Pulse Amperometric Detection of Lithium in Artificial Serum Using a

Anal. Chem. 1998, 70, 4286-4290

Pulse Amperometric Detection of Lithium in Artificial Serum Using a Flow Injection System with a Liquid/Liquid-Type Ion-Selective Electrode Shigeo Sawada,*,† Hirotsugu Torii,† Toshiyuki Osakai,*,‡ and Takashi Kimoto§

Department of Chemistry, Faculty of Science and Technology, Kinki University, Higashiosaka, Osaka 577-8502, Japan, Department of Chemistry, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan, and Research Institute of Oceano-Chemistry, Osaka office, 3-1 Funahashi-cho, Tennoji, Osaka 543-0024, Japan

A voltammetric ion-selective electrode based on the facilitated transfer of Li+ by dibenzyl-14-crown-4 at the o-nitrophenyl phenyl ether/water interface was applied to the amperometric detector in a FIA system for Li+ with a cation-exchange column. The current response was stably detected by pulse amperometry in which voltage pulses of short duration (50 ms) were applied at the interval of 2 s. With this FIA system, 0.2-2.0 mM Li+ in aqueous solution could be determined even in the presence of 120-160 mM Na+, the detection limit being 0.15 mM. A test using artificial serum demonstrated the practical applicability of this system.

Lithium salts such as Li2CO3 have been extensively used as medicine for manic depressive and hyperthyroidism patients.1 The therapeutic range of the Li+ concentration is generally accepted to be 0.5-1.5 mM in blood serum. An excess dose can produce irreversible damage to the nervous system and the kidneys. Therefore, accurate measurements of Li+ in patients are very important. Although flame photometry and atomic absorption spectrometry are still employed for this purpose, a more convenient method using an ion-selective electrode (ISE) is earnestly desired. Instruments based on ISEs are compact, require small sample volumes, and have a reduced analysis time and a low operating cost. For serum Li+ assay, a variety of Li+ ISEs (potentiometric) have been developed, which are based on neutral ionophores, such as 14-crown-4 derivatives2-6

and acyclic ligands.7-9 However, any ISEs are still inadequate due to the coexistence of high concentrations of Na+ (∼140 mM) in blood. Although it was reported that a rather high Li+/Na+ selectivity (more than 1000) was obtained for the ISE using a 14crown-4 derivative,6 a higher selectivity of over 10 000 seems to be desired for accurate determination of Li+ at the millimolar level.7 In the recent decade, a new type of ISE has been proposed, being based on the amperometric (or voltammetric) detection of the ion-transfer current across the liquid/liquid interface (or the oil (O)/water (W) interface).10-13 The amperometric ISE, giving a current response directly proportional to analyte concentration, is more suitable for detecting small changes in analyte concentration than the usual potentiometric ISE, giving a potential response proportional to the logarithm of analyte activity (concentration). It should be noted that when the amperometric ISE is used for detection in flow injection analysis (FIA), its linear response to analyte concentration is of great advantage. Previous authors14-19 have already developed amperometric flow detectors for some ions based on the O/W interface. We would like to add that another important advantage of the amperometric ISE is that its ion selectivity can be altered by controlling the galvani potential W - φO) of the O/W interface using a difference (∆W Oφ t φ potentiostat. A suitable choice of ∆W O φ could render the best ion selectivity to the electrode.

* Corresponding authors: (e-mail) [email protected]; [email protected]. † Kinki University. ‡ Kobe University. § Research Institute of Oceano-Chemistry. (1) Amdisen, A. D. Handbook of Lithium Therapy; MTP Press: Lancaster, U.K., 1986. (2) Kitazawa, S.; Kimura, K.; Yano, H.; Shono, T. Analyst 1985, 110, 295-299. (3) Kimura, K.; Yano, H.; Kitazawa, S.; Shono, T. J. Chem. Soc., Perkin Trans. 2 1986, 1945-1951. (4) Kimura, K.; Oishi, H.; Miura, T.; Shono, T. Anal. Chem. 1987, 59, 23312334. (5) Kataky, R.; Nicholson, P. E.; Parker, D.; Covington, A. K. Analyst 1991, 116, 135-140. (6) Suzuki, K.; Yamada, H.; Sato, K.; Watanabe, K.; Hisamoto, H.; Tobe, Y.; Kobiro, K. Anal. Chem. 1993, 65, 3404-3410.

(7) Metzger, E.; Ammann, D.; Asper, R.; Simon, W. Anal. Chem. 1986, 58, 132-135. (8) Metzger, E.; Dohner, R.; Simon, W.; Vonderschmitt, D. J.; Gautschi, K. Anal. Chem. 1987, 59, 1600-1603. (9) Bochenska, M.; Simon, W. Mikrochim. Acta 1990, 111, 277-281. (10) Senda, M.; Osakai, T.; Kakutani, T.; Kakiuchi, T. Nippon Kagaku Kaishi 1986, 956-963. (11) Osakai, T. Kagaku to Kogyo (Tokyo) 1990, 43, 184-188. (12) Senda, M.; Kakiuchi, T.; Osakai, T. Electrochim. Acta 1991, 36, 253-262. (13) Senda, M.; Yamamoto, Y. In Liquid-Liquid Interfaces, Theory and Methods; Volkov, A. G., Deamer, D. W., Eds.; CRC Press: Boca Raton, FL, 1996; Chapter 12. (14) Marecek, V.; Ja¨nchenova´, H.; Colombini, M. P.; Papoff, P. J. Electroanal. Chem. 1987, 217, 213-219. (15) Ji, H.; Wang, E. Analyst 1988, 113, 1541-1543. (16) Wang, E.; Ji, H. Electroanalysis 1989, 1, 75-80. (17) Wang, E.; Ji, H.; Hou, W. Electroanalysis 1991, 3, 1-11. (18) Wilke, S.; Franzke, H.; Mu ¨ ller, H. Anal. Chim. Acta 1992, 268, 285-292. (19) Wilke, S.; Wang, H.; Muraczewska, M.; Mu ¨ ller, H. Fresenius J. Anal. Chem. 1996, 356, 233-236.

4286 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998

S0003-2700(98)00534-4 CCC: $15.00

© 1998 American Chemical Society Published on Web 09/12/1998

Figure 1. Principle of the voltammetric Li+ ISE based on the facilitated transfer of Li+ at the o-NPPE/W interface by DBz14C4.24

Facilitated transfer of Li+ across the O/W interface by neutral carriers such as crown ethers,20-24 acyclic ligands,21,24-26 and 1,10-phenanthroline27 has extensively been studied. In the preceding study,24 we prepared voltammetric Li+ ISEs using several neutral ionophores and organic solvents and then showed that a combination of dibenzyl-14-crown-4 (DBz14C4) and o-nitrophenyl phenyl ether (o-NPPE) led to the best Li+/Na+ selectivity (∼240 28). Figure 1 shows the principle of the voltammetric Li+ ISE. If the ionophore is not present in the O-phase, Li+ as well as Na+ is not readily transferred to the O-phase due to the extreme hydrophilicity. In the presence of DBz14C4, however, the transfer of Li+ to the O-phase is facilitated by the selective complexation of Li+ and DBz14C4 in o-NPPE. The observable current flowing through the O/W interface due to the facilitated Li+ transfer is proportional to the Li+ concentration in the W-phase (i.e., the sample solution), provided that the ionophore is added in large excess. In the previous paper,24 we confirmed a certain applicability of this system to the serum Li+ assay by voltammetric measurements with artificial serum under quiescent conditions (i.e., in batch measurements). Nevertheless, there still remained a serious problem with the interference from Na+. In this study, we have applied this Li+ ISE to an amperometric detector of the FIA system in which a cation-exchange column is employed for separating Li+ and Na+ to some degree in advance. EXPERIMENTAL SECTION Reagents. DBz14C4 and o-NPPE (both available from Dojindo Laboratories) were used as received. Tetrabutylammonium tetrakis(4-chlorophenyl)borate (TBATClPB) was prepared as described previously.29 Analytical grade tetrabutylammonium chloride (TBA-Cl) was occasionally contaminated by a trace amount (20) Samec, Z.; Papoff, P. Anal. Chem. 1990, 62, 1010-1015. (21) Kudo, Y.; Takeda, Y.; Hiratani, K.; Matsuda, H. Anal. Sci. 1991, 7, 549553. (22) Kudo, Y.; Kobayashi, T.; Ezaki, T.; Refaat, I. H.; Takeda, Y.; Matsuda, H. Anal. Sci. 1994, 10, 129-131. (23) Kudo, Y.; Takeda, Y.; Matsuda, H. J. Electroanal. Chem. 1995, 396, 333338. (24) Sawada, S.; Osakai, T.; Senda, M. Anal. Sci. 1995, 11, 733-738. (25) Shao, Y.; Tan, S. N.; Devaud, V.; Girault, H. H. J. Chem. Soc., Faraday Trans. 1993, 89, 4307-4312. (26) Kudo, Y.; Miyakawa, T.; Takeda, Y.; Matsuda, H.; Hiratani, K. Anal. Sci. 1994, 10, 375-378. (27) Yudi, L. M., Baruzzi, A. M.; Solis, V. M. J. Electroanal. Chem. 1992, 328, 153-164. (28) Simply evaluated from the difference (140 mV) of the reversible half-wave potentials24 of the transfers of Li+ and Na+ as 10140/59 ≈ 240. (29) Osakai, T.; Kakutani, T., Nishiwaki, Y.; Senda, M. Anal. Sci. 1987, 3, 499503.

Figure 2. Diagram of the thin-layer flow-through cell: (1) o-NPPE containing 20 mM DBz14C4 and 0.1 M TBATClPB, (2) 0.1 M TBACl aqueous solution, (3) 0.05 M MgCl2 aqueous solution, (4) and (5) dialysis membranes, (6) and (7) Ag/AgCl electrodes, (8) and (9) glass tubes, (10) polyester gasket (0.1 mm thick), (11) and (12) Teflon blocks, (13) detector inlet, and (14) detector outlet.

of iodide ion, which was removed by metathesis with silver chloride in the aqueous solution; the concentration of TBA-Cl was determined by potentiometric titration with a standard silver nitrate solution. Artificial serum (Pathonorm L) was obtained from Nycomed Pharma AS. The samples containing 0.30-2.0 mM Li+ and 140 mM Na+ were prepared by adding standard solutions of Li+ and Na+ to the artificial serum containing no Li+ and 120 mM Na+. The other reagents were of analytical grade. Construction of a Thin-Layer Flow-Through Cell. Figure 2 shows the diagram of the amperometric detector. A polyester gasket (an OHP film) of 0.1-mm thickness, with a channel being cut out in the central part, was sandwiched between two Teflon blocks. The upper one is the holder of the amperometric Li+ ISE based on the o-NPPE/W interface. The o-NPPE phase (20 µL) contained 20 mM DBz14C4 and 0.1 M TBATClPB as the supporting electrolyte and was held at the cut end of a glass tube (3-mm inner diameter) using a dialysis membrane (Viskase, 20 µm thick). Since the dialysis membrane is hydrophilic, the o-NPPE/W interface is formed at the inner surface of the membrane.30 An internal solution (0.1 M TBACl aqueous solution) was gently applied onto the o-NPPE phase. A spiral Ag/AgCl electrode was then immersed in the internal solution. From the lower part of the detector, another Ag/AgCl electrode immersed in a 0.05 M MgCl2 aqueous solution (degassed with ultrasonic wave) was in contact through a dialysis membrane to the channel. The two Ag/AgCl electrodes were connected to a homemade potentiostat with a positive feedback iR compensation in order to control the ∆W O φ of the o-NPPE/W interface and detect the iontransfer current. The solution resistance between the two electrodes (∼48 kΩ) was measured in advance using a Yanaco MY-9 conductivity meter, and the measured value was used for adjustment of the feedback amount. (30) Sawada, S.; Osakai T., Senda, M. Bunseki Kagaku 1990, 39, 539-545.

Analytical Chemistry, Vol. 70, No. 20, October 15, 1998

4287

Figure 3. Scheme of the FIA system for the determination of Li+ in blood serum: (D) amperometric detector, (C) cation-exchange column, (P) pump, (SI) sample injector, (R) 0.5 M MgSO4 aqueous solution, (W) waste, (PS) potentiostat, and (PC) personal computer.

Thus, the electrochemical cell used can be expressed as

The mobile phase (IV) contained 0.5 M MgSO4 as the supporting electrolyte.31 The interfacial potential difference (∆W O φ) between phases I and II is determined by the distribution equilibrium of TBA+ between the two phases.32 Since TBA+ was added to the respective phase in large excess (0.1 M), the current flowing across the interface, if not very large, should hardly influence the distribution potential of TBA+. Supposing that liquid junction potentials of the dialysis membranes are negligible, the potential difference (E) between the two Ag/AgCl electrodes is given approximately by II E ) ∆W O φ + ∆I φ

(1)

Note that the electrode potentials of the two Ag/AgCl electrodes would be canceled out in E, because they were immersed in solutions of the same concentrations of Cl-. Apparatus. Figure 3 shows the scheme of the FIA system, which comprised a HPLC pump (PM-60, BAS), a sample injector (model 7125, 100 µL, Rheodyne), a cation-exchange column (TSKgel SCX, 5 µm, 4.6 mm i.d. × 150 mm, Tosoh), and the amperometric detector. The cation-exchange column, being distributed in the Na+ form, was treated successively with 1 M H2SO4, distilled water, and the eluent (0.5 M MgSO4 aqueous solution). The flow rate of the eluent was 0.5 mL/min. As shown in Figure 3, the column and the detector were placed in a Faraday cage. For control of the voltage applied to the detector and acquisition of the current response, a 16-bit personal computer (NEC, PC9801VM) equipped with a 12-bit analog-to-digital converter and a 16-bit digital-to-analog converter was used. (31) A preliminary study showed the optimum concentration of MgSO4 to be 0.5 M; the lower concentrations prolonged the elution time of Li+ (e.g., 31 min at 0.05 M), while the higher concentrations worsened the separation of Li+ and Na+. (32) Kakutani, T.; Osakai, T.; Senda, M. Bull. Chem. Soc. Jpn. 1983, 56, 991996.

4288 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998

Figure 4. Principle of pulse amperometry. The upper part shows cyclic voltammograms for the facilitated transfers of 1.0 mM Li+ and 1.0 mM Na+ by 20 mM DBz14C4 at the o-NPPE/W interface, which were recorded using the flow-through cell (Figure 2) under quiescent conditions. τ ) 50 ms; ∆t ) 2 s.

Pulse Amperometry. In the upper part of Figure 4, cyclic voltammograms of the facilitated transfers of Li+ and Na+ by DBz14C4 are shown, which were recorded using the flow-through cell under quiescent conditions. In pulse amperometry,33-35 the electrode is held at a base potential (here, Eb ) 240 mV) at which negligible ion transfer occurs. On this base potential, voltage pulses of short duration (τ ) 50 ms) and the same amplitudes (∆E ) Es - Eb ) 155 mV, unless noted otherwise) are applied at an interval ∆t ) 2 s. Each voltage pulse gives a nonfaradaic current (dashed line) for recharging the O/W interface, which occurs immediately after the voltage step, rapidly disappears, and then gives a faradaic current (solid line) due to the ion transfer (for Li+, and only partially for Na+) from W to O. The faradaic current, being proportional to the ion concentration,36 is sampled at the end of the voltage pulse. Returning the electrode potential to the base potential leads to a negative faradaic current for the reverse ion transfer from O to W. Before the next pulse is applied after ∼2 s, the electrode conditions return to the beginning state. Such a reversal pulse technique would enable us to obtain stable and reproducible current responses for the amperometric detector. RESULTS AND DISCUSSION Response Characteristics. Figure 5 shows a typical chromatogram obtained by using an aqueous solution containing 1.0 (33) Osakai, T.; Nuno, T.; Yamamoto, Y.; Saito, A.; Senda, M. Bunseki Kagaku 1989, 38, 479-485. (34) Yamamoto, Y.; Nuno, T.; Osakai, T.; Senda, M. Bunseki Kagaku 1989, 38, 589-595. (35) Katano, H.; Osakai, T.; Himeno, S.; Saito, A. Electrochim. Acta 1995, 40, 2935-2942. (36) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, Fundamentals and Applications; Wiley: New York, 1980; Chapter 5.

Figure 5. Chromatogram of an aqueous solution containing 1.0 mM Li+ and 140 mM Na+. Es ) 395 mV; Eb ) 240 mV.

Figure 7. Effects of the concentration of Na+ on the Li+ peak. [Li+] ) 1.0 mM; Es ) 395 mV; Eb ) 240 mV.

Figure 8. Calibration graph. [Na+] ) 140 mM; Es ) 395 mV; Eb ) 240 mV.

Figure 6. Variation of the chromatograms with the step potential (Es). [Li+] ) 0.2 mM; [Na+] ) 140 mM; Eb ) 240 mV.

mM Li+ and 140 mM Na+ as the sample solution. A well-defined current peak for Li+ was observed 15 min after the sample injection, though its peak was followed by a comparatively large peak for Na+. In view of the high concentration of Na+, we may recognize again the excellent Li+/Na+ selectivity of the amperometric detector. As reported previously,24 this excellent selectivity is ascribed to the difference between the complex formation constants (K°ML) of Li+ and Na+ with DBz14C4 in o-NPPE; log(K°ML/M-1) ) 5.3 and 2.5 for Li+ and Na+, respectively. It should also be noted that in a classical ion chromatographic separation with conductometric detection, it would be practically impossible to determine Li+ in the presence of such a large amount of Na+. The current responses for Li+ and Na+ were affected by the amplitude of the voltage pulse. Figure 6 shows the variation of the chromatograms with the step potential (Es). With increasing Es, both peaks were enlarged, but at different rates. At higher Es’s, the Na+ peak grew more progressively than the Li+ peak, so that the latter peak finally became a shoulder at Es ) 430 mV. Such dependences may be expected from the voltammetric

current-potential curves shown in Figure 4. Thus, in principle, we can enhance the Li+/Na+ selectivity by lowering Es. However, when Es was made much lower than 395 mV, the Li+ peak could not be detected with adequate sensitivity. Consequently, Es was determined to be 395 mV. The chromatogram shown in Figure 5 was obtained with this optimized Es. Inhibitory effects of the Na+ concentration were also examined. It is known that the concentration of Na+ in human blood has little variation (for reference data,37 [Na+] ) 130-145 mM). For insurance, however, chromatograms were obtained with five different [Na+]’s in the range of 120-160 mM. As shown in Figure 7, the Li+ peak rose with the increase in [Na+]. Nevertheless, there was no significant variation in the peak height (Ip ) 0.51 ( 0.02 µA) which was measured with the help of an additional line as shown in Figure 5. Calibration Graph. Figure 8 shows the dependence of Ip on [Li+], i.e., the calibration graph. As seen in the figure, Ip was proportional to [Li+] in the range of 0.2-2.0 mM. This concentration range fully covers the therapeutic range of 0.5-1.5 mM. From the calibration curve, the detection limit38 was determined to be 0.15 mM. The observed linear dependence of Ip on [Li+] clearly shows that, in the presence of excess ionophore, the ion-transfer current is controlled by diffusion of Li+ in the dialysis membrane. The (37) Worth, H. G. J. Analyst 1988, 113, 373-384. (38) Currie, L. A.; Svehla, G. Pure Appl. Chem. 1994, 66, 595-608.

Analytical Chemistry, Vol. 70, No. 20, October 15, 1998

4289

Figure 9. Chromatogram for (A) an artificial serum containing 0.6 mM Li+ and 140 mM Na+ and (B) an aqueous solution containing the same concentrations of the ions. Es ) 395 mV; Eb ) 240 mV. Table 1. Determination of Li+ in Artificial Serum Containing 140 mM Na+ [Li+], mM

a

added

founda

recovery, %

0.30 0.60 1.0 1.5 2.0

0.27 ( 0.05 0.48 ( 0.14 1.11 ( 0.14 1.66 ( 0.12 1.96 ( 0.36

91 80 111 111 98

Mean of four repeat measurements.

m 1/2 ) 2.7 diffusion-layer thickness is estimated as δ ) (πDLi +τ) m + µm with DLi+ (the diffusion coefficient of Li in the membrane)24 ) 4.7 × 10-7 cm2 s-1 and τ ) 50 ms. Thus, the diffusion layer should be confined within the membrane of 20-µm thickness. A Test Using Artificial Serum. Figure 9 shows a typical chromatogram for the artificial serum, which is almost identical with that obtained for an aqueous solution containing the same concentrations of Li+ and Na+. In the amperometric detector used, the ion-selective O/W interface is covered with a dialysis membrane (Figure 2), which may serve to prevent possible interference from polymeric materials such as protein in blood serum. Table 1 shows the result of determination of Li+ (0.302.0 mM) in artificial serum containing 140 mM Na+. The found values of [Li+] were calculated using the calibration graph in Figure 8. As seen in the table, a satisfactory result was obtained, although the accuracy is still insufficient, possibly because of some variation of the detector sensitivity. At the present stage, the origin of the instability is not clear. However, since a regular

4290 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998

membrane-covered electrode can be used repeatedly for more than two weeks,30 the other parts (e.g., counter electrode) of the detector are possibly responsible for the instability. A study to improve of the detector stability is currently under way. Li+/Na+ Selectivity. In the chromatogram shown in Figure 5, the difference in the elution time (∆te) between Li+ and Na+ is 2.8 min, and the base widths (W1 and W2) of their peaks are almost identical, being 3.3 min. Accordingly, the resolution39 (Rs) is given as Rs ) 2(∆te)/(W1 + W2) ) 0.85. A simulation assuming a Gaussian peak for each ion has revealed that the area of the crosscontamination is ∼4% of the total peak area, when the two ions have the same peak areas. This shows that the Li+/Na+ selectivity inherent in the column system is ∼25 () 1/0.04) in case the peak area is measured. In this study, however, the peak height was measured as shown in Figure 5 to minimize the interference from Na+. Accordingly, the overall Li+/Na+ selectivity of this FIA system can be expected to exceed 6000 () 25 × 240), since the selectivity of the amperometric detector is ∼240.28 Thus, the present system seems to achieve the Li+/Na+ selectivity close to that (over 10 000) desirable for serum Li+ assay. CONCLUSIONS The present amperometric Li+ ISE based on the O/W interface is promising for the FIA of Li+ in blood serum. When the ISE having a Li+/Na+ selectivity of 240 is employed as the amperometric detector for the FIA system with a cation-exchange column, the overall Li+/Na+ selectivity is >6000, which is sufficient to determine Li+ in blood serum at the millimolar level. The proposed method (pulse amperometry) seems to be suitable for detecting the current response of such a liquid/liquid-type ISE. However, there is still great room for improvement in the present FIA system. For example, it is desirable to obtain a more excellent Li+ ionophore having a higher Li+/Na+ selectivity, which will vest higher sensitivity and accuracy in the FIA system or may possibly remove the cation-exchange column from the system. In the course of the preparation of this paper, the authors were informed of a new report on spectrophotometric determination of Li+ using a water-soluble porphyrin.40 According to the paper, the proposed method can be applied to a serum Li+ assay. The practical utility of the spectrophotometric method as well as our electrochemical method should hereafter be examined. Received for review May 14, 1998. Accepted July 30, 1998. AC9805347 (39) Done, J. N.; Knox, J. H.; Loheac, J. Applications of High-Speed Liquid Chromatography, Wiley: London, 1974; Chapter 3. (40) Tabata, M.; Nishimoto, J.; Kusano, T. Talanta 1998, 46, 703-709.