betadex and 2-3-naphthalenedicarboxaldehyde

This study proposes a sensitive method for the simultaneous separation and concentration of 9 pairs of amino acid enantiomers by combining poly(ethylene oxide) (PEO)-based stacking, β-cyclodextrin (β-CD)-mediated micellar electrokinetic chromatography (MEKC), and 9-fluoroenylmethyl chloroformate (FMOC) derivatization. The 9 pairs of FMOC-derivatized amino acid enantiomers were baseline separated using a discontinuous system, and the buffer vials contained a solution of 150 mM Tris-borate (TB), 12.5% (v/v) isopropanol (IPA), 0.5% (w/v) PEO, 35 mM sodium taurodeoxycholate (STDC), and 35 mM β-CD, and the capillary was filled with a solution of 1.5 M TB, 12.5% (v/v) IPA, 35 mM STDC, and 35 mM β-CD. Based on the difference in viscosity between the sample zone and PEO solution and because of the STDC sweeping, the discontinuous system effectively stacked 670 nL of the 9 pairs of FMOC-derivatized amino acid enantiomers without losing chiral resolution. Consequently, the limits of detection for the 9 pairs of FMOC-derivatized amino acid enantiomers were reduced to 40-60 nM. This method was successfully used to determine d-Tryptophan (Trp), l-Trp, d-Phenylalanine (Phe), l-Phe, d-Glutamic acid (Glu), and l-Glu in various types of beers.

Topics: Amino Acids; beta-Cyclodextrins; Chromatography, Micellar Electrokinetic Capillary; Fluorenes; Naphthalenes; o-Phthalaldehyde; Online Systems; Polyethylene Glycols; Stereoisomerism; Taurodeoxycholic Acid

A rapid analytical method was developed for the quantitative determination of glutathione (GSH) using capillary electrophoresis and fluorescence detection. A fluorescence derivatization reagent, naphthalene-2,3-dicarboxaldehyde (NDA) was successfully applied to label GSH. The optimal derivatization reaction was performed with 5.0 mM NDA, 20 mM borate buffer (pH 9.2) with the reaction time of 4 min at room temperature. The capillary electrophoresis analysis of GSH could be achieved in less than 120 s using 10 mM sodium tetraborate (pH 9.2) containing 2.5 mM beta-cyclodextrin (beta-CD) as the running buffer, and the detection limit of 2.5 x 10(-9) M (S/N=3) was obtained. This method was successfully applied to analyze the content of GSH in tobacco BY-2 cells.

Topics: beta-Cyclodextrins; Buffers; Cell Line; Electrophoresis, Capillary; Fluorescence; Glutathione; Limit of Detection; Naphthalenes; Nicotiana

A rapid and sensitive capillary electrophoresis (CE) method coupled with fluorescence detection was developed for identification of protein phosphorylation by determination of phosphoamino acids. Naphthalene-2,3-dicarboxaldehyde (NDA), a fluorescence derivatization reagent, was used to label protein hydrolysate. The optimal derivatization reaction was performed with 3.5mM NDA, 40 mM NaCN and 20mM borate buffer (pH 10.0) for 15 min. The baseline separation of three phosphorylated amino acids could be obtained in less than 180 s with good repeatability by using 30 mM borate (pH 9.2) containing 2.0mM beta-cyclodextrin (beta-CD) as the running buffer. The detection limits for phosphothreonine, phosphotyrosine and phosphoserine were 7.0 x 10(-9)M, 5.6 x 10(-9)M and 7.2 x 10(-9)M, respectively (S/N=3). Also, the interference from other protein amino acids with large molar excess over that of phosphoamino acids was studied. With beta-casein as the analysis protein, this method was successfully validated.

Topics: beta-Cyclodextrins; Calibration; Caseins; Electrophoresis, Capillary; Hydrolysis; Indicators and Reagents; Naphthalenes; Phosphoamino Acids; Phosphorylation; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence

gamma-Aminobutyric acid (GABA), glutamate (Glu), and L-aspartate (L-Asp) are three major amino acid neurotransmitters in the central nervous system. In this work, a method for the separation of these three neurotransmitters in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. Molecules were tagged on their primary amine function with the fluorogene agent naphthalene-2,3-dicarboxaldehyde (NDA), and, after separation by micellar electrokinetic chromatography, were detected by laser-induced fluorescence using a 442 nm helium-cadmium laser. The separation conditions for the analysis of derivatized neurotransmitters in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical basis. The separation of GABA, Glu, and L-Asp takes less than 10 min by using a 75 mmol/L borate buffer, pH 9.2, containing 70 mmol/L SDS and 10 mmol/L hydroxypropyl-beta-cyclodextrin and + 25 kV voltage. The detection limits were 3, 15 nmol/L and, 5 nmol/L for GABA, Glu, and L-Asp, respectively. Moreover, submicroliter samples can be analyzed. This method allows a simple, rapid and accurate measurement of the three amino acid neurotransmitters for the in vivo brain monitoring using microdialysis sampling.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Aspartic Acid; beta-Cyclodextrins; Brain Chemistry; Buffers; Chromatography, Micellar Electrokinetic Capillary; Cyclodextrins; Dialysis Solutions; Electrophoresis, Capillary; Fluorescence; gamma-Aminobutyric Acid; gamma-Cyclodextrins; Glutamic Acid; Hydrogen-Ion Concentration; Lasers; Microdialysis; Naphthalenes; Neurotransmitter Agents; Rats; Rats, Sprague-Dawley; Sodium Dodecyl Sulfate; Time Factors

The use of appropriate fluorometric derivatization procedures is of considerable importance for accurate determination of amino acids in biological samples and in metal-assisted peptide hydrolysis reactions. It is especially critical for the relative fluorescence intensities (RFI) of equal amounts of amino acids to be as similar as possible. While fluorescamine and naphthalene-2,3-dicarboxaldehyde (NDA) have proven to be excellent fluorogenic reagents for amino acid detection, the effects of various factors such as organic solvent, buffer, and pH have never been rigorously evaluated with respect to normalizing the relative fluorescence intensities of individual amino acids. To this end, here we describe optimized fluorescamine and NDA derivatization reactions that enhance the accuracy of microplate-based detection of amino acids. For both fluorescamine and NDA, we have shown that the RFI values of 16 of 19 amino acids are greater than 70%. Although determination of tryptophan is problematic, this difficulty is overcome by the addition of beta-cyclodextrin to the NDA reaction. In principle, the optimized fluorescamine and NDA microplate procedures reported here can be utilized as complementary techniques for the detection of 19 of 20 naturally occurring amino acids.

Topics: Amino Acids; beta-Cyclodextrins; Cyclodextrins; Dipeptides; Fluorescamine; Fluorescent Dyes; Fluorometry; Hydrolysis; Naphthalenes; Platinum; Reproducibility of Results; Tryptophan