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Quantitative determination of erythromycin and its hydrolysis products by cyclic voltammetry at the interface between water and 1,2-dichloroethane
211
J. Electroanal. Chem., 360 (1993) 211-219 Elsevier Sequoia S.A., Lausanne
JEC 02819
Quantitative determination of erythromycin and its hydrolysis products by cyclic voltammetry at the interface between water and 1,2-dichloroethane L.M. Yudi, A.M. Baruzzi and V. Solis INFIQC, Departamento de Fisico Quimicq Facultad de Ciencias Quimicas, Uniuersidad National de Cordoba, Sucursal 16, C.C. 61, 5016 Cordoba (Argentina)
(Received 10 November 1992; in revised form 15 March 1993)
Abstract
An electrochemical method for quantifying erythromycin (Er) salts and their hydrolysis products is described. Cyclic voltammetry at the water/l,2-dichloroethane interface in a four-electrode system was used. A linear relationship between ZP and the erythromycin concentration was obtained up to the solubility limit of the antibiotic. The various hydrolysis products which can be obtained in acid or basic solutions were characterized by their respective transfer potentials. Similar hydrolysis products were observed for aged solutions of Er lactobionate. It is concluded that the proposed method is suitable for the quantification of Er and for distinguishing it from its hydrolysis products.
INTRODUCTION
Erythromycin A (Er) is a macrolide antibiotic composed of a polyfunctionalized 1Cmembered lactone ring (erythronolide) substituted with an amino sugar (desosamine) and a neutral sugar (cladinose) [l].
0022-0728/93/$06.00
0 1993 - Elsevier Sequoia S.A. All rights reserved
212
The tertiary amine present in desosamine makes Er a weak base with a pK, of 8.6. It dissolves easily in most common organic solvents as well as in dilute aqueous acids, where it forms crystalline salts. The pharmaceutical products are usually salts such as the glucoheptonate or the lactobionate (ErH+L-) [2]. The salts are unstable in acidic or alkaline solutions and show their maximum stability between pH 6.0 and pH 9.5. IR and UV spectra of Er and its degradation products are well described in the literature [31. Early work on Er has shown that the biological activity is lost rapidly when the salt of the antibiotic is placed in either acidic or alkaline solutions. The loss of one or both sugar units as well as the opening of the lactone ring have been reported as responsible for the inactivation of Er as a bactericide. In aqueous solutions buffered at pH 7.0-8.0 Er salts are stable for about two weeks under refrigeration
121. Quantitative analysis of Er is usually performed by UV spectroscopy after developing a chromophore on the molecule by alkaline degradation. For this reason, the technique, although very good for determining initial concentrations of Er, is not suitable for quantification of the unhydrolysed fraction in aged solutions. This fraction has to be determined by microbiological analysis, i.e. evaluating the bactericide action of the samples 141. Voltammetry at the interface of two immiscible electrolyte solutions (ITIES) has been shown to be a very suitable technique for the investigation of ionophore antibiotics as ion carriers [5-71. The aim of the present paper is to apply voltammetry at ITIES as an analytical method for the simultaneous determination of Er and its degradation products under different conditions of hydrolysis. In this way, the microbiological active fraction of pharmaceutical samples can be determined by a fast, reliable and relatively simple technique. EXPERIMENTAL
The four-electrode potentiostat with dynamic ZR drop compensation, the electrolytic cell and the electrodes have been described in earlier publications [8]. The potential values E reported are the applied potentials which include A@ = 0.364 V for the transfer of the reference ion TPAs+. The base electrolyte solutions were 1 X lo-’ M KC1 (Mallinckrodt A.R.) in ultrapure water and 1 x lo-’ M tetraphenyl arsonium dicarbollyl cobaltate (TPAsDCC) in 1,2-dichloroethane (DCE) (Merck p.A). TPAsDCC was prepared as described in ref. 8. The pH of the aqueous phase was varied in the range 2-11 by the addition of HCl (Merck p.a>, KOH or KHCO, (Carlo Erba rpe>. The salt erythromycin lactobionate (ErH+L-1 was added to the aqueous phase at different concentrations. This salt is one of the therapeutical products commonly used. In some experiments the solutions were prepared just before use so as to avoid hydrolysis. In others, samples of ErH+L- which had previously been hydrolysed were employed. Hydrolyses were performed in solutions of different
213
pH at various temperatures, following the procedures described in ref. 3. The hydrolysis products obtained in each case were analysed by UV spectroscopy using a Shimadzu UV-260 spectrophotometer. The same technique was used to analyse aged solutions of ErH+L- kept at different temperatures. RESULTS AND DISCUSSION
We have analysed the ability of Er to mediate the transfer of ions through the water/DCE interface. Figure 1 shows cyclic voltammograms (CVs) for the transfer processes taking place at the water/DCE interface for aqueous solutions of 1 X 10e3 M ErH+L- + 1 x 10e2 M KC1 with various pH values in contact with an organic solution containing 1 x 10e2 M TPAsDCC. The aqueous solutions were prepared just before use in order to avoid hydrolysis. After subtraction of the blank currents, either one or two transfer processes were observed, depending on pH, the first at Eil = 0.41 V and the second at EC2= 0.62 V, close to the positive potential limit. Only EL was observed in acid solutions. The peak potential difference AE,, = Eg -EL, = 60 mV was independent of the potential sweep rate u and pH, for pH < pK, (where K, is the equilibrium constant for ErH+ dissociation in the aqueous phase). The positive peak current Zzt was proportional to u112 and to the equilibrium concentration of ErH+, which remained almost constant for pH < 5 (pH -=zzpK,). According to these results, ZA should be assigned to the transfer of ErH+ (W+ 0). For pH = 9 (close to or greater than pK,), the second process at Eg2 was apparent together with a decrease in ZzI. Both Zp: and Z& increased steadily with pH, but the total positive current Z,’ = Z& + Zp: remained almost constant and was controlled by the analytical concentration of ErH+L-. The
0
I
I
0.1
02
pi
,
I
I
I
0.3
0.4
0.5
0.6
0.7
Fig. 1. Cyclic voltammograms for the transfer of ErH+ at different pH values: . . pH 2; - - pH 5; .- .-. pH 9. Organic phase composition, 10m2 M TPAsDCC, aqueous phase PH 4; composition: 10m2 M KCI+ lop3 M ErHf L-; u = 0.05 V s-l.
214
m--
wpH-4.m
ao-w-4n--
IO3c/M Fig. 2. Plot of I: vs. c for the ErHf transfer at: (a) pH 4 and (b) pH 7. Aqueous phase composition, 10e2 M KCI+ xM M ErHf L-; organic phase composition: lo-2 M TPAsDCC, L’= 0.05 V s-l.
second process is assigned to the transfer of K+ cations mediated by Er. This assignment is supported by the observation of a positive shift in Ez2 when KC1 is substituted by 10e2 M LiCl. The same tendency is observed for the transfer of the hydrated cations, indicating that the stability of ErK+ and ErLi+ complexes is not very high. Whether the transfer is direct or facilitated is a matter for further research [93. The competition between K+ and H+ for Er determined the relative values of the peak currents and their dependence on pH. Electroanalytical determination of the antibiotic
In order to check whether cyclic voltammetry was a suitable electroanalytical technique for ErH+L- the linearity of the ZP vs. C plot and the reproducibility of the measurements were analysed. Figure 2 shows plots of Zz, vs. ErH+L- concentration at pH values of 4 and 7 and u = 0.05 V s-l. For both calibration plots the linear relationship between current/PA and ErH+L- concentration/mol dmP3 holds up to the limit of the solubility of the antibiotic (2.5 x 10m3 M). The lowest detection limit was 1 x lop5 M. The linear current-concentration plots for u = 0.05 V s-* had slopes of (3.03 + 0.04) X lo4 Z_LA(mol dm-3)-’ and (2.31 f 0.02) X lo4 Z.LA(mol dm-3)-1 with regression coefficients of 0.9996 and 0.9998, for pH 4 and pH 7 respectively, evaluated using the least-squares method. Sets of eight CVs for different ErH+Lconcentrations at both pH values were used to average the values of Ez, and ZrJ1 and to calculate the corresponding standard deviations, as shown in Fig. 2. Similar analytical parameters were obtained at u = 0.1 V s-‘. Hydrolysed solutions of erythromycin
Early degradation studies of Er had established the structure of the products obtained when the antibiotic was hydrolysed in acid or in basic media. In our case,
215
30d =720O-10-2o0
I I 0.1 02
I I I I 0.3 0.4 0.5 0.6 EIV
Fig. 3. Transfer of acidic hydrolysis products: CV for fresh ErHf L- solutions; - - - CV after 24 h at pH 2. Aqueous phase composition, lo-* M KCI+ 10m3 M ErH+ L- (pH 2); organic phase composition: lo-* M TPAsDCC, u = 0.05 V s-l.
the experimental conditions of the hydrolyses described in the literature [3] were carefully reproduced and the presence of the corresponding products was checked by UV spectroscopy. The transfer of these products through the water/DCE interface was studied using cyclic voltammetry under different experimental conditions. Hydrolysis in acid media
According to ref. 3, the hydrolysis of ErH+L- in dilute acid solutions yields two compounds, erythralosamine (C,,H,,NO,) and cladinose, a neutral sugar with the molecular formula CsH,,O,. Figure 3 shows the CVs for a sample of a 1 X 10e3 M ErH+L- solution treated with 0.01 M HCI for 24 h at room temperature and recorded at pH 2. A CV for a fresh ErH+L- solution of the same initial concentration was included for comparison. No changes in the current-potential profiles were observed when the pH of the hydrolysed solution was varied between 2 and 5 prior to recording the CV. After hydrolysis, a new transfer process is detected at E& = 0.47 V, independent of u. A shoulder at Eg is indicative of a remaining unhydrolysed fraction of Er. UV spectrophotometric analysis of the hydrolysed samples of ErH+L- reproduced the literature results [3]. Thus a significant decrease in the absorption band at 270 nm was observed in good agreement with the fact that neither cladinose nor erythralosamine absorb in the UV region of the spectrum. According to the above results, the species transferred at E& is the protonated form of erythralosamine, EryH+.
216
Hydrolysis in basic media
Figure 4 shows the CVs obtained for samples treated with 0.01 M NaOH for 24 h at room temperature. According to the literature [3], under these conditions the lactone function of the ring is saponified, yielding a zwitterion whose acidic and basic groups have pK, values of 4.3 and 9.1 respectively. The UV absorption curve obtained for the hydrolysed sample no longer showed the 270 nm band
-201 0
,
,
,
,
,
(
0.1
02
0.3
0.4
0.5 E/V
0.6
I 0.5
I 0.6
(a)
0 Cc)
0.1
I 02
I 0.3
1 0.4
0 (b)
0.1 0.2
0.3
0.L
0.5
0.6
0.7
E/V
I 0.7
E/V
Fig. 4. (a) Cyclic voltammograms obtained at pH 2 before ( -) and after (- - -_) hydrolysis at pH 11 for 24 h; . . . deconvoluted I-E profile. Aqueous phase composition, lo-’ M KCl+ 10m3 M ErH+ L-; organic phase composition, lo-* M TPAsDCC; u = 0.05 V s-t. (b) Cyclic voltammograms obtained at pH 7 before ( ---I and after (--_) hydrolysis at pH 11 for 24 h; . . deconvoluted I-E profile. Aqueous phase composition, lo-* M KCl+ 10m3 M ErH+L-; organic phase composition, lo-* M TPAsDCC; u = 0.05 V s-‘. (c) Cyclic voltammograms obtained at pH 7 after hydrolysis at pH 11 for 24 h at different sweep rates u/V s-‘: .-.-. 0.01; - - - 0.05; . 0.25. Aqueous phase composition, lo-* M KCI+ 10m3 M ErH+ L-; organic phase composition, lo-* M TPAsDCC.
present for Er, but there was a new absorption maximum at 230 nm which has been attributed to the formation of the grouping [3] 0
x=c-_c-
II
After the antibiotic had been hydrolysed, the pH of the solution was changed by the addition of HCI in order to analyse the effect of pH on the transfer processes of the hydrolysis products. Figure 4(a) shows the CVs recorded at pH 2 for a 1 x 10e3 M ErH+L- solution before and after alkaline hydrolysis. The following behaviour was observed. The peak current 1: at 0.43 V due to the transfer of ErH+ almost vanished after hydrolysis. A new peak system at EC4= 0.54 V was observed; E& and A EPI = E& - EP, = 60 mV were independent of U. The peak current Z& at 0.54 V was proportional to u112 and to a concentration value of 87% of the initial concentration of ErH+L- (before hydrolysis) if the same value for the diffusion coefficient was used. A small peak at Ez, with I;, = 3 PA is only observed if the Z-E profile is deconvoluted as shown in Figs. 4(a) and 4(b). This current is estimated to be proportional to the remaining 13% of unhydrolysed Er, according to Fig. 2. Reversible transfer was assumed for the deconvolution procedure. On the basis of the above results, the species transferred at E& is the cationic form Z+ of the zwitterion which predominates at pH 2. The results obtained for the transfer of Z+ at pH 7, close to the isoelectric point of 6.7, are shown in Fig. 4(b). The transfer currents at E;, and Ez, (in the deconvoluted CV) indicated the presence of some unhydrolysed fraction of the antibiotic. f- - EP4 + - EP, and Zpf4/Z;don u at pH 7 is complex, as The dependence of A EPI can be seen in Fig. 4(c). For u 3 0.25 V s-l, AEPd = 60 mV and Z&/Z& = 1. This ratio increased for decreasing c’, and for u G 0.01 V s-l no peak at EP, corresponding to the transfer of Zf (0 -+ W) was detected. The process at E;, was clearly seen when no overlapping with Z+ transfer took place, i.e. it is the only peak for u G 0.010 V s-l whereas for intermediate values of U, typically 0.05 V s-r, both transfer processes (E;, and E;J were observed. The complex equilibria present at this pH, involving other ions such as Kf, must be responsible for the observed behaviour. An unambiguous assignment of the transferred species at each peak is not possible, and so no reliable quantitative information can be obtained at this pH. Aged solutions of erythromycin lactobionate Cyclic voltammetry was used to analyse the degradation processes taking place in aged solutions of ErH+L-. Solutions were prepared in therapeutic concentrations (1 x lop3 M) in 1 x lo-* M KC1 (pH 4) or 1 X lop2 M KC1 + 1 X lop3 M KHCO, (pH 7) and incubated at 5°C and 40°C for 2 weeks. No appreciable changes were detected in the CV for the solutions aged at 5°C. Figure 5 shows the
218
-301 , , , , , , , 0
0.1
0.2
0.3
0.1
0.5
0.6 0.7
EIV Fig. 5. Cyclic voltammograms obtained after ageing ErH+ L- solution at pH 4 and T = 40°C for different times t: t = 0; .-.-. t = 7 days; - - - t = 14 days. Aqueous phase composition, lo-* M KCI+ 10m3 M ErHf L- (pH 4); organic phase composition, lo-* M TPAsDCC; u = 0.05 V s-1.
CVs for fresh and aged solutions at pH 4 after one and two weeks of incubation at 40°C. A decrease in ZzI as a function of time was observed together with a new + peak system at EP2. A comparison of Figs. 3 and 5 suggests that the hydrolysis products of Er under these conditions were erythralosamine and cladinose; this conclusion was confirmed by UV spectroscopy of the aged solution.
2 O21 OO-
-1 O-2 O-
0
0.1
cl2
0.3 0.4
0.5
0.6
0.7
El'.' Fig. 6. Cyclic voltammograms obtained after ageing ErHf L- solution at pH 7 and T = 40°C for different times t: t = 0; -. -. t = 7 days; - - - t = 14 days. Aqueous phase composition, lo-* M KCI+ low3 M ErHf L- (pH 7); organic phase composition, lo-’ M TPAsDCC; u = 0.05 V SC’.
219
The behaviour of solutions aged under the same conditions but at pH 7 is shown in Fig. 6. In this case, the decrease in 1c1 with time was accompanied by a corresponding increase in Zpa. Comparison with Fig. 4 shows that the hydrolysis product was the zwitterion, and this was also verified by UV spectroscopy. The voltammetric response obtained at different sweep rates is the same as that shown in Fig. 4(c). In order to evaluate the degree of hydrolysis of the antibiotic under different conditions as a function of time, a semi-integral analysis [lo] of the CV was performed. After 1 week the activity of the antibiotic had decreased to 42% and 49% of its original value at pH 4 and pH 7 respectively. After two weeks, the degree of hydrolysis of ErH+L- was 71% of its original value at pH 4 and 67% at pH 7. These results agree with those obtained using biological methods. CONCLUSIONS
Cyclic voltammetry at ITIES is a suitable analytical technique not only for the quantitative determination of ErH+Lbut also for determining the type and extent of hydrolytic degradation of the antibiotic, provided that an appropriate choice of pH is made in order to avoid the production of multiple equilibria. ACKNOWLEDGEMENTS
Financial support from the Consejo National de Investigaciones Cientificas y Tecnologicas (CONICET) and the Consejo de Investigaciones de la Provincia de Cdrdoba (CONICOR) is gratefully acknowledged. REFERENCES 1 J.R. Everett and J.W. Tyler, J. Chem. Sot. Perkin Trans. II, 11 (19871 1659. 2 D. Perlamn in W.O. Foye (Ed.), Principios de Quimica FarmacCutica, Editorial Revert& Barcelona, 1984, Ch. 32, p. 794. 3 E.H. Flynn, M.V. Sigal, P.F. Wiley and K. Gerzon, J. Chem. Sot., 76 (19541 3121. 4 W.L. Koch in K. Florey (Ed.), Analytical Profiles of Drug Substances, Vol. 8, Academic Press, New York, 1979, p. 159. 5 J. Koryta, Electrochim. Acta, 33 (19881 189. 6 G. Duo, J. Koryta, W. Ruth and P. Vanysek, J. Electroanal. Chem., 159 (19831413. 7 E. Wang and Y. Liu, J. Electroanal. Chem., 214 (1986) 459. 8 L.M. Yudi, A.M. Baruzzi and V.M. Solis, J. Electroanal. Chem., 328 (19921 153. 9 L.M. Yudi, A.M. Baruzzi and V.M. Solis, to be published. 10 M. Grenness and K.B. Oldham, Anal. Chem., 44 (19721 1121.
J. Electroanal. Chem., 360 (1993) 211-219 Elsevier Sequoia S.A., Lausanne
JEC 02819
Quantitative determination of erythromycin and its hydrolysis products by cyclic voltammetry at the interface between water and 1,2-dichloroethane L.M. Yudi, A.M. Baruzzi and V. Solis INFIQC, Departamento de Fisico Quimicq Facultad de Ciencias Quimicas, Uniuersidad National de Cordoba, Sucursal 16, C.C. 61, 5016 Cordoba (Argentina)
(Received 10 November 1992; in revised form 15 March 1993)
Abstract
An electrochemical method for quantifying erythromycin (Er) salts and their hydrolysis products is described. Cyclic voltammetry at the water/l,2-dichloroethane interface in a four-electrode system was used. A linear relationship between ZP and the erythromycin concentration was obtained up to the solubility limit of the antibiotic. The various hydrolysis products which can be obtained in acid or basic solutions were characterized by their respective transfer potentials. Similar hydrolysis products were observed for aged solutions of Er lactobionate. It is concluded that the proposed method is suitable for the quantification of Er and for distinguishing it from its hydrolysis products.
INTRODUCTION
Erythromycin A (Er) is a macrolide antibiotic composed of a polyfunctionalized 1Cmembered lactone ring (erythronolide) substituted with an amino sugar (desosamine) and a neutral sugar (cladinose) [l].
0022-0728/93/$06.00
0 1993 - Elsevier Sequoia S.A. All rights reserved
212
The tertiary amine present in desosamine makes Er a weak base with a pK, of 8.6. It dissolves easily in most common organic solvents as well as in dilute aqueous acids, where it forms crystalline salts. The pharmaceutical products are usually salts such as the glucoheptonate or the lactobionate (ErH+L-) [2]. The salts are unstable in acidic or alkaline solutions and show their maximum stability between pH 6.0 and pH 9.5. IR and UV spectra of Er and its degradation products are well described in the literature [31. Early work on Er has shown that the biological activity is lost rapidly when the salt of the antibiotic is placed in either acidic or alkaline solutions. The loss of one or both sugar units as well as the opening of the lactone ring have been reported as responsible for the inactivation of Er as a bactericide. In aqueous solutions buffered at pH 7.0-8.0 Er salts are stable for about two weeks under refrigeration
121. Quantitative analysis of Er is usually performed by UV spectroscopy after developing a chromophore on the molecule by alkaline degradation. For this reason, the technique, although very good for determining initial concentrations of Er, is not suitable for quantification of the unhydrolysed fraction in aged solutions. This fraction has to be determined by microbiological analysis, i.e. evaluating the bactericide action of the samples 141. Voltammetry at the interface of two immiscible electrolyte solutions (ITIES) has been shown to be a very suitable technique for the investigation of ionophore antibiotics as ion carriers [5-71. The aim of the present paper is to apply voltammetry at ITIES as an analytical method for the simultaneous determination of Er and its degradation products under different conditions of hydrolysis. In this way, the microbiological active fraction of pharmaceutical samples can be determined by a fast, reliable and relatively simple technique. EXPERIMENTAL
The four-electrode potentiostat with dynamic ZR drop compensation, the electrolytic cell and the electrodes have been described in earlier publications [8]. The potential values E reported are the applied potentials which include A@ = 0.364 V for the transfer of the reference ion TPAs+. The base electrolyte solutions were 1 X lo-’ M KC1 (Mallinckrodt A.R.) in ultrapure water and 1 x lo-’ M tetraphenyl arsonium dicarbollyl cobaltate (TPAsDCC) in 1,2-dichloroethane (DCE) (Merck p.A). TPAsDCC was prepared as described in ref. 8. The pH of the aqueous phase was varied in the range 2-11 by the addition of HCl (Merck p.a>, KOH or KHCO, (Carlo Erba rpe>. The salt erythromycin lactobionate (ErH+L-1 was added to the aqueous phase at different concentrations. This salt is one of the therapeutical products commonly used. In some experiments the solutions were prepared just before use so as to avoid hydrolysis. In others, samples of ErH+L- which had previously been hydrolysed were employed. Hydrolyses were performed in solutions of different
213
pH at various temperatures, following the procedures described in ref. 3. The hydrolysis products obtained in each case were analysed by UV spectroscopy using a Shimadzu UV-260 spectrophotometer. The same technique was used to analyse aged solutions of ErH+L- kept at different temperatures. RESULTS AND DISCUSSION
We have analysed the ability of Er to mediate the transfer of ions through the water/DCE interface. Figure 1 shows cyclic voltammograms (CVs) for the transfer processes taking place at the water/DCE interface for aqueous solutions of 1 X 10e3 M ErH+L- + 1 x 10e2 M KC1 with various pH values in contact with an organic solution containing 1 x 10e2 M TPAsDCC. The aqueous solutions were prepared just before use in order to avoid hydrolysis. After subtraction of the blank currents, either one or two transfer processes were observed, depending on pH, the first at Eil = 0.41 V and the second at EC2= 0.62 V, close to the positive potential limit. Only EL was observed in acid solutions. The peak potential difference AE,, = Eg -EL, = 60 mV was independent of the potential sweep rate u and pH, for pH < pK, (where K, is the equilibrium constant for ErH+ dissociation in the aqueous phase). The positive peak current Zzt was proportional to u112 and to the equilibrium concentration of ErH+, which remained almost constant for pH < 5 (pH -=zzpK,). According to these results, ZA should be assigned to the transfer of ErH+ (W+ 0). For pH = 9 (close to or greater than pK,), the second process at Eg2 was apparent together with a decrease in ZzI. Both Zp: and Z& increased steadily with pH, but the total positive current Z,’ = Z& + Zp: remained almost constant and was controlled by the analytical concentration of ErH+L-. The
0
I
I
0.1
02
pi
,
I
I
I
0.3
0.4
0.5
0.6
0.7
Fig. 1. Cyclic voltammograms for the transfer of ErH+ at different pH values: . . pH 2; - - pH 5; .- .-. pH 9. Organic phase composition, 10m2 M TPAsDCC, aqueous phase PH 4; composition: 10m2 M KCI+ lop3 M ErHf L-; u = 0.05 V s-l.
214
m--
wpH-4.m
ao-w-4n--
IO3c/M Fig. 2. Plot of I: vs. c for the ErHf transfer at: (a) pH 4 and (b) pH 7. Aqueous phase composition, 10e2 M KCI+ xM M ErHf L-; organic phase composition: lo-2 M TPAsDCC, L’= 0.05 V s-l.
second process is assigned to the transfer of K+ cations mediated by Er. This assignment is supported by the observation of a positive shift in Ez2 when KC1 is substituted by 10e2 M LiCl. The same tendency is observed for the transfer of the hydrated cations, indicating that the stability of ErK+ and ErLi+ complexes is not very high. Whether the transfer is direct or facilitated is a matter for further research [93. The competition between K+ and H+ for Er determined the relative values of the peak currents and their dependence on pH. Electroanalytical determination of the antibiotic
In order to check whether cyclic voltammetry was a suitable electroanalytical technique for ErH+L- the linearity of the ZP vs. C plot and the reproducibility of the measurements were analysed. Figure 2 shows plots of Zz, vs. ErH+L- concentration at pH values of 4 and 7 and u = 0.05 V s-l. For both calibration plots the linear relationship between current/PA and ErH+L- concentration/mol dmP3 holds up to the limit of the solubility of the antibiotic (2.5 x 10m3 M). The lowest detection limit was 1 x lop5 M. The linear current-concentration plots for u = 0.05 V s-* had slopes of (3.03 + 0.04) X lo4 Z_LA(mol dm-3)-’ and (2.31 f 0.02) X lo4 Z.LA(mol dm-3)-1 with regression coefficients of 0.9996 and 0.9998, for pH 4 and pH 7 respectively, evaluated using the least-squares method. Sets of eight CVs for different ErH+Lconcentrations at both pH values were used to average the values of Ez, and ZrJ1 and to calculate the corresponding standard deviations, as shown in Fig. 2. Similar analytical parameters were obtained at u = 0.1 V s-‘. Hydrolysed solutions of erythromycin
Early degradation studies of Er had established the structure of the products obtained when the antibiotic was hydrolysed in acid or in basic media. In our case,
215
30d =720O-10-2o0
I I 0.1 02
I I I I 0.3 0.4 0.5 0.6 EIV
Fig. 3. Transfer of acidic hydrolysis products: CV for fresh ErHf L- solutions; - - - CV after 24 h at pH 2. Aqueous phase composition, lo-* M KCI+ 10m3 M ErH+ L- (pH 2); organic phase composition: lo-* M TPAsDCC, u = 0.05 V s-l.
the experimental conditions of the hydrolyses described in the literature [3] were carefully reproduced and the presence of the corresponding products was checked by UV spectroscopy. The transfer of these products through the water/DCE interface was studied using cyclic voltammetry under different experimental conditions. Hydrolysis in acid media
According to ref. 3, the hydrolysis of ErH+L- in dilute acid solutions yields two compounds, erythralosamine (C,,H,,NO,) and cladinose, a neutral sugar with the molecular formula CsH,,O,. Figure 3 shows the CVs for a sample of a 1 X 10e3 M ErH+L- solution treated with 0.01 M HCI for 24 h at room temperature and recorded at pH 2. A CV for a fresh ErH+L- solution of the same initial concentration was included for comparison. No changes in the current-potential profiles were observed when the pH of the hydrolysed solution was varied between 2 and 5 prior to recording the CV. After hydrolysis, a new transfer process is detected at E& = 0.47 V, independent of u. A shoulder at Eg is indicative of a remaining unhydrolysed fraction of Er. UV spectrophotometric analysis of the hydrolysed samples of ErH+L- reproduced the literature results [3]. Thus a significant decrease in the absorption band at 270 nm was observed in good agreement with the fact that neither cladinose nor erythralosamine absorb in the UV region of the spectrum. According to the above results, the species transferred at E& is the protonated form of erythralosamine, EryH+.
216
Hydrolysis in basic media
Figure 4 shows the CVs obtained for samples treated with 0.01 M NaOH for 24 h at room temperature. According to the literature [3], under these conditions the lactone function of the ring is saponified, yielding a zwitterion whose acidic and basic groups have pK, values of 4.3 and 9.1 respectively. The UV absorption curve obtained for the hydrolysed sample no longer showed the 270 nm band
-201 0
,
,
,
,
,
(
0.1
02
0.3
0.4
0.5 E/V
0.6
I 0.5
I 0.6
(a)
0 Cc)
0.1
I 02
I 0.3
1 0.4
0 (b)
0.1 0.2
0.3
0.L
0.5
0.6
0.7
E/V
I 0.7
E/V
Fig. 4. (a) Cyclic voltammograms obtained at pH 2 before ( -) and after (- - -_) hydrolysis at pH 11 for 24 h; . . . deconvoluted I-E profile. Aqueous phase composition, lo-’ M KCl+ 10m3 M ErH+ L-; organic phase composition, lo-* M TPAsDCC; u = 0.05 V s-t. (b) Cyclic voltammograms obtained at pH 7 before ( ---I and after (--_) hydrolysis at pH 11 for 24 h; . . deconvoluted I-E profile. Aqueous phase composition, lo-* M KCl+ 10m3 M ErH+L-; organic phase composition, lo-* M TPAsDCC; u = 0.05 V s-‘. (c) Cyclic voltammograms obtained at pH 7 after hydrolysis at pH 11 for 24 h at different sweep rates u/V s-‘: .-.-. 0.01; - - - 0.05; . 0.25. Aqueous phase composition, lo-* M KCI+ 10m3 M ErH+ L-; organic phase composition, lo-* M TPAsDCC.
present for Er, but there was a new absorption maximum at 230 nm which has been attributed to the formation of the grouping [3] 0
x=c-_c-
II
After the antibiotic had been hydrolysed, the pH of the solution was changed by the addition of HCI in order to analyse the effect of pH on the transfer processes of the hydrolysis products. Figure 4(a) shows the CVs recorded at pH 2 for a 1 x 10e3 M ErH+L- solution before and after alkaline hydrolysis. The following behaviour was observed. The peak current 1: at 0.43 V due to the transfer of ErH+ almost vanished after hydrolysis. A new peak system at EC4= 0.54 V was observed; E& and A EPI = E& - EP, = 60 mV were independent of U. The peak current Z& at 0.54 V was proportional to u112 and to a concentration value of 87% of the initial concentration of ErH+L- (before hydrolysis) if the same value for the diffusion coefficient was used. A small peak at Ez, with I;, = 3 PA is only observed if the Z-E profile is deconvoluted as shown in Figs. 4(a) and 4(b). This current is estimated to be proportional to the remaining 13% of unhydrolysed Er, according to Fig. 2. Reversible transfer was assumed for the deconvolution procedure. On the basis of the above results, the species transferred at E& is the cationic form Z+ of the zwitterion which predominates at pH 2. The results obtained for the transfer of Z+ at pH 7, close to the isoelectric point of 6.7, are shown in Fig. 4(b). The transfer currents at E;, and Ez, (in the deconvoluted CV) indicated the presence of some unhydrolysed fraction of the antibiotic. f- - EP4 + - EP, and Zpf4/Z;don u at pH 7 is complex, as The dependence of A EPI can be seen in Fig. 4(c). For u 3 0.25 V s-l, AEPd = 60 mV and Z&/Z& = 1. This ratio increased for decreasing c’, and for u G 0.01 V s-l no peak at EP, corresponding to the transfer of Zf (0 -+ W) was detected. The process at E;, was clearly seen when no overlapping with Z+ transfer took place, i.e. it is the only peak for u G 0.010 V s-l whereas for intermediate values of U, typically 0.05 V s-r, both transfer processes (E;, and E;J were observed. The complex equilibria present at this pH, involving other ions such as Kf, must be responsible for the observed behaviour. An unambiguous assignment of the transferred species at each peak is not possible, and so no reliable quantitative information can be obtained at this pH. Aged solutions of erythromycin lactobionate Cyclic voltammetry was used to analyse the degradation processes taking place in aged solutions of ErH+L-. Solutions were prepared in therapeutic concentrations (1 x lop3 M) in 1 x lo-* M KC1 (pH 4) or 1 X lop2 M KC1 + 1 X lop3 M KHCO, (pH 7) and incubated at 5°C and 40°C for 2 weeks. No appreciable changes were detected in the CV for the solutions aged at 5°C. Figure 5 shows the
218
-301 , , , , , , , 0
0.1
0.2
0.3
0.1
0.5
0.6 0.7
EIV Fig. 5. Cyclic voltammograms obtained after ageing ErH+ L- solution at pH 4 and T = 40°C for different times t: t = 0; .-.-. t = 7 days; - - - t = 14 days. Aqueous phase composition, lo-* M KCI+ 10m3 M ErHf L- (pH 4); organic phase composition, lo-* M TPAsDCC; u = 0.05 V s-1.
CVs for fresh and aged solutions at pH 4 after one and two weeks of incubation at 40°C. A decrease in ZzI as a function of time was observed together with a new + peak system at EP2. A comparison of Figs. 3 and 5 suggests that the hydrolysis products of Er under these conditions were erythralosamine and cladinose; this conclusion was confirmed by UV spectroscopy of the aged solution.
2 O21 OO-
-1 O-2 O-
0
0.1
cl2
0.3 0.4
0.5
0.6
0.7
El'.' Fig. 6. Cyclic voltammograms obtained after ageing ErHf L- solution at pH 7 and T = 40°C for different times t: t = 0; -. -. t = 7 days; - - - t = 14 days. Aqueous phase composition, lo-* M KCI+ low3 M ErHf L- (pH 7); organic phase composition, lo-’ M TPAsDCC; u = 0.05 V SC’.
219
The behaviour of solutions aged under the same conditions but at pH 7 is shown in Fig. 6. In this case, the decrease in 1c1 with time was accompanied by a corresponding increase in Zpa. Comparison with Fig. 4 shows that the hydrolysis product was the zwitterion, and this was also verified by UV spectroscopy. The voltammetric response obtained at different sweep rates is the same as that shown in Fig. 4(c). In order to evaluate the degree of hydrolysis of the antibiotic under different conditions as a function of time, a semi-integral analysis [lo] of the CV was performed. After 1 week the activity of the antibiotic had decreased to 42% and 49% of its original value at pH 4 and pH 7 respectively. After two weeks, the degree of hydrolysis of ErH+L- was 71% of its original value at pH 4 and 67% at pH 7. These results agree with those obtained using biological methods. CONCLUSIONS
Cyclic voltammetry at ITIES is a suitable analytical technique not only for the quantitative determination of ErH+Lbut also for determining the type and extent of hydrolytic degradation of the antibiotic, provided that an appropriate choice of pH is made in order to avoid the production of multiple equilibria. ACKNOWLEDGEMENTS
Financial support from the Consejo National de Investigaciones Cientificas y Tecnologicas (CONICET) and the Consejo de Investigaciones de la Provincia de Cdrdoba (CONICOR) is gratefully acknowledged. REFERENCES 1 J.R. Everett and J.W. Tyler, J. Chem. Sot. Perkin Trans. II, 11 (19871 1659. 2 D. Perlamn in W.O. Foye (Ed.), Principios de Quimica FarmacCutica, Editorial Revert& Barcelona, 1984, Ch. 32, p. 794. 3 E.H. Flynn, M.V. Sigal, P.F. Wiley and K. Gerzon, J. Chem. Sot., 76 (19541 3121. 4 W.L. Koch in K. Florey (Ed.), Analytical Profiles of Drug Substances, Vol. 8, Academic Press, New York, 1979, p. 159. 5 J. Koryta, Electrochim. Acta, 33 (19881 189. 6 G. Duo, J. Koryta, W. Ruth and P. Vanysek, J. Electroanal. Chem., 159 (19831413. 7 E. Wang and Y. Liu, J. Electroanal. Chem., 214 (1986) 459. 8 L.M. Yudi, A.M. Baruzzi and V.M. Solis, J. Electroanal. Chem., 328 (19921 153. 9 L.M. Yudi, A.M. Baruzzi and V.M. Solis, to be published. 10 M. Grenness and K.B. Oldham, Anal. Chem., 44 (19721 1121.