Quantitative in vivo monitoring of primary amines in rat caudate nucleus using microdialysis coupled by a flow-gated interface to capillary electrophoresis with laser-induced fluorescence detection.
A method for monitoring primary amines in vivo using microdialysis coupled on-line with capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection was explored. Dialysates were derivatized on-line with o-phthaldialdehyde/beta-mercaptoethanol and automatically transferred to a separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nL/min, resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits were in the 20-50 nM range, and the response was linear up to 50 microM. Temporal resolution was between 45 s and 3 min and was limited by separation time or broadening of sample zones during transfer to the separation capillary, depending on the operational parameters. The system was applied to measurement of primary amines in the caudate nucleus of anesthetized rats. Using CZE for separation, it was possible to resolve and monitor several compounds, including aspartate and glutamate. The measured basal concentrations of aspartate and glutamate were 1.2 +/- 0.1 and 5.0 +/- 0.4 microM, respectively, which agrees well with literature values. Increases in in vivo aspartate and glutamate were monitored with 90 s temporal resolution during K+ depolarization using dialysis flow rates of 79 nL/min; however, temporal resolution of 45 s was possible at the expense of lower relative recovery if the dialysis flow rate was increased to 155 nL/min. The use of MEKC as the separation mode significantly increased the number of compounds that could be resolved and detected. Using MEKC to separate the dialysate samples allowed aspartate, glutamate, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, tyrosine, and valine to be resolved and detected. The basal concentrations for these compounds using MEKC were 1.9 +/- 0.2, 4.1 +/- 0.2, 4.6 +/- 0.7, 2.6 +/- 0.3, 5.4 +/- 0.4, 1.8 +/- 0.2, 2.0 +/- 0.2, 11.3 +/- 1.3, 3.3 +/- 0.9, and 5.3 +/- 0.3 microM, respectively. The concentrations of these primary amines in the striatum were monitored after K+ depolarization with 3 min temporal resolution. This is the first microdialysis system to generate high relative recoveries and good temporal resolution simultaneously for multiple neurotransmitters.
Lada
M W
MW
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Kennedy
R T
RT
D000818:Animals; D001679:Biogenic Amines / Q000032:analysis*; D002421:Caudate Nucleus / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D007834:Lasers; D008297:Male; D017551:Microdialysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence
D001679:Biogenic Amines