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Ion-selective electrode for procainamide determination in blood serum
ANALmcA CHIMICA ACTA
ELSEVIER
Analytica ChimicaActa 312 (1995) 35-38
Ion-selective electrode for procainamide determination in blood serum Takashi Katsu a3*, Katsushi Furuno b, Syoichi Yamashita b, Yutaka Gomita b aFaculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama 700, Japan b Department of Hospiial Pharmacy, Okayama University Medical School, Shikata-cho, Okayama 700, Japan Received 17 January
1995; revised 27 February
1995; accepted 4 April 1995
Abstract A procainamide-selective electrode was constructed and applied for the determination of procainamide concentration in blood serum. The detection limit was 1.5 pg ml-‘, but determination down to 0.5 pg ml-’ was possible with an appropriate calibration. The results correlated well with those obtained by a fluorescence polarization immunoassay which is widely used for the determination of serum procainamide concentration. The present method is simple, rapid, economical and is unaffected by common cations present in blood and only slightly by N-acetylprocainamide, a metabolite of the drug. It is therefore useful for therapeutic drug monitoring in a clinical setting. Keywords: Ion selective electrodes;
Procainamide;
Blood analysis;
Serum; Drug monitoring
1. Introduction
Ion-selective electrodes are widely applied for the determination of biologically and clinically important substances [1,2]. We have been interested in applying this technique for therapeutic drug monitoring in a clinical setting [3]. Such monitoring is fundamental to pharmacotherapy, since it can be used for adjusting the dose of drugs whose therapeutic and toxic concentration ranges are very similar. So far, a lithium ion-selective electrode has been successfully applied for monitoring the blood concentration of lithium carbonate, which is administered to patients with manic disorder [4], and cur-
* Corresponding
author.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10003-2670(95)00196-4
rently this electrode is very widely used in clinical laboratories because of its easy handling. We have recently succeeded in introducing a bromide ionselective electrode for determination of serum bromide concentrations in patients with epilepsy [5]. We were particularly interested in extending the use of ion-selective electrodes to therapeutic monitoring of organic pharmaceutical agents, because application has hitherto been limited to inorganic medicines such as lithium or bromide, as described above. Among the many organic medicines, we chose procainamide for the present study, because this drug requires a rather high dose (4-8 pg ml-’ > for effective therapy [3,6] and has a low ability to bind with serum protein [3], resulting in a high free concentration in serum. Thus it was expected that determination of this drug would be readily accomplished using the ion-selective electrode.
36
T. Katsu et al. /Analytica Chimica Acta 312 (1995) 35-38
The procainamide-selective electrode for use in this study was constructed by incorporating sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-propyl)pheny&orate (NaHFPB) as the ion exchanger and 2-fluoro-Z-nitrodiphenyl ether (FNDPE) as the membrane solvent into a poly(viny1 chloride) (PVC) membrane matrix. NaHFPB was chosen because of its highly lipophilic character and high stability [7] and FNDPE was found by screening to be the most suitable membrane solvent for determination of procainamide in serum. To our knowledge, this is the first attempt to apply an organic ion-sensitive electrode for therapeutic drug monitoring.
2. Experimental 2.1. Reagents The sources of the reagents used were as follows: NaHFPB and FNDPE from Dojindo Labs.; PVC (degree of polymerization, 1020) from Nacalai Tesque; procainamide hydrochloride and Nacetylprocainamide hydrochloride from Sigma. All the other chemicals used were of analytical reagent grade. 2.2. Electrode system The procainamide-selective membrane electrode was based on a PVC membrane [8,9] and its components were 0.5 mg of NaHFPB, 60 ~1 of FNDPE and 30 mg of PVC. The materials were dissolved in tetrahydrofuran, poured into a flat petri dish (30 mm diameter) and the solvent was allowed to evaporate at room temperature. The resulting membrane was cut out and stuck to a PVC tube (4 mm o.d., 3 mm i.d.) using tetrahydrofuran as an adhesive. The PVC tube was filled with an internal solution of 1 mM procainamide hydrochloride and 10 mM NaCl, and the sensor membrane was conditioned for several hours. The electrochemical cell arrangement was: Ag, AgCl/intemal solution/sensor membrane/sample solution/l M NH,NO, (salt bridge)/10 mM KCl/Ag, AgCl. The electromotive force (e.m.f.1 between the silver/silver chloride electrodes was measured using a voltmeter of high input impedance made with a field-effect transistor operational ampli-
fier (LF356, National Semiconductor; input resistance > 1012 fI) and recorded. The selectivity coefficients of the electrode, k!q’, were determined by the separate solution method using the respective chloride salts at 10 mM [8,9] and calculated from the equation, log kz’ = ( Ej - Ei)/ S + log ci - log c!/‘j, where Ei and Ej represent the e.m.f. readings measured for procainamide and interfering ion, respectively, S is the slope of the calibration graph for procainamide, ci and cj are the concentrations of procainamide and interfering ion, respectively, and zj is the charge of interfering ion. A typical procedure for determining procainamide in serum was as follows. The electrodes were placed into 100 ~1 of semm under constant stirring with a stirring bar. It should be emphasized that the present electrode system, including the reference electrode [lo], is compact, and therefore assay solution volumes as small as 100 ~1 can be used. Procainamide-containing serum samples were prepared by adding procainamide hydrochloride to human serum. All measurements were carried out at a constant temperature of 25°C. 2.3. Fluorescence polarization immunoassay Procainamide concentrations in sera were also determined by a fluorescence polarization immunoassay using a TDX@ Automated Fluorescence Polarization Analyzer (Abbott) [3,11].
3. Results and discussion Using NaHFPB as an ion exchanger, the effect of membrane solvents was first examined to obtain the most suitable electrode for the present purpose. The membrane solvents tested were FNDPE, onitrophenyl octyl ether, dioctyl phthalate, bis(Zethylhexyl) sebacate, tris(Zethylhexy1) phosphate and tricresyl phosphate. Among them, FNDPE had the highest sensitivity to procainamide in blood serum. A combination of NaHFPB and FNDPE was used, and Fig. 1 shows the calibration graph of this electrode for procainamide in serum. The slope within the linear range was 57 mV per decade. The lower limit of detection was 1.5 Fg ml-‘; the linear regions of the calibration graph were extrapolated and
31
T. Katsu et al./Analytica Chimica Acta 312 (1995) 35-38
-60
Table 1 Selectivity
coefficients,
Interfering
ion
log k$’ 0 log k:’
(j) -80
s
-6.1 -5.6 - 4.4 -3.3 -3.0 -0.9 -0.8 1.0
Mg*+ Ca*+ Na+ K’ CH,NH;
S
-100
Ill
(cH~)~N+ N-Acetylprocainamide
-120
-140
clinical range
I 0.2
0.5
1
2
5
(C2H5),N+
1
I
10
20
a i = Procainamide
and j = interfering
ion.
[Procainamide] (pg ml-l) Fig. 1. Response of the procainamide-selective electrode in human serum. The clinical concentration range for procainamide is also shown.
the intersection point was taken as the detection limit [8,9]. It should be emphasized, however, that the determination of procainamide down to 0.5 pg ml-’ was still easy with an appropriate calibration, as shown in Fig. 1. The sensitivity of the electrode was adequate for measurement of the procainamide concentration range required for pharmacotherapy (4-8 pg ml-‘) [3,6]. The response time (90% final signal) of the electrode was less than 10 s when the concentration of procainamide was changed from 0.5 to 5 pg ml- ‘. The selectivity coefficients of the electrode are shown in Table 1. The electrode suffered no serious interference from Na+, K+ and other cations present in blood serum. It is worth noting that the
Immunoassay (pg ml-l) Fig. 2. Correlation of procainamide concentrations in 20 serum samples determined by potentiometry using a procainamide-selective electrode and by fluorescence polarization immunoassay.
present electrode did not respond strongly to Nacetylprocainamide, which is a metabolic product of procainamide, and thus the electrode escaped serious interference from this metabolite. A series of 12 successive determinations of a sample containing 5 pg ml-’ or 10 pg ml-’ procainamide was used to evaluate the precision of the measurement. The mean e.m.f. value at 5 pg ml-’ was - 103.5 mV (0.7% relative standard deviation) with a range of - 103 to - 105 mV, while that at 10 pg ml-’ was - 88.9 mV (0.5% relative standard deviation) with a range of -88 to -89 mV. We compared the present method with a fluorescence polarization immunoassay [3,111 commonly used in clinical laboratories, and a good correlation was obtained, as shown in Fig. 2. As already mentioned by many workers, the use of an ion-selective electrode has inherent advantages over various other analytical methods because it requires no special sample pretreatment, the analysis time is shorter, and the equipment necessary is not expensive. Monitoring of procainamide in whole blood is also possible, since the procedure is independent of sample color or turbidity. It is obvious that this new method will reduce markedly the workload involved in therapeutic drug monitoring for procainamide in a clinical setting.
Acknowledgements This work was supported by a Grant-in-Aid from the Takeda Science Foundation and a Grant-in-Aid
38
T. Katsu et al./Analytica
for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References [l] A. Lewenstam, M. Maj-Zurawska and A. Hulanicki, Electroanalysis, 3 (1991) 727. [2] J. Wang, Anal. Chem., 65 (1993) 450R. [3] T. Iga and Y. Saito (Eds.), TDM No Jissai (Practice in TDM), Yakugyo Jiho Sha, Tokyo, 1993.
Chimica Acta 312 (1995) 35-38 [4] R.L. Bertholf, M.G. Savory, K.H. Winbome, J.C. Hundley,
G.M. Plummer and J. Savory, Clin. Chem., 34 (1988) 1500. [5] T. Katsu, K. Furuno, S. Yamashita, H. Kawasaki, Y. Gomita, Y. Ohtsuka and S. Ohtahara, Clin. Chim. Acta, 234 (1995) 157. [6] J. Koch-Weser and S.W. Klein, JAMA, 215 (1971) 1454. [7] G.H. Zhang, T. Imato, Y. Asano, T. Sonoda, H. Kobayashi and N. Ishibashi, Anal. Chem., 62 (1990) 1644. [8] T. Katsu, T. Kayamoto and Y. Fujita, Anal. Chim. Acta, 239 (1990) 23. [9] T. Katsu, Anal. Chem., 65 (1993) 176. [lo] T. Katsu, H. Kobayashi and Y. Fujita, Biochim. Biophys. Acta, 860 (1986) 608. [ll] M. Hayashi and S. Awazu, Saishin Kensa, 3 (1985) 11.
ELSEVIER
Analytica ChimicaActa 312 (1995) 35-38
Ion-selective electrode for procainamide determination in blood serum Takashi Katsu a3*, Katsushi Furuno b, Syoichi Yamashita b, Yutaka Gomita b aFaculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama 700, Japan b Department of Hospiial Pharmacy, Okayama University Medical School, Shikata-cho, Okayama 700, Japan Received 17 January
1995; revised 27 February
1995; accepted 4 April 1995
Abstract A procainamide-selective electrode was constructed and applied for the determination of procainamide concentration in blood serum. The detection limit was 1.5 pg ml-‘, but determination down to 0.5 pg ml-’ was possible with an appropriate calibration. The results correlated well with those obtained by a fluorescence polarization immunoassay which is widely used for the determination of serum procainamide concentration. The present method is simple, rapid, economical and is unaffected by common cations present in blood and only slightly by N-acetylprocainamide, a metabolite of the drug. It is therefore useful for therapeutic drug monitoring in a clinical setting. Keywords: Ion selective electrodes;
Procainamide;
Blood analysis;
Serum; Drug monitoring
1. Introduction
Ion-selective electrodes are widely applied for the determination of biologically and clinically important substances [1,2]. We have been interested in applying this technique for therapeutic drug monitoring in a clinical setting [3]. Such monitoring is fundamental to pharmacotherapy, since it can be used for adjusting the dose of drugs whose therapeutic and toxic concentration ranges are very similar. So far, a lithium ion-selective electrode has been successfully applied for monitoring the blood concentration of lithium carbonate, which is administered to patients with manic disorder [4], and cur-
* Corresponding
author.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10003-2670(95)00196-4
rently this electrode is very widely used in clinical laboratories because of its easy handling. We have recently succeeded in introducing a bromide ionselective electrode for determination of serum bromide concentrations in patients with epilepsy [5]. We were particularly interested in extending the use of ion-selective electrodes to therapeutic monitoring of organic pharmaceutical agents, because application has hitherto been limited to inorganic medicines such as lithium or bromide, as described above. Among the many organic medicines, we chose procainamide for the present study, because this drug requires a rather high dose (4-8 pg ml-’ > for effective therapy [3,6] and has a low ability to bind with serum protein [3], resulting in a high free concentration in serum. Thus it was expected that determination of this drug would be readily accomplished using the ion-selective electrode.
36
T. Katsu et al. /Analytica Chimica Acta 312 (1995) 35-38
The procainamide-selective electrode for use in this study was constructed by incorporating sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-propyl)pheny&orate (NaHFPB) as the ion exchanger and 2-fluoro-Z-nitrodiphenyl ether (FNDPE) as the membrane solvent into a poly(viny1 chloride) (PVC) membrane matrix. NaHFPB was chosen because of its highly lipophilic character and high stability [7] and FNDPE was found by screening to be the most suitable membrane solvent for determination of procainamide in serum. To our knowledge, this is the first attempt to apply an organic ion-sensitive electrode for therapeutic drug monitoring.
2. Experimental 2.1. Reagents The sources of the reagents used were as follows: NaHFPB and FNDPE from Dojindo Labs.; PVC (degree of polymerization, 1020) from Nacalai Tesque; procainamide hydrochloride and Nacetylprocainamide hydrochloride from Sigma. All the other chemicals used were of analytical reagent grade. 2.2. Electrode system The procainamide-selective membrane electrode was based on a PVC membrane [8,9] and its components were 0.5 mg of NaHFPB, 60 ~1 of FNDPE and 30 mg of PVC. The materials were dissolved in tetrahydrofuran, poured into a flat petri dish (30 mm diameter) and the solvent was allowed to evaporate at room temperature. The resulting membrane was cut out and stuck to a PVC tube (4 mm o.d., 3 mm i.d.) using tetrahydrofuran as an adhesive. The PVC tube was filled with an internal solution of 1 mM procainamide hydrochloride and 10 mM NaCl, and the sensor membrane was conditioned for several hours. The electrochemical cell arrangement was: Ag, AgCl/intemal solution/sensor membrane/sample solution/l M NH,NO, (salt bridge)/10 mM KCl/Ag, AgCl. The electromotive force (e.m.f.1 between the silver/silver chloride electrodes was measured using a voltmeter of high input impedance made with a field-effect transistor operational ampli-
fier (LF356, National Semiconductor; input resistance > 1012 fI) and recorded. The selectivity coefficients of the electrode, k!q’, were determined by the separate solution method using the respective chloride salts at 10 mM [8,9] and calculated from the equation, log kz’ = ( Ej - Ei)/ S + log ci - log c!/‘j, where Ei and Ej represent the e.m.f. readings measured for procainamide and interfering ion, respectively, S is the slope of the calibration graph for procainamide, ci and cj are the concentrations of procainamide and interfering ion, respectively, and zj is the charge of interfering ion. A typical procedure for determining procainamide in serum was as follows. The electrodes were placed into 100 ~1 of semm under constant stirring with a stirring bar. It should be emphasized that the present electrode system, including the reference electrode [lo], is compact, and therefore assay solution volumes as small as 100 ~1 can be used. Procainamide-containing serum samples were prepared by adding procainamide hydrochloride to human serum. All measurements were carried out at a constant temperature of 25°C. 2.3. Fluorescence polarization immunoassay Procainamide concentrations in sera were also determined by a fluorescence polarization immunoassay using a TDX@ Automated Fluorescence Polarization Analyzer (Abbott) [3,11].
3. Results and discussion Using NaHFPB as an ion exchanger, the effect of membrane solvents was first examined to obtain the most suitable electrode for the present purpose. The membrane solvents tested were FNDPE, onitrophenyl octyl ether, dioctyl phthalate, bis(Zethylhexyl) sebacate, tris(Zethylhexy1) phosphate and tricresyl phosphate. Among them, FNDPE had the highest sensitivity to procainamide in blood serum. A combination of NaHFPB and FNDPE was used, and Fig. 1 shows the calibration graph of this electrode for procainamide in serum. The slope within the linear range was 57 mV per decade. The lower limit of detection was 1.5 Fg ml-‘; the linear regions of the calibration graph were extrapolated and
31
T. Katsu et al./Analytica Chimica Acta 312 (1995) 35-38
-60
Table 1 Selectivity
coefficients,
Interfering
ion
log k$’ 0 log k:’
(j) -80
s
-6.1 -5.6 - 4.4 -3.3 -3.0 -0.9 -0.8 1.0
Mg*+ Ca*+ Na+ K’ CH,NH;
S
-100
Ill
(cH~)~N+ N-Acetylprocainamide
-120
-140
clinical range
I 0.2
0.5
1
2
5
(C2H5),N+
1
I
10
20
a i = Procainamide
and j = interfering
ion.
[Procainamide] (pg ml-l) Fig. 1. Response of the procainamide-selective electrode in human serum. The clinical concentration range for procainamide is also shown.
the intersection point was taken as the detection limit [8,9]. It should be emphasized, however, that the determination of procainamide down to 0.5 pg ml-’ was still easy with an appropriate calibration, as shown in Fig. 1. The sensitivity of the electrode was adequate for measurement of the procainamide concentration range required for pharmacotherapy (4-8 pg ml-‘) [3,6]. The response time (90% final signal) of the electrode was less than 10 s when the concentration of procainamide was changed from 0.5 to 5 pg ml- ‘. The selectivity coefficients of the electrode are shown in Table 1. The electrode suffered no serious interference from Na+, K+ and other cations present in blood serum. It is worth noting that the
Immunoassay (pg ml-l) Fig. 2. Correlation of procainamide concentrations in 20 serum samples determined by potentiometry using a procainamide-selective electrode and by fluorescence polarization immunoassay.
present electrode did not respond strongly to Nacetylprocainamide, which is a metabolic product of procainamide, and thus the electrode escaped serious interference from this metabolite. A series of 12 successive determinations of a sample containing 5 pg ml-’ or 10 pg ml-’ procainamide was used to evaluate the precision of the measurement. The mean e.m.f. value at 5 pg ml-’ was - 103.5 mV (0.7% relative standard deviation) with a range of - 103 to - 105 mV, while that at 10 pg ml-’ was - 88.9 mV (0.5% relative standard deviation) with a range of -88 to -89 mV. We compared the present method with a fluorescence polarization immunoassay [3,111 commonly used in clinical laboratories, and a good correlation was obtained, as shown in Fig. 2. As already mentioned by many workers, the use of an ion-selective electrode has inherent advantages over various other analytical methods because it requires no special sample pretreatment, the analysis time is shorter, and the equipment necessary is not expensive. Monitoring of procainamide in whole blood is also possible, since the procedure is independent of sample color or turbidity. It is obvious that this new method will reduce markedly the workload involved in therapeutic drug monitoring for procainamide in a clinical setting.
Acknowledgements This work was supported by a Grant-in-Aid from the Takeda Science Foundation and a Grant-in-Aid
38
T. Katsu et al./Analytica
for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References [l] A. Lewenstam, M. Maj-Zurawska and A. Hulanicki, Electroanalysis, 3 (1991) 727. [2] J. Wang, Anal. Chem., 65 (1993) 450R. [3] T. Iga and Y. Saito (Eds.), TDM No Jissai (Practice in TDM), Yakugyo Jiho Sha, Tokyo, 1993.
Chimica Acta 312 (1995) 35-38 [4] R.L. Bertholf, M.G. Savory, K.H. Winbome, J.C. Hundley,
G.M. Plummer and J. Savory, Clin. Chem., 34 (1988) 1500. [5] T. Katsu, K. Furuno, S. Yamashita, H. Kawasaki, Y. Gomita, Y. Ohtsuka and S. Ohtahara, Clin. Chim. Acta, 234 (1995) 157. [6] J. Koch-Weser and S.W. Klein, JAMA, 215 (1971) 1454. [7] G.H. Zhang, T. Imato, Y. Asano, T. Sonoda, H. Kobayashi and N. Ishibashi, Anal. Chem., 62 (1990) 1644. [8] T. Katsu, T. Kayamoto and Y. Fujita, Anal. Chim. Acta, 239 (1990) 23. [9] T. Katsu, Anal. Chem., 65 (1993) 176. [lo] T. Katsu, H. Kobayashi and Y. Fujita, Biochim. Biophys. Acta, 860 (1986) 608. [ll] M. Hayashi and S. Awazu, Saishin Kensa, 3 (1985) 11.