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HPLC determination of acidic d ‐amino acids and their N ‐methyl derivatives in biological tissues
Author(s) -
Tsesarskaia Mara,
Galindo Erika,
Szókán Gyula,
Fisher George
Publication year - 2009
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1156
Subject(s) - chemistry , high performance liquid chromatography , amino acid , reagent , chromatography , elution , derivatization , organic chemistry , biochemistry
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‐NH 2 , 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.