Determination of amino acids in urine by cyclodextrin-modified capillary electrophoresis-laser-induced fluorescence detection.
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 alpha-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 80 mM borate buffer containing 45 mM alpha-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 10 microM to 100 microM. The concentration limits of detection for all of the amino acids are estimated to be 160-330 nM. Conversely, the limit of quantitation (LOQ) was approximately 10 microM 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.
Kaneta
Takashi
T
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Motooka, Fukuoka 819-0395, Japan. kaneta@cstf.kyushu-u.ac.jp
Maeda
Hiromasa
H
Miyazaki
Mari
M
Miyake
Rina
R
Izaki
Hirotomo
H
Sakoda
Yuji
Y
Kinoshita
Shohei
S
Imasaka
Totaro
T
D000596:Amino Acids / Q000652:urine*; D002138:Calibration; D003505:Cyclodextrins / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D007834:Lasers; D012015:Reference Standards; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000596:Amino Acids; D003505:Cyclodextrins; D016650:Fluorescein-5-isothiocyanate