HPLC-fluorescence detection and MEKC-LIF detection for the study of amino acids and catecholamines labelled with naphthalene-2,3-dicarboxyaldehyde.

Naphthalene-2,3-dicarboxyaldehyde (NDA) is commonly used for detection of primary amines in conjunction with their separation with HPLC and CE. The fluorescence of the derivatives can be measured by a conventional fluorometer or via LIF. NDA is a reactive dye, which can replace o-phthaldehyde (OPA) and provides for derivatives which are considerably more stable than OPA derivatives. In addition, NDA can be used to derivatize primary amines at concentrations as low as 100 pM. In this work, HPLC/fluorescence and MEKC/LIF experiments were performed to separate/detect six neuroactive compounds, the amino acids, Gly, Glu, Asp, gamma-aminobutyric acid (GABA) and the catecholamines, dopamine and noradrenaline. The two methods were compared in terms of performance of separation. The amino acids can be separated in HPLC in less than 30 min and an identical separation is obtained in CE using MEKC and lithium salts with greater resolution (the number of theoretical plates was approximately 5000 for HPLC and 200 000 for MEKC). The lowest detected concentration was in the range of 0.1 nM for CE/LIF. The presence of a high salt concentration does not affect the separation of the samples. Examples of the analysis of microdialysate samples as well as amino acids in Ringer's solution are presented.

Analysis of tryptophan and tyrosine in cerebrospinal fluid by capillary electrophoresis and "ball lens" UV-pulsed laser-induced fluorescence detection.

For the purpose of this study, we used a "ball lens" UV laser-induced fluorescence (LIF) detector comprising a pulsed laser and a collinear optical arrangement. The fluorescence signal is induced by a pulsed laser and detected by a photomultiplier tube. When coupling the high-frequency pulsed laser to the LIF detector we used, the electronics which is designed for continuous wavelength (CW) lasers, "viewed" the laser as a continuous source. Despite this mismatch between the laser and the "ball lens" UV LIF detector, the sensitivity we obtained with tryptophan is comparable to the one obtained with the best "laboratory-made" detector described in the literature which used a CW UV laser. Limits of detection of 0.15 nM for tryptophan and 50 nM for tyrosine were estimated. As an application of this technology, we studied tryptophan and tyrosine in cerebrospinal fluids (CSFs). The analysis is very simple and works on very small samples (5 microl). It consists of using a 10 mM 3-cyclohexylamino-1-propanesulfonic acid, 15 mM sodium tetraborate, pH 9.2 buffer and injecting CSF diluted 20 times in water prior to injection. 5-Hydroxyindoleacetic acid was used as an internal standard. The separation is completed in less than 12 min. The capillary electrophoresis method which we chose is rapid, resolutive and allows accurate measurements. Recovery experiments in CSFs show recoveries between 97 and 102%. We investigated 14 different CSFs from patients who suffered from neurological disorders. Most of the concentrations vary in a range of 1.7 to 3.7 microM for Trp and 6.6 to 13.7 microM for Tyr, which is in the range observed in the literature. One patient who suffers from Huntington disease had a higher concentration of Tyr at 17.3 microM.

Automated large-volume sample stacking procedure to detect labeled peptides at picomolar concentration using capillary electrophoresis and laser-induced fluorescence detection.

We have developed an automated large-volume sample stacking (LVSS) procedure to detect fluorescein isothiocyanate-labeled peptides in the picomolar range. The injection duration is 10 min at 50 mbar to fill 62% of the capillary volume to the detection cell. The calculated limit of detection (S/N=3), filling 1% of the capillary volume, is 74 pM for bradykinin and 45 pM for L-enkephalin with samples diluted in water and analyzed in a 50 mM borate buffer, pH 9.2. With the automated LVSS system, the limits of detection are 7 pM for bradykinin, 3 pM for L-enkephalin and 2 pM for substance P. LVSS is shown to be quantitative from 500 to 10 pM.