Applications of Untargeted Chemical Analysis to Determine Psilocybin and Psilocin Content in Multiple Psilocybe cubensis Mushroom Strains Using LC–MS/MS: An Interview with Kevin Schug

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Untargeted analysis is designed to capture as much information about samples (including, but not limited to, classes of molecules, such as volatiles, lipids, proteins, metals, and metabolites) as possible. Preparation of samples and methods of analysis are designed to profile a particular sample dimension and provide a reproducible fingerprint which can be used to compare and potentially differentiate different sample types using a variety of data treatment strategies. Differing chemical features between samples are then identified and used as markers for discrimination. Untargeted analysis often involves the use of techniques such as liquid chromatography coupled with high resolution mass spectrometry (LC–HRMS), so that complex mixtures can be resolved, and specific features of interest can be noted with the resulting information. At the 2024 Pittcon Conference, Kevin Schug discussed the efforts of he and his colleagues to use untargeted analysis in the study and differentiation of psilocybin mushroom strains; shortly after, we discussed these efforts with Schug in greater detail.

Kevin Schug is with the Department of Chemistry & Biochemistry at the University of Texas at Arlington

Kevin Schug is with the Department of Chemistry & Biochemistry at the University of Texas at Arlington

What made you decide to focus on psilocybin mushrooms in your research?

Our laboratory has been long interested in less traditional research areas. For example, we have spent a great deal of time researching the potential environmental impacts of unconventional oil and gas extraction since 2012. At that time, little was known about how this massive industrial process might affect the environment. Further, and still to this date, it is extremely difficult to obtain research funding from traditional sources to carry out such research.

The legalization of cannabis in the United States brought about another potential interest. However, the schedule 1 status of cannabis has made it difficult to perform cutting edge research using the wide variety of cultivars being produced. Cannabis is a plant rich in bioactive chemical components. Modern analytical methods are ideal to investigate the wide variety of cannabinoids, terpenes, and flavonoids; however, limited access had us initially working with hemp (low-THC) plants. While we did make some substantive contributions, the inability to obtain a rich set of varied cannabis plants, especially high-THC variants, without Drug Enforcement Administration (DEA) approval, still has limited what could be done in a state like Texas. It is important to note that not only does one need a DEA license to perform research, the supplier of plants for research must also be a DEA approved manufacturer, and few of these exist, even with the massive landscape of cannabis cultivation that exists today.

The potential to research psilocybin mushrooms came about due to our being connected with Sue Sisley at the Scottsdale Research Institute. A medical doctor, she was the first to receive DEA approval for the manufacture of psilocybin mushrooms. With that source available, we obtained our own DEA schedule 1 research license, with the intent to help develop the analytical research landscape around magic mushrooms. Compared to cannabis, it felt like we could step into the magic mushroom space and be at a place where cannabis research was 15 years ago. Very few research studies have been published on psilocybin mushrooms and many of these publications have been centered around forensics evaluations of mushrooms and biological metabolite analysis associated with illegal consumption. Sisley is interested in promulgating clinical studies to investigate the potential therapeutic benefit of psilocybin mushrooms, especially for palliative (end-of-life) care. Though some interesting clinical work has been reported on the therapeutic benefit of psilocybin, all that work has used synthetic psilocybin. Just as cannabis is known to have a variety of different bioactive compounds, which modulate the effect of its use on individuals–think, sativa vs. indica vs. hybrid strains–it is believed that psilocybin mushrooms delivered in their natural state could also provide benefit through experiences that were more natural, presumably modulated by the presence of other compounds besides just the psychedelic psilocybin and psilocin molecules.

With the analytical techniques we have in hand, we are well situated to characterize the different molecules in psilocybin mushrooms, and we can obtain a variety of different cultivars from our connection to the Scottsdale Research Institute. Moreover, the potential to contribute to meaningful clinical treatment of mental illnesses and end-of-life care for individuals is fulfilling. It is exciting to think that our work could sit at the forefront of a new paradigm in mental health treatments based on the use of psychedelic substances.

You utilized liquid chromatography-quadrupole time of flight mass spectrometry (LC–QTOF-MS) in your analysis. What made you choose this analysis technique?

Our initial work was to develop a potency test for psilocybin and psilocin in mushrooms using liquid chromatography–triple quadrupole-mass spectrometry. Such a method provides good sensitivity and specificity for targeted analysis, and a reliable potency method was needed to help advance strategies for formulation and dosing for subsequent clinical testing. However, to gain a better understanding of the full spectrum of potential bioactive components in psilocybin mushrooms, we would need to cast a wider net. Untargeted chemical analysis methods provide this net, and a key piece to obtaining reliable chemical information is high resolution mass spectrometry. Thus, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry became the appropriate choice. We are just now scratching the surface to understand the molecular composition of psilocybin mushrooms. Much more work needs to be done to compare the potential variations in chemical content amongst different strains of psilocybin mushrooms, but it is exhilarating to be in a position now to start to do that.

How does your work differ from what has been previously done by yourself or others?

While some excellent baseline investigations have been published in the literature, much of that work has had a forensic focus. Foremost in our effort is to advance science to support the use of psilocybin mushrooms in a clinical context, as a potential therapy for mental disorders, such as PTSD, depression, and anxiety. The development of new, accurate, efficient, and reliable sample preparation and chromatographic methods are needed to support a paradigm shift in the use of a substance long viewed as an illegal drug (hence, the greater forensic context of prior studies), as opposed to recognizing them as potential medicines with rigorous quality control requirements. Reliable potency determinations are needed to formulate proper dosing of the therapeutic. Regulatory agencies require well-established and reproduced methods for quality control and product safety. Our potency study adds significantly to a growing consensus on how to best analyze psilocybin mushrooms. Our continued work into the untargeted analysis of psilocybin mushroom content is very new. There are many types of psilocybin mushroom strains that can be grown, but next to nothing has been reported in the scientific literature about how these strains could vary in their broader chemical content.

Please summarize your findings.

We have in hand and have published a reliable methodology for potency determinations of mushrooms. Moving on, our initial untargeted analytical work has shown clearly that the chemical profiles of psilocybin mushrooms differ greatly from that which you would find in standard mushrooms that people consume daily. That work is still preliminary. We initially utilized reversed phase separations; however, our studies have shown that we need to explore other modes of separations, such as HILIC, because many of the key compounds, which differentiate magic mushrooms from regular mushrooms, are not well retained in reversed phase separations. While reversed phase separations may be sufficient for characterizing and profiling the wider range of tryptamine alkaloids, which have similar structure to psilocybin and psilocin, we seek a more comprehensive understanding of the chemical content of psilocybin mushrooms. It will take us some time to get there.

Were there any limitations or challenges you encountered in your work?

A significant challenge in the analysis of psilocybin mushrooms is maintaining the stability of active components. Psilocybin will readily hydrolyze and degrade into psilocin in the presence of water and humidity. Thus, it was crucial to develop reliable sample milling and extraction techniques that would minimize degradation. Psilocin is also not particularly stable. When it degrades, it forms blue quinoid dimers–the blue color of extracts is a hallmark of psilocybin mushrooms, but once a significant blue color is observed, potency has been lost, because these quinoid dimers are presumably inactive. Moving forward to formulating reliable indications, which can be used for clinical testing, it will be essential to develop a process where reliable doses can be created, which are stable over enough time, such that we know what a patient is receiving during treatment.

What best practices that can you recommend in this type of analysis for both instrument parameters and data analysis?

Maintaining the stability of extracts means that one must work quickly and avoid exposure of the material to a significant amount of water. Acidification of extracts is important to improve stability. Also, proper milling of the samples prior to extraction is important to obtain a repeatable and homogeneous material for extraction. One of the aspects we are also studying using our untargeted methods is the stability of extracts. While this work is still in its infancy, we can use the sample analysis and data treatment techniques, which would be used to differentiate different mushroom strains, also to monitor how the chemical composition of extracts change over time. And they do change over time, which emphasizes the need to be careful and to work quickly, to obtain accurate profiles of the chemical constituents in the mushrooms that represent their natural state.

Can you please summarize the feedback that you have received from others regarding this work?

Feedback has been overwhelmingly positive. We have the feeling that we are doing some important work and venturing into the unknown using analytical measurements. While positive feedback from outside interests is nice, it is also nice to see the strong interest of students who want to work with us and pursue studies in this topic. It is an excellent topic for the development of solid analytical skills, since one must take close care to make reliable extractions, but also in chemical separations, measurement, and curating the data to make sure meaningful results are obtained.

What are the next steps in this research?

While we have been working on this topic for some months, it is still in its infancy. We need to work through our untargeted data sets and publish that work. Next steps might be to consider how we can create some extracts of psilocybin mushrooms that could be useful for study in the clinical setting. Of course, just exploring the rich variety of chemical components present in psilocybin mushrooms is interesting, but it will also be important to eventually understand how compounds other than psilocybin and psilocin might have some therapeutic benefit.

What other hallucinogen or psychedelic natural products might you analyze going forward?

Our DEA license is not limited to cannabis and psilocybin mushrooms. Eventually, we would like to be able to spend efforts investigating peyote cactus for its chemical content. Peyote cactus contains the psychedelic mescaline and there is anecdotal evidence that treatments using this natural product could be beneficial for treatment of addiction and pain. Thus, work in that area could contribute substantially to providing alternatives for opioid and opioid addiction treatments. In the end, we are interested in breaking new ground and performing non-traditional research. I am extremely interested in how we can expand the work that we have done in cannabis and psilocybin mushrooms to other psychedelic substances. We need to develop robust clinical collaborations soon to take the analytical research to the next step.

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