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

Abstract

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 which 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 are separated by HPLC on an ODS-Hypersil column eluted with TFA/water – TFA/MeCN. UV absorption at 340 nm permits detection levels as low as 5–10 picomoles. D-Asp, NMDA and NMDG peaks are not obscured by other primary or secondary amino acids; hence pretreatment of tissues with OPA is not required. This method is highly reliable and fast (less than 40 minutes HPLC run). Using this method, we have 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.

Use of Marfey's reagent and analogs for chiral amino acid analysis: Assessment and applications to natural products and biological systems

“…For the determination of N-methyl-d-(or l-)-aspartic acid and N-methyl-d-(or l-) glutamic acid in the mollusk Scapharca broughtonii, Tsesarskaia et al [86] used derivatization with FDNP-l-(and d-)-Val-NH 2 for the quantification down to 5-10 pmol levels. Authors showed an HPLC of a standard composed of mixtures of dl-AAs to which N-Methyl-d-Asp and N-Methyl-l-Asp as well as N-Methyl-d-Glu and N-Methyl-l-Glu had been added along with a chromatogram of a tissue extract from the mollusk.…”

Quantitative determination of free D-Asp, L-Asp and N-methyl-D-aspartate in mouse brain tissues by chiral separation and Multiple Reaction Monitoring tandem mass spectrometry

“…However, the use of derivatising agents is often not recommended due to the enantiomeric impurities and incomplete reactions giving false positive results and quantification errors. Moreover, LOD obtained for derivatised amino acids analysed by different HPLC methods resulted to be at least 10 times higher than the values obtained by MRM measurements [35–37]. As already demonstrated, the LOD for acidic amino acid by MS analysis was 100 times lower than that by HPLC-UV method and it was much lower for other amino acids [26].…”

Simultaneous determination of d-aspartic acid and d-glutamic acid in rat tissues and physiological fluids using a multi-loop two-dimensional HPLC procedure

“…These LOD values were better than those of most of the already reported methods. Concerning the selectivity, d-Asp and d-Glu often co-eluted with some unidentified compounds in the tissues by one-dimensional chromatographic methods [23,30,37]. Although the already reported methods enable sensitive and rapid determination of d-Asp and dGlu, a more selective method is required for the determination of trace amounts of the d-amino acids, especially d-Glu in mammalian tissues and physiological fluids to avoid the co-elution of various unknown intrinsic substances in the complex biological matrices.…”

“…For the HPLC method, using various chiral derivatization reagents, the detection sensitivity depends on the reagents used. The N-␣-(5-fluoro-2,4-dinitrophenyl)-(d or l)-valine amide (FDNP-Val-NH 2 ) derivatization gave an LOD of 5-10 pmol [37]. Using the OPA plus chiral thiols as the derivatization reagents, the LOD of dAsp was reported to be 500 fmol [38] or 20 fmol [21], and the LOD of d-Glu was reported to be 10 fmol [21].…”