Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept.
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.
Hamase
Kenji
K
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. hamase@phar.kyushu-u.ac.jp
Miyoshi
Yurika
Y
Ueno
Kyoko
K
Han
Hai
H
Hirano
Junzo
J
Morikawa
Akiko
A
Mita
Masashi
M
Kaneko
Tsuneaki
T
Lindner
Wolfgang
W
Zaitsu
Kiyoshi
K
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry / Q000652:urine; D000818:Animals; D054022:Cerebrum / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D000596:Amino Acids