TECHNICAL NOTE

J o h n F. Casale, 1 B.S.

An Aqueous-Organic Extraction Method for the

Isolation and Identification of Psilocin from
Hallucinogenic Mushrooms

REFERENCE: Casale, J. F., "An Aqueous-Organic Extraction Method for the Isolation and
Identification of Psiloein from Hallucinogenic Mushrooms," Journal of Forensic Sciences,
JFSCA, Vol. 30, No. 1, Jan. 1985, pp. 247-250.

ABSTRACT: A simple aqueous extraction method for the isolation and identification of psilocin
from Psilocybe cubensis mushrooms is reported. This method employs a dephosphorylation of
the phosphate ester to psilocin, which facilitates a greater product yield and simplifies identifica-
tion. Psilocin extracted by this method is sufficiently concentrated and free of cocontaminants to
allow identification by infrared spectroscopy and gas chromatography/mass spectrometry.

KEYWORDS: toxicology, psilocin, extraction

The tryptamines are one of four categories of hallucinogenic indoles in more than 20
classes of indole compounds comprising approximately 600 alkaloids [1]. Considerable re-
search has been conducted with psilocin and psilocybin since their isolation by Hofmann et
al [2]. Several extraction techniques [1,3-6] have been used to isolate psilocin and psilocybin
from more than two dozen species of mushrooms in four genera (Conocybe, Panaeolus, Psi-
locybe, Stropharia). The techniques that use methanol coextract other compounds such
as urea, ergosterol, ergosteral peroxide, a,c~-trehalose, baeocystin, and norbaeocystin
[3,4, 7]. At present, a useful aqueous extraction procedure has not been reported for psilocin
and psilocybin.
The dephosphorylation of psilocybin to psilocin in vivo has been well documented [1, 8. 9]
and is thought to account for most or all of its central nervous system activity [8]. Conversion
of psilocybin to psilocin is also necessary for aqueous extraction with organic solvents be-
cause of the very low lipid solubility of psilocybin. Extraction of only one compound also
permits infrared analysis of the extract.
Concentration and detectability of psilocin and psilocybin are dependent on several vari-
ables, including:
1. The absence of glucose, which will prevent the production of psilocybin [10].
2. Low levels of a m m o n i u m succinate, which will give poor yields of psiloeybin [10].
3. The growing medium, which requires a pH of less than 7 [10].
4. Timing: m a x i m u m protection of psilocybin occurs on the seventh day after germina-
tion, while m a x i m u m production of the mycelium is reached by the ninth day [10].
Received for publication 24 March 1984; accepted for publication 21 May 1984.
1Forensic drug chemist, North Carolina State Bureau of Investigation, Raleigh, NC.

247

Copyright © 1985 by ASTM International

248 JOURNAL OF FORENSIC SCIENCES

5. Temperature: complete loss of psilocin and psilocybin will occur in harvested mush-
rooms left at room temperature for an extended period of time [3].
6. Oxidation: psilocin will oxidize to a blue product (possibly accounting for the bluing
color in the four genera containing psilocin and psilocybin) [9].
Because of the increasing popularity of these mushrooms and kits available from drug-
oriented publications for growing mushrooms containing psilocin and psilocybin in cow ma-
nure a simple aqueous extraction procedure has been developed that extracts reasonably
pure psilocin from mature mushrooms. This extraction method greatly simplifies the identi-
fication of psilocin from those mushrooms by infrared spectroscopy and gas chromatogra-
phy/mass spectrometry (GS/MS).

Experimental Procedure
A representative sample of 2 to 10g of dried mushrooms is ground to a fine powder by
mortar and pestle. The powder is mixed with 100 mL of dilute acetic acid in a 250-mL
beaker. The pH is readjusted to pH 4 with glacial acetic acid. After standing 1 h, the beaker
is placed in a boiling water bath for 8 to 10 min or until the internal temperature of the acid
mixture reaches 70~ The beaker is removed and cooled to room temperature under run-
ning water. The acid mixture is separated from the mushroom powder by suction filtration
using glass wool. The filtrate is brought to pH 8 with concentrated ammonium hydroxide
and quickly extracted with two 50-mL portions of diethyl ether. Gentle mixing instead of
shaking should be used to prevent an emulsion. The ether is dried over sodium sulfate, fil-
tered, and evaporated under nitrogen with no applied heat.
Crude psilocin will appear as a greenish residue. Recrystalization from chloroform/n-
heptane (1 : 3) yields white crystals. The resulting powder can then be submitted to infrared
and mass spectral analyses.
Infrared spectra were obtained from potassium bromide disks on a Beckman Microlab
600 spectrophotometer. A Finnigan Model 3200 GC/MS with Model 6000 data system was
used for producing the mass spectra.

Results and Discussion

This method permits rapid isolation of psilocin from hallucinogenic mushrooms by coex-
traction of both psilocin and psilocybin. Dilute acetic acid is an excellent solvent for this
purpose, because both compounds are very soluble in acetic acid [11] and very little of other
interfering substances are extracted. It is most likely some other compounds are coextracted
but are removed from psilocin in the ether extraction from the aqueous base. Psilocybin is
completely dephosphorylated to psilocin by heating the acid extract. After addition of the
base, extraction into ether should be performed promptly, because of decomposition of psi-
locin at a greater than 7 [12]. The extraction and dephosporylation steps produce reasonably
pure psilocin from a small amount of mushroom material. Two grams of mushrooms will
often be sufficient to obtain an infrared spectrum of psilocin (Fig. 1). Smaller mushrooms
exhibits provide ample psilocin for mass spectral analysis (Fig. 2).
This method has been used in our laboratory for six months and has given excellent results
in seperating psilocin from methanol-soluble compounds. Other identification techniques
such as gas chromatography and microcrystalline tests are possible on psilocin extracted by
this method.

Acknowledgments
The author would like to thank J. R. Daniel and M. S. Nelson for their help in the prepa-
ration of this manuscript.
I00

90

ft.
80

70
\ ~h
F'I
/
k.11 J.D V
60
l
I^111
50
~A / I ~,lllll
H C~
40

30
/v r[ I II,I ~ r
Fn
o~N~l CH2CH~NC~
20 m
V X
"11
t0
C~

0
4000 3000 2000 1800 1600 1400 1200 I000 800 600
Z
0
FIG. 1--1nfrared spectrum of psilocin.
"0
r"
0
Z
250 JOURNAL OF FORENSIC SCIENCES

IO0
H

~N ~ CH2CH2N\
C
/H3 IO
CH 3
OH

..... I ......... I ......... I .... ' .... [ .....

25 50 100 150 200

FIG. 2--Electron impact mass spectrum of psilocin.

References
[1] Schultes, R. E., "Indole Alkaloids in Plant Hallucinogens," Journal of Psychedelic Drugs, Vol. 8,
No. 1, Jan.-March 1976, pp. 7-25.
[2] Hofmann, A., Heim, R., Barck, A., Kobel, H., Frey, A., et al, "Psilicybin and Psilocin,"
Helvetica Chimica Acta, Vol. 42, No. 2, 1959, pp. 1557-1572.
[3] Beug, M. W. and Bigwood, J., "Quantitative Analysis of Psilocybin and Psilocin in PsiLicybe
Baeocystis (Singer and Smith) by High-Performance Liquid Chromatography and by Thin-Layer
Chromatography," Journal of Chromatography, Vol 207, No. 3, March 1981, pp. 379-385.
[4] Koike, Y., Wada, K,, Kusano, G., Nozoe, S., and Yokoyama, K., "Isolation of Psilocybin form
Psilocybe Argentypes and Its Determination in Specimens of Some Mushrooms," Journal of Natu-
ralProducts, Vol. 44, No. 3, May-June 1981, pp. 362-365.
[5] Ott, J. and Guzm~in, G., "Detection of Psilocybin in Species of Psilocybe Panaeolus and Psathy-
rella, "Lloydia, Vol. 39, No. 4, July-Aug. 1976, pp. 258-260.
[6] Guzm~tn, G. and Ott, J., "Description and Chemical Analysis of a New Species of Hallucinogenic
Psilocybe from the Pacific Northwest," Mycologia, Vol. 68, No. 6, Nov. 1976, pp. 1261-1267.
[7] Leuny, A. W. and Paul, A. G., "Baeocystin and Norbaeocystin: New Analogs of Psilocybin form
Psilocybe Baeocystis," Journal of Pharmaceutical Sc&nces, Vol. 57, No. 10, Oct. 1968, pp. 1667-
1671.
[8] Horita, A. and Weber, L. J., "Dephophorylation of Psilocybin in the Intact Mouse," Tox&ology
and Applied Pharmacology, Vol. 4, No. 6, Nov. pp. 730-737.
[9] Horita, A. and Weber, L. J., "The Enzymatic Dehposphorylation and Oxidation of Psilocybin and
Psilocin by Mammalian Tissue Homogenates," Biochemical Pharmacology, Vol. 7, No. I, 1961,
pp. 47-54.
[10] Catalfomo, P. and Tyler, V. E., "The Production of Psilocybin in Submerged Culture by Psilocybe
Cubensis, "Lloydia, Vol. 27, No. 1, March, 1964, pp. 53-63.
[11] Clarke, E. G. C., Isolation and Identification of Drugs, Pharmaceutical Press, London, 1974, p.
526.
[12] Agurell, S. and Eilsson, L., "Biosynthesis of Psilocybin Part 11. Incorporation of Labeled Trypta-
mine Derivatives," Acta Chemica Scandinav&a, Vol. 22, No. 4, 1968, pp. 1210-1218.

Address requests for reprints or additional information to

J. F. Casale
North Carolina State Bureau of Investigation
3320 Old Garner Rd.
P.O. Box 29500
Raleigh, NC 27626