Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
46, Number 8, September 2008, pp. 712-716
Determination of Amino Acids in Urine by Cyclodextrin-Modified
Capillary Electrophoresis–Laser-Induced Fluorescence Detection
Takashi Kaneta1,2, Hiromasa Maeda3,
Mari Miyazaki3,
Rina Miyake3, Hirotomo Izaki3, Yuji Sakoda3,
Shohei Kinoshita3,
and Totaro Imasaka1,2 1Department of Applied Chemistry, Graduate School of Engineering,
Kyushu University, Motooka, Fukuoka 819-0395, Japan; 2Division
of Translational Research, Center for Future Chemistry, Kyushu
University, Motooka, Fukuoka 819-0395, Japan; and 3Department
of Material Science and Engineering, Faculty of Engineering,
Kyushu University, Motooka, Fukuoka 819-0395, Japan
Capillary electrophoresis (CE) combined with laser-induced
fluorescence detection is applied to the determination of amino
acids in urine samples. The urine samples are first ultrafiltered,
to remove proteins and large peptides, and the filtrates are
then directly labeled by reaction with fluorescein isothiocyanate
(FITC). Cyclodextrin-modified CE using α-cyclodextrin
is employed for the separation of the FITC-labeled amino acids.
Seven amino acids are clearly separated from side reaction products
produced during the labeling reaction, when an 80mM borate buffer
containing 45mM a-cyclodextrin is used as the running buffer.
For quantitative analysis, rhodamine B is added to the labeled
urine samples as an internal standard. The calibration curves
for phenylalanine, glutamine, proline, glycine, serine, alanine,
and valine are linear in the range of 10µM to 100µM.
The concentration limits of detection for all of the amino acids
are estimated to be 160~330nM. Conversely, the limit of quantitation
(LOQ) was ~10µM and the limitations are due to the labeling
efficiency rather than the sensitivity of the detector. Three
amino acids in urine samples, glutamine, glycine, and alanine,
are readily quantitated, while the concentrations of the others
are below the LOQ. The present method would permit the determination
of seven amino acids in urine successfully.
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