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Analysis of amino acids in human vascular endothelial (ECV‐304) cells by microchip electrophoresis with fluorescence detection
Author(s) -
Shi Baoxian,
Huang Weihua,
Cheng Jieke
Publication year - 2008
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200700529
Subject(s) - fluorescence , chromatography , capillary electrophoresis , chemistry , amino acid , biochemistry , physics , quantum mechanics
A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg‐lamp excitation fluorescence detection. Fluorescein‐isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit ( S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho ‐phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA‐labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM β‐cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38–1.0 μM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV‐304). The average amount of amino acids in single ECV‐304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.