Determinação de aminoácidos neuroativos em amostras de microdiálise utilizando eletroforese capilar e microchips com detecção por fluorescência

Abstract

Specific amino acids (i.e. arginine, citrulline, aspartic acid, histamine, glutamic acid and taurine) play significant roles in a number of physiological processes such as neurotransmission, inflammation and cell proliferation. Analytical methods that are capable of measuring these compounds in small volumes are important for in vivo sampling strategies and single cell analysis. Electrophoresis-based methods (e.g. capillary (CE) and microchip (ME) electrophoresis) with fluorescence detection have been applied to determine various biological compounds due to the high separation efficiency, simplicity, very low sample and solvent volume consumption and short analysis time. In this study, selected amino acids were off-line derivatized with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN-). NDA itself is not fluorescent, but can react with primary amines in the presence of cyanide to produce N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives, which are fluorescent. Run conditions (e.g. buffer additives, organic solvent and separation voltage) were optimized for both methods (CE and ME) to obtain baseline separation of the six analytes. Excitation was accomplished using a diode laser (λex = 445 nm and λem = 490 m). Based on five-point calibration curves, both methods showed good linearity in the range of 10 to 0.1 µmolL-1 for each amino acid. Precision with %RSDs of less than 6.9 and 13.2% was obtained from CE and ME, respectively. For CE, the number of plates (N) was greater than 150 000/m, resolution values were more than 3.4 and migration time was between 11.2 to 18.2 min. ME offered a better efficiency (N > 300 000/m) and resolution (Rs > 3.9) with a much shorter analysis time (3.8 min) than the CE method. Limits of detection and quantification were significantly lower than the concentrations measured in microdialysis sample (MD) for both methods. According to the results with MD, it was possible to identify and quantify all analytes using CE and ME method. A portable fluorescence detection system for use with microchip electrophoresis was developed. Using the portable system with ME, limits of detection for the analytes of interest were 250 nmolL-1 – 1.3 μmolL-1, which were adequate for most analyte detection in brain microdialysis samples. (P.S.: some expressions without training)