Determination of free disopyramide in blood serum in situ using a disopyramide-sensitive membrane electrode

ANALYTIC4 CHIMICA ACTA

ELSEVIER

Analytica

Chimica Acta 343 (1997) 79-83

Determination of free disopyramide in blood serum in situ using a disopyramide-sensitive membrane electrode Takashi Katsu*, Yuki Mori Faculty of Pharmaceutical Sciences, Okayam University, Tsushima, Okayama 700, Japan

Received 6 November 1996; accepted 6 January 1997

Abstract Free disopyramide levels in blood serum, the monitoring of which is especially effective for antiarrhythmic therapy, were determined in situ using a disopyramide-sensitive membrane electrode. Sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-

propyl)phenyl]borate was incorporated as the ion-exchanger and 2-fluoro-2’-nitrodiphenyl ether as the solvent mediator in a poly(viny1 chloride) membrane matrix to form the electrode. The lower limit of the detection for free disopyramide in serum was 0.8 PM. The free disopyramide concentrations measured by the ion-selective electrode agreed well with those determined by an established fluorescence polarization immunoassay that requires an additional ultrafiltration procedure to remove disopyramide bound to serum protein. Keywords: Ion-selective

electrode;

Disopyramide;

Blood analysis;

Serum; Drug monitoring

1. Introduction Ion-selective electrodes are used routinely in biomedical instruments to measure clinically important inorganic ions such as Na+, Kf, Ca2+ and Cl- in blood [l-3]. Several electrodes have been developed that completely cover the clinical concentration range of various drugs. These include the psychotherapeutic lithium [4,5], the antiepileptic bromide [6,7], the ant&rhythmic procainamide [8] and bretylium [9], along with the anti-inflammatory salicylate [lo]. Electrodes require high drug sensitivity and high

*Corresponding author. Tel.: (+81) 86-251-7955;

fax: (+81) 86-

255.7456. 0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(97)00029-9

selectivity against Na+ or Cl- present at high levels in serum and thus few are available. While developing electrodes that respond to various biologically active amines, we found that procainamide and bretylium electrodes can be prepared using a combination of the lipophilic ion-exchanger, sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-propyl)phenyllborate (NaHFPB), and the solvent mediator, 2fluoro-2’-nitrodiphenyl ether (FNDPE) [8,9]. We found that this combination was also suitable for constructing a disopyramide electrode. The electrode was quite effective for determining free disopyramide levels in serum, the monitoring of which is especially important in an&rhythmic therapy (free is defined as the unbound disopyramide concentration in undiluted serum samples). Similar antiarrhythmic drugs except for bretylium, did not interfere.

I: Katsu. Y Mori/Analytica

80

2. Experimental 2.1. Reagents The sources of the reagents were as follows: NaHFPB and FNDPE from Dojindo Laboratories (Kumamoto, Japan); poly(viny1 chloride) (PVC; degree of polymerization, 1020) from Nacalai Tesque (Kyoto, Japan); disopyramide phosphate, N-acetylprocainamide hydrochloride, bretylium tosylate, lidoCaine hydrochloride, procainamide hydrochloride and quinidine hydrochloride monohydrate from Sigma (St. Louis, MO); mexiletine hydrochloride from Nippon Boehringer Ingelheim (Hyogo, Japan); sotalol hydrochloride from Bristol-Myers Squibb (Tokyo); tocainide hydrochloride from Astra Hassle (Molndal, Sweden); mouse anti-disopyramide monoclonal antibody from Chemicon lntemational (Temecula, CA). All the other chemicals were of analytical reagent grade. 2.2. Electrode

system

The disopyramide electrode was constructed similarly to that for bretylium [9], The components of the sensor membrane were 0.1 mg of NaHFPB, 60 ~1 of FNDPE and 30 mg of PVC. The materials were dissolved in tetrahydrofuran (about 1 ml), poured into a flat Petri dish (30 mm diameter), and the solvent was evaporated at room temperature. The resulting membrane was excised and attached to a PVC tube (4 mm outer diameter, 3 mm inner diameter) with tetrahydrofuran adhesive. PVC membranes containing other solvent mediators were prepared in the same manner. Each PVC tube was filled with an internal solution comprising 1 mM disopyramide phosphate, 10 mM NaCl and 10 mM NaH2P04/Na2HP04 (pH 7.4) and the sensor membrane was conditioned overnight. The electrochemical cell arrangement was: Ag, AgCllinternal solutionlsensor membranelsample solution11 M NH4N0s (salt bridge)110 mM KClIAg, AgCl. The electromotive force (e.m.f.) between the silver/silver chloride electrodes was measured using a voltmeter with high input impedance produced by a field-effect transistor operational amplifier (LF356; National Semiconductor, Sunnyvale, CA; input resistance >1012 0 and recorded. The detection limit was defined as the intersection

Chimica Acta 343 (1997) 79-83

of the extrapolated linear regions of the calibration graph [8,9]. The selectivity coefficients of the electrode, k:“, were determined by the separate solution method [8,9] using the respective chlorides, except for disopyramide and bretylium, for which we used the phosphate and tosylate, respectively. The values were calculated from the equation, log$J’=

(E,-Ei)/S+logci-lOgC:‘“,

where Ei and Ej represent the e.m.f. readings measured for disopyramide and the interfering ion, respectively, S is the slope of the calibration graph for disopyramide, ci and cj are the concentrations of disopyramide and the interfering ion, respectively, and zj is the charge of the interfering ion. Test reagents were dissolved in a buffer comprising 0.5 M Whydroxymethyl)aminomethane-HCl (pH 7.4) at a concentration of 10 mM, except for quinidine, which was adjusted to 5 mM because of low solubility in the buffer. A typical serum disopyramide assay proceeded as follows. The electrodes were placed in 100 pl of serum and constantly stirred with a bar. This electrode system, including the reference electrode [ll], is compact. Therefore, volumes as low as 100 pl can be assayed. Samples containing disopyramide were prepared by adding disopyramide phosphate to human serum. Between measurements, the electrode was soaked in distilled water for 20-30 s, rinsed several times with distilled water and wiped. The electrode was stored in 1 mM disopyramide phosphate containing 10 mM NaCl and 10 mM NaH2POfla2HPOd (pH 7.4) when not in use. All measurements were performed at a constant temperature of 25°C.

2.3. Fluorescence

polarization

immunoassay

Disopyramide concentrations in sera were also determined by a fluorescence polarization immunoassay using a TDX@ Automated Fluorescence Polarization Analyzer (Abbott, Abbott Park, IL). The serum samples were ultrafiltered through Centricon-30 (Amicon, Beverly, MA; molecular weight cut-off >30 000) to remove disopyramide bound to serum protein, and free disopyramide concentrations in the serum samples were determined in 50 pl of filtrate.

81

T Katsu, Y Mori/Analytica Chimica Acta 343 (1997) 79-83

3. Results and discussion Line a (0-o)

3.1. Response characteristics deproteinized

in serum and

Line b (O-O)

serum

To select the most suitable electrode, we initially determined the effect of solvent mediators on the response to disopyramide in serum, since these largely affect the response characteristics of the electrode [8,9,12-151. Calibration graphs were obtained by measuring known amounts of disopyramide phosphate added to serum samples from a normal donor and by plotting the concentrations against the corresponding e.m.f. values. The solvent mediators tested were FNDPE, o-nitrophenyl octyl ether, dioctyl phthalate, bis(2-ethylhexyl) sebacate, tris(2-ethylhexyl) phosphate and tricresyl phosphate. Of these, the electrode containing FNDPE was the most sensitive to disopyramide in serum. The sensitivity of the electrode was also dependent on the amount of ion-exchanger; 0.1 mg NaHFPB gave the highest sensitivity to disopyramide in serum, similarly to the bretylium electrode [9]. Therefore, a combination of NaHFPB and FNDPE with the membrane composition described in the experimental section was prepared and Fig. 1 (line a) shows the calibration graph for this electrode for disopyramide in serum. The electrode calibration completely covered the clinical concentration range of disopyramide in serum required for antiarrhythmic therapy (2-6 pg ml-‘; 618 PM) [16]. This concentration range describes the total disopyramide concentration of both free and bound drug in serum. The sensitivity of the electrode response was 63 mV per concentration decade and the lower limit of serum disopyramide detection was 1 PM. The response time of the electrode (90% final signal) was below 10 s when the concentration of disopyramide was changed from 5 to 50 pM. We constructed a calibration graph after the serum sample was ultrafiltered through Centricon(Fig. 1, line b). The response slope was reduced to 55 mV per concentration decade and the detection limit for disopyramide was slightly improved to 0.8 PM. As disopyramide binds to serum protein [16], the free concentration of disopyramide decreases in the presence of serum, and thus the calibration graph from the serum sample (line a) shifted to a higher concentration than that from the deproteinized sample (line b). The

Line c (X---X)

-lOOt_

9

5

-150

w

I

-200 I I

-I

1

-8

-7

-6

Log [Disopyramide]

-5

-4

(M)

Fig. 1. Comparison of the electrode response to disopyramide in (a) serum (O), (b) deproteinized serum (0) and (c) physiological saline containing 0.15 M NaCl and 5 mM 4-(2_hydroxyethyl)-2piperazineethanesulfonic acid-NaOH (pH 7.4) (x). The clinical concentration range required for antiarrhythmic therapy is also shown.

higher slope (63 mV) obtained using serum can be explained by the concentration dependence of disopyramide binding to serum protein, as in salicylate binding in serum [lo]. That is, at lower concentrations of disopyramide, a large portion of the drug is bound to serum proteins, while at the higher ranges, the portion of the drug bound to proteins is decreased, thus generating a steeper slope. The percentage of disopyramide bound to protein can be estimated by comparing these two calibration graphs (lines a and b), as the concentration of free disopyramide can be determined by line b and the total disopyramide (the sum of the concentrations of free disopyramide and that bound to protein) can be estimated from line a. For example, when 5 and 20 C {p } } M disopyramide were added to the serum, 66% and 45% of it was bound to serum protein, respectively. 3.2. Application in serum

to monitoring free disopyramide

For therapeutic drug monitoring, determining the free concentration of disopyramide is especially

82

I: Katsu, Y. Mori/Analytica

Table 1 Selectivity coefftcients, Interfering

log k$’ a

ion fj)

log k$’

Mg2+ Ca2+ Nat K’ CHsNH: Tocainide Sotalol

-5.6 -5.6 -4.6 -4.5 -4.2 -3.5 -3.5 -2.9 -2.8 -2.2 -2.1 -1.1 -1.0 -0.9 0.2 0.7

+ (CHskN N-Acetylprocainamide Mexiletine Procainamide + (&H&N Quinidine Lidocaine Bretylium + (CsH7)4N a i=Disopyramide

Chimica Acta 343 (1997) 79-83

and j=interfering

ion.

effective, as the degree of binding of this drug to serum protein is variable [ 17,181. Thus, our method is useful as the electrode is responsive only to the free concentration. Although the free concentration of disopyramide can be determined using the calibration graph shown in Fig. 1 (line b), we wished to construct a calibration graph from a much simpler solution, as to do so for each deproteinized serum sample is difficult. We found that the calibration graph in physiological saline containing 0.15 M NaCl and 5 mM 4-(2-hydroxyethyl)-2-piperazineethanesulfonic acid-NaOH (pH 7.4) shown in Fig. 1 (line c), was virtually superimposable on that of the deproteinized serum sample (line b), indicating that the free concentration of disopyramide can be estimated from line c. Table 1 summarizes the selectivity coefficients of the electrode and shows that it was sufficiently selective against Na+, K+ and 0 th er cations in serum. Furthermore, this electrode was not seriously affected by similar antiarrhythmic drugs such as N-acetylprocainamide, lidocaine, mexiletine, procainamide, quinidine, sotalol and tocainide. Only bretylium interfered. An anti-disopyramide antibody can distinguish the electrode response between disopyramide and an interfering drug. When serum contained disopyramide, the electrical potential significantly changed after an anti-disopyramide antibody was added, since the concentration of free disopyramide decreased upon binding with the specific antibody,

0

2

4

0

Time

t

4

(min)

Fig. 2. Changes in the electrical potential caused by an antidisopyramide antibody. Mouse anti-disopyramide antibody (10 pl) was added at time zero in serum (200 ~1) containing (a) 5 pM disopyramide phosphate and (b) 2 pM bretylium tosylate, respectively. The latter potential change was monitored using a bretylium electrode [9].

while serum containing an interfering drug such as bretylium did not induce such a large change, as shown in Fig. 2. Thus, the participation of disopyramide in the electrode response can be determined. We compared the free concentrations of disopyramide in serum samples determined by potentiometry using a calibration graph (Fig. 1, line c) with those determined by fluorescence polarization immunoassay after serum samples were ultrafiltered. Linear regression analysis of free disopyramide levels (OS-25 PM) obtained by potentiometry against values obtained by immunoassay showed good correlation. The slope and the intercept of the line were 0.911 and -0.123 c(M, respectively (-0.996; n=20). When we used a calibration graph measured in deproteinized serum (Fig. 1, line b), the correlation was further improved. The slope of the line was 0.970 and the intercept 0.022 uM (-0.996; n=20). The potentiometric method does not require ultrafiltration to remove disopyramide bound to serum protein. Thus, the method is much simpler, more rapid and more economical than the immunoassay. Free disopyramide concentrations in whole blood can also be monitored, as the procedure is independent of sample color or turbidity. This new method will markedly reduce the workload involved in therapeutic drug monitoring for disopyramide in a clinical setting.

Acknowledgements We thank Nippon Boehringer Ingelheim, BristolMyers Squibb and Astra Hassle for providing us with

T. Katsu, Z Mori/Analytica Chimica Acta 343 (1997) 79-83

mexiletine hydrochloride, sotalol hydrochloride and tocainide hydrochloride, respectively. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. References [l] U. Oesch, D. Ammann and W. Simon, Clin. Chem., 32 (1986) 1448. [2] T.P. Byrne, Sel. Electrode Rev., 10 (1988) 107. [3] A. Lewenstam, M. Maj-Zurawska and A. Hulanicki, Electroanalysis, 3 (1991) 727. [4] K. Kimura, H. Oishi, T. Miura and T. Shono, Anal. Chem., 59 (1987) 2331. [5] R.L. Bertholf, M.G. Savory, K.H. Winbome, J.C. Hundley, G.M. Plummer and J. Savory, Clin. Chem., 34 (1988) 1500. [6] T. Katsu, K. Furuno, S. Yamashita, H. Kawasaki, Y. Gomita, Y. Ohtsuka and S. Ohtahara, Clin. Chim. Acta, 234 (1995) 157.

83

171 T. Katsu, Y. Mori, N. Matsuka and Y. Gomita, J. Pharm. Biomed. Anal., in press. PI T. Katsu, K. Furuno, S. Yamashita and Y. Gomita, Anal. Chim. Acta, 312 (1995) 35. [91 T. Katsu, Y. Mori and H. Kataoka, Anal. Lett., 29 (1996) 1281. UOI T. Katsu and Y. Mori, Talanta, 43 (1996) 755. [Ill T. Katsu, H. Kobayashi and Y. Fujita, Biochim. Biophys. Acta, 860 (1986) 608. [W T. Katsu, T. Kayamoto and Y. Fujita, Anal. Chim. Acta, 239 (1990) 23. iI31 K. Watanabe, K. Okada and T. Katsu, Anal. Chim. Acta, 274 (1993) 59. 1141 K. Watanabe, K. Okada, H. Oda, K. Furuno, Y. Gomita and T. Katsu, Anal. Chim. Acta, 316 (1995) 371. u51 T. Katsu, K. Yamanaka, S. Hiramaki, T. Tanaka and T. Nagamatsu, Electroanalysis, in press. [161 W.G. Clark, D.C. Brater and A.R. Johnson, Goths Medical Pharmacology, 13th edn., Mosby-Year Book, St. Louis, MO, 1992. 1171 J. Koch-Weser, N. Engl. J. Med., 300 (1979) 957. U81 T. Iga and Y. Saito (Eds.), TDM No Jissai (Practice in TDM), Yakugyo Jiho Sha, Tokyo, 1993.