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Anal. Chem. 1990. 62, 932-936
Figures 8, 10, and 11are different, as well as their relative size. Under certain sets of experimental conditions the second signal may disappear, by cancellation of the responses of the detector for the two concentration perturbations. We have discussed above the properties of solutions of one or two solutes dissolved in a pure solvent used as mobile phase. We have shown that the velocity of each pulse can be related to the derivatives of the isotherm. It is easy to generalize our results to the case of a N component solution. Because of the coupling effect between the components of the mobile phase, the velocity eigenvalues are related to the slopes of the tangents to the multidimensional surface in the N - 1composition path directions. These slopes can be calculated when the isotherm surface is known. Conversely, the systematic measurement of the retention times of very small vacancy pulses for various compositions of the mobile phase may permit the determination of competitive equilibrium isotherms, provided a suitable set of isotherm equations is available. Least-squares fitting of the set of slope data on the isotherm equations allow the calculation of the isotherm parameters. Finally, the method discussed here for the prediction of system peaks and of vacancy chromatograms can be used as well for the calculation of the column response to more complex injections combining positive pulses of certain compounds and negative pulses of others in a mobile phase containing some of these compounds. Only the initial and boundary conditions have to be changed.
ACKNOWLEDGMENT We gratefully acknowledge support of our computational effort by the University of Tennessee Computing Center. LITERATURE CITED (1) Solms, D. J.; Smuts, T. W.; Pretorius, V. J. Chromatogr. Sci. 1971, 9 , 600.
(2) Zhukhovitskli, A. A.; Turkel'taub. N. M. Wl.Akad. Nauk 1962, 743, 646. (3) McCormick, R. M.; Karger, B. L. J . Chromatogr. 1980, 199, 259. (4) Berek, D.; Bleha, T.; Pevana, Z. J . Chromatogr. Scl. 1978, 14, 560. (5) Perlman, S.; Kirschbaum, J. J. J . Chromatogr. 1988, 357, 39. (6) Levin, S.; Grushka, E. Anal. Chem. 1988, 5 8 , 1602. (7) Levin, S.; Grushka, E. Anal. Cbem. 1987, 5 9 , 1157. (8) Hemerich, F.; Klein, G. Multicomponent Chromatography. A Theory of Interferences; M. Dekker: New York, 1970. (9) Knox, J. H.; Kaliszan, R. J. Chromatogr. 1985, 349, 211. (10) Crommen, J.; Schiil, G.; Herne, P. Chromatographie 1988, 2 5 , 397. (11) Crommen, J.; Schill. G.; Westerlund, D.; Hackzell. L. Chromatographla 1987, 24, 252. (12) Stahlberg, J.; Aimgren, M. Anal. Ch8m. 1989, 67. 1109. (13) Sokolowski, A. Chromatographla 1986, 2 2 , 177. (14) Arvidsson, E.; Crommen, J.; Schill, G.; Westerlund, D. J. Chromatogr. ism. 461.429. . - .. (15) Bilingmeyer, 8. A.: Deming, S. N.; Price Jr., W. P.; Sachok, 9.; Petrusek. M. J. Chromatoor. 1979. 786. 419. (16) Denkert, M.; HackzelcL.; Schill, G.; Sjogren, E. J . Chromatogr. 1981, 218, 31. (17) Golshan-Shirazi, S.; Guiochon, G. J . Chromatogr. 1989, 461, 1. (18) Golshan-Shirazi, S.; Guiochon, G. Anal. Chem. 1989, 6 7 , 2373. (19) Golshan-Shirazi, S.; Guiochon, G. J . Chromatogr. 1989, 467, 19. (20) Golshan-Shirazl, S.; Guiochon, G. Anal. Chem. 1989, 67,2360. (21) Guiochon, G.; Golshan-Shirazi, S.; Jaulmes, A. Anal. Chem. 1988, 6 0 , 1856. (22) Lin, 9.; Guiochon. G. S e p . Sci. Techno/. 1989, 2 4 , 31. (23) Glover, C. J.; Lau, W. R. AIChf J. 1983. 2 9 , 73. (24) Gnanasambandan, T.; Freiser, H. Anal. Chem. 1982, 5 4 , 1262. (25) Small, H.; Miller, T. E., Jr. Anal. Chem. 1982, 54, 462. (26) Heren. P.; Renson, M.; Crommen, J. Chromatographie 1984. 79, 274. (27) Reilley, C. N.; Hildebrand, G. P.; Ashley, J. W., Jr. Anal. Chem. 1982, 34, 1198. (28) Jauimes, A.; VidaCMadjar, C.; Ladurelli, A,; Guiochon, G. J Phys. Chem. 1984, 8 8 , 5379. (29) Helfferich, F.; Peterson, D. L. Science 1963, 742, 661.
----.
RECEIVED for review November 29,1989. Accepted January 24, 1990. This work has been supported in part by Grant CHE-8901382 of the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory.
Flow System for Direct Determination of Enzyme Substrate in Undiluted Whole Blood T. Buch-Rasmussen Radiometer Medical AIS, Emdrupvej 72, DK-2400 Copenhagen, Denmark
Enzyme sensors requiring reagent and contrdled pH to detect substrate at non-steady-state conditions are described. Hematocrit dependence in sample transportatlon, in sample and reagent mixing, and In sample dialysis Is mlnknired in the system by the use of segmented sample injection, membrane deposited reagent, and membranes of low permeablllty. The clinical features of the flow system are illustrated by a B-0glucose determination. Other aspects of the flow system are illustrated by L-lactate and creatinine detennlnatkns. All three assays end up with a NADH detection at a chemically modified electrode (CME).
INTRODUCTION A time-consuming step in blood analysis is the often necessary separation of blood cells and plasma. This separation
is done to avoid the blood cells, which influence many sample operations in flow systems. Harrow et al. (1,2),when determining pH and COPcontent in whole blood, were the first to point out the difficulties associated with the varying hematocrit level in blood samples, observing that the measurement was unreliable unless minimum dispersion in a flow system was accomplished. However, at minimum dispersion the power to do chemistry is limited. The errors are related to a dilution effect and a hydrodynamic effect of the red blood cells on the dispersion. Petersson et al. (3)demonstrated in a urea determination that it is possible to perform chemistry on whole blood samples without interference from the blood cells, by use of time/space kinetic discrimination in a flow injection analysis (FIA) system with minimal dispersion. Another interference, the blood cell volume effect, is seen in dialysis of whole blood. The cells reduce the active sample diffusion volume in front of the dialysis membrane. Risinger
0003-2700/90/0362-0932$02.50/00 1990 American Chemical Society
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b
Flgure 1. CME and the combined auxiliary (A) and reference (REF) electrodes placed in two connected measuring cells (7 pL each).
et al. (4) reduced this problem by using dialysis membranes with low permeabilities. In this work a general method for the direct determination of metabolites in undiluted whole blood, using memhranedeposited reagent in a single line flow system, is described. Segmented sample injection (5) was used to transport the sample, in a nondispered mode, through a measuring cell with a membrane-covered detector. The membrane was equilibrated with reagent from the d e r solution before the sample was introduced, and while sample contacts the membrane, the required quantity of substrate diffuses in, and chemical reaction can take place under controlled conditions. Membranes of low permeability are used to decrease the hematocrit dependency in the dialysis step (5). A D-glucose determination demonstrates the minimized hematocrit dependency of the flow system. A L-lactate determination shows how an unfavorable enzyme equilibrium reaction can be forced by a second enzyme reaction to give an increased response. A creatinine determination displays an indirect subtrate determination, where the enzyme product is used in a second enzyme reaction to determine the desired enzyme substrate.
EXPERIMENTAL SECTION Electrode System. The working electrode was prepared according to the procedure given in ref 6. Graphite rods (Type RWOOl from Ringsdorff-Werke GmbH, Bonn-Bad Godesherg) were cut and polished on wet fine emery paper and thoroughly washed with water. They were heated in a muffle furnace at 700 "C for 90 s and allowed to eo01 in a desiccator. The pbenoxazine mediator, BPT (his(henzophenoxaziny1)derivative of terephtaloic acid) was synthesized hy reacting 2 mol of the imino form of oxidized Nile Blue with 1mol of terephthaloyl chloride according to refs 7 and 8. It was adsorbed on the flat circular end of the graphite rod (area 3.14 mm2)from a diethyl ether solution until the surface coverage was hetween 2 and 10 nmol/cm2, as determined by cyclic voltammetry. The electrodes were pressed into PVC holders, so that only the flat circular ends were exposed. Enzyme solutions were placed at the electrode surface, and a membrane was fixed on the top with an O-ring in order to encapsulate the enzyme, to protect against sample contamination, and to act as a diffusion harrier. In the glucose determination the enzyme glucose dehydrogenase (GDH) was immobilized with glutardialdehyde at the optimal conditions described in ref 7. In the L-lactate determination 2 pL of lactate dehydrogenase (LDH, 5000 units/mL suspension from Sigma) and 2 +L of glutamic pyruvic transaminase (GPT, lo00 units/mL in 1.8 M (NH,),SOI, pH 6.0) were placed at the CME top and entrapped with a cuprophan membrane. In the creatinine determination 2 pL of creatinine iminohydrolase (CIH, 1000 units/mL in 0.1 M phosphate buffer, pH 7.0) and 2 pL glutamate dehydrogenase (GlDH, 1000 units/mL in 2.0 M (NH,),SO,, pH 7.0) were also placed on the CME top and entrapped by a membrane. The auxiliary and reference electrodes were platinum and Ag/AgCI pins (both 0.5 mm 0.d.) which were integrated into a PVC body with an O-ring. The three electrodes were placed in two connected measuring cells (7 pL each), the auxiliary and the reference in the first and the working electrode in the second; see Figure la. Segmented Sample Injection. Samples (30 pL) were injected into a flow system, in such a way that it was surrounded by air bubbles, each 10 +Land hy the carrier stream; see Figure 2a. The injection is made by means of a six-port valve with connections as shown on Figure 2h. In position 1, the carrier stream is pumped
e
AIR
WASTE
AP Y
POT
REC
Figure 2. (a)Segmented stream sequence between reagent buffer. air (IO +L), and sample (30 +L) used in the flow system. (b) Air and
the sample are pumped into the injection valve by peristaltic pumps in posfmn 1, When fumed clockwise the sequence cisscribed in Figure l a is pumped out by a syringe pump in position 2. (c) Flow system for detection of enzyme substrates comprising sample pump (SP), air pump (AP), reagent pump (RP). an injection valve (I). a detector (D). a potentiostat (POT), and a recorder (REC). throngh the measuring ceb at 0.35 mL/min, in order to equilibrate the membrane and the electrode film with reagent; the sample and the air loops are filled simultaneously. In position 2, the carrier stream propels the air segmented sample through the measuring cells at 0.35 mL/min. Part of the sample diffuses into the membrane. The flow system is shown in Figure 2c. For the lactate and the creatinine determinations it was necessary to stop the sample zone, when the second cell had just filled, to get a measurable amount of substrate into the sensor. After a sufficiently long diffusion time, the cells are washed with the carrier solutions and the system is ready for a new sample. The electrode system was mnnected to a potentiostat (Taeussel, amperometric unit) and the current was recorded as a function of time (Radiometer A/S Servograph REA 61, with a REA 112 high-sensitivity interface). The peak height at peak maximum served as the analytical readout for the D-glucose and L-lactate determination. The time to obtain a 50% decrease in the response after sample introduction was used for the quantification of the creatinine. Enzymes and Chemicals. Glucose dehydrogenase (GDH) EC 1.1.1.47 (from Bncillus megaterium), glutamate dehydrogenase (GlDH) EC 1.4.1.3 (from bovine liver in 2.0 M (NH,),SOI, pH 7.0), NAD+, NADH, ADP, Nile Blue, and Kathon were obtained from Merck. L-Lactate dehydrogenase (LDH) EC 1.1.1.27 (from pig muscle, suspension) and glutamicpyruvic transaminase (GFW EC 2.6.1.2 (from porcine heart in 1.8 M (NH,),SO,, pH 6.0) were from Sigma, Creatinine iminohydrolase (CIH) EC 3.5.4.21 was from Kodak. All other chemicals were of analytical grade. The mediator BPT was synthesized from Nile Blue and terephtaloyl chloride according to ref 7. The carrier buffer for glucose determinations was 0.5 M phosphate, 0.5 M NaCI, 3 mM NAD', and 50 ppm Kathon as preservative, pH 7.40. The carrier buffer for L-lactate determinations was 0.5 M phmphate, 0.5 M NaCI, 2.5 mM NAD', 30 mM L-glutamate, and 50 ppm Kathon, pH 7.40. The d e r huffer for creatinine determinations was 0.5 M phosphate, 0.5 M NaC1, 5 mM n-ketoglutaricacid, 4 mM NADH, 20 mM EDTA, 0.5 mM ADP, and 50 ppm Kathon, pH 7.40. Aqueous wglucose, L-lactate, and creatinine standards were made from stock solutions containing 100 mM D-glume, 50 mM blactate, and 10 mM creatinine
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 9, MAY 1, 1990
I/I (plasmal
H+
D-gluconic acid Figure 3.
J
Reaction scheme for the determination of glucose. 20'o
I/nA
I5O' 100.
1 I
40*0
6 0 * o %hematocrit
Normalized glucose signal as a function of the hematocrit value displays a decrease in the response with an increase in the hematocrit level. This is shown for two membranes with different Permeability. 0 is a Spectralpor and X is a Gambro membrane. Figure 5.
A'
L-lactate
glutamate
10.0 20.0 IUM glucose Calibration curve for glucose standards in the clinical concentration range 1 to 40 mM using the system described in Fig 1: (0) with the Gambro membrane; ( X ) with the Spectra/Por membrane. Figure 4.
(10 mM phosphate, 140 mM NaC1, pH 7.40), respectively. A corresponding buffer was used for dilution. The different membranes used to cover the working electrodes were Spectra/Por no. 3 (molecular weight cut-off approximately 3500 made of cellulose) from Spectrum, Inc., and Lundia IC (molecular weight cut-off approximately 5000 made of cuprophan) from Gambro AB for the glucose assay. In the lactate and the creatinine assays only the Gambro membrane was used. R E S U L T S AND DISCUSSION Three analyte determinations were adapted to the flow system. The D-glucose determination was tested on blood, whereas the L-lactate and the creatinine determinations were tested on aqueous analyte standards. The D-Glucose Determination. D-Glucose was determined in undiluted whole blood by the reaction sequence shown in Figure 3. The assay was based on a final electrochemical detection of NADH production with the use of a chemically modified electrode, CME (9). A phenoxazinium ion, M (7), is used to mediate the oxidation of NADH at an applied potential at 0 mV vs the Ag/AgCl reference electrode. D-Glucose was degraded enzymatically by means of immobilized glucose dehydrogenase (GDH). The nicotinamide coenzyme (NAD+),present in the membrane, was reduced in the enzymatic reaction, and reoxidized amperometically at the CME. Hydrolysis caused a fast formation of D-gluconic acid from D-ghcono-&lactone, and the equilibrium of the overall reaction is displaced toward the product side. The glucose diffusion through the dialysis membrane should be the rate-limiting step in the system in order to give a linear relation between concentration and response. In this system, an exact amount of reagent was used for the reaction, namely the quantity that was deposited in the membrane, and in the electrode film, when it was equilibrated with the carrier solution. The air segmented sample was pumped through the measuring cell and some of the glucose diffused into the membrane during the 5-s contact time. The glucose that had entered the membrane was oxidized in the enzyme reaction and some of the reduced NADH diffused from the enzyme to the electrode surface, where it was reoxidized. The response maximum which occurred 15 s after sample introduction was used as the analytical readout. After maximum, the signal decreases slowly due to the elution of glucose and NADH from the membrane into the carrier stream, which simultaneously equilibrate the sensor with reagent. The total analysis time, 2 min for the Gambro membrane and 3.5 min for the Spec-
=-ketoglutarate
Figure 6.
\GPTf
i
pyruvate
J
LNm,
Lalanine
Reaction scheme for the determination of L-lactate.
tra/Por membrane, was limited by the relatively slow diffusion of NAD+/NADH through the low permeability dialysis membrane. A calibration curve for glucose standards between 0 and 40 mM is shown in Figure 4 using either the Gambro membrane with high permeability or the Spectra/Por no. 3 membrane with low permeability. The variation from linearity at high concentrations is due to either a limited amount of NAD+ in the membrane or kinetic limitations. This explains the extented linearity when the Spectra/Por membrane, which decreases the glucose concentration in the enzyme matrix, is used. Both the enzyme and the mediator kinetics are pH dependent, the optimum has been determined to pH 6.6 in ref 7 . The buffer capacity of the carrier solution should be sufficient to make the pH around the electrodes independent of the pH changes in blood samples between 7.3 and 7.5. This was achieved with a 0.5 M phosphate buffer at pH 7.4. The difference between carrier and sample buffer capacity was necessary to compensate for the dilution of the reagent phase by the relatively large sample volume. The dialysis of inhomogeneous samples such as whole blood is affected by its content of blood cells. When the diffusion zone in front of the dialysis membrane is filled up with an unspecified number of blood cells, the distance of the diffusing species to he membrane is extended. The glucose diffusion through the erythrocyte membrane is negligible compared to the dialysis time. The dialysis dependence of the blood cells can be minimized if the permeability of the dialysis membrane is small, related to the diffusion resistance in blood. This is illustrated in Figure 5, where the hematocrit dependence of the dialyzer is shown for two membranes with different permeability. In the clinical range of hematocrit values (30-70%), the Spectra/Por membrane has a response variation of 5% and the Gambro membrane has a response variation of 15%. The CME was stable for 3-4 days, when operated at 25 "C, and 1-2 weeks when operated daily and stored at 4 "C during nights. T h e L-Lactate Determination. L-Lactate was oxidized to pyruvate by lactate dehydrogenase (LDH) in the presence of NAD+, and the produced NADH was detected at the modified electrode. The low equilibrium constant of the reaction (2.76 X lo4 at 25 "C, pH 7.0 (10))favors the substrate side and can only be slightly higher by increasing the pH and the NAD+ concentration. Gorton et al. (11)showed how the reaction was forced to the product side when coupled with
ANALYTICAL CHEMISTRY, VOL. 62, NO. 9, MAY 1, 1990
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creatinine CIH
N-methylhydantion
d
\NH4+
a-ketoglutarate
H20 + glutamate
5.00
10.0 mM l a c t a t e . Flgure 7. Calibration curves for L-lactate in the clinical range from 0.08 t r 20 mM. For operations conditions see text.
an additional enzymatic reaction which could remove the produced pyruvate. Glutamic-pyruvic transaminase (GPT) was used to force the forward reaction. The moderate equilibrium constant of this reaction, 2.2 at pH 8.0 (IO),is enough to ge a detectable reaction in the clinical range, if a large surplus of L-glutamate is used; see Figure 6. Optimal conditions of operation for the L-lactate analysis system were evaluated with aqueous samples containing 1mM lactate. An acceptable response was obtained with a carrier buffer including 30 mM glutamate and 2.5 mM NAD+. Increases in the glutamate concentration had only a minor positive effect, while an increase in the NAD+ concentration decreases the response due to substrate inhibition of the reaction. The pH optimum of the combined LDH/GPT reaction was determined by Gorton et al. (11)to 8.8 and the pH optimum of the mediator reaction was determined by Appelqvist et al. (7) to 5.5. In this system, only a small increase in the response was observed when changing the pH between 7.0 and 8.5. The operational pH was, therefore, chosen to 7.4, close to expected sample pH. In order measure lactate concentrations at 0.1 mM, the samples were stopped in front of the sensor to let a measurable amount of substrate diffuse into the reaction zone. A maximum response approximately 15 s after sample introduction was used as the analytical readout. This response was affected by the lactate diffusion through the dialysis membrane, the enzyme kinetics, the equilibrium of the two reactions, and the dilution of the reaction zone by the sample. Calibration values for lactate concentrations between 0.1 and 10 mM is shown in Figure 7 . In order to get a pseudo-first-order reaction rate, it is necessary to have a limiting diffusion of lactate through the membrane, a lactate concentration in the enzyme membrane less than the K , value, and a 100% conversion in the LDH reaction to pyruvate. The low equilibrium constant for the G P T reaction could not fulfill the later condition, which is reflected in the nonlinear relation shown in Figure 7. As the conversion factor was less than loo%, a pyruvate dependency was observed. The Creatinine Determination. The creatinine determination is used to illustrate an indirect substrate detection, where membrane-deposited reagent is the main feature. Creatinine is almost exclusively determined by procedures based on the Jaffe reaction, which involves the formation of a red-yellow complex with picric acid. However, many substances in blood interfere with this reaction, and more specific enzyme reactions have been suggested. The ammonium ion, produced by the creatinine iminohydrolase (CIH) reaction, can be detected in several ways (12). A sensitive procedure, such as the glutamate dehydrogenase (GlDH) reaction, must be chosen in order to measure the creatinine in the clinical range between 25 and 200 pM. In these experiments the problem with endogenous ammonium has not been treated. It could, however, be solved by the use of a differential measurement where a corresponding sensor without CIH was used to detect endogent NH4+or with an additional enzyme
Figure 8. Reaction scheme for the determination of creatinine.
sec t o 50% NADH response
1 250
500 triov6 M c r e a t i n i n e
Figure 9. Calibration curve for creatinine in the clinical range from 25 to 800 pM. For operation condition, see text.
reaction, such as a carbamate kinase membrane, which could remove NH4+from the sample before the creatinine enters the CIH/GlDH matrix in front of the CME. The creatinine assay illustrates how the flow system was used for an indirect determination of enzyme substrate. Creatinine was degraded by CIH, the ammonium ion produced converted into glutamate by GlDH, and NADH was oxidized to NAD+. The consumption of NADH was detected a t the CME, Figure 8. Optimal operational conditions for the creatinine assay was evaluated with aqueous samples containing 100 pM creatinine. The carrier contained 20 mM EDTA and 0.5 mM adenosine-5-diphosphate (ADP) to stabilize and activate the GlDH enzyme (12). The product side was favored in the G l D H reaction and 5 mM a-ketoglutaric acid was, therefore, sufficiently high. The continuous consumption of NADH, at the CME, generated a concentration gradient over the dialysis membrane, and in order to have an excess during the enzyme reaction, 4 mM NADH in the carrier was required. A calibration graph of creatine concentrations between 25 and 800 pM is shown in Figure 9. The response from the CME is a function of the creatinine concentration and the reagent dilution into the sample. In order not to be limited by the NADH consumption in the reaction or the dilution, a non-steady-state response was used as the analytical readout. The reaction time, necessary to decrease the NADH response to 50% of the start level, was used for quantification. This level was reached after 72 s for 25 pM creatinine and after 16 s for the 800 pM creatinine. After 80 s the sample was washed out and the membrane was equilibrated with NADH for 160 s.
CONCLUSIONS The results show that a direct non-steady-state measurement with a reagent requiring enzyme in undiluted whole blood is possible if a controlled indirect addition of reagent and buffer in a flow system is used. The interference from the blood cells can be avoided in the sample transportation step and in the sample and reagent mixing step and can be decreased in the dialysis step in the flow system. When the described glucose sensor is compared with the well-known glucose oxidase sensors, the major improvement is that easy oxidizable substances from blood are eliminated by the use of a CME working at 0 mV vs Ag/AgCl, thus giving a more
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accurate result ( 5 ) . The lactate and creatinine sensors illustrate that complex enzymatic systems can be used under nonoptimal conditions and with non-steady-state responses to give a fast analytical readout, which can be important in many cases. For systems independent of reagent addition, it will be an improvement to control the pH of the sensor by the use of buffer deposition in the sensor membrane.
ACKNOWLEDGMENT The author thanks Professor Gillis Johansson of the Department of Analytical Chemistry, University of Lund, Sweden, for valuable discussions. LITERATURE CITED (1) Harrow, J. J.; Janata, J. Anal. Chim. Acta 1985, 774, 115. (2) Harrow, J. J.; Janata, J. Anal. Chim. Acta 1985, 774, 123.
(3) Petersson, B. PhD. Thesis, The Technical Universlty of Denmark, 1988. (4) Risinger, L.; Buch-Rasmussen, T.; Johansson, 0.Anal. Chim. Acta, in press. (5) Buch-Rasmussen, T.; unpublished results. (6) Appelqvist, R.; Marko-Varga, G.; Gorton, L.; Torstensson, A,; Johansson, G. Anal. Chim. Acta 1985, 769, 237. (7) Appelqvist, R. Thesis, University of Lund, Sweden, 1988. L. Proceedings of ElectroFinnAnalysis, An International $onGorton, (8) ference on Electroanalytical Chemistry, June 6-9, 1988, Turku-Abo, Finland. (9) Gorton, L. J . Chem. SOC.,Faraday Trans. 7 1988, 82, 1245. (10) Barman, T. E. In Enzyme Handbook; Rauen, H. M.,Ed.; Springer: Heidelberg, 1969; Vol. 1. (11) Gorton, L.; Hedlund, A. Anal. Chim. Acta 1988, 273,91. M. T.; Hansen, E. H. Anal. Chim. Acta 1989, 274, 147. Jeppesen. (12)
RECEIVED for review September 11,1989. Accepted January 19,1990. This work was in part supported by the Committee on Nordic Industrial Research Education Programme.
Design and Synthesis of Lithium Ionophores for an Ion-Selective Electrode by Chemical Modification of Natural Carboxylic Polyether Antibiotic Monensin Koji Tohda, Koji Suzuki,* Nobutaka Kosuge, Kazuhiko Watanabe, Hitoshi Nagashima, Hidenari Inoue, and Tsuneo Shirai'
Department of Applied Chemistry, Keio Uniuersity, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223, Japan
Ion-selective electrodes were prepared wlth 12 kinds of monensln derivatives obtained by chemical modlficatlon of natural antlMotk monensin, and the relatlonsMp between the chemlcai structures of the derhmtlves and the Ion selectlvltles of those electrodes was investigated for designlng Ll+-selectlve ionophores. Lactonlzatlon of monensin and subsequent acylatlon of Its tertlary hydroxyl group are effective for obtaining highly LI+-selective ionophores. Of all the monensln derlvatlves synthesized, macrocyclic monensln monoisobutyrate proved to have the highest LI+ selectlvity. The sewgs -1.8 In the lectivity coefflclent of Li+ to Na+ (log G,) poly(vinyl chkride) (PVC) matrix membrane electrode, whkh was prepared by using the macrocyclic monensln derivative and dlbenzyl ether (DBE) as the membrane solvent.
INTRODUCTION Natural ionophores generally have fairly complicated chemical structures by which they can coordinate ions tridimensionally into themselves by means of oxygen or nitrogen atoms existing in their molecules (I, 2). These structures are stereospecifically accurate and have firm ionic coordination. They are attractive as starting materials for creating many ionophores that have excellent specific ion selectivity. Furthermore, to examine physicochemical properties of the derivatives obtained by chemical modifiation of natural ionophores, it is helpful to understand the complicated and skillful structure of natural molecules. I t has been reported from the crystallographical data (3, 4) that monensin (Figure la), possessing some furans, pyrans, Deceased.
and a spiro ring, forms a lipophilic and stable complex with Na+ by forming a pseudocyclic molecule with a hydrogen bond between the carboxyl group a t one end of the molecule and the hydroxyl group at the other end. Some functional groups such as a carboxyl group (Cl, see Figure la) and three hydroxyl groups (primary, C26; secondary, C7; tertiary, C25) in the monensin molecule can be modified with relative ease; hence, we can produce a number of monensin derivatives. We previously reported (5) that the ion-selective electrode based on Cl-C26 lactone monensin (macrocyclic monensin; see Figure lh) exhibits high Li+ selectivity, although natural monensin based electrodes show high Na+ selectivity. In the present report, 12 kinds of monensin derivatives were synthesized to create highly Li+-selective ionophores, and the relationship between the molecular structures of the derivatives (discussed mainly with NMR spectroscopy) and their ion selectivities measured by potentiometry was investigated to design Li+-selective ionophores for an ion-selective electrode. As a result, macrocyclic monensin monoisobutyrate (Figure lm), which was synthesized from the lactonization of monensin followed by the acylation (isobutyrylation) to the tertiary hydroxyl group, showed excellent Li+ selectivity.
EXPERIMENTAL SECTION Reagents. All chemicals used were reagents of the highest grade commercially available. Distilled deionized water had resistivities greater than 1.5 X lo' Q cm at 25 "C. Monensin Derivatives. The chemical structures of the derivatives synthesized by chemical modification of natural monensin are indicated in Figure lb-m. The NMR spectra for structural analysis of these molecules were measured with GX 400 (400MHz for 'H) and GSX 270 (270 MHz for 'H, 68 MHz for 13C)NMR spectroscopy (JEOL Co., Ltd., Tokyo). Monensin methyl ester (b)was synthesized by treating monensin (Sigma Chem. Co., St. Louis, Mo) with methyl iodide and 1,8-diazabicyclo[5.4.O]un-
0003-2700/90/0362-0936$02.50/0 0 1990 American Chemical Society
