HPLC determination of acidic D-amino acids and their N-methyl derivatives in biological tissues

Biomed Chromatogr. 2009 Jun;23(6):581-7. doi: 10.1002/bmc.1156.


D-Aspartate (D-Asp) and N-methyl-D-aspartate (NMDA) occur in the neuroendocrine systems of vertebrates and invertebrates, where they play a role in hormone release and synthesis, neurotransmission, and memory and learning. N-methyl-d-glutamate (NMDG) has also been detected in marine bivalves. Several methods have been used to detect these amino acids, but they require pretreatment of tissue samples with o-phthaldialdehyde (OPA) to remove primary amino acids that interfere with the detection of NMDA and NMDG. We report here a one-step derivatization procedure with the chiral reagent N-alpha-(5-fluoro-2,4-dinitrophenyl)-(D or L)-valine amide, FDNP-Val-NH2, a close analog of Marfey's reagent but with better resolution and higher molar absorptivity. The diastereomers formed were separated by HPLC on an ODS-Hypersil column eluted with TFA/water-TFA/MeCN. UV absorption at 340 nm permitted detection levels as low as 5-10 pmol. D-Asp, NMDA and NMDG peaks were not obscured by other primary or secondary amino acids; hence pretreatment of tissues with OPA was not required. This method is highly reliable and fast (less than 40 min HPLC run). Using this method, we detected D-Asp, NMDA and NMDG in several biological tissues (octopus brain, optical lobe and bucchal mass; foot and mantle of the mollusk Scapharca broughtonii), confirming the results of other researchers.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds / chemistry
  • Chromatography, High Pressure Liquid / economics
  • Chromatography, High Pressure Liquid / methods*
  • D-Aspartic Acid / analogs & derivatives
  • D-Aspartic Acid / analysis*
  • D-Aspartic Acid / chemistry
  • Glutamates / analysis*
  • Glutamates / chemistry
  • Isomerism
  • N-Methylaspartate / analogs & derivatives
  • N-Methylaspartate / analysis*
  • N-Methylaspartate / chemistry
  • Sensitivity and Specificity
  • Time Factors
  • Valine / analogs & derivatives
  • Valine / chemistry


  • Aniline Compounds
  • Glutamates
  • N-alpha-(5-fluoro-2,4-dinitrophenyl)valinamide
  • N-methylglutamate
  • D-Aspartic Acid
  • N-Methylaspartate
  • Valine