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

J Chromatogr A. 2010 Feb 12;1217(7):1056-62. doi: 10.1016/j.chroma.2009.09.002. Epub 2009 Sep 6.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / analysis*
  • Amino Acids / chemistry
  • Amino Acids / urine
  • Animals
  • Cerebrum / chemistry
  • Chromatography, High Pressure Liquid / instrumentation
  • Chromatography, High Pressure Liquid / methods*
  • Equipment Design
  • Hydrophobic and Hydrophilic Interactions
  • Male
  • Rats
  • Rats, Wistar
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Stereoisomerism


  • Amino Acids