10 1002@dta 2950
10 1002@dta 2950
10 1002@dta 2950
Klára Gotvaldováa, Kateřina Hájkováa,b, Jan Borovičkac,d, Radek Juroka,b,e, Petra Cihlářováa,b,
Martin Kuchařa,f*
a
Forensic Laboratory of Biologically Active Substances, Department of Chemistry of Natural Compounds,
University of Chemistry and Technology Prague, Technická 3, 166 28 Praha 6 – Dejvice, Czech Republic
b
Department of Brain Electrophysiology, National Institute of Mental Health, Topolová 748, 250 67 Klecany,
Czech Republic
c
Institute of Geology of the Czech Academy of Sciences, Rozvojová 269, 165 00 Praha 6, Czech Republic
d
Nuclear Physics Institute of the Czech Academy of Sciences, Hlavní 130, 250 68 Husinec, Czech Republic
e
Department of Organic Chemistry, University of Chemistry and Technology Prague, Technická 3, 166 28
Praha 6 – Dejvice, Czech Republic
f
Department of Experimental Neurobiology, National Institute of Mental Health, Topolová 748, 250 67
Klecany, Czech Republic
Corresponding author*:
Martin Kuchař
University of Chemistry and Technology Prague, Technická 3, 166 28 Praha 6 – Dejvice, Czech Republic
Tel.: (+420) 220 444 432
Fax: (+420) 220 444 422
E-mail: martin.kuchar@vscht.cz
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/dta.2950
Keywords:
Mushrooms, Psilocybe, stability, psilocybin, tryptamines, LC-MS
Repeated extraction
The next step after choosing the extraction solvent was to evaluate whether the efficiency
of the whole process was sufficient. We decided to use a subsequent extraction of the matrix
with another solvent. To evaluate the efficiency of the extraction, subsequent extractions of
the matrix were performed. Approximately 20 % of the analytes were found in the second
extract, but no further re-extractions improved recovery. Methanol was chosen as the best
solvent for a re-extraction of the matrix. The re-extraction solvents were selected in the same
way as the extraction solvents, i.e. using Agilent software MassHunter Qualitative Analysis.
During the re-extraction of the residual pellet, acidification of the solvent was probably no
longer necessary.
For the minor alkaloids (aeruginascin, baeocystin, norbaeocystin, and psilocin) the changes in
concentrations were negligible after the first week. After one month of storage, a slight
decrease in the concentration of all of the remaining alkaloids was seen at a similar time
under all of the defined conditions. After two months of storage, all of the alkaloids were
reduced, except for psilocin, which decomposed more only in the light at 20°C. After 15
months of storage, no further changes occurred, except for psilocin, whose concentration
4 Conclusion
An extraction procedure and a UHPLC-MS/MS analytical method for the analysis of
psilocybin, psilocin, baeocystin, norbaeocystin, and aeruginascin in fungal biomass were
developed and validated. All of these analytes were present in P. cubensis. Concentrations of
aeruginascin are low and have not been reported before. It was found that the degradation
of indole alkaloids occurs when fresh fungal fruiting bodies are mechanically damaged
(sliced). Conversely, better extraction yields for dried fungal fruiting bodies were achieved
after the mushrooms were homogenized to a powder form. Higher amounts of
phosphorylated tryptamines (e.g., psilocybin) were found in the dried fungal fruiting bodies,
while higher amounts of the dephosphorylated forms of indoles (e.g., psilocin) were found in
the fresh fruiting bodies.
To prevent the degradation of fungal tryptamines during sample processing, the most suitable
conditions were to dry the fruiting bodies in the dark at room temperature. The total content
of tryptamines was strikingly reduced when they were stored under different conditions.
Mushrooms that were stored in the freezer (-80°C) lost almost 90 % of tryptamines.
Regardless of the storage conditions, rapid degradation of all analytes was observed when the
dried fungal powder was stored. To improve the storage of dried mushrooms, they should be
stored in an inert gas environment.
5 Acknowledgments
This work was supported by the long-term development projects RVO67985831,
RVO61389005, and the Ministry of the Interior of the Czech Republic
(MV0/VI20172020056).