8. Lam, Y. W. F.; Duroux, M. H.; Gambertoglio, T. G.; Barriere, S. L.; Gugliemo, B. T. Antimicrob. Agents Chemother., 1988, 32, 298. 9. Ferreira, V. S.; Zanoni, M. V. B.; Furlan, M.; Fogg, A. G. Anal. Chim. Acta, in press. 10. El-Maali, N. A.; Ali, M. M.; Maala, M. K.; Ghandour, M. A. Bioelectrochem. Bioenerg., 1991, 26, 485. 11. Ali, A. M. M.; El-Maali, N. A.; Ghandour, M. A. Electroanalysis, 1991, 6, 92. 12. Ogorevc, B.; Krasna, A.; Hudnik, V.; Gomiscek, S. Mikrochim. Acta, 1991, 1, 131. 13. Flores, J. R.; Smyth, M. R. J. Electroanal. Chem., 1987, 235, 317. 14. Tyczkowka, K. L.; Seay, S. S.; Stoskopf, M. K. S.; Aucoin, D. P. J. Chromatogr., 1992, 576, 305.
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MJ971516
Cathodic Stripping Voltammetric Determination of Ceftazidime in Urine at a Hanging Mercury Drop Electrode Valdir S. Ferreira,* M. Valnice B. Zanoni,* and Arnold G. Fogg† *Departamento de Quimica Analitica, Instituto de Quimica, Universidade Estadual Paulista, Caixa Postal 355, 14800-900, Araraquara, Sa˜o Paulo, Brazil; and †Chemistry Department, Loughborough University, Loughborough, Leicestershire LE11 3TU, United Kingdom Received May 2, 1997; accepted May 12, 1997 A method was developed for the differential-pulse cathodic stripping voltammetric determination of ceftazidime with a hanging mercury drop electrode using its reduction peak at 00.43 V in Britton–Robinson buffer pH 4.0. The optimum accumulation potential and time were 00.15 V and up to 60 s, respectively. Linear calibration graphs were obtained from 1 1 1008 M and 1.5 1 1007 M. The limit of determination was calculated to be 5 1 1009 M. The coefficient of variation was 4% (n Å 7) at 1 1 1007 M ceftazidime. The effect of various components of urine on the voltammetric response was studied, and creatinine, uric acid, urea, and glucose were shown to interfere in the method. Ceftazidime bound to human albumin gives a unique stripping peak at 00.48 V. Recoveries of 87% { 2% of the ceftazidime (n Å 5) were obtained from urine spiked with 1.27 mg ml01 using C-18 solid phase extraction cartridges. q 1997 Academic Press
INTRODUCTION
Ceftazidime [7-[2-(2-aminothiazol-4-yl)-2-(1-carboxy-1-methylethoxyimino) acetamido]-3(1-pyridiniomethyl)-3-cephem-4-carboxylate pentahydrate] is a b-lactam antibiotic of the third-generation cephalosporin family, active against a wide variety of bacterial infections (1). Although ceftazidime has shown an excellent record of clinical success, rigorous control of dosage is required since high dosage can cause renal tubular necrosis in humans (2). Its therapeutic and nephrotoxic effects need rapid and sensitive methods for determination of trace levels. Various analytical methods have been employed for this purpose. Fluorescence ELISA assay and liquid chromatographic methods with spectrophotometric and electrochemical detection for the determination of ceftazidime in human serum, urine, and bile are reported (3–8). Stripping voltammetry has been used successfully for the determination of other cephalosporin drugs (10 – 12). A differential pulse polarographic method of determining ceftazidime was reported previously by the present authors (9). Indications of the adsorption of ceftazidime on the mercury electrode, particularly in acidic conditions, were observed. The aim of the present study was to establish suitable experimental conditions for the determination of ceftazidime by adsorptive stripping voltammetry at the hanging mercury drop electrode, and, particularly, its determination in human urine. EXPERIMENTAL
Adsorptive stripping voltammetry was carried out using a Metrohm (Herisau, Switzerland) E-506 Polarecord and Voltammetric Scanner E612 and a Houston Instruments 115 0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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2000 X–Y recorder. A Metrohm 663 VA Stand was used in the hanging mercury drop electrode (HMDE) mode. The three electrode system was completed by means of a glassy carbon auxiliary electrode and an Ag/AgCl (3 M KCl) reference electrode: all potentials are quoted relative to this electrode. Supporting electrolytes and buffers were prepared using Suprapur grade reagents supplied by Merck and demineralized water from a Milli-Q system (Millipore, Milford, MA). Ceftazidime stock standard solutions (1 1 1004 –1 1 1005 M) were prepared from the pure compound (after drying) supplied by Glaxo Pharmaceuticals in demineralized water. Britton–Robinson (B–R) buffer (pH 2.0–8.0) was prepared by mixing 0.004 M orthophosphoric acid, 0.004 M acetic acid, and 0.004 M boric acid with the appropriate amount of 0.02 M sodium hydroxide solution. Hydrochloric acid solution (0.01– 2.5 M) was used for voltammetric measurements at pH õ 2.0. Solutions were deoxygenated for 12 min initially. Adsorptive accumulation was carried out while the solution was stirred, and cathodic scans were carried out after 15 s to allow the solution to become quiescent. Scan rates of 50 and 5 mV s01 were used with linear sweep stripping voltammetry and differential pulse stripping voltammetry, respectively. A pulse amplitude of 50 mV s01 and a pulse interval of 1 s were used with the differential pulse mode. Extraction Procedure for the Determination of Ceftazidime in Human Urine A Waters Associates Sep-Pak C18 extraction cartridge was prewashed once with 10 ml of methanol and then with 10 ml of water. For the extraction, 50 ml of human urine containing 63.7 ng of ceftazidime diluted in 5 ml of water was passed through the Sep-Pak cartridge, the ceftazidime being adsorbed on the Sep-Pak column. The column was then rinsed with 4 ml of water. For elution of the drug, 4-ml portions of water/ethanol 50% (v/v) were passed through the matrix. The total eluent was dried under a stream of nitrogen. The residue was reconstituted with 10 ml of B – R buffer at pH 4.0. The voltammograms were recorded under the optimum instrumental conditions. RESULTS AND DISCUSSION
Ceftazidime in acidic solution is polarographically reduced in two reduction processes (9). The first cathodic step is attributed to the reduction of the methylethoxyimino group and the second cathodic step to the reductive elimination of the pyridine group. At slightly less acidic pH values both reduction peaks show the existence of adsorption processes at the mercury electrode (9), which indicates that adsorption could be used as an effective preconcentration step before voltammetric measurements. Typical cyclic voltammograms of a 5 1 1007 M solution of ceftazidime in 0.004 M Britton – Robinson (B – R) buffer at pH 4.0 after 30 s accumulation at 00.15 V are shown in Fig. 1. A single well-defined peak at 00.43 V, corresponding to an irreversible process, can be seen. The peak obtained in the second scan, without further accumulation, is much smaller, indicating the adsorptive nature of the process. The second reduction peak previously observed polarographically is masked by the electrolyte discharge. Ceftazidime was shown to be stable in these solutions for at least 6 h. Comparison of the voltammetric responses obtained with
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FIG. 1. Cyclic voltammograms for ceftazidime (5 1 1007 M) in Britton–Robinson buffer at pH 4.0. Accumulation potential Å 00.15 V. (I) First scan after accumulation time 30 s. (II) Second scan without further accumulation.
FIG. 2. Effect of pH on differential-pulse adsorptive stripping peak current for 5 1 1007 M ceftazidime in Britton–Robinson buffer with 30 s accumulation at 00.15 V.
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FIG. 3. Effect of accumulation time on peak current for ceftazidime. (A) 5 1 1008 M, (B) 1 1 1007 M at 00.15 V.
linear scan and differential-pulse waveforms showed that the use of the pulse technique improves the sensitivity. The effect of pH on the differential pulse adsorptive stripping voltammograms of ceftazidime (5 1 1007 M ) is shown in Fig. 2. Maximum signal is observed between pH 3.0 and 5.5. Outside this pH range the signal is very small. This is in broad agreement with the polarographic results, as the polarographic wave for the first process becomes increasingly small at pH ú 4. For analytical purposes pH 4.0 was therefore chosen for the adsorptive stripping voltammetric determination of ceftazidime. The effect of accumulation potential on the stripping current at potentials from 0 to 00.30 V was minimal and an accumulation potential of 00.15 V was chosen as satisfactory. The peak current increased with the drop size on the voltammetric stand and the larger drop size (nominally 0.4 mm2) was chosen for use. Increased stirring rate (positions 0 to 6 on the voltammetric stand) produced a linear increase of the signal up to stirring speed 4; stirring speed 3 (approximately 1500 rev min01) was chosen as giving the best results. Variations of pulse amplitude (5–100 mV) and scan rate (5–20 mV s01) established that for 1 1 1007 M ceftazidime solutions with an accumulation time of 30 s the peak current increased with an increase of either parameter. A pulse amplitude of 50 mV/s and a scan rate of 5 mV/s produced the best signal in intensity and resolution. A rest time between 0 and 30 s after the stirring ended and the potential scan began had no effect on the size of the peak current. A rest time of 15 s was chosen as the measurements provided good reproducibility. The influence of accumulation time on the peak heights observed with 5 1 1008 M and 1 1 1007 M solutions of ceftazidime at pH 4.0 is shown in Fig. 3. A rectilinear
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FIG. 4. Effect of various urine components on the stripping peak current of 1 1 1007 M ceftazidime in Britton–Robinson buffer pH 4.0, with an accumulation time of 60 s. (a) creatinine; (b) glucose; (c) urea; (d) uric acid.
relationship is observed up to 200 and 90 s, respectively. Above these times marked decreases in the peak current are seen: these may be caused by multilayer adsorption or some reorientation of the molecules on the surface (12). Hence, care has to be taken in choosing accumulation times and shorter accumulation times are recommended for determining ceftazidime. Linear calibration graphs were obtained from voltammograms recorded at accumulation times of 60 s from 1.0 1 1008 to 1.5 1 1007 M ceftazidime in B – R buffer at pH 4.0 with an accumulation potential of 00.15 V. With an accumulation time of 60 s, limits of determination around 5 1 1009 M could be obtained. Mean standard deviations of 4% were obtained by measuring seven ceftazidime solutions (1 1 1007 M ). In order to investigate the possibility of applying cathodic stripping voltammetry to the determination of ceftazidime in urine samples, a study was carried out to determine the influence of various urine constituents on the peak current. Certain urine
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FIG. 5. Differential pulse adsorptive stripping voltammograms of 1 1 1007 M ceftazidime in Britton– Robinson buffer at pH 4.0 for 30 s at 00.15 V (Voltammogram I) and after addition of albumin 2.4 mg ml01 (Voltammogram III); 3.2 mg ml01 (Voltammogram IV) and 10 mg ml01 (Voltammogram V). Albumin (4.0 mg ml01) in Britton–Robinson buffer pH 4.0 (Voltammogram II).
components, viz., uric acid, urea, glucose, and creatinine, were found to lower the voltammetric response, as shown in Fig. 4. The presence of human albumin has a very interesting effect on the stripping voltammograms of ceftazidime in Britton–Robinson buffer at pH 4.0 after 30 s accumulation at 0 V. Stripping voltammograms for human albumin and ceftazidime separately are shown in Fig. 5, voltammograms I and II, respectively: those for mixtures of the two are shown in voltammograms III–V. The stripping voltammetric behavior of human albumin is well known (13). Human albumin gives a stripping peak at 00.34 V (marked in Fig. 5 as peak C), compared with the stripping peak of ceftazidime at 00.40 V (marked in Fig. 5 as peak A). The addition of 2.4 mg ml01 of albumin to the ceftazidime solution (voltammogram III) causes a slight decrease in the ceftazidime peak and the appearance of a new peak at 00.48 V (marked in Fig. 5 as peak B). The addition of 3.2 mg ml01 of albumin again decreases peak A and enhances peak B, and a shoulder corresponding to peak C becomes apparent. With 10 mg ml01 of albumin peak C becomes predominant with only a slight sign of peak B, and none of peak A, remaining. The heights of peaks A, B, and C are plotted in Fig. 6 against albumin concentration. Consideration of the results suggests that the appearance of peak B probably can be attributed to the interaction of ceftazidime with albumin, i.e. the protein binding of ceftazidime. The addition of excess albumin swamps the signals of the bound ceftazidime. These results with albumin are in agreement with information in the literature (8, 14): the extent of protein binding of ceftazidime in serum was previously determined
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FIG. 6. Effect of albumin concentration on the height of the stripping peaks of ceftazidime (1 1 1007 M) (peak A), free albumin (peak C), and ceftazidime bound with albumin (peak B).
by HPLC, and associated with the pharmacological efficacy of ceftazidime (3). In human plasma, at least 21 { 6% of ceftazidime has been shown to be bound to proteins. From the results given above it is apparent that the direct determination of the drug in urine by cathodic stripping voltammetry is not possible. For that reason a prior extraction of ceftazidime using the solid-phase extraction procedure described in the experimental section was applied. Sep-Pak C18 cartridges were used successfully for the extraction of the drug. Urine samples with differing ceftazidime contents were analysed. The stripping peaks obtained after extraction were of the same form as those obtained for aqueous solutions prepared directly from the pure drug. A standard addition method was used for the determination in urine. At 1.27 mg ml01 of ceftazidime in urine, the recovery was 87% { 2% (5 determinations) using a more rapid accumulation time of 15 s and an accumulation potential of 00.25 V. In conclusion, ceftazidime is adsorbed spontaneously onto a hanging mercury drop
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electrode, and the method of cathodic stripping voltammetry can be applied to the determination of ceftazidime in urine samples in conjunction with a Sep-Pak C18 cartridge. ACKNOWLEDGMENTS The authors thank CAPES/The British Council and CNPq for financial support for this collaboration.
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