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  • Dustin Theibert

Stability of tryptamines in Psilocybe cubensis mushrooms


Not unlike the fruiting body of mushrooms; tryptamines unveil as enthralling, but elusive. Rapid environmental deterioration of these fungal alkaloids are well documented. As is the concern for sample processing and long-term storage, giving rise to questions of optimal storage conditions and analysis methodology. Gotvaldová et al. of this 2020 study, seek out to answer such questions. Their best results came from storage as whole mushrooms in cool (but not COLD) conditions with no light or water exposure; reaffirming anecdotal evidence.

#psilocybin #psilocin #baeocystin #norbaeocystin #aeruginascin #psilocybe #alkaloids #storage

Hypothesis and Takeaways

The study ultimately draws from the questions of what is the best methodology to analyze tryptamines in these fungal structures as well as the best method to store Psilocybe cubensis mushrooms. Individual parts of the fruiting bodies (caps, stipes, and basidiospores) were gauged along with mycelium. This effort was performed in Prague through funding from the Czech Academy of Sciences.

As tryptamine concentrations are variable throughout the fungal structures, this study concluded the caps contained roughly double the amount of tryptamine alkaloids as the stems. However, this result was not statistically significant due to large variance in tryptamine concentrations. The mycelium showed only psilocin while the spores contain no detectable tryptamines.

In a quest for improved storage methods, dried Psilocybe cubensis mushrooms were examined after three months in varying conditions. Analysis was performed on tryptamines including psilocybin, psilocin, baeocystin, norbaeocystin, and aeruginascin. Their results demonstrate dark room, and dry and temperate storage best for maintaining integrity with cold storage proving far worse. The cold-associated change includes likely conversion of psilocybin through dephosphorylation to psilocin. The conversion to psilocin reduces stability during storage due to rapid oxidation and formation of blue quinoid dye, as previously discussed on the blog.

Testing Methodology

Analysis was done via ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) for improved resolution and detection. These methods are the mainstay of analysis as they have the advantages of accuracy, repeatability, and generally lower detection limits. Liquid chromatography being the standard for tryptamine analysis due to natural volatility and heat instability and has been discussed on this blog multiple times. [1-3]

Tryptamine Extraction

Based on prior studies [4-9] a small trial determined acidified methanol was the best extraction solvent, supporting the increased solubility of phosphorylated tryptamine alkaloids in acidic environments.[10] A second extraction with methanol alone yielded 20% additional analytes with no yield from additional extractions. Vortexing samples in solvent increased extraction efficiency over soaking. However, after a short minimum time of mixing there was no additional increase in detectable levels. Extraction of larger pieces of fresh mushrooms was found to be more effective than chopping these mushrooms into small pieces, which noted extensive bluing. Unprocessed fresh mushrooms contained about 30% more tryptamines than the chopped counterparts.

Tryptamine Stability

Thermal stability as well as photo degradation are a concern for through exposure to air and light.[11-13] To understand this effect whole mushrooms as well as a powdered samples were stored under various conditions presented in Table 1.

After only a week in the dark, the powdered form psilocybin reduced from 1.5% to 1.3% and the sample exposed to light had degraded by a third. This downward trend continued through the fifteen month mark as seen below.

In the first month the greatest decrease occurred and through the second month the overall trend continued, albeit at a slower rate. By fifteen (15) months and excessive degradation, only a third of original content remaining, no further changes occurred except for psilocin whose concentration slightly increased likely due to its conversion from psilocybin.

To measure the effect of thermal stability, fungal powder was heated for thirty (30) minutes. Thermal treatment showing rapid decay in the phosphorylated tryptamines above 100°C. However, psilocin concentration increased at higher temperatures, again explained by dephosphorylation. Based on this room temperature was used for further extractions.

Tryptamine Content

Mycelium of P. cubensis was vacuum dried for six (6) hours with a 89% reduction in weight and analysis detected only psilocin at 0.15 %. Mycelial concentrations are notably variable with psilocin previously ranging up to 0.2% and psilocybin up to 2.0%.14-16 As expected from previous studies, no tryptamines were detected in the basidiospores.

Dried mushrooms tested as powder and mushrooms in whole pieces for the greatest possible yield. The moisture content in the fresh mushrooms was 90% and average tryptamine content of 0.87% of dry weight.

Analysis of the individual parts of the fruiting bodies concluded that tryptamine alkaloids in the stipes were approximately 50% less than in the caps. However, these results were not statistically significant as there was a drastic difference between individual mushrooms. Although the measured tryptamine content in caps was higher than in stems, this statement cannot be broadly applied to all mushrooms The variability of tryptamines in individual fruiting bodies has been noted in numerous works [17-18] as well as recent analysis presented on the blog.

Conclusion and Caveats

To prevent the degradation of alkaloids, the most suitable conditions were to dry the mushrooms whole and store in the dark at room temperature. Regardless of storage, rapid degradation of all tryptamines was observed in powder form. In order to maintain alkaloid content in dried mushrooms, it is recommended to store in an inert gas environment. Degradation of tryptamines occurs rapidly when fresh mushrooms are mechanically damaged. However, better extraction yield for measurement was achieved for dried mushrooms in a powdered form. Furthermore, this work developed and validated an extraction procedure and analytical method for analysis for tryptamines in fungal biomass via UHPLC-MS. In addition, this work was conceivably the first published to include aeruginascin in its characterization due to the low natural concentrations. This will hopefully lead to future improvements in standard testing and analysis methods for including a broader range of tryptamines.


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