Analysis of a psilomethoxin sample

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In 2021, the Church of Psilomethoxin was founded, an organization that claims to have developed a new unregulated compound. According to its founders, by adding 5-MeO-DMT to the growing substrate of Psilocybe cubensis, the fungus transforms it into Psilomethoxin. But is it really a new compound?
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Since the foundation of the Church of Psilomethoxin and the announcement of the development of a supposed new unregulated compound, various analyses of samples from this organization have been carried out, generating great controversy on the matter [1, 2]. Analyses conducted by various organizations and using different methods rule out that their new sacrament is Psilomethoxin, but they agree in pointing out psilocin and psilocybin as the main compounds of their sacred product [2, 3].

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In their defense, the Church argues that:

• GC-MS methods are not capable of directly analyzing drugs that are not volatile (they do not vaporize easily), polar (water-soluble), or thermally labile (destroyed by heat).
• We believe that Psilomethoxin is not volatile, polar, and thermally labile and, therefore, is a poor candidate for testing by this process.
• Our church personally conducts bioassays on each batch of our sacrament to determine its efficacy and safety.

In addition, other members question the results of the analyses carried out, arguing the lack of a reference standard and the supposed need to synthesize psilomethoxin in the laboratory to obtain said pattern.

In order to provide data and shed light on the case, in this article, we will explain the methods and techniques used to analyze a sealed sample from this organization that was sent to us anonymously by one of its members. At the same time, we will try to answer the questions raised by the Church.

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Sample Analysis

All commonly known alkaloids from Psilocybe sp., such as Psilocin, Psilocybin, Baeocystin. Norbaeocystin, etc., can be isolated from dried fungal biomass with Methanol [4, 5]. 5-MeO-DMT is readily soluble in methanol as well [6]. Thus we can safely assume that Psilomethoxin is fairly soluble in Methanol too, which we used as an untargeted extraction solvent for the sample we had received. The sample appears to be dried ground fungal biomass. It has the characteristic mushroom smell and tan grey look. 

To unambiguously identify potential Psilomethoxin without a reference standard at hand we used a targeted mass spectrometry (MRM -- multiple reaction monitoring) approach. Here is a quick break down of the technique and explanation of why Psilomethoxin can be detected even without a reference standard if truly present in the sample:

Targeted Mass Spectrometry: Mass spectrometry (MS) is a technique used to identify and quantify the amount of specific molecules in a sample. In targeted mass spectrometry, the instrument is set up to detect only specific molecules of interest, rather than scanning for all molecules present in a sample. This makes the process faster and more precise for those specific molecules. 

MRM (Multiple Reaction Monitoring): MRM is a mode of targeted mass spectrometry. Within MRM, the mass spectrometer is set to detect a particular "parent" molecule and subsequently monitor its fragmentation into designated "daughter" molecules. Monitoring both the parent and daughter molecules ensures a heightened level of confidence in the detection of the molecule of interest.

Why can it be used to identify analytes via LCMS without reference standards?

• Specificity: MRM is highly specific. By monitoring the transition from a parent molecule to a daughter molecule, there's a double-check system in place. It's like having a two-factor authentication system; the chance of a random molecule having both the correct parent and daughter mass is exceedingly low.
• Sensitivity: As the mass spectrometer is exclusively observing specific transitions, it exhibits heightened sensitivity to those particular molecules, even if they are present in minuscule amounts.
• Predictable Fragmentation: In the absence of a reference standard, it is feasible to anticipate how certain molecules will fragment based on their inherent structure and the conditions within the mass spectrometer. This facilitates the prediction of the daughter ions to monitor.
• LCMS Integration: When combined with liquid chromatography (LC), the time at which a molecule comes out (elutes) of the chromatograph (its retention time) provides another layer of identification. Distinct molecules exhibit different retention times; thus, if a molecule demonstrates the anticipated MRM transitions and elutes at the expected time, there is a high degree of confidence in its identification.  

Details of which transitions we have chosen can be found in our analytical report below. 
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Results

Only Psilocin and Psilocybin could be detected in the sample. No peaks were observed for Psilomethoxin's parent ion or MRM transitions at its predicted retention time [3]. Additionally, trace amounts of Baeocystin have been found (data not shown).

On the other hand, according to Church, 5-MeO-DMT is supplied to Psilocybe sp. which then biosynthetically introduces a hydroxy group at position four of the indole scaffold producing Psilomethoxin (4-OH-5-MeO-DMT). If the precursor 5-MeO-DMT is supplied to the fungus, it has to be taken up by the cells and, at a minimum, traces should be detected within the biomass [7]. However, we tested the sample for the presence of 5-MeO-DMT, the result of which was also negative.

Here is our full analytical report:

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Conclusion

In recent years, the Church of Psilomethoxin, now called the Church of Sacred Synthesis, claims to have developed an exclusive sacrament containing psilomethoxin, an unregulated compound.

A claim that has generated skepticism and controversy in the scientific and psychedelic community. Despite the defenses and arguments presented by the Church, the results obtained by our laboratory, in line with those obtained by other independent laboratories, question the actual existence of psilomethoxin in the Church's sacraments, and instead, have identified the presence of psilocin and psilocybin.

Finally, we also found no trace of 5-MeO-DMT in the sample, which is further evidence that the alleged biosynthetic production of psilomethoxin is not true.

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References

1. Nickels, D. (2023). Church of Psilomethoxin, Part 1: Sacramental Skepticism. Is the Church in Denial? Psymposia. Link 
2. Nickles, D. (2023). The Church of Psilomethoxin Part 2: Unraveling the Sacred Chemistry. Psymposia. Link 
3. Williamson S, Sherwood A. Fungi Fiction: Analytical Investigation into the Church Of Psilomethoxin's Alleged Novel Compound Using UPLC-HRMS. ChemRxiv. Cambridge: Cambridge Open Engage; 2023; This content is a preprint and has not been peer-reviewed. 
4. Patent US3183172A - Obtaining psilocybin and psilocin from fungal material (US 3183172 A); Owner: Albert Hofmann; Filed: 02/16/1959 
5. Casale, John. (1985). An Aqueous-Organic Extraction Method for the Isolation and Identification of Psilocin from Hallucinogenic Mushrooms. Journal of forensic sciences. 30. 247-50. 10.1520/JFS10989J. 
6. K. Trout, Some Simple Tryptamines, 2nd ed. Trout’s Notes & Mydriatic Productions, 2012
7. Mario de la Fuente (2023). Opinions | The Church of Psilomethoxin: Fantasy Chemistry Gets Fact Checked. Psychedelic Alpha. Link

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