Extraction and GC/MS Methodology for Identification of Psilocyn in shroom/Chocolate

[triptamine]

For all interested, here is the "simple" psilocin extraction which can yield fairly pure psilocin crystals after recrystalizing in chloroform/heptane (1:3).




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:

1.
About 0.2 to 0.5 gram of mushrooms are transferred into a mortar.
2.
The mushrooms are covered with 10 % acetic acid, and ground with the help of a pestle.
3.
Another 5 mL of deionized water are added and the mixture is ground into a fine slurry.
4.
The slurry is then transferred into a test tube and centrifuged for about 3 minutes.
5.
The supernatant is transferred into a small beaker
6.
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.
7.
The resulting solution is transferred into a test tube and extracted with an equal amount of chloroform.
8. The biphasic solution is centrifuged, and the chloroform layer collected in a shell vial.
9. 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. 1.0 to 2.0 gram(s) of sample is transferred into a mortar and ground with a pestle.
2. The resulting powder is covered with 10 % acetic acid, and the sample is further ground with a pestle.
3. An additional 5 to 7 mL deionized water is added, and the mixture is ground for about 2 minutes, creating a thin slurry.
4. This slurry is divided into two equal portions, and each is transferred into a test tube.
5. An equal amount of chloroform is added to each tube, and the tubes are centrifuged for 3 minutes.
6. The aqueous layer is pipetted into a beaker from both of the test tubes.
7. 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.
8. The aqueous solution in the beaker is neutralized by slowly adding sodium bicarbonate until the effervescence stops.
9. A little excess bicarbonate is added, and the pH is checked with pH paper to make sure it lies between 8-8.5.
10. The resulting solution is then transferred into two test tubes, and each extracted with an equal amount of chloroform.
11. The tubes are centrifuged for about 5 minutes.
12. The chloroform layers are collected into two new test tubes.
13. An excess of 2% sodium bicarbonate solution is added to each test tube.
14. After vigorous shaking, the test tubes are centrifuged for 5 minutes.
15. The chloroform layers are combined in a small beaker.
16. 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.


Figure 1. Total Ion Chromatogram of Mushrooms (Only) using Acetic Acid
and Sodium Bicarbonate (4.84 Minutes = Psilocyn).


Figure 2. Total Ion Chromatogram of Mushroom/Chocolate Concoctions using Acetic Acid
and Sodium Bicarbonate (4.22 Minutes = Caffeine; 4.50 Minutes = Theobromine; and
4.84 Minutes = Psilocyn).


Figure 3. Total Ion Chromatogram of Mushroom/Chocolate Concoctions when the
Chloroform Extract was Washed with 2 Percent Aqueous Sodium Bicarbonate.


Figure 4. Total Ion Chromatogram of Mushroom/Chocolate Concoctions when the
Chloroform Extract was Washed with Water.


Figure 5. Mass Spectrum of Caffeine.


Figure 6. Mass Spectrum of Theobromine


Figure 7. Mass Spectrum of Psilocyn.

Acknowledgements

The authors thank Forensic Scientists Joseph Gillono and Paula Bosco Szum, this laboratory, for their technical assistance.

References

1. Drugs of Abuse. US Department of Justice, Drug Enforcement Administration, 1997 Edition.

2. Aboul Enein HY. Psilocybin: A pharmaceutical profile. Am Journal of Pharmacy. 1974;146(3):91 95.

3. Kaul TN. Introduction to mushroom science. Enfield, New Hampshire: Science Publishers, Inc., 1972.

4. McKnight KH, McKnight VB. A field guide to mushrooms. Boston: Houghton Mifflin Co., 1987.

5. Ammirati JF, Traquair JA, Horgen PA. Poisonous mushrooms of northern United States and Canada. Minneapolis: University of Minnesota Press, Inc., 1985.

6. Editor. “Homemade” chocolates containing psilocybin mushrooms appearing across the United States. Microgram Bulletin 2003;36(6):111 et seq.

7. Gross TS. Detecting psychoactive drugs in the developmental stages of mushrooms. J Forensic Sci 2000;45(3):527 537.

8. Gross TS. Psychotropic drugs in developmental mushrooms: A case study review. J Forensic Sci 2002;47(6):1298 1302.

9. Lee RE. Technique for the rapid isolation and identification of psilocyn from psilocybin containing mushrooms. J Forensic Sci 1985;30(3):931 941.

10. Thomson BM. Analysis of psilocybin and psilocyn in mushroom extracts by reversed phase HPLC. J Forensic Sci 1980;25(4):779 785.

11. Christiansen AL, Rasmussen KE, Tonnesen F. Determination of psilocybin in psilocybe Semilanceata using HPLC on silica column. Journal of Chromatography 1981;210:163 167.

12. Beug MW, Bigwood J. Quantitative analysis of psilocybin and psilocyn in psilocybe Baeocystis by HPLC and TLC. Journal of Chromatography 1981;207(3):163-167.

13. Timmons JE. Identification of psilocyn and psilocybin using gas chromatography and mass spectrometry. Microgram 1984;17(2):28 31.

14. Casale JF. An aqueous organic extraction method for the isolation and identification of psilocyn from hallucinogenic mushrooms. J Forensic Sci 1985;30(1):247 250.

15. Microgram Bulletin. 2003; 36(6):113.

16. Microgram Bulletin 2003;36(8):174.

17. The Merck Index, 13th Ed., Merck and Co., Inc., Whitehouse Station, NJ, No.8016, p. 1418.

18. Sottolano SM, Lurie IS. The quantitation of psilocybin in hallucinogenic mushrooms using HPLC. J Forensic Sci 1983;28(4):929 935.

19. Gartz J. Extraction and analysis of indole derivatives from fungal biomass. Journal of Basic Microbiology 1994;34:17-22.

20. PDR for Herbal Medicines, 2nd Ed., Medical Economics Company, Motvale, NJ. 2000:199.

[euphorius]

Very nice write-up triptamine.

None of your images are working however.


A quantitative analysis of the sample before aqueous extraction and after would be a really good idea as well though. I'm particularly interested in this enzyme "phosphatase". Is it present in all psychoactive species? What is it's method of action? I'm guessing it just grabs the phosphate and churns out phosphoric acid(seems like the easiest option(energy wise), and nature loves easy most of the time)

But I would think that would be something that would need to be checked out before this method becomes officially viable. Because you don't really know whether the psilocybin was converted or destroyed (or rendered otherwise inactive) if you don't have an accurate before and after measurement of the concentrations.

I personally prefer psilocin to psilocybin, so it'd be great news to me if it was in fact fully converted.

(Being able to see the graphs would help a lot in furthering this discussion)

[triptamine]

According to another paper, it says, "Psilocybin is completely dephosphorylated to psilocin by heating the acid bath to 70 C for 8-10 minutes". There definately is at least some conversion, since the yields are fairly good (about 50% recovered). I would imagine that yields can be higher if a more careful filtration and basification was used. Another note: Using sodium bicarbonate to basify seems like a good idea, but it causes quite a foam to form which really interferes with the rest of the process. NaOH can be used, as long as dilute and added very slowly while mixing to achieve a pH of ~8. Also, try to take up the alkaloids quickly into the nonpolar solvent, as they are quite unstable at this pH. Does anyone here know how well ascorbic acid stabilizes psilocin?

Triptamine

[triptamine]

Euphorius, to answer your question about the dephosphorylation, look at the discussion section below (and just before it).

Triptamine


Extraction and analysis of indole derivatives from fungal biomass

Journal of Basic Microbiology
Vol 34, 1994; 17-22
by Jochen Gartz

The occurence and extraction of indole derivatives in six species from four genera of higher fungi were investigated. By using pure methanol for extraction of the mushrooms analysis revealed the highest concentrations of psilocybin and baeocystin. The psilocin content of the species was higher by using aqueous solutions of alcohols than with methanol alone but was an artificial phenomenon caused by enzymatic destruction of psilocybin. The extraction with dilute acetic acid yielded better results than with the water containing alcohols. The simlpe one-step procedure with methanol for the quantitative extraction is still the safest method to obtain the genuine alkaloids from funghal biomass.

Comments:
The abstract says it, if you are planning to extract the alkaloids from either dries and pulverisized fruiting bodies or from mycelium it is best to use pure methanol. Superior to aqueous solutions of alcohols (which is wet alcohol, the one you are likely to have!) is dilute acetic acid which means simple vinegar (better: vinegar essence diluted with same amount of water) which is quite nice because there is no problem obtaining it. The problem with wet alcohol is that the enzymes which dephosphorylise Psilocybin to the instable Psilocin are also extracted from the biomass. This also occures with acetic acid but to a smaller amount and does not occure at all with pure methanol (ethanol?). The recommended extraction time (magnetical stirring) is with methanol 12h at room temperature or 1h at 45 deg.Celsius, no times given for the acetic acid method. And the moral: Dont use clandestine-quality alcohols for extraction, use vinegar ! Or dry the alcohol by adding salts like MgSO4, CaCl2, NaSO4 which were previously dried in an oven and decand or filter the solvent from them after a day or longer.

PLEASE NOTE:
For those new to extractions, it is extremely important to note that methanol is poisonous to humans and if it is used for any extractions, it must be thoroughly evaporated before the material is used.

Full Text #
In the last 15 years many papers have been published about the occurance and determination of psychotropic tryptamine derivatives like psilocybin, psilocin and baeocystin in fungi (Gartz 1992, 1993).

Various extraction procedures of these substances from mushrooms have been ised mainly with methanol as solvent (Beug and Bigwood 1982, Gartz 1987, Sottolano and Lurie 1983).

In 1985 an aqueous-organic extraction method with acetic acid for these compounds was described (Casale 1985). Recently, Czech analysts have used aqueous solutions of methanol and ethanol (pure or in presence of potassium-nitrate) for extraction of the indole derivatives in Psilocybe bohemica Sebek (Kysilka and Wurst 1990, Wurst et al 1992). They claimed that it was possible to have found more psilocin with aqueous ethanol extraction than with pure methanol and that a dissimilar extraction of the alka loids by using both new systems could be achieved. In this work the extraction procedures of psilocybin, psilocin and baeocystin from varoius mushroom species including P. bohemica Sebek were studied by using methanol and the recommended mixtures of sol vents (Casale 1985, Kysilka and Wurst 1990, Wurst et al. 1992), respectively.

Materials & Methods #
Fungal material: Cultivated mushrooms: Psilocybe semilanceata (FR.) Kumm from horse manure compost (Gartz 1991); Psilocybe cubensis (Earle) Singer grown on cow dung/rice grain mixture (Gartz 1989a); P. bohemica from rice grain/water (Gartz and Mueller 1989; Gymnopilus purpuratus (Cooke and Mass) Singer from rice grain/saw dust medium (Gartz and Mueller 1990, Gartz 1991).

Naturally grown mushrooms: Panaeolus cyanescens (BK & BK) SACC. (leg. Oahu, Hawaii 13.11.88); Inocybe aeruginascens Babos (leg. Potsdam 20.05.1987); P. bohemica Sebek (leg. near Sazava, Czech Republic 15.11.89).

All basidiocarps were dried at room temperature. Possible present residual water was removed from the mushrooms by freeze-drying. Voucher speciments of each species have been deposited in the herbarium of the Univeristy of Leipzig (LZ).

Extraction: (Samples (0.01 - 0.1 g) of dried ground mushrooms were extracted with 5 to 20 ml of methanol for 0.5 to 12 hours by using a magnetic stirrer at room temperature. Under equal conditions the mixtures with aqueous acetic acid (Casale 1985) and a queous ethanol (psilocin) and methanol (psilocybin) (Kysilka and Wurst 1990, Wurst et al. 1992) were used for extraction of the same batch of mushrooms. In the cases with aqueous alcohols as solvent a different extraction time for psilocybin (10 min) and psilocin (160 min) was performed (Kysilka and Wurst 1990). By using of dilute acetic acid the solution was placed in a boiling water bath for 10 min after extraction and anaysis and was then analysed 10 min after extraction and analysis and was then anal ysed again (Casale 1985).

The filtration and analysis of the indole derivatives by using HPLC and TLC were described elsewhere (Gartz 1987, Semerdzieva et al. 1986, Wurst et al. 1992). An analysis of the extracts for enzymes of the phosphatase type was also carried out (Weber a nd Horita 1963).

Results #
In this investigation the extraction of psilocin, psilocybin and baeocystin with pure methanol was not completely after 30 min in all species and even 6 hours in analysis of P. cubensis and G. purpuratus. But the full extraction of the alkaloids from al l mushrooms was reached after 12 hours. After this time no traces of indole derivatives could be detected after subsequent extraction of the fungal material with aqueous solutions of ethanol/methanol or acetic acid as well as with chloroform for psilocin.

Baeocystin as incompletely methylated counterpart and possible precursor of psilocybin (Gartz 1989a) was found in all species by using methanol but in some cases only in very small amounts (Table 1).

The psilocybin and psilocin content was in the same order of magnitude as that found earlier (Gartz 1992, 1993). This substance seems to be a phosphoric acid ester like psilocybin and baeocystin. Similar concentrations of psilocin were detected in the extracts of P. cubensis and G. purpuratus by using an aqueous solution of acetic acid versus pure methanol (Table 2).

Table 1 #
Amount of indole alkaloids in fruiting bodies of different species by using pure methanol as solvent (%, dry weight).

Species Psilocybin Psilocin Baeocystin
P. semilanceata 0.98 - 0.34
P. bohemica 0.85 0.02 0.04
P. bohemica (cultivated) 0.93 0.04 0.02
P. cubensis 0.63 0.11 0.02
G. purpuratus 0.34 0.29 0.05
I. aeruginacens 0.40 - 0.21
P. cyanescens 0.32 0.51 0.02

Table 2 #
Concentraction of alkaloids by using acetic acid for extraction of the dried mushrooms (%, dry weight).

Species Psilocybin Psilocin Baeocystin
P. semilanceata 0.97 0.15 0.11
P. bohemica 0.60 0.21 -
P. bohemica (cultivated) 0.65 0.28 -
P. cubensis 0.45 0.25 -
G. purpuratus 0.24 0.35 0.01
I. aeruginacens 0.32 0.05 0.15
P. cyanescens 0.20 0.61 -

Table 3 #
Results of the mushroom extraction of six species using aqueous mixtures of methanol and ethanol (%, dry weight).

Species Psilocybin Psilocin Baeocystin
P. semilanceata 0.80 0.15 0.11
P. bohemica 0.60 0.21 -
P. bohemica (cultivated) 0.65 0.28 -
P. cubensis 0.45 0.25 -
G. purpuratus 0.24 0.35 0.01
I. aeruginacens 0.32 0.05 0.15
P. cyanescens 0.20 0.61 -



By using the new solvent mixtures containing ethanol and methanol for extraction it was found that more psilocin could be detected in extracts of every species but always smaller amounts of psilocybin than with pure methanol (Table 3).

Additionally, a high activity of enzymes of the phosphatase type could be detected in these aqueous solutions from all species. In contrast to these results only the extracts of P. cubensis and P. cyanescens showed a significant enzymatic activity b y using acetic acid as solvent. In these cases psilocybin was completely dephosphorylated to psilocin by heating the acid extracts and no baeocystin could be detected in P. cyanescens.

Discussion #
It is well known that an extraction procedure with methanol needs much time (up to 12 hours) at room temperature (Beug and Bigwood 1982, Gartz 1987, Semerdzieva et al. 1986) or one hour at 45 C (SCOTTOLANO and Lurie 1983) for complete extrraction.

In our investigations psilocin could be found in high concentrations as well as psilocybin after simple extraction with methanol from various species (Gartz 1987, 1989c, 1991). When undertaking quantitave analysis of levels of indole derivatives after biotransformation of tryptamine and similar compounds in fruiting mycelia of P. cubensis the highest concentrations of psilocin in every mushroom for example could be detected by using methanol (Gartz 1989a, b). By using aqueous methanol and ethanol as so lvent for analysis of P. bohemica the Czech analysts have not always analyzed the same batch of mushrooms during their comparative study of extraction methods (Kysilka, pers. communication 1989).

We generally found variations from one mushroom to another in every species even within P. bohemica from a single location (Gartz and Mueller 1989) and also in controlled cultures (Gartz 1991). Additionally, the high activity of enzymes of the phosphat ase type in the aqueous solutions of alcohols was already described in aqueous mycelial extracts of P. cubensis and other psilocybin containing mushrooms many years ago (BOCKS 1968, Gartz 1993, Weber and Horita 1963). These enzymes were also extracted wit h the water containing solvents and caused a partial dephosphorylation of psilocybin to psilocin (Tables 1 and 3). By using these aqueous soluions it was also observed that in some cases bluish mixtures have been resulted after extraction as a sign of par tial oxydation of psilocin (BOCKS 1968, Gartz 1989a, Weber and Horita 1963). It is also interesting that most of the baeocystin was destroyed during the extraction procedure with water containing alcohols (Tables 1 and 3).

Casale (1985) described the rapid formaion of psilocin after complete dephosphorylation of psilocybin by heating the dilute acetic acid extract. It is now quite clear that the decomposition under these conditions is an enzymatic reaction and was not ca used by the acid alone. For example the phosphoric acid ester psilocybin, baeocystin and aeruginascin in these acidic extracts from I. aeruginacens were stable during heating in contrast to the behaviour of the same alkaloids in solutions of P. cubensis a nd P. cyanescens. It seems that active enzymes of the phosphatase type could be extracted with aqueous acetic acid only in these two species in contrast to water containing alcohols as extraction method. In the past attempts at the sparation of psilocybin and psilocin simply using mixtures of organic solvents and water were also unsatisfactory (THOMSON 1980).

This investigation shows that the high percentage of psilocin detected in P. bohemica (Kysilka and Wurst 1990, Wurst et al. 1992) and not found earlier (Gartz and Mueller 1989) was an artificial phenomenon casued by enzymatic destrucion of psilocybin w hich is common in different species by using water containing organic solvents. Extraction with pure methanol is the safest method to obtain the genuine indole derivatives from mushroom species of various genera.

Acknowledgements
The author thanks the following persons: G. Drewitz, J. Allen, G.K. Mueller and M. Semerdzieva who geneously supplied herbarium material and/or valuable information.

References #
# Beug MW, Bigwood J. 1982. Psilocybin and psilocin levels in twenty species from several genera of wild mushrooms in the Pacific Northwest, U.S.A. J. Ethnopharm, 5, 271-289.
# Bocks SM. 1968. The metabolism and psilocin and psilocybin by fungal enzymes. Biochem. J., 106, 12-13.
# Casale JF. 1985. An aqueous-organic extraction method for the isolation and identification of psilocin from hallucinogenic mushrooms. J. Forensic Sci., 30, 247-250.
# Gartz J. 1985. Zur Isolierung des Baeocystins aus den Fruchtkoerpern einer Psilocybe-Art. Pharmazie, 40, 274.
# Gartz J. 1987. Variation deer Indolalkaloide von Psilocybe cubensis durch unterschiedliche Kultivierungsbedingungen. Beitraege z. Kenntnis d. Pilze Mitteleuropas, 3, 275-281.
# Gartz J. 1989a. Biotransformation of tryptamine derivatives in mycelial cultures of Psilocybe. J. Basic Microbiol., 29, 347-352.
# Gartz J. 1989b. Biotransformation of tryptamine in fruiting mycelia of psilocybe cubensis. Planta Med., 55, 249-250.
# Gartz J. 1989c. Occurence of psilocybin, psilocin and baeocystin in Gymnopilus purpuratus. Persoonia, 14, 19-22.
# Gartz J, Mueller GK. 1990. Analysis and cultivation of fruit bodies and mycelia of Psilocybe bohemica. Biochem. Physiol. Pflanzen, 184, 337-341.
# Gartz J, Mueller GK. 1990. Versuche zur Kultur von Gymnopilus purpuratus, Purpurflaemmling. Myk. Mitt. blatt (Halle), 33, 29-30.
# Gartz J. 1991. Further investigations on psychoactive mushrooms of the genera Psilocybe, Gymnopilus and Conocybe. Ann. Mus. civ. Rovereto (Italy), Sez. sc. nat.,7, 265-274.
# Gartz J. 1992. New aspects of the occurance chemistry and cultivaion of European hallucinogenic mushrroms. Ann. Mus. civ. Rovereto (Italy), Sez. sc. nat., 8, 107-124.
# Gartz J. 1993. Narrenschwaemme. Psychotrope Pilze in Europa in Europa. Herausforderung an Forschung und Wertsystem. Editions Heuwinkel. Genf/Neuallschwill.
# Kysilka R, Wurst M. 1990. A novel extraction procedure for psilocybin and psilocin determination in mushroom samples. Planta Med., 56, 327-328.
# Semerdzieva M, Wurst M, Koza T, Gartz J. 1986. Psilocybin in Fruchtkoerpern von Inocybe aeruginascens. Planta Med., 47, 83-85.
# Sottolano SM, Lurie IS. 1983. The quantitation of psilocybin in hallucinogenic mushrooms using high performance liquid chromatography. J. Forensic Sci., 28, 931-935.
# Thomson BM. 1980. Analysis of psilocybin and psilocin in mushroom extracts by reversed-phase high performance liquid chromatography. J. Forensic Sci., 25, 779-785.
# Weber LJ, Horita A. 1963. Oxydation of 4 and 5-hydroxy-indole derivatives by mammalian cytochrome oxydase. Life Sciences 1, 44-49.
# Wurst M, Kysilka R, Koza T. 1992. Analysis and islolation of indole alkaloids of fungi by high-performance liquid chromatography. J. Chromatogr., 593, 201-208.

[euphorius]

Is the "acid bath" you are referring to the acetic acid solution?

-Bicarb is always messy, especially with acetic (homemade volcano, anyone?)

-I would use NaOH, as suggested, probably nothing above 0.05-0.1M, but make sure to add the nonpolar before you get above (or right around) neutral pH, so the alky's can migrate over when they are ready.

--Also, it would be interesting to try the extraction with a small amount of phosphoric acid (or anything else that donates some phosphate to solution) to see if it affects the conversion from cybin to psilocin by the enzyme.(just be careful with the pH of course)

[euphorius]

Ok, took me a bit to read it all. Good stuff though, really good stuff.

The only thing that strikes me as odd is P. semilanceata. It actually loses psilocin AND psilocybin and makes baeocystin (in acetic acid soln).

It just sucks that they didn't do a pure, well 95%, EtOH extraction as well, so we'd have a direct comparison.

[triptamine]

95% EtOH is not the way to go, as the actives are not very soluable in this. 70% EtOH is far better (in terms of extraction efficiency). And speaking of efficiency, someone should optimize this process for maximum efficiency and minimum decomposition. Does anyone know what psilocin decomposes into and what the properties of this might be? Some say that psilocin oxidises to a blue product. Is this true, and what is it?

Triptamine

[Hippie3]

bluing isn't psilocin as far as we know.