Quantification of Psilocybin and Psilocin in Psilocybe Cubensis Mushrooms

Psilocybin and psilocin are naturally occurring psychedelic compounds found in numerous mushroom species, including Psilocybe cubensis—more commonly known as the magic mushroom.

These mushrooms have gained significant attention as pyscohoplastogens for the treatment of brain-related conditions, including mental health disorders (such as anxiety, depression, bipolar, autism, and psychoses) and substance-use disorders (such as alcohol- and tobacco-dependence).¹

Interest in the therapeutic potential of these compounds is growing. Standardized methodologies for the reliable and effective quantification of psilocybin and psilocin in mushroom matrices are therefore required.

Previous quantitative methodologies have been based on standard- and food-grade mushroom matrices rather than on actual psilocybin mushrooms. Challenges such as low sensitivity and sample degradation also exist. To overcome this, authentic analytical methods that are simple, sensitive, and robust are required.²

Innovations in psilocybin and psilocin analysis will be discussed at this year’s Pittcon. Alongside a range of talks from experts in psychedelics quantification, Pittcon 2024 will host market-leading scientific equipment manufacturers. This includes GenTech Scientific, which will showcase its novel systems for preparing and evaluating psilocybin and psilocin.

Setting the stage for Pittcon 2024, this article outlines the techniques used for psilocybin and psilocin analysis and highlights the latest developments in the psychedelics field.

Analyzing psilocybin and psilocin in mushrooms: Methods and challenges

The most common method of determining the concentration of psilocybin and psilocin in mushrooms is high-performance liquid chromatography (HPLC).³ To detect these compounds, HPLC is often coupled with fluorescence, electromechanical, voltammetric detection, ultraviolet (UV), and mass spectrometry (MS).⁴ These techniques assess the response of a molecule to various energy inputs.⁵

In HPLC-MS, a molecule is showered with high-energy electrons and passed through a magnetic field, enabling masses within a sample to be measured via their mass-to-charge ratio.⁶ In HPLC-UV, a molecule is positioned in a beam of UV light and the amount of light absorption is measured. The electronic structure of a sample can then be determined, allowing identification of the compounds within.⁷

These methods are high-throughput, accurate, repeatable, robust, and selective.³ While they have the potential to achieve low detection limits, efforts to refine their detection capabilities are underway.³ This is because psilocin and psilocybin are only present in Psilocybe cubensis in trace quantities (0.5% and 2% m/m, respectively).⁴

Proper sample preparation is, therefore, crucial for obtaining accurate results. Extraction of psilocybin and psilocin involves drying fresh Psilocybe cubensis, milling processes, and extraction with a solvent.⁸

Drying breaks down cell walls, enabling solvent to capture the molecules inside, making extracting the psychoactive compounds easier. Dried biomass is also more stable; mushrooms with a moisture level above 80% will degrade quickly.⁸ The amount of moisture within the raw Psilocybe cubensis and the quantity of raw mushrooms determine the drying time and temperature of the oven.

The dried biomass is then placed into a mill for grinding and cutting. Following drying and milling, the psychedelic compounds are extracted with a solvent, such as methanol, dichloromethane, acetone, and diethyl ether. According to the literature, a simple methanolic extraction is most effective.⁹

However, sample stability remains a challenge in this field. Therefore, innovations in sample preparation techniques are required.

Pittcon 2024: Discover innovations in psilocybin and psilocin quantification

One of the six educational tracks at Pittcon 2024, ‘Cannabis and Psychedelics,’ is committed to the advancement of analytical techniques for characterizing psychedelic drugs.

One of the experts presenting this year is Dr. Jonathan Ferguson, New Strategy Scientific Business Leader at Shimadzu Scientific Instruments. Leveraging his expertise in HPLC-MS and small molecule characterization, Dr. Ferguson and his team developed HPLC-MS and HPLC-UV methods for accurately quantifying psilocybin and psilocin. He will discuss these methods further in his talk at Pittcon.

Sample preparation approaches (drying, milling, and extraction) were investigated to make homogenous samples with reproducible extractions for introduction into HPLC-MS or HPLC-UV. Numerous UV wavelengths were used to perform an Intelligent Peak Deconvolution Analysis (i-PDeA), a data analysis method that extracts an individual peak from co-eluted peaks and quantifies it.¹⁰ This, along with the selectivity of MS, reduced matrix interferences.

Dr. Ferguson will discuss the outcomes of his work, including establishing an appropriate sample preparation protocol and fully resolved analytes for quantitation. He will highlight which sample preparation processes are crucial for achieving accuracy, sensitivity, and precision. This includes sample dilution, which enables mobile phase matching while retaining sample concentration within detection limits.

These methods represent a turn-key approach for routine analysis and are a foundation for additional, in-depth interrogation in the future. The need for standardized methodologies will also be discussed, as will the possibility of expanding the application of these techniques for other psychedelic molecules.

Chris Biamonte, Application Laboratory Specialist at Fritsch Milling and Sizing, will also present at Pittcon. In his talk, Mr. Biamonte will introduce problems when incorporating homogenized mushroom samples into different food matrices, such as gummies and chocolates.

For instance, a lack of heterogeneity may lead to inaccurate quantification, thus impairing analytical measurements and the efficacy of clinical trials. His talk will also highlight the outcomes of recent investigations that compared wet-, dry-, and cryo-milling techniques using the Fritsch Pulverisette 11.

To summarize, milling mitigated the causes of sample degradation, such as heat build-up. Compared to rudimentary grinding methods, dry milling was superior as it is quicker, cheaper, offers greater reproducibility, and provides higher particle size uniformity.

Learn more at Pittcon 2024

The use of psilocybin and psilocin in neuropsychopharmacological studies is growing thanks to their therapeutic potential in treating brain-related disorders. Methods to improve their quantification in psilocybe cubensis are underway, with HPLC-MS and HPLC-UV representing turn-key solutions for routine analysis.

To harness the full therapeutic potential of these psychoactive compounds, standardized methodologies are needed, as is the exploration of these methods for other psychedelic molecules. Precise and reproducible sample preparation is imperative for dosing accuracy and clinical trial success.

Detailed discussions on psilocybe cubensis analysis will take place at Pittcon 2024, providing attendees with a comprehensive perspective on the future of psychedelics. For more information and registration, visit the Pittcon website.

References and further reading

1. Lowe, H., et al. (2021). The Therapeutic Potential of Psilocybin. Molecules. doi.org/10.3390/molecules26102948.
2. Oetjen, KA., et al. Quantification of Psilocybin and Psilocin in Mushroom by LC-MS/MS. [Online] SCIEX. Available at: https://sciex.com/tech-notes/food-beverage/food-and-beverage/quantification-of-psilocybin-and-psilocin-in-mushroom-by-lc-ms-m (Accessed on 30 November 2023).
3. Gotvaldová, K., et al. (2020). Stability of psilocybin and its four analogs in the biomass of the psychotropic mushroom Psilocybe cubensis. Drug Testing and Analysis. doi.org/10.1002/dta.2950.
4. Anastos, N., et al. (2006). The Determination of Psilocin and Psilocybin in Hallucinogenic Mushrooms by HPLC Utilizing a Dual Reagent Acidic Potassium Permanganate and Tris(2,2′-bipyridyl)ruthenium(II) Chemiluminescence Detection System. Journal of Forensic Sciences. doi.org/10.1111/j.1556-4029.2005.00033.x.
5. Neuman, R. (2013). Organic Chemistry Textbook. [Online] University of California Riverside. Available at: https://chem.ucr.edu/curricular-materials/textbook (Accessed on 1 December 2023).
6. ATA Scientific. (2020). Spectrometry And Spectroscopy: What’s The Difference? [Online] ATA Scientific. Available at: https://www.atascientific.com.au/spectrometry/ (Accessed on 1 December 2023).
7. Maloney, J. (2023). 5 Different Types of Spectroscopy. [Online] Platypus Technologies. Available at: https://www.platypustech.com/5-different-types-of-spectroscopy (Accessed on 1 December 2023).
8. Lightburn, B. (2021). Canadian Patent 3, 088, 384. Canada: Extraction of Psychoactive Compounds From Psilocybin Fungus. 
9. Cody, JT. (2008). Hallucinogens. Handbook of Analytical Separations. doi.org/10.1016/s1567-7192(06)06004-9.
10. Shimadzu. (no date). Intelligent Peak Deconvolution Analysis (I-PDEA II). [Online] Shimadzu. Available at: https://www.shimadzu.eu/i-pdea-analysis (Accessed on 2 December 2023).

About Pittcon

Pittcon is the world’s largest annual premier conference and exposition on laboratory science. Pittcon attracts more than 16,000 attendees from industry, academia and government from over 90 countries worldwide.

Their mission is to sponsor and sustain educational and charitable activities for the advancement and benefit of scientific endeavor.

Pittcon’s target audience is not just “analytical chemists,” but all laboratory scientists — anyone who identifies, quantifies, analyzes or tests the chemical or biological properties of compounds or molecules, or who manages these laboratory scientists.

Having grown beyond its roots in analytical chemistry and spectroscopy, Pittcon has evolved into an event that now also serves a diverse constituency encompassing life sciences, pharmaceutical discovery and QA, food safety, environmental, bioterrorism and cannabis/psychedelics. 

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