In vivo simultaneous monitoring of γ‐aminobutyric acid, glutamate, and L‐aspartate using brain microdialysis and capillary electrophoresis with laser‐induced fluorescence detection: Analytical developments and in vitro/in vivo validations

Abstract

gamma-Aminobutyric acid (GABA), glutamate (Glu), and L-aspartate (L-Asp) are three major amino acid neurotransmitters in the central nervous system. In this work, a method for the separation of these three neurotransmitters in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. Molecules were tagged on their primary amine function with the fluorogene agent naphthalene-2,3-dicarboxaldehyde (NDA), and, after separation by micellar electrokinetic chromatography, were detected by laser-induced fluorescence using a 442 nm helium-cadmium laser. The separation conditions for the analysis of derivatized neurotransmitters in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical basis. The separation of GABA, Glu, and L-Asp takes less than 10 min by using a 75 mmol/L borate buffer, pH 9.2, containing 70 mmol/L SDS and 10 mmol/L hydroxypropyl-beta-cyclodextrin and + 25 kV voltage. The detection limits were 3, 15 nmol/L and, 5 nmol/L for GABA, Glu, and L-Asp, respectively. Moreover, submicroliter samples can be analyzed. This method allows a simple, rapid and accurate measurement of the three amino acid neurotransmitters for the in vivo brain monitoring using microdialysis sampling.

MEKC: An update focusing on practical aspects

“…Naphthalene-2,3-dicarboxaldehyde (NDA) is a another typical labeling reagent for amino compounds (especially amino acids). NDA derivatives have been detected by LIF after separation by MEKC, using different types of laser, including argon-ion [202], He-Cd (442 nm) [203], and krypton ion (413 nm) [204]. As a rule, the separation of the labeled analytes requires the addition of b-CD [204,205] or even methanol [202] to a BGE consisting of SDS and borate buffer at pH ca.…”

Mice Lacking Brain/Kidney Phosphate-Activated Glutaminase Have Impaired Glutamatergic Synaptic Transmission, Altered Breathing, Disorganized Goal-Directed Behavior and Die Shortly after Birth

“…On the day of the analysis, 30 l of sample and 30 l of standard solutions were derivatized at room temperature by adding 12 l of a mixture (1:2:1 v/v/v) containing the internal standard (10 Ϫ4 mol/L cysteic acid in 0.117 mol/L perchloric acid), a borate/NaCN solution [mixed solution (100:20, v/v) of 500 mM borate buffer, pH 8.7, and 87 mM NaCN in water] and the fluorogen naphthalene-2,3-dicarboxaldehyde solution (2.925 mM in acetonitrile/water; 50:50, v/v). The electrophoretic system and optimized separation procedure were as described by Sauvinet et al (2003). Separations were performed with a 50 m inner diameter ϫ 63 cm fusedsilica capillary (effective length, 52 cm) using 75 mM sodium borate containing 10 mM HP-␤-CD and 70 mM SDS, pH 9.2, as the running buffer, an applied voltage of 25 kV and hydrodynamic injections of samples.…”

Capillary electrophoresis combined with microdialysis in the human spinal cord: A new tool for monitoring rapid peroperative changes in amino acid neurotransmitters within the dorsal horn

“…In the first part of this work, the CE protocol, recently developed in our laboratory for the analysis of amino acids in microdialysates obtained from rat brain [9], was optimized and validated for human microdialysates according to good clinical practices. In the second part of this study, we report that a 1 min sampling rate microdialysis can be used to monitor amino acids in the dorsal horn of human spinal cord, mainly because our dialysis sampling technique is coupled to CE.…”