Comparison of quantitative performance of three fluorescence labels in CE/LIF analysis of aspartate and glutamate in brain microdialysate.
Three different fluorescent tags have been compared for the quantitative analysis of aspartate and glutamate in brain microdialysate samples. Separation conditions have been optimized to achieve short analysis time using reversed polarity separation in coated capillary. Method validation has revealed similar quantification limit of 0.1 μM of analytes using either of the labels, although LOD values were different: 7.8-9.8 nM for 4-fluoro-7-nitro-2,1,3-benzoxadiazole, 3.5 nM for fluorescein-5-isothiocyanate and 1.3-1.5 nM for carboxyfluorescein succinimidyl ester derivatives. The almost two orders of magnitude difference between LOD and LOQ values is likely due to the unreliable derivatization reaction at low sample concentration. Based on the superior stability, FITC derivatization was used for the analysis of biological samples. The applicability of the method has been demonstrated by analyzing basal and potassium evoked amino acid concentrations in individual brain microdialysate samples.
Department of Pharmacodynamics, Semmelweis University, Budapest, Hungary.
Zsolt
Z
Wagner
Tamás
T
Tábi
Gergely
G
Zachar
András
A
Csillag
Eva
E
Szöko
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000032:analysis*; D001923:Brain Chemistry; D002645:Chickens; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D018698:Glutamic Acid / Q000032:analysis*; D017551:Microdialysis; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D013388:Succinimides / Q000737:chemistry
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D001224:Aspartic Acid; D018698:Glutamic Acid; D009327:4-Chloro-7-nitrobenzofurazan; D016650:Fluorescein-5-isothiocyanate