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clampconnected
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HPLC results 11
#28106324 - 12/20/22 05:14 PM (2 years, 28 days ago) |
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Hi!
I wanted to share some of the results i've been generating from my HPLC analyses of Psilocybe. I test wild finds and cultivated strains, I thought some of you might like to see!
Background: High Performance Liquid Chromatography (HPLC) is a analytical chemistry instrument used to separate and quantify chemical components (analytes) in a sample (matrix). It relies on principles of chromatography, the science of separation, and it occurs in the liquid phase, hence liquid chromatography. High performance really means high pressure, usually the process is performed at 200-400 bar (2900-5800 psi). First, for our intention, mushrooms are extracted in an appropriate solvent for an appropriate amount of time. Then, the extract is filtered. This filtered extract then gets injected into the system. Essentially, the HPLC system is like an obstacle course for molecules, and some molecules have innate advantages over others in this course. Initially, all the analytes are mixed together in the extract. Once injected, the extract is introduced into the liquid, known as the mobile phase. The components in the extract flow through the system path in the mobile phase, eventually reaching a column, packed with a specialized material, known as the stationary phase. The analytes passing through the stationary phase have varying affinities to it, some linger on the stationary phase longer than others. This lingering time for the different analytes can be tuned by varying factors such a mobile or stationary phase composition, temperature, and flow rate. One by one (ideally) the analytes exit the column and pass to a detector, which in my instance is a spectrum of light. The molecules are detected based on how much light they absorb, hence causing an electrical potential difference at the photon sensor, and this difference is plotted on some interface, in today's world a computer. Back in the day this was recorded on a printer. At the interface, the signal output is called a chromatogram, and visually looks like a line graph. As compounds that absorb light pass the detector, the line goes up, then comes back down as the molecule leaves the detector. This looks like a peak on the chromatogram. Bringing in some algebra and calculus, we can quantify the amount of molecule based on the area of the peak. We can take pure compound that we're looking for, say psilocin, and run it through the system at various known concentrations. This gives us two things, 1 - what time psilocin passes the detector (elutes) and 2 - what the response level is. The signals are integrated (area under the curve) and the different levels can be plotted as signal against concentration. A linear regression can be applied, generating a line of best fit and an equation to calculate unknown values. Now, there's many uncovered intricacies here and I maybe should've explained the math part in more depth, but that should give one some context to understand what the technology used is and how these results came to be. There's always wikipedia and google if you'd like to learn more, I'm also open to discussion.
To kick things off, here is an analysis of Psilocybe semilanceata. I collected these with a friend in coastal Oregon, U.S.A.
https://www.inaturalist.org/observations/141588727
I've included a couple images here, including a bar chart comparing different sample results, a chromatogram, and a table of results. In total, 23 samples were analyzed, from two collections of 15 total fruit bodies, some split into cap and stem portions. Some key takeaways for me: The potency can vary drastically, 3x in the most extreme case. The Baeocystin content can be quite high, 0.79:1 in the most extreme case, and they are quite potent, averaging somewhere around 15 mg/g psilocybin with some over 20 mg/g. Psilocin was not reported because it was at ug/g levels and I need to rerun the samples to see analytes that low. But working towards that, i recognise the importance of aeruginascin and other 'minor' compounds.
I've got a lot more to share, and I will do my best to keep this active. Happy solstice Shroomery, you guys rock!


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jm2bso
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I wanted to be the first to say thank you for your work. Fantastic.
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Imatryin
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Re: HPLC results [Re: jm2bso]
#28106458 - 12/20/22 06:49 PM (2 years, 28 days ago) |
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That is fantastic work. Thank you!
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Stipe-n Cap
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Land Trout
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Re: HPLC results [Re: jm2bso] 1
#28106518 - 12/20/22 07:30 PM (2 years, 28 days ago) |
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bakedbeings
orbiter of truth


Registered: 09/01/20
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-------------------- Confused? Well now you can!
HHG - cheapest way to start - how i roll
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hummingbird

Registered: 06/29/14
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Awesome!
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Baba Yaga
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Fuck yeah, I really appreciate this. Good sample size as well.
Great work, thank you very much.
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SingularFusion


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Thanks for your contribution CC
Please keep them coming
Edited by SingularFusion (12/20/22 10:35 PM)
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Dandurn777



Registered: 12/09/19
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Awesome work
-------------------- Prying open my Allenii
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jm2bso
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Are you able to get a reference sample of Aeruginascin (4-HO-TMT)? David Manke at Dartmouth should be able to provide a sample. If not I would contact CaaMTech's Andrew Chadeayne. There is biochemical evidence that this compound binds the SERT transporter, and weaker evidence that Aeruginascin is associated with euphoria after Psilocybe ingestion. There is further speculation that Aeruginascin is the cause of wood loverse paralysis. If you take a look at the Psilocybe natalensis thread you will see a lot of discussion about a gentler or more euphoric effect compared to cubensis. It would be fascinating to add these other tryptamine metabolites to your assay. If you can obtain a natural sample of Inocybe aeruginascens this would be a good positive control.
Thanks again!
https://pubs.acs.org/doi/10.1021/acsomega.2c03476
Edited by jm2bso (12/21/22 04:46 PM)
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clampconnected
Researcher



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Re: HPLC results [Re: jm2bso] 1
#28106932 - 12/21/22 07:21 AM (2 years, 27 days ago) |
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Yes, I do have aeruginascin and 4-HO-TMT. I will be going back and reanalyzing samples to look for aeruginascin, it is in very small concentrations.
I was skeptical of aeruginascin being the cause of WLP, as it is mostly occurring at really low concentration. But maybe even at these ug/g concentrations, they can have some effects. I'm curious if it has action at acetylcholine receptors, as these systems are often targeted for intentionally inducing temporary paralysis in surgery, and the compounds used share the quaternary ammonium moiety.
It is certainly on my to-do list to get better at seeing the small things.
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MLPismyOPSEC
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Absolutely love to see this crossposted and spread further! Thank you for your work
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clampconnected
Researcher



Registered: 04/07/20
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Psilocybe baeocystis from New Hampshire! This has got to be one of the coolest temperate north american species, at least to me.
https://www.inaturalist.org/observations/140076884

dup stands for duplicate.
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Bismillah
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This is really cool! Are you publishing any of this? This group in Prague is also doing great work on characterizing the tryptamines in different species: https://www.mdpi.com/1422-0067/23/22/14068
In this paper, they show that psilocybin content in powdered cubensis is reduced by about 50% after a month in a ziplock. I almost find this hard to believe:

It would be interesting to test samples stored in vacuum mylar bags, or with oxygen absorbers..
In a recent paper, decades old samples are analyzed, showing almost no tryptamines in cubes, while semis and cyans were still very active: https://journals.asm.org/doi/epdf/10.1128/aem.01498-22
Quote:
One possible explanation for the generally better chemical preservation in P. cyanescens and P. semilanceata is that these species have sporocarps that are comparatively lower in biomass than those of P. cubensis.This may result in a more rapid desiccation during heat-assisted drying, minimizing the opportunity for spontaneous and/or enzymatic degradation of psilocybin and psilocin. Another possible explanation may be from differences in the expression or catalytic activity of the enzymes involved in conversion of psilocybin and psilocin (22). However, the overall pattern of degradation for the other metabolites we measured mirrored that of psilocybin and psilocin (Fig. 2), suggesting spon-taneous rather than enzyme-assisted degradation.
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Icyurmt
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-------------------- 👁️ 🌊 why you are empty.
Hunt for the habitat not the mushroom.
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AyePlus
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Re: HPLC results [Re: Icyurmt]
#28116656 - 12/29/22 06:31 PM (2 years, 19 days ago) |
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And here i was expecting to open up a thread about horse poo liquid culture and instead i get this gem..
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bakedbeings
orbiter of truth


Registered: 09/01/20
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Re: HPLC results [Re: AyePlus]
#28117234 - 12/30/22 08:16 AM (2 years, 18 days ago) |
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-------------------- Confused? Well now you can!
HHG - cheapest way to start - how i roll
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OlSk00lFarmer
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Wow
-------------------- LAGM 2.023
Man, who gave that Shroomery?
Who taught him how that works?
Someone tell him when he mentions shit do research first!
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clampconnected
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I am still working on this one, but figure the info that’s present now may be of interest. Psilocybe subtropicalis, mostly from Veracruz, and one from the cultivated strain ‘hoogshagenii var convexa’ or ‘semperviva’. Background images of Psilocybe subtropicalis by Alan.


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Baba Yaga
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Great work. Thanks.
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YoshiTrainer
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Awesome job!
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Adas
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Cool! Any additional info? Such as the time from colonization to pinning, and from pinning to harvest?
It still seems like on paper they should be less potent than Pans.
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joze



Registered: 11/10/20
Posts: 1,056
Loc: PNW
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Quote:
clampconnected said: I am still working on this one, but figure the info that’s present now may be of interest. Psilocybe subtropicalis, mostly from Veracruz, and one from the cultivated strain ‘hoogshagenii var convexa’ or ‘semperviva’. Background images of Psilocybe subtropicalis by Alan.



Hello and thank you for sharing. I'm a fellow lab rat in Portland.
Are you working independently or with a research facility?
Could you please share more on your method?
What detector are you using?
What is your extraction method?
Have you run into any unique challenges?
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clampconnected
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Registered: 04/07/20
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Loc: Portland Oregon
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Degradation studies are of interest. I do think it’s highly dependent on the container used, temperature, dry method, and species. Mushrooms are sponges, and if not stored properly will absorb water, which can really accelerate degradation. I personally think glass is the best container material to preserve for long periods. I’ll have to do some testing with that.
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clampconnected
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Re: HPLC results [Re: Adas] 1
#28136415 - 01/12/23 09:29 AM (2 years, 5 days ago) |
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Most of those subtropicalis tests were ran with wild collected specimens. The one on the right was cultivated from Yoshi Amano. I’m not sure of exact times, they were mature fruits. I think pans can be much more potent than this. I am taking pan submissions if anybody is interested in sending some material in for analysis.
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clampconnected
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Re: HPLC results [Re: joze] 2
#28136433 - 01/12/23 09:54 AM (2 years, 5 days ago) |
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Awesome to have some local rats! I’m working as Tryp Labs, my independent company. Mushrooms are desiccated prior to analysis. Sample size is 50mg. I extract in 1mL methanol. Extraction is done with one hour in an ultrasonic bath around 30c, then an 18hr shake. Extracts are then filtered and a dilution is made. I’ll run the dilution for psilocybin and the undiluted extract for the minors. I use a reverse phase system with 0.1% formic acid in water and 0.1% formic acid in acetonitrile, on a aqueous stable c18 column. The detector is a DAD. Peak identity is by retention time and confirmed with uv spectra. I calibrate with standards from an ISO 17034:2016 chemical supplier using a 6 point calibration curve for each analyte. Right now challenges are coelutions that can make the results for the minor tryptamines biased high. The spectra do not always come out looking like indole, and it hinders my ability to report them in some cases. Aeruginascin is also tricky to fully separate. I need to continue working on method development. I may try going back to HILIC, or try other columns, gradient elutions, different buffer systems, it will take some Time to resolve these issues and have a perfect method! Methanol is not the most efficient extraction solvent but it represents the mushrooms true profile the best, according to some studies. I’m not running internal standards, I need to incorporate that to begin to understand reproducibility in my method over time.
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joze



Registered: 11/10/20
Posts: 1,056
Loc: PNW
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Quote:
clampconnected said: Awesome to have some local rats! I’m working as Tryp Labs, my independent company. Mushrooms are desiccated prior to analysis. Sample size is 50mg. I extract in 1mL methanol. Extraction is done with one hour in an ultrasonic bath around 30c, then an 18hr shake. Extracts are then filtered and a dilution is made. I’ll run the dilution for psilocybin and the undiluted extract for the minors. I use a reverse phase system with 0.1% formic acid in water and 0.1% formic acid in acetonitrile, on a aqueous stable c18 column. The detector is a DAD. Peak identity is by retention time and confirmed with uv spectra. I calibrate with standards from an ISO 17034:2016 chemical supplier using a 6 point calibration curve for each analyte. Right now challenges are coelutions that can make the results for the minor tryptamines biased high. The spectra do not always come out looking like indole, and it hinders my ability to report them in some cases. Aeruginascin is also tricky to fully separate. I need to continue working on method development. I may try going back to HILIC, or try other columns, gradient elutions, different buffer systems, it will take some Time to resolve these issues and have a perfect method! Methanol is not the most efficient extraction solvent but it represents the mushrooms true profile the best, according to some studies. I’m not running internal standards, I need to incorporate that to begin to understand reproducibility in my method over time.
Thank you for taking the time to share this information. I'm looking forward to seeing more of your results, and seeing how your method and laboratory grow over time. It will also be nice to have another scientist on the forum who can help answer chemistry questions.
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clampconnected
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Re: HPLC results [Re: joze] 4
#28676843 - 02/26/24 11:43 AM (10 months, 15 days ago) |
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Low resolution screen shot, but still nice none the less, of some cultivated Psilocybe caerulescens from a Georgia USA population. Nice potency on these indoor ones. When I visited GA in 22 I tried some fresh and was taken by surprise with their strong potency. I’ve also heard they can be mild, especially from trip reports in Mexico. Environmental factors such as rain, sun, or herbivory can likely affect potency, and wild mushrooms will inherently be more variable than cultivated. But if you are considering trying your hand at cultivating this species, here’s some data to inform you it is likely well worth your efforts. I believe workman just listed some on his site not too long ago, as well as yoshi, and maybe if you’re lucky you can get a wild print from some our fellow GA or MX shroomerites.
Feel free to reply with caerulescens trip reports or link To them.
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jrc5
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very cool.. thank you for sharing!!
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