Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1217, Issue 7, Pages 1056-1062Publisher
ELSEVIER
DOI: 10.1016/j.chroma.2009.09.002
Keywords
Hydrophilic amino acid; Enantiomer separation; Two-dimensional HPLC; Fluorescence
Funding
- New Energy and Industrial Technology Development Organization (NEDO) of Japan
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A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the non-chiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The D-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios. (c) 2009 Elsevier B.V. All rights reserved.
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