HPLC methods for determination of D-aspartate and N-methyl-D-aspartate.

D-Amino acids are stereoisomers or optical isomers of naturally occurring L-amino acids and thus possess the same chemical structure, but may differ in their biological/physiological properties. Until a half century ago, D-amino acids had been considered to be unnatural substances found only in microorganisms. However, improvements in analytical instruments and methods have revealed that D-amino acids are present in invertebrates and vertebrates, including humans, and that they possess important physiological functions. D-Aspartate (D-Asp) and its methylated form N-methyl-D-aspartate (NMDA) possess neuroendocrine properties in many species. Several methods have been developed for determination of D- and L-enantiomers of amino acids by high performance liquid chromatography (HPLC). We report here improved HPLC methods for the specific determination of D-Asp and NMDA in biological tissues.

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

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.