4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorogenic labeling reagent for the in vivo analysis of amino acid neurotransmitters using online microdialysis-capillary electrophoresis.
4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been characterized as a fluorogenic labeling reagent for high speed, online microdialysis-capillary electrophoresis (MD-CE) assays for amino acid neurotransmitters. NBD-F labeled amines are efficiently excited using 488 nm light allowing more common lasers to be used for excitation than has been previously possible. Amino acids were sampled using microdialysis, derivatized using NBD-F in an online reaction, and directly coupled to a high-speed CE separation with laser induced fluorescence (LIF) detection. Separation conditions were optimized allowing baseline separation of 16 amino acids, including glutamate, GABA, glycine, taurine, and D-serine, in 21.5 s. Parameters of the assay, including reaction temperature (80 degrees C), reaction time (5 min), NBD-F concentration (20 mM), and laser power (20 mW), were optimized. Limits of detection for glutamate and GABA were improved over previous assays by 4-fold and 25-fold, respectively. In vivo measurements were made in the rat striatum to demonstrate the effectiveness of the assay. Changes in in vivo concentrations as small as 8-9% could be observed for glycine, d-serine, and l-serine with statistical confidence. Changes in neurotransmitter concentrations induced by stimulation with high-K+ artificial cerebral spinal fluid (aCSF) were observed.
Klinker
Chanda Ciriacks
CC
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
Bowser
Michael T
MT
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000032:analysis; D000596:Amino Acids / Q000032:analysis* / Q000302:isolation & purification; D000818:Animals; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D008297:Male; D017551:Microdialysis / Q000295:instrumentation* / Q000379:methods*; D009862:Online Systems / Q000295:instrumentation*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D000596:Amino Acids; D005456:Fluorescent Dyes; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D009327:4-Chloro-7-nitrobenzofurazan