A Technique for the Rapid Isolation and Identification of Psilocin from Psilocin/Psilocybin- Containing Mushrooms

TECHNICAL NOTE

Robert Earl Lee, l B.S.

REFERENCE: Lee, R. E.. "A Technique for the Rapid Isolation and Identification of Psiloeln from Psilocln/Psilocybln-Contalning Mushrooms," Journal of Forensic Sciences, JFSCA, Vol. 30, No. 3. July 1985, pp. 931-941.

ABSTRACT: A method has been developed for the rapid isolation and identification of psiloein from psilocin/psilocybin-containing mushrooms. Based on the difference in the solubility properties in butyl chloride of psiloein and other constituents present in psilocin/psilocybin-containing mushrooms, psilocin was easily separated in pure form.

KEYWORDS: toxicology, psilocin, psiloeybin, solubility

The analysis of psilocin/psilocybin-containing mushrooms has been done by thin-layer chromatography (TLC) [1], ultraviolet (UV) spectrophotometry [2], infrared (IR) spectroscopy [3], and, more recently, high performance liquid chromatography (HPLC) [4]. In each case, lengthy procedures are needed to prepare the mushrooms for analysis. When ultraviolet spectrophotometry or infrared spectroscopy are used, thin-layer chromatography is the method relied upon to isolate and obtain the desired component. The technique developed here involves a method which takes advantage of the solubility property difference between psilocin and the other constituents present in psilocin/psilocybin mushrooms in the solvent butyl chloride. Psiolocin and psilocybin in psilocin/psilocybin mushrooms are soluble in methanol and in dilute sulfuric acid and sodium hydroxide solutions. However, only psilocin is soluble in butyl chloride. Because of this psilocin can be extracted from mushrooms, purified by solvent/solvent extraction methods using butyl chloride, and identified using ultraviolet spectrophotometry or infrared spectroscopy or both in less than 1 h. The ease of extraction and purification using this method eliminates the need for any special time-consuming preparation of the mushrooms and allows analysis to proceed rapidly.

Received for publication 28 Sept. 1984; revised manuscript received 17 Nov. 1984; accepted for publication 19 Nov. 1984. t Formerly, criminalist, Ventura Sheriff's Department, Ventura, CA; presently, member of the technical staff, Rockwell International, Anaheim, CA.

931 932 JOURNAL OF FORENSIC SCIENCES

Experimental Procedures

Ultraviolet and Infrared Spectroscopy The UV and IR spectra of the samples were recorded using a Beckman model DB-G grating spectrophotometer and Perkin-Elmer model 1430 ratio recording infrared spectrophotometer with a model 3600 data station.

Reagents Samples of pure psilocybin, psilocin, and N,N-diethyltryptamine fumarate were obtained from Applied Science Laboratories, State College, PA. Pure bufotenine monooxalate was obtained from Sigma Chemical Co. Methanol and butyl chloride were high purity grade solvents. All chemicals were used as received.

Mushroom Extraction A weighed amount of mushroom material, about 2 g, consisting of stool caps and stems were placed into a closed vial with 15 mL of methanol. The material was allowed to soak for t/2 h, after which, the methanol was removed and evaporated to dryness. The residue was taken up in 25 mL of 0.1N sodium hydroxide (NaOH) and extracted with 25 mL of butyl chloride. The butyl chloride was then extracted with 15 mL of 0. I N sulfuric acid (H2SO 4) and the UV spectrum of the resulting aqueous fraction was recorded at both acid and basic pH. After recording the UV spectrum at basic pH, this solution was re-extracted with 25 mL of butyl chloride. This was followed by the separation of the phases and evaporation of the butyl chloride layer to dryness in a mortar for the preparation of a KBr pellet. An IR spectrum was then recorded from the KBr pellet.

Results Figure 1 shows the UV spectrum obtained after the solvent/solvent purification of the mushroom extract which was obtained after only V2 h of contact with the mushrooms. The UV spectra were recorded at both acid and basic pH. For comparison purposes, Fig. 2 shows the UV spectra from a standard of pure psilocin which was also recorded at acid and basic pH. After recording the UV spectra at basic pH from both the mushroom sample and the standard psilocin solution, each material was re-extracted into butyl chloride and prepared for an IR analysis. Figure 3, spectra (a) and (b), shows the spectrum of the purified material from the mushroom and the spectrum of the pure psilocin standard, respectively.

Discussion In the past, attempts to separate psilocin from psilocybin by solvent systems were generally unsatisfactory [4]. The similar structure of psilocin and psilocybin (Fig. 4) suggests that the two materials would be similar in their solubility properties in given solvents. For example, both materials are soluble in methanol, and also in dilute acids and in dilute bases, a result that is reasonable considering the fact that both the structures exhibit similar polarity characteristics and have present in their structures electronegative atoms of oxygen and nitrogen. Also, both substances have replaceable hydrogen atoms in their molecular structures as well as having basic amine groups which can probably be attributed to their solubility in dilute acids and in dilute bases. LEE 9IDENTIFICATION OF PSILOCIN 933

i_0_

0.9

0.8

0.7

0.2

I I 0.1

! %

t 2~ -~o 30O 350 Wavelength (r~n)

FIG. l--Solvent/solvent purification of mushroom extract where solid line is O. 1 N H2SO 4 and broken line is NaOH. 934 JOURNAL OF FORENSIC SCIENCES

_.1,-o

0.9

it I ,.t). { , C-,"/ll '~-<...-<... _2" , \~.,

0.6 I /'1 II ,a I' 0..5 i /li', / I',,! , I //, I /I\/tl IIv o., ttZ/

I).,~

t).l

250I ~o Wdvele2~gth ( r~l~ )

FIG. 2--Pure psilocht 35 #g/mL where solid lbte is O. 1 N H2SO 4 and broken Ihte is NaOH. LEE 9 IDENTIFICATION OF PSILOCIN 935

100 MBX=I88.88

75 ,,A . ~..,~

E so /

SPECTRUM A ]

25 ~ 90 LT'ZC~'Z~O~

Mltl:8.88I T 0 4808 3588 3888 2588 2898 1588 1888 588 WAVENUMBER (Cm"1)

'| I /ir~~ ~.7 /MflX=I88,88 T

J A

BO- i + SPECTRUM B oL | ,IM:8.88 T ~ 4888 3588 3888 2588 2888 1588 1888 588

WAVENUMBER ( Cm"1 )

FIG. 3--(a) Solvent/solvent purific.tion J~'om mushroom-KBr pellet and (b) pure psiloein standard--KBr pellet.

H H I I /N _N/CH~ H3 o.. _ CH 2 1 CH 2 1 N I \ I \ O CH3 OH CH 3 I HO-- p =O I OH

[ [i

FIG. 4--Structure I is psiloeybin and Structure H is psilocin. 936 JOURNAL OF FORENSIC SCIENCES

1.0

0.9

0.8

0.'/

0.2

0.I

I 225 150 30~0 350 Wavelength (nm)

FIG. 5--Pure psilocin 31 tLg/mL ill butyl chloride. LEE . IDENTIFICATION OF PSILOCIN 937

1.0

0.9

0.8

U.7

~,6

0.5

O.4

o.-

9,1

I I I 225 250 300 350 Wavelength ( r~u )

FIG. 6--Solvent/solvent purification of mushroom extract--the butyl chloride phase. 938 JOURNAL OF FORENSIC SCIENCES

1.0 _.

).9

0.8

o_2__ .o .Oh \,"

0.6

.5__.L_ -

0.4 ,\ o.___/_~ I 0.2 LJ

0.1

2J5 2Jo 3~o Wavelength ( ran )

FIG. 7--Pure bufotenine 30 #g/mL where solid line is O. 1 N H2SO 4 and broken li.e is NaOH. LEE . IDENTIFICATION OF PSILOCIN 939

0.9

0.8

0.7 ,,I_.._..._./ I \ o I 14o- P=O I OH 0.6

0.4

U.5

[.2

0.1 I |

2~ 2L Wavelength (nm )

FIG. 8--Pure psilocybbt 30 Bg/mL where solid line is 0.1 N H2SO 4 and broke, li.e is NaOH. 940 JOURNAL OF FORENSIC SCIENCES

.U__

0.9

0.8

0. "/

_ -- -- ~/| --a~l C]~ .z .. N\ o. 6 esl --~

U.4

0.~ ',//A Ii

(~. 2

0.1

Wavelength ( nm )

FIG. 9--Pure N,N-diethyltyptamhle 31 t~g/mL where solid line is O. 1 N H2SO 4 and broke, line is NaOH. LEE 9IDENTIFICATION OF PSILOCIN 941

On further inspection, it appears that there is nothing in both of the structures that would suggest that only psilocin should be soluble in butyl chloride or other such halogenated hydro- carbon solvents and, indeed, be extracted into a butyl chloride solution from an aqueous basic solution. Normally, it would be expected that psilocin should be extracted, if at all, into butyl chloride from a neutral solution. However, on a closer look at the two structures, it can be seen that the ionic properties of psiiocybin is greater than that of psilocin. This is believed to be the reason why psilocin is solubilized in a solvent of low dielectric constant such as butyl chloride or chloroform while psilocybin is not. Figure 5 shows a UV spectrum of the butyl chloride phase after extraction of a basic solution containing mushroom-extracted material. It indicates at this point that there is an apparent absence of the other mushroom constituents and that psilocin is present in relatively pure form. For comparison purposes, Fig. 6 shows a UV spectrum of a pure sample of psiiocin in butyl chloride. Finally, in comparing the UV spectrum of materials having somewhat similar molecular structures to psilocin, such as N,N-diethyltryptamine, psilocybin, and, indeed, psilocin's isomer bufotenine, Figs. 7, 8, and 9 show that the UV spectrum of psilocin can be differentiated from the spectra of these other substances.

References

[1] Hein, R., Genest, K., Hughes, D. W., and Belec, G., "Botanical and Chemical Characterization of a Forensic Mushroom Specimen of the Genus Psilocybe," Forensic Science Society Journal, Vol. 6, 1966, pp. 192-201. [2] Hofmann, A., Frey, A., Ott, H., Petrizolka, Th., and Troxler, F., "Konstitionsaufklarung und Syn- theses yon Psilocybin," Experienta, Vol. IS, 1958, pp. 397-399. [3] Mestey, R. J. and Evans, W. H.. "Infrared Identification of Some Hallucinogenic Derivatives of Tryptamine and Amphetamine," Journal of Pharmacy and Pharmacology, Vol. 22, 1970, pp. 321-332. [4] Thomas, B. M., "Analysis of Psilocybin and Psilocin in Mushrooms Extracts by Reverse-Phase High Performance Liquid Chromatography," Journal of Forensic Sciences, Vol. 25, No. 4, Oct. 1980, pp. 779-785.

Address requests for reprints or additional information to Robert Earl Lee Rockwell International 3370 Miraloma Ave. P.O. Box 4921 Anaheim, CA 92803