Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2025 Nov 10;10(46):55725–55732. doi: 10.1021/acsomega.5c07092

Novel qNMR Method to Quantify Psilocybin and Psilocin in Psychedelic Mushrooms

Luisina Rodríguez †,⊥, Guillermo Morera ‡,⊥, Sandra Lupo ‡, Danilo Davyt §, Ignacio Carrera †,⊥,*, Gonzalo Hernández Dossi §,∥,⊥,*
PMCID: PMC12658598  PMID: 41322617

Abstract

Psychedelic mushrooms of the Psilocybe genus contain the psychoactive tryptamines psilocybin and psilocin, compounds currently under clinical investigation for the treatment of depression and other psychiatric conditions. However, the accurate quantification of these alkaloids in fungal matrices remains analytically challenging. Here, we report an optimized extraction protocol and a robust, nondestructive quantification method based on quantitative nuclear magnetic resonance (qNMR) spectroscopy. Using a combination of 1H- and 31P NMR, we achieved simultaneous detection and quantification of psilocin and psilocybin in dried Psilocybe cubensis samples with high accuracy and reproducibility. Our method revealed significant variability in tryptamine content and psilocybin-to-psilocin ratios among user-provided and laboratory-grown samples, underscoring the potential influence of storage conditions on alkaloid stability. Compared with conventional chromatographic approaches, qNMR offers a rapid and calibration-free alternative for the routine analysis of psychedelic fungi. This approach may facilitate quality control in emerging clinical and regulatory contexts of psychedelic mushrooms.

graphic file with name ao5c07092_0006.jpg

graphic file with name ao5c07092_0004.jpg

Introduction

Psychedelic mushrooms, primarily belonging to the Psilocybe genus, have been extensively utilized throughout history for medicinal, religious, and spiritual purposes. , In the 1960s, they were widely known as “magic mushrooms” due to the intense psychedelic effects induced by their consumption, attributed later to the presence of the psychoactive alkaloids psilocybin (PSB, 1) and psilocin (PS, 5) (Figure ). , Years later in 1970, psilocybin was included as a Schedule 1 drug, adversely impacting scientific research of their medicinal properties. Since the 1990s, there has been renewed interest in exploring the medicinal potential of psychedelic substances for treating psychiatric disorders. Recent clinical studies indicate the safety and efficacy of PSB-assisted psychotherapy in producing immediate and enduring antidepressant effects in patients with major depressive disorder, results that appear to surpass the efficacy of conventional antidepressants. , PSB has also shown promise as a therapeutic intervention for various conditions, including substance use disorder, and demoralization and depression in patients with serious illnesses.

1.

1

Open in a new tab

Tryptamines are described for Psilocybe and related psychedelic mushrooms. Reprinted with permission from Anaclara Cabrera Varela.

PSB-producing mushrooms are distributed worldwide and grow in various substrates. The biosynthesis of PSB and PS stems from a complex evolutionary trajectory shaped by gene-clustering events, convergent evolution, and horizontal gene transfer (HGT) driven by interactions among cohabiting species, alongside selective ecological pressures such as predation by mycophagous animals. , It has been determined that PSB is a pro-drug for PS, which is the actual active compound that targets the animal central nervous system and, given its close structural relationship with serotonin, intervenes in various physiological functions. The removal of the phosphate group can be undertaken by several phosphatases present in the mushrooms, and in the digestive system and liver of animals. , PS interacts with most serotonin receptors, and it is well established that its psychedelic effects are due to activation as a partial agonist of the 5HT2A receptor, which could also mediate some of its therapeutic effects.

Although the main psychoactive alkaloids found in “magic mushrooms” species are PSB and PS, recent studies have shown that the universe of secondary metabolites present is far from being fully known and characterized. Structural variants of PSB have recently been described in several species of psychedelic mushrooms (Figure ), such as baeocystin (4-OP-NMT, 2), norbaeocystin (4-OP-T, 3), aeruginascin (4-OP-TMT, 4), and norpsilocin (4-OH-NMT, 6). − In addition, the presence of several β-carbolines, such as harmine, harmane, norharmane, perlolyrine, and cordycinin C/D, has been found in Psilocybe. A recent genomic analysis revealed the presence of genes encoding the biosynthesis of various families of secondary metabolites in some Psilocybe species, demonstrating unexplored metabolic diversity from the perspective of chemical analysis. Genes involved in the synthesis of terpenes, nonribosomal peptide synthetases (NRPSs), nonreducing polyketide synthases (NR-PKS), and halogenases were detected. Additionally, two new products were reported as metabolites of Panaeolus cyanescens and Psilocybe mexicana: lumichrome (an isoalloxazine) and Verpacamide A, a diketopiperazine with strong Chitinase activity.

Regarding chemical analysis of psychedelic mushrooms, previous work (see Table S1 in the Supporting Information) has used numerous taxa to extract, detect, and quantify PSB and PS from samples of dried mushrooms, primarily from the Psilocybe genus. Regarding the extraction protocol, methanol has consistently been the solvent of choice, but with highly variable concentrations, ranging from 1 to 200 mg of dried mushroom per mL of solvent. Other experimental parameters, like the number of extraction cycles and conditions (e.g., mechanical agitation, ultrasound), show high variability. In terms of chemical and analytical quantification methods, previous studies analyzing psilocybin-producing mushrooms have relied primarily on chromatographic methods such as HPLC and LC-MS, with no reports utilizing NMR spectroscopy. In this regard, a recent study from our group successfully applied NMR-based quantification of tryptamines in ayahuasca, demonstrating its capacity for simultaneous detection and quantification of multiple components in a single spectrum. This method is noninvasive, nondestructive, and rapid, with a linear response spanning 6 orders of magnitude (μM to M), making it highly advantageous for complex natural product matrices. In this regard, a recent report describes the metabolomic profile of edible mushrooms and quantifies several metabolites using qNMR. In this manuscript, we developed and validated an optimized method for exhaustive extraction of PSB and PS from dried mushrooms and their detection and quantification by 1H and 31P NMR spectroscopy.

Results and Discussion

Biological Samples and Identification

For this study, six samples of psychedelic mushrooms were analyzed, five of those were donated by users in Uruguay between March 2023 and February 2025 (labeled according to the name stated by the donor as “Golden Teacher”, “Thai Pink Buffalo”, “Cambodia”, “B+” and “Libelula”) and the remaining one was isolated from cow dung in a field in the Rocha department (Uruguay, 2022) and grown in the laboratory (labeled as “RA” by the collector). Samples were identified as belonging to the Psilocybe cubensis species, as described in the Methods section.

Optimization of Alkaloid Extraction

The optimization of alkaloid extraction from P. cubensis focused on maximizing the recovery of PSB and PS while minimizing sample degradation and interference from matrix components, such as sugars. The selected method, involving four consecutive methanolic extractions with ultrasound assistance (Figure S1A), demonstrated high efficiency by recovering 95% of the total PSB and PS content (from now on, total tryptamine content, TTC) by the fourth extraction (Figure S1B).

This optimized protocol was informed by a comprehensive comparison with previously published extraction methodologies (Table S1). Historically, a variety of solvents and extraction approaches have been used, ranging from single macerations with methanol at room temperature to Soxhlet extractions and, in some cases, acidified solvent systems. For example, early protocols employed thin-layer chromatography (TLC) analysis with single methanol extractions at concentrations as low as 2.5–10 mg of dry mushroom per mL of solvent, with no validation of alkaloid recovery. , More recent studies utilized more refined HPLC and UHPLC-MS/MS approaches with similar methanolic extraction protocols but limited the number of extraction cycles, often stopping after one or two extractions, potentially leaving a significant portion of the alkaloid content in the residual fungal matrix. ,

Our study addressed this limitation through exhaustive sequential extractions, confirming that alkaloid recovery increased with each successive cycle until plateauing at the fourth extraction (Figure S1). This iterative approach contrasts with several previously reported methods that, while simpler, likely underestimate PSB and PS concentrations. Notably, methods using dynamic maceration, , or overnight extractions, introduce a higher risk of degradation of the alkaloids, as evidenced by the formation of blueish psilocin oligomers as reported in the literature and observed during this work.

One drawback of using methanol is the high trehalose content present in all of the methanol-based extracts. Trehalose, a sugar abundant in fungal matrices, is the major component of the extract (Figures and S2A), and its signals, especially the hydroxyl groups between 4 and 5 ppm, complicated NMR analysis of the much weaker alkaloid signals due to baseline distortions (Figure S2B). An acetone precipitation method was assayed to selectively reduce sugar interference while preserving alkaloid integrity (data not shown). However, to streamline sample preparation, minimize analyte loss, and reduce processing time, the final optimized method employed methanolic extraction without trehalose removal, as a robust baseline correction procedure was developed to obtain precise integrations, vide infra (Figure S2B). To the best of our knowledge, this is the first report that identifies trehalose as the main component of the methanolic extracts. A probable reason for this could be the difference in detection methods: previous studies relied on various LC-MS techniques, whereas the present work employed NMR spectroscopy. The difference between these methods resides in that the identification of analytes in the former is based on a targeted approach (where the researcher looks for evidence of specific compounds), while an NMR spectrum shows all components at once. For example, we found trehalose in the LC-MS ion trace as ammonium adducts (Figure S3), something that might easily be missed if one is not specifically looking for it.

2.

2

Open in a new tab

1H NMR (DMSO-d 6, 400 MHz) spectra of a typical methanolic extract of P. cubensis mushroom with signals from PS (red triangles), PSB (blue triangles), and trehalose (green triangles) identified. The signal used for PS quantification is marked with a yellow star.

Qualitative Analysis of Alkaloid Extracts

Qualitative analysis of the alkaloid content in the P. cubensis RA extract was performed using a combination of 1H NMR, 31P NMR, and LC-MS techniques. Methanolic extracts were redissolved in DMSO-d 6 after solvent evaporation. As mentioned before, the proton NMR spectra (Figure ) revealed major signals in the aliphatic region, especially in the region between 3 and 5 ppm, suggesting the presence of sugars, especially trehalose, as was later confirmed. Most important for this work is the examination of the aromatic region where PS and PSB characteristic signals can be clearly distinguished and represent the major aromatic components, as shown in Figure . There are, nevertheless, other minor aromatic components that did not correspond to other reported tryptamines (Figure S4) or β-carbolines (Figure S5), as was evidenced by comparison with authentic samples of these compounds.

The chromatographic analysis with LC-MS further validated these findings, detecting both major alkaloids, which were corroborated through a comparison with authentic standards (Figure S6). In the same manner, no minor tryptamines or β-carbolines were found in the methanolic extract. As mentioned before, trehalose was also identified as the ammonium adduct in the extracted ion chromatogram, corroborating its presence by comparing the mass spectrum with that of the corresponding standard (Figure S3). The qualitative profiles for all samples were consistent across multiple extraction replicates, indicating the robustness of the optimized extraction protocol.

Quantitative NMR Determination of Alkaloids

Quantification of PS and PSB in P. cubensis extracts was performed by using quantitative NMR (qNMR), combining both 1H NMR and 31P NMR techniques. Psilocin was successfully quantified by using 1H NMR, taking advantage of its isolated and well-resolved signal at 6.27 ppm, corresponding to the H-5 proton of the indole ring (Figure ). As mentioned before, the methanolic extract was redissolved in DMSO-d 6 after solvent evaporation; solvents with exchangeable deuterium, i.e., MeOD and D2O, were purposely avoided since 4-OH indoles can participate in hydrogen exchange mediated by the hydroxyl group. Since psilocin quantification depends on integration of the H5 signal, its exchange with deuterium would invalidate the method. On the other hand, PSB could not be quantified using 1H NMR due to the absence of an isolated peak in the proton spectrum. Instead, 31P NMR was employed to quantify PSB by targeting the phosphate group’s signal. Comparing the phosphate resonance of an authentic PSB standard with the corresponding signal in the 31P NMR spectrum of an extract, both externally referenced to 85% phosphoric acid, reveals a pronounced matrix effect, evidenced by a chemical-shift deviation of up to 0.4 ppm. We believe this is due to slight differences in the protonation states of the phosphate group. Since the samples are dissolved in DMSO-d 6 and have different amounts of remnant water, it is difficult to assess the pH of the sample. Hence, to confirm the pH dependence of the 31P chemical shift, we have added small amounts of TFA (10 uL total) to the NMR tube for two different extracts. Signal attributed to PSB shows an upfield shift of about 1.97 ppm, confirming the pH dependence of the 31P chemical shift (Figure S7). Furthermore, to unequivocally assign the PSB phosphate group signal, we first looked for a long-range correlation with indole protons through 1H–31P Heteronuclear Multiple Bond Correlation spectroscopy (HMBC). We can observe a cross-peak between the 31P signal at −2.875 ppm and H5 and H7 of PSB, which links the phosphate group to the indole ring (Figures S8 and S9). Also, we performed an additional spiking experiment in which pure PSB was added to the same NMR tube that was used previously to record the 31P NMR spectra of the extract. In this experiment, we look for an increase in the PSB phosphate signal without the appearance of new signals. As shown in Figure , we can see a clear increase in the signal at −2.875 ppm, unambiguously assigning this signal to the PSB phosphate group (see also Figure S10).

3.

3

Open in a new tab

31P NMR (DMSO-d 6, 161.976 MHz) spectra comparison between a typical P. cubensis methanolic extract (maroon line) and the same extract with the addition of pure PSB (cyan line). An increase in the signal at −2.875 confirms its assignment to the PSB phosphate group. This signal was used for quantification purposes (yellow star).

The use of 31P NMR offered several advantages over 1H NMR for PSB quantification: a clean spectral window, minimal signal overlap, and a flat baseline free from interfering signals. These characteristics allowed for straightforward and reliable integration of the PSB signal.

In contrast, 1H NMR spectra exhibited broad and overlapping peaks between 3 and 5 ppm, where the hydroxyl-rich structure of trehalose gives rise to broad, intense resonances that compromise integration accuracy. Since accurate NMR quantification requires a flat baseline, this interference posed a challenge. To address this issue, a first-order polynomial baseline correction was applied locally to the integration region of interest, effectively improving the psilocin quantification reliability (Figure S2B).

Quantitative results for all samples are summarized in Table , expressed as a percentage of the initial mass of dry material. Since it is well-known that PSB can be hydrolyzed to PS under acidic or humid conditions due to the activation of phosphatases present in fungal tissue, we calculated the TTC as the sum of PS and PSB. This provides a more comprehensive metric for the total bioactive alkaloid load in each sample.

1. Amounts of PS and PSB Found in P. cubensis Samples by 1H and 31P Quantitative NMR.

sample name % PS % PSB % TTC
1 Cambodian 0.160 ± 0.016 0.033 ± 0.003 0.193
2 RA 0.016 ± 0.005 0.440 ± 0.019 0.456
3 Libelula 0.098 ± 0.001 0.056 ± 0.001 0.154
4 Golden Teacher 0.050 ± 0.002 0.250 ± 0.011 0.30
5 B+ 0.360 ± 0.001 ND 0.36
6 Thai Pink Buffalo 0.13 ± 0.006 0.14 ± 0.007 0.27
Open in a new tab

Results revealed significant variation in both the TTC and the relative proportions of psilocybin (PSB) and psilocin (PS) in the six P. cubensis samples. TTC values ranged from 0.154 to 0.456% of dry biomass, aligning with the mid-to-lower end of values reported in the literature, where TTCs as high as 1.5% of dry weight have been observed under optimal cultivation and storage conditions. ,

At the higher end, RA and B+ samples showed TTC values of 0.456 and 0.362%, respectively. Interestingly, the relative proportions of PS and PSB diverged between these two. RA contained almost exclusively PSB, whereas B+ consisted predominantly of PS. This contrast likely reflects differences in origin and storage: B+ was obtained from users and possibly exposed to environmental moisture, promoting PSB hydrolysis, while RA was cultivated and dried under controlled laboratory conditions that preserved the phosphorylated form. In contrast, samples like Cambodian and Libélula were at the lower end of the TTC spectrum, exhibiting low concentrations of both PS and PSB. This coincides with anecdotal and qualitatively reports on the Cambodian strain being of moderate potency. We found no published information about the “Libelula” strain, suggesting it is a locally coined name. As others have noted, even well-known strain labels are often misapplied and chemically inconsistent, underscoring the need for rigorous characterization of locally named clones.

Finally, Golden Teacher and Thai Pink Buffalo occupy an intermediate position in terms of TTC, with values of 0.30 and 0.27% of dry biomass, respectively. In the case of Golden Teacher, a strain known for its substantial psychotropic effects, the majority of tryptamines were present as psilocybin (0.25%), with only a minor fraction as psilocin (0.05%). On the other hand, Thai Pink Buffalo exhibited a more balanced distribution, with approximately equal contributions from psilocybin (0.14%) and psilocin (0.13%). Together, these results highlight how even samples with comparable TTC values can differ markedly in their psilocybin/psilocin ratio, underscoring the importance of controlling storage conditions to preserve the chemical integrity of these alkaloids, as other authors have also noted. ,

Validation of the qNMR Method for Alkaloid Determination

The qNMR method was validated by determining for each analyte linearity, accuracy, and precision; limit of detection (LOD) and limit of quantification (LOQ); and recovery and extraction efficiency.

Linearity and Precision

External calibration standards were used to construct concentration curves for both alkaloids to confirm the linearity. Benzoic acid (5.26 mg/mL) was used as an external standard for psilocin (1H NMR), while dimethyl methylphosphonate (86 mM) served as the standard for psilocybin (31P NMR). Both calibration curves exhibited excellent linearity throughout the range of concentrations measured, confirming the accuracy of the method (Figures S11 and S12).

The precision of the method was assessed through five independent extractions of P. cubensis, each analyzed in triplicate by 1H and 31P NMR (Table S2). The relative standard deviation (RSD) values were 11.3% for psilocin and 16.1% for psilocybin, indicating acceptable reproducibility for a method that includes extraction from a complex natural matrix.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

To determine the LOD and LOQ, serial dilutions of P. cubensis extracts were analyzed by 1H NMR for psilocin and 31P NMR for psilocybin, and each measurement was performed in triplicate. The LOD was defined as the lowest concentration presenting an RSD ≤ 20%, while the LOQ was set as the lowest concentration with an RSD ≤ 10%. The LOD and LOQ values obtained were 0.11 and 0.21 mM for psilocin (Figure S13 and Table S3), and 0.06 and 0.16 mM for psilocybin (Figure S14 and Tables S4 and S5).

Recovery Studies and Extraction Efficiency

To evaluate the efficiency of the methanolic extraction, recovery studies were performed using P. cubensis samples spiked with commercial psilocin and psilocybin standards, which were obtained as acetonitrile/water solutions. Initial trial results showed a complete disappearance of the psilocybin peak and a corresponding increase in psilocin concentration, suggesting complete enzymatic dephosphorylation of psilocybin by phosphatases present in the fungal matrix. To investigate whether the solvent of the standards was responsible for activating these enzymes, additional tests were conducted using acetonitrile and acetonitrile/water mixtures, which confirmed that the presence of water induced psilocybin hydrolysis (Figures S15–S17 and the text therein).

To circumvent this issue, a final recovery experiment was performed using PSB and PS standards dissolved in pure methanol, avoiding the presence of water to ensure minimal enzymatic activity. Under these conditions, recovery percentages of 92% for psilocin and 91% for psilocybin were achieved, confirming the efficiency of the extraction protocol and the reliability of the qNMR quantification.

Comparison with LC-MS Analysis

The accuracy of the qNMR method was further validated through a comparative analysis with LC-MS quantification using the same fungal extracts. The results summarized in Table showed a strong correlation between the two methods. Notably, 31P NMR provided an especially robust and interference-free approach for psilocybin quantification, highlighting its potential as a rapid and solvent-efficient alternative to chromatographic methods.

2. Comparison of qNMR and LC-MS Quantification Results for Four Mushroom Samples.

sample % PS NMR % PS LC-MS % PSB NMR % PSB LC-MS
P. cyanescens S1 0.31 ± 0.03 0.36 ± 0.03 0.282 ± 0.016 0.271 ± 0.007
P. cyanescens S2 0.110 ± 0.007 0.131 ± 0.005 0.044 ± 0.007 0.012 ± 0.003
P. cubensis RA 0.016 ± 0.005 0.018 ± 0.004 0.44 ± 0.02 0.52 ± 0.10
P. cubensis Cambodian 0.160 ± 0.016 0.190 ± 0.001 0.033 ± 0.003 0.044 ± 0.002
Open in a new tab

Conclusions

This study presents an optimized extraction protocol that ensures reliable and exhaustive recovery of psilocybin and psilocin from mushroom biomass. The method improves upon the efficiency of previously reported approaches while preserving the alkaloid stability and reducing sample preparation complexity for both qualitative and quantitative analyses.

The developed qNMR method, employing 1H NMR for psilocin and 31P NMR for psilocybin, demonstrated high accuracy, reproducibility, and robustness in the quantification of these psychoactive alkaloids. The use of 31P NMR for psilocybin quantification significantly simplified the analysis, overcoming the limitations associated with matrix interferences in 1H NMR spectra.

The method was successfully applied to a panel of P. cubensis samples obtained from different sources, revealing significant variability in alkaloid content across specimens. These differences may reflect genetic diversity, growth conditions, or postharvest handling, underscoring the importance of robust analytical tools for consistent product characterization.

Together, the combined extraction and qNMR approach offers a rapid, nondestructive, and reagent-free alternative to chromatographic techniques. This workflow is particularly well-suited for the routine analysis of psychoactive fungi, supporting efforts in strain profiling, the standardization of therapeutic materials, and regulatory quality control.

Methods

Reagents and Chemicals

Chemicals and reagents, including authentic samples of psilocin and psilocybin, were purchased from Sigma-Aldrich and used as received or were synthesized as described below. All solvents were distilled prior to use.

Biological Samples, Cultivation, and Identification

For this study, six samples of psychedelic mushrooms were analyzed. Five of these were donated by users in Uruguay between March 2023 and February 2025. These specimens were provided dehydrated in aluminum jars and were stored at room temperature and in the dark. The remaining sample was obtained through the isolation and cultivation of a native P. cubensis strain collected from cow dung during a sampling conducted in the Rocha Department, Uruguay, in 2022. In this case, tissue samples were taken from fresh basidiomata and cultivated on Petri dishes containing Potato Dextrose Agar (PDA). Then, pieces of pure culture were grown (4 weeks at 25 °C) on sterilized, hydrated oat grains (120 °C, 1 h). These cultures were used as inoculum at a 10% ratio in sterilized, hydrated bovine dung bags. The bags were incubated in the dark at 25 °C for 4 weeks. When the substrate was fully colonized, the bags were exposed to light, 80% humidity, and airflow to stimulate the formation of primordia. The mushrooms were subsequently harvested, dried, and stored at 20 °C in the dark and under airflow for future assays.

For the identification of the mushrooms, macroscopic and microscopic observations of basidiomes were conducted using the methodology described by Guzman (1995), Singer and Smith (1958), and Wright and Albertó (2002). − Macroscopic elements were analyzed, including basidiome dimensions, shape, color, margin characteristics, context, gills, and stipe coverage and base. Microscopic features, including basidiospores and cystidia, were examined by using an optical microscope, and small sections of basidiomes were hydrated with 5% KOH and stained with Congo Red (1%).

NMR Experiments

The qualitative and quantitative analyses of alkaloid extracts were recorded at 25 °C on a Bruker Neo 400 equipped with a BBO z-gradient probe operating at 1H, 13C, and 31P frequencies of 400.13, 100.62, and 161.98 MHz, respectively.

qNMR analyses were carried out using the PUlse length-based CONcentration (PULCON) method. Concentration measurements with PULCON are based on the reciprocity principle, which states that the sensitivity of a measurement and the radio frequency magnetic field generated in the coil are correlated. Since the rf field is inversely proportional to the pulse lengths of a 90 or 360° pulse, the concentration of an unknown sample can be determined as long as the concentration of a standard is precisely known and the 90° pulse of all of the samples has been calibrated correctly. To this end, a 20 mM benzoic acid in DMSO-d 6 sample was used for calibration, and spectra were recorded with 16 scans, a relaxation delay of 40 to 60s, and a 90° pulse length of 13 μs. All extract samples were redissolved in DMSO-d6 after solvent evaporation.

LC-MS/MS Analysis

For the LC-MS analysis of psilocybin and psilocin, an HPLC-DAD-ESI-MS/MS system was used, specifically a Shimadzu LCMS-8040, equipped with an LC-20AD HPLC pump, a solvent degassing module DGU-20A5R, a DAD SPD-M20A detector, a CTO-20A oven, a SIL-20A injector, and a FCV-32AH high-pressure switching valve with a split connection to the ESI-MS. The software used to process data was LabSolutions LC-MS. For chromatographic separation, the column used was a 100 × 4.6 mm Phenomenex Kinetex C18 Nucleosil with a particle size of 5 μm, a flow rate of 1.25 mL/min, and a temperature of 40 °C. UV–vis spectra were recorded in the range of 220–360 nm with detection at nm. Gradient systemMobile phase: (A) 0.1% formic acid +2 mM ammonium formate and (B) 0.1% formic acid +2 mM ammonium formate in methanol. The gradient program was as follows: t0′, 0% B, t4′, 50% B, t7′, 90% B, t11′, 90% B, and t11.01′, 0% B. The mass spectrometer was programmed to carry out a full scan over m/z 100–600 (MS1) in positive ion detection mode. The heat-block and desolvation line (DL) temperatures were set to 400 and 250 °C, respectively. The nebulizer gas flow rate, drying gas flow rate, CID gas pressure, and ion spray voltage were 3.0, 15.0, 230, and 4.5 kV, respectively.

Supplementary Material

ao5c07092_si_001.pdf (1.4MB, pdf)

Acknowledgments

This work is dedicated to Prof. D.D., who passed away while this manuscript was in preparation. His guidance and example, not only in chemistry but in the human values he embodied, have been, and remain, a profound source of inspiration to us. This work stemmed from the Interdisciplinary Group on Psychedelics Studies “Arché” (arche.ei.udelar.edu.uy), and was funded by the Espacio Interdisciplinario (Universidad de la República) and the Comisión Sectorial de Investigación Científica (Universidad de la República, Proyecto CSIC Grupos I + D, 1063). We thank Agenacia Nacional de Investigación Científica (ANII, Uruguay) and Comisión Académica de Posgrado (CAP, UdelaR) for the PhD scholarships of L.R. and G.M.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c07092.

  • Mushroom identification procedure, extraction protocols, and validation of the qNMR method (PDF)

#.

L.R. and G.M. contributed equally to this work and share the first authorship

The authors declare no competing financial interest.

References

  1. Ott, J. Pharmacotheon: Entheogenic Drugs, Their Plant Sources and History, 1st ed.; Natural Products Co: Kennewick, WA, 1993; pp 1–639. [Google Scholar]
  2. Van Court R. C., Wiseman M. S., Meyer K. W., Ballhorn D. J., Amses K. R., Slot J. C., Dentinger B. T. M., Garibay-Orijel R., Uehling J. K.. Diversity, Biology, and History of Psilocybin-Containing Fungi: Suggestions for Research and Technological Development. Fungal. Biol. 2022;126:308–319. doi: 10.1016/j.funbio.2022.01.003. [DOI] [PubMed] [Google Scholar]
  3. Hofmann A., Heim R., Breack A., Kobel H.. Psilocybin, Ein Psychotroper Wirkstoff Aus Dem Mexikanischen Rauschpilz Psilocybe Mexicana Heim. Experientia. 1958;14:107–109. doi: 10.1007/BF02159243. [DOI] [PubMed] [Google Scholar]
  4. Wasson R. G.. Seeking the Magic Mushroom. Life. 1957;42:100–120. [Google Scholar]
  5. Carhart-Harris R. L., Goodwin G. M.. The Therapeutic Potential of Psychedelic Drugs: Past, Present, and Future. Neuropsychopharmacology. 2017;42:2105–2113. doi: 10.1038/npp.2017.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carhart-Harris R., Giribaldi B., Watts R., Baker-Jones M., Murphy-Beiner A., Murphy R., Martell J., Blemings A., Erritzoe D., Nutt D. J.. Trial of Psilocybin versus Escitalopram for Depression. N. Engl. J. Med. 2021;384:1402–1411. doi: 10.1056/NEJMoa2032994. [DOI] [PubMed] [Google Scholar]
  7. Haikazian S., Chen-Li D. C. J., Johnson D. E., Fancy F., Levinta A., Husain M. I., Mansur R. B., McIntyre R. S., Rosenblat J. D.. Psilocybin-Assisted Therapy for Depression: A Systematic Review and Meta-Analysis. Psychiatry Res. 2023;329:115531. doi: 10.1016/j.psychres.2023.115531. [DOI] [PubMed] [Google Scholar]
  8. van der Meer P. B., Fuentes J. J., Kaptein A. A., Schoones J. W., de Waal M. M., Goudriaan A. E., Kramers K., Schellekens A., Somers M., Bossong M. G., Batalla A.. Therapeutic Effect of Psilocybin in Addiction: A Systematic Review. Front. Psychiatry. 2023;14:1134454. doi: 10.3389/fpsyt.2023.1134454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Whinkin E., Opalka M., Watters C., Jaffe A., Aggarwal S.. Psilocybin in Palliative Care: An Update. Curr. Geriatr. Rep. 2023;12:50–59. doi: 10.1007/s13670-023-00383-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Reynolds H. T., Vijayakumar V., Gluck-Thaler E., Korotkin H. B., Matheny P. B., Slot J. C.. Horizontal Gene Cluster Transfer Increased Hallucinogenic Mushroom Diversity. Evol. Lett. 2018;2:88–101. doi: 10.1002/evl3.42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Meyer, M. ; Slot, J. . The Evolution and Ecology of Psilocybin in Nature SSRN Electronic Journal 2023. 10.2139/ssrn.4384673. [DOI] [PubMed]
  12. Geiger H. A., Wurst M. G., Daniels R. N.. DARK Classics in Chemical Neuroscience: Psilocybin. ACS Chem. Neurosci. 2018;9:2438–2447. doi: 10.1021/acschemneuro.8b00186. [DOI] [PubMed] [Google Scholar]
  13. Gartz J.. Extraction and Analysis of Indole Derivatives from Fungal Biomass. J. Basic Microbiol. 1994;34:17–22. doi: 10.1002/jobm.3620340104. [DOI] [PubMed] [Google Scholar]
  14. Kalberer F., Kreis W., Rutschmann J.. The Fate of Psilocin in the Rat. Biochem. Pharmacol. 1962;11:261–269. doi: 10.1016/0006-2952(62)90050-3. [DOI] [PubMed] [Google Scholar]
  15. Horita A., Weber L. J.. The Enzymic Dephosphorylation and Oxidation of Psilocybin and Pscilocin by Mammalian Tissue Homogenates. Biochem. Pharmacol. 1961;7:47–54. doi: 10.1016/0006-2952(61)90124-1. [DOI] [PubMed] [Google Scholar]
  16. Repke D. B., Leslie D. T., Guzman G.. Baeocystin in Psilocybe, Conocybe and Panaeolus. Lloydia. 1977;40:566–578. [PubMed] [Google Scholar]
  17. Leung A. Y., Paul A. G.. Baeocystin and Norbaeocystin: New Analogs of Psilocybin from Psilocybe Baeocystis. J. Pharm. Sci. 1968;57:1667–1671. doi: 10.1002/jps.2600571007. [DOI] [PubMed] [Google Scholar]
  18. Jensen N., Gartz J., Laatsch H.. Aeruginascin, a Trimethylammonium Analogue of Psilocybin from the Hallucinogenic Mushroom Inocybe Aeruginascens. Planta Med. 2006;72:665–666. doi: 10.1055/s-2006-931576. [DOI] [PubMed] [Google Scholar]
  19. Lenz C., Wick J., Hoffmeister D.. Identification of ω-N-Methyl-4-Hydroxytryptamine (Norpsilocin) as a Psilocybe Natural Product. J. Nat. Prod. 2017;80:2835–2838. doi: 10.1021/acs.jnatprod.7b00407. [DOI] [PubMed] [Google Scholar]
  20. Blei F., Dörner S., Fricke J., Baldeweg F., Trottmann F., Komor A., Meyer F., Hertweck C., Hoffmeister D.. Simultaneous Production of Psilocybin and a Cocktail of β-Carboline Monoamine Oxidase Inhibitors in “Magic” Mushrooms. Chem. - Eur. J. 2020;26:729–734. doi: 10.1002/chem.201904363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dörner S., Rogge K., Fricke J., Schäfer T., Wurlitzer J. M., Gressler M., Pham D. N. K., Manke D. R., Chadeayne A. R., Hoffmeister D.. Genetic Survey of Psilocybe Natural Products. ChemBioChem. 2022;23:e202200249. doi: 10.1002/cbic.202200249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rodríguez L., López A., Moyna G., Seoane G. A., Davyt D., Vázquez Á., Hernández G., Carrera I.. New Insights into the Chemical Composition of Ayahuasca. ACS Omega. 2022;7:12307–12317. doi: 10.1021/acsomega.2c00795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pacheco-Hernández Y., Villa-Ruano N., Lozoya-Gloria E., de Jesús Terán-Sánchez E., Becerra-Martínez E.. Revealing the 1H-NMR Profiling of Six Edible Mushrooms Consumed in the Northeastern Highlands of Puebla, Mexico. Chem. Biodivers. 2024;21:e202301851. doi: 10.1002/cbdv.202301851. [DOI] [PubMed] [Google Scholar]
  24. Beug M. W., Bigwood J.. Quantitative Analysis of Psilocybin and Psilocin in Psilocybe Baeocystis (Singer and Smith) by High-Performance Liquid Chromatography and by Thin-Layer Chromatography. J. Chromatogr. 1981;207:379–385. doi: 10.1016/S0021-9673(00)88741-5. [DOI] [PubMed] [Google Scholar]
  25. Catalfomo P., Tyler V. E.. The Production of Psilocybin in Submerged Culture by Psilocybe Cubensis . Lloydia. 1964;27:53–63. [Google Scholar]
  26. Saito K., Toyo’oka T., Kato M., Fukushima T., Shirota O., Goda Y.. Determination of Psilocybin in Hallucinogenic Mushrooms by Reversed-Phase Liquid Chromatography with Fluorescence Detection. Talanta. 2005;66:562–568. doi: 10.1016/j.talanta.2004.11.031. [DOI] [PubMed] [Google Scholar]
  27. Gotvaldová K., Borovička J., Hájková K., Cihlářová P., Rockefeller A., Kuchař M.. Extensive Collection of Psychotropic Mushrooms with Determination of Their Tryptamine Alkaloids. Int. J. Mol. Sci. 2022;23:14068–14083. doi: 10.3390/ijms232214068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Christiansen A. L., Rasmussen K. E.. Analysis of Indole Alklaoids in Norwegian Psilocybe Semilanceata Using High-Performance Liquid Chromatography and Mass Spectrometry. J. Chromatogr. 1982;244:357–364. doi: 10.1016/S0021-9673(00)85700-3. [DOI] [Google Scholar]
  29. Koike Y., Wada K., Kusano G., Nozoe S., Yokoyama K.. Isolation of Psilocybin from Psilocybe Argentipes and Its Determination in Specimens of Some Mushrooms. J. Nat. Prod. 1981;44:362–365. doi: 10.1021/np50015a023. [DOI] [Google Scholar]
  30. Gartz J., Wiedemann G.. Discovery of a New Caerulescent Psilocybe Mushroom in Germany: Psilocybe Germanica sp.nov. Drug Test. Anal. 2015;7:853–857. doi: 10.1002/dta.1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lenz C., Dörner S., Sherwood A., Hoffmeister D.. Structure Elucidation and Spectroscopic Analysis of Chromophores Produced by Oxidative Psilocin Dimerization. Chem. - Eur. J. 2021;27:12166–12171. doi: 10.1002/chem.202101382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kurzbaum E., Páleníček T., Shrchaton A., Azerrad S., Dekel Y.. Exploring Psilocybe Cubensis Strains: Cultivation Techniques, Psychoactive Compounds, Genetics and Research Gaps. J. Fungi. 2025;11:99–123. doi: 10.3390/jof11020099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Bradshaw A. J., Backman T. A., Ramírez-Cruz V., Forrister D. L., Winter J. M., Guzmán-Dávalos L., Furci G., Stamets P., Dentinger B. T. M.. DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites. Appl. Environ. Microbiol. 2022;88:e0149822. doi: 10.1128/aem.01498-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Guzmán, G. Supplement to the Monograph of the Genus Psilocybe* Taxonomic Monographs of Agaricales. Bibliotheca Mycologica 1995; Vol. 159, pp 91–141. [Google Scholar]
  35. Wright, J. ; Albertó, E. . Guía de Hongos de La Región Pampeana. I. Hongos Con Laminillas, 1st ed.; L.O.L.A. 2002. [Google Scholar]
  36. Singer R., Smith A. H.. Mycological Investigations on Teonanacatl, the Mexican Hallucinogenic Mushroom. Part II. A Taxonomic Monograph of Psilocybe, Section Caerulescentes. Mycologia. 1958;50:262–303. doi: 10.1080/00275514.1958.12024726. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao5c07092_si_001.pdf (1.4MB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES