Anal. Chem. 1995, 67, 1326-1331
Applications of LLWiredgg Peroxidase Electrodes for Peroxide Determination in Liquid Chromatography Coupled to Oxidase Immobilized Enzyme Reactors Liu Yang, Elsa Janle, Tiehua Huang, James Gitzen, and Peter T. Kissinger* Bioanalyiiml Systems, Inc., 270 1 Kent Avenue, West Lafayette, Indiana 47906
Mark Vreeke and Adam Heller Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712-1062
An osmium poly(viny1pyridine) redox polymer "wired"
horseradishperoxidase electrode has been used to detect H202 for the determination of glucose, lactate, and acetylcholine/choline with liquid chromatography (E) and postcolumn immobilized enzyme reactors (IMERs). The redox polymer 6lm containing peroxidase is coated on the surface of a glassy carbon electrode and operated at +lo0 mV vs Ag/AgCl for the reduction of H202. Compared with a conventional platinum electrode oxidizing H202 at $500 mVvs Ag/AgCl, the peroxidase cathode exhibits a 2-10-fold improvement in sensitivity and detection limit. The enzyme electrode also shows better operational stability than the Pt electrode. When the enzyme electrodeis coupled to LC oxidase WIER systems, the initial stabilization of the background ament is significantly faster than that for the conventional Pt electrode. The enzyme electrode has been used to determine glucose and lactate concentrations in rat subcutaneous microdialysates. After 8 days of continuous injection of the dialysate samples, the enzyme electrode sensitivity dropped by only 5%. The electrochemical determination of hydrogen peroxide formed in the presence of various oxidase enzymes has been employed to measure a number of oxidase Amperometric oxidative methods are most widely used to determine the hydrogen peroxide produced during enzymatic reactions as follows:
H,O,
-
0,
+ 2Hf + 2e
(2)
where S(red) and S(ox) are the reduced and oxidized forms of the substrate, respectively. The k s t reaction is catalyzed by an oxidase enzyme; in the second reaction, HzOz is oxidized at the working electrode, which is often a platinum electrode operating (1) Umana, M.; Waller, J. Anal. Chem. 1986,58, 2979-2983. (2) Wang, J.: Fang, L.; Lopez, D.; Tobias, H. Anal. Left. 1993,26, 1819-1830. (3) Palleschi, G.; Lavamini, M. G.; Compagnone, D.; Bertocchi, P. Electroanalysis 1992,4 , 851-857. (4) Hu, Y.: Zhang, Y.; Wilson, G. Anal. Chim. Acta 1993,281, 503-511.
1326 Analytical Chemistry, Vol. 67, No. 8, April 75, 7995
at +500 to +700 mV (vs Ag/AgC1).3z5j6 This method has found wide application in LC coupled with postcolumn oxidase reactors, where HzOz, the product from the oxidase immobilized enzyme reactor (IMER), is electrochemically detected7" (Figure 1). The combination of LC and IMER with electrochemical detection (EC) results in high overall selectivity and sensitivity. The detection of HzOz with the Pt electrode alone has an inherent problem. The analyte solution usually contains other compounds which can be oxidized nonselectively at the applied potential, resulting in significant interference with the signal from the substrate under study. Nevertheless, this problem can be minimized by an LCEC system, in which the components are separated prior to the electrochemical step. The Pt electrode in the LC-EC systems, however, often suffers from the liabilities of baseline drift following even a minor change in the system and then a long reequilibration time after the change. A freshly polished Pt electrode requires an even longer time for its initial equilibration,usually overnight. Recently, various amperometric electrodes with immobilized peroxidase have been developed for the detection of H202?-14 These enzyme electrodes usually have the advantages of high sensitivity and mild operating potential. There are two conventional types of the peroxidase enzymemodified electrodes: "mediated'' and "mediatorless". Mediated enzyme electrodes are based on monitoring the electroreduction of the oxidized mediator generated by the peroxidase enzymatic reaction, while mediatorless enzyme electrodes are based on monitoring direct electroreduction of peroxidase active centers oxidized by HzOz. Mediated enzyme electrodes employ the high efficiency of the electron transfer by effective mediators, while mediatorless enzyme electrodes have the apparent advantage of simplicity. In a recently (5) Lundback, H.; Johansson, G.; Holst, 0. Anal. Chim.Acta 1983,155, 4756. (6) Hendji, A N.; Bataillard, P.; Jaffrezic-Renault, N. Sens. Actuators B 1993, 15-16, 127-134. (7) Huang, T.;Kissinger, P. Cum Separ, 1989,9,9-13. (8) Fossati, T.; Colombo, M.; Castiglioni, C.; Abbiati, G.]. Chromatogr. B 1994, 656, 59-64. (9) Vreeke, M.; Maidan, R; Heller, A Anal. Chem. 1992,64, 3084-3090. (10) Ohara, T. J.; Vreeke, M. S.; Battaglini, F.; Heller, A Electroanalysis 1993, 5, 825-831. (11) Wang, J.; Ciszewski, A: Naser, N. Electroanalysis 1992,4, 777-782. (12) Marko-Varga, G.; Johansson, K; Gorton, L. J Chromatogr. A 1994,660, 153- 167. (13) Kacaniklic, V.; Johansson, K; MarkeVarga, G.; Gorton, L.: JonssonPettersson, G.; Csoregi, E. Electroanalysis 1994,6, 381-390. (14) Csoregi, E.; Gorton, L.; Marko-Varga, G.; Tudos, A J.; Kok, W. T. Anal. Chem. 1994,66, 3604-3610. 0003-2700/95/0367-1326$9.00/0 0 1995 American Chemical Society
Intedace
Detector
Pump
Waste
Mobile Phase
Figure 1. LC-EC system coupled with an immobilized enzyme reactor (IMER).
developed type of “wired” enzyme electrode, the enzyme peroxidase is covalently bound to a redox hydrogel coated on the surface of an electrode and can be electroreduced effectively through the redox centers of the polymer network?JO Electrodes based on the wired peroxidase have shown the advantage of absence of leachable components over the conventionalmediated peroxidase electrode and also have advantages of sensitivity and stability over the conventional mediatorless peroxidase electrode. The wired peroxidase has been coupled to oxidase enzymes in electrochemical sensors for flow injection analy~is.~OJ~ The wired peroxidase electrode, however, has not previously been used to detect HzOz in liquid chromatography systems with oxidase IMERs. The IMER approach has the potential advantage of a much greater loading of an active catalyst in a rugged format, and a single peroxidase electrode design can be used with a number of different oxidase IMERs. In the present study, a redox polymer, derived from poly(vinylpyridine) partly complexed to [Os(bpy)~Cl]*+/~+ redox centers, wired the peroxidase to the ele~trode.~ This electrode was coupled with LC and compared with a conventional Pt electrode for the determination of four biologically important substances: glucose, lactate, acetylcholine, and choline. Glucose oxidase, lactate oxidase, and acetylcholine esterase/choline oxidase IMERs are used for the detection of their respective substrates. The sensitivity, selectivity, linearity, and stability of these LCEC methods are discussed. EXPERIMENTAL SECTION Materials. Horseradish peroxidase (HRP) (type VI, EC 1.11.1.7) was purchased from Sigma. The osmium poly(viny1pyridine) redox polymer (Os(€”)) was synthesized as described previously.16 lacti lactic acid, lithium salt, was purchased from Calbiochem Corp. All other chemicals were analytical reagent grade from Sigma or Aldrich. Ringer’s solution was prepared as follows: 147 mM Na+, 2.3 mM Caz+,4 mM K+, 155 mM C1-, pH 6.0. (15) Garguilo, M. G.; Michael, A C.Anal. Chem. 1994,66, 2621-2629. (16)Gregg, B. A; Heller, A 1.Phys. Chem. 1991,95, 5970-5975.
Microdialysis Sampling. The rat was anesthetized with an intraperitoneal injection of 0.01 mL/kg of KX (10 mL of ketamine (100 mg/mL) 1 mL of xylazine (100 mg/mL)). The microdialysis probe @L2, PN MF-7050, BAS) was placed in an introducer cannula (PN MF-7021, BAS). The animal‘s fur was clipped from the insertion site on the back at the base of the neck and at the exit site 7 cm distal to the insertion point. Small incisions were made at the insertion and exit sites. The cannula was inserted under the skin at the neck and advanced to the distal incision. The cannula was pulled through the exit site, leaving the fiber portion of the probe under the skin. The probe was sutured in place and perfused with Ringer’s solution at 7 pL/min with a syringe pump (I” MF-5102, BAS). The in vitro recovery of the probe at this perfusion speed was determined to be 20%. The dialysate was collected in small Eppendorf tubes ( C W 1 4 2 fraction collector, BAS) and was injected into the LC system without any further dilution. The rat was kept awake during the sample collection. Electrochemistry and Chromatography. The rotating disk electrode @DE) voltammetric measurements were performed at ambient temperature with a BAS Model lOOB/W potentiostat, REAg/AgCl reference electrode, and Pt wire auxiliary electrode. Both Pt and glassy carbon RDEs were 3 mm in diameter. The glassy carbon enzyme electrode was prepared as described previously? All chromatography was performed using a BAS 480 liquid chromatography system (PM-80 pump, LC4C amperometric detector, CG5 flow cell and column compartment, DA-5 data acquisitioninterface, and ChromGraph software for data collection and analysis). Peroxidase-redox polymercoated glassy carbon electrodes were prepared as described previouslYg and operated at +lo0 mV vs Ag/AgC1; the Pt electrode was operated at +500 mV vs Ag/AgCl. The thin-layer electrochemical cell was used in a cross-flow mode. A BAS glucose applicationskit (PN MF-8925), including an ODS column (3 pm particle diameter, 40 x 3.2 mm) and a glucose oxidase enzyme reactor, was used for glucose analysis. The mobile phase was 20 mM NaHzP04 and 0.05% dimethylhexylamine, pH 5.5, and the flow rate was 0.8 mL/min.
+
Analytical Chemistry, Vol. 67, No. 8,April 15, 1995
1327
C
A
a
I
I
1 1 .o
1
I
I
0.8
I
0.6
l
I
0.4
1
'
0.2
'
I
0.0
Potential I V
L
0.6
I
1
0.5
I
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0.4
I
I
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0.3 0.2 PotentlalI V
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0.1
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Figure 2. Rotating disk cyclic voltammograms of (A) a Pt electrode in the mobile phase for lactate; (B) a Pt electrode in the mobile phase for glucose; (C) an HRP electrode in the mobile phase for lactate; and (D) an HRP electrode in the mobile phase for glucose with (a) no H202 and (b) 0.1 mM H202. For the Pt electrode, the scan rate is 5 mV/s; 200 rpm. For the HRP electrode, the scan rate is 5 mV/s; 1000 rpm.
The precision of the analysis with the enzyme electrode was determined as 1.1%(n = lo), and that with the Pt electrode was 0.7% (n = 10). For lactate analysis, an ion exchange column and a lactate oxidase enzyme reactor were used. The mobile phase was 50 mM Na2HP04and 0.5 mM EDTA at pH 8.0, and the flow rate was 1mWmin. The precision of the analysiswith the enzyme electrode was determined as 3.9% (n = lo), and that with the Pt electrode was 2.4%(n = 10). A BAS microbore acetylcholinecholine kit, including a SepStik acetylchodne analytical column (1 x 530 mm) and a SepStik acetylcholine/choline IMER was used for acetylcholine/choline analysis. The mobile phase was the same as that in the lactate experiments, and the flow rate was 140 pL/min. Kathon CG reagent (1%)0" CF-2150, BAS) was added to all mobile phases at 5 mL/L to retard bacterial growth. AU chromatography was carried out at room temperature. RESULTS AND DISCUSSION
t'F and HRP-Os(PW) Electrodes for HzOz Detection. Figure a B displays cyclic voltammograms for the oxidation of H ~ 0 2at a rotating Pt disk electrode. The oxidation current of H202 is buffer-dependent. In a buffer at pH 8, the oxidation starts at +ZOO mV (Figure 2A), while in a buffer at pH 5.5, the oxidation starts at +300 mV (Figure 2B). When a Pt working electrode is used for the measurement of Hz02in LC-EC, the applied potential is usually set within $0.5 to $0.7 V vs Ag/AgCL7 It can be 1328 Analytical Chemistry, Vol. 67,No. 8,April 15, 1995
expected that the sensitivity of HzOz detection with a Pt electrode may vary as a function of the buffer solution and applied potential. Figure 2C,D shows cyclic voltammograms for the reduction of HzOz at a rotating H W enzyme disk electrode in the two different buffers. The cyclic voltammograms (voltammograms a in Figure 2C,D) of the Os redox center of the polymer in these two buffers are quite close, except that the redox potential of the Os redox center in the buffer at the lower pH is 20 mV more positive. The catalytic reduction current of H2Oz (voltammograms b in Figure 2C,D), however, shows significantbuffer dependence. This buffer dependence of the current is possibly due to the buffer dependence of the enzyme activity or the buffer dependence of the conformation of the wiring polymer and thus the electron transport via the redox polymer. Previous studies of HRP have shown significant pH dependence of the enzyme reaction kinetics, which slow at high pH.I7-l9 The smaller reduction current of HzOz in the buffer of a higher pH agrees with the previous results. It should be noticed that these two buffers are also different in ionic strength, which could affect both the enzyme activity and the redox polymer conformation. In both buffers, the HzOz reduction current reaches a plateau negative of $200 mV (Figure 2C,D). (17) Smit, M. H.; Cass, A E. G. Anal. Chem. 1990,62, 2429-2436. (18) Back, A. IC; Wart, H. E. V.J. Am. Chem. SOC. 1992,114, 718-725. (19) Matsumura-Inoue, T.; Kuroda, K; Umezawa, Y.; Achiba, Y. J. Chem. SOC., Faraday Trans. 1989,85, 857-866.
Figure 3. Chromatograms of a standard of 1 nmol of glucose (G), 0.23 nmol of ascorbic acid (AA), and 0.24 nmol of uric acid (UA) with (A) the Pt electrode as an anode and (B) the enzyme electrode as a cathode.
Therefore, the assay of HzOz with the enzyme electrode can be performed at a potential of +200 mV or less, while positive of the potential where the 02 reduction is significant. Application of HRP-Os(PVP) Electrode in Glucose Determination. The coupling of a glucose oxidase catalytic reaction to electrochemical oxidation of the enzymatic reaction product HzOz has been the most common method for the detection of glucose in biological samples. A platinum working electrode is often used for the electrochemical measurement of HzOz at a potential of f0.5 to +0.7 V vs Ag/AgCl.? Under these conditions, reducing agents such as ascorbic acid and uric acid, which are fairly common in biological samples, can also be oxidized easily and therefore cause interference. This has been a problem in many glucose oxidase amperometric sensors for glucose and also in the flow injection analysis of glucose employing the glucose oxidase catalytic reaction.20 In the present study, however, ascorbic acid and uric acid were separated from glucose by using an LC ODS column and an ion pairing reagent.? As shown in Figure 3A, obtained from a glucose assay using a Pt electrode at f500 mV vs Ag/AgCl, glucose barely has any retention, while both ascorbic acid and uric acid show retention and elute after glucose. When the Pt working electrode is replaced by a wired peroxidase-coated glassy carbon electrode, the current for HzOz reduction can be measured. A new enzyme electrode coupled to the LC-MER system requires less than 1h for equilibration,while (20) Janle, E. M.; Ash, S. R; Zopp. W. E.; Kissinger, P. T.Cum Separ. 1993, 12, 14-17.
a freshly polished Pt electrode requires at least an overnight equilibration to achieve a stable baseline when it is coupled to the LC system. The applied potential for the HRP enzyme electrode has been set at +lo0 mV (vs Ag/AgCl), where the Os redox center of the redox polymer is fully reduced, while a background current possibly due to the reduction of 02 is minimized. With the peroxidase electrode at +lo0 mV, ascorbic acid shows an oxidation current response at almost the same level as with the Pt electrode, and uric acid shows no significant response; on the other hand, the glucose response is 9 times greater than that for the Pt electrode (Figure 3B). The separation of ascorbic acid and uric acid from glucose can be changed by varying the content of the ion-pairing reagent in the mobile phase. Better separation, however, requires a longer operation time. In many cases when a large number of samples need to be analyzed, especially when the samples are facing degradation, fast assay is desired. Therefore, the ascorbic acid peak is usually set very close to the glucose peak, as shown in Figure 3, to save time. This could affect the accuracy of the glucose measurement when the ascorbic acid response is relatively high. Since the peroxidase electrode is much more sensitive to HzOz but not to ascorbic acid as compared with a Pt electrode, it offers better selectivity than the Pt electrode, which is important in the case of a high ascorbic acid concentration. In previous studies, efforts have been made to apply various permselective membranes to the electrode surface to prevent ascorbic acid and uric acid from reaching the electrode.20s21In this study, as a comparison, Nafion coating was also applied to both Pt and peroxidase electrodes. It resulted in the elimination of the ascorbic acid and uric acid responses and also in a 60% drop in glucose response for both coated electrodes. While much attention is given to this issue, generally speaking,glucose is 1W fold more concentrated than ascorbic acid in most biological samples, and thus the problem is actually minimal. In another glucose analysis of this study with Pt and enzyme electrodes, where the applied potential for the Pt electrode was set at +700 mV, the Pt electrode showed a higher sensitivity than in the above results, and thus the enzyme electrode sensitivity was only 4 times higher than the Pt electrode sensitivity. The higher sensitivity of the Pt electrode at f 7 0 0 mV is consistent with the significant potential dependence of the Pt electrode kinetics. At +700 mV, however, the Pt electrode also showed a much greater response to ascorbic acid. The dependencies of the peak currents on the amount of glucose injected were determined in this study using both the Pt and the enzyme electrodes. It was found that the peak currents from both electrodes initially increased linearly with the amount of glucose injected and then reached a plateau at the same glucose amount. The consistency of the upper limit of the linear range of the two electrodes (Table 1) indicates independence of the response linearity on the electrode under the experimental condition. The range of linearity observed in this study may be a function of the enzyme loading of the glucose oxidase IMER One of the goals of this study was to determine the glucose concentration in rat subcutaneous microdialysate. The microdialysis perfusion medium for this study was Ringer's solution, a biologically isotonic liquid that has been widely used for microdialysis. Ringer's solution can give a response at the same retention time as glucose, and this response is usually a limiting factor in (21) Zhang, Y.;
Hu,Y.; Wilson, G . S.Anal. Chem. 1994,66,1183-1188.
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Lac
Table I.Comparison of the Wired Peroxidase Electrode and the Pt Electrode in Glucose, Lactate, and Acetylchoiine/Choiine Determinations
assay
sensitivity (nA/nmol)
electrode
Pt
glucose
84.5 917 138 356 42 1 1790
enzyme
F’t
lactate
enzyme
Pt
ACh/Ch
enzyme
detection limit (pmol) 25W 25a 50b
2@ 0.05” 0.01”
linear range 0.25-10 nmol 0.025-10 nmol 0.05-5 nmol 0.02-5 nmol 0.05-2 pmol 0.01-2 pmol
Measured with a signal-to-noise ratio of 3:l. Calculated with a signal-to-noise ratio of 3:l. (I
G
0
4 min
A
B
Figure 5. Chromatograms of (a) Ringer’s solution and (b) a standard of 0.5 nmol of lactate (Lac) with (A) the Pt electrode as an anode and (B) the enzyme electrode as a cathode. Arrows indicate injections.
I
1
0
8
I 16
I 24
I
I
I
32
40
48
Vmin
Figure 4. Chromatograms of continuous injections of rat subcutaneous microdialysate samples with the enzyme electrode. Arrows indicate injections, and G is for glucose.
the detection limit for glucose determination of undiluted dialysate.20 The magnitude of this response from Ringer’s solution has been determined to be about the same with either the Pt electrode or the enzyme electrode, but because the enzyme electrode has a much higher glucose sensitivity, it can reliably detect a much smaller amount of glucose in the presence of Ringer’s solution. Overall, the enzyme electrode offers a lower detection limit and better sensitivity for the determination of glucose than a Pt electrode (Table 1). Figure 4 shows the measurement of glucose in rat subcutaneous microdialysate using an HRP electrode. The average glucose concentration in the dialysate over 8 days was determined as 0.7 mM. After 8 days of continuous injection of the dialysate (50 injectiodday), there was only a 5% decrease in the analysis sensitivity from the calibration with standard glucose solutions. Previous experimentsz2with Pt or Nafioncoated Pt electrodes have shown a much larger variation in sensitivity during the glucose assay of microdialysate. Application of HRP-Os(PVP) Electrode in Lactate Determination. In this study, the LC-EC system for lactate analysis, including an ion exchange LC column, a lactate oxidase IMER, and an HzOz sensing electrochemical detector, is analogous to that for glucose analysis. Again, both the enzyme electrode and the Pt electrode were used and compared for the measurement of HzOz, and the enzyme electrode showed a higher sensitivity (Figure 5), similar to the results for glucose. When currents were measured for different amounts of lactate injected, the enzyme electrode and the Pt electrode showed the same upper limit of the linear range of the response (Table l),suggesting again that (22) Janle, E. M. Unpublished results.
1330 Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
the lactate oxidase loading of the IMER is probably an important factor in determining the range of linearity. In this LC system, uric acid is very well separated from lactate, while the retention of ascorbic acid is quite close to that of lactate. As in the glucose assay, the ascorbic acid oxidation current responses at the enzyme electrode and the Pt electrode are approximately the same. Therefore, the enzyme electrode is again more selective due to its higher sensitivity for lactate. Injections of water, mobile phase, or Ringer’s solution all give small responses at the same retention as lactate, restricting the detection limit. Since the magnitudes of these blank solution responses are the same with the Pt and enzyme electrodes, the higher lactate sensitivity of the enzyme electrode leads to a lower detection limit (Table 1). The lactate concentration in a rat subcutaneousmicrodialysate sample has been determined as 0.6 mM by using the wired peroxidase enzyme electrode. No signiscant decrease in response was observed after 7 days of continuous operation of the enzyme electrode with both standard lactate solutions and dialysate samples. Applicationof HRP-Os(PVP) Electrode in Acetylcholine/ Choline Determination. Acetylcholine is an important neurotransmitter. Because the extracellular concentration of acetylcholine in rat brain is typically very low due to its fast conversion to choline by acetylcholine esterase, a very low detection limit for acetylcholine is desired. Such a low detection limit would allow the investigator to determine the basal level of acetylcholine in rat brain dialysate without using an esterase inhibit0r,2~1~~ which may seriously affect the physiology of the system. Figure 6 shows the acetylcholine/choline responses at the Pt and peroxidase electrodes. As with the glucose and lactate analyses, the enzyme electrode is more sensitive than the Pt electrode. In the meantime, the baseline noise levels at the enzyme and Pt electrodes are about the same. Therefore, the enzyme electrode offers a better detection limit than the Pt electrode (Table 1).The (23) Storella, R J.; Begen, S. Cum Separ. 1993,12,8-9. (24) Damsma, G.; Westerink, B. H. C. In Microdialysis in the Neurosciences; Robinson, T. E., Justice, J. B., Jr., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; pp 237-252.
ACh Ch
0.2 nA
0
1 4
Minules
A
8
7 0
4
8
Minutes
B
Figure 6. Chromatograms of a standard of 0.5 pmol of acetylcholine (ACh)/choline (Ch) with (A) the Pt electrode as an anode and (B) the enzyme electrode as a cathode.
linear ranges of the acetylcholine/choliine responses at these two electrodes are very close (Table l), again suggesting that the IMER part is most possibly determining the linear range. The sensitivity differentials between the enzyme electrode and the Pt electrode in glucose, lactate, and acetylcholine/choline assays are different and are possibly due to variations in the buffer solutions and the preparation of electrodes. Unlike the measurements of glucose and lactate, where in vivo samples of relatively high analyte concentrations are available and
therefore relatively high current scales are used, acetylcholine/ choline determination in rat brain dialysate needs to be conducted on a much more sensitive, lowcurrent scale. To conduct measurements on a very lowcurrent scale, a stable baseline is especially important. As mentioned earlier, with a Pt electrode, even minor variation of the system may cause significant baseline drifting and a rather long time to. return to a stable baseline. The peroxidase electrode, on the other hand, is less susceptible to changes of the system and can return to steady state more rapidly after any disturbance. The response of a Pt electrode to acetylcholine/choline may decrease significantly over time. In order to achieve a very low detection limit, one might reactivate the Pt electrode by polishing, or wiping with methanol, or operating between extreme potentials. This will inevitably lead to a very noisy and drifting baseline and then a very long equilibration of the electrode. The acetylcholine/choline analysis with a Pt electrode is therefore often limited by the dual requirements of a high response and a stable baseline. The enzyme electrode, however, can remain viable for a fairly long time; also, a new enzyme electrode requires a much shorter time to give a stable baseline. CONCLUSION A wired peroxidase electrode was developed to detect HzOz in glucose, lactate, and acetylcholine/choline analyses with LC coupled to oxidase IMERs. This electrode offers good selectivity, high sensitivity, low detection limits, and superior stability. Applications of the peroxidase electrode for glutamate, oxalate, amino acid, polyamine, and alcohol analyses with LC coupled with oxidase IMERs are now being investigated. Tentatively, it appears that the advantage of the wired polymer film peroxidase electrode will be general for all cases where an oxidase IMER is used. Received for review November 7, 1994. Accepted January
29, 1995.@ AC941080Q Abstract published in Advance ACS Abstracts, March 1, 1995.
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