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InvisibleStonehenge
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Extraction of psilocybin
    #3265030 - 10/22/04 06:14 PM (12 years, 4 months ago)

There was a great thread, actually two of them, that were deleted in the recent mix up. It was about extracting from shrooms or from mycellium to get fairly pure psilocybin and psilocin. I can't even remember who started the thread but he was working on an experiment. I hope to hear how it came out. There was also a link to a dea website where they talked about using acetic acid to extract the goodies. The person who started the thread planed to use hcl to extract and then do a wash or two along with standard acid base procedure. I'd sure like to see that thread restarted and hopefully hear some good news on the extraction. Are you out there, whoever it was?

Stoney


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InvisibleEgo Death
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Re: Extraction of psilocybin [Re: Stonehenge]
    #3265259 - 10/22/04 07:09 PM (12 years, 4 months ago)

That would be some powerful and dangerous stuff!!!

Psilocybin is an unstable chem. Is the salt anymore stable? If not it would be annoying to use and 1 may as well stick to shrooms. They are potent enough in there natural form for me!


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OfflineDadeMurphy
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Re: Extraction of psilocybin [Re: Ego Death]
    #3265477 - 10/22/04 08:16 PM (12 years, 4 months ago)

There's an old one somewhere that shows a method of extracting fairly pure without using acid/base method. Ill try and dig it up.

it looks like this
http://fanaticus.fungifun.org/mycoalki.htm


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Edited by DadeMurphy (10/22/04 08:25 PM)


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OfflinemotamanM
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Re: Extraction of psilocybin [Re: Stonehenge]
    #3266643 - 10/23/04 02:24 AM (12 years, 4 months ago)

Technical Note

A Rapid Extraction and GC/MS Methodology for the Identification
of Psilocyn in Mushroom/Chocolate Concoctions

Mohammad Sarwar*, Ph.D., and John L. McDonald, B.S.
Illinois State Police
Division of Forensic Services
Forensic Science Center at Chicago
1941 W. Roosevelt Road
Chicago, IL 60608
[email: msarwar36 -at- yahoo.com]

ABSTRACT: A simple, convenient, and rapid method for the identification of psilocyn in hallucinogenic mushroom/chocolate concoctions is presented. A 10% solution of acetic acid is used to extract psilocyn from the mushrooms. The acidic solution is then basified with solid sodium bicarbonate, then extracted with chloroform. The resulting extract is then back-washed to remove theobromine and caffeine from the chocolate, then concentrated and analyzed by TLC and GC/MS. The method takes about 30 minutes for mushroom/chocolate concoctions. A more simplified version of the method can be used for mushrooms, and takes about 15 minutes.

KEYWORDS: Forensic Science, Psilocyn, Extraction, Psilocybe Mushrooms, Mushroom/Chocolate Concoctions.

Introduction

Psilocyn and psilocybin, and the mushrooms containing these substances, are Schedule I substances under both Federal and Illinois state law. Psilocyn and psilocybin are hallucinogens, which act on the central nervous system to produce changes in perception, mood, and thinking ability. The effects produced by psilocyn and psilocybin are similar to those produced by LSD and mescaline (1-2). Since the mushrooms that produce these hallucinogens are easily cultivated, and spores, growing kits, and information are readily available through the Internet, increasing numbers of mushrooms and mushroom containing preparations (especially mushroom/chocolate concoctions, vide infra) are being encountered in forensic laboratories.

The life cycle of mushrooms has four stages, namely spores, the mycelium, pinhead or the primordial, and the mature fruit. The spores are actually the seeds of the fungi. Mushrooms cannot be classified as plants because they lack a root system, and do not have leaves, flowers, or the main constituent of plants, chlorophyll. Plants get their food through roots and leaves through photosynthesis, while mushrooms get their food or nutrients from the surrounding environment. The four species of mushrooms that contain psilocyn and psilocybin are strophariaceae, bolbitiaceae, coprinaceae, and cortinariaceae (3-5).

Psilocyn, psilocybin, and various other alkaloids are found naturally in all four above listed species of mushrooms. The mature fungi are sold in the underground market in both whole and powdered forms. More recently, various mushroom-containing concoctions have become popular, especially grated or powdered mushrooms in chocolate (6). A number of such cases have been received at this laboratory over the past year.

The most common analytical techniques reported in the literature for analysis of hallucinogenic mushrooms are all based on methanol extraction. In the most common procedure, the mushrooms are simply soaked in methanol overnight, and the resulting extracts condensed to near dryness and then analyzed using TLC and GC/MS (7-8). A more rapid technique involves placing the mushrooms in a closed vial with methanol, heating for a half an hour, then heating to dryness; the resulting residue is taken into 0.1 N sodium hydroxide, then extracted with butyl chloride. The butyl chloride extract is then back-extracted with 0.1 N sulfuric acid, and the UV spectrum recorded in acidic and basic media. The basic solution is further extracted with butyl chloride, and the extract evaporated to dryness; the resulting powder is then analyzed by IR (9). In another, longer method, the mushrooms are dried at 40 ?C in an oven for 16 hours, ground, and then soaked in methanol for 24 hours. The volume is reduced and then analyzed by HPLC (10). In a more rapid method using a buffer extraction, ground mushrooms are triturated in a rotary mixer with 10% ammonium nitrate in methanol for 30 minutes, then two methanol extractions are performed, and the combined methanol extracts analyzed by HPLC (11). Quantitative determination of psilocybin and psilocyn is accomplished by stirring freeze dried mushrooms in methanol for 12 hours, followed by analysis by HPLC and TLC (12). In a more refined method, the mushrooms are extracted with methanol, and the co-extracted sugars then precipitated with acetone; the resulting solution is concentrated prior to analysis by GC/MS (13). The aqueous extraction of psilocyn was achieved by using dilute acetic acid, adjusting the pH with glacial acetic acid, and heating the contents for one hour. The pH of the solution was then raised by the addition of ammonium hydroxide, and psilocyn extracted with diethyl ether. The latter method was also applicable to pure mushrooms but was more time consuming (14).

As noted above, mushroom/chocolate concoctions have become popular. The isolation and identification of psilocyn and psilocybin from mushrooms is somewhat problematic when the mushrooms have been grated or powdered and mixed with chocolate, because chocolate is a complex matrix containing a wide variety of components, many of which are soluble in methanol. Thus, the standard methanolic extraction techniques detailed above are almost inapplicable to mushroom/chocolate concoctions. In one recently described method, the concoction is soaked in dilute sulfuric acid and then washed with chloroform or methylene chloride. The aqueous layer is then basified with sodium hydroxide to pH 10, then extracted with chloroform (15-16). However, a clean peak of psilocyn was not obtainable even after multiple washings. Moreover, psilocyn is unstable at higher pH values (17). A short review on the methods of extraction for psilocyn can be read elsewhere (18).

In general, methanolic extraction procedures are very time consuming. Most procedures either involve an ?overnight? extraction or heating. In addition, methanolic extractions of psilocybe mushrooms usually co-extract other indolic compounds (and other methanol soluble components), some of which can mask the psilocyn and psilocybin peaks in GC or GC/MS analyses. And as noted above, methanolic extraction is poorly suited for mushroom/chocolate concoctions. Herein, we present a new method for the extraction of psilocyn from such concoctions. The extraction takes about fifteen minutes for pure mushrooms, and about half an hour for mushroom/chocolate concoctions. In addition, large number of samples can be analyzed in a relatively short period of time.

Materials and Methods

Reagents: (1) A 10% acetic acid by volume (Analytical Reagent); ( 2) Chloroform (A.R.); (3) Sodium bicarbonate (A.R.); (4) Deionized water; and (5) Ehrlichs reagent.

Equipment: GC/MS (HP 6890/5973), centrifuge, pestle and mortar.

Procedure for Pure Mushrooms:

About 0.2 to 0.5 gram of mushrooms are transferred into a mortar.
The mushrooms are covered with 10 % acetic acid, and ground with the help of a pestle.
Another 5 mL of deionized water are added and the mixture is ground into a fine slurry.
The slurry is then transferred into a test tube and centrifuged for about 3 minutes.
The supernatant is transferred into a small beaker
The supernatant is neutralized by adding small amounts of sodium bicarbonate (neutralization is judged to be complete when the foamy effervescence stops). A little excess bicarbonate is then added.
The resulting solution is transferred into a test tube and extracted with an equal amount of chloroform.
The biphasic solution is centrifuged, and the chloroform layer collected in a shell vial.
The chloroform extract is concentrated under air, transferred to a micro vial, and analyzed on the GC/MS.
Total extraction takes around 15 minutes. The results are shown in Figure 1.

Procedure for Mushroom/Chocolate Concoctions:

1.0 to 2.0 gram(s) of sample is transferred into a mortar and ground with a pestle.
The resulting powder is covered with 10 % acetic acid, and the sample is further ground with a pestle.
An additional 5 to 7 mL deionized water is added, and the mixture is ground for about 2 minutes, creating a thin slurry.
This slurry is divided into two equal portions, and each is transferred into a test tube.
An equal amount of chloroform is added to each tube, and the tubes are centrifuged for 3 minutes.
The aqueous layer is pipetted into a beaker from both of the test tubes.
2 or 3 drops of this solution are placed in a test tube, and treated with the Ehrlich?s reagent; a deep purple color is indicative of presence of indolic compounds.
The aqueous solution in the beaker is neutralized by slowly adding sodium bicarbonate until the effervescence stops.
A little excess bicarbonate is added, and the pH is checked with pH paper to make sure it lies between 8-8.5.
The resulting solution is then transferred into two test tubes, and each extracted with an equal amount of chloroform.
The tubes are centrifuged for about 5 minutes.
The chloroform layers are collected into two new test tubes.
An excess of 2% sodium bicarbonate solution is added to each test tube.
After vigorous shaking, the test tubes are centrifuged for 5 minutes.
The chloroform layers are combined in a small beaker.
The chloroform extract is concentrated under air, transferred to a micro vial, and analyzed on the GC/MS.
The results are shown in Figure 2.

Results and Discussion

The presented acetic acid facilitated extraction of psilocyn from mushrooms is more rapid and convenient versus traditional methanolic extraction procedures, which require long time frames or potentially destructive heating. In addition, the use of sodium bicarbonate as a neutralization agent keeps the pH below 8.5, thereby avoiding base-facilitated destruction of psilocyn. The Total Ion Chromatogram (TIC) of the mushroom only sample shows a clean psilocyn peak (Figure1). Analysis by TLC also shows only psilocyn. No psilocybin was detected - this is perhaps due to the activity of the phosphatase enzymes present in the mushrooms, which can dephosphorylate psilocybin to psilocyn in aqueous medium (19).

Analysis of mushroom/chocolate concoctions requires additional cleanup steps. Analysis of the chloroform extract at Step 12 (that is, before the extract was washed with sodium bicarbonate) showed three peaks in the TIC (Figure 2). The small peak at 4.223 minutes is due to caffeine, the broad peak at 4.50 minutes is due to theobromine, and the sharp peak at 4.837 minutes is due to psilocyn. Caffeine and theobromine (both purine alkaloids) result from the chocolate; theobromine is the main alkaloid in chocolate (2.8-3.5 % in cocoa), and caffeine is another major alkaloid (0.1-0.4 %) (20). After washing the chloroform extract (at Step 12) with 2 % sodium bicarbonate solution, the amounts of theobromine and caffeine are very low (see Figure 3), resulting in a nearly clean TIC showing only psilocyn. However, the mass spectrum of psilocyn depicted in Figure 3 showed an extraneous ion 109, probably resulting from trace theobromine. When the chloroform extract was washed with water only, the peak due to theobromine nearly disappears, and there is no extraneous 109 fragment, but the peak due to caffeine is still present (see Figure 4).

Quantitation was not performed in this study; however, the procedure allows facile identification of psilocyn in mushroom/chocolate concoctions.

The mass spectra acquired in this study for caffeine, theobromine, and psilocyn are presented in Figures 5 - 7.


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OfflinemotamanM
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Re: Extraction of psilocybin [Re: Stonehenge]
    #3266661 - 10/23/04 02:33 AM (12 years, 4 months ago)



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InvisibleStonehenge
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Re: Extraction of psilocybin [Re: motaman]
    #3268085 - 10/23/04 04:14 PM (12 years, 4 months ago)

Thanks very much, motaman. Those are interesting techs. DadeMurphy linked to an older tech that is used a lot. What I see here is that the active ingredients can be dissolved in methanol and to a lesser extent in ethanol. I found where I can order methanol for about $4 a quart which isn't bad. However, the stuff is toxic and while proper procedure will keep you from being poisoned, you still have some danger of breathing the fumes and you have to get every trace of the stuff out of what you will consume. I have come to accept the fact that I screw up on a regular basis so I'm going to avoid anything that'll kill me when I do.

What the dea info tells us is that it also can be extracted with acids. They mention sulphuric acid and acetic (vinegar). I saw in one of the techs that they did it with cranberry juice and ascorbic acid (vit c). This indicates that most any acidic water will probably work to some extent. There was someone who posted who was going to do an extract with hcl and then defat and a/b extract to get pure psilly crystals. That would be nice but if the extract is 25% pure, that's plenty pure enough for me particularly since the impurities are likely to be sugars and a tiny bit of mycellium. I've eaten colonised cakes and am not afraid of a tiny bit of mycellium though it's nice to get the volume reduced. This is also supposed to reduce the nausea and stomach cramps you sometimes get with shrooms.

The part that is likely to be a pain is the part where you evaporat the extract so that you have a powder. With alcohol it's fast and easy. With water solutions it's going to be time consuming. Boiling will likely destroy the psilocybin and will certainly destroy the psilocyn. If you can get the goodies to precipitate you have saved a lot of work. Alternately, you could have pans of solution sitting out evaporating over a period of days. Dust would likely get into it and maybe bugs. Does anyone know if basifying the acidic extraction will make the actives precipitate out? I might try the defatting step later but I'd like to come up with a simple tech that gives you a dry powder.


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“A democracy cannot exist as a permanent form of government. It can only exist until the voters discover that they can vote themselves largesse from the public treasury. From that moment on, the majority always votes for the candidates promising the most benefits from the public treasury with the result that a democracy always collapses over loose fiscal policy, always followed by a dictatorship.” (attributed to Alexis de Tocqueville political philosopher Circa 1835)

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