title: 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.
abstract: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. hamase@phar.kyushu-u.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Hamase
authors.forename: Yurika
authors.initials: Y
authors.lastname: Miyoshi
authors.forename: Kyoko
authors.initials: K
authors.lastname: Ueno
authors.forename: Hai
authors.initials: H
authors.lastname: Han
authors.forename: Junzo
authors.initials: J
authors.lastname: Hirano
authors.forename: Akiko
authors.initials: A
authors.lastname: Morikawa
authors.forename: Masashi
authors.initials: M
authors.lastname: Mita
authors.forename: Tsuneaki
authors.initials: T
authors.lastname: Kaneko
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lindner
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Zaitsu
mesh_terms: 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
chemical_list: D000596:Amino Acids