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The electrochemical behavior of erythromycin A on a gold electrode
Electrochimica Acta 54 (2008) 649–654
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
The electrochemical behavior of erythromycin A on a gold electrode M.L. Avramov Ivic´ a,∗,1 , S.D. Petrovic´ b,c , D.Zˇ . Mijin b , F. Vanmoos a , D.Zˇ . Orlovic´ d , D.Zˇ . Marjanovic´ d , V.V. Radovic´ c a
ICTM, Institute of Electrochemistry, University of Belgrade, Njegoˇseva 12, Belgrade, Serbia Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, Belgrade, Serbia c Hemofarm, Pharmaceutical and Chemical Industry, Vrˇsac, Serbia d Zorka Pharma, Hemofarm, Pharmaceutical and Chemical Industry, Sˇ abac, Serbia b
a r t i c l e
i n f o
Article history: Received 24 April 2008 Received in revised form 23 June 2008 Accepted 4 July 2008 Available online 15 July 2008 Keywords: Erythromycin A Gold electrode Cyclic voltammetry HPLC FTIR
a b s t r a c t The reactivity of erythromycin (pure) was investigated on a gold electrode in neutral electrolyte by cyclic voltammetry. The resulting structural changes were observed with HPLC and FTIR spectroscopy by analyzing the bulk electrolyte after the electrochemical reactions. The results were compared with those previously obtained for azithromycin and clarithromycin under the same experimental conditions. It was found that the electrochemical behavior of erythromycin A differs from that of azithromycin dihydrate. Comparison with the electrochemical activity of basic clarithromycin suggests that the electrochemical activity of erythromycin is similar but more pronounced than that of clarithromycin. HPLC analysis confirmed these observations and showed that during the electrochemical oxidation of erythromycin A, the amount of starting macrolide decreased while the amount of starting impurities increased. Also some new products were observed. FTIR spectroscopy confirmed that erythromycin A is more reactive than clarithromycin, although similar changes in their molecular structures were observed. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Erythromycin (Fig. 1) is a macrolide antibiotic often administered to people who are allergic to penicillins [1]. Erythromycin A is the major and the most active microbiological component in erythromycin mixtures produced by Streptomyces erythreus [2–6]. Erythromycin is very rapidly absorbed and diffuses into most tissues and phagocytes and is actively transported to the site of infection. Erythromycin is mostly metabolized by demethylation in the liver and a small portion in the urine [1]. In the last decade, semi-synthetic derivatives of erythromycin exhibited better oral bioavailability, and a more favorable pharmacokinetic behavior [7,8]. For threating upper respiratory tract infection, lower respiratory tract infection and otitis media, amoxicillin and erythromycin were the most commonly prescribed antibacterials in 1998–2001 period in UK. For sore throat, penicillin, amoxicillin and erythromycin were the most common prescriptions, whereas for sinusitis, amoxicillin, tetracycline and erythromycin were most commonly prescribed in 1998–2001 period in UK. This demonstrates that most often erythromycin is one of the recommended first-line antibiotics [9].
∗ Corresponding author. Tel.: +381 11 3370389; fax: +381 11 3370389. ´ E-mail address: [email protected] (M.L. Avramov Ivic). 1 ISE member. 0013-4686/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2008.07.010
Before the advent of chromatography, the macrolides were extracted from plasma into organic solvents or separated by solidphase extraction [10]. The first paper on the determination of a macrolide, namely erythromycin, by HPLC and electrochemical detection (EC) appeared in 1983. Various HPLC methods using electrochemical detectors have been reported for quantifying erythromycin in serum, plasma and urine but little has been published on its quantification in animal tissues [11,12]. Being cheaper and faster than chromatography, electrochemical methods using the oxidation behavior for the determination of erythromycin on various glassy carbon electrodes have been reported [13–15]. Our previously published results showed that a gold electrode could be successfully applied for the qualitative and quantitative electrochemical determination of azithromycin dihydrate and azithromycin from capsules (Hemomycin® ) and for the separation of azithromycin from one of the excipients, i.e., lactose monohydrate [16–19]. The good catalytic activity of gold electrodes was employed for the qualitative electrochemical determination of pure clarithromycin by the appearance of one cathodic and four anodic reactions, which enabled structural changes in this molecule during electrochemical reactions to be studied [17]. Commercial clarithromycin, Clathrocyn® , was qualitatively determined by the one reproducible anodic reaction. The activity of one of the excipients, Avicel, observed as a cathodic peak at a potential different from that obtained with pure clarithromycin was employed for the determination of its presence in Clathrocyn® tablets [18].
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Fig. 1. Structures of macrolide antibiotics.
FTIR analysis showed apparent changes in the structure of the pure clarithromycin molecule, i.e., changes in the bands arising from the ester bond of the lactone and in ether and acetal bonds [17]. The changes were observed in the molecule structure of clarithromycin in Clathrocyn® tablets, indicating the disappearance of the lactone structure and changes of the carbonyl group at position 9 [18]. HPLC analysis showed a significant decrease in the concentration of azithromycin, Hemomycin® clarithromycin and Clathrocyn® after the electrochemical reactions [16–19]. The fact that clarithromycin when investigated under the same experimental conditions as azithromycin exhibited quite different behavior was attributed to the differences in their molecular structure. Both macrolide antibiotics were synthesized from the parent molecule, erythromycin. If the 6-hydroxy group of erythromycin is methylated, clarithromycin is obtained. Azithromycin has a methylated nitrogen atom at position number nine on the macrolide lactone ring (Fig. 1). To confirm the influence of the differences in the molecular structure of macrolides on their electrochemical behavior on a gold electrode, it is necessary to determine the behavior of the parent molecule, erythromycin. The aims of present work were to investigate the electrochemical behavior of erythromycin under the same experimental conditions as were employed in the study of azithromycin and clarithromycin, to detect structural changes in the molecule by FTIR spectroscopy and HPLC and to compare its behavior with those exhibited by clarithromycin and azithromycin. Commercial erythromycin, Erythromycin® , was separately tested under the same experimental conditions. 2. Experimental Erythromycin ethyl succinate, kindly provided by Hemofarm, ˇ Pharmaceutical and Chemical Industry “Zorka Pharma” a.d. (Sabac, Serbia) was examined separately as a pure substance. Also, experi-
ments were performed with erythromycin in tablet form marketed by Hemofarm as Erythromycin® , which, in addition to pure erythromycin, contains the excipients: corn starch, microcrystalline cellulose, magnesium stearate, hypromellose, macrogol and titanium(IV) oxide [20]. The pure erythromycin ethyl succinate and the content of the tablets were added directly, in the concentrations given in detail in the figure captions, into the electrolyte, which was purged with nitrogen for 20 min before each measurement. The sodium bicarbonate used for the supporting electrolyte was of analytical grade (Merck). The pH value of the solution was adjusted by adding diluted sulfuric acid. Used sulfuric acid was of analytical grade (Merck). The solutions were prepared with 18 M water. Standard equipment and a three-electrode electrochemical cell, as previously described [16–19], were used for the cyclic voltammetry measurements. The polycrystalline gold (surface area 0.500 cm2 ), which served as the working electrode, was polished with diamond paste and cleaned with a mixture of 18 M water and sulfuric acid. A platinum wire was used as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode. All the potentials are given vs. the SCE. The electrode surface was controlled by cyclic voltammetry before each experiment. Prior to the control of the electrode surface and before the addition of antibiotics, the electrolyte was purged with nitrogen. All the experiments were performed at room temperature. The pH of the electrolyte was measured using a PHM 93 reference pH meter, Radiometer Copenhagen. The characteristics of the employed HPLC instrument are as follows: HPLC Instrument Agilent 1100 Series, pump G1312A, UV VIS detector DAD G1315B, injector ALC G1313A, column SupelcosilTM LC-8 (150 mm × 4.6 mm, stationary support: silica, particle shape: spherical, bonded phase: octylsilyl, particle size: 5 m), mobile phase: acetonitrile mixed with buffer (potassium dihydrogen phosphate, triethylamine, deionized water, pH 3, diluted with H3 PO4 ) 350:650 (v/v), flow rate 1.0 ml/min, detection wavelength 195 nm and injection volume 10 l.
M.L. Avramov Ivi´c et al. / Electrochimica Acta 54 (2008) 649–654
The IR spectra were obtained using a FTIR BOMEM MB 100 Hartmann Braun FTIR spectrometer. The samples were analyzed in the form of KBr pellets after removal of the liquid under high vacuum at low temperatures. 3. Results and discussion A polycrystalline gold electrode was selected as an optimal working electrode and in order to avoid the influence of organic molecules (by their direct oxidation/reduction or adsorption on the gold electrode) either as the solvent or a component of the buffer solution in the electrolyte and hence to obtain only the electrochemical reactivity of the examined macrolides, 0.05 M NaHCO3 (pH 8.55) was chosen as the optimal supporting electrolyte for azithromycin and clarithromycin [16–19]. Study of erythromycin A decomposition products in aqueous solution by solid-phase microextraction/liquid chromatography/tandem mass spectrometry shown that erythromycin A was relatively stable at pH values >4 and <10 but became increasingly labile at both low and very high pH values, especially at pH < 3 [21]. Study of the stability of erythromycin in neutral and alkaline solutions by liquid chromatography on poly(styrene-divinylbenzene) pointed out that degradation of erythromycin A at 20 ◦ C at pH 8–9 during 10 h does not occurred [22]. Investigation of the chemical stability of an erythromycin-tretionin lotion by the use of an optimization system shown that optimal stability was found in the pH range of 8.2–8.6 for erythromycin [23]. This data indicated that 0.05 M NaHCO3 can be safely used and for the examination of electrochemical behavior of erythromycin A and that observed reactions at gold electrode should be considered only as the processes of electrochemical degradation. Under the same experimental conditions, the cyclic voltammogram of erythromycin A exhibits three anodic and one cathodic reaction and one apparent anodic activity in the whole range of the formation of oxides (Fig. 2). From Fig. 2, it is clear that erythromycin A causes a suppression of the anodic and cathodic currents in the region from −0.35 V to 0.0 V and a suppression of hydrogen evolution on a gold electrode. After the addition of antibiotic, pH of the electrolyte was 8.63 and did not change during the further electrochemical examination. No concentration dependency of any of the
Fig. 2. Cyclic voltammogram of an Au electrode in 0.05 M NaHCO3 (- - ) and with the addition of 0.40 mg cm−3 erythromycin succinate (full line), sweep rate: 50 mV s−1 .
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cathodic and anodic reaction was observed in the examined range of concentrations (from 0.235 mg cm−3 to 0.588 mg cm−3 ). Beckmann rearrangement of erythromycin 9-oxime results in ring expansion to a 15-membered intermediate, the subsequent reduction and N-methylation of which produce azithromycin with a quite different structure (Fig. 1) [24]. These structural differences cause the electrochemical behavior of erythromycin A to differ greatly from that of azithromycin dihydrate, which is active only in the oxide formation region and this activity is concentration dependent in the range 0.235–0.588 mg cm−3 [16]. Comparing the voltammogram of basic clarithromycin [17] with that of basic erythromycin A presented in Fig. 2, it is clear that the three anodic peaks appear at the same potentials. The cathodic reaction is shifted a little to more negative potentials in a case of erythromycin. The activity of clarithromycin is apparent only in the beginning of the region of oxide formation but that of erythromycin covers the whole region. A suppression of the anodic and cathodic currents in the region from −0.35 V to 0.0 V is more apparent in a case of erythromycin, as well as a suppression of hydrogen evolution. From the decrease of the capacitive currents and the absence of concentration dependence it was supposed that at the electrode surface exists an adsorbed layer of erythromycin/active intermediate species that undergoes transformation. The fouling of the currents after cyclic voltammetry is not observed during 4 h of cycling. Most probable explanation is that the electrode is cleaned in the region of the oxide reduction after each cycle and that erythromycin/active intermediates are not strongly adsorbed. Taking into account that the 6-hydroxy group of erythromycin was methylated to obtain the clarithromycin (Fig. 1), it can be assumed that the comparable but more pronounced electrochemical activity of erythromycin is caused by the free hydroxyl group. The degradation ability of erythromycin A in different pH solutions [21–23] suggested that it would be interesting to test its electrochemical behavior in the region of its low stability, acid solution: pH 2.17 and in the region of its high stability, neutral solution, pH 7.14. The second one is close to the pH value of the tested solution (pH 8.55) and is very important for the possible analyses of biological samples with the same or similar pH values: human blood, urine and plasma [25–29]. Erythromycin A in electrolyte at pH 2.12 undergoes immediately to the spontaneous degradation and under the experimental conditions presented in Fig. 2 additionally causes the reactions with strong gas evolution with obvious bubbling and complete blocking of the electrode surface. At pH 7.14, erythromycin A exhibited the same electrochemical activity as was observed at pH 8.55. In order to compare the activity of the entire tested macrolides one can suggest to continue with the using of 0.05 M NaHCO3 in the further erythromycin A examination. The electrochemical behavior of commercial erythromycin, Erythromycin® , was studied in the concentration range 0.235– 0.588 mg cm−3 . The obtained data are useful for the producers of erythromycin A in a case that their commercial products in capsules and tablets contain the same or similar excipients. Due to the presence of excipients (listed in Section 2), the obtained electrochemical activity differed greatly from that of the basic erythromycin ethyl succinate (Fig. 2). It is clear from Fig. 3 that the anodic activity at the beginning of the region of the formation of oxides is only small. The appearance of an additional cathodic peak at −0.25 V and an expanded cathodic activity in the region from −0.55 V to −1.00 V was observed. The peak of the oxide reduction is expanded and shifted to more negative potentials compared with the clean gold electrode (dashed line). All the excipients in Erythromycin® were studied separately and the observed cathodic activity can be attributed to their presence but can not be used for the separation of the basic erythromycin from any of them.
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Fig. 3. Cyclic voltammogram of an Au electrode in 0.05 M NaHCO3 (- - ) and with the addition of 0.40 mg cm−3 Erythromycin® (full line) in a concentration of 0.40 mg cm−3 , sweep rate: 50 mV s−1 .
As in the study of azithromycin and clarithromycin, samples for HPLC analysis were taken at the beginning and after 5 h of the electrochemical reaction [16,17]. HPLC analysis of electrolyte with added erythromycin A was performed before the electrochemical experiment and after 5 h of potential cycling under the conditions presented in Fig. 2. The results of this analysis are presented in Fig. 4 and in Table 1. As can be seen, the starting molecule, which represents a commercial product, is very pure, containing only very small amount of impurities. Impurities in commercial erythromycin are known and several papers on this subject were published [30,31]. Also, the decomposition products of erythromycin A were studied [21]. Different compounds, such as derivatives of erythromycin A (erythromycin B–F) were found as impurities as well as N-demethylerythromycin A, erythromycin A N-oxide, N-demethylerythromycin E, erythromycin E N-oxide,
N-demethylerythromycin B, anhydroerythromycin C, anhydro-Ndemethylerythromycin A and pseudoerythromycin E enol ether [30,31]. Anhydroerythromycin A was shown to be the major reaction product of erythromycin decomposition in both acidic and basic aqueous solutions [21]. In addition, HPLC–MS/MS as well as HPLC–ESI–MS methods for determination of erythromycin were developed [26–28]. The second method [28] was developed for simultaneous determination of erythromycin propionate and its active metabolite, erythromycin A. No other studies to our knowledge concerning erythromycin metabolites were published except for a certain erythromycin derivative, namely de(N-methyl)-Nethyl-8,9-anhydroerythromycin A 6,9-hemiacetal, which has no antibiotic activity [29]. During the electrochemical oxidations of erythromycin A, the amount of starting macrolide decreased while the amount of starting impurities increased and some new products were observed. At zero time, impurities A and B were detected as well as erythromycin A (compound D). As reaction proceeded, new products were detected, namely compounds C, E and F. The amounts of all compounds are given in Table 1 as the % area of the chromatogram. At the end of the electrochemical oxidations of erythromycin A (5 h), approximately 70% of the starting compound was recovered. From the obtained results, it is obvious that erythromycin A underwent oxidative degradation. Probably, the first step in the oxidation process is the removal of the electron from one of the nitrogen atoms to form an aminium cation radical [32]:
(1) where R is a sugar moiety. Formed aminium radical cation is a very reactive species and rapidly reacts with the environment to form stable products. The formed radical cation can abstract hydrogen atom from the water resulting in salt formation (reaction 2) in an overall one-electron process. Hence, the formed cation inhibits further electrochemical oxidation [32].
(2) In addition to reaction 2, it is probable that the amine group underwent a demethylation reaction, resulting in the corresponding secondary amines. This reaction proceeds via an overall two-electron transfer step (ECE mechanism) (reactions 3–5) [32]. The rate determining steps in these mechanisms are the removal of the ␣-proton and the formation of an enamine as an intermediate (reactions 3 and 4) [33].
(3)
(4)
(5) Fig. 4. HPLC chromatogram before (grey line) and after (black line) electrochemical oxidation of erythromycin A.
From the results presented in Table 1, the products of the oxidation of erythromycin A are not only the protonated salt and
M.L. Avramov Ivi´c et al. / Electrochimica Acta 54 (2008) 649–654
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Table 1 HPLC data of the electrochemical oxidation of erythromycin A Time (h)
Compound A (%)
Compound B (%)
Compound C (%)
Compound D (%)
Compound E (%)
Compound F (%)
0 5
3.34 11.12
1.43 4.35
0 2.05
95.22 70.22
0 5.34
0 6.90
Fig. 5. FTIR spectra of pure clarithromycin (a), pure clarithromycin mixed with carbonate (NaHCO3 ) (b) and reaction mixture after 5 h of electroreduction at +0.18 V vs. SCE (c).
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demethylated erythromycin, but also the recorded peaks might correspond to anhydro and/or demethylated products, such as anhydro-N-demethylerythromycin A and anhydroerythromycin C. The possible formation of erythromycin C or D occurred by cleavage of the ether bond. To obtain more information about other possible products, FTIR analysis of bulk electrolyte was performed. As in our previous paper [17], the FTIR spectra of pure erythromycin and erythromycin mixed with carbonates before the electrochemical experiment served as references for further analysis. The observed changes in the molecule of erythromycin were tracked with these data. The potential was held at selected values corresponded to all the observed current peaks for 4 h. The first sweep after holding the potential was recorded by cyclic voltametry and sample was analyzed by FTIR. The potential was held at −0.70 V, −0.63 V, −0.57 V, +0.18 V and +0.32 V vs. SCE. Also, FTIR analysis was performed after cyclic voltametry after a 5 h reaction time. The first sweep after release, shown increasing currents at the all mentioned values of the potential, for example after the hold at −0.7 V, the increased currents between +0.1 V and 0.3 V were observed. After the hold at −0.63 V, in the area of the oxide formation the currents slightly increased. After two or three cycles the cyclic voltammogram shown the electrochemical activity presented in Fig. 2. This suggests that erythromycin A and active intermediates are adsorbed at the electrode surface in a way which enables the electrochemical reactions during the holding of the potential. Significant changes in the erythromycin A structure were observed at all potentials. In most experiments, the FTIR spectra (Fig. 5) revealed the following changes: the disappearance (at −0.70 V, +0.18 V and +0.32 V vs. SCE) or an intense reduction (at −0.63 V, −0.57 V vs. SCE and 5 h of cycling) of the 1736 cm−1 peak, corresponding to the carbonyl group vibration of the lactone moiety and the disappearance of the 1164 cm−1 peak, probably corresponding to the C O vibration of the lactone moiety (in all experiments). Carbonyl group absorption was not detected as separate peak. No absorption was recorded in the 1000–1100 cm−1 range (−0.70 V and +0.18 V vs. SCE), which could be the result of changes in the ether and acetal bonds. In other experiments, lower absorptions were detected at 1059 cm−1 or 1049 cm−1 , except in the 5 h cycling experiment, when a peak was detected at 1089 cm−1 . The FTIR results indicate, as did cyclic voltammetry, that erythromycin A is more reactive than clarithromycin, although similar changes in the molecular structure were observed. The FTIR spectra of Erythromycin® showed the same pattern as pure erythromycin A. 4. Conclusions It was shown that the electrochemical behavior of erythromycin A differs greatly from that of azithromycin dehydrate, which was caused by structural differences. Comparison of the electrochemical activity of basic erythromycin with that of clarithromycin showed that the electrochemical activity of erythromycin is more pronounced than that of clarithromycin. Taking into account that the 6-hydroxy group of erythromycin was methylated to obtain the clarithromycin, it can be assumed that the comparable but more pronounced electrochemical activity of erythromycin is caused by the free hydroxyl group. HPLC analysis confirmed this observation and showed that during the electrochemical oxidations of erythromycin A, the amount of starting macrolide decreased while the amount of star-
ing impurities increased and some new products were observed. The products of the oxidation of erythromycin A are not only the protonated salt and demethylated erythromycin. According to the obtained results, the recorded peaks might also correspond to anhydro and/or demethylated products, such as anhydro-Ndemethylerythromycin A and anhydroerythromycin C. The possible formation of erythromycin C or D occurred by cleavage of the ether bond. FTIR results indicate, as did cyclic voltammetry and HPLC, that erythromycin A is more reactive than clarithromycin, although similar changes in molecular structure were observed. Acknowledgment We are grateful to the Ministry of Science of Serbia for financial support (Project 142063). References [1] W.A. Ray, K.T. Murray, S. Meredith, S.S. Narasimhulu, K. Hall, C.M. Stein, N. Engl. J. Med. 351 (2004) 1089. [2] Z. Wang, J. Wang, M. Zhang, L. Dang, J. Chem. Eng. Data 51 (2006) 1062. [3] J. Linares, J. Garau, C. Dominguez, J.L. Perez, Antimicrob. Agents Chemother. 23 (1983) 545. [4] H. Seppälä, A. Nissinen, H. Järvinen, S. Huovinen, T. Henriksson, E. Herva, S.E. Holm, M. Jahkola, M.L. Katila, T. Klaukka, S. Kontiainen, O. Liimatainen, S. Oinonen, L. Passi-Metsomaa, P. Huovinen, N. Engl. J. Med. 326 (1992) 292. [5] R. Vanhoof, B. Gordts, R. Dierickx, H. Coignau, J.P. Butzler, Antimicrob. Agents Chemother. 18 (1980) 118. [6] L.D. Bechtol, V.C. Stephens, C.T. Pugh, M.B. Perkal, P.A. Coletta, Curr. Ther. Res. Clin. Exp. 20 (1976) 610. [7] B.J. Anders, B.A. Lauer, J.W. Paisley, L.B. Reller, Lancet 1 (1982) 131. [8] A.S. Dajani, K.A. Taubert, W. Wilson, A.F. Bolger, A. Bayer, P. Ferrieri, M.H. Gewitz, S.T. Shulman, JAMA 277 (1997) 1794. [9] I. Petersen, A.C. Hayward, J. Antimicrob. Chemother. 60 (2007) 43. [10] F. Kees, S. Spangler, M. Wellenhofer, J. Chromatogr. A 812 (1998) 287. [11] H. Toreson, B.-M. Eriksson, J. Chromatogr. B 673 (1995) 81. [12] E. Dreeassi, P. Corti, F. Bezzini, S. Furianetto, Analyst 125 (2000) 1077. [13] H. Wang, A. Zhang, H. Cui, D. Liu, R. Liu, Microchem. J. 64 (2000) 67. [14] L.M. Yudi, A.M. Baruzzi, V. Solis, J. Electroanal. Chem. 369 (1993) 211. [15] N.H.S. Ammida, G. Volpe, R. Draisci, F. Delli Quadri, L. Palleschi, G. Palleschi, Analyst 129 (2004) 15. ´ S.D. Petrovic, ´ D.Zˇ . Mijin, P.M. Zˇ ivkovic, ´ I.M. Kosovic, ´ K.M. [16] M.L. Avramov Ivic, ´ M.B. Jovanovic, ´ Electrochim. Acta 51 (2006) 2407. Drljevic, ´ S.D. Petrovic, ´ V. Vonmoos, D.Zˇ . Mijin, P.M. Zˇ ivkovic, ´ K.M. [17] M.L. Avramov Ivic, ´ Electrochem. Commun. 9 (2007) 1643. Drljevic, ´ S.D. Petrovic, ´ V. Vonmoos, D.Zˇ . Mijin, P.M. Zˇ ivkovic, ´ K.M. [18] M.L. Avramov Ivic, ´ Russ. J. Electrochem. 44 (2008) 931. Drljevic, ´ S.D. Petrovic, ´ D.Zˇ . Mijin, J. Serb. Chem. Soc. 72 (2007) [19] M.L. Avramov Ivic, 1427. [20] Fiedler Encyclopedia of Excipients for Pharmaceuticals, Cosmetics and Related Areas, vol. 2, 5th ed., Cantor Vorlay, Aulendorf, 2002. [21] D.A. Volmer, J.P.M. Hui, Rapid Commun. Mass Spectrom. 12 (1998) 123. [22] J. Paesen, K. Khan, E. Roets, J. Hoogmartens, Int. J. Pharm. 113 (1995) 215. [23] M. Brisaert, M. Gabriels, J. Plaizier-Vercammen, Int. J. Pharm. 197 (2000) 153. [24] Burger’s Medicinal Chemistry and Drug Discovery, vol. 2, 5th ed., John Wiley & Sons, New York, 1996. [25] A.C. Guyton, J.E. Hall, Textbook of Medical Physiology, 11th ed., W.B. Saunders Co, Philadelphia, 2005. [26] P. Velagaleti, S. McComish, F. McCush, M. Raub, Proceedings of the 2002 AAPS Annual Meeting and Exposition, Toronto, Canada, November 10–14, 2002; AAPS PharmSci., 4 (S1) (2002) Abstract 2850. [27] A. Mize, S. McComish, N. Premkumar, Proceedings of the 2003 AAPS Annual Meeting and Exposition, Salt Lake City, USA, October 26–30, 2003; AAPS PharmSci. 5 (S1) (2002) Abstract 88. [28] W. Xiaoa, B. Chena, S. Yaoa, Z. Cheng, J. Chromatogr. B 817 (2005) 153. [29] H. Monji, M. Yamaguchi, I. Aoki, H. Ueno, J. Chromatogr. B 690 (1997) 305. [30] C. Govaerts, H.K. Chepkwony, A. Van Schepdael, E. Roets, J. Hoogmartens, Rapid Commun. Mass Spectrom. 14 (2000) 878. [31] S.K. Chitneni, C. Govaerts, E. Adams, A. Van Schepdael, J. Hoogmartens, J. Chromatogr. A 1056 (2004) 111. ´ Z. Weitner, M. Ilijas, J. Pharmaceut. Biomed. Anal. 33 (2003) 647. [32] Z. Mandic, [33] P.J. Smith, C.K. Mann, J. Org. Chem. 34 (1969) 1821.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
The electrochemical behavior of erythromycin A on a gold electrode M.L. Avramov Ivic´ a,∗,1 , S.D. Petrovic´ b,c , D.Zˇ . Mijin b , F. Vanmoos a , D.Zˇ . Orlovic´ d , D.Zˇ . Marjanovic´ d , V.V. Radovic´ c a
ICTM, Institute of Electrochemistry, University of Belgrade, Njegoˇseva 12, Belgrade, Serbia Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, Belgrade, Serbia c Hemofarm, Pharmaceutical and Chemical Industry, Vrˇsac, Serbia d Zorka Pharma, Hemofarm, Pharmaceutical and Chemical Industry, Sˇ abac, Serbia b
a r t i c l e
i n f o
Article history: Received 24 April 2008 Received in revised form 23 June 2008 Accepted 4 July 2008 Available online 15 July 2008 Keywords: Erythromycin A Gold electrode Cyclic voltammetry HPLC FTIR
a b s t r a c t The reactivity of erythromycin (pure) was investigated on a gold electrode in neutral electrolyte by cyclic voltammetry. The resulting structural changes were observed with HPLC and FTIR spectroscopy by analyzing the bulk electrolyte after the electrochemical reactions. The results were compared with those previously obtained for azithromycin and clarithromycin under the same experimental conditions. It was found that the electrochemical behavior of erythromycin A differs from that of azithromycin dihydrate. Comparison with the electrochemical activity of basic clarithromycin suggests that the electrochemical activity of erythromycin is similar but more pronounced than that of clarithromycin. HPLC analysis confirmed these observations and showed that during the electrochemical oxidation of erythromycin A, the amount of starting macrolide decreased while the amount of starting impurities increased. Also some new products were observed. FTIR spectroscopy confirmed that erythromycin A is more reactive than clarithromycin, although similar changes in their molecular structures were observed. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Erythromycin (Fig. 1) is a macrolide antibiotic often administered to people who are allergic to penicillins [1]. Erythromycin A is the major and the most active microbiological component in erythromycin mixtures produced by Streptomyces erythreus [2–6]. Erythromycin is very rapidly absorbed and diffuses into most tissues and phagocytes and is actively transported to the site of infection. Erythromycin is mostly metabolized by demethylation in the liver and a small portion in the urine [1]. In the last decade, semi-synthetic derivatives of erythromycin exhibited better oral bioavailability, and a more favorable pharmacokinetic behavior [7,8]. For threating upper respiratory tract infection, lower respiratory tract infection and otitis media, amoxicillin and erythromycin were the most commonly prescribed antibacterials in 1998–2001 period in UK. For sore throat, penicillin, amoxicillin and erythromycin were the most common prescriptions, whereas for sinusitis, amoxicillin, tetracycline and erythromycin were most commonly prescribed in 1998–2001 period in UK. This demonstrates that most often erythromycin is one of the recommended first-line antibiotics [9].
∗ Corresponding author. Tel.: +381 11 3370389; fax: +381 11 3370389. ´ E-mail address: [email protected] (M.L. Avramov Ivic). 1 ISE member. 0013-4686/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2008.07.010
Before the advent of chromatography, the macrolides were extracted from plasma into organic solvents or separated by solidphase extraction [10]. The first paper on the determination of a macrolide, namely erythromycin, by HPLC and electrochemical detection (EC) appeared in 1983. Various HPLC methods using electrochemical detectors have been reported for quantifying erythromycin in serum, plasma and urine but little has been published on its quantification in animal tissues [11,12]. Being cheaper and faster than chromatography, electrochemical methods using the oxidation behavior for the determination of erythromycin on various glassy carbon electrodes have been reported [13–15]. Our previously published results showed that a gold electrode could be successfully applied for the qualitative and quantitative electrochemical determination of azithromycin dihydrate and azithromycin from capsules (Hemomycin® ) and for the separation of azithromycin from one of the excipients, i.e., lactose monohydrate [16–19]. The good catalytic activity of gold electrodes was employed for the qualitative electrochemical determination of pure clarithromycin by the appearance of one cathodic and four anodic reactions, which enabled structural changes in this molecule during electrochemical reactions to be studied [17]. Commercial clarithromycin, Clathrocyn® , was qualitatively determined by the one reproducible anodic reaction. The activity of one of the excipients, Avicel, observed as a cathodic peak at a potential different from that obtained with pure clarithromycin was employed for the determination of its presence in Clathrocyn® tablets [18].
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Fig. 1. Structures of macrolide antibiotics.
FTIR analysis showed apparent changes in the structure of the pure clarithromycin molecule, i.e., changes in the bands arising from the ester bond of the lactone and in ether and acetal bonds [17]. The changes were observed in the molecule structure of clarithromycin in Clathrocyn® tablets, indicating the disappearance of the lactone structure and changes of the carbonyl group at position 9 [18]. HPLC analysis showed a significant decrease in the concentration of azithromycin, Hemomycin® clarithromycin and Clathrocyn® after the electrochemical reactions [16–19]. The fact that clarithromycin when investigated under the same experimental conditions as azithromycin exhibited quite different behavior was attributed to the differences in their molecular structure. Both macrolide antibiotics were synthesized from the parent molecule, erythromycin. If the 6-hydroxy group of erythromycin is methylated, clarithromycin is obtained. Azithromycin has a methylated nitrogen atom at position number nine on the macrolide lactone ring (Fig. 1). To confirm the influence of the differences in the molecular structure of macrolides on their electrochemical behavior on a gold electrode, it is necessary to determine the behavior of the parent molecule, erythromycin. The aims of present work were to investigate the electrochemical behavior of erythromycin under the same experimental conditions as were employed in the study of azithromycin and clarithromycin, to detect structural changes in the molecule by FTIR spectroscopy and HPLC and to compare its behavior with those exhibited by clarithromycin and azithromycin. Commercial erythromycin, Erythromycin® , was separately tested under the same experimental conditions. 2. Experimental Erythromycin ethyl succinate, kindly provided by Hemofarm, ˇ Pharmaceutical and Chemical Industry “Zorka Pharma” a.d. (Sabac, Serbia) was examined separately as a pure substance. Also, experi-
ments were performed with erythromycin in tablet form marketed by Hemofarm as Erythromycin® , which, in addition to pure erythromycin, contains the excipients: corn starch, microcrystalline cellulose, magnesium stearate, hypromellose, macrogol and titanium(IV) oxide [20]. The pure erythromycin ethyl succinate and the content of the tablets were added directly, in the concentrations given in detail in the figure captions, into the electrolyte, which was purged with nitrogen for 20 min before each measurement. The sodium bicarbonate used for the supporting electrolyte was of analytical grade (Merck). The pH value of the solution was adjusted by adding diluted sulfuric acid. Used sulfuric acid was of analytical grade (Merck). The solutions were prepared with 18 M water. Standard equipment and a three-electrode electrochemical cell, as previously described [16–19], were used for the cyclic voltammetry measurements. The polycrystalline gold (surface area 0.500 cm2 ), which served as the working electrode, was polished with diamond paste and cleaned with a mixture of 18 M water and sulfuric acid. A platinum wire was used as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode. All the potentials are given vs. the SCE. The electrode surface was controlled by cyclic voltammetry before each experiment. Prior to the control of the electrode surface and before the addition of antibiotics, the electrolyte was purged with nitrogen. All the experiments were performed at room temperature. The pH of the electrolyte was measured using a PHM 93 reference pH meter, Radiometer Copenhagen. The characteristics of the employed HPLC instrument are as follows: HPLC Instrument Agilent 1100 Series, pump G1312A, UV VIS detector DAD G1315B, injector ALC G1313A, column SupelcosilTM LC-8 (150 mm × 4.6 mm, stationary support: silica, particle shape: spherical, bonded phase: octylsilyl, particle size: 5 m), mobile phase: acetonitrile mixed with buffer (potassium dihydrogen phosphate, triethylamine, deionized water, pH 3, diluted with H3 PO4 ) 350:650 (v/v), flow rate 1.0 ml/min, detection wavelength 195 nm and injection volume 10 l.
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The IR spectra were obtained using a FTIR BOMEM MB 100 Hartmann Braun FTIR spectrometer. The samples were analyzed in the form of KBr pellets after removal of the liquid under high vacuum at low temperatures. 3. Results and discussion A polycrystalline gold electrode was selected as an optimal working electrode and in order to avoid the influence of organic molecules (by their direct oxidation/reduction or adsorption on the gold electrode) either as the solvent or a component of the buffer solution in the electrolyte and hence to obtain only the electrochemical reactivity of the examined macrolides, 0.05 M NaHCO3 (pH 8.55) was chosen as the optimal supporting electrolyte for azithromycin and clarithromycin [16–19]. Study of erythromycin A decomposition products in aqueous solution by solid-phase microextraction/liquid chromatography/tandem mass spectrometry shown that erythromycin A was relatively stable at pH values >4 and <10 but became increasingly labile at both low and very high pH values, especially at pH < 3 [21]. Study of the stability of erythromycin in neutral and alkaline solutions by liquid chromatography on poly(styrene-divinylbenzene) pointed out that degradation of erythromycin A at 20 ◦ C at pH 8–9 during 10 h does not occurred [22]. Investigation of the chemical stability of an erythromycin-tretionin lotion by the use of an optimization system shown that optimal stability was found in the pH range of 8.2–8.6 for erythromycin [23]. This data indicated that 0.05 M NaHCO3 can be safely used and for the examination of electrochemical behavior of erythromycin A and that observed reactions at gold electrode should be considered only as the processes of electrochemical degradation. Under the same experimental conditions, the cyclic voltammogram of erythromycin A exhibits three anodic and one cathodic reaction and one apparent anodic activity in the whole range of the formation of oxides (Fig. 2). From Fig. 2, it is clear that erythromycin A causes a suppression of the anodic and cathodic currents in the region from −0.35 V to 0.0 V and a suppression of hydrogen evolution on a gold electrode. After the addition of antibiotic, pH of the electrolyte was 8.63 and did not change during the further electrochemical examination. No concentration dependency of any of the
Fig. 2. Cyclic voltammogram of an Au electrode in 0.05 M NaHCO3 (- - ) and with the addition of 0.40 mg cm−3 erythromycin succinate (full line), sweep rate: 50 mV s−1 .
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cathodic and anodic reaction was observed in the examined range of concentrations (from 0.235 mg cm−3 to 0.588 mg cm−3 ). Beckmann rearrangement of erythromycin 9-oxime results in ring expansion to a 15-membered intermediate, the subsequent reduction and N-methylation of which produce azithromycin with a quite different structure (Fig. 1) [24]. These structural differences cause the electrochemical behavior of erythromycin A to differ greatly from that of azithromycin dihydrate, which is active only in the oxide formation region and this activity is concentration dependent in the range 0.235–0.588 mg cm−3 [16]. Comparing the voltammogram of basic clarithromycin [17] with that of basic erythromycin A presented in Fig. 2, it is clear that the three anodic peaks appear at the same potentials. The cathodic reaction is shifted a little to more negative potentials in a case of erythromycin. The activity of clarithromycin is apparent only in the beginning of the region of oxide formation but that of erythromycin covers the whole region. A suppression of the anodic and cathodic currents in the region from −0.35 V to 0.0 V is more apparent in a case of erythromycin, as well as a suppression of hydrogen evolution. From the decrease of the capacitive currents and the absence of concentration dependence it was supposed that at the electrode surface exists an adsorbed layer of erythromycin/active intermediate species that undergoes transformation. The fouling of the currents after cyclic voltammetry is not observed during 4 h of cycling. Most probable explanation is that the electrode is cleaned in the region of the oxide reduction after each cycle and that erythromycin/active intermediates are not strongly adsorbed. Taking into account that the 6-hydroxy group of erythromycin was methylated to obtain the clarithromycin (Fig. 1), it can be assumed that the comparable but more pronounced electrochemical activity of erythromycin is caused by the free hydroxyl group. The degradation ability of erythromycin A in different pH solutions [21–23] suggested that it would be interesting to test its electrochemical behavior in the region of its low stability, acid solution: pH 2.17 and in the region of its high stability, neutral solution, pH 7.14. The second one is close to the pH value of the tested solution (pH 8.55) and is very important for the possible analyses of biological samples with the same or similar pH values: human blood, urine and plasma [25–29]. Erythromycin A in electrolyte at pH 2.12 undergoes immediately to the spontaneous degradation and under the experimental conditions presented in Fig. 2 additionally causes the reactions with strong gas evolution with obvious bubbling and complete blocking of the electrode surface. At pH 7.14, erythromycin A exhibited the same electrochemical activity as was observed at pH 8.55. In order to compare the activity of the entire tested macrolides one can suggest to continue with the using of 0.05 M NaHCO3 in the further erythromycin A examination. The electrochemical behavior of commercial erythromycin, Erythromycin® , was studied in the concentration range 0.235– 0.588 mg cm−3 . The obtained data are useful for the producers of erythromycin A in a case that their commercial products in capsules and tablets contain the same or similar excipients. Due to the presence of excipients (listed in Section 2), the obtained electrochemical activity differed greatly from that of the basic erythromycin ethyl succinate (Fig. 2). It is clear from Fig. 3 that the anodic activity at the beginning of the region of the formation of oxides is only small. The appearance of an additional cathodic peak at −0.25 V and an expanded cathodic activity in the region from −0.55 V to −1.00 V was observed. The peak of the oxide reduction is expanded and shifted to more negative potentials compared with the clean gold electrode (dashed line). All the excipients in Erythromycin® were studied separately and the observed cathodic activity can be attributed to their presence but can not be used for the separation of the basic erythromycin from any of them.
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Fig. 3. Cyclic voltammogram of an Au electrode in 0.05 M NaHCO3 (- - ) and with the addition of 0.40 mg cm−3 Erythromycin® (full line) in a concentration of 0.40 mg cm−3 , sweep rate: 50 mV s−1 .
As in the study of azithromycin and clarithromycin, samples for HPLC analysis were taken at the beginning and after 5 h of the electrochemical reaction [16,17]. HPLC analysis of electrolyte with added erythromycin A was performed before the electrochemical experiment and after 5 h of potential cycling under the conditions presented in Fig. 2. The results of this analysis are presented in Fig. 4 and in Table 1. As can be seen, the starting molecule, which represents a commercial product, is very pure, containing only very small amount of impurities. Impurities in commercial erythromycin are known and several papers on this subject were published [30,31]. Also, the decomposition products of erythromycin A were studied [21]. Different compounds, such as derivatives of erythromycin A (erythromycin B–F) were found as impurities as well as N-demethylerythromycin A, erythromycin A N-oxide, N-demethylerythromycin E, erythromycin E N-oxide,
N-demethylerythromycin B, anhydroerythromycin C, anhydro-Ndemethylerythromycin A and pseudoerythromycin E enol ether [30,31]. Anhydroerythromycin A was shown to be the major reaction product of erythromycin decomposition in both acidic and basic aqueous solutions [21]. In addition, HPLC–MS/MS as well as HPLC–ESI–MS methods for determination of erythromycin were developed [26–28]. The second method [28] was developed for simultaneous determination of erythromycin propionate and its active metabolite, erythromycin A. No other studies to our knowledge concerning erythromycin metabolites were published except for a certain erythromycin derivative, namely de(N-methyl)-Nethyl-8,9-anhydroerythromycin A 6,9-hemiacetal, which has no antibiotic activity [29]. During the electrochemical oxidations of erythromycin A, the amount of starting macrolide decreased while the amount of starting impurities increased and some new products were observed. At zero time, impurities A and B were detected as well as erythromycin A (compound D). As reaction proceeded, new products were detected, namely compounds C, E and F. The amounts of all compounds are given in Table 1 as the % area of the chromatogram. At the end of the electrochemical oxidations of erythromycin A (5 h), approximately 70% of the starting compound was recovered. From the obtained results, it is obvious that erythromycin A underwent oxidative degradation. Probably, the first step in the oxidation process is the removal of the electron from one of the nitrogen atoms to form an aminium cation radical [32]:
(1) where R is a sugar moiety. Formed aminium radical cation is a very reactive species and rapidly reacts with the environment to form stable products. The formed radical cation can abstract hydrogen atom from the water resulting in salt formation (reaction 2) in an overall one-electron process. Hence, the formed cation inhibits further electrochemical oxidation [32].
(2) In addition to reaction 2, it is probable that the amine group underwent a demethylation reaction, resulting in the corresponding secondary amines. This reaction proceeds via an overall two-electron transfer step (ECE mechanism) (reactions 3–5) [32]. The rate determining steps in these mechanisms are the removal of the ␣-proton and the formation of an enamine as an intermediate (reactions 3 and 4) [33].
(3)
(4)
(5) Fig. 4. HPLC chromatogram before (grey line) and after (black line) electrochemical oxidation of erythromycin A.
From the results presented in Table 1, the products of the oxidation of erythromycin A are not only the protonated salt and
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Table 1 HPLC data of the electrochemical oxidation of erythromycin A Time (h)
Compound A (%)
Compound B (%)
Compound C (%)
Compound D (%)
Compound E (%)
Compound F (%)
0 5
3.34 11.12
1.43 4.35
0 2.05
95.22 70.22
0 5.34
0 6.90
Fig. 5. FTIR spectra of pure clarithromycin (a), pure clarithromycin mixed with carbonate (NaHCO3 ) (b) and reaction mixture after 5 h of electroreduction at +0.18 V vs. SCE (c).
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demethylated erythromycin, but also the recorded peaks might correspond to anhydro and/or demethylated products, such as anhydro-N-demethylerythromycin A and anhydroerythromycin C. The possible formation of erythromycin C or D occurred by cleavage of the ether bond. To obtain more information about other possible products, FTIR analysis of bulk electrolyte was performed. As in our previous paper [17], the FTIR spectra of pure erythromycin and erythromycin mixed with carbonates before the electrochemical experiment served as references for further analysis. The observed changes in the molecule of erythromycin were tracked with these data. The potential was held at selected values corresponded to all the observed current peaks for 4 h. The first sweep after holding the potential was recorded by cyclic voltametry and sample was analyzed by FTIR. The potential was held at −0.70 V, −0.63 V, −0.57 V, +0.18 V and +0.32 V vs. SCE. Also, FTIR analysis was performed after cyclic voltametry after a 5 h reaction time. The first sweep after release, shown increasing currents at the all mentioned values of the potential, for example after the hold at −0.7 V, the increased currents between +0.1 V and 0.3 V were observed. After the hold at −0.63 V, in the area of the oxide formation the currents slightly increased. After two or three cycles the cyclic voltammogram shown the electrochemical activity presented in Fig. 2. This suggests that erythromycin A and active intermediates are adsorbed at the electrode surface in a way which enables the electrochemical reactions during the holding of the potential. Significant changes in the erythromycin A structure were observed at all potentials. In most experiments, the FTIR spectra (Fig. 5) revealed the following changes: the disappearance (at −0.70 V, +0.18 V and +0.32 V vs. SCE) or an intense reduction (at −0.63 V, −0.57 V vs. SCE and 5 h of cycling) of the 1736 cm−1 peak, corresponding to the carbonyl group vibration of the lactone moiety and the disappearance of the 1164 cm−1 peak, probably corresponding to the C O vibration of the lactone moiety (in all experiments). Carbonyl group absorption was not detected as separate peak. No absorption was recorded in the 1000–1100 cm−1 range (−0.70 V and +0.18 V vs. SCE), which could be the result of changes in the ether and acetal bonds. In other experiments, lower absorptions were detected at 1059 cm−1 or 1049 cm−1 , except in the 5 h cycling experiment, when a peak was detected at 1089 cm−1 . The FTIR results indicate, as did cyclic voltammetry, that erythromycin A is more reactive than clarithromycin, although similar changes in the molecular structure were observed. The FTIR spectra of Erythromycin® showed the same pattern as pure erythromycin A. 4. Conclusions It was shown that the electrochemical behavior of erythromycin A differs greatly from that of azithromycin dehydrate, which was caused by structural differences. Comparison of the electrochemical activity of basic erythromycin with that of clarithromycin showed that the electrochemical activity of erythromycin is more pronounced than that of clarithromycin. Taking into account that the 6-hydroxy group of erythromycin was methylated to obtain the clarithromycin, it can be assumed that the comparable but more pronounced electrochemical activity of erythromycin is caused by the free hydroxyl group. HPLC analysis confirmed this observation and showed that during the electrochemical oxidations of erythromycin A, the amount of starting macrolide decreased while the amount of star-
ing impurities increased and some new products were observed. The products of the oxidation of erythromycin A are not only the protonated salt and demethylated erythromycin. According to the obtained results, the recorded peaks might also correspond to anhydro and/or demethylated products, such as anhydro-Ndemethylerythromycin A and anhydroerythromycin C. The possible formation of erythromycin C or D occurred by cleavage of the ether bond. FTIR results indicate, as did cyclic voltammetry and HPLC, that erythromycin A is more reactive than clarithromycin, although similar changes in molecular structure were observed. Acknowledgment We are grateful to the Ministry of Science of Serbia for financial support (Project 142063). References [1] W.A. Ray, K.T. Murray, S. Meredith, S.S. Narasimhulu, K. Hall, C.M. Stein, N. Engl. J. Med. 351 (2004) 1089. [2] Z. Wang, J. Wang, M. Zhang, L. Dang, J. Chem. Eng. Data 51 (2006) 1062. [3] J. Linares, J. Garau, C. Dominguez, J.L. Perez, Antimicrob. Agents Chemother. 23 (1983) 545. [4] H. Seppälä, A. Nissinen, H. Järvinen, S. Huovinen, T. Henriksson, E. Herva, S.E. Holm, M. Jahkola, M.L. Katila, T. Klaukka, S. Kontiainen, O. Liimatainen, S. Oinonen, L. Passi-Metsomaa, P. Huovinen, N. Engl. J. Med. 326 (1992) 292. [5] R. Vanhoof, B. Gordts, R. Dierickx, H. Coignau, J.P. Butzler, Antimicrob. Agents Chemother. 18 (1980) 118. [6] L.D. Bechtol, V.C. Stephens, C.T. Pugh, M.B. Perkal, P.A. Coletta, Curr. Ther. Res. Clin. Exp. 20 (1976) 610. [7] B.J. Anders, B.A. Lauer, J.W. Paisley, L.B. Reller, Lancet 1 (1982) 131. [8] A.S. Dajani, K.A. Taubert, W. Wilson, A.F. Bolger, A. Bayer, P. Ferrieri, M.H. Gewitz, S.T. Shulman, JAMA 277 (1997) 1794. [9] I. Petersen, A.C. Hayward, J. Antimicrob. Chemother. 60 (2007) 43. [10] F. Kees, S. Spangler, M. Wellenhofer, J. Chromatogr. A 812 (1998) 287. [11] H. Toreson, B.-M. Eriksson, J. Chromatogr. B 673 (1995) 81. [12] E. Dreeassi, P. Corti, F. Bezzini, S. Furianetto, Analyst 125 (2000) 1077. [13] H. Wang, A. Zhang, H. Cui, D. Liu, R. Liu, Microchem. J. 64 (2000) 67. [14] L.M. Yudi, A.M. Baruzzi, V. Solis, J. Electroanal. Chem. 369 (1993) 211. [15] N.H.S. Ammida, G. Volpe, R. Draisci, F. Delli Quadri, L. Palleschi, G. Palleschi, Analyst 129 (2004) 15. ´ S.D. Petrovic, ´ D.Zˇ . Mijin, P.M. Zˇ ivkovic, ´ I.M. Kosovic, ´ K.M. [16] M.L. Avramov Ivic, ´ M.B. Jovanovic, ´ Electrochim. Acta 51 (2006) 2407. Drljevic, ´ S.D. Petrovic, ´ V. Vonmoos, D.Zˇ . Mijin, P.M. Zˇ ivkovic, ´ K.M. [17] M.L. Avramov Ivic, ´ Electrochem. Commun. 9 (2007) 1643. Drljevic, ´ S.D. Petrovic, ´ V. Vonmoos, D.Zˇ . Mijin, P.M. Zˇ ivkovic, ´ K.M. [18] M.L. Avramov Ivic, ´ Russ. J. Electrochem. 44 (2008) 931. Drljevic, ´ S.D. Petrovic, ´ D.Zˇ . Mijin, J. Serb. Chem. Soc. 72 (2007) [19] M.L. Avramov Ivic, 1427. [20] Fiedler Encyclopedia of Excipients for Pharmaceuticals, Cosmetics and Related Areas, vol. 2, 5th ed., Cantor Vorlay, Aulendorf, 2002. [21] D.A. Volmer, J.P.M. Hui, Rapid Commun. Mass Spectrom. 12 (1998) 123. [22] J. Paesen, K. Khan, E. Roets, J. Hoogmartens, Int. J. Pharm. 113 (1995) 215. [23] M. Brisaert, M. Gabriels, J. Plaizier-Vercammen, Int. J. Pharm. 197 (2000) 153. [24] Burger’s Medicinal Chemistry and Drug Discovery, vol. 2, 5th ed., John Wiley & Sons, New York, 1996. [25] A.C. Guyton, J.E. Hall, Textbook of Medical Physiology, 11th ed., W.B. Saunders Co, Philadelphia, 2005. [26] P. Velagaleti, S. McComish, F. McCush, M. Raub, Proceedings of the 2002 AAPS Annual Meeting and Exposition, Toronto, Canada, November 10–14, 2002; AAPS PharmSci., 4 (S1) (2002) Abstract 2850. [27] A. Mize, S. McComish, N. Premkumar, Proceedings of the 2003 AAPS Annual Meeting and Exposition, Salt Lake City, USA, October 26–30, 2003; AAPS PharmSci. 5 (S1) (2002) Abstract 88. [28] W. Xiaoa, B. Chena, S. Yaoa, Z. Cheng, J. Chromatogr. B 817 (2005) 153. [29] H. Monji, M. Yamaguchi, I. Aoki, H. Ueno, J. Chromatogr. B 690 (1997) 305. [30] C. Govaerts, H.K. Chepkwony, A. Van Schepdael, E. Roets, J. Hoogmartens, Rapid Commun. Mass Spectrom. 14 (2000) 878. [31] S.K. Chitneni, C. Govaerts, E. Adams, A. Van Schepdael, J. Hoogmartens, J. Chromatogr. A 1056 (2004) 111. ´ Z. Weitner, M. Ilijas, J. Pharmaceut. Biomed. Anal. 33 (2003) 647. [32] Z. Mandic, [33] P.J. Smith, C.K. Mann, J. Org. Chem. 34 (1969) 1821.