Analytical and Bioanalytical Chemistry

, Volume 386, Issue 7–8, pp 2035–2042 | Cite as

Determination of aspartate and glutamate in rabbit retina using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

Original Paper


A simple, sensitive and low-cost method was developed for the determination of aspartate (Asp) and glutamate (Glu) in rabbit retina. Polymer monolith microextraction (PMME) using a poly(acrylamide–vinylpyridine–N,N′-methylene bisacrylamide) (AA–VP–Bis) monolithic column was combined with derivatization of Asp and Glu using 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu), and this was used to analyze the derivatives of Asp and Glu by high-performance liquid chromatography (HPLC) with fluorescence detection. The conditions for the derivatization and the subsequent extraction of Asp and Glu derivatives were optimized. The enrichment factors for the derivatives of Asp and Glu were found to be 14.1 and 14.7, respectively, by PMME. The limits of detection of Asp and Glu were 0.14 and 0.53 nmol/L, respectively. The precision and recovery were evaluated with spiked retina. The inter- and intraday relative standard deviations were less than 10%. The proposed method was successfully applied to the determination of Asp and Glu levels in rabbit retina samples with different stages of intraocular hypertension.


Polymer monolith microextraction Derivatization Excitatory amino acids 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu) HPLC 



This work was partly supported by grants from the National Natural Science Foundation of China (Grant No. 20475040) and the Program for New Century Excellent Talents in University (NCET-05-0616) of MOE, China.


  1. 1.
    Qureshi GA, Qureshi AR (1989) J Chromatogr 491:281–289Google Scholar
  2. 2.
    Hemelrijck AV, Sarre S, Smolders I, Michotte Y (2005) J Neurosci Methods 144:63–71CrossRefGoogle Scholar
  3. 3.
    Tcherkas YV, Kartsova LA, Krasnova IN (2001) J Chromatogr A 913:303–308CrossRefGoogle Scholar
  4. 4.
    Wang CL, Zhao SL, Yuan HY (2006) J Chromatogr B 833:129–134CrossRefGoogle Scholar
  5. 5.
    Olney JW (1969) J Neuropathol Exp Neurol 28:455–474Google Scholar
  6. 6.
    Lipton SA, Rosenberg PA (1994) N Engl J Med 330:613–622CrossRefGoogle Scholar
  7. 7.
    Glovinsky Y, Quigley HA, Dunkelberger GR (1991) Invest Ophthalmol Vis Sci 32:484–491Google Scholar
  8. 8.
    Quigley HA, Sanchez RM, Dunkelberger GR, Baginski TA (1987) Invest Ophthalmol Vis Sci 28:913–920Google Scholar
  9. 9.
    Weinreb RN, Levin LA (1999) Arch Ophthalmol 117:1540–1544Google Scholar
  10. 10.
    Dreyer EB, Zurakowski D, Schumer RA (1996) Arch Ophthalmol 114:299–305Google Scholar
  11. 11.
    Sucher NJ, Lipton SA, Dreyer EB (1997) Vision Res 37:3483–3493CrossRefGoogle Scholar
  12. 12.
    Heinrikson RL, Meredith SC (1984) Anal Biochem 136:65–74CrossRefGoogle Scholar
  13. 13.
    Negro A, Garbisa S, Gotte L, Spina M (1987) Anal Biochem 160:39–46CrossRefGoogle Scholar
  14. 14.
    Kutlán D, Molnár-Perl I (2003) J Chromatogr A 987:311–322CrossRefGoogle Scholar
  15. 15.
    Einarsson S, Josefsson B, Lagerkvist S (1983) J Chromatogr 282:609–618CrossRefGoogle Scholar
  16. 16.
    Cohen SA, Michaud DP (1993) Anal Biochem 211:279–287CrossRefGoogle Scholar
  17. 17.
    Bosch L, Alegría A, Farré R (2006) J Chromatogr B 831:176–183CrossRefGoogle Scholar
  18. 18.
    Wael EV, Pardoen J, Van Koeveringe JA, Lugtenburg J (1977) Recl Trav Chim Pays-Bas 96:306–309Google Scholar
  19. 19.
    Li JS, Wang H, Cao LW, Zhang HS (2006) Talanta 69:1190–1199CrossRefGoogle Scholar
  20. 20.
    Wieczorek P, Jönsson JÅ, Mathiasson L (1997) Anal Chim Acta 337:183–189CrossRefGoogle Scholar
  21. 21.
    Dövyap Z, Bayraktar E, Mehmetoğlu Ü (2006) Enzyme Microb Technol 38:557–562CrossRefGoogle Scholar
  22. 22.
    Haensels R, Halwach W, Schugerl K (1996) Chem Eng Sci 41:1811–1815CrossRefGoogle Scholar
  23. 23.
    Arthur CL, Pawliszyn J (1990) Anal Chem 62:2145–2148CrossRefGoogle Scholar
  24. 24.
    Alpendurada M (2000) J Chromatogr A 889:3–14CrossRefGoogle Scholar
  25. 25.
    Stashenko EE, Martýnez JR (2004) Trends Anal Chem 23:553–561Google Scholar
  26. 26.
    Herráez-Hernández R, Cháfer-Pericás C, Campíns-Falcó P (2004) Anal Chim Acta 513:425–433CrossRefGoogle Scholar
  27. 27.
    Zhao YY, Cai LS, Jing ZZ, H Wang, Zhang HS (2003) J Chromatogr A 1021:175–181CrossRefGoogle Scholar
  28. 28.
    Hilder EF, Svec F, Frechet JMJ (2004) J Chromatogr A 1044:3–22CrossRefGoogle Scholar
  29. 29.
    Ikegami T, Tanaka N (2004) Curr Opin Chem Biol 8:527–533CrossRefGoogle Scholar
  30. 30.
    Shintani Y, Zhou X, Furuno M, Minakuchi H, Nakanishi K (2003) J Chromatogr A 985:351–354CrossRefGoogle Scholar
  31. 31.
    Quirino JP, Dulay MT, Zare RN (2001) Anal Chem 73:5557–5563CrossRefGoogle Scholar
  32. 32.
    Zhang M, Wei F, Zhang YF, Nie J, Feng YQ (2006) J Chromatogr A 1102:294–301CrossRefGoogle Scholar
  33. 33.
    Zhang HJ, Huang JF, Wang H, Feng YQ (2006) Anal Chim Acta 565:129–135CrossRefGoogle Scholar
  34. 34.
    Xie SF, Svec F, Frechet JMJ (1998) Chem Mater 10:4072–4078CrossRefGoogle Scholar
  35. 35.
    Fan Y, Zhang M, Feng YQ (2005) J Chromatogr A 1099:84–91CrossRefGoogle Scholar
  36. 36.
    Fan Y, Zhang M, Da SL, Feng YQ (2005) Analyst 130:1065–1069CrossRefGoogle Scholar
  37. 37.
    Rivas B, Maturana H, Luna M (1999) J Appl Polym Sci 74:1557–1562Google Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Hui-Juan Zhang
    • 1
  • Jin-Shu Li
    • 1
  • Hong Wang
    • 1
  • Yu-Qi Feng
    • 1
  1. 1.Department of ChemistryWuhan UniversityWuhanPeople’s Republic of China

Personalised recommendations