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A fully automated amino acid analyzer using NBD‐F as a fluorescent derivatization reagent
Author(s) -
Aoyama Chiaki,
Santa Tomofumi,
Tsunoda Makoto,
Fukushima Takeshi,
Kitada Chieko,
Imai Kazuhiro
Publication year - 2004
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.365
Subject(s) - derivatization , chemistry , chromatography , reagent , calibration curve , amino acid , high performance liquid chromatography , detection limit , fluorescence , linear range , organic chemistry , biochemistry , physics , quantum mechanics
A fully automated amino acid analyzer using NBD‐F (4‐uoro‐7‐nitro‐2,1,3‐benzoxadiazole) as a uorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re‐evaluated and optimized. Amino acids were derivatized by NBD‐F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and uorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8–20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefcients of 0.999. The coefcients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method. Copyright © 2004 John Wiley & Sons, Ltd.