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Electrochemical behaviour of the monobactam antibiotic aztreonam at different electrodes and in biological fluids
Bioelectrochemistry and Bioenergetics 45 Ž1998. 281–286
Short communication
Electrochemical behaviour of the monobactam antibiotic aztreonam at different electrodes and in biological fluids Nagwa Abo El-Maali
)
Chemistry Department, Faculty of Science, Assiut UniÕersity, 71516 Assiut, Egypt Received 22 October 1997; revised 10 February 1998; accepted 23 February 1998
Abstract An investigation of the electrochemical reduction, oxidation, and determination of the monobactam antibiotic, aztreonam ŽAzm. at different types of electrodes namely: dropping mercury electrode ŽDME., static mercury drop electrode ŽSMDE., glassy carbon electrode ŽGCE., carbon paste electrode ŽCPE., and modified carbon paste electrode ŽMCPE. is introduced. The electrode mechanism is found to be comparable to that of the metabolic reaction. Two different techniques were utilized for the drug determination in either aqueous medium or in urine samples, differential pulse stripping voltammetry ŽDPSV. and Osteryoung square-wave stripping voltammetry ŽOSWSV.. These allow the drug determination in a concentration range as low as 5 = 10y8 M after preconcentration for 60 s. Modification of the CPE with gelatin allows better selectivity of the drug in urine matrix. Detection limits of 2 = 10y8 M and 8 = 10y8 M Azm were achieved in aqueous and urine samples, respectively. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Azactam; Electrode mechanism; Modified electrode; Determination in biological fluids
1. Introduction Aztreonam ŽAzm. is a synthetic, monocyclic b-lactam antimicrobial agent, active against Gram negative organism and belonging to a relatively new class of antibiotics, the monobactams w1,2x. In aqueous solution, the most important source of instability of Azm over the whole pH range is hydrolysis of the b-lactam ring w3x. In weakly acid solutions ŽpH 2 to 5., hydrolysis is preceded by isomerization of the side chain w3,4x. Azm is most stable in the pH 5–6 range w3x. In aqueous, buffer-free solution, Azm is about five times more stable in the pH 4–7 range than most penicillins and cephalosporins, including ampicillin and cephradine 1. At pH 5–7, Azm shows 10% degradation after 300–500 h. Phosphate, tris, borate and carbonate buffers accelerate degradation. Degradation of Azm to dimers and trimers in aqueous solution has also been described w5x. Azm is stable in human serum and urine for
at least 10 months when kept at y788C w1x 2 . However, it is more stable in urine than in serum. It looses about 2.5%r24 h at 258C w1x. After either intravenous or intramuscular administration, Azm was eliminated primarily by urinary excretion of unchanged Azm w1x. The most prominent biotransformation product of Azm was the derivative resulting from the hydrolytic opening of the b-lactam ring w1,6x. Microbiological and high pressure liquid chromatography ŽHPLC. assay methods give similar values for Azm concentration in human serum and urine, indicating the absence of microbiologically active metabolites w7x. Azm was detected in serum at a concentration of 0.625 m grml when samples were extracted with MeOH w8x. Results obtained agreed well with those obtained by HPLC. Many authors determined the drug using HPLC, spectrophotometry, and colorimetry w9–16x. These analyzes include the drug determination in serum and urine in tissues and body fluids. The aim of the present work is to give a scope on the electrochemical behaviour Želectrode mechanism. of the drug since no data are present in literature dealing with it, and then to use the very sensitive method ‘adsorptive
)
Corresponding author. J.D. Pipkin, E.P. Barry, The Squibb Institute, personal communication, 1981. 1
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 9 3 - 2
2
M.A. Leitz, S. Wind, The Squibb Institute, personal communication, 1983.
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N.A. El-Maalir Bioelectrochemistry and Bioenergetics 45 (1998) 281–286
stripping voltammetry’ for drug determination in aqueous and biological fluids.
2. Experimental 2.1. Apparatus A BAS CV-50W Voltammetric Analyzer running Windowse software was used for oxidation, coulometry bulk electrolysis ŽBE. and analytical determination of the drug. Three electrodes used are a carbon paste working electrode ŽMF-2010, diameter 3 mm. or a glassy carbon electrode ŽMF-2012, diameter 3 mm., a silverrsilver chloride reference electrode ŽMF-2063. and a platinum wire auxiliary electrode ŽMW-1032.. Voltammograms are collected using a hp Hewlett Packard LaserJet 4L printer. For coulometric measurements in the cell required for ŽBE., the working electrode was a mercury pool of large surface area 4.9 cm2 , the separated auxiliary electrode was a platinum coil with a large surface area and an AgrAgCl electrode was used for reference. The solution of 10 ml was stirred, before a BE experiment can be run, the potential had been chosen 200 mV more negative than the redox potential so that the rate of electrolysis is controlled by the rate of mass transport to the working electrode. The total charge Ž Q . passing during the BE experiment is related to the number of electrons transferred per molecule Ž n. and the number of moles of the oxidized species initially present Ž N . according to Faraday’s law. Direct current ŽDC. polarograms were obtained with a Sargent-Welch voltammetric analyser ŽMFD model 3001. designed by Sargent-Welch Scientific as described in previous publication w17x. An EG & G Princeton Applied Research microprocessor-controlled ŽPAR. Model 264A stripping analyzer, coupled with a PAR 303A SMDE Žmedium drop size, 0.014 cm2 . served for recording voltammograms as mentioned previously w18x.
2.3. Electrode preparation A 10-ml aliquot of the supporting electrolyte was tested as a blank after exposure to the proper potential for an appropriate time. Reproducibility was checked by repeating the procedure many times or by choosing the order multirun in the control menu of the CV-50W software. A fresh smoothed surface was used for each test Žin case of the carbon paste electrode. or polishing the glassy carbon electrode between each run. The antibiotic was then added and voltammograms were recorded. In experiments—including urine samples—either directly measured in the cell or the preconcentration-medium exchange voltammetric scheme was used. The modified electrodes were prepared by mixing the carbon paste with either gelatin of gum acacia, individually, in a mortar in the presence of a small amount of their appropriate organic solvent and allowing the solvent to evaporate overnight. Three modifier contents were tested: 3 wt.%, 6 wt.% and 10 wt.%. In the preconcentration step, the electrode was immersed in the cell, the potential was applied for a given time and the system was allowed to equilibrate for 10 s before recording the voltammograms. In experiments involving medium exchange, the preconcentration step was performed under open circuit conditions, the electrode was transferred to the electrolyte blank solution and the voltammograms were recorded. Quantitative measurements of the drug in the urine samples were carried out by replacing the supporting electrolyte blank solution with another containing a urine sample diluted 10 times with the same supporting electrolyte and then applying different spikes of the drug under investigation.
3. Results and discussion The structure of the antibiotic under investigation is shown in Fig. 1.
2.2. Reagents and solutions
3.1. Electrochemical reduction and oxidation
A fresh solution of the antibiotic Azm was prepared daily in doubly distilled water. Azm was generously provided by SQUIBB ŽBristol-Myers Squibb, Egypt.. It was used without any further purification. The supporting electrolytes were either phosphoric or acetic acid ŽMerck quality. adjusted to the desired pH using sodium hydroxide ŽMerck quality.. All other reagents were of analytical grade quality. The carbon paste was purchased from BAS CPO-Carbon Paste Žoil base, BAS CF-1010.. The modifier was dissolved in carbon tetrachloride by gentle heating. Gelatin and Gum acacia were laboratory samples ŽHopkin, Williams, England.. Urine samples were taken from healthy persons. All measurements were carried out at 25 " 18C.
3.1.1. Electrochemical reduction of the antibiotic As it is well-known w3x, in aqueous solution, the most important source of instability of Azm over the whole pH range is the hydrolysis of the b-lactam ring. This mechanism resembles the electrochemical one previously elucidated in our laboratory w18x for Pipracillin. Preliminary investigation of the electroreduction of Azm at mercury electrode in 0.1 M phosphate buffer at different pH values gives rise to only one reduction wave in acidic pH values at about f y0.35 V vs. SCE. This wave is shifted to more electronegative potentials when the pH value exceeds 3.0 ŽFig. 2a.. The morphology of the wave changes starting from pH f 7.0 until it disappears at about pH 8.5 while an appearance of another wave at more
N.A. El-Maalir Bioelectrochemistry and Bioenergetics 45 (1998) 281–286
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Fig. 1. Structure of Azm and contribution of various substructures to its activity.
negative potentials Ž; y0.8. was found as the pH reaches 9.5. Analysis of these waves elucidate the irreversibility of the electrochemical process calculated from the following equation w19x: 0.0542 i E s E1r2 y log an i1 y I
a s 03. According to coulometry ŽBE., the number of the electrons consumed was found to be two. Therefore, the number of protons consumed in the reduction step is calculated from the equation w19x: D E1r2 2.3p RT s D pH 2aF Ž to be zero i.e., no proton consumption. in the pH range 1–3 ŽFig. 2b. while two protons are consumed in the pH
Fig. 2. Effect of the pH on the DC-polarogram of 1.6=10y4 M Azm, Ža. 0.1 M phosphate buffer, Žb. Ža.qAzm, pH: Žb. 1.24, Žc. 2.04, Žd. 3.10, Že. 4.48, Žf. 5.02. DC mode 4 mV sy1 . The E1r 2 –pH relation could be seen inside the figure.
range 3–6, the distortion of this peak at pH values higher than 6.5 makes it difficult to calculate such parameters. The following reduction mechanism therefore is proposed.
3.1.2. Voltammetric measurements Fig. 3 examines the dependence of the time on the cyclic voltammogram of 5.2 = 10y6 M Azm. The drug exhibits significant peak current decrease curve b then
Fig. 3. Multiple cyclic voltammograms of 5.2=10y6 M Azm in 0.1 M HClO4 , pH 1.84, scan rate 100 mV sy1 , starting potential y0.2 V vs. AgrAgCl, KCl s. Ža. First, Žb. second, Žc. third, Žd. fourth, Že. fifth, . . . cycle.
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N.A. El-Maalir Bioelectrochemistry and Bioenergetics 45 (1998) 281–286
almost a steady state is obtained on the same drop Žcycles b, c, d, . . . . with a little shift in the peak potential. This fast desorption behaviour of the drug is obtained in strong acidic pH’s Ž1–2. while in the pH range Ž2–4. this phenomenon disappears. The accumulation process was optimized by varying a number of experimental parameters including the supporting electrolyte, pH, accumulation potential, etc. The best conditions were found to be 0.1 M HClO4 , pH 1–3 and an accumulation potential of y0.2 V vs. AgrAgCl. A linear plot in obtained in the concentration range 2 = 10y9 –7 = 10y6 ŽAzm. having the correlation coefficient 0.996 applying an accumulation time of 30 s. The lower limit of this linearity is decreased without preconcentration of the drug to be 4 = 10y8 –7 = 10y6 M correlation coefficient 0.992. Therefore, adsorptive stripping voltammetry is helpful for the drug determination at low concentration levels until 2 = 10y9 M. 3.1.3. Electrooxidation of the antibiotic at glassy carbon electrode (GCE) and carbon paste electrodes (CPE) Preliminary investigation of the oxidation of Azm in 0.05 M acetic acid, pH 2.5 at GCE and CPE is shown by the voltammograms represented in Fig. 4. One peak is obtained at q0.925 V and q0.82 V at GCE and CPE, respectively. The well-defined differential pulse voltammogram obtained at the CPE is much better in sensitivity, morphology than that at the GCE. Also it is far enough—
Fig. 4. A comparison of the oxidation of Azm at the GCE, curve Žb. and the CPE Žcurve a.. Acetate buffer, pH 2.5, other experimental parameters as cited in the figure.
Fig. 5. Effect of the Ep and Ip on the pH of the oxidation of 1=10y5 M Azm at the CPE in 0.052 M acetate buffer, pH: Ža. 2.1, Žb. 3.17, Žc. 4.02, Žd. 5.13, Že. 6.9, and Žf. 8.1.
under these conditions—from the limit of the oxidation of the water molecules. The effect of the pH on the differential pulse voltammograms of 1 = 10y5 M Azm in 0.052 M acetic acid at the CPE shows one oxidation peak shifted to less oxidation potentials from pH 2–4, while no shift is observed from pH 4.0 to 7.0. A steady decrement ŽFig. 5. of the current is obtained with a complete disappearance of the peak when the pH reaches a value of more than 7.5, probably due to complete hydrolysis of the drug in alkaline media. From these results, we postulate that the oxidation of the drug occurs at the b-lactam ring because it is the weakest side in the molecule as it has been mentioned elsewhere w18x. This scheme is in accordance to that of the drug metabolism in biological fluids w3x; it is, therefore concluded that the electrode mechanism is identical to that of the metabolic reaction illustrated by the following scheme w1x:
3.1.4. Cyclic Õoltammetric measurements The nature of the electrochemical process has been carried out applying cyclic voltammetry at different pH values. One irreversible peak is obtained, the peak is highly affected by changing the pH until it completely disappears at about pH 7.0. The effect of the scan rate on this peak has also been tested on 5 = 10y5 M Azm, pH 2.26 at the CPE, the irreversibility of the electrochemical
N.A. El-Maalir Bioelectrochemistry and Bioenergetics 45 (1998) 281–286
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process decreases at higher scan rates viz. 100, 200 mV sy1 . 3.2. Analytical aspects 3.2.1. Analytical aspects at the GCE and CPE From the above results, it is concluded that, for analytical purposes, the best pH value for the drug determination lies between 1.5–5.0. Therefore, the effect of the different experimental parameters has been tested in this pH-range at CPE and GCE. Fig. 6 shows a comparison between the DPSV curve Ža., OSWSV curve Žb. and the LSSV, curve Žc.. It is obvious that at the CPE, OSWSV and DPSV are much better than the LSSV, and that the sensitivity of the OSWSV is better than that of DPSV whereas, the morphology of the latter regarding to the peak shape is better. Its potential is far enough from any perturbing oxidation current. Therefore, for further investigation at the CPE’s the DPSV mode will always be used. At the GCE, OSWSV is found to be much more sensitive. Therefore, for the drug quantitation, this mode will be used. The dependence of the OSWS on the accumulation time for 1 = 10y6 M Azm has been studied at pH 1.75 and 5.0 at the GCE. A significant peak current enhancement is
Fig. 7. Standard addition of Azm. CPE, accumulation potential q0.6 V vs. AgrAgCl, accumulation time 60 s, pH 2.6, Azm concentration: Ža. 1=10y6 , Žb. 2=10y6 , Žc. 4=10y6 , Žd. 5=10y6 , Že. 1=10y5 , Žf. 2=10y5 M.
obtained by longer accumulation periods. For example, accumulation for 5 min allows preconcentration of the drug and therefore 26, 21-fold enhancements at pH 1.75 and 5.0 were measured, respectively. This allows the drug determination to reach a level as low as 5 = 10y8 M. On the CPE, the accumulation step must always be performed on a new paste surface as depression in the current is obtained. Standard addition procedure was done with and without preconcentration. Fig. 7 shows the voltammograms obtained applying 60 s accumulation. A linear plot is obtained up to 6 = 10y5 M with the characteristics cited in Table 1. The reproducibility of the process was evaluated by performing 10 measurements on the 3 = 10y6 M Azm after 60 s accumulation on fresh prepared CPE. A mean value of 0.255 m A with a relative standard deviation of 1.8%. A detection limit of 8 = 10y8 M Azm in 10 ml has been established meaning that 8.71 ng Azm are detectable in the 10 ml solution.
Fig. 6. Comparison between DPSV Ža., OSWSV Žb., and LSSV Žc. of 5=10y5 M Azm, parameters for each mode: DPSV: scan rate 20 mV sy1 , pulse amplitude 50 mV, sample width 17 m s, pulse width 50 m s, pulse period 200 m s, Quiet time 10 s, OSWSV: S.W. amplitude 25 mV, S.W. frequency 15 Hz, step E 4 mV, Quiet time 10 s, LSSV: scan rate 100 mV sy1 , Quiet time 10 s, ŽA. at the CPE, pH 2.26 and Žb. at the GCE, pH 1.75.
Table 1 Characteristic features of the linearity limit for Azm at the CPE) and Gel MCPEq Drug ŽM.
Equation Ž m A m My1 .
Correlation Limit of coefficient linearity ŽM.
1=10y6 –6=10y6 ) Y s 0.260 X y0.25 0.9996 4=10y6 –4=10y5 q Y s 0.176 X q0.75 0.9926
6=10y5 4=10y5
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N.A. El-Maalir Bioelectrochemistry and Bioenergetics 45 (1998) 281–286
3.2.2. Analytical aspects at chemically modified electrodes Three different modifiers were tested in this work trying to enhance both sensitivity and selectivity of the method: sodium dodecyl sulphate, gum and gelatin. The three tested modifiers are mixed, individually, in three percentages namely 3, 6, and 10% with the carbon paste, then the entire pH-range is tested for choosing the best one which may give rise to better sensitivity and selectivity. A 10% gelatin modified carbon paste electrode ŽGel MCPE. gave rise to better sensitivity. The DPS voltammograms of Azm at 10% Gel MCPE in 0.03 M acetate buffer, pH 2.0 shows a relatively small desorption of the antibiotic from the electrode surface comparing with that carried out at the CPE, indicating that the modifier plays a role in the accumulation of the drug. Also, a shift in the oxidation potential of the drug under investigation is observed at the Gel MCPE. For the drug quantitation in urine, there is a peak obtained in the base line—due to the existence of the potentially interfering substances in urine—at the same oxidation potential of Azm. The shift of the peak at the Gel MCPE allows its determination in such medium without any interference and also by applying the method of medium exchange—i.e., accumulating the drug from urine solution and then transferring the electrode to another cell containing a 0.03 M acetate buffer, pH 2.0. Voltammograms of the drug at 10% Gel MCPE are obtained, their characteristic features are cited in Table 1. Detection limit of the drug in the urine samples reaches 8 = 10y8 M Azm in 10 ml solution.
References w1x K. Florey, Aztreonam, Analytical profiles of drug substances, Vol. 17, 1988. w2x C.M. Cimarusti, R.B. Sykes, Discovery and synthesis of the monobactam antibiotic: aztreonam, Med. Res. Rev. 4 Ž1984. 1. w3x J.D. Pipkin, in: K.A. Connors et al. ŽEds.., Chemical Solubility of Pharmaceuticals, 2nd edn., Wiley, New York, 1986, p. 250. w4x J. Adamovics, S. Unger, Degradation of aztreonam, off-line HPLC combined with FAB, J. Liq. Chromatogr. 9 Ž1986. 141.
w5x J.D. Pipkin, S.A. Ranadive, N.H. Chang, S.E. Unger, T.J. McCormick, M. Porubcan, E.P. Barry, S.A. Varia, J. Adamovics, Degradation of aztreonam to dimers and trimers in aqueous solution, Am. Pharm. Assoc. Acad. Sci. Abstr. 15 Ž1985. 135–142. w6x E.A. Swabb, S.M. Singhvi, M.A. Leitz, M. Frantz, A. Sugerman, Antimicrob. Agents Chemother. 24 Ž1983. 394. w7x E.A. Swabb, A.A. Sugerman, T.B. Platt, G. Pilkiewiczf, M. Frantz, Single-dose pharmacokinetics of the monobactam aztreonam ŽSG 26,776. in healthy subjects, Antimicrob. Agents Chemother. 21 Ž1982. 944–949. w8x Y. Koyama, N. Namiki, C.H. Ozaki, Microbiological methods for the assay of aztreonam concentration in body fluids, Chemotherapy ŽTokyo. 33 Ž1985. 118–125. w9x F.G. Pilkiewicz, B.J. Remsburg, S.M. Fisher, R.B. Sykes, Highpressure liquid-chromatographic analysis of aztreonam in serum and urine, Antimicrob. Agents Chemother. 23 Ž1983. 852–856. w10x J. Adamovics, S. Unger, Preparative liquid chromatography of pharmaceuticals using silica gel with aqueous, J. Liq. Chromatogr. 9 Ž1986. 141–155. w11x A. Meulemans, J. Mohler, D. Vittecoq, G. Haroche, M.A. Rosset, M. Vulpillat, J. Modai, High performance liquid chromatographic analysis of aztreonam in tissues and body fluids, J. Chromatogr. Biomed. Appl. 50 Ž1986. 466–470. w12x F. Jehl, P. Birckel, H. Monteil, Hospital routine analysis of penicillins, third-generation cephalosporins and aztreonam by conventional and high-speed high-performance liquid chromatography, J. Chromatogr. Biomed. Appl. 57 Ž1987. 109–119. w13x A.A.M. Wahbi, M.A. Abounassif, E.R.A. Gad-Kariem, M.E. Ibrahim, H.Y. Aboul-Enein, Determination of aztreonam and Larginine combination in parenteral formulations, J. Assoc. Off. Anal. Chem. 71 Ž1988. 31–33. w14x M.E. Mohamed, M.A. Abounassif, H.A. Al-Khamees, Spectrophotometric determination of aztreonam in pharmaceutical formulations, Anal. Lett. 21 Ž1988. 1579. w15x B. Morelli, Simultaneous derivative spectrophotometric determination of aztreonam and L-arginine in injections, J. Pharm. Sci. 79 Ž1990. 261–265. w16x M.E. Mohamed, M.A. Abounassif, H.A. Al-Khamas, H. Kandil, H.Y. Aboul-Enein, Colorimetric determination of aztreonam and conductometric determination of L-arginine in injectable form, J. Pharm. Belg. 43 Ž1998. 429–436. w17x N.A. El-Maali, M.A. Ghandour, J.M. Kauffmann, Cephalosporin antibiotics at carbon paste and modified carbon paste electrodes in both aqueous and biological media, Bioelectrochem. Bioenerg. 38 Ž1995. 91–97. w18x N.A. El-Maali, M.A. Ghandour, M. Khodari, Electroreduction and determination of Pipril ŽPiperacillin. in both aqueous and biological samples, Talanta 40 Ž1993. 1833–1839. w19x J. Heyrovsky, Principles of Polarography, Chap. XIV, Academic Press, New York, 1966, p. 257.
Short communication
Electrochemical behaviour of the monobactam antibiotic aztreonam at different electrodes and in biological fluids Nagwa Abo El-Maali
)
Chemistry Department, Faculty of Science, Assiut UniÕersity, 71516 Assiut, Egypt Received 22 October 1997; revised 10 February 1998; accepted 23 February 1998
Abstract An investigation of the electrochemical reduction, oxidation, and determination of the monobactam antibiotic, aztreonam ŽAzm. at different types of electrodes namely: dropping mercury electrode ŽDME., static mercury drop electrode ŽSMDE., glassy carbon electrode ŽGCE., carbon paste electrode ŽCPE., and modified carbon paste electrode ŽMCPE. is introduced. The electrode mechanism is found to be comparable to that of the metabolic reaction. Two different techniques were utilized for the drug determination in either aqueous medium or in urine samples, differential pulse stripping voltammetry ŽDPSV. and Osteryoung square-wave stripping voltammetry ŽOSWSV.. These allow the drug determination in a concentration range as low as 5 = 10y8 M after preconcentration for 60 s. Modification of the CPE with gelatin allows better selectivity of the drug in urine matrix. Detection limits of 2 = 10y8 M and 8 = 10y8 M Azm were achieved in aqueous and urine samples, respectively. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Azactam; Electrode mechanism; Modified electrode; Determination in biological fluids
1. Introduction Aztreonam ŽAzm. is a synthetic, monocyclic b-lactam antimicrobial agent, active against Gram negative organism and belonging to a relatively new class of antibiotics, the monobactams w1,2x. In aqueous solution, the most important source of instability of Azm over the whole pH range is hydrolysis of the b-lactam ring w3x. In weakly acid solutions ŽpH 2 to 5., hydrolysis is preceded by isomerization of the side chain w3,4x. Azm is most stable in the pH 5–6 range w3x. In aqueous, buffer-free solution, Azm is about five times more stable in the pH 4–7 range than most penicillins and cephalosporins, including ampicillin and cephradine 1. At pH 5–7, Azm shows 10% degradation after 300–500 h. Phosphate, tris, borate and carbonate buffers accelerate degradation. Degradation of Azm to dimers and trimers in aqueous solution has also been described w5x. Azm is stable in human serum and urine for
at least 10 months when kept at y788C w1x 2 . However, it is more stable in urine than in serum. It looses about 2.5%r24 h at 258C w1x. After either intravenous or intramuscular administration, Azm was eliminated primarily by urinary excretion of unchanged Azm w1x. The most prominent biotransformation product of Azm was the derivative resulting from the hydrolytic opening of the b-lactam ring w1,6x. Microbiological and high pressure liquid chromatography ŽHPLC. assay methods give similar values for Azm concentration in human serum and urine, indicating the absence of microbiologically active metabolites w7x. Azm was detected in serum at a concentration of 0.625 m grml when samples were extracted with MeOH w8x. Results obtained agreed well with those obtained by HPLC. Many authors determined the drug using HPLC, spectrophotometry, and colorimetry w9–16x. These analyzes include the drug determination in serum and urine in tissues and body fluids. The aim of the present work is to give a scope on the electrochemical behaviour Želectrode mechanism. of the drug since no data are present in literature dealing with it, and then to use the very sensitive method ‘adsorptive
)
Corresponding author. J.D. Pipkin, E.P. Barry, The Squibb Institute, personal communication, 1981. 1
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 9 3 - 2
2
M.A. Leitz, S. Wind, The Squibb Institute, personal communication, 1983.
282
N.A. El-Maalir Bioelectrochemistry and Bioenergetics 45 (1998) 281–286
stripping voltammetry’ for drug determination in aqueous and biological fluids.
2. Experimental 2.1. Apparatus A BAS CV-50W Voltammetric Analyzer running Windowse software was used for oxidation, coulometry bulk electrolysis ŽBE. and analytical determination of the drug. Three electrodes used are a carbon paste working electrode ŽMF-2010, diameter 3 mm. or a glassy carbon electrode ŽMF-2012, diameter 3 mm., a silverrsilver chloride reference electrode ŽMF-2063. and a platinum wire auxiliary electrode ŽMW-1032.. Voltammograms are collected using a hp Hewlett Packard LaserJet 4L printer. For coulometric measurements in the cell required for ŽBE., the working electrode was a mercury pool of large surface area 4.9 cm2 , the separated auxiliary electrode was a platinum coil with a large surface area and an AgrAgCl electrode was used for reference. The solution of 10 ml was stirred, before a BE experiment can be run, the potential had been chosen 200 mV more negative than the redox potential so that the rate of electrolysis is controlled by the rate of mass transport to the working electrode. The total charge Ž Q . passing during the BE experiment is related to the number of electrons transferred per molecule Ž n. and the number of moles of the oxidized species initially present Ž N . according to Faraday’s law. Direct current ŽDC. polarograms were obtained with a Sargent-Welch voltammetric analyser ŽMFD model 3001. designed by Sargent-Welch Scientific as described in previous publication w17x. An EG & G Princeton Applied Research microprocessor-controlled ŽPAR. Model 264A stripping analyzer, coupled with a PAR 303A SMDE Žmedium drop size, 0.014 cm2 . served for recording voltammograms as mentioned previously w18x.
2.3. Electrode preparation A 10-ml aliquot of the supporting electrolyte was tested as a blank after exposure to the proper potential for an appropriate time. Reproducibility was checked by repeating the procedure many times or by choosing the order multirun in the control menu of the CV-50W software. A fresh smoothed surface was used for each test Žin case of the carbon paste electrode. or polishing the glassy carbon electrode between each run. The antibiotic was then added and voltammograms were recorded. In experiments—including urine samples—either directly measured in the cell or the preconcentration-medium exchange voltammetric scheme was used. The modified electrodes were prepared by mixing the carbon paste with either gelatin of gum acacia, individually, in a mortar in the presence of a small amount of their appropriate organic solvent and allowing the solvent to evaporate overnight. Three modifier contents were tested: 3 wt.%, 6 wt.% and 10 wt.%. In the preconcentration step, the electrode was immersed in the cell, the potential was applied for a given time and the system was allowed to equilibrate for 10 s before recording the voltammograms. In experiments involving medium exchange, the preconcentration step was performed under open circuit conditions, the electrode was transferred to the electrolyte blank solution and the voltammograms were recorded. Quantitative measurements of the drug in the urine samples were carried out by replacing the supporting electrolyte blank solution with another containing a urine sample diluted 10 times with the same supporting electrolyte and then applying different spikes of the drug under investigation.
3. Results and discussion The structure of the antibiotic under investigation is shown in Fig. 1.
2.2. Reagents and solutions
3.1. Electrochemical reduction and oxidation
A fresh solution of the antibiotic Azm was prepared daily in doubly distilled water. Azm was generously provided by SQUIBB ŽBristol-Myers Squibb, Egypt.. It was used without any further purification. The supporting electrolytes were either phosphoric or acetic acid ŽMerck quality. adjusted to the desired pH using sodium hydroxide ŽMerck quality.. All other reagents were of analytical grade quality. The carbon paste was purchased from BAS CPO-Carbon Paste Žoil base, BAS CF-1010.. The modifier was dissolved in carbon tetrachloride by gentle heating. Gelatin and Gum acacia were laboratory samples ŽHopkin, Williams, England.. Urine samples were taken from healthy persons. All measurements were carried out at 25 " 18C.
3.1.1. Electrochemical reduction of the antibiotic As it is well-known w3x, in aqueous solution, the most important source of instability of Azm over the whole pH range is the hydrolysis of the b-lactam ring. This mechanism resembles the electrochemical one previously elucidated in our laboratory w18x for Pipracillin. Preliminary investigation of the electroreduction of Azm at mercury electrode in 0.1 M phosphate buffer at different pH values gives rise to only one reduction wave in acidic pH values at about f y0.35 V vs. SCE. This wave is shifted to more electronegative potentials when the pH value exceeds 3.0 ŽFig. 2a.. The morphology of the wave changes starting from pH f 7.0 until it disappears at about pH 8.5 while an appearance of another wave at more
N.A. El-Maalir Bioelectrochemistry and Bioenergetics 45 (1998) 281–286
283
Fig. 1. Structure of Azm and contribution of various substructures to its activity.
negative potentials Ž; y0.8. was found as the pH reaches 9.5. Analysis of these waves elucidate the irreversibility of the electrochemical process calculated from the following equation w19x: 0.0542 i E s E1r2 y log an i1 y I
a s 03. According to coulometry ŽBE., the number of the electrons consumed was found to be two. Therefore, the number of protons consumed in the reduction step is calculated from the equation w19x: D E1r2 2.3p RT s D pH 2aF Ž to be zero i.e., no proton consumption. in the pH range 1–3 ŽFig. 2b. while two protons are consumed in the pH
Fig. 2. Effect of the pH on the DC-polarogram of 1.6=10y4 M Azm, Ža. 0.1 M phosphate buffer, Žb. Ža.qAzm, pH: Žb. 1.24, Žc. 2.04, Žd. 3.10, Že. 4.48, Žf. 5.02. DC mode 4 mV sy1 . The E1r 2 –pH relation could be seen inside the figure.
range 3–6, the distortion of this peak at pH values higher than 6.5 makes it difficult to calculate such parameters. The following reduction mechanism therefore is proposed.
3.1.2. Voltammetric measurements Fig. 3 examines the dependence of the time on the cyclic voltammogram of 5.2 = 10y6 M Azm. The drug exhibits significant peak current decrease curve b then
Fig. 3. Multiple cyclic voltammograms of 5.2=10y6 M Azm in 0.1 M HClO4 , pH 1.84, scan rate 100 mV sy1 , starting potential y0.2 V vs. AgrAgCl, KCl s. Ža. First, Žb. second, Žc. third, Žd. fourth, Že. fifth, . . . cycle.
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almost a steady state is obtained on the same drop Žcycles b, c, d, . . . . with a little shift in the peak potential. This fast desorption behaviour of the drug is obtained in strong acidic pH’s Ž1–2. while in the pH range Ž2–4. this phenomenon disappears. The accumulation process was optimized by varying a number of experimental parameters including the supporting electrolyte, pH, accumulation potential, etc. The best conditions were found to be 0.1 M HClO4 , pH 1–3 and an accumulation potential of y0.2 V vs. AgrAgCl. A linear plot in obtained in the concentration range 2 = 10y9 –7 = 10y6 ŽAzm. having the correlation coefficient 0.996 applying an accumulation time of 30 s. The lower limit of this linearity is decreased without preconcentration of the drug to be 4 = 10y8 –7 = 10y6 M correlation coefficient 0.992. Therefore, adsorptive stripping voltammetry is helpful for the drug determination at low concentration levels until 2 = 10y9 M. 3.1.3. Electrooxidation of the antibiotic at glassy carbon electrode (GCE) and carbon paste electrodes (CPE) Preliminary investigation of the oxidation of Azm in 0.05 M acetic acid, pH 2.5 at GCE and CPE is shown by the voltammograms represented in Fig. 4. One peak is obtained at q0.925 V and q0.82 V at GCE and CPE, respectively. The well-defined differential pulse voltammogram obtained at the CPE is much better in sensitivity, morphology than that at the GCE. Also it is far enough—
Fig. 4. A comparison of the oxidation of Azm at the GCE, curve Žb. and the CPE Žcurve a.. Acetate buffer, pH 2.5, other experimental parameters as cited in the figure.
Fig. 5. Effect of the Ep and Ip on the pH of the oxidation of 1=10y5 M Azm at the CPE in 0.052 M acetate buffer, pH: Ža. 2.1, Žb. 3.17, Žc. 4.02, Žd. 5.13, Že. 6.9, and Žf. 8.1.
under these conditions—from the limit of the oxidation of the water molecules. The effect of the pH on the differential pulse voltammograms of 1 = 10y5 M Azm in 0.052 M acetic acid at the CPE shows one oxidation peak shifted to less oxidation potentials from pH 2–4, while no shift is observed from pH 4.0 to 7.0. A steady decrement ŽFig. 5. of the current is obtained with a complete disappearance of the peak when the pH reaches a value of more than 7.5, probably due to complete hydrolysis of the drug in alkaline media. From these results, we postulate that the oxidation of the drug occurs at the b-lactam ring because it is the weakest side in the molecule as it has been mentioned elsewhere w18x. This scheme is in accordance to that of the drug metabolism in biological fluids w3x; it is, therefore concluded that the electrode mechanism is identical to that of the metabolic reaction illustrated by the following scheme w1x:
3.1.4. Cyclic Õoltammetric measurements The nature of the electrochemical process has been carried out applying cyclic voltammetry at different pH values. One irreversible peak is obtained, the peak is highly affected by changing the pH until it completely disappears at about pH 7.0. The effect of the scan rate on this peak has also been tested on 5 = 10y5 M Azm, pH 2.26 at the CPE, the irreversibility of the electrochemical
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process decreases at higher scan rates viz. 100, 200 mV sy1 . 3.2. Analytical aspects 3.2.1. Analytical aspects at the GCE and CPE From the above results, it is concluded that, for analytical purposes, the best pH value for the drug determination lies between 1.5–5.0. Therefore, the effect of the different experimental parameters has been tested in this pH-range at CPE and GCE. Fig. 6 shows a comparison between the DPSV curve Ža., OSWSV curve Žb. and the LSSV, curve Žc.. It is obvious that at the CPE, OSWSV and DPSV are much better than the LSSV, and that the sensitivity of the OSWSV is better than that of DPSV whereas, the morphology of the latter regarding to the peak shape is better. Its potential is far enough from any perturbing oxidation current. Therefore, for further investigation at the CPE’s the DPSV mode will always be used. At the GCE, OSWSV is found to be much more sensitive. Therefore, for the drug quantitation, this mode will be used. The dependence of the OSWS on the accumulation time for 1 = 10y6 M Azm has been studied at pH 1.75 and 5.0 at the GCE. A significant peak current enhancement is
Fig. 7. Standard addition of Azm. CPE, accumulation potential q0.6 V vs. AgrAgCl, accumulation time 60 s, pH 2.6, Azm concentration: Ža. 1=10y6 , Žb. 2=10y6 , Žc. 4=10y6 , Žd. 5=10y6 , Že. 1=10y5 , Žf. 2=10y5 M.
obtained by longer accumulation periods. For example, accumulation for 5 min allows preconcentration of the drug and therefore 26, 21-fold enhancements at pH 1.75 and 5.0 were measured, respectively. This allows the drug determination to reach a level as low as 5 = 10y8 M. On the CPE, the accumulation step must always be performed on a new paste surface as depression in the current is obtained. Standard addition procedure was done with and without preconcentration. Fig. 7 shows the voltammograms obtained applying 60 s accumulation. A linear plot is obtained up to 6 = 10y5 M with the characteristics cited in Table 1. The reproducibility of the process was evaluated by performing 10 measurements on the 3 = 10y6 M Azm after 60 s accumulation on fresh prepared CPE. A mean value of 0.255 m A with a relative standard deviation of 1.8%. A detection limit of 8 = 10y8 M Azm in 10 ml has been established meaning that 8.71 ng Azm are detectable in the 10 ml solution.
Fig. 6. Comparison between DPSV Ža., OSWSV Žb., and LSSV Žc. of 5=10y5 M Azm, parameters for each mode: DPSV: scan rate 20 mV sy1 , pulse amplitude 50 mV, sample width 17 m s, pulse width 50 m s, pulse period 200 m s, Quiet time 10 s, OSWSV: S.W. amplitude 25 mV, S.W. frequency 15 Hz, step E 4 mV, Quiet time 10 s, LSSV: scan rate 100 mV sy1 , Quiet time 10 s, ŽA. at the CPE, pH 2.26 and Žb. at the GCE, pH 1.75.
Table 1 Characteristic features of the linearity limit for Azm at the CPE) and Gel MCPEq Drug ŽM.
Equation Ž m A m My1 .
Correlation Limit of coefficient linearity ŽM.
1=10y6 –6=10y6 ) Y s 0.260 X y0.25 0.9996 4=10y6 –4=10y5 q Y s 0.176 X q0.75 0.9926
6=10y5 4=10y5
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3.2.2. Analytical aspects at chemically modified electrodes Three different modifiers were tested in this work trying to enhance both sensitivity and selectivity of the method: sodium dodecyl sulphate, gum and gelatin. The three tested modifiers are mixed, individually, in three percentages namely 3, 6, and 10% with the carbon paste, then the entire pH-range is tested for choosing the best one which may give rise to better sensitivity and selectivity. A 10% gelatin modified carbon paste electrode ŽGel MCPE. gave rise to better sensitivity. The DPS voltammograms of Azm at 10% Gel MCPE in 0.03 M acetate buffer, pH 2.0 shows a relatively small desorption of the antibiotic from the electrode surface comparing with that carried out at the CPE, indicating that the modifier plays a role in the accumulation of the drug. Also, a shift in the oxidation potential of the drug under investigation is observed at the Gel MCPE. For the drug quantitation in urine, there is a peak obtained in the base line—due to the existence of the potentially interfering substances in urine—at the same oxidation potential of Azm. The shift of the peak at the Gel MCPE allows its determination in such medium without any interference and also by applying the method of medium exchange—i.e., accumulating the drug from urine solution and then transferring the electrode to another cell containing a 0.03 M acetate buffer, pH 2.0. Voltammograms of the drug at 10% Gel MCPE are obtained, their characteristic features are cited in Table 1. Detection limit of the drug in the urine samples reaches 8 = 10y8 M Azm in 10 ml solution.
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