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.
Tsesarskaia
Mara
M
Department of Chemistry, Barry University, Miami Shores, FL 33161, USA.
Galindo
Erika
E
Szókán
Gyula
G
Fisher
George
G
D000814:Aniline Compounds / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000379:methods*; D026603:D-Aspartic Acid / Q000031:analogs & derivatives / Q000032:analysis* / Q000737:chemistry; D005971:Glutamates / Q000032:analysis* / Q000737:chemistry; D007536:Isomerism; D016202:N-Methylaspartate / Q000031:analogs & derivatives / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D013997:Time Factors; D014633:Valine / Q000031:analogs & derivatives / Q000737:chemistry
D000814:Aniline Compounds; D005971:Glutamates; C542373:N-alpha-(5-fluoro-2,4-dinitrophenyl)valinamide; C476559:N-methylglutamate; D026603:D-Aspartic Acid; D016202:N-Methylaspartate; D014633:Valine