Anal. Chem. 1999, 71, 4486-4492
An Integrated Decoupler for Capillary Electrophoresis with Electrochemical Detection: Application to Analysis of Brain Microdialysate Jianghong Qian, Yunqing Wu, Hua Yang, and Adrian C. Michael*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
An approach to capillary electrophoresis with electrochemical detection (CE-EC) suitable for determination of dopamine in 1-min brain microdialysate samples is described. The CE-EC system includes an electrochemical detection cell that permits easy, precise, and permanent alignment of a carbon fiber microelectrode with a separation capillary (30-µm i.d., 75-cm length). Amperometric detection was performed at a constant applied potential of 600 mV with respect to a Ag/AgCl reference electrode. Decoupling of the electrophoretic current from the amperometric detector was accomplished with an integrated end-column decoupler prepared by etching the capillary outlet with HF. The decoupler produces baseline noise of 50 fA, or less, in the presence of 10-20-µA current in the separation capillary. The low baseline noise affords low mass (attomoles) and low concentration (nanomolar) detection limits for dopamine and 4-methylcatechol. A peak attributable to dopamine was identified in electropherograms of brain microdialysate samples obtained from anesthetized rats. Identification of the dopamine peak was confirmed by pharmacological methods. Dopamine was readily detected in 1-min brain microdialysate samples. The dopamine concentration in 1-min brain microdialysis samples was significantly altered by drug treatments and by brief electrical stimulation of dopaminergic axons. Microdialysis has been used extensively to monitor the neurotransmitter content of the extracellular space of mammalian brain.1 The neurotransmitter dopamine,2 a biogenic amine, has frequently been the target of such research, due to its central importance in many aspects of normal brain function as well as its role in brain disorders such as Parkinson’s disease,3 schizophrenia,4 and substance abuse.5 Determination of dopamine in * Corresponding author: (phone) (412) 624-8560; (fax) (412) 624-8611; (email)
[email protected]. (1) Robinson, T. E., Justice, J. B., Jr., Eds. Techniques in the Behavioral and Neural Sciences, Vol. 7: Microdialysis in the Neurosciences; Elsevier: Amsterdam, 1991. (2) Cooper, J. R.; Bloom, F. E.; Roth, R. H. The Biochemical Basis of Neuropharmacology, 6th ed.; Oxford University Press: New York, 1991; Chapter 10. (3) Hornykiewicz, O.; Kish, S. J. In Advances in Neurology; Yahr, M. D., Bergmann, K. J., Eds.; Raven Press: New York, 1986; Vol. 45, pp 19-34. (4) Grace, A. A. Neuroscience 1991, 41, 1-24. (5) Koob, G. F.; Bloom, F. E. Science 1988, 242, 715-723.
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brain microdialysate is challenging because the concentration of dopamine is generally in the low-nanomolar range, even in microdialysate collected from brain regions heavily innervated by dopaminergic terminals.6 To quantify these low concentrations, sufficient microdialysate volume must be collected so that the quantity of dopamine in individual samples exceeds the available mass detection limit, which is often in the femtomole range.7 As a result, the sample collection time in microdialysis studies is usually several minutes in duration, which prevents the use of microdialysis for the investigation of important phenomena that occur on a shorter time scale.8 As a step toward the goal of increasing the temporal resolution of microdialysis-based investigations of extracellular dopamine in mammalian brain, we report herein that capillary electrophoresis with electrochemical detection (CE-EC) provides both the requisite mass and concentration detection limits for the determination of dopamine in 1-min brain microdialysate samples. Although both CE-EC and capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) have been applied to analysis of brain microdialysate,9-15 prior effort in this area has focused mainly on the excitatory amino acid neurotransmitters, glutamate and aspartate. Nanomolar concentration detection limits for dopamine have been achieved with CE-LIF16,17 and attomole mass detection limits have been achieved with CE-EC.18,19 Nevertheless, the CEbased determination of dopamine in brain microdialysate has not yet been described. (6) Sam, P. M.; Justice, J. B., Jr. Anal. Chem. 1996, 68, 724-728. (7) Karreman, M.; Moghaddam, B. J. Neurochem. 1996, 66, 589-98. (8) Lu, Y.; Peters, J. L.; Michael, A. C. J. Neurochem. 1998, 70, 584-593. (9) Hogan, B. L.; Lunte, S. M.; Stobaugh, J. F.; Lunte, C. E. Anal. Chem. 1994, 66, 569-602. (10) Robert, F.; Bert, L.; Lambas-Senas, L.; Denoroy, L.; Renaud, B. J. Neurosci. Methods 1996, 70, 153-162. (11) Tucci, S.; Rada, P.; Sepulveda, M. J.; Hernandez, L. J. Chromatogr., B 1997, 694, 343-349. (12) Lada, M. W.; Vickroy, T. W.; Kennedy, R. T. Anal. Chem. 1997, 69, 45604565. (13) Lada, M. W.; Vickroy, T. W.; Kennedy, R. T. J. Neurochem. 1998, 70, 617625. (14) Robert, F.; Bert, L.; Parrot, S.; Denoroy, L.; Stoppini, L.; Renaud, B. J. Chromatogr., A 1998, 817, 195-203. (15) 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. (16) Robert, F.; Bert, L.; Denoroy, L.; Renaud, B. Anal. Chem. 1995, 67, 18381844. (17) Bert, L.; Robert, F.; Denoroy, L.; Renaud, B. Electrophoresis 1996, 17, 523525. (18) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990, 62, 1872-1876. (19) Olefirowicz, T. M.; Ewing, A. G. J. Neurosci. Methods 1990, 34, 11-15. 10.1021/ac990338f CCC: $18.00
© 1999 American Chemical Society Published on Web 09/18/1999
High-performance CE-EC demands that steps be taken to prevent the electrophoretic current from returning to ground via the electrochemical detector, which causes baseline noise and detector instability. This has been prevented in two ways, which are called end-column and off-column detection. In the former, a small-diameter capillary is used to decrease the electrophoretic current to the point where electrochemical noise can be tolerated.20-26 This has produced low mass detection limits, but the concentration detection limits remain above those needed here. The off-column approach permits the use of larger capillaries by using a decoupling joint.27-36 These joints, which are fragile, must be constructed carefully to avoid fluid leakage and dead volume. Precise and stable positioning of the detection electrode near the outlet of the separation capillary also presents a challenge. Several methods for aligning the electrodes with the separation capillary have been described,33,37-39 but extremely low detection limits have not yet been established by these methods. Lunte40 and Baldwin41 have described on-column electrodes that ensure precise and permanent electrode placement but that are not easy to replace as needed. Recently, Hu et al.42 reported that a decoupling joint could be prepared by etching a short section of the capillary wall to a thickness of ∼10 µm, rather than by fracturing the capillary. The etched joint was fragile and had to be carefully protected from strain. Nevertheless, etching appears to be a simple and effective approach to decoupling. Furthermore, Lunte described an endcolumn decoupler constructed with cast Nafion.43,44 The endcolumn decoupling approach is valuable, in that it eliminates concerns about the alignment and fragility of on-column joints. However, the fabrication of the Nafion decoupler was not easy. (20) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189192. (21) Lu, W.; Cassidy R. M.; Baranski A. S. J. Chromatogr. 1993, 640, 433-440. (22) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 65, 577-581. (23) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994, 66, 527A536A. (24) Wen, J.; Cassidy R. M. Anal. Chem. 1996, 68, 1047-1053. (25) Gerhardt, G. C.; Cassidy, R. M.; Baranski, A. S. Anal. Chem. 1998, 70, 2167-2173. (26) Wallenborg, S. R.; Nyholm, L.; Lunte, C. E. Anal. Chem. 1999, 71, 544549. (27) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (28) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1988, 60, 258-263. (29) 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. (30) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1993, 65, 247-250. (31) O’Shea, T. J.; Lunte, S. M.; LaCourse, W. R. Anal. Chem. 1993, 65, 948951. (32) Kristensen, H. K.; Lau, Y. Y.; Ewing, A. G. J. Neurosci. Methods 1994, 51, 183-188. (33) Chen, M.-C.; Huang, H.-J. Anal. Chem. 1995, 67, 4010-4014. (34) Matysik F.-M.; Meister, A.; Werner G. Anal. Chim. Acta. 1995, 305, 114120. (35) Park, S.; Lunte, S. M.; Lunte, C. E. Anal. Chem. 1995, 67, 911-918. (36) Park, S.; McGrath, M. J.; Smyth, M. R.; Diamond, D.; Lunte, C. E. Anal. Chem. 1997, 69, 2994-3001. (37) Kuhr, W. G. U.S. Patent 5244560, 1993. (38) Colon, L. A.; Dadoo, R.; Whitted, W. H.; Zare, R. N.; Ewing, A. G.; Ferris, S. S.; Woelker, J. U. U.S. Patent 5480525, 1996. (39) Fermier, A. M.; Gostkowski, M. L.; Colon, L. A. Anal. Chem. 1996, 68, 1661-1664. (40) Zhong, M.; Lunte, S. M. Anal. Chem. 1996, 68, 2488-2493. (41) Voegel, P. D.; Zhou, W. H.; Baldwin, R. P. Anal. Chem. 1997, 69, 951957. (42) Hu, S.; Wang, Z. L.; Li, P. B.; Cheng, J. K. Anal. Chem. 1997, 69, 264-267. (43) Park, S.; Lunte, C. E. Anal. Chem. 1995, 67, 4366-4370. (44) Park, S.; Lunte, C. E. FACSS 1996, Abstract 89.
Figure 1. Schematic of the etched end-column decoupler and the rotatable detection cell for CE-EC (a, stationary support collar; b, rotatable detection cell; c, butt connector; d, capillary; e, micropositioner mount; f, three translation stages of the micropositioner; g, carbon fiber microelectrode).
We report herein that etching provides a very simple way to construct a highly effective integrated fused-silica end-column decoupler. The integrated end-column decoupler is prepared by etching just the very outlet of the capillary. This decreases the detector noise to ∼50 fA, and as low as 10 fA in ideal cases, even in the presence of 10-20-µA electrophoretic current. Although the wall of the end-column decoupler is delicate, it is not subjected to strain in the system described here. To easily position a carbon fiber detection electrode at the decoupler outlet, we constructed a rotatable detection cell that enables the electrode-to-capillary alignment to be conveniently viewed from multiple angles with a microscope. A miniature micropositioner is rigidly mounted to the cell so that the alignment is maintained permanently. Application of this CE-EC system to the determination of dopamine in 1-min brain microdialysate samples collected from anesthetized rats is described. EXPERIMENTAL SECTION Integrated End-Column Decoupler. Figure 1 shows a schematic of the geometry of the integrated end-column decoupler. Fused-silica capillaries with an inner diameter of 30 µm and an outer diameter of 365 µm (Polymicro Technologies, Phoenix, AZ) were cut into 75-cm lengths. A flame was used to remove a short section of the polyimide coating from the outlet end of the capillary. The outlet end was immersed in 48% HF to a depth of 2 mm and held in that position for 10 min. This procedure enlarged the inner diameter of the capillary outlet to 50-60 µm, which simplified the task of positioning the detection electrode (see below). The inlet of the capillary was connected to a syringe pump that was used to pump water through the capillary at 1.5 µL/min to prevent further etching of the inner bore of the capillary. The outlet end of the capillary was again immersed in 48% HF to a depth of 2 mm and held in that position for 90 min, during which time the thickness of the capillary wall decreased to ∼10 µm, as estimated by examination under a calibrated microscope. The etched portion of the capillary outlet was stored under water until used. Rotatable Detection Cell. The rotatable detection cell was constructed to aid the task of aligning the detector electrode with Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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the capillary. The cell allows the alignment of the capillary and the electrode to be easily viewed from multiple angles under a fixed-position microscope (StereoZoom 6, Fisher Scientific, Pittsburgh, PA). The cell was constructed with plexiglass in the machine shop of the Department of Chemistry at the University of Pittsburgh. Figure 1 shows a cross-sectional schematic of the cell. The circular detection cell was mounted in a stationary collar, in which the cell could rotate. A support rod attached the stationary collar to a ring stand. A capillary butt connector (23797, Supelco, Inc., Bellefonte, PA) was mounted through the center of the floor of the detection cell to hold the capillary along the axis of rotation of the cell. A three-dimensional miniature micropositioner (No. 25100-01, Fine Science Tools, Foster City, CA) was rigidly attached to the rotatable detection cell with jewelers’ screws and cement. An electrode holder was attached to the micropositioner. The placement of the electrode holder was such that the axis of the electrode was also aligned with the axis of rotation of the cell. During use, the detection cell was housed in a grounded copper mesh Faraday cage. Electrochemical Detection. Detection was performed with carbon fiber microcylinder electrodes, prepared as described for in vivo voltammetry experiments.8 Individual carbon fibers (Thornell T300, Amoco Performance Products, Greenville, SC) with a radius of 3.5 µm were placed into 1-mm-o.d. glass tubes. The tubes were pulled to a fine tip and sealed with epoxy (Spurr embedding medium, Polyscienes, Warrington, PA). The exposed fiber was cut to a length of 200-300 µm. Before use, the electrodes were electrochemically pretreated in the electrophoresis run buffer (described below) by application of a triangular potential waveform (0-2 V vs Ag/AgCl delivered at 200 V/s for 2-3 s). The electrode was placed into the electrode holder of the rotatable cell and aligned with the center of the capillary. The end of the carbon fiber was placed level with the end of the capillary. A Ag/AgCl reference, a Pt auxiliary, and a Pt ground electrode were each placed in contact with the solution in the detection cell. Amperometric detection was carried out at a potential of +600 mV vs Ag/AgCl, which was applied with a home-built potentiostat. Current from the carbon fiber microelectrode was amplified at a sensitivity of 109 V/A and a rise time of 300 ms (model 428, Keithley Instruments, Cleveland, OH). The output of the current amplifier was monitored with a computer via a 12-bit analog-todigital converter (LabMaster, Scientific Solutions, Solon, OH) operated at a 20-kHz conversion rate. Each data point in the electropherogram comprised a boxcar average of the ADC values obtained over discrete 100-ms time windows. Data points were generated at 120-ms intervals. Capillary Electrophoresis. Before initial use, the capillaries were rinsed for 10 min with 0.1 M NaOH, followed by water for 10 min, and then run buffer (20 mM phosphate, pH 7.4) for 2 h. The rinsing solutions were delivered with a syringe pump at 1.5 µL/min. Rinsing was repeated every 2-3 days. During electrophoresis, the inlet of the capillary was held at an applied voltage of 24 kV (CZE1000R, Spellman High Voltage Electronics, Corp., Hauppauge, NY). Except where noted otherwise, electrokinetic sample injection was carried out at 24 kV for 5 s. Samples were prepared in run buffer or the artificial cerebrospinal fluid used for microdialysis, described below. 4488
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Other Electrochemical Detection Strategies. The performance of the integrated end-column decoupler was compared with other electrochemical detection strategies using identical capillary columns, electrophoresis paramaters, and data acquisition methods. End-column detection was performed in unetched capillaries, and improved end-column detection was performed in capillaries etched only on the inside.25 Microdialysis. Concentric-style microdialysis probes were prepared as described before.45 A short section of fused-silica capillary (75 µm i.d., 142 µm o.d, 10 cm long, Polymicro Technologies, Pheonix, AZ) served as the probe outlet line. The active portion of the probe was constructed with a short section of dialysis hollow fiber (4 mm long, 220 µm o.d, 13 000 MWCO, SpectraPore RC, Fisher Scientific, Pittsburgh, PA). The probes were perfused at 0.56 µL/min with artificial cerebrospinal fluid (aCSF) delivered from a microliter syringe pump (Harvard Apparatus, Inc., Holliston, MA). The aCSF was prepared according to the following recipe: 145 mM Na+, 2.7 mM K+, 1 mM Mg2+, 1.2 mM Ca2+, 152 mM Cl-, and 2.0 mM phosphate at pH 7.4.46 Microdialysate samples were collected into small plastic vials and stored on crushed ice under an atmosphere of dry N2 until used. Experiments involving animals were conducted under the approval of the Institutional Animal Care and Use Committee of the University of Pittsburgh. Male Sprague-Dawley rats were anesthetized with chloral hydrate (400 mg/kg ip) and placed in a stereotaxic surgical frame (Kopf Instruments, Tujunga, CA). Microdialysis probes were slowly implanted into the striatal region of the brain and perfused in place for 2-4 h before samples were collected. In some experiments, rats received 20 mg/kg nomifensine, a dopamine uptake inhibitor,47 or 400 mg/kg γbutyrolactone (GBL), a compound that decreases the activity of dopaminergic axons projecting to the striatum.48 Both drugs were administered by intraperitoneal (ip) injection. Finally, some rats received electrical stimulation of dopaminergic axons in the medial forebrain bundle. Electrical stimulation procedures are described in detail elsewhere.49 The stimulation waveform was a constant current biphasic square wave with following parameters: frequency, 45 Hz; pulse width, 2ms; pulse height, 280 µA; duration, 25 s. Reagents. All reagents were used as received from their respective supplier. Hydrofluoric acid was obtained as a 48% aqueous solution from Aldrich. Salts were obtained from Fisher. Nomifensine was obtained from Research Biochemicals, Inc. (Natick, MA). All other chemicals were obtained from Sigma (St. Louis, Mo.). All solutions were prepared in ultrapure water (Nanopure) and were filtered through 0.2-µm pore size Nylon filters before use (Supelco, Inc., Bellefonte, PA). Safety Considerations. Following instructions provided by the manufacturer of the high-voltage power supply, the highvoltage electrode at the capillary inlet was housed inside a plexiglass box equipped with an interlock switch that disabled the high voltage if the box was opened. (45) Abercrombie, E. D.; Bonatz, A. E.; Zigmond, M. J. Brain Res. 1990, 525, 36-44. (46) Moghaddam, B.; Bunney, B. S. J. Neurochem. 1989, 53, 652-654. (47) Fielding, S.; Szewczak, M. R. J. Clin. Psychiatry 1984, 45, 12-20, (48) Walters, J. R.; Roth R. H. J. Pharmacol. Exp. Ther. 1974, 191, 82-91. (49) Yang, H.; Peters, J. L.; Michael, A. C. J. Neurochem. 1998, 71, 684-692.
Figure 2. CE-EC electropherograms of mixtures of dopamine (DA) and 4-methylcatechol (4-MC) dissolved in run buffer at a concentration of (A) 50 and (B) 5 nM. In (B), the amount of dopamine and 4-methylcatechol is 38 and 27 amol, respectively.
RESULTS AND DISCUSSION General Operational Characteristics of the Integrated Decoupler. The integrated end-column decoupler is simple to prepare. The end of the capillary is just dipped in HF to a measured depth for a measured time. The etching process is highly reproducible, does not need to be monitored, and requires no specialized skill or apparatus. The etched portion of the integrated end-column decoupler is delicate but is not subjected to any strain in the system described here. Mounting the capillary into the detection cell is straightforward. The end-column decouplers do not break during normal use and have remained operational for more than 1 month with consistent daily use. Capillary lifetime is shortened to 2-3 weeks by analysis of microdialysis samples. The rotatable detection cell aids the task of aligning the capillary and detection electrode because the alignment can be checked from multiple angles with a microscope. The alignment procedure requires 2-3 min to complete. Since the micropositioner is rigidly mounted to the detection cell, there has been no need to realign the electrode during use. As with the decoupler itself, the electrode is not subjected to strain and the electrodes do not break during normal use. CE-EC of Dopamine and 4-Methylcatechol Standards. Figure 2 shows electropherograms of dopamine and 4-methylcatechol standards that illustrate the routine performance of the CEEC system described here. Figure 2A shows an electropherogram of a 50 nM mixture of dopamine and 4-methylcatechol prepared in the CE run buffer. Figure 2B shows an electropherogram of a 5 nM mixture. In Figure 2B, the peaks are easily distinguished from the baseline noise and the injected amounts for each solute are 38 and 27 amol, respectively. Figure 2 demonstrates that CEEC system described here provides both low mass (attomoles) and low concentration (nanomolar) detection limits.
Figure 3. (A) Illustration of baseline noise that appears after extended, continuous use of the integrated end-column decoupler. (B) The return to low baseline noise after the application of -24 kV to the capillary inlet for 1 min, followed by a 10-min reequilibration of the system.
The low baseline absolute current value and low baseline noise evident in Figure 2 are not maintained indefinitely with the integrated end-column decoupler. Figure 3A shows that, after extended continuous use, both the baseline current and the baseline noise may suddenly increase. Without corrective measures, the noise illustrated in Figure 3A increases continuously over time. This is attributed to polarization of the fused-silica decoupler, which causes it to become nonconductive. We have found, however, that this is easily eliminated by a brief reversal of the polarity of the high-voltage power supply. Figure 3B shows that low baseline current and the low baseline noise returned after application of -24 kV to the capillary inlet for 1 min, followed by reequilibration of the system for ∼10 min. Delivery of the negative voltage was accomplished by means of a voltage polarity switch on the front panel of the power supply. We now apply the voltage reversal procedure as a precaution once each day or each other day of operation. Table 1 reports the baseline peak-to-peak noise and dopamine detection limits obtained with end-column detection (no etching of the capillary), improved end-column detection (etching of the inner capillary wall only), and the integrated end-column decoupler. All measurements were performed with 30-µm-i.d. by 75-cmlong capillaries, 5-s electrokinetic sample introduction, a 20 mM phosphate run buffer, and boxcar averaging of the amplifier output. In each case, the tip of the carbon fiber electrode was aligned with the center of the capillary and level with the capillary outlet. Table 1 shows that, in our hands, the integrated end-column decoupler produced 1 order of magnitude less baseline noise than the other detection methods, with all other experimental conditions being equal. This, together with Figure 3, demonstrates that the conductivity of the thin wall of the integrated end-column Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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Table 1. Effect of Different CE-EC Designs on Baseline Noise and Dopamine Concentration Detection Limitsa design
noise (pA)
dopamine detection limit (nM)
end-column (n ) 4) improved end-column (n ) 9) integrated decoupler (n ) 6)
1-1.5 0.5-1 0.01-0.05
50 20 5
a The n values are the number of separate capillaries included in this test. The dopamine detection limit is given as the lowest dopamine concentration that gave rise to an observed peak amplitude at least 3 times larger than the base line noise.
decoupler plays an important role in decreasing the contribution of the electrophoretic current to the baseline noise. The decrease in baseline noise yielded a concomitant decrease in the dopamine concentration detection limit, defined in Table 1 as the lowest dopamine concentration that produced a peak at least 3 times larger than the baseline noise. Calibration curves for dopamine, based on the heights of the respective peaks in the electropherogram, were constructed using the CE-EC system with the integrated decoupler. A representative calibration curve was linear over the 5-50 nM concentration range (r2 ) 0.992), had a slope of 0.02 pA/µM, and had an intercept very close to the origin (-0.05 pA). Again, this illustrates the typical performance of this CE-EC system. CE-EC of Dopamine in Brain Microdialysate. Figure 4A shows electropherograms recorded by CE-EC following the electrokinetic injection of dopamine and 4-methycatechol dissolved in aCSF, rather than run buffer. Several spikes are evident that are also present in the electropherogram of blank aCSF (Figure 4B), which are derived from the additional metal ions in the aCSF (K+, Ca2+, Mg2+). One of these spikes occurs at a migration time similar to that of dopamine and occasionally interfered with the quantification of the dopamine peak. Addition of 2 mM EDTA to the run buffer mostly eliminated this interference (Figure 4C). The remainder of the work described below was carried out with EDTA in the run buffer. The spike close to 4-methylcatechol appears in all electropherograms and serves as a marker for electroosmotic flow. Figure 5 shows a CE-EC electropherogram recorded after a 5-s electrokinetic injection of brain microdialysate collected from the striatum of an anesthetized rat. The feature near a migration time of 750 s is the spike that appears close to neutral substances. The most prominent features of the electropherogram are several late-eluting anionic substances. By comparison of the migration times with standards and by evaluation of the elution order, these anions have been identified as homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), and uric acid (UA). These anions are commonly found in striatal dialysate, the first two being acidic dopamine metabolites and the latter two being ubiquitous in brain. The inset in Figure 5 is the detector response recorded near the migration time of 500 s plotted on an expanded scale. A small peak is evident on this expanded scale. The peak is clearly due to a cation present at low concentration with a migration time similar to that of dopamine. Pharmacological experiments, described below, provide further indication that this peak is dopamine. 4490 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 4. (A) CE-EC electropherogram of 500 nM dopamine (DA) and 4-methylcatechol (4-MC) standards prepared in the aCSF solution used for microdialysis perfusion. (B) CE-EC electropherogram of blank aCSF. (C) CE-EC electropherogram of 500 nM DA and 4-MC prepared in aCSF after addition of 2-mM EDTA to the run buffer.
Figure 5. CE-EC electropherogram of a basal brain microdialysate sample obtained from the striatum of a rat anesthetized with chloral hydrate. The inset is the detector response near a migration time of 500 s plotted on an expanded scale. Abbreviations: DA, dopamine; HVA, homovanillic acid; DOPAC, dihydroxyphenylacetic acid; AA, ascorbic acid; UA, uric acid.
Figure 6A is the same data presented in the inset of Figure 5 after treatment with a moving average smoothing routine. Figure 6B shows the electropherogram of a brain microdialysate sample collected 20 min after the rat had received a dose of nomifensine (20 mg/kg ip) to block dopamine uptake. The peak in the electropherogram near a migration time of 500 s was increased ∼50-fold by this pharmacological treatment, which is consistent
Figure 6. (A) CE-EC electropherogram of a brain microdialysate sample collected from a rat anesthetized with chloral hydrate (data from the inset in Figure 5 after treatment with a moving average). (B) CE-EC electropherogram of a brain microdialysate sample collected 20 min after the rat received a dose of the dopamine uptake inhibitor, nomifensine (20 mg/kg ip). (C) CE-EC electropherogram of a brain microdialysate sample collected after the rat was euthanized with a lethal dose of anesthetic.
with previous reports of the action of nomifensine on dialysate dopamine levels.50 We have not yet been able to identify the substance that elutes near 560 s after nomifensine administration. Nomifensine has some potency as an inhibitor of norepinephrine and serotonin uptake,51 but these neurotransmitters do not elute near 560 s (data not shown). Figure 6C shows the electropherogram of a brain microdialysate sample collected for 20 min immediately after the rat was euthanized by an overdose of anesthetic. A very large peak appears near a migration time of 510 s, which is consistent with the well-known massive postmortem release of neurotransmitter stores. A smaller peak near a migration time of 505 s is attributed to serotonin, which is present at a low level in the striatum and which elutes just before dopamine in experiments with standards. The peak that we attribute to dopamine steadily increases slightly in migration time from A to C in Figure 6. This increase in migration time is attributed to changes in the electroosmotic flow rate during the analysis of microdialysate. The migration time of dopamine standards examined before and after a series of microdialysate samples shows the same slight increase (data not included). We have not specifically examined the cause of this change in electroosmotic flow rate, but we tentatively attribute it to adsorp(50) Church, W. H.; Justice, J. B., Jr.; Byrd, L. D. Eur. J. Pharmacol. 1987, 139, 345-348. (51) Schacht, U.; Leven, M.; Gerhards, H. J.; Hunt, P.; Raynaud, J.-P. Int. Pharacopsychiat. 1982, 17 (Suppl. 1), 21-34.
Figure 7. (A) CE-EC electropherogram of a 5 nM dopamine standard prepared in aCSF. Field-amplified stacking was used. The standard was diluted 10-fold with distilled water and the electrokinetic sample injection lasted for 30 s. (B) CE-EC electropherogram of a 1-min brain microdialysate sample collected from the striatum of a rat anesthetized with chloral hydrate. The microdialysate was diluted 10-fold in distilled water and introduced to the column with a 30-s electrokinetic injection. Inset: Time course of the change in the height of the suspected dopamine peak after rats received a dose of GBL (symbols are the mean ( the standard deviation of results obtained from four rats).
tion of biomolecules to the capillary wall. More extensive column reconditioning between microdialysis samples may stabilize the electroosmotic flow rate. The dopamine peak obtained by a 5-s electrokinetic introduction of brain microdialysate is only slightly above detection limits. Figure 7 shows that the detection limit can be significantly lowered by field amplification stacking. Figure 7A shows the CE-EC electropherogram of a 5 nM dopamine standard prepared in aCSF. The 5 nM dopamine standard was diluted 10-fold with distilled water, and the electrokinetic injection time was increased to 30 s. The width of the dopamine peak in this case was considerably decreased and the amplitude significantly increased. Figure 7B is the electropherogram of a 1-min brain microdialysate sample that was also diluted 10-fold with distilled water and introduced to the separation capillary by means of a 30-s electrokinetic injection. To collect the sample, the outlet of the microdialysis probe was transferred for 1 min to a sample vial containing 4.5 µL of distilled water, i.e., enough water to dilute the microdialysate sample ∼10-fold. Figure 7 demonstrates, therefore, the successful application of this CE-EC system for the monitoring dopamine in short-duration (1-min) microdialysis samples collected from animals in the resting state. Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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Panels A and B of Figure 8 show the electropherograms of 1-min microdialysis samples collected before and during, respectively, electrical stimulation of dopaminergic axons passing through the medial forebrain bundle as they project to the striatal region of the brain. The samples were again diluted by 10-fold with water and introduced to the capillary by means of a 30-s electrokinetic injection. The inset panel in Figure 8B reports the mean and the standard deviation of the dopamine concentration found in the microdialysate samples. The brief stimulation (25 s in duration) caused a significant increase in the dopamine concentration in 1-min microdialysate samples. Figure 8 demonstrates that the CE-EC system described here can be used to monitor the effects of brief changes in neuronal activity on the concentration of dopamine in brain microdialysate samples.
Figure 8. CE-EC electropherograms of 1-min microdialysis samples collected from striatum of an anesthetized rat (A) before and (B) during electrical stimulation of dopaminergic axons in the medial forebrain bundle. In both cases, the microdialysate samples were diluted 10fold in distilled water and introduced to the capillary with a 30-s electrokinetic injection. Inset: The mean ( the standard deviation of the dopamine concentration found in 1-min brain microdialysate samples collected before and during electrical stimulation.
We used a second pharmacological approach to further confirm that the lone peak in Figure 7B is due to dopamine. The inset in Figure 7B is the time course of the change in the height of the peak in the electropherograms of brain microdialysis samples before and after the rat received a dose of GBL (400 mg/kg ip), which causes a decrease in the electrical activity of dopaminergic neurons.48 Over the course of 1 h following administration of GBL, the peak height decreased to below detectable levels, providing clear pharmacological evidence that the peak can be attributed to dopamine.
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Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
CONCLUSION In this work, we have focused attention on establishing low mass (attomoles) and low concentration (nanomalar) detection limits for dopamine in short duration (1-min) brain microdialysate samples via CE-EC. Low detection limits were achieved through the development of a decoupler design that reduces baseline electrochemical noise to levels normally only achieved with much smaller inner diameter capillaries. The CE-EC strategy reported here is distinct from the improved end-column detection described by Ewing22,23 and the end-column decoupler described by Lunte,43,44 neither of which employed a conductive section in the fused-silica capillary wall. Although low detection limits have been established, this is only an initial step toward high speed in vivo monitoring of dopamine. Further refinements of this CE-EC system are needed to decrease and stabilize the dopamine migration time and to incorporate on-line coupling to the microdialysis probe.52,53 ACKNOWLEDGMENT Financial support was provided by NIH (Grant NS31442) and by The National Parkinson’s Foundation Center of Excellence at the University of Pittsburgh.
Received for review March 31, 1999. Accepted August 18, 1999. AC990338F (52) Lada, M. W.; Schaller, G.; Carriger, M.H.; Vickroy, T. W.; Kennedy, R. T. Anal. Chim. Acta 1995, 307, 217-225. (53) Lada, M. W.; Kennedy, R. T. J. Neurosci. Methods 1997, 72, 153-159.
