HPLC Methods for Determination of d-Aspartate and N-methyl-d-Aspartate

  • George H. FisherEmail author
  • Mara Tsesarskaia
Part of the Methods in Molecular Biology book series (MIMB, volume 794)


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.

Key words

d-aspartate N-methyl-d-aspartate N-methyl-l-aspartate HPLC 



We wish to acknowledge the Barry University students who helped develop the experimental procedures described here: Erika Galindo, Susana Lopez, and Collins Boston. Separate reports of these methods have previously been published in references (19, 20, 21).


  1. 1.
    D’Aniello A, Giuditta A (1977) Identification of D-aspartic acid in the brain of Octopus vulgaris. J Neurochem 29, 10531057.Google Scholar
  2. 2.
    Hashimoto A, Oka T (1997) Free d-aspartate and d-serine in the mammalian brain and periphery. Prog Neurobiol 52, 325–353.PubMedCrossRefGoogle Scholar
  3. 3.
    D’Aniello A, DiFiore M M, Fisher G (1998) Occurrence of d-aspartic acid in animal tissues and its role in the nervous and endocrine systems. Trends Comp Biochem Physiol 4, 1–24.Google Scholar
  4. 4.
    Spinelli P, Brown E et al. (2006) D-Aspartic acid in the nervous system of Aplysia limacina: Possible role in neurotransmission. J Cell Physiol 206, 672–681.PubMedCrossRefGoogle Scholar
  5. 5.
    D’Aniello A, DiFiore M M, Fisher G H et al. (2000) Occurrence of d-aspartic acid and N-methyl-d-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J 14, 699–714.PubMedGoogle Scholar
  6. 6.
    Fisher G H, D’Aniello A et al. (1991) Free d-aspartate and d-alanine in normal and Alzheimer brain. Brain Res Bull 26, 983–985.PubMedCrossRefGoogle Scholar
  7. 7.
    D’Aniello A (2007) D-Aspartic acid: An endogenous amino acid with an important neuroendocrine role. Brain Res Rev 53, 215–234.PubMedCrossRefGoogle Scholar
  8. 8.
    Watkins J C, Evans R H (1981) Excitatory amino acid transmitters. Ann Rev Pharmacol Toxicol 21, 165–204.CrossRefGoogle Scholar
  9. 9.
    Monaghan D T, Cotman C W (1986) Identification and properties of N-methyl-d-aspartate receptors in rat brain synaptic plasma membranes. Proc Natl Acad Sci USA 83, 176–179.CrossRefGoogle Scholar
  10. 10.
    Mondadori C, Weiskrantz L et al. (1989) NMDA receptor antagonists can enhance or impair learning performance in animals. Exp Brain Res 75, 449–456.PubMedCrossRefGoogle Scholar
  11. 11.
    Aswad D W (1984) Determination of d- and l-aspartate in amino acid mixtures by high performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde. Anal Biochem 137, 405–407.PubMedCrossRefGoogle Scholar
  12. 12.
    Nimura N, Kinoshita T (1986) o-Phthaldialdehyde-N-acetyl-l-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatog 352, 169–177.CrossRefGoogle Scholar
  13. 13.
    Nishikawa T, Oka T et al. (1992) Determination of free amino acid enantiomers in rat brain and serum by HPLC after derivatization with N-tert.-butoxycarbonyl-L-cysteine and o-phthaldialdehyde. J Chromatog 582, 41–48.CrossRefGoogle Scholar
  14. 14.
    Brüchner H, Haasmann S et al. (1994) Liquid chromatographic determination of d- and l-amino acids by derivatization with o-phthaldialdehyde and chiral thiols. J Chromatog 666, 259–273.CrossRefGoogle Scholar
  15. 15.
    Todoroki N, Shibata K et al. (1999) Determination of N-methyl-d-aspartic acid in tissues of bivalves by HPLC. J Chromatog B 728, 41–47.CrossRefGoogle Scholar
  16. 16.
    D’Aniello A, De Simone A et al. (2002) A specific high-performance liquid chromatography method to determine N-methyl-d-aspartic acid in biological tissues. Anal Biochem 308, 42–51.PubMedCrossRefGoogle Scholar
  17. 17.
    Skine M, Fukuda H, Nimura N et al. (2002). Automated column-switching high-performance liquid chromatography system for quantifying N-methyl-d- and -L-aspartate. Anal Biochem 310, 114–121.CrossRefGoogle Scholar
  18. 18.
    Szókán G, Mezö G, Hudecz F (1988) Application of Marfey’s reagent in racemization studies of amino acids and peptides. J Chromatog 444, 115–122.CrossRefGoogle Scholar
  19. 19.
    Galindo E, Tsesarskaia M, Fisher G et al. (2009) An Improved HPLC Method for Determination and Quantification of D- and L-Aspartic Acid. In: Konno, Brueckner, D’Aniello, Fisher, Fujii, Homma (eds), D-Amino acids: practical methods and protocols, Volume 1: Analytical methods for D-amino acids. Nova Science Publishers, New York. pp. 43–48.Google Scholar
  20. 20.
    Tsesarskaia M, Galindo E, Fisher G, Szókán G (2009) A Sensitive One Step HPLC Method for Simultaneous Determination of N-Methyl-(D and L)-Aspartate, N-Methyl-(D and L)-Glutamate and (D and L)-Aspartate in Biological Tissues. In: Konno, Brueckner, D’Aniello, Fisher, Fujii, Homma (eds), D-Amino acids: practical methods and protocols, Analytical methods for D-amino acids. Nova Science Publishers, New York, pp 25–31.Google Scholar
  21. 21.
    Tsesarskaia M, Galindo E, Szókán G, Fisher G (2009) HPLC determination of acidic d-amino acids and their N-methyl derivatives in biological tissues. Biomed Chromatog 23, 581–587.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of ChemistryBarry UniversityMiami ShoresUSA

Personalised recommendations