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

• Published in: Biomedical chromatography Vol. 23; no. 6; pp. 581 - 587
• Main Authors: Tsesarskaia, Mara, Galindo, Erika, Szókán, Gyula, Fisher, George
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01-06-2009
• Subjects: Aniline Compounds - chemistry; Chromatography, High Pressure Liquid - economics; Chromatography, High Pressure Liquid - methods; d-Asp; D-Aspartic Acid - analogs & derivatives; D-Aspartic Acid - analysis; D-Aspartic Acid - chemistry; Glutamates - analysis; Glutamates - chemistry; HPLC; Isomerism; l-Asp; l-Glu; N-methyl-d; N-Methylaspartate - analogs & derivatives; N-Methylaspartate - analysis; N-Methylaspartate - chemistry; NMDA; N‐methyl‐d,l‐Asp; N‐methyl‐d,l‐Glu; Octopus; Scapharca broughtonii; Sensitivity and Specificity; Time Factors; Valine - analogs & derivatives; Valine - chemistry
• ISSN: 0269-3879; 1099-0801
• 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‐α‐(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. Copyright © 2009 John Wiley & Sons, Ltd.