Amino Acids Analysis Using in Loop Derivatization with 4-fluoro-7-nitro- 2,1,3-benzoxadiazole
Makoto Tsunoda, Yanting Song and Takashi Funatsu
Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
A determination method for amino acids in mouse plasma was developed by HPLC with fluorescence detection.
The developed methodology is based on a pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole
(NBD-F) carried out in the sample injection loop. The optimized derivatization time and temperature were 10 min and 60
°C, respectively. The separation of 21 amino acid derivatives was achieved on a monolithic silica column with gradient
elution. The amino acid derivatives were detected with excitation and emission at 470 nm and 530 nm, respectively. The
lower limit of detection for the individual amino acids was 1.52 to 47.0 fmol. The calibration curves for all the amino acids
were linear over the tested range of 0.03 to 30 pmol. The validation data showed that the assay is sensitive, selective
and reproducible for determination of amino acids. The analytical method was successfully applied to determine the
amino acids in a mouse plasma sample.
Keywords: Mouse, plasma, NBD-F, fluorescence.
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