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  • Ian Bollinger

Preliminary tryptamine potency analysis from dried, homogenized fruit bodies of Psilocybe mushrooms

Updated: Feb 2


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We worked with our client Oakland Hyphae to develop a testing method for analysis of tryptamines in various Psilocybe cubensis species cultivars and two samples of, what were accepted as, liberty caps (Psilocybe semilanceata). The following data is based on preliminary research and development methods, does not represent final data and requires further peer review before being taken more seriously than 'interesting'. However, this does represent meaningful, comparable data to the cultivators, to the consumers, and to the public that can help push the conversation about proper testing into the forefront. One of the largest issues within the Cannabis industry is the variability in testing, and the sequestering of testing as IP. Both our client and we believe that testing should not be a proprietary method, but instead an open source procedure to ensure 'apples-to-apples' comparisons instead of the rampant 'lab shopping' for the highest results observed in Cannabis potency testing. Instead, we hope to push for a standardized test that is reproducible, clear, and readily accessible to everyone desiring it. This data represents the first steps in that direction.



Preliminary HPLC Analysis of Psilocybe spp.

All samples tested were dried fruit bodies that were homogenized, then had 1 gram extracted in methanol with a warm sonicator bath, the extract was then filtered and run on an Agilent 1100 High Performance Liquid Chromatograph (HPLC) with an attached Variable Wavelength Detector (VWD) analysis array. These extracts were compared to standard curves of Psilocybin (PCB), Psilocin (PCN), and a mixture of both 1:1 produced from the same run. This was later used to calculate values for sample potency based on the dry weight extracted. Detection of Harmane and Harmine (as stated in the graphic) presence are theorized based on published research. This is a set of three different runs, with the same lots for all reagents and products used. Previously posted data depicts the highest potency for Psilocybin in Psilocybe cubensis at the time was 0.63% by dry weight (Stamets 1996); thus our grading system places that in the A-range, and we graded everything else in 0.20% ranges (D-range: 0.00-0.19%, C-range: 0.20%-0.39, B-range: 0.40-0.59, A-range: 0.60-0.80), anything testing above 0.80% was considered superlative (+S-range).


There is a wide variety of samples present spanning the range of 'established' cultivars like 'Golden Teacher' and 'Burma', to the more exotic 'True Albino Golden Teacher' and 'Star Gazer'. Samples ranged from marble-size to finger-width P. cubensis, while the liberty caps were all-together different in structure from the 'cubes. Some samples were multiple smaller mushrooms, others were single large fruit bodies. The potency ranges were just as variable. Psilocybin potency ranges for example, ranged from low, but detectable (0.14%) all the way to three times the expected 'maximum' (1.98%). Psilocin potency was observed typically in a fractions (to be expected) relative to psilocybin, and the ranges were from below detectable limits (0.00%) to almost double what would be expected of psilocybin in a single outlier. Noticeably, this outlier (HUN) was submitted with unknown heritage and was resampled multiple times to ensure it wasn't an error.


There are a lot of pieces to note: from variability noticed in different flushes of same cultivars from the same cultivator, samples testing way above expected levels, and hidden insights like mushroom size and potency. The last point, is hidden and will hopefully be documented later, lies in deeper analysis of fruit body's dimensions. Samples that were as small as the average marble, about 35mm diameter (CPAP sample series) were arguably some of the most potent samples tested. Furthermore, multiple flushes of 'Burma' and 'Hawaiian' were tested side-by-side and were revealed to have varying potency amongst themselves. As far as potency ranges reaching far above expected ranges could be due to numerous circumstances. One possibility is the age of the data, it is literally 25 years old of writing; since then cultivators have been mastering growing these organisms, so increases to the upper ends of potency ranges are very likely. Another outcome might be from cell density, some samples that were observed to have the same general proportions were later observed to have different properties after being ground. Some samples were observed to be far more 'dense' and 'fibrous' than others while others ranged from indigo-blue to off-white. These differences are all areas that require further investigation and documentation to better understand and track how these factors impact potency and downstream cultivation.



Shedding light on Caps versus Stems


Among the runs, a preliminary comparison of Caps, Stems, and Caps & Stems were also made. A division of caps, cut at the point of attachment to the stem, were separately weighed out; as well as a separation of whole (caps and stem) fruit bodies as well. Once, separated, each were individually homogenized, weighed out to 1g and extracted as previously mentioned. These extracts were then sent through for analysis and processed as the rest of the samples. The data points towards unique facts, but the most important is the lack of consistency between caps from different samples, even the Burma from two different flushes produced two noticeably different sets of results.

Based off of this data I would say that if you were to split all of your fruit bodies into piles of caps and stems, and consumed the same weight of each, the likelihood of you have a more subdued experience from the caps relative to a more potent experience with the stems is high; however there is not enough evidence to state conclusively that stems are more potent than caps. Stems also seemed to have the least amount of variability relative two the other two sample types, but that variability is still wide at around 50%. Important to note: variability in potency, while present individually pointing towards stems being more potent, is likely wider between flushes than it is between caps and stems of the same flush. More data will increase our ability to answer this question!


Notable Caveats

There are an number of steps we need to take to make this system more robust, but the largest is the use of a VWD array, as this requires multiple runs of the same sample/standard to analyze multiple frequencies to ensure accuracy of reads. Upgrading to a Diode Array Detector (DAD) would allow for multiple wavelengths within the UV-to-visible spectrum at the same time, on a single run. Another process we are adding in once development is complete is a Matrix-Matched Calibration Curve which will help to better understand the interactions of fungal cells and other metabolites on analysis.

item to note is the low potency values for liberty caps (0.46%) observed compared to the 0.98% expected (Stamets 1996), this could be due to other tryptamines of interest not being present in the method for analysis, degradation of product, or even a underdeveloped method. We also recognize that our method might not have the best separation of more psilo-like tryptamines (i.e. baeocystin, norbaeocystin, aeruginascin, etc.), and further development with those standard compounds is in the works. Also of note, the differences in mushroom provided; this variation, noticeable even within cultivars, might point to wide variability in harvests due or other unknown factors. Deeper analysis into same-cultivar/different-harvests caps & stems, as well as across cultivars caps & stems is also in the works. There are so many questions we are hoping to answer and we appreciate you and your support in our efforts!



References:

Stamets, P. Psilocybin mushrooms of the world: An identification guide. Berkeley, Calif: Ten Speed Press 1996; p. 39.

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