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OfflineCloneufc
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Re: Potency Project [Re: LogicaL Chaos] * 1
    #12125689 - 03/02/10 03:37 AM (14 years, 10 months ago)

I havent looked into everything yet but I did look at used equipment, even the GCMS was affordable. New equipment is another story. I have to wait for the money, then do the research. I dont see why this couldnt be done in a homemade lab under sterile conditions? I would be using professional grade lab equipment that would make a cave suitable. The thing Im most worried about is not being able to obtain the right chemicals if they are needed.

Im not sure if you understand me correctly but I want to use this machine to make a more potent cubensis. If I have to make an isolate of it I will. They can be stored almost indefinitely. I really dont want to have many people counting on me. Im not being selfish or mean but I just dont want the pressure,I want to enjoy what Im doing. I would be pressured for time and I dont like that idea.For the project, I will take notes and pictures. I will post them in the forum when I get setup. My guess is that all cubensis sub strains(except PE) are going to vary mushroom to mushroom,flush by flush and substrain is not gonna matter. I think it will come down to genetics on potency and genetics is a game of chance.

Edited by Cloneufc (03/02/10 03:45 AM)

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OfflineLogicaL ChaosM
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Re: Potency Project [Re: Cloneufc]
    #12125724 - 03/02/10 04:05 AM (14 years, 10 months ago)

All good points, you sure know your stuff....

But would you consider someone paying you a small fee to have you analyze their shrooms? I think a lot of people would be interested in it, as long as you provide some good results.

And even a couple samples of PE with a couple samples of some Control cubenis (i.e. "B+") would help this project tremendously!

As for chemicals, is that required? I thought you could just use the material straight-up, as long as its small enough....any GCMS experts here?

I just hope you get the money, because I would pay up to $20 to have shrooms analyzed by your machine, as long as I saw some trials on your mushrooms first.

There May be hope
~ LogicaL Chaos ~

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Re: Potency Project [Re: LogicaL Chaos]
    #12127663 - 03/02/10 01:10 PM (14 years, 10 months ago)

I like the way you guys are going with this. Good work.


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Re: Potency Project [Re: LogicaL Chaos] * 1
    #12134496 - 03/03/10 01:02 PM (14 years, 10 months ago)

Quote:

LogicaL Chaos said:
As for chemicals, is that required? I thought you could just use the material straight-up, as long as its small enough....any GCMS experts here?

I just hope you get the money, because I would pay up to $20 to have shrooms analyzed by your machine, as long as I saw some trials on your mushrooms first.




I've done a fair amount of GCMS, but I wouldn't call myself an expert. You don't need any chemicals other than the extraction solvent and the inert gasses. The solvent used depends on the column in the machine and the analyte; I usually used methylene chloride, but for the solvent to be appropriate the analyte must also be soluble in it. It's been a while since I looked up psilocybin solubilities. Water can't be injected into a GC without messing it up, it will clog the column. A GC will constantly have a stream of heated inert gas passing through it, so a tank of such a gas would be necessary (and easily found at your local welding/gas supplier). Tiny amounts of solvent are used, so I'm sure you could get any that you would need and still fall under the radar.

When injecting into a GC, you use a microliter syringe. The amount of solvent injected is minuscule, the amount of analyte you inject into the machine is incredibly small.

About sending Clone samples to analyze, I think that is a bad idea. It is impossible to receive a package without giving out an address, and that wouldn't be good for all of us to have his address especially if he is doing something that illegal. It would be cool, but an impractical aspiration.


--------------------

"Now ether was substituted for chloroform, and the difference of their phenomena noted, and now some other exhilarant, in the form of an opiate or stimulant, was the instrument of my experiments, until I had run through the whole gamut of queer agents within my reach..."          I can do everything!!

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Offlinelibertaire
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Re: Potency Project [Re: Necco]
    #12134571 - 03/03/10 01:15 PM (14 years, 10 months ago)

Indeed, don't send out your address if you're undertaking such a project as this.  Although, mass producing mushrooms is probably way more illegal than chemically analyzing mushrooms, and people send out their addresses all the time to give and receive prints.  As long as it's from a trusted member who has been around for a fair amount of time and has good trade ratings, I think it should be safe.

For a solvent, methanol seems to be the most reliable.

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OfflineCloneufc
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Re: Potency Project [Re: libertaire]
    #12134811 - 03/03/10 02:04 PM (14 years, 10 months ago)

Thats the main reasons why I will not do anything like that through the mail. Posting results and testing the results will suffice. Even if I was caught and I had a GCMS, I would probably just get a fine. I mean how many people have these in theirs homes? With a good lawyer, I could claim medical research and get off with a fine for practicing without a schedule 1 license.

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OfflineLogicaL ChaosM
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Re: Potency Project [Re: Cloneufc]
    #12136977 - 03/03/10 06:57 PM (14 years, 10 months ago)

What a bummer, but you do have a good reason for not wanting to (ya know, legal concerns).

How about this:

WE grow some mushrooms, take a spore print, send you the print and you fruit them. Hows that? No legal problems and I don't mind giving out my address to a few people as long as they are responsible with it and don't black mail (small pun intended) me later for growing mushrooms.

Would you consider that Cloneufc? I'd still pay like a small fee to help with growing costs. I could even send some powdered brown rice flour and vermicilite with it to reduce costs on you.

That would still be good for this project, but that means *you* will be doing most the work. With you being the only one with a GCMS machine, this project could get some reliable and interesting results...

And since you will be recieving spores from all over the world, you can clone the most potent mushroom you grew and tested for yourself and start a business. Pretty good deal right?

Would you consider my idea?
~ LogicaL Chaos ~

Edited by LogicaL Chaos (03/03/10 07:05 PM)

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Invisiblebiologys
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Re: Potency Project [Re: LogicaL Chaos]
    #12137072 - 03/03/10 07:12 PM (14 years, 10 months ago)

since each print has millions of different genetic's, i dont think that would do anything at all to actually tell how potent YOUR shrooms are...every batch will be different.

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OfflineLogicaL ChaosM
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Re: Potency Project [Re: biologys]
    #12137222 - 03/03/10 07:32 PM (14 years, 10 months ago)

Well then he would have to do multi-batches, powder ALL the dried shrooms into one large collection, and then take a sample from that.

Again, maybe too much work for the Clone Man, but he's willing, I'm willing, ya know?

~ LogicaL Chaos ~

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InvisibleNecco
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Re: Potency Project [Re: libertaire]
    #12139868 - 03/04/10 02:06 AM (14 years, 10 months ago)

Quote:

libertaire said:
...Although, mass producing mushrooms is probably way more illegal than chemically analyzing mushrooms...

For a solvent, methanol seems to be the most reliable.




Growing mushrooms is often less offensive than chemically purifying them/or altering them by extraction. One person would be seen as a "cultivator" the other would be seen as "a drug cook with a clandestine lab." It depends on how bad they want to get you. I'm sure the DEA would love to take down somebody managing to have any success.

Sometimes methanol is too polar for GC, it would have to be a column designed for its use.

GC and IR have to be my favorite analytical methods, I would love to have those machines. I've been too afraid of trouble fixing old machinery, or the cost of upkeep. Then I'd want nmr, and hplc, fluorescence, pressure columns, sequencers... gawww  :drooling:


--------------------

"Now ether was substituted for chloroform, and the difference of their phenomena noted, and now some other exhilarant, in the form of an opiate or stimulant, was the instrument of my experiments, until I had run through the whole gamut of queer agents within my reach..."          I can do everything!!

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Offlinelibertaire
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Re: Potency Project [Re: Necco]
    #12140459 - 03/04/10 07:50 AM (14 years, 10 months ago)

Quote:

Necco said:
Quote:

libertaire said:
...Although, mass producing mushrooms is probably way more illegal than chemically analyzing mushrooms...

For a solvent, methanol seems to be the most reliable.




Growing mushrooms is often less offensive than chemically purifying them/or altering them by extraction. One person would be seen as a "cultivator" the other would be seen as "a drug cook with a clandestine lab." It depends on how bad they want to get you. I'm sure the DEA would love to take down somebody managing to have any success.




I'm not sure that's true, and I've heard plenty of "cultivators" demonized as mad scientists who were operating a drug lab, even though nothing they were doing was remotely as technical as anything that goes on in a lab.  Cultivating has way more risk of getting found out as well, because there is always a possibility that someone you give the mushies to will squeal, but obviously analyzing mushrooms has no customers or other parties involved that could lead to you getting busted except the members of this community.  Like I said, as long as the people sending samples are well trusted.

I also don't think we should rule out the use of plain alcohol or methanol extractions for analysis, so as to keep the sample population at least a bit larger than one person in their home with a machine.  We should work on coming up with a protocol for how we can test an extraction method for it's reliability against other extraction methods so we can start getting lots of other members involved.  I think if we have a set protocol that we know works, and lay it out like a tek, more people will be inclined to provide samples.  For example, if the alcohol is pure, nothing other than psilocybin should be extracted into that alcohol, correct, at least in theory?  If this is the case, we should grow out a clone or an isolate, so we know the genetics of the flush will be absolutely uniform, and do several extraction trials with several different samples from each flush.  If the method is consistent and effective, every trial should come up with the same amount of extract, correct?  Then the amount extracted can be weighed and then the original wet weight of  the mushrooms divided by that number to figure out the percentage of psilocin/psilocybin content.  Does this sound like a start?

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OfflineLogicaL ChaosM
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Re: Potency Project [Re: libertaire]
    #12143188 - 03/04/10 04:16 PM (14 years, 10 months ago)

On the subject of extraction Liber, alcohol and methanol extractions aren't exactly that clean and precise....

And I learned that from personal extractions I've done myself (see my journal) and from this:

Here's what chemistry-expert FastFred (should he have the title "Trusted Chemist"?) Said from my own thread I made in the "Chemistry & Pharamcology" forum
called Regarding Psilocybin Extraction: Is This Statement True?
(Note: questions with " > " were original written by me, and answered by FF.)

Quote:

FastFred said:

> So the extracted crystals are not just psilocybin, but other sugars and carbohydrates that "cling" onto the psychoactive chemicals?

It's likely that the crystals ARE sugars and other bio-gunk and that a small amount of actives co-precipitate with them or simply fall on top of them as the solution is evaporated.

> I thought crystals had to be pure if they are clear...

Nope.  Most pure chemicals are generally whitish or clear.  And those crystals look whitish to me.  Almost anything can form a whitish crystal.

> Also, do you know what kinds of organic chemicals (beside the active ones) are in mycelium?

Anything living is going to have a complex biochemistry.  Life is water based so a great deal of the biomass is water soluable.  Alcohol is also polar so it dissolves mostly the same stuff.

You can seperate everything that living matter is made out of mainly into polar or non-polar.  Fats, cell membranes, etc. are non-polar.  Sugars, starches, etc. are polar.

Any exraction that simply extracts the polar or non-polar components is called a crude extract.  It will contain a ton of uninteresting things and be a complex mixture.

AB extractions are the most common non-crude extractions used.  It uses the differing solubity at different pH's to move alkaloids from polar to non-polar solvents which you then seperate and evaporate.

Here's a thread I noticed was dealing with AB actives extraction...
http://www.shroomery.org/forums/showflat.php/Number/11897461




So, as you can see from his answers, alcohol and methanol extractions are very crude. The resulting extract from mushrooms might even have a higher ratio of non-actives than mycelium (just a logical theory I have, since mushrooms are more complex organically than mycelium).

But, as long as its consistent between ALL mushrooms, meaning some sub-strains (like PE) won't have *more* of these non-active organic protiens and carbohydrates than others, the results should give us a rough idea of how much actives they have.

The best part about is that its simple and pretty expensive, and you don't need a lot of tools/utentiles to do it (i.e. you don't need a fully-stocked lab), which means nearly anyone who can grow shrooms can do these simple extractions....

I think this is the best bet, unless someone here wants to volenteer to do a full-on, psilocybin-only extract and has a fully-equiped lab to do so, which I seriously doubt (but you never know).

Keepin' it simple has its downsides..
~ LogicaL Chaos ~

Edited by LogicaL Chaos (03/04/10 09:19 PM)

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Offlinelibertaire
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Re: Potency Project [Re: LogicaL Chaos]
    #12144930 - 03/04/10 08:50 PM (14 years, 10 months ago)

Interesting, for some reason I thought A/B extractions were completely out for psilocybin extraction.  I guess it does make sense though that it would work better than just one solvent.  If I can get my hands on some of the chemicals involved, I would definitely be willing to try out an A/B extraction.  I wonder how NOKHTA's extractions turned out...

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Offlinelibertaire
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Re: Potency Project [Re: libertaire]
    #12145031 - 03/04/10 09:10 PM (14 years, 10 months ago)

Oh yea, forgot to mention, I've posted this project across two of other mushroom cultivation forums.

They can be found here:

http://forums.mycotopia.net/fungi-growing-edible-medicinal-magic-mushrooms/64687-potency-project.html

and here:

http://shroomotopia.net/index.php?showtopic=9075&st=0&gopid=110525&#entry110525

If anyone knows of any other mushroom cultivation or drug chemistry forums, feel free to post them there as well.  I'll probably post them at the dmt nexus once I get posting privileges there.

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Re: Potency Project [Re: libertaire]
    #12147318 - 03/05/10 08:57 AM (14 years, 10 months ago)

My extractions didn't turn out so well lol. I messed up a bunch of stuff. I'm waiting to get some ph calibration liquid for my new ph pen before I go at it again.


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Offlinelibertaire
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Re: Potency Project [Re: NoOneKnowsHowToAct]
    #12147762 - 03/05/10 10:51 AM (14 years, 10 months ago)

Damn, that stinks.  Hopefully you'll get better results next time, and also hopefully others will try the methods out as well.

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OfflineCloneufc
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Re: Potency Project [Re: libertaire] * 2
    #12353536 - 04/08/10 03:39 AM (14 years, 9 months ago)

I just found this article on how to do it. It took awhile because the study was done in Holland and the PDF file was in Dutch. I cant read dutch so I had to find a google translator to convert it to English. I haven't read it yet but Im excited to read it. Original file here: http://www.lycaeum.org/mv/TMC/021118_paddo.pdf


Page 1

SAZD/01/30/21 - psilocybin and psilocin in magic mushrooms
SAZD/01/30/21
Page 1 of 17
Southern Inspection Services
Division Multiplex Signaling Products
Sector: Signaling Lab
Psilocybin and psilocin IN
Shrooms
LFJ van de Laak, JCA van der Wielen and PH in 't Veld
Inspection Service of Goods
PO Box 2280
5202 CG 's-Hertogenbosch
Tel: 040-29 11 500
Fax: 040-29 11 600
E-mail: @ Leo.van.de.laak kvw.nl
Date: July 2002
Project identification or report SAZD/01/30/21
Copyright 2002
1
1
Copyright 2002 Southern Food Inspection / Detection Division.  Reproduction is
authorized by written permission of the author (s) and source
________________________________________
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SAZD/01/30/21 - psilocybin and psilocin in magic mushrooms
SAZD/01/30/21
Page 2 of 17
CONTENTS
TABLE OF CONTENTS
2
SUMMARY
3
1
INTRODUCTION
4
1.1
General
4
1.2
Shrooms
4
1.3
Research Techniques
5
2
MATERIALS AND METHODS
5
2.1
Methodology
5
2.1.1
Chemicals
5
2.1.2
Sample Preparation (sample preparation)
6
2.1.3
Standard and sample solutions
6
2.1.4
LC
6
2.1.5
Robustness Study
7
2.1.6
Validation Study
7
2.2
Monitoring Shrooms
7
3
RESULTS
8
3.1
Methodology
8
3.2
Monitoring Project
8
4
DISCUSSION
9
4.1
Methodology
9
4.2
Monitoring Project
9
5
CONCLUSION
10
6
THANKS
11
7
LITERATURE
11
8
ANNEXES
12
8.1
Chromatograms 1 and 2
12
8.2
Tables 1 and 2
13
8.3
Charts 1, 2 and 3
15
________________________________________
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SAZD/01/30/21 - psilocybin and psilocin in magic mushrooms
SAZD/01/30/21
Page 3 of 17
SUMMARY
For the project "Maintaining and monitoring the smartshop industry" was a
method for the determination of the hallucinogenic substance psilocybin and psilocin in
mushrooms developed.  Because of the simple sample preparation and good separations were
an LC method Muss Hoff [7] as a starting point for research.  By
conditioning problems of the LC system was finally chosen and psilocybin
psilocin through separate isocratic analysis to determine.  Both assessments were
subject to a robust research which examined factors (vibration time and
temperature in the ultrasonic bath and the percentage of acetonitrile in the mobile phase) within the
margins were found to be not critical.  The validation study showed that the analysis with
Recovery and quantification limits of respectively 93.4% and 0.009% for psilocybin and
of 95.8% and 0.005% for psilocin good results.
Using this analysis were validated by the smart shops planned
monitoring samples (mainly fresh and dried mushrooms) and a mushroom grower
sampled fresh mushrooms investigated.
Some Fresh Panaeolus cyanescensmonsters was a much higher total psilocin content
found, than in the smart shops sampled dried variety (2.5% versus 1.5%).  The
form in which the psyco-active substance is present (psilocybin or psilocin) is this mushroom-type
completely arbitrary.  This applies to both fresh and dried the samples.
The Psilocybe cubensis mexican, whether or not dried, had a total psilocin between 0.5 and 0.9
%, This value was for the fresh Psilocybe cubensis thai only marginally below 0.5 - 0.7%.  In
the Psilocybe Tampanensis (truffle), a total of about 0.3% psilocin measured.  The
measured rates of psilocybin and psilocin in dried mushrooms or not be
correspond to the values in the literature are listed.  Only the occasional but very
high psilocybin and psilocin levels (and therefore the total psilocinegehalte) in freshly picked
Panaeolus cyanescens are striking.
The investigation also revealed that the psycho-active substances in fresh mushrooms,
except in the Psilocybe Tampanensis, much less stable than in the dried form.  The
hallucinogenic effect is due to degradation of the two very different psychoactive substances.
KEYWORDS:
psilocybin, psilocin, smart shop, mushrooms, indole alkaloids
________________________________________
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SAZD/01/30/21 - psilocybin and psilocin in magic mushrooms
SAZD/01/30/21
Page 4 of 17
1.
INTRODUCTION
1.1
General
Shrooms (mushrooms with hallucinogenic properties) are among the oldest known strip means.
Trade (through smart shop) and the use of hallucinogenic mushrooms since 1995
strongly challenged in the Netherlands.  Lately, however, the use of mushrooms all over again
its peak to be [7.15].
The active (mind-altering) substances psilocin and psilocybin mushrooms are.  Both substances
are tier 1 of the Opium Act and are therefore regarded as hard drugs.  Growing
psilocybin and psilocin-containing mushrooms with the intention to prepare,
edit and sell (etc.) is a criminal act under Article 10a of the
Opium and is punishable.  Trade in fresh mushrooms and growing trays to grow
magic mushrooms is not prohibited, these activities fall under the Commodities Act.
In a study of the risks that these mushrooms to public health, came
The Ministry of Health Assessment and Monitoring Coordination Committee established
new drugs (CAM) concluded that this must be assessed as low.  Reactions
Acute toxicity limited to panic and anxiety attacks.  In chronic toxicity can
most of the action called flashbacks.  There is also no
physical or mental dependence [7.15].
In the section of Signaling KvW South in the context of the project "Preservation and
monitoring in smartshop industry "developed an analytical method, which limits
psilocin and psilocybin in magic mushrooms can be determined.
In 2001, a study of mushrooms in the smart shop branch out.  In two periods
of 3 months (March to May and September to November), there are 20 samples twice
mushrooms for a monitoring sampled.  In these samples were the newly developed
method of psilocybin and psilocin levels determined.  The purpose of this monitoring was to
a clear picture of the current situation in the world smartshop occurs (eg
fresh / dried mushrooms, psilocybin and psilocin levels occurring in mushrooms, types
mushrooms, etc.).  The results of this market survey in the future as a basis
at any higher levels of psilocin and psilocybin in magic mushrooms, obtained by
Applying changed farming methods, to identify.
1.2.
Shrooms
In recent years, the mushrooms as "natural" eco-drugs, partly due to the rise of the
house culture is very popular.  In the Netherlands the following mushrooms are most
used:
a)
Psilocybe cubensis Stropharia cubensis or ('Mexican' or 'Thai' magic mushrooms).
b)
Psilocybe semilanceata (indigenous pointy bald head).
c)
Panaeolus cyanescens cyanescens or Copelandia ("Hawaiian" or "Balinese"
mushrooms).
d)
Psilocybe Tampanensis (truffles), root-like mass of hardened mycelium with a
dry matter content of about 30 percent.
The active substance in various mushroom species varies from almost nil to 15 mg / g
dry weight.  The fresh mushrooms, this is a strength factor of 10 lower, as a fresh
mushroom (excluding truffles) for approximately 90% water.
The mushrooms may be either fresh or dried, often in combination with other foods,
consumed.  As usual dose for a mushroom trip is a quantity of 8
to 10 mg of psilocybin / psilocin.
Average mushroom contains approximately 90% water, 3% protein and additionally small
nitrogen-containing compounds, carbohydrates, fats, salts, minerals and
vitamins.  Especially the proteins and nitrogen compounds play a major role in
mushroom poisoning.  Many of the fungi occurring toxins
________________________________________
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SAZD/01/30/21
Page 5 of 17
amino acids (building blocks of proteins) or from the amino acids formed.  This includes
the indole alkaloids psilocybin and psilocin, which are formed from the amino acid tryptophan.
Psilocybin and psilocin are organic compounds, the heterocyclic built
nitrogen atom is responsible for basic properties.  These compounds are
mainly in plants and have specific effects on the nervous system.
The structure formulas of psilocybin and psilocin are very similar.  Psilocybin (4-fosforyloxy-
N, N-dimethyltryptamine) can be regarded as the phosphoric ester of psilocin (4-hydroxy-N, N-
dimethyltryptamine).  When psilocybin is ingested by the enzyme alkaline
phosphatase rapidly and completely converted into psilocin.  This is the psychoactive effect completely
due to psilocin.
+ H
3
PO
4
============>
psilocybin
enzyme + H
2
O
psilocin
The working volume of 'toxin' in a mushroom should better be displayed in
a total psilocin content.  A better comparison between the various mushroom species for the
hallucinogenic effects is also possible.
1.3.
Research Techniques
Various techniques have been applied to psilocybin and psilocin in magic mushrooms to identify.
Besides the paper chromatography [7.1] and thin layer chromatography [7.2 and 7.3] were later
instrumental techniques developed to quantitatively analyze indole alkaloids such as:
conventional column chromatography with UV-spectrometry and colorimetry,
gas chromatography (GC) and mass spectrometry (GC-MS).  These instrumental techniques are
however, quite time consuming.  In GC and GC-MS techniques [7.4], both compounds
also be derivatized, and degradation of the components can not be determined
can be ruled out.
Currently, the LC method (reversed phase) combined with UV, fluorescence or
electrochemical detection [7.2, 7.5, 7.6 and 7.9] more frequently used for the quantitative
determination of psilocybin and psilocin in magic mushrooms.  The advantage of the LC technique is very
simple extraction of both substances from the crushed dried sample material
methanol [7.5 and 7.7].  An additional advantage is that the derivatized extracts are not required to
and readily lend themselves to research (analysis).
Because of the simple sample preparation, separations and good results was a
LC method (reversed phase, UV detection at 266 nm) [7.7] as the basis for
research.
2
MATERIALS AND METHODS
2.1
Methodology
2.1.1
Chemicals

Standard psilocybin A: Sandoz Switzerland, received from Dr.  WG van der Sluis of
Faculty of Pharmacy, Department of Pharmacognosy, University of Utrecht (purity 99%).

Psilocin standard: Prepare the standard using a psilocybin by Dr.  WG van der
Sluis been handed procedure involving psilocybin quickly and completely using the enzyme
alkaline phophatase can be converted into psilocin ..
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Standard psilocybin B: Alltech / Applied Science USA.  (Ampoules containing 100 micrograms / ml in
methanol).

Enzyme phosphatase, alkaline: Sigma.

Other chemicals: analytical.
Psilocybin and psilocin are the Opium Act.  For possession of these substances, but also
for the acts to be performed in order to process and mushrooms to
analysis, an opium for these substances (site specific) to be present.  For
orders of standards psilocybin and psilocin abroad is also a
Import license (KvW South is owned by an opium for both substances).
2.1.2
Sample Preparation (sample preparation)
The dried mushrooms were sampled using an analytical mill ground and in air-tight
preserved specimen jars.
Fresh mushrooms were sampled immediately after receiving cut into small pieces and a
Petri dish for at least 5 days to air dry.  Simultaneously, the
moisture loss (in%) of these mushrooms determined.  In Table 2 (8.2) is the percentage of dry residue
(Dry residue =% 100% - moisture%) of the various dried mushroom samples (specimens)
displayed.  Then the dried mushrooms with an analysis mill
ground.  Some samples were thus not homogeneous samples;
these samples were then ground in a ball mill.  The specimen was sealed
preserved specimen jars.
In the above prepared sample was determined by dry matter content of approximately 100 mg
This specimen 150 minutes to dry in an oven with no fan at 102-105
° C.  With this level, the percentage of psilocybin and psilocin are calculated on the dry
and to the levels of both substances in fresh and dried mushrooms are compared
be (8.2: 1 and Table 2).
The mushrooms were not further characterized, the species name was marked on the
label used.
2.1.3
Standard and sample solutions
Standard psilocybin:
Weighing just prior to analysis 1.0 -1.5 mg psilocybin at 0.01mg accurately in a
10 ml flask.  Dissolve and make up to 10 ml.  Prepared from this solution other desired
concentrations for the selection of the calibration line (uitverdunnen).
Standard psilocin:
Prepared shortly before the analysis using the standard psilocybin
the enzyme alkaline phosphatase psilocin the standard.  Prepared from this solution
other desired concentrations for the selection of the calibration line (uitverdunnen).
Sample Solutions:
Weigh 250 mg of dried sample in a flask of 25 ml.  Add 20 ml of methanol to
and place the flask during 120 minutes in an ultrasonic bath.  Make sure the temperature
in the ultrasonic bath is not above 40 ° C rises.  Check the temperature in the bath and add ice
increases as the temperature is higher than (>) 40 ° C border.  Dilute with methanol to 25 ml
mix.  Centrifuge (about 16000x g) a portion of the extract and transfer the clear extract
two vials (see 2.1.4).
2.1.4.
LC
The use of reversed phase chromatography is very suitable for biological materials
separate and analyze.  The LC separation was performed on a Merck RP-60 Lichrospher
select B (250 x 4 mm, 5 μm) column with a 4-4 LiChroCART Merck, LiChrospher 60 RP-select B
(5 μm) guard column.  The components were detected at 266 nm with a UV detector.
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Page 7 of 17
Initially it was the intention of both components by gradient elution
In an analysis run to determine.  The above LC conditions were used: Solvent A: 20
mM KH
2
PO
4
and solvent B: acetonitrile, flow rate constant size1 ml / min., gradient: 5% B for
8 minutes, then rising to 20% B in 20 minutes and then for 10 minutes 20%
B (injection volume 5 column oven temperature sterile glycerol 30 ° C).
Both substances were well separated.  Conditioning and stabilizing
the column after the gradient run, however, lasted very long.  As a result, the retention times
of psilocybin and psilocin after 5 to 10 injections were more or less constant.
Based on this experience we decided to separate the two substances by means of
isocratic analysis to determine.
The psilocybin was using the mobile phase 4% acetonitrile in 20 mM potassium phosphate solution
(V / v) and psilocin using the mobile phase 12% acetonitrile in 20 mM potassium phosphate solution
(V / v) was determined.  The other parameters such as wavelength, injection volume and column temperature
remained the same as the gradient.  The analysis in this report using these
custom analysis obtained.
2.1.5
Robustness Study
Both assessments were on a robust examination.  As a possible critical
factors were considered:

The vibration time of sample extracts in an ultrasonic bath (120 minutes).
For both psilocybin and psilocin analysis for the "+" level of vibration time
135 minutes and the '-' chosen level 115 minutes.

The temperature in the ultrasonic bath (up to 40 ° C).
For the "+" level was up to 45 ° C and the '-' level up to 35 ° C is selected.
When the experiment was checked whether these temperatures during the extraction in
ultrasonic bath were achieved, but also has ensured that indicated
limits were not exceeded.  This factor was critical for both analysis
investigated.

The content of acetonitrile in the eluent (psilocybin analysis: 4% acetonitrile in 20 mM
buffer solution (v / v); psilocin analysis: 12% acetonitrile in a 20 mM buffer solution (v / v).
For the "+" level was both analyzes the content of acetonitrile in the buffer 1% higher
and "-" level 1% lower than the declared value chosen.
2.1.6
Validation Study
The methods for psilocybin and psilocin were subjected to such validatestudie
described in SOP-KAM05 WV102, version 2 of the KvW "Validation of (physico-) chemical and
(Physico-) mechanical methods ".
The specificity and selectivity of the method were discussed in some detail.  Also, the
detection limits (3 * Noise), the detection limits (6 * Noise), the correlation
coefficients, the recovery and the standard deviations of the two analysis determined.
Because the standards were difficult to obtain, an alternative way to apply
recovery and standard deviation of each analysis to determine:
Extracts of sample survey of mushrooms containing high levels of psilocybin were
collected, and mushroom extracts containing high levels of psilocin.  These solutions were
psilocybin or psilocin content of the quantitatively determined in duplicate.  To blank
samples were 3 different amounts of these solutions added in duplicate and
analyzed.  The added amounts of psilocybin / psilocin (known concentration)
and analyzed the levels of psilocybin / psilocin could recovery programs at the levels 3
computing (possibly after correction for the blank).
2.2
Monitoring Shrooms
In 2001 the project "Maintaining and monitoring the smartshop branch" out.  A
part of this project was a smart shop in the surveillance industry and in the context of this
activity was the sampling and analysis of 40 psilocybin and psilocin or
________________________________________
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SAZD/01/30/21
Page 8 of 17
samples (mushrooms) are planned.  The products were two periods of three months (1
e
period: March-April-May and 2
e
September-October-November) by specially
trained inspectors sampled (20 samples per planning period).  The levels
psilocybin and psilocin in these samples were determined with the developed method.
An inspection by an inspector at a mushroom farm showed that the mushrooms and
mushrooms flights (growth stages) can be picked.  On this farm were 4 types of mushrooms
(Types) mushrooms grown, namely the Psilocybe cubensis mexican, the Psilocybe cubensis thai, the
Panaeolus cyanescens and Psilocybe Tampanensis.  The first three grades were 3
Flights picked, while Psilocybe Tampanensis was harvested at once.
With the willing cooperation of the grower could harvest samples immediately after each
taken from these types of mushrooms.  Because these samples immediately after entering the
laboratory to work, could such a reliable picture of the levels and psilocybin
psilocin in the fresh mushrooms in various stages of growth are obtained.
3
RESULTS
3.1
Methodology
-
LC separations
Psilocybin and psilocin were determined by separate LC analysis.  Both provisions
separations were good with the other components in the observed matrix (see
chromatograms 1 and 2, see Annex 8.1).  Retention times of psilocybin / psilocin were
under these circumstances, consistently good.
-
Robustness Study
The investigated factors (vibration time, temperature and% acetonitrile in the mobile phase) were both
determination of psilocybin and psilocin in determining (both with 2 different
Sample extracts performed) within the margins are not critical.
-
Selectivity and Specificity
The methods for psilocybin and psilocin are only performed in mushrooms and
directly related products (eg mushroom tea).  Matrix of issues (eg caused
by fats and carbohydrates) is thus no question.
Including interference effects, which could potentially arise with related components
the extract, can be excluded.  The only indole alkaloids in solution both to
investigate materials and given the difference in polarity of the two compounds, no
interference effects to be expected.
-
Detection and the detection limits
The detection limit (3 * Noise) and the limit (6 * Noise) for determining
psilocybin in magic mushrooms, respectively 0.005% and 0.009% (w / w).
For psilocinebepaling mushrooms in these parameters 0.002% and 0.003% (w / w).
-
Correlation coefficients
The psilocybin and psilocin were over the monitoring analysis and robustness and
valdatieonderzoek respectively 7 and 5 once.  The calculated calibration lines were
linear.  The correlation coefficients of psilocybin calibration lines were between 0.9997 and 1.0000.
In the calibration lines of psilocin determination were 0.9994 and 0.9999 between the values
found.  The range of both psilocybin and psilocin in the analysis ranged
0.001 - 0.1 mg per ml of psychoactive (5 points).
-
Recovery and relative standard deviations
The recovery of the provision for the working psilocybin (j
w
) Is 93.4% with a
relative standard deviation (RSD
w
) Of 0.7%.  When psilocin provision for recovery
area (j
w
) 95.8% and relative standard deviation (RSD
w
) 1.5%.
3.2
Monitoring Project
In total for the monitoring project in 11 smartshops 19
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SAZD/01/30/21
Page 9 of 17
dried mushroom samples (whether or not ground) and 3 mushrooms containing products sampled.
The vast majority of these (19 dried mushrooms and mushroom-containing product 1) was
the 1
e
period sampled.
Besides the sampling of dried mushrooms, fresh mushrooms were also sampled.  In the
2
e
sampling period, a total of 7 samples taken from 4 fresh mushrooms
several smart shops.  It was remarkable, all the smart shops sampled fresh mushrooms
also the mushroom grower came.
This magic mushroom grower were also sent 22 samples of fresh mushrooms taken.
All results of the dried mushrooms are shown in Table 1, the results of the fresh
mushrooms in Table 2 (8.2).
Samples 37085286 and 37099821 are in the table twice.  The reason is that
Both samples consisted of 2 cups, whose mushroom size, color and
consistency differed much from that reprocessing as separate sample was desired.
In tables and charts (8.2 and 8.3) are listed codes, which the research
laboratory have been applied.  These codes correspond to ISI numbers.
4.
DISCUSSION
4.1
Methodology
Extracting the hallucinogenic substances from the crushed sample with methanol
an ultrasonic bath is simple to perform and reproducible.  Using both
isocratic analysis psilocybin and psilocin were well separated from the other
matrix components in the mushroom.  Found with recovery programs (determined using the alternative
validation procedure § 2.1.6) of 93.4% to 95.8% psilocybin and psilocin is for
concluded that both substances properly extracted from the matrix can be mushrooms.
The limit of psilocybin is 0.009% w / w and psilocin 0.005% w / w.
Muss Hoff [7.7] found a similar method with a limit of quantification of 0.003%
0.01% psilocybin and psilocin on.  Both components were in his research, however,
an analytical run by applying gradient elution determined.
4.2
Monitoring Project
The 2
e
sampling period, in contrast to the 1
e
period, a joint action by
the KvW with the police, all dried samples (Opium Act violation) were the
Courts seized.  This has the 2
e
found significantly less
samples (only fresh mushrooms and two tea samples) for research received.
The overview of the dried mushrooms in the 1
e
sampling period shows that mushrooms
Panaeolus cyanescens type of the highest rates of hallucinogenic substances.
For total psilocin in dry matter (the best measure for the hallucinogenic effect of
mushrooms to be measured) values were between 1.0 and 1.6% were found.  Striking was the
changing contribution of psilocybin (0.2-0.9%) and psilocin (0.4-1.3%) to total fatty
psilocin.  Other researchers [7.2 and 7.7] found similar results for psilocybin and
psilocin in the mushroom species.
Most dried mushrooms (7.9) were called Psilocybe cubensis mexican
type, the levels found ranged between 0.7 and 1.2% psilocybin and psilocin in
between 0 and 0.3% (dry matter).  These values were at the same level as the
results by Muss Hoff [7.7] for this type of mushroom found.  Total psilocin
(On dry matter) of this type of mushroom is between 0.5 and 0.9%.  For the sample Psilocybe
semilanceata (bald head) were found identical values as for Psilocybe cubensis
mexican.
In the sample and a azurens Psilocybe cubensis Thai Psilocybe of values were between
the 0.5 and 0.9% for total psilocin (on dry weight) measured.  The participation in these psilocin
samples is considerably higher than the Psilocybe cubensis mexican.
The remaining samples, including the mushroom tea samples contain only minor
quantities of hallucinogenic substance (<0.05%).
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SAZD/01/30/21
Page 10 of 17
The psilocybin and psilocin levels in fresh samples of Panaeolus cyanescens
mushroom grower showed a varied picture.  Some samples were very high
psilocybin rates (up to 3% dry matter) combined with relatively low rates
to psilocin (up 0.5%) were measured.  In contrast, other samples were high percentages
psilocin (up 1.8% on dry substance) were found and the very low rates of psilocybin
(<0.1%).  The form in which hallucinogenic substances are present, is random, at one time
the hallucinogenic substance psilocybin largely in the form of existing, sometimes almost
entirely in the form of psilocin.  One explanation for these large variations could not be
given.  The total psilocin (on dry matter) varies widely, from 0.8% to 2.5%.  This
rates of hallucinogenic substances, in whatever form, are some freshly picked
Panaeolus cyanescensmonsters is significantly higher than other investigators [7.2 and 7.8]
published in the past (and also higher than in the investigated variant of dried
This type of mushroom).  The applied drying and harvest time are the possible causes
lower rates of psychoactive substances in the dried form.  When comparing the
Panaeolus cyanescensmonsters, originating from different flights (growth stages, and the mushrooms
picked) were derived, no association between the levels
psilocybin / psilocin and the flight from which the samples were obtained (Chart 3).
The psilocybin, psilocin, and the total psilocinegehalte (0.5-0.9%) in fresh Psilocybe
mexican cubensis samples were at the same level as in the dried samples from
smartshops.
The Psilocybe cubensis thai appears not only in appearance but also in the analysis
concerns, much like the mexican cubensis Psilocybe.  The detected levels of psychoactive substances
In this type of mushrooms were only marginally lower (total psilocin: 0.5 -0.7%).
The levels of psychoactive substance in the fresh Psilocybe cubensis thai, taken in a
smartshop (code 41 and 43), however, from the same mushroom grower, were particularly
low.  This indicates that psilocybin (psilocin that breaks through) and psilocin in the fresh
mushrooms much less stable than in the dried mushrooms.
This is also consistent with experience gained with demo samples.  Two new demo samples (1
Psilocybe cubensis Thai Psilocybe cubensis mexican and 1), both sampled the mushroom grower
(For an instruction session), were dried and analyzed after 4 weeks.  In both
samples could be demonstrated only traces hallucinogenic substance.
There were also the Psilocybe cubensis mexican and thai no relationship was observed between
both the percentages psilocybin, psilocin and total psilocin and the flight, which
the samples were taken (8.3: Chart 3).
Total psilocin (the solids) in Psilocybe Tampanensis samples was approximately 0.3%.
Hallucinogenic substances (and psilocybin) mushrooms in this species much more stable than the
other investigated fresh mushrooms.  The smart shops in the sampled Psilocybe Tampanensis
samples contain fact, despite the dry residue by wastage (during storage in
smartshop) was increased to 60-65%, almost as many psychoactive substance and fresh
harvested samples.  A demo-sample, which, like the above samples even after 4
weeks were dried and analyzed, had a total of nearly 0.3% psilocin.
5.
CONCLUSION
The indole alkaloids psilocybin and psilocin can by using methanol extraction
with the reversed phase LC technique quickly and accurately determined mushrooms.
The levels of psychoactive substance same type layers in the dried mushrooms and in the
fresh form roughly the same level and agreed to in the literature
values.  Exception is the occasional but very high levels of psilocybin and psilocin
in the fresh Panaeolus cyanescens.
There were no relationships observed between the psilocybin, psilocin and total
psilocinegehalte and the flight from which the mushrooms came.
The investigation also revealed that the psychoactive substances psilocybin and psilocin
fresh mushrooms far less stable than in the dried form.
________________________________________
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SAZD/01/30/21 - psilocybin and psilocin in magic mushrooms
SAZD/01/30/21
Page 11 of 17
6.
THANKS
The authors of this report to Dr.  WG van der Sluis of the Faculty of Pharmacy, Department
Pharmacognosy, University of Utrecht to thank for his advice, free
availability of standard equipment and the procedure for psilocybin psilocin
from this standard can be prepared.
7.















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OfflineLogicaL ChaosM
Ascension Energy & Alien UFOs
Male User Gallery


Folding@home Statistics
Registered: 05/12/07
Posts: 71,179
Loc: The Inexpressible... Flag
Last seen: 3 hours, 18 minutes
Trusted Cultivator
OG Cultivator
Re: Potency Project [Re: Cloneufc]
    #12353554 - 04/08/10 03:53 AM (14 years, 9 months ago)

Wow Cloneufc, that is an amazing article find. Good job! :yesnod::thumbup:
Those Dutch always get the cool illegal experiments.

I'm going to read the whole thing later, but the second graph on the bottom, comparing the active concentrations of 3 types of psychoactive species, is incredible! Its exactly what I want to see done for *just* for Ps. Cubenis compared to Penis Envy...that would be the ultimate experiment result.

Also, how's that Gas/Liquid Spectrometry machine doing? Are you still considering buying one?

Excellent On-Topic Article!
~ LogicaL Chaos ~

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OfflineCloneufc
Master Exploder!
Male


Registered: 11/15/07
Posts: 1,237
Loc: Las Vegas Flag
Last seen: 1 year, 5 months
Re: Potency Project [Re: LogicaL Chaos]
    #12354436 - 04/08/10 10:37 AM (14 years, 9 months ago)

I have the money to buy the machine now. Im holding off buying it for two reasons. First I need to buy new stuff,house, cars, toys ect. Second I want to make sure I buy the correct equipment, I dont want to waste money buying the wrong machine. Before I buy it I need to make sure 100% that it will do what I need it to do.

Wow the Pan Cyan hawaii sample was way stronger than the Azure sample. That surprises me. The P. tampanensis was weak as hell.

Am I reading this correctly? Is it this easy to prepare the sample? Why do you need to transfer the sample to two vial instead of one?

Weigh 250 mg of dried sample in a flask of 25 ml.  Add 20 ml of methanol to
and place the flask during 120 minutes in an ultrasonic bath.  Make sure the temperature
in the ultrasonic bath is not above 104 ° F rises.  Check the temperature in the bath and add ice
increases as the temperature is higher than (>) 104 ° F border.  Dilute with methanol to 25 ml
mix.  Centrifuge (about 16000x g) a portion of the extract and transfer the clear extract
two vials.

Edited by Cloneufc (04/08/10 11:48 AM)

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Offlinelibertaire
liberator
Male


Registered: 08/06/08
Posts: 4,204
Last seen: 4 years, 6 months
Re: Potency Project [Re: Cloneufc]
    #12461643 - 04/26/10 07:05 PM (14 years, 8 months ago)

So who's game for a revival of this?  Shea25 posted this recently, looks pretty legit:

http://www.thenook.org/nooki/index.php?title=Psilocin_Extraction_triptamine

It's an ab extraction, and the end result gets crystals.  The writer of the tek says that it is 70% efficient, which is pretty damn efficient if you ask me.

I intend to try this on the OI and OR I just made lc's of once I have fruits from them, and then later down the line try it on PE whenever that grow materializes, which probably won't be for at least a few months.  Anyone have any fruit on hand they'd like to try this on?  Everything is available at your local grocery store or hardware store. 

So whadya think, are we back in business?  Anyone wanna try this out?

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