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

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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.

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References

  1. Wasson RG (1959) Soma, divine mushroom of immortality. Harcourt, Brace & World, New York

    Google Scholar 

  2. Samorini G (1992) The oldest representations of hallucinogenic mushrooms in the world (Sahara Desert, 9000-7000 B.P.). Integration 2(3):69–78

    Google Scholar 

  3. Saar M (1991) Ethnomycological data from Siberia and North-East Asia on the effect of Amanita muscaria. J Ethnopharmacol 31:157–173

    Article  PubMed  CAS  Google Scholar 

  4. Wasson WP, Wasson RG (1957) Mushrooms, Russia and History. Pantheon Books, New York

    Google Scholar 

  5. Takemoto T, Nakajima T (1964) Structure of ibotenic acid. Yakugaku Zasshi 84:1232–1233

    PubMed  CAS  Google Scholar 

  6. Bowden K, Drysdale AC (1965) A novel constituent of Amanita muscaria. Tetrahedron Lett 6:727–728

    Article  Google Scholar 

  7. Eugster CH, Müller GFR, Good R (1965) Wirkstoffe aus Amanita muscaria: Ibotensäure und Muscazon. Tetrahedron Lett 6:1813–1815

    Article  Google Scholar 

  8. Good R, Müller GFR, Eugster CH (1965) Isolierung und Charakterisierung von Prämuscimol und Muscazon aus Amanita muscaria (L. ex Fr.). Helv Chim Acta 48:927–930

    Article  PubMed  CAS  Google Scholar 

  9. Michelot D, Melendez-Howell LM (2003) Amanita muscaria: chemistry, biology, toxicology, and ethnomycology. Mycol Res 107(2):131–146

    Article  PubMed  CAS  Google Scholar 

  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 2:168–174

    Article  PubMed  CAS  Google Scholar 

  11. Verdoorn TA, Dingledine R (1988) Excitatory amino acid receptors expressed in Xenopus oocytes: agonist pharmacology. Mol Pharmacol 34:298–307

    PubMed  CAS  Google Scholar 

  12. Nestler EJ, Hyman SE, Malenka RC (2001) Molecular pharmacology: a foundation for clinical neuroscience. McGraw-Hill, New York

    Google Scholar 

  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 419–439

    Google Scholar 

  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 416–419

    Google Scholar 

  15. Benjamin DR (1992) Mushroom poisoning in infants and children: the Amanita pantherina/muscaria group. J Toxicol-Clin Toxic 30(1):13–22

    Article  CAS  Google Scholar 

  16. Satora L, Pach D, Ciszowski K, Winnik L (2006) Panther cap Amanita pantherina poisoning case report and review. Toxicon 47:605–607

    Article  PubMed  CAS  Google Scholar 

  17. Benedict RG, Tyler VE, Brady LR (1966) Chemotaxonomic significance of isoxazole derivatives in Amanita species. Lloydia 29:333–342

    CAS  Google Scholar 

  18. Chilton WS, Ott J (1976) Toxic metabolites of Amanita pantherina, A. cothurnata, A. muscaria and other Amanita species. Lloydia 39:150–157

    PubMed  CAS  Google Scholar 

  19. Repke DB, Leslie DT, Kish NG (1978) GLC–mass spectral analysis of fungal metabolites. J Pharm Sci 67:485–487

    Article  PubMed  CAS  Google Scholar 

  20. Lund U (1979) Estimation of muscimol and ibotenic acid in Amanita muscaria using high-performance liquid chromatography. Arch Pharm Chem Sci, Edition 7:115–118

    CAS  Google Scholar 

  21. Gore MG, Jordan PM (1982) Microbore single-column analysis of pharmacologically active alkaloids from the fly agaric mushroom Amanita muscaria. J Chromatogr 243:323–328

    Article  CAS  Google Scholar 

  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 164:172–178

    Article  PubMed  CAS  Google Scholar 

  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 852:430–435

    Article  CAS  Google Scholar 

  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):744–748

    PubMed  Google Scholar 

  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 2:23–43

    Article  Google Scholar 

  26. Hušek P (1991) Amino acid derivatization and analysis in five minutes. FEBS Lett 280(2):354–356

    Article  PubMed  Google Scholar 

  27. Husek P (1997) Urine organic acid profiling by capillary gas chromatography after a simple sample pretreatment. Clin Chem 43(10):1999–2001

    PubMed  CAS  Google Scholar 

  28. Husek P, Liebich HM (1994) Organic acid profiling by direct treatment of deproteinized plasma with ethyl chloroformate. J Chromatogr B 656(1):37–43

    Article  CAS  Google Scholar 

  29. Husek P (1992) Gas chromatographic determination of amines, aminoalcohols and acids after treatment with alkyl chloroformates. Anal Chim Acta 259:185–192

    Article  CAS  Google Scholar 

  30. Satora L, Pach D, Butryn B, Hydzik P, Balicka-Slusarczyk B (2005) Fly agaric (Amanita muscaria) poisoning, case report and review. Toxicon 45:941–943

    Article  PubMed  CAS  Google Scholar 

  31. Ott J, Preston SW, Chilton WS (1975) Fate of muscimol in the mouse. Physiol Chem Phys 7:381–384

    PubMed  CAS  Google Scholar 

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Acknowledgments

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

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Authors and Affiliations

  1. Military Institute of Forensic Medicine, Central Military Hospital Prague, Prague, Czech Republic

    Jan Stříbrný & Miloš Sokol

  2. Department of Toxicology, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic

    Jan Stříbrný

  3. Department of Medical Chemistry and Biochemistry, Palacký University, Olomouc, Czech Republic

    Barbora Merová

  4. Faculty Hospital, Institute of Forensic Medicine and Medical Law, Palacký University, Olomouc, Czech Republic

    Peter Ondra

Authors
  1. Jan Stříbrný
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  2. Miloš Sokol
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  3. Barbora Merová
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  4. Peter Ondra
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Correspondence to Jan Stříbrný.

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Stříbrný, J., Sokol, M., Merová, B. et al. GC/MS determination of ibotenic acid and muscimol in the urine of patients intoxicated with Amanita pantherina . Int J Legal Med 126, 519–524 (2012). https://doi.org/10.1007/s00414-011-0599-9

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  • DOI: https://doi.org/10.1007/s00414-011-0599-9

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