Methods for the Detection of Muscarinic, Muscimol and Ibotenic Acid in Fungi and Biological Matrices Using the HPLC-MS/MS Method

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Abstract

An algorithm for two-step detection of muscarinic, ibotenic acid and muscimol in urine samples is described. A two-step method for the detection of muscarinic, muscimol and ibotenic acid in data-dependent MRM analysis mode with simultaneous registration of complete mass spectra of the target substances was developed. Detection of analytes was carried out by HPLC with a three-quadrupole mass spectrometric detector (LC-MS/MS Shimadzu 8050). Chromatographic separation was performed on a column with reversed-phase sorbent Phenomenex Kinetex C18. At the first stage, muscarine was detected in a diluted urine sample; at the second stage, muscimol and ibotenic acid were detected using the bimolecular dansylation method. The method was successfully applied in practice for routine chemical-toxicological studies of urine samples of patients delivered with suspected mushroom poisoning.

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About the authors

M. Sh. Aigumov

Noyabrsk Psychoneurological Dispensary

Author for correspondence.
Email: aygumov.m@yandex.ru
Russian Federation, Noyabrsk

A. P. Novikov

Surgut Clinical Psychoneurological Hospital

Email: aygumov.m@yandex.ru
Russian Federation, Surgut

M. V. Vishnevskii

Center for Innovative Mycological Research

Email: aygumov.m@yandex.ru
Russian Federation, Moscow

N. A. Chernova

Surgut Clinical Psychoneurological Hospital

Email: aygumov.m@yandex.ru
Russian Federation, Surgut

I. V. Maidanec

Surgut Clinical Psychoneurological Hospital

Email: aygumov.m@yandex.ru
Russian Federation, Surgut

M. A. Gofenberg

Sverdlovsk Regional Clinical Psychiatric Hospital

Email: aygumov.m@yandex.ru
Russian Federation, Ekaterinburg

D. V. Kuznecov

Volgograd Regional Clinical Narcological Dispensary

Email: aygumov.m@yandex.ru
Russian Federation, Volgograd

N. V. Samyshkina

Novourengoy Psychoneurological Dispensary

Email: aygumov.m@yandex.ru
Russian Federation, Novy Urengoy

L. N. Rizvanova

Nizhnevartovsk Psychoneurological Dispensary

Email: aygumov.m@yandex.ru
Russian Federation, Nizhnevartovsk

A. Z. Temerdashev

Kuban State University

Email: aygumov.m@yandex.ru

Educational, scientific and production team of the Scientific and Production Complex “Analit”

Russian Federation, Krasnodar

S. A. Savchuk

Association of Specialists in Chemical-Toxicological and Forensic Chemical Analysis; The Institute of Physical Chemistry and Electrochemistry RAS (IPCE RAS)

Email: aygumov.m@yandex.ru
Russian Federation, St. Petersburg; Moscow

References

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  5. Hasegawa K., Gonmori K., Fujita H., Kamijo Y., Nozawa H., Yamagishi I. et al. Determination of ibotenic acid and muscimol, the Amanita mushroom toxins, in human serum by liquid chromatography–tandem mass spectrometry // Forensic Toxicol. 2013. V. 31. P. 322.
  6. Ginterová P., Sokolová B., Ondra P., Znaleziona J., Petr J., Ševčík J., Maier V. Determination of mushroom toxins ibotenic acid, muscimol and muscarine by capillary electrophoresis coupled with electrospray tandem mass spectrometry // Talanta. 2014. V. 125. P. 242.
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  8. Tsujikawa K., Kuwayama K., Miyaguchi H., Kanamori T., Iwata Y., Inoue H. et al. Determination of muscimol and ibotenic acid in Amanita mushrooms by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry // J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007. V. 852. № 2. P. 430.
  9. Xu X.M., Zhang J.S., Huang B.F., Han J.L., Chen Q. Determination of ibotenic acid and muscimol in plasma by liquid chromatography-triple quadrupole mass spectrometry with bimolecular dansylation // J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2020. V. 1146. P. 122. https://doi.org/10.1016/j.jchromb.2020.122128

Supplementary files

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2. Scheme 1. Structural formulas of muscimol, ibotenic acid and muscarin.

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3. Fig. 1. (a) Chromatographic profile of the mushroom extract. Dilution 1: 20 with mobile phase A. MRM chromatograms: 159.03 > 113.00, 159.03 > 42.10. ( b) Mass spectrum of ibothenic acid at impact energy CE-15 (positive).

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4. Fig. 2. (a) Chromatographic profile of the mushroom extract. Dilution 1: 20 with mobile phase A. MRM chromatograms: 115.40 > 97.95, 115.40 > 39.00. ( b) The mass spectrum of muscimol at the impact energy of CE-15 (positive).

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5. Fig. 3. (a) Chromatographic profile of the mushroom extract. Dilution of 1 : 20 with urinary matrix. MRM chromatograms: 174.15 > 43.05, 174.15 > 57.05, 174.15 > 115.00. ( b) The mass spectrum of muscarin at the impact energy of CE-15 (positive).

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6. Fig. 4. Fragmentation scheme of bidancyl derivatives of muscimol and ibothenic acid.

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7. Fig. 5. (a) Chromatographic profile of the mushroom extract. Dilution of 1 : 20 with urinary matrix. MRM chromatograms: 581.20 > 347.10, 581.20 > 234.10, 581.20 > 170.10. ( b) The mass spectrum of the bidancyl derivative of muscimol at the impact energy CE-15 (positive).

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8. Fig. 6. (a) Chromatographic profile of the mushroom extract. Dilution of 1 : 20 with urinary matrix. MRM chromatograms: 625.20 > 391.10, 625.20 > 346.10, 625.20 > 234.10, 625.20 > 170.10. ( b) The mass spectrum of the bidancyl derivative of ibothenic acid at an impact energy of CE-25 (positive).

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9. Fig. 7. (a) Chromatographic profile of the patient's urine sample. MRM chromatograms: 174.15 > 43.05, 174.15 > 115.05, 174.15 > 57.05. Peak 1 corresponds to muscarine, peak 2 to an unknown component of the urinary matrix. (b) The mass spectrum of muscarin at the impact energy of CE-15 (positive).

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10. Fig. 8. (a) Chromatographic profile of the urine sample. MRM chromatograms: 581.20 > 347.10, 581.20 > 234.10, 581.20 > 170.10. ( b) Chromatographic profile of the muscimol standard at a concentration of 100 ng/ml. MRM chromatograms: 581.20 > 347.10, 581.20 > 234.10, 581.20 > 170.10. ( c) The mass spectrum of the bidancyl derivative of muscimol at the CE-15 impact energy (positive) in the urine sample.

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