, Volume 68, Supplement 1, pp 57–62

Measurement of Hydroxyl-Radical Formation in the Rat Striatum by In Vivo Microdialysis and GC-MS



A GC-MS method was developed for measuring hydroxyl-radical capture products of salicylic acid, a common trapping agent for this reactive oxygen species, in samples obtained by in vivo cerebral microdialysis experiments. The assay employed liquid–liquid extraction followed by derivatization of 2,3- and 2,5-dihydroxybenzoic acid, along with 3,5-dihydroxybenzoic acid added as an internal standard. Due to their simple electron ionization mass spectra featuring [M–57]+ ions through the loss of tertiary alkyl group from the corresponding molecular ions, tert-butyldimethylsilyl (TBDMS) derivatives afforded straightforward method development based on selected-ion monitoring. In addition, tandem mass spectrometry probing collision-induced dissociation of [M–57]+ ions obtained from the isomeric tert-butyldimethylsilyl derivatives revealed characteristic differences in the resultant product-ion spectra. Our work has demonstrated the applicability of GC-MS for the assay of microdialysates for 2,3- and 2,5-dihydroxybenzoic acid by confirming that local administration of the excitotoxic glutamate into the rat striatum significantly increased in vivo hydroxyl-radical production in this brain region and that subsequent systemic administration of α-phenyl-tert-butylnitrone reversed glutamate-induced oxidative stress.


Gas chromatography-mass spectrometry In vivo intracranial microdialysis Derivatization Oxidative stress Dihydroxybenzoic acid 


  1. 1.
    Kaur H, Halliwell B (1996) In: Free radicals, a practical approach (Punchard NA, Kelly FJ, Eds), Oxford University Press, Oxford, pp 101–116Google Scholar
  2. 2.
    Nakao N, Brundin P (1998) Prog Brain Res 116:245–263. doi:10.1016/S0079-6123(08)60441-0 CrossRefGoogle Scholar
  3. 3.
    Tabner BJ, Turnbull S, El-Agnaf OMA, Allsop D (2002) Free Radic Biol Med 32:1076–1083. doi:10.1016/S0891-5849(02)00801-8 CrossRefGoogle Scholar
  4. 4.
    Halliwell B (2006) J Neurochem 97:1634–1658. doi:10.1111/j.1471-4159.2006.03907.x CrossRefGoogle Scholar
  5. 5.
    Ferger B, Van Amsterdam C, Seyfried C, Kuschinsky K (1998) J Neurochem 70:276–280CrossRefGoogle Scholar
  6. 6.
    Teismann P, Ferger B (2000) Brain Res Protoc 5:204–210. doi:10.1016/S1385-299X(00)00014-3 CrossRefGoogle Scholar
  7. 7.
    Themann C, Teismann P, Kuschinsky K, Ferger B (2001) J Neurosci Methods 108:57–64. doi:10.1016/S0165-0270(01)00370-3 CrossRefGoogle Scholar
  8. 8.
    Halliwell B, Kaur H, Ingelman-Sundberg M (1991) Free Radic Biol Med 10:439–441. doi:10.1016/0891-5849(91)90052-5 CrossRefGoogle Scholar
  9. 9.
    Molnar-Perl I, Horvath K, Bartha R (1998) Chromatographia 48:101–110. doi:10.1007/BF02467525 CrossRefGoogle Scholar
  10. 10.
  11. 11.
    Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. Academic Press, San DiegoGoogle Scholar
  12. 12.
    Prokai L, Kim HS, Zharikova A, Roboz J, Ma L, Deng L et al (1998) J Chromatogr A 800:59–68. doi:10.1016/S0021-9673(97)01295-8 CrossRefGoogle Scholar
  13. 13.
    Thanikachalam S, Rajappan M, Kannappan V (2008) Chromatographia 67:41–47. doi:10.1365/s10337-007-0452-y CrossRefGoogle Scholar
  14. 14.
    Ungerstedt U, Herrera-Marschitz M, Jungnelius U, Ståhle L, Tossman U, Zetterström T (1982) Adv Biosci 37:219–231Google Scholar
  15. 15.
    Okazaki S, Nishida Y, Kawai H, Saito S (1996) Neurochem Res 21:1201–1207. doi:10.1007/BF02532396 CrossRefGoogle Scholar
  16. 16.
    Weber GF (1999) Neurosci Biobehav Rev 23:1079–1086. doi:10.1016/S0149-7634(99)00041-X CrossRefGoogle Scholar
  17. 17.
    Glantz SA (2002) Primer of Biostatistics, 5th edn. McGraw-Hill, New York, pp 110–111Google Scholar
  18. 18.
    Wan FJ, Tung CS, Shiah IS, Lin HC (2006) Eur Neuropsychopharmacol 16:147–153. doi:10.1016/j.euroneuro.2005.07.002 CrossRefGoogle Scholar
  19. 19.
    Prokai L, Prokai-Tatrai K, Perjesi P, Zharikova D, Perez J, Liu R et al (2003) Proc Natl Acad Sci USA 100:11741–11746. doi:10.1073/pnas.2032621100 CrossRefGoogle Scholar

Copyright information

© Vieweg+Teubner | GWV Fachverlage GmbH 2008

Authors and Affiliations

  1. 1.Department of Molecular Biology and ImmunologyUniversity of North Texas Health Science CenterFort WorthUSA

Personalised recommendations