International Journal of Legal Medicine

, Volume 126, Issue 4, pp 519–524 | Cite as

GC/MS determination of ibotenic acid and muscimol in the urine of patients intoxicated with Amanita pantherina

  • Jan Stříbrný
  • Miloš Sokol
  • Barbora Merová
  • Peter Ondra
Original Article
First Online:
Received:
Accepted:

Abstract

Ibotenic acid and muscimol are substances which mostly participate in psychotropic properties of Amanita pantherina and Amanita muscaria. They are rapidly absorbed from the gastrointestinal tract and readily excreted in urine. The poisoning with A. pantherina is in the majority of cases accidental because it can be easily mistaken for the edible species (Amanita rubescens, Amanita spissa and Macrolepiota procera). Intoxication with A. muscaria is mostly intentional for recreational purposes. Prognosis of the poisoning is generally good; lethal cases are rare. Mushroom poisoning is often proved by microscopic examination of spores in the stomach or intestinal content. Authors of this article introduce an instrumental method of proving A. pantherina or A. muscaria poisoning. The article describes the isolation of ibotenic acid and muscimol from urine, the derivatization step and the determination of these compounds by gas chromatography/mass spectrometry. Isolation of these alkaloids from urine was performed on a strong cation exchanger (Dowex® 50W X8), and the elution and derivatization of the alkaloids were made in one step with ethyl chloroformate in aqueous solution of sodium hydroxide with the addition of ethanol and pyridine. Cycloserine was used as internal standard. By this method, concentrations of ibotenic acid and muscimol in the urine of four persons intoxicated with A. pantherina were determined. In this study, mass spectra of derivatized ibotenic acid and muscimol are shown, and validation of the method is described.

Keywords

Ibotenic acid Muscimol Amanita muscaria Amanita pantherina Intoxication GC/MS 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was supported by the Ministry of Health of the Czech Republic (IGA NS10269-3).

References

  1. 1.
    Wasson RG (1959) Soma, divine mushroom of immortality. Harcourt, Brace & World, New YorkGoogle Scholar
  2. 2.
    Samorini G (1992) The oldest representations of hallucinogenic mushrooms in the world (Sahara Desert, 9000-7000 B.P.). Integration 2(3):69–78Google Scholar
  3. 3.
    Saar M (1991) Ethnomycological data from Siberia and North-East Asia on the effect of Amanita muscaria. J Ethnopharmacol 31:157–173PubMedCrossRefGoogle Scholar
  4. 4.
    Wasson WP, Wasson RG (1957) Mushrooms, Russia and History. Pantheon Books, New YorkGoogle Scholar
  5. 5.
    Takemoto T, Nakajima T (1964) Structure of ibotenic acid. Yakugaku Zasshi 84:1232–1233PubMedGoogle Scholar
  6. 6.
    Bowden K, Drysdale AC (1965) A novel constituent of Amanita muscaria. Tetrahedron Lett 6:727–728CrossRefGoogle Scholar
  7. 7.
    Eugster CH, Müller GFR, Good R (1965) Wirkstoffe aus Amanita muscaria: Ibotensäure und Muscazon. Tetrahedron Lett 6:1813–1815CrossRefGoogle Scholar
  8. 8.
    Good R, Müller GFR, Eugster CH (1965) Isolierung und Charakterisierung von Prämuscimol und Muscazon aus Amanita muscaria (L. ex Fr.). Helv Chim Acta 48:927–930PubMedCrossRefGoogle Scholar
  9. 9.
    Michelot D, Melendez-Howell LM (2003) Amanita muscaria: chemistry, biology, toxicology, and ethnomycology. Mycol Res 107(2):131–146PubMedCrossRefGoogle Scholar
  10. 10.
    Walker RJ, Woodruff GN, Kerkut GA (1971) The effect of ibotenic acid and muscimol on single neurons of the snail, Helix aspersa. Comp Gen Pharmacol 2:168–174PubMedCrossRefGoogle Scholar
  11. 11.
    Verdoorn TA, Dingledine R (1988) Excitatory amino acid receptors expressed in Xenopus oocytes: agonist pharmacology. Mol Pharmacol 34:298–307PubMedGoogle Scholar
  12. 12.
    Nestler EJ, Hyman SE, Malenka RC (2001) Molecular pharmacology: a foundation for clinical neuroscience. McGraw-Hill, New YorkGoogle Scholar
  13. 13.
    Waser FG (1979) The pharmacology of Amanita muscaria. In: Efron DH, Holmstedt B, Kline NS (eds) Ethnopharmacological search for psychoactive drugs. US Public Health Service, Washington, DC, pp 419–439Google Scholar
  14. 14.
    Eugster CH (1979) Isolation structure and synthesis of central active compounds from Amanita muscaria (L. ex Fr.) Hooker. In: Efron DH, Holmstedt B, Kline NS (eds) Ethnopharmacological search for psychoactive drugs. US Public Health Service, Washington, DC, pp 416–419Google Scholar
  15. 15.
    Benjamin DR (1992) Mushroom poisoning in infants and children: the Amanita pantherina/muscaria group. J Toxicol-Clin Toxic 30(1):13–22CrossRefGoogle Scholar
  16. 16.
    Satora L, Pach D, Ciszowski K, Winnik L (2006) Panther cap Amanita pantherina poisoning case report and review. Toxicon 47:605–607PubMedCrossRefGoogle Scholar
  17. 17.
    Benedict RG, Tyler VE, Brady LR (1966) Chemotaxonomic significance of isoxazole derivatives in Amanita species. Lloydia 29:333–342Google Scholar
  18. 18.
    Chilton WS, Ott J (1976) Toxic metabolites of Amanita pantherina, A. cothurnata, A. muscaria and other Amanita species. Lloydia 39:150–157PubMedGoogle Scholar
  19. 19.
    Repke DB, Leslie DT, Kish NG (1978) GLC–mass spectral analysis of fungal metabolites. J Pharm Sci 67:485–487PubMedCrossRefGoogle Scholar
  20. 20.
    Lund U (1979) Estimation of muscimol and ibotenic acid in Amanita muscaria using high-performance liquid chromatography. Arch Pharm Chem Sci, Edition 7:115–118Google Scholar
  21. 21.
    Gore MG, Jordan PM (1982) Microbore single-column analysis of pharmacologically active alkaloids from the fly agaric mushroom Amanita muscaria. J Chromatogr 243:323–328CrossRefGoogle Scholar
  22. 22.
    Tsujikawa K, Mohri H, Kuwayama K, Miyaguchi H, Iwata Y, Gohda A, Fukushima S, Inoue H, Kishi T (2006) Analysis of hallucinogenic constituents in Amanita mushrooms circulated in Japan. Forensic Sci Int 164:172–178PubMedCrossRefGoogle Scholar
  23. 23.
    Tsujikawa K, Kuwayama K, Miyaguchi H, Kanamori T, Iwata Y, Inoue H, Yoshida T, Kishi T (2007) Determination of muscimol and ibotenic acid in Amanita mushrooms by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. J Chromatogr B 852:430–435CrossRefGoogle Scholar
  24. 24.
    Merová B, Ondra P, Staňková M, Válka I (2008) Isolation and identification of the Amanita muscaria and Amanita pantherina toxins in human urine. Neuroendocrinol Lett 29(5):744–748PubMedGoogle Scholar
  25. 25.
    Hušek P, Šimek P (2006) Alkyl chloroformates in sample derivatization strategies for GC analysis review on a decade use of the reagents as esterifying agents. Curr Pharm Anal 2:23–43CrossRefGoogle Scholar
  26. 26.
    Hušek P (1991) Amino acid derivatization and analysis in five minutes. FEBS Lett 280(2):354–356PubMedCrossRefGoogle Scholar
  27. 27.
    Husek P (1997) Urine organic acid profiling by capillary gas chromatography after a simple sample pretreatment. Clin Chem 43(10):1999–2001PubMedGoogle Scholar
  28. 28.
    Husek P, Liebich HM (1994) Organic acid profiling by direct treatment of deproteinized plasma with ethyl chloroformate. J Chromatogr B 656(1):37–43CrossRefGoogle Scholar
  29. 29.
    Husek P (1992) Gas chromatographic determination of amines, aminoalcohols and acids after treatment with alkyl chloroformates. Anal Chim Acta 259:185–192CrossRefGoogle Scholar
  30. 30.
    Satora L, Pach D, Butryn B, Hydzik P, Balicka-Slusarczyk B (2005) Fly agaric (Amanita muscaria) poisoning, case report and review. Toxicon 45:941–943PubMedCrossRefGoogle Scholar
  31. 31.
    Ott J, Preston SW, Chilton WS (1975) Fate of muscimol in the mouse. Physiol Chem Phys 7:381–384PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jan Stříbrný
    • 1
    • 2
  • Miloš Sokol
    • 1
  • Barbora Merová
    • 3
  • Peter Ondra
    • 4
  1. 1.Military Institute of Forensic MedicineCentral Military Hospital PraguePragueCzech Republic
  2. 2.Department of Toxicology, Faculty of Military Health SciencesUniversity of DefenceHradec KrálovéCzech Republic
  3. 3.Department of Medical Chemistry and BiochemistryPalacký UniversityOlomoucCzech Republic
  4. 4.Faculty Hospital, Institute of Forensic Medicine and Medical LawPalacký UniversityOlomoucCzech Republic

Personalised recommendations