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Membrane electrode with a pseudoliquid potential-determining phase for cloxacillin determination
0039.9140/89 $3.00 + 0.00
Talan~, Vol. 36, No. 4, pp. 509-512, 1989 Printed in Great Britain. All rights rc.scrvcd
copyright 0 1989Pergamon Press plc
MEMBRANE ELECTRODE WITH A PSEUDOLIQUID POTENTIAL-DETERMINING PHASE FOR CLOXACILLIN DETERMINATION RYSZARD DUMKIEWICZ Department of Analytical Chemistry and Instrumental Analysis, Chemistry Institute, UMCS 20031 Lubhn, Poland (Received 10 February 1988. Revised 2 May 1988. Accepted I November 1988) Summary-An ion-selective electrode with a pseudoliquid potential-determining phase for cloxacillin determination has been prepared. The basic electrode analytical parameters (measuring range, slope, detection limit, response time, lifetime, and selectivity coefficients in relation to penicillins and some
inorganic ions) have been determined.
Determination of penicillin compounds proves to be very difficult because of their complex structure and the imprecisely stated chemical composition of medical preparation (e.g., Penicillium Crystalisatum). Penicillins have been determined by iodometric,‘-3 spectrophotometric4-6 and polarographic’ methods. Recently, indirect potentiometric methods with ionselective electrodes”” have been developed. Penicillins that can be decomposed by enzymes have been determined by using enzymatic electrodes with penicillinase immobilized in polyacrylamide gel on the surface of a glass electrode.‘“” Better results are usually achieved with direct methods, but electrodes selective for penicillin have not yet been described. The ability of quaternary ammonium salts to form ion-association complexes with cloxacillin anions has been used to prepare electrodes selective for cloxacillin (6-{(I3-(2-chlorophenyl)-5-methyl-4-isoxazolylJcarbonyl)amino)-3,3-dimethyl-7-oxo-4-thia-l-azobicyclo[3.2.0]heptane-2-carboxylic acid, monosodium salt). This complex may be an active substance in an ISE membrane phase. This paper presents the preparation and properties of the electrode. EXPERIMENTAL
acts as the sensor. The electrode structure is shown in Fig. 1. The penicillin electrode potential can be described by the equation: E=E,+
y
x log [penicillin]
Electrodes of this type, which do not contain an inner internal reference solution, possess the same advantages as the coated wire electrodes but have longer lifetime’* because the modified PVC plasticizer used is a reservoir of the active substances necessary for functioning of the electrode. The liquid exchanger The- liquid exchanger was the Aliquat 336-cloxacillin comulex, DmDwcd by shakina 10 ml of Ahouat 336 and 20 n? of aql;eous O.iM petri&in solution in-a separating funnel for 10 min. The extraction was continued until the aqueous phase was chloride-free. After drying, the Aliquat 3364oxacillin complex was kept in a dry vessel at 5”. Preparation of potential-determining phase The potential-determining phase was prepared by mixing 0.2 g of the Aliquat 3364oxacillin complex, 1.1 g of dibutyl phthalate, of 0.2 g of tributyl phosphate and 0.6 g of PVC. After deaeration, the mixture was used to fill the Teflon container of the electrode and was then gelled by heating at 80-90” for 30min. After cooling, the electrode was mounted, and conditioned for 2 hr in O.lM cloxacillin solution
before use. Potential measurement The emf of the ion-selective pencillin electrode and refer-
Reagent Analytical grade salts (PGCh, Gliwice) were used. Cloxacillin, potassium benxylpenicillin (Penicillium Crystahsatum) and ampicillin sodium, were obtained from PGLFA, and amoxicillin from Laboratoires de Recherche Reecham, Sevigna. Aliquat 336 was obtained from General Mills, USA. Freshly prepared pencillin solutions were used, and were kept in the refrigerator (5”) between successive measurements.
ence electrode svstem was measured in a vessel keDt at
Electrode construction The electrode consists of a cylindrical Teflon container connected to the poly(viny1 chloride) (PVC) body by a screw-thread. The pseudoliquid potential-determining phase of the electrode, into which the Ag/AgCl electrode is introduced, is placed in a cylindrical PTFE container which
Calibration graphs
25 f 0.1”. The &-bridge of the Orion 90-02 Ag/AgCl reference electrode was filled with O.OSM sodium acetate
adjusted to pH 7.0 with O.OSMacetic acid. Measurements were made with an Orion 901 Ionometer and a Radiometer PHM 62 pH-meter. RESULTS
The behaviour of the cloxacillin electrode in solutions of penicillins and interfering inorganic ions was studied over the concentration range lo-‘-lo-‘M. The calibration plots are shown in Fig. 2. The slope 509
RYSZMD DUMKIEWICZ
510
Table 1. Analytical parameters of the cloxacillin electrode 60.2
Slope, mV/Pc&x std. devn., mV corr. coeff. Intercept, m V Limit of detection. M
2.1 0.9991 238.2 7 x 1o-6
3.5
figcglml
10-S-10-’ O.oo444-44 0.5 3
Measurement range, M pgg/m[ Response time, min Lifetime, months
in Table 2. For penicilloate the electrode has a response slope of 30 mV/decade in the concentration range 10-3-10-‘M. The selectivity coefficient KCp$pn is 0.15. For 6-APA the electrode gives a response only in the range 0.01-O. lM, with a slope of 26 mV/decade and a selectivity coefficient KCp&.ApA= 0.005.
Fig. 1. Structure of the cloxacillin electrode: l-body, 3-FIFE sensor, 4-Ag/AgCI 2--table, electrode, 5---pseudoliquid potential-determining phase.
of the cloxacillin electrode response is 60 mV/decade, and the detection limit is 7 x 10e6M. Other analytical parameters of the electrode are given in Table 1. Selectivity The selectivity coefficients of the cloxacillin electrode for a number of anions were determined by the separate solution method. The values found are given
Response time Response time was determined by measuring the change in the emf after injection of v, ml of cloxacillin solution of concentration c, into upml of stock solution of concentration cp, with vigorous stirring; the concentration and volume ratios were kept constant at c,/c, = 100 and v&v, = 20. The solution was then diluted with an equal volume of water and the emf recorded as a function of time. The results obtained are shown in Fig. 3 and Table 1. During the stirring the emf changes by N 1mV for O.Ol-O.lM solutions, but by up to 5 mV for more dilute solutions. The electrode is not suitable for use in flow-through systems, on account of washing away of the liquid exchanger.
I 5
I
4
I
3
1
I
2
1
PC Fig. 2. Calibration curves of cloxacillin electrode. A: l-cloxacillin; 2-benzylpenicillin; 4-amoxycillin; B: I-cloxacillin; I-nitrate; 6-chloride; ‘I-acetate.
3-ampicillin;
Membrane electrode for cloxacillin Table 3. Results of cloxacillin determination
Table 2. Selectivity coefficients Species x (O.OlM)
KZ:, r 0.096 0.019 0.014
Benxylpenicillin Ampicillin Amoxicillin
6-APA acid Penicilloate Nitrate Chloride Acetate
Name of penicillin
1
Cloxacillin std. devn., mgll.
z3
79
399 40.8
51.9
Cloxacillin Ampicillin
250 250
6
251
2
Cloxacillin Ampicillin
500 250
7
513
12
Cloxacillin Amnicillin
250 500
7
264
4
40:o
0.05 0.0018 0.001
Assay of pharmaceuticals The standard addition technique was used, for samples at an ionic strength of 0.05M and a pH of 7.0. The results are given in Table 3. DISCUSSION As follows from Tables l-3, the cloxacillin electrode is characterized by good analytical parameters. The response time of 30 set, advantageous detection
Injettrd
c,. V,
"P
?
11OmV G
Dilution l+l
5z.c Time Fig. 3. Response time of cloxacillin electrode: c,, = 10-‘&f; c,=O.lM; v,=20ml; u,=lml; dilution l:l.
. r
6
limit of 5 x 10b5M and relatively good selectivity coefficients for inorganic ions make it useful for cloxacillin determination. The selectivity coefficients of the cloxacillin electrode for penicillins decrease in the order cloxacillin > benzylpenicillin > ampicillin > amoxicillin, and this order can be explained in items of the change of substituent character in the penicillin sidechain. Of the penicillins examined, cloxacillin has the strongest affinity for the ion-exchanger owing to the presence of a heterocyclic isoxazolyl substituent. Amoxicillin, which has amine and hydroxyl groups on the benzyl substituent, has the lowest affinity for the ion-exchanger; the atIinity of ampicillin, which has only an amine group as the substituent, is slightly higher. The affinity of benzylpenicillin is only slightly smaller than that of cloxacillin. Cloxacillin has been determined with the electrode (Table 3) in both pure preparations of the penicillin and in solutions corresponding to commercial formulations containing cloxacillin and ampicillin (Ampliclox and Ampiclox Neonatal, Beecham). The selectivity is sufficient for determination of cloxacillin in such mixtures. The results show that the electrode can be employed for control analysis of antibiotics or for determinations made in hospital laboratories. The potentiometric determination of cloxacillin (a pencillin which is insensitive to enzyme action) creates new possibilities for application of ion-selective electrodes. It has been found to give satisfactory results for determination of cloxacillin in serum, and its use for control of cloxacillin production is being investigated.
1Omv
REFERENCES l
.--OF\, . \
PH Fig. 4. Effect of pH on response of electrode to cloxacillin. TAL 3614-F
n
Found, mgll. 601
Cloxacillin
0.15
The dependence of the electrode potential on the pH of the solution was examined by adding 0.05M hydrochloric acid or sodium hydroxide dropwise to 20 ml of a stirred lo-‘M solution of cloxacillin. After each addition of acid or base, the electrode potential and pH were measured. The cloxacillin electrode is effective at pH 5-9 (Fig. 4).
t
Taken, mgll.
0.005
Eflect of pH
cD’
511
1. Farmakopea Polska, 1974. 2. British Pharmacoweia, The
Pharmaceutical Press.
London, 1973. s 3. T. Higuchi and E. Brochmann-Hanssen. Pharmaceutical Analvsis, Interscience, New York, 1961. 4. B. Casu and-P. Yentura, J. jharm. Sci. 1974, 63,211. 5. M. Gttis and M. Mtit, Cesk. Farm., 1986, 35, 119. 6. M. M. Ellaithy and M. G. El-Bardicy, Phurm. Sci., 1985, 47, 1974.
512
RYSZARDDUMKIEWICZ
7. J. A. Squella and L. J. Nufiez-Vergara, J. Electroanal.
8. 9. 10. 11. 12.
Chem. Znterfic. Electrochem., 1981, 130, 361. B. Karlberg and U. Forsman, Anal. Chim. Acta, 1976, 83,309. S. S. M. Hassan, M. T. M. Zaki and M. H. Eldesouki, Talanta, 1979, 26, 91. D. L. Simpson and R. K. Kobos, Anul. Chem., 1983,55, 1974. Z&m, Anal. Chim. Acta, 1984, 164, 273. L. F. Cullen, J. F. Rusling, A. Schleifer and G. J. Papariello, Anal. Chem., 1974, 46, 1955.
13. G. J. Papariello, A. K. Mukjerji and C. M. Shearer, &id., 1973, 45, 790. 14. A. Junichi and 0. Tetsuo, Chem. Charm. Bull., 1986,34, 3522. 15. M. T. Flanagan and N. J. Carroll, Biotechnol. Bioeng., 1986, 28, 1093; Chem. Abstr., 1986, lOS,77456. 16. A. G. Dobrolyubov, S. B. Itsygin and T. P. Levadnaya, Antibiot. Med. Biotechnol., 1986, 31, 336. 17. R. Tor and A. Freeman, Anal. Chem., 1986,58, 1042. 18. K. Sykut, R. Dumkiewin and J. Dumkiewicz, Ann. Univ. M. Curie-Sklodowska, Sect. AA, 1978, 13, 1.
Talan~, Vol. 36, No. 4, pp. 509-512, 1989 Printed in Great Britain. All rights rc.scrvcd
copyright 0 1989Pergamon Press plc
MEMBRANE ELECTRODE WITH A PSEUDOLIQUID POTENTIAL-DETERMINING PHASE FOR CLOXACILLIN DETERMINATION RYSZARD DUMKIEWICZ Department of Analytical Chemistry and Instrumental Analysis, Chemistry Institute, UMCS 20031 Lubhn, Poland (Received 10 February 1988. Revised 2 May 1988. Accepted I November 1988) Summary-An ion-selective electrode with a pseudoliquid potential-determining phase for cloxacillin determination has been prepared. The basic electrode analytical parameters (measuring range, slope, detection limit, response time, lifetime, and selectivity coefficients in relation to penicillins and some
inorganic ions) have been determined.
Determination of penicillin compounds proves to be very difficult because of their complex structure and the imprecisely stated chemical composition of medical preparation (e.g., Penicillium Crystalisatum). Penicillins have been determined by iodometric,‘-3 spectrophotometric4-6 and polarographic’ methods. Recently, indirect potentiometric methods with ionselective electrodes”” have been developed. Penicillins that can be decomposed by enzymes have been determined by using enzymatic electrodes with penicillinase immobilized in polyacrylamide gel on the surface of a glass electrode.‘“” Better results are usually achieved with direct methods, but electrodes selective for penicillin have not yet been described. The ability of quaternary ammonium salts to form ion-association complexes with cloxacillin anions has been used to prepare electrodes selective for cloxacillin (6-{(I3-(2-chlorophenyl)-5-methyl-4-isoxazolylJcarbonyl)amino)-3,3-dimethyl-7-oxo-4-thia-l-azobicyclo[3.2.0]heptane-2-carboxylic acid, monosodium salt). This complex may be an active substance in an ISE membrane phase. This paper presents the preparation and properties of the electrode. EXPERIMENTAL
acts as the sensor. The electrode structure is shown in Fig. 1. The penicillin electrode potential can be described by the equation: E=E,+
y
x log [penicillin]
Electrodes of this type, which do not contain an inner internal reference solution, possess the same advantages as the coated wire electrodes but have longer lifetime’* because the modified PVC plasticizer used is a reservoir of the active substances necessary for functioning of the electrode. The liquid exchanger The- liquid exchanger was the Aliquat 336-cloxacillin comulex, DmDwcd by shakina 10 ml of Ahouat 336 and 20 n? of aql;eous O.iM petri&in solution in-a separating funnel for 10 min. The extraction was continued until the aqueous phase was chloride-free. After drying, the Aliquat 3364oxacillin complex was kept in a dry vessel at 5”. Preparation of potential-determining phase The potential-determining phase was prepared by mixing 0.2 g of the Aliquat 3364oxacillin complex, 1.1 g of dibutyl phthalate, of 0.2 g of tributyl phosphate and 0.6 g of PVC. After deaeration, the mixture was used to fill the Teflon container of the electrode and was then gelled by heating at 80-90” for 30min. After cooling, the electrode was mounted, and conditioned for 2 hr in O.lM cloxacillin solution
before use. Potential measurement The emf of the ion-selective pencillin electrode and refer-
Reagent Analytical grade salts (PGCh, Gliwice) were used. Cloxacillin, potassium benxylpenicillin (Penicillium Crystahsatum) and ampicillin sodium, were obtained from PGLFA, and amoxicillin from Laboratoires de Recherche Reecham, Sevigna. Aliquat 336 was obtained from General Mills, USA. Freshly prepared pencillin solutions were used, and were kept in the refrigerator (5”) between successive measurements.
ence electrode svstem was measured in a vessel keDt at
Electrode construction The electrode consists of a cylindrical Teflon container connected to the poly(viny1 chloride) (PVC) body by a screw-thread. The pseudoliquid potential-determining phase of the electrode, into which the Ag/AgCl electrode is introduced, is placed in a cylindrical PTFE container which
Calibration graphs
25 f 0.1”. The &-bridge of the Orion 90-02 Ag/AgCl reference electrode was filled with O.OSM sodium acetate
adjusted to pH 7.0 with O.OSMacetic acid. Measurements were made with an Orion 901 Ionometer and a Radiometer PHM 62 pH-meter. RESULTS
The behaviour of the cloxacillin electrode in solutions of penicillins and interfering inorganic ions was studied over the concentration range lo-‘-lo-‘M. The calibration plots are shown in Fig. 2. The slope 509
RYSZMD DUMKIEWICZ
510
Table 1. Analytical parameters of the cloxacillin electrode 60.2
Slope, mV/Pc&x std. devn., mV corr. coeff. Intercept, m V Limit of detection. M
2.1 0.9991 238.2 7 x 1o-6
3.5
figcglml
10-S-10-’ O.oo444-44 0.5 3
Measurement range, M pgg/m[ Response time, min Lifetime, months
in Table 2. For penicilloate the electrode has a response slope of 30 mV/decade in the concentration range 10-3-10-‘M. The selectivity coefficient KCp$pn is 0.15. For 6-APA the electrode gives a response only in the range 0.01-O. lM, with a slope of 26 mV/decade and a selectivity coefficient KCp&.ApA= 0.005.
Fig. 1. Structure of the cloxacillin electrode: l-body, 3-FIFE sensor, 4-Ag/AgCI 2--table, electrode, 5---pseudoliquid potential-determining phase.
of the cloxacillin electrode response is 60 mV/decade, and the detection limit is 7 x 10e6M. Other analytical parameters of the electrode are given in Table 1. Selectivity The selectivity coefficients of the cloxacillin electrode for a number of anions were determined by the separate solution method. The values found are given
Response time Response time was determined by measuring the change in the emf after injection of v, ml of cloxacillin solution of concentration c, into upml of stock solution of concentration cp, with vigorous stirring; the concentration and volume ratios were kept constant at c,/c, = 100 and v&v, = 20. The solution was then diluted with an equal volume of water and the emf recorded as a function of time. The results obtained are shown in Fig. 3 and Table 1. During the stirring the emf changes by N 1mV for O.Ol-O.lM solutions, but by up to 5 mV for more dilute solutions. The electrode is not suitable for use in flow-through systems, on account of washing away of the liquid exchanger.
I 5
I
4
I
3
1
I
2
1
PC Fig. 2. Calibration curves of cloxacillin electrode. A: l-cloxacillin; 2-benzylpenicillin; 4-amoxycillin; B: I-cloxacillin; I-nitrate; 6-chloride; ‘I-acetate.
3-ampicillin;
Membrane electrode for cloxacillin Table 3. Results of cloxacillin determination
Table 2. Selectivity coefficients Species x (O.OlM)
KZ:, r 0.096 0.019 0.014
Benxylpenicillin Ampicillin Amoxicillin
6-APA acid Penicilloate Nitrate Chloride Acetate
Name of penicillin
1
Cloxacillin std. devn., mgll.
z3
79
399 40.8
51.9
Cloxacillin Ampicillin
250 250
6
251
2
Cloxacillin Ampicillin
500 250
7
513
12
Cloxacillin Amnicillin
250 500
7
264
4
40:o
0.05 0.0018 0.001
Assay of pharmaceuticals The standard addition technique was used, for samples at an ionic strength of 0.05M and a pH of 7.0. The results are given in Table 3. DISCUSSION As follows from Tables l-3, the cloxacillin electrode is characterized by good analytical parameters. The response time of 30 set, advantageous detection
Injettrd
c,. V,
"P
?
11OmV G
Dilution l+l
5z.c Time Fig. 3. Response time of cloxacillin electrode: c,, = 10-‘&f; c,=O.lM; v,=20ml; u,=lml; dilution l:l.
. r
6
limit of 5 x 10b5M and relatively good selectivity coefficients for inorganic ions make it useful for cloxacillin determination. The selectivity coefficients of the cloxacillin electrode for penicillins decrease in the order cloxacillin > benzylpenicillin > ampicillin > amoxicillin, and this order can be explained in items of the change of substituent character in the penicillin sidechain. Of the penicillins examined, cloxacillin has the strongest affinity for the ion-exchanger owing to the presence of a heterocyclic isoxazolyl substituent. Amoxicillin, which has amine and hydroxyl groups on the benzyl substituent, has the lowest affinity for the ion-exchanger; the atIinity of ampicillin, which has only an amine group as the substituent, is slightly higher. The affinity of benzylpenicillin is only slightly smaller than that of cloxacillin. Cloxacillin has been determined with the electrode (Table 3) in both pure preparations of the penicillin and in solutions corresponding to commercial formulations containing cloxacillin and ampicillin (Ampliclox and Ampiclox Neonatal, Beecham). The selectivity is sufficient for determination of cloxacillin in such mixtures. The results show that the electrode can be employed for control analysis of antibiotics or for determinations made in hospital laboratories. The potentiometric determination of cloxacillin (a pencillin which is insensitive to enzyme action) creates new possibilities for application of ion-selective electrodes. It has been found to give satisfactory results for determination of cloxacillin in serum, and its use for control of cloxacillin production is being investigated.
1Omv
REFERENCES l
.--OF\, . \
PH Fig. 4. Effect of pH on response of electrode to cloxacillin. TAL 3614-F
n
Found, mgll. 601
Cloxacillin
0.15
The dependence of the electrode potential on the pH of the solution was examined by adding 0.05M hydrochloric acid or sodium hydroxide dropwise to 20 ml of a stirred lo-‘M solution of cloxacillin. After each addition of acid or base, the electrode potential and pH were measured. The cloxacillin electrode is effective at pH 5-9 (Fig. 4).
t
Taken, mgll.
0.005
Eflect of pH
cD’
511
1. Farmakopea Polska, 1974. 2. British Pharmacoweia, The
Pharmaceutical Press.
London, 1973. s 3. T. Higuchi and E. Brochmann-Hanssen. Pharmaceutical Analvsis, Interscience, New York, 1961. 4. B. Casu and-P. Yentura, J. jharm. Sci. 1974, 63,211. 5. M. Gttis and M. Mtit, Cesk. Farm., 1986, 35, 119. 6. M. M. Ellaithy and M. G. El-Bardicy, Phurm. Sci., 1985, 47, 1974.
512
RYSZARDDUMKIEWICZ
7. J. A. Squella and L. J. Nufiez-Vergara, J. Electroanal.
8. 9. 10. 11. 12.
Chem. Znterfic. Electrochem., 1981, 130, 361. B. Karlberg and U. Forsman, Anal. Chim. Acta, 1976, 83,309. S. S. M. Hassan, M. T. M. Zaki and M. H. Eldesouki, Talanta, 1979, 26, 91. D. L. Simpson and R. K. Kobos, Anul. Chem., 1983,55, 1974. Z&m, Anal. Chim. Acta, 1984, 164, 273. L. F. Cullen, J. F. Rusling, A. Schleifer and G. J. Papariello, Anal. Chem., 1974, 46, 1955.
13. G. J. Papariello, A. K. Mukjerji and C. M. Shearer, &id., 1973, 45, 790. 14. A. Junichi and 0. Tetsuo, Chem. Charm. Bull., 1986,34, 3522. 15. M. T. Flanagan and N. J. Carroll, Biotechnol. Bioeng., 1986, 28, 1093; Chem. Abstr., 1986, lOS,77456. 16. A. G. Dobrolyubov, S. B. Itsygin and T. P. Levadnaya, Antibiot. Med. Biotechnol., 1986, 31, 336. 17. R. Tor and A. Freeman, Anal. Chem., 1986,58, 1042. 18. K. Sykut, R. Dumkiewin and J. Dumkiewicz, Ann. Univ. M. Curie-Sklodowska, Sect. AA, 1978, 13, 1.