ABSTRACT
Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.
Subject(s)
Brain Chemistry , Electrophoresis, Capillary/methods , Serotonin/analysis , Serotonin/isolation & purification , Animals , Brain Chemistry/drug effects , Citalopram/pharmacology , Fenclonine/pharmacology , Hydrogen-Ion Concentration , Lasers , Male , Microdialysis/methods , Rats , Rats, Sprague-Dawley , Sensitivity and Specificity , Spectrometry, Fluorescence/methodsABSTRACT
Oxidative stress is present in cardiovascular diseases and hyperhomocysteinemia, an independent risk factor for these diseases. It may play a role by inducing production of oxygen free radicals. Reduced glutathione is the most abundant intracellular low-molecular-weight thiol and plays an essential role in protecting cells from toxic species. The thiol-containing compounds which are the most often considered in biological analysis, are homocysteine (Hcy), cysteine (Cys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), gamma-glutamyl-cysteine (gammaGlu-Cys), and their derivatives. These aminothiols are present in body fluids or cells, associated with proteins or occur free (reduced and oxidized). These free forms may play a role in the pathogenesis of disease. Because Hcy (with Cys) exhibits pro-oxidative properties and GSH (with Cys-Gly) antioxidative properties, and because there is extensive interconversion between these metabolites, their simultaneous analysis in biological samples is necessary to examine their role in human disease. Capillary electrophoresis (CE) seems to be a solution to reach this goal. No extensive review reports the analysis of aminothiols using CE. This review describes the different CE approaches which have been used to separate and assay aminothiols, and the different obtained datas.
Subject(s)
Body Fluids/metabolism , Cells/metabolism , Glutathione/analysis , Oxidation-Reduction , Sulfhydryl Compounds/analysis , Cysteine/analysis , Electrophoresis, Capillary , Fluorescent Dyes/chemistry , Homocysteine/analysis , HumansABSTRACT
Amino acids are studied extensively using capillary electrophoresis. In a previous article, we reviewed applications reported in the period 1999-early 2001 (Prata, C., Bonnafous, P., Fraysse, N., Treilhou, M., Poinsot, V., Couderc, F., Electrophoresis 2001, 22, 4129-4138). In this article we follow on with this review for the period end of 2001-beginning of 2003. We will report the developments of detection methods, separations of enantiomers, the new medical applications, and amino acids in food and plants. This review shows that CE is more and more important for the amino acid analysis.
Subject(s)
Amino Acids/analysis , Homocysteine/analysis , Sulfhydryl Compounds/analysis , Anti-Bacterial Agents/chemistry , Electrophoresis, Capillary , Fluorescent Dyes/chemistry , Lasers , Mass SpectrometryABSTRACT
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.
Subject(s)
Electrophoresis, Capillary/methods , Tryptophan/cerebrospinal fluid , Tyrosine/cerebrospinal fluid , Buffers , Humans , Hydrogen-Ion Concentration , Lasers , Reference Standards , Spectrometry, Fluorescence , Ultraviolet RaysABSTRACT
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.
Subject(s)
Electrophoresis, Capillary/methods , Peptides/analysis , Spectrometry, Fluorescence/methods , Automation , Lasers , Sensitivity and SpecificityABSTRACT
We are interested in the detection of DNA adducts and other trace analytes by labeling them with a fluorescent tag followed by use of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for high resolution and sensitivity. Towards this goal, here we report the following: (1) synthesis and handling properties of a near-IR, carboxyl-substituted heptamethine cyanine dye; (2) modification of an existing ball lens LIF detector to provide near-LIF detection with excitation at 785 nm for CE; and (3) corresponding handling and detection of as little as 0.8 amol of the dye by enrich-injection of 4.7 microl of 1 x 10(-13) mol/l dye in methanol from an 8-microl volume into a corresponding CE-LIF system. The electrolyte for the separation was methanol-40 mmol/l aqueous sodium borate (98:2, v/v). This finding encourages further exploration of the dye by functionalization of its carboxyl group for chemical labeling purposes.
