Anal. Chem. 1995,67, 594-599
Continuous in Vivo Monitoring of Amino Acid Neurotransmitters by Microdialysis Sampling with On-Line Derivatization and Capillary Electrophoresis Separation Simon Yuji Lhou, Hong Zuo, John F. Stobaugh, Craig E. Lunte, and Susan M. Lunte* Center for Bioanalytical Research and Departments of Chemistry and Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047
A separation-based biosensor has been developed that is capable of near-real-time analysis of aspartate and glutamate with a temporal resolution of less than 2 min in anesthetized or awake, freely moving animals. The instrument consists of a microdialysis sampling system, an on-line reactor, an injection interface, and a CE-LIF system. Primary amine analytes are derivatized with NDA/CN following microdialysis sampling using an online reactor to produce fluorescent CBI derivatives. The reaction takes approximately 1 min. The derivatized sample then travels to a microinjection valve which altemately sends CE running buffer and reacted microdialysis sample to the CE column via an injection interface. The interface allows a controllable volume of 10-20 nL to be injected onto the CE separation capillary. Separation of aspartate and glutamate from the other amino acids present in the microdialysis sample was achieved within 70 s. Detection limits for glutamate and aspartate using laser-induced fluorescence detection were 0.1 pM. The linear dynamic range was acceptable for the determination of aspartate and glutamate in dialysate samples where the levels are between 1 and 10 pM. Full automation of the system was achieved by computer control of the valve, the interface, and the data collection system. The performance of this system was demonstrated in an anesthetized rat by monitoring ECF levels of aspartate and glutamate released in brain after stimulation with high concentrations of K+. Microdialysis sampling has been shown to be a powerful technique for pharmacokinetic and neurochemical Liquid chromatography (LC) is the technique most commonly employed for the analysis of microdialysis samples. Dialysates are protein-free, allowing direct injection into the chromatographic system. However, while microdialysis is a continuous sampling (1) Robinson, T. E.; Justice, J. B., Jr., Eds. Microdialysis in the Neurosciences: Techniques in the Behavioral and Neural Sciences; Elsevier: Amsterdam, 1991; VOl. 7. (2) Lunte, C. E.; Scott, D. 0.; Kissinger, P. T. Anal. Chem. 1991, 63, 773A779A. (3) Telting-Diaz, M.; Scott, D. 0.; Lunte, C. E. Anal. Chem. 1992, 64, 806810. (4) Palsmeier, R K; Lunte, C. E. Life Sci. 1994,55, 815-825. (5) Menacherry, S.; Hubert, W.; Justice, J. B., Jr. Anal. Chem. 1992,64,577583. (6) Sable, L.; Segersvard, S.; Ungerstedt, U. Life Sci. 1991, 49, 1843-1852.
594 Analytical Chemistry, Vol. 67, No. 3, February 7, 7995
method, LC requires discrete samples. The dialysate must, therefore, be collected over a fixed time interval to provide the required sample volume, and each sample represents an average concentration value obtained over this time interval. Thus, the temporal resolution becomes dependent upon the sample require ments of the chromatographic system. To minimize the sample volume required, thereby increasing the temporal resolution, capillary electrophoresis (CE) has been explored as an alternative to LC for analysis of dialysate^.^-'^ One difficulty encountered with CE analysis of microdialysis samples is the collection and injection of sample volumes of 1,uL or less. Even though only a few nanoliters of sample is actually injected onto the CE system, at least 1pL of sample is generally needed to obtain reproducible injections. Sample loss due to evaporation or adhesion of liquid to the tube during sample transfer is a major limitation in the analysis of smaller sample volumes and necessitates the use of sophisticated sample collectors and autosamplers. It is possible to couple microdialysis directly to CE because microdialysis samples are protein-free and, therefore, no protein precipitation step is necessary prior to injection. Such an on-line system also eliminates problems associated with evaporation and sample transfer. On-line coupling of microdialysis sampling with CE analysis has been reported previo~sly.'~A rotary valve and an on-line injection interface were used to isolate the experimental animal from the high voltage employed for the CE separation and to convert the dialysate with a flow rate of pL/min into discrete nanoliter volume samples for injection onto the CE. The major shortcoming of this system is that detection is limited to laser-inducedfluorescence (LIF'),which dramatically restricts the range of analytes that can be determined. This paper reports the development of an on-line derivatization system for microdialysis/CE. By derivatizing the dialysate after (7) O'Shea, T. J.; Weber, P. L.; Bammel, B. P.; Lunte, C. E.; Lunte, S. M.; Smyth, M. R J. Chromatogr. 1992,608, 189-195. (8)O'Shea, T. J.; Telting-Diu, M.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R Electroanalysis 1991, 4 , 463-468. (9) Hemandez, L.; Escalona, P. V.; Guzman, N. A. J Liq. Chromatogr. 1993, 16, 2149-2160. (10) Tellez, S.; Forges, N.; Roussin, A; Hernandez, L. /. Chromatogr. Biomed. Afifil. 1992,581, 257-266. (11) Advis, J.; Guzman, N. J. Liq. Chromatogr. 1993, 16, 2129-2148. (12) Hernandez, L.; Tucci, S.; Guzman, N.; Paez, X.J.Chromatogr. 1993,652, 393-398. (13) Hemandez, L.; Joshi, N.; Murz, E.; Verdeguer, P.; Mistud, J.J. Chromatogr. 1993,652, 399-405. (14) Roussin, A; Verdeguer, P.; Hemandez, L. Analysis 1993, M42-M45. (15) Hogan, B. L.; Lunte, S. M.; Stobaugh,J. F.; Lunte, C. E.Anal. Chem. 1994, 66. 596-602.
0003-2700/95/0367-0594$9.00/0 0 1995 American Chemical Society
sampling from the experimentalanimal but prior to injection into the CE system, continuous monitoring of compounds that are not naturally fluorescent is possible. Using this system, it is possible to monitor the levels of aspartate and glutamate in brain dialysate samples with a temporal resolution of less than 2 min. Aspartate and glutamate were chosen as test compounds since they have been shown to be involved in neurodegeneration and stroke and are, therefore, of particular interest to neuroscientists.16 However, the instrumentation described should be generally applicable to any primary amine analyte present at greater than 0.1 pM concentration that can be separated by CE. The utilization of this system for monitoring the release of aspartate and glutamate following high K+ stimulation of the brain is demonstrated. EXPERIMENTAL SECTION
Reagents. Naphthalene-2,3-dicarboxyaldehyde(NDN was received from BAS (West Lafayette, IN). Sodium cyanide was purchased from Fluka (Ronkonkoma, NY), sodium borate from Fisher Scientific (Pittsburgh, PA), and tris(hydroxymethy1)aminomethane (Tris) from Aldrich (Milwaukee,WI). Aspartate, glutamate, and phosphoserine were obtained from Sigma (St. Louis, MO). Laser-grade sodium fluorescein was purchased from Kodak (Rochester, MI). All chemicals were used as received from the manufacturer. Capillary Electrophoresis System. The CE system consisted of a CZE l00Or high-voltage power supply (Spellman, Plainview, NY) and a separation capillary of 25 pm i.d. and 360 pm 0.d. (Polymicro Technologies, Phoenix, AZ) having a total length of 30 cm and an effective length of 14 cm. The timer used to make off-line electrokinetic injections has been described previ0us1y.l~ A window was made by removing the polyimide coating on the surface, and the capillary was glued onto a positioning glass. In these experiments, the injection end of the capillary was grounded and the detection end held at a negative potential. The high-potential end (cathode) was housed in an isolation box. Capillaries were flushed with 100 mM NaOH prior to use. A separation potential of -25 to -30 kV was employed in these studies. The CE run buffer was 100 mM Tris unless otherwise indicated. Detection and Data Collection System. LIF detection was accomplished using the 442 nm line of a 5-7 mW He-Cd laser (Model 4300, Liconix, Santa Clara, CA) . The LIF detection system has been described previ0us1y.l~ Data collection was achieved using an Isco hardware/software package (Lincoln, NE), which was also capable of controlling the switching valve. Signal amplification, variable offset, and RC filtering were carried out via a locally constructed circuit. Optimization of Reaction Conditions. The effect of borate concentration on the reaction of aspartate and glutamate with NDNCN was determined. Cyanide solutions (10 mM) were prepared in pH 9.3 borate buffers which were varied in concentration between 20 and 100 mM in 20 mM increments. A 7 mM solution of NDA was prepared in methanol, and aspartate and glutamate (5 pM) were dissolved in water. For the reaction, 2 pL of the aspartate/glutamate solution and 2 pL of the cyanide solution were mixed with 2 pL of NDA The reaction was allowed to proceed for 3 min. Samples were analyzed off-line by CE-LIF. (16)Choi, D.M.;Rothman,S. M. Annu. Rev. Neurosci. 1990,13,171-182. (17) O’Shea, T.J.; Greenhagen, R D.; Lunte, S. M.; Lunte, C. E.; Smyth,M. R; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992,593,305-312.
Syringe Pump I2
Syringe Pump #3
Injection
RMoOnb
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IntMFPce
Figure 1. Schematic of microdialysis/CE system with on-line derivatization.
The effect of NDA concentration on reaction yield was also evaluated. For these experiments, the reaction conditions were the same as above except that the cyanide was dissolved in 50 mM borate and the concentration of the NDA solution was varied from 2 to 10 mM in increments of 2 mM. As before, the reaction was allowed to proceed for 3 min, and samples were analyzed off-line using the CELIF system. Reaction profiles for aspartate and glutamate were also determined under the optimal reaction conditions. In this case, 2 p L of 7 mM NDA, 2 p L of 10 mM cyanide in 50 mM borate (PH 9.3),and 2 p L each of 5 pM aspartate and glutamate were mixed together. Every 2 min, an aliquot of this reaction solution was injected into the CELIF system. The reaction was monitored over a period of 20 min. Microdialysis System. Microdialysis was accomplished using a CMA 102 microinfusion pump and a C W 1 2 microdialysis probe (Bioanalytical Systems Inc./CMA, West Lafayette, IN). The probe consisted of a polycarbonate ether dialysis membrane which was 3 mm long and had a molecular weight cutoff of 20 OOO. Connection of the microinjection pump to the inlet of the microdialysis probe was accomplished with 580 pm i.d. PE-50 tubing. The outlet of the microdialysis probe was connected to the on-line system using a fused silica capillary of 75 pm i.d. and 150 pm 0.d. &-Line Derivatization of the Dialysate. A diagram of the entire microdialysis/CE system with on-line derivatization is shown in Figure 1. The microdialysis perfusate was pumped through the probe using a CMA 102 microinfusion pump at a flow rate of 1pL/min and was directly transferred into the mixing cross via a capillary of 75 pm i.d. and 150 pm 0.d. NDA and CN- were delivered into the premixing cross by two separate syringes using a CMA 102 microinfusion pump operating at 1 pL/min. The resulting mixture was then transferred into the second mixing cross (reactor) to react with the dialysate. The crosses, with an internal channel of 0.25 mm, were the smallest commercially available (Model ZX.lC, Valco Instruments, Houston, ?x). The transfer lines from the micropumps containing NDA and CN- to the premixing cross consisted of 10-50 cm of 240pm i.d. standard HPLC PEEK tubing (Upchurch Scientific, Oak Harbor, WA). These larger inside diameter transfer lines were necessary to minimize system backpressure. Two 10 cm lengths of the same size tubing were employed to transfer the mixture of NDA and CN- from the premixing cross to the reactor. For these experiments, the NDA was dissolved in a 5446 mixture of ACN and water rather than methanol to minimize bubble formation in the reactor. Analytical Chemistry, Vol. 67, No. 3, February 1, 7995
595
Microdialysis Sample I
NDA t C N
J
Figure 2. Schematic diagram of the on-line derivatization apparatus. Inset: expanded view of on-line reactor.
It was found to be very important to use PEEK tubing and standard HPLC fittings rather than fused silica capillaries and capillary fittings in the reactor. Large inside diameter fused silica capillaries have a very small outside diameter and are thus quite susceptible to fracture. Teflon tubing was also found to be unsuitable since it has a tendency to crimp. Since the animal experiments described here are usually performed over several hours, it was important that the system be as rugged as possible. An enlarged diagram of the reactor is shown in Figure 2. The small outside diameter capillary which carries the microdialysis sample was threaded into the larger reaction capillary made of PEEK tubing of 240 pm i.d. Reaction time was measured as the transit time of the dialysate/NDA/CN mixture through the length of reaction capillary. At a reaction mixture flow rate of 4-8 pL/ min using a reaction coil of 240 pm i.d. and 300 pm o.d., the minimum reaction capillary was found to be between 17.7 and 35.4 cm. This length was less than the length of capillary necessary for mechanically connectingthe mixer to the interface, and, therefore, a reaction capillary of 45 cm was employed in all subsequent experiments unless otherwise indicated. The derivatized sample traveled to the 60 nL microinjection valve, which alternately sends CE running buffer and reacted dialysate samples to the CE capillary via the injection interface. A more detailed description of the operation of the interface has been p~b1ished.l~The gap distance between the tubing carrying the reacted dialysate and the CE separation column was 50 pm unless otherwise indicated. The valve was switched every 2 min using the 1 x 0 software. The injection time was 3 s. CE running buffer was delivered to the injection interface by a CMA 102 microinjection pump with the flow rate ranging from 2 to 7 pL/ min, depending on the sensitivity required. The apparatus was washed after each use in order to remove any residual reagent or buffer. Probe and System Calibration. In order to be able to estimate the in vivo concentrations of glutamate and aspartate, it was necessary to determine the recovery of the microdialysis probe and the linearity of the microdialysis/CE-LIF system. This was accomplished by placing the microdialysis probe in a solution of a known concentration of glutamate and aspartate and perfusing at 1pL/min. The NDA and CN- were pumped into the reactor at the same flow rate. The sample was collected in the 60 nL loop of the sampling valve and then injected into the CE separation capillary. The results of this experiment were compared with those of an experiment run under similar conditions except that the standard solution was pumped directly into the reactor. The system linearity was tested in the same fashion using a series of concentrations of glutamate and aspartate standard solution. 596 Analytical Chemistry, Vol. 67,No. 3,February 1, 1995
In V i 0 Neurochemical Studies. Male Sprague-Dawleyrats weighing 0.34-0.39 kg were anesthetized with urethane (1.5 g/kg ip) with small additional doses throughout the experiment to maintain stable anesthesia. The microdialysis probe (CMA/12, 3 mm) was stereotaxically implanted into the hippocampus using a previously described procedure.18 The probes were perfused with an artificial cerebrospinal fluid (ACSF, which contains 120 mM NaC1, 3 mM KC1,20 mM NaHC03, 1.2 mM CaC12,l.O mM MgCl2, and 0.25 mM NazHPOa). The flow rate of the perfusate was 1 pllmin. The probe was connected with the on-line analytical system approximately 3 h after surgery. Basal levels of aspartate and glutamate were monitored for at least 1h prior to K+ stimulation. After that time, the perfusate was changed to a high K+ ACSF (which contains 20 mM NaCl, 103 mM KCl, 20 mM NaHC03, 1.2 mM CaClz, 1.0 mM MgC12, and 0.25 mM Na2HPO4), which was delivered through the microdialysis probe for 10 min. Sampling was continued during the stimulation and for 10 min after stimulation ceased. RESULTS AND DISCUSSION Optimization of Derivabtion Parameters. In order to accurately determine aspartate and glutamate in microdialysis samples using an on-line reactor system, a rapid reaction which produces highly fluorescentproducts is required. NDA/CN was found to meet this criterion once the optimal reaction conditions were idenaed. The resulting CBI derivatives are fluorescent and possess an excitation maximum of 440 nm, which exactly matches the emission wavelength of the He-Cd laser. Because the final on-line derivatization system is completely automated and the levels of aspartate and glutamate in microdialysis samples are well above the limit of detection obtainable by CE-LIF, it is not necessary to achieve 100%yield. It is more important that the reaction is reproducible and that the response is linear over the concentration range commonly found in brain dialysate samples. It has been previously observed that the rate of the NDAICN reaction is dependent on the borate buffer con~entration.'~ Typical concentrations of glutamate and aspartate in brain microdialysis samples are in the range of 0.1-10 PM.~O In order to determine the optimal borate concentration for the NDA/CN reaction, 5 pM aspartate and glutamate were reacted with 7 mM NDA and 10 mM CN-. The borate buffer concentration was varied between 6 and 33 mM (final concentration). After a 3 min reaction time, the peak heights were monitored using CE-LIF. Figure 3A shows a plot of peak height vs borate buffer concentration. It can be seen that the yield increases dramatically with increasing borate concentration up to about 17 mM. Figure 3B shows the reaction profiles of glutamate and aspartate with NDA using a final borate concentration of 17 mM. As is seen in Figure 3 4 the yield can be improved by further increasing the borate concentration; however, it was found that injection of samples with buffer concentrations greater than 33 mM had a negative effect on the separation. Therefore, 17 mM borate was chosen as the appropriate reaction buffer for all subsequent experiments. The effect of NDA/CN concentration on peak height was also investigated. Again, 5 pM aspartate and glutamate were reacted with NDMCN. The final NDA concentration was varied between 0.7 and 3.3 mM. The reaction was run in 17 mM borate (final (18) Zhang, J.; Benveniste, H.; Piantadosi, C. A Neurosci. Lett. 1993,23, 179182. (19) Wong, 0. S.; Lunte, S. M. LC-CC 1989,7, 908-913. (20) Donzanti, B. A; Yamamoto, B. K. Lik Sci. 1988,43, 913-922.
0’
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Figure 3. Optimization of reaction conditions. (A) plot of peak height vs borate concentration; (8) plot of peak height vs reaction time; (C) plot of peak height vs NDA concentration. -m- glutamate, -0- aspartate. Reaction mixture consisted of 2 pL of NDA (7 mM) and 2 pL of CN- (10 mM) dissolved in borate plus 2 ,uL each of 5 pM aspartate and glutamate. Separation conditions: 100 mM Tris, pH 8.65, as the separation buffer; total capillary length, 27 cm; length from injection end to detection window, 14 cm; applied voltage, 25 kV, PMT = 300 V.
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Figure 4. Separation of CBI aspartate and CBI glutamate using (A) 10 mM borate and (8) 10 mM Tris as the running buffer. Reaction conditions are the same as in Figure 3, with a final borate concentration of 17 mM. Separation conditions are identical to those in Figure 3.
concentration), and the cyanide concentrationwas always 1.2 times that of the NDA Figure 3C shows the plot of peak height obtained using CELIF vs NDA concentration. It can be seen that the yield continues to increase slightly with increasing NDA concentration. The best yield was obtained when concentrations of NDA greater than 3.3 mM were employed. Optimization of &e Separation. NDA is not fluorescent, and no peaks were observed when a methanolic solution of NDA was injected onto the separation capillary. A large peak was apparent when NDA was dissolved in 50 mM borate prior to injection, probably due to reaction with amine contaminants in the borate
buffer. This peak increased in size over time. If NDA concentrations above 2.3 mM (final concentration) were employed for the derivatization, this peak was so large that it interfered with the determination of aspartate and glutamate. Therefore, a 7 mM solution of NDA (2.3 mM final concentration postreactor) was used in the on-line reactor. With the on-line microdialysis/CE system, the CE voltage is always on and the sample is injected electrokinetically onto the capillary. Therefore, in order to get the best injection precision, it is necessary to use a run buffer of ionic strength higher than that of the sample. Initially, borate buffers identical to that used for the NDA/CN reaction were evaluated. However, it was found that the high conductivity of the borate led to Joule heating, which caused the separation to deteriorate. Much better results were obtained with Tris buffer, which has been shown to be of lower conductivity. Figure 4 compares the separation of the same CBI derivatives using the two different buffers. When Tris was used, a much more efficient separation was achieved. On-Iine Derhtization. Unlike o-phthalaldehyde/mercap toethanol, NDA cannot be stored in the presence of CN- for long periods of time because of the potential formation of cyanohydrin intermediates that degrade to numerous fluorescent products. In our design, NDA and CN- are stored in separate syringes and are mixed in a premixing cross less than 10 s prior to the reaction. This premixing was done to assure even mixing of the reagent prior to reaction with the microdialysis sample. The reaction capillary (42 cm length) was connected directly to the injection valve. The linearity of the system was evaluated from 0.1 to 50 pM aspartate and glutamate. The system was linear for both compounds between 0.1 and 10 pM, with correlation coefficients of 0.9960 (n = 5) and 1.000 (n = 6) for aspartate and glutamate, respectively. Above 10 pM, the system showed a slight negative deviation from linearity. The limit of detection for both aspartate and glutamate using the on-line reactor microdialysis/CE system was 0.03 pM (estimated from peak-to-peak noise at a S/N of 3). This was approximately Sfold higher than detection limits obtained off-line. In the microdialysis/CE system, samples are injected electrokinetically using the injection interface. Electrokinetic injection has been shown to be less precise than other CE injection modes Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
597
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120
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Figure 6. Electropherograms obtained (a) prior to and (b) after stimulation with high-concentration potassium ACSF. (A) Fluorescein (internal standard), (B) glutamate, (C) aspartate. Dialysate flow rate, 1 pUmin; NDA (7 mM) fluorescein (10 mM) flow rate, 1 pUmin; NaCN (IO mM) flow rate, 1 pUmin; CE run buffer flow rate, 3.5 pU min. Separation conditions are the same as in Figure 3.
+
pM), (C) aspartate (5 pM), and (D) phosphoserine (10 pM). Separation
conditions are the same as in Figure 3.
due to its large dependence on sample ionic Therefore, in most cases where electrokinetic injection is employed, an internal standard is used to correct for differences in sample conductivity and electroosmoticflow. In our system, the on-line reactor provides a good mechanism by which to introduce an internal standard. Sodium fluorescein was chosen as the internal standard because its charge and mass (and, therefore, electrophoretic mobility) are similar to those of CBI glutamate. It was introduced into the system by adding it to the NDA reagent reservoir in the on-line reactor. The use of fluorescein to correct for injection variability greatly improved the precision of the technique. The relative standard deviation for 12 separate injections of glutamate was reduced from greater than 10%to less than 4%. The lack of precision in the derivatization due to uneven mixing within the reactor was also tested by continuously pumping phosphoserine into the reaction coil while derivatizing standard amino acids solutions. The peak height of the CBI derivative of phosphoserine is an indicator of any imprecision due to uneven mixing or variations in reaction time. Figure 5 shows an electropherogram containing both internal standards. It was found that most of the imprecision was due to the use of electrokinetic (21) Weinberger, R Practical Capillay Electrophoresis; Academic Press: New York, 1993; pp 197-221.
598 Analytical Chemistry, Vol. 67, No. 3,February 7, 7995
injection, with less than 1%due to variability in the reactor. Therefore, in subsequent experiments, only sodium fluorescein was added to correct for imprecision in electrokinetic injection. The total delay in the response of this system to a biochemical event is a sum of the following: (1)the time it takes for the sample to be collected and transferred to the derivatization apparatus, which is determined by the length of the transfer line; (2) the time the sample spends in the reactor, which is dependent on the length of the reaction capillary; (3) the time the sample spends traveling from the injection valve to the injection interface capillary; and (4)the CE separation time. In our case, (1) was 0.5-2 min, depending on the length of dialysate transfer line; (2) was 2.5 min; (3) was 0.5 min; and (4) was 2 min. Thus, the total delay time between the in vivo biochemical event and final analysis was 5-7 min. Shorter delay times are possible by using a shorter reaction capillary and transfer lines. In Vi0 Studies. This system was evaluated for monitoring aspartate and glutamate in brain. Glutamate is known to be released and taken up by specialized neurons and is thought to play an important role in learning and memory.22 The release of large amounts of glutamate is believed to be at least partially responsible for the neurodegeneration seen in stroke. High K+ stimulation of brain tissue is known to increase the overtlow of (22) McGeer, P. L.; McGeer, E. G. In Basic Neurochemisty: Molecular, Cellular and Medical Aspects, 4th ed.; Agranoff, B. W., Albers, R W., Molmoff, P. B., Eds.; Raven: New York, 1989; pp 311-331.
several amino a ~ i d s . In ~ ~order . ~ ~to determine the response time of this system to the release of aspartate and glutamate, an experiment involving high K+ stimulation of the brain was performed. Basal levels of aspartate and glutamate were determined at 2 min intervals for 80 min. The perfusate was then changed to a high K+ ACSF. Figure 6 shows electropherograms obtained prior to and after stimulation. An approximately Sfold increase in the response for both aspartate and glutamate was observed. This is the same magnitude of change observed previously using NDA/CN derivatization and analysis off-line by capillary electrophoresis/electrochemistry? The high K+ stimulation lasted 10 min, after which time the perfusate was switched back to ACSF. The concentration of aspartate and glutamate then returned to basal levels. A plot of the entire experiment is shown in Figure 7. Although this system has been demonstrated specifically for the determination of aspartate and glutamate, it can potentially be applied to any primary amine analyte that occurs at concentrations above 0.1 pM in dialysate samples. By changing the separation conditions, other amino acids could be detected along with aspartate and glutamate. In addition, by using other derivatization chemistries, systems can be developed for the detection of analytes possessing other reactive functional groups such as thiols or alcohols. Future studies will focus on the development of new separation-based biosensors for analytes that cannot be detected by conventional biosensor technology. (23)Tossmann, U.; Jonsson, G.; Ungerstedt. U. Acta Physiol. Scand. 1986.127, 533-545. (24)Tossmann, U.;Segovia, J.; Ungerstedt, U. Acta Physiof. Scand. 1986,127, 547-551.
.End stimulation L
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Figure 7. Plot of aspartate and glutamate release during stimulation of brain by high K+. Data obtained under conditions given in Figure 6.
ACKNOWLEDGMENT The support of this work by Bioanalytical Systems, Kansas
Technology Enterprise Corp., and the National Science Foundation via Grant EHR 92-55223 is gratefully acknowledged. The authors thank Barry Hogan for helpful discussions and Nancy Harmony for editorial assistance in the preparation of the manuscript. Received for review August 23, 1994. Accepted November 18, 1994.@ AC9408361 @
Abstract published in Aduance ACS Abstracts, December 15, 1994.
Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
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