Development of a method for the determination of amanitin and phaloidin toxins in Amanita fungi and in human urine
Abstract
At the temperature (20 ± 1) °C, the extraction of amanitin (α-amanitin, β-amanitin) and phalloidin (phalloidin and phallocidin) toxins with butanol from water and with butanol and isopropanol from aqueous solutions of ammonium sulfate and potassium hydrogen phosphate was studied. On the basis of the obtained experimental data, the distribution constants of toxins were calculated, which were used to develop a technique for extraction sample preparation in the process of determining toxins in fungal material and human urine. It has been shown that for the extraction of amanitin and phalloidin toxins from various objects with a high water content, it is advisable to use extraction with butanol using salting out with ammonium sulfate up to saturated solutions. Extraction techniques have been developed for sample preparation of fungal material and human urine for the subsequent determination of toxins in them by liquid chromatography with mass spectrometric detectors such as «triple quadrupole» and «quadrupole – orbital trap». The technique for the determination of toxins in urine is characterised by a limit of quantification of about 1 ng/mL of urine, the technique for the determination of toxins in mushroom material – 600 ng in 1 g of dry mushrooms. The relative standard deviation of the determination of toxins is about 25 %.
References
- Ward J, Kapadia K, Brush E, Salhanick SD. Amatoxin poisoning: case reports and review of current therapies. Journal of Emergency Medicine. 2013;44(1):116–121. DOI: 10.1016/j.jemermed.2012.02.020.
- Musselius SG, Ryk AA. Otravleniya gribami [Mushroom poisoning]. Moscow: Miklosh; 2002. 324 p. Russian.
- Garcia J, Costa VM, Carvalho A, Baptista P, Guedes de Pinho, de Lourdes Bastos M, et al. Amanita phalloides poisoning: mechanisms of toxicity and treatment. Food and Chemical Toxicology. 2015;86:41–55. DOI: 10.1016/j.fct.2015.09.008.
- Flament E, Guitton J, Gaulier J-M, Gaillard Y. Human poisoning from poisonous higher fungi: focus on analytical toxicology and case reports in forensic toxicology. Pharmaceuticals. 2020;13(12):454. DOI: 10.3390/ph13120454.
- Wong JH. Fungal toxins. In: Kastin AJ, editor. Handbook of biologically active peptides. 2nd edition. [S. l.]: Elsevier; 2013. p. 166–168. DOI: 10.1016/B978-0-12-385095-9.00025-7.
- Baumann K, Muenter H, Faulstich. Identification of structural features involved in binding of α-amanitin to a monoclonal antibody. Biochemistry. 1993;32(15):4043–4050. DOI: 10.1021/bi00066a027.
- Vetter J. Toxins of Amanita phalloides. Toxicon. 1998;36(1):13–24. DOI: 10.1016/S0041-0101(97)00074-3.
- Wong JH, Ng TB. Toxins from Basidiomycete fungi (mushroom): amatoxins, phallotoxins, and virotoxins. In: Kastin AJ, editor. Handbook of biologically active peptides. [S. l.]: Elsevier; 2006. p. 131–135. DOI: 10.1016/B978-012369442-3/50023-4.
- Bever CS, Hnasko RM, Cheng LW, Stanker LH. A rapid extraction method combined with a monoclonal antibody-based immunoassay for the detection of amatoxins. Toxins. 2019;11(12):724. DOI: 10.3390/toxins11120724.
- Wieland T, Faulstich H, Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Critical Reviews in Biochemistry. 1978;5(3):185–260. DOI: 10.3109/10409237809149870.
- Enjalbert F, Gallion C, Jehl F, Monteil H, Faulstich H. Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography. Journal of Chromatography A. 1992;598(2):227–236.
- Tanahashi M, Kaneko R, Hirata Y, Hamajima M, Arinobu T, Ogawa T, et al. Simple analysis of α-amanitin and β-amanitin in human plasma by liquid chromatography-mass spectrometry. Forensic Toxicology. 2010;28(2):110–114.
- Nomura M, Suzuki Y, Kaneko R, Ogawa T, Hattori H, Seno H, et al. Simple and rapid analysis of amatoxins using UPLC-MS-MS. Forensic Toxicology. 2012;30(2):185–192.
- Ali Ahmed WH, Gonmori K, Suzuki M, Watanabe K, Suzuki O. Simultaneous analysis of α-amanitin, β-amanitin, and phalloidin in toxic mushrooms by liquid chromatography coupled to time-of-flight mass spectrometry. Forensic Toxicology. 2010;28(2):69–76.
- Yoshioka N, Akamatsu S, Mitsuhashi T, Todo C, Asano M, Ueno Y. A simple method for the simultaneous determination of mushroom toxins by liquid chromatography – time-of-flight mass spectrometry. Forensic Toxicology. 2014;32(1):89–96.
- Zhang S, Zhao Y, Li H, Zhou S, Chen D, Zhan Y, et al. A simple and high-throughput analysis of amatoxins and phallotoxins in human plasma, serum and urine using UPLC-MS/MS combined with PRiME HLB µ-elution platform. Toxins. 2016 May 4;8(5):128. DOI: 10.3390/toxins8050128.
- Vujović M, Ilić I, Kilibarda V. Determination of Mushroom toxin alpha-amanitin in serum by liquid chromatography – mass spectrometry after solid-phase extraction. Acta Medica Medianae. 2015;54(1):12–19. DOI: 10.5633/amm.2015.0102.
- Zayats MF. Novye podkhody v primenenii ekstraktsii pri opredelenii mikrokolichestv pestitsidov v sel’skokhozyaistvennoi produktsii i ob’ektakh okruzhayushchei sredy khromatograficheskimi metodami [New approaches in the application of extraction in the determination of microquantities of pesticides in agricultural products and environmental objects by chromatographic methods]. Minsk: Belarusian State University; 2019. 355 p. Russian.
Copyright (c) 2023 Journal of the Belarusian State University. Chemistry

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
The authors who are published in this journal agree to the following:
- The authors retain copyright on the work and provide the journal with the right of first publication of the work on condition of license Creative Commons Attribution-NonCommercial. 4.0 International (CC BY-NC 4.0).
- The authors retain the right to enter into certain contractual agreements relating to the non-exclusive distribution of the published version of the work (e.g. post it on the institutional repository, publication in the book), with the reference to its original publication in this journal.
- The authors have the right to post their work on the Internet (e.g. on the institutional store or personal website) prior to and during the review process, conducted by the journal, as this may lead to a productive discussion and a large number of references to this work. (See The Effect of Open Access.)