PittCon 2000: Abstract #553

DETERMINATION OF DOPAMINE IN BRAIN MICRODIALYSIS SAMPLES BY CAPILLARY ELECTROPHORESIS WITH ELECTROCHEMICAL DETECTION

JIANGHONG QIAN, HUA YANG AND ADRIAN C. MICHAEL, Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260

Microdialysis and voltammetry have been used extensively in efforts to monitor extracellular neurotransmitter levels in the central nervous system. This work requires detailed knowledge of the relationship between the results obtained and the neurochemical properties of the tissue under investigation. Comparison of the results generated simultaneously by voltammetry and microdialysis provides a valuable new approach to investigating this relationship.

Electrically evoked dopamine in rat striatum was measured simultaneously by microdialysis and in vivo voltammetry. Microdialysis samples (1-min duration) were collected during electrical stimulation and analyzed by capillary electrophoresis with electrochemical detection (CE-EC). The CE-EC system includes an electrochemical detector that permits easy, precise alignment of a detection electrode with a separation capillary. Amperometric detection was performed at a constant applied potential of 600 mV vs. Ag/AgCl. Decoupling of the electro-phoretic current from the electrochemical 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-mA separation current. The low baseline noise affords low mass (attomoles) and low concentration (nanomolar) detection limits for dopamine. Amplified-field stacking was applied for detecting dopamine in 1-min brain microdialysis samples.

The evoked dopamine concentration recorded by voltammetry was used as an index of the dopamine concentration external to the microdialysis probe. The ratio of the stimulus-evoked change in dopamine concentration observed by voltammetry in the brain to that determined in 1-min microdialysis sample is used as an apparent value of the in vivo relative recovery for dopamine. The apparent relative recovery of dopamine is two orders of magnitude smaller than values determined during in vitro probe calibration. The very low recovery is attributed to the combined effects of probe-induced trauma of the tissue and the ability of the uptake process to remove dopamine from the extracellular space and thereby prevent its diffusion to the probe. The apparent relative recovery value leads to an estimate of basal extracellular dopamine in rat striatum of 1.4 mM. The nanomolar dialysate dopamine concentration does not directly reflect the extracellular dopamine concentration: the magnitude of the dopamine relative recovery must be considered.