Anal. Chern. 1987, 59, 1600-1603
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Lithium/Sodium Ion Concentration Ratio Measurements in Blood Serum with Lithium and Sodium Ion Selective Liquid Membrane Electrodes Erich Metzger, Rent5 Dohner, and Wilhelm Simon*
Department of Organic Chemistry, Swiss Federal Institute of Technology ( E T H ) , CH-8092 Zurich, Switzerland Dieter J. Vonderschmitt and Kurt Gautschi
University Hospital, Zurich, Switzerland
A flow-through electrode system requiring 50.1 mL of sample solution has been developed to determine Li+/Na+ concentration ratlos in aqueous solutlons and Mood serum by uslng a cell assembly consisting of a Li+-selectlve and a Na+-selective ilquld membrane electrode. Assays in serum samples of manic-depresslve patlents under ilthlum treatment were executed and the results were compared wlth atomic absorptlon spectrometry/flame photometry measurements.
Very recently the efforts to develop Li+-selectiveelectrodes for the determination of Li+ activities in blood serum of patients under a lithium treatment of manic-depressive psychosis have been intensified (for a review see ref 1). Only a few of the proposed ionophores can be utilized in clinically relevant sensors (1-6). The ionophore N,N-dicyclohexyl-N',N'-diisobutyl-ciscyclohexane-1,2-dicarboxamide(ETH 1810, 1 in Figure 1) exhibits the best Li+/Na+ selectivities in clinically relevant sensors found to date (log K[& = -2.45 in o-NPOE (onitrophenyl octyl ether)/KT-p-C1PB (potassium tetrakis(pchlorophenyl)borate)/PVC (poly(viny1chloride)) membranes) (3, 5 ) . Sensors based on this ionophore allow absolute Li+ assays in blood serum within the clinical Li+ concentration range by using a fixed ion background calibration method (5). The use of a relatively polar plasticizer of low lipophilicity (0-NPOE, 4 in Figure 1)however imposes certain limitations in respect to sensor response time and membrane lifetime ( I , 7, 8). Li+ electrodes based on the ionophore 5-butyl-N,N,N',N'-tetracyclohexyl-5-ethyl-3,7-dioxaazelaic acid diamide (ETH 2137,2 in Figure 1)show a lower Li+/Na+selectivity (log K:PA, = -1.9 in BBPA (bis(1-butylpentyl) adipate)/PVC membranes) but have the advantage of fast electromotive force (emf) response, high electrode potential stability, and improved membrane lifetime due to the use of a more lipophilic plasticizer (BBPA, 5 in Figure 1) ( I ) . The highest Li+/Na+ selectivities so far achieved are still considerably away from the value that is required for a contribution of less than 1%by the activity of the interfering ion (Na+) to the activity of the primary ion (Li+) (log K:& = -4.3 ( 9 ) ,see Nikolsky-Eisenman equation (10)).The clinical Li+ concentration range (0.7-1.5 mM (11-13)) therefore covers the curved part of the electrode response function of these sensors. With the use of the Li+ ionophores of the highest known (so far) Li+/Na+ selectivity, this range is slightly above the detection limit ( I , 5,10,14) so that Li+ assays are possible if a fixed ion background calibration is used. Due to the curvature of the electrode response function in the clinical Li+ concentration range, the emf difference between a Li+ concentration of 0.7 and 1.5 mM decreases from ideally 19.3
mV (no Na+ interference) to 13.0 mV and 7.2 mV for ETH 181O/KT-p-ClPB/o-NPOE/PVC membranes and E T H 2137/BBPA/PVC membranes, respectively. As a consequence, high requirements with respect to the reproducibility of the cell emf have to be met (1). This is achieved for both types of membranes; membranes based on ETH 2137 being superior. Even if a fixed ion background (140 mM Na+) calibration is used, there remain uncertainties in the emf of the sensor cell assembly of up to about 0.74 mV (ETH 1810) and 1.3 mV (ETH 2137) due to varying sodium levels in blood over the normal physiological range of 135-150 mM ( I , 5 ) . This results in uncertainties in the Li' assay of up to about 5.1% (ETH 1810) and 18.0% (ETH 2137). This problem can be overcome if no longer the absolute Li+ activity but the Li+/Na+ concentration ratio is determined. A cell assembly consisting of a Li+-selective electrode and a Na+-selective electrode as a reference (instead of a conventional reference electrode with liquid junction) exhibits an emf which depends on the Li+/ Na+ concentration ratio. The Li+/Na+ concentration ratio seems to be even the more distinctive parameter in clinical lithium therapy because a patient with a low sodium level in blood reaches a dangerous lithium ion concentration at lower lithium levels than someone normal in sodium (11-13, 15). Here we report on Li+/Na+ concentration ratio measurements in aqueous solutions and blood serum by using different 1 2 sensors, which are based on our most promising ionophores, and a suitable Na+-selective solvent polymeric membrane electrode. Blood serum samples of patients under Li+ treatment were assayed and the results were compared with measurements by atomic absorption spectrometry and flame photometry.
EXPERIMENTAL SECTION Reagents, Sample Preparation. The aqueous electrolyte solutions for the potentiometric measurements were prepared with doubly quartz distilled water and chloride salts of high purity (purum p.a. or puriss. p.a., Fluka AG, Buchs, Switzerland, and pro analysis, E. Merck, Darmstadt, Germany). The blood serum for the measurement of the electrode response functions (three samples of about 100 mL) was pooled serum from the MedicoChemical Central Laboratory, University Hospital, Zurich, Switzerland, where sodium, potassium, total calcium, and magnesium ion concentrationswere determined by flame photometry (AES) or atomic absorption spectrometry (AAS). The serum was proven to be less than 3 X M in Li+. The serum samples were then prepared for the measurements by multiple addition of small volumes of an aqueous solution being 0.05 M in LiCl and 0.135 M in NaCl to the same serum stock solution. After each addition and a short mixing period, volumes of 0.1 mL were taken for emf measurements. The comperative studies of measurements with ISE's (ion selective electrodes) and with AAS/AES were executed with blood serum samples of patients suffering from manic-depressive psychosis and under lithium treatment. Their 1 2 concentrations were determined by AAS and the Na+ concen-
0003-2700/87/0359-1600$01.50/0 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
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-7 Figure 1. Structures of the Li+ ionophores (1 (ETH 1810)and 2 (ETH 2137)),the Na+ Ionophore (3 (ETH 2120)),the membrane plasticizers (4 (0-NPOE), 5 (BBPA), and 6 (ETH 469)), and the membrane additive (7 (KT-p-CIPB)).
trations by AES. The calibration solutions for the corresponding ISE assays were prepared from serum stock solutions by adding small volumes of LiClO.05 M/NaCl 0.135 M. Poly(viny1chloride) (PVC S704 hochmolekular) originated from Lonza AG, Visp, Switzerland (now available from Fluka AG); o-nitrophenyl octyl ether (0-NPOE, puriss. p.a., 4 in Figure l), bis(1-butylpentyl) adipate (BBPA, purum p.a., 5 in Figure l), decane-1,lO-diyl bis[ (1-butylpentyl) glutarate] (ETH 469, 6 in Figure l), and potassium tetrakis(pchloropheny1)borate (KTp-ClPB, purum p.a., 7 in Figure 1)were supplied by Fluka AG. The synthesis of the Li+ ionophores 1and 2 (Figure 1) is described in refs 5 and 1, respectively. The preparation of the Na+ ionophore 3 (Figure 1) is given in ref 16. Cell Assemblies for the emf Measurements. All emf studies were performed with the cell (see Figure 2) Ag; AgC1, internal filling solution11 Na+-selective membranellsample solution11 Li+-selective membranellinternal filling solution, AgC1; Ag (i) The Li+-selective [9], the Na+-selective [6], and a common (platinum wire) [4] poly(methy1 methacrylate) electrode module are mounted together with a fourth poly(methy1 methacrylate) module [lo] (equipped with a Cheminert (LDC/Milton Roy, Riviera Beach, FL) drain tube junction [12,13]) on an aluminum plate [ll]. The actual flow-through electrode system (Figure 2) has a volume of 15 WL(modules [4,6,9]). The sample solutions are injected through a platinum net [3] with a glass syringe and fill the vertical sample channel (0.8-mm i.d.) and the tube [13], which is fixed at the end of the channel and is bent upward. The sample is thus contacted simultaneously with the platinum wire of the common electrode [19], the Na+-selectivemembrane [18], and the Li+-selectivemembrane [17]. The sample volume required is about 0.1 mL. The solution is removed by applying vacuum. The internal filling solutions of the ISEs are 0.001 M LiC1/0.14 M NaCl in aqueous 0.5% Agar [7] and are in contact with the chlorinated silver wires and the plugs [16] (Lemo SA., 1110 Morges, Switzerland). Buna-nitrile O-rings [5, 14, 151 are employed for sealing purposes. The whole system is shielded and grounded by an aluminum cylinder [2,8, 111 and an aluminum cap [l], which is placed during the emf measurements. Three different Li+-selective membranes of the following approximate compositions were used (a) 2.6 w t % carrier ETH 1810
Flgure 2. Li+/Na+Now-thrwgh electrode system for the concentration
ratio measurements (schematic): [ 11 aluminum cap; [2] aluminum plate; [3] platinum net; [4] common electrode (platinum wire) module; [5] buna-nitrile O-rings; [6] Na+-selective electrode module; [7] internal filling solutions; [8] aluminum cylinder: [9] Li+-selectlve electrode module; [ 101 fourth module; [ 111 aluminum plate; [ 121 drain tube junction; [ 131 drain tube; [ 141 buna-nitrile O-ring; [ 151 buna-nitrile O-rings; [ 161 chlorinated silver wires, plugs; [ 171 Li+-selective membrane; [ 181 Na+-selective membrane; [ 191 common electrode (platinum wire), plug. (l), 0.4 wt % KT-P-ClPB (7),64.7 wt % O-NPOE(4), 32.3 wt % PVC; (b) 2.6 wt % carrier ETH 1810 (l),0.6 wt % KT-p-C1PB (7),64.5 w t % ETH 469 (6), 32.3 wt % PVC; (c) 2.0 wt % carrier ETH 2137 (2), 65.6 wt % BBPA ( 5 ) , 32.4 wt % PVC. The composition of the Na+-selective membrane was as follows: (d) 1.0 wt % carrier ETH 2120 (3), 66.0 wt % ETH 469 (6), 33.0 wt % PVC. The preparation of the membranes and of the flowthrough ion selectiveelectrode modules is described in ref 17. The electrodes were conditioned before use for about 8 h with an aqueous LiCl(O.OO1M)/NaCl(O.14 M) solution reinjected several times into the sample channel. emf Measurements. They were performed by using the common mode rejection technique (17). The flow-through system was first rinsed with one or two sample injections. The emf for the sample injected afterward was measured during 4 min at 21-23 "C, taking readings in 20-s intervals. The mean of the last six values was taken for further evaluation (see results and discussion). Determination of Li+ Concentrations in Serum by AAS. The serum sample was diluted 10-fold with 0.2 M 2-amino-2methyl-1-propanol/O.Ol M ethylenediaminetetraacetic acid, containing 1%0by volume of a 30% solution of Brij 35, and directly aspirated into the flame. The measurements were carried out on a Varian atomic absorption spectrophotometer,Model AA-875, at 670.8 nm. The enhancement of the signal by Na+ and K+ was compensated by employing lithium standards containing these ions in physiological concentrations. Determination of Na+ Concentrations in Serum by AES. The Na+ concentrations were determined by AES in the same way as K+ in ref 18.
RESULTS AND DISCUSSION Based on Nicolsky-Eisenman equations (10) for the re-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Table I. Parameters Obtained with Three Different Li+-Selective'Membranes in Li+/Na+ Concentration Ratio Measurements"
membrane a b C
cell response function log KEQ, residual std dev, mV aqueous undil serum aqueous undil serum -2.2 -1.8 -1.9
-2.2 -1.7 -1.9
response time,*s aqueous undil serum
0.6 0.2 0.06
0.3 0.06 0.1
-100 15-20 30
-
100 20-25 15-20
reproducibility of cell emf (std dev, mV) aqueous undil serum 0.2 0.05 0.03
0.2 0.07 0.03
"The Na+-selective membrane d was used. bThe time to reach the final emf to within a value being equivalent to a 5-fold subdivision of the clinical range with a 95% confidence limit (19) (0.5,0.3, and 0.4 mV for membranes a, b, and c, respectively). The concentration changes covered the clinical range. Table 11. Li+/Na' Concentration Ratio Determinations in Human Blood Serum by ISE's (Membranes c/d) and AAS/AES"
sample
rLi/cSaby cell ( i ) X IO3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
4.19 (0.4)e 3.25 (0.4) 5.48 (0.3) 2.64 (3.5) 4.64 (0.1) 4.63 (0.6) 1.83 (0.0) 4.51 (0.7) 1.99 (4.7) 4.46 (0.2) negative (-1 5.17 (0.2) 3.69 (0.4) 11.62 (0.7) 4.30 (1.7) 6.32 (0.7) 1.71 (3.3) 4.05 (0.9) 4.54 (1.0) 2.54 (0.9) 5.38 (0.0) 9.78 (0.4) 8.97 (0.2) 8.00 (0.1)
error calcd for uncertainty in emf of 0.1 mV, 9~
re1 accuracy, cLI by AAS, mM (std dev. 0.03 mM)
1.7 2.1 1.4 2.5 1.6 1.6 3.4 1.6 3.1 1.6
0.64 0.52 0.81 0.41 0.59 0.67 0.24 0.68 0.31 0.56
