
Journal of Chromatography, 207 (1981) 379-385

Quantitative Analysis of Psilocybin and Psilocin in Psilocybe Baeocytis by
High-Performance Liquid Chromatography and by Thin-Layer Chromatography

TLC:

TLC was carried out on three different types (and sizes) of plates: 5 x 20
cm coated with a 0.25 mm layer of silica gel 60F(256) (Merck, Darmstadt, 
G.F.R.); 10 x 20 cm coated with a 0.25-mm layer of microcrystalline 
cellulose (Q2F, Quantum Industries, Fairfield, NJ, U.S.A.); and 5 x 10 cm 
coated with a 0.25-mm layer of alumina K (Q3F, Quantum Industries). All 
plates were oven-dried and stored in a dessicator (damp plates resulted in
inconsistent results). Psilocybin and psilocin standards were spotted on 
each plate, together with the methanolic extracts used in the HPLC work.

The plates were developed for 15 cm at room temperature in the dark in 
either a covered development tank or a half-gallon covered Mason jar with 
one or more filter-paper wicks to ensure vapor saturation in the tanks. 
Nine solvent systems were used: (1) butanol-acetic acid-water (12:3:5); 
(2) butanol-acetic acid-water (2:1:1); (3) pure methanol; (4) 1.5% conc. 
aqueous ammonia in methanol (1.5 ml of reagent-grade ammonium hydroxide in
98.5 ml of methanol); (5) conc. ammonia-water-n-propanol (12:188:500); 
(6) methanol saturated with conc. aqueous ammonia in chloroform (5:95); 
(7) water-saturated n-butanol; (8) pure n-propanol; and (9) ammonia-water-
n-propanol (10:50:150). Development times ranged from 1.5 h with methanol 
to more than 7 h with aqueous ammonia-n-propanol.

Developed plates were air-dried and then placed under a short-wave UV lamp
(Mineralight, Ultra-Violet Products). Any spots visible to the naked eye 
or under short-wave UV radiation were encircled with a pencil. Ehrlich's 
(or Van Urk's) reagent [10% p-dimethylaminobenzaldehyde (pDAB) in conc. 
hydrochloric acid] was freshly prepared and sprayed in an acetone solution 
(1 part of Ehrlich's reagent to 4 parts of acetone), and color was allowed
to develop overnight by wrapping the plate in the paper towel upon which 
it had been sprayed. On other plates, color was developed by dipping the 
plates ina 20% solution of pure anhydrous toluene-p-sulfonic acid (pTSA) 
in methanol. The plates were then photographed, and the photographs and 
plates were stored for future reference.

Plate Type: 20 x 5 cm silica gel w/0.25-mm layer silica gel
Solvent System: butanol-acetic acid-water (12:3:5)

  Rf           Upon Drying    Short-Wave UV   Ehrlich's reagent 
 0.83           not visible      visible         yellow-brown
 0.76           not visible      visible         strong light purple
 0.61           not visible      visible         very light blue
 0.56           not visible      visible         orange
 0.50           not visible      visible         pink-red becoming grey
 0.48           not visible      visible         yellowish brown (merged with .50)
 0.44            sky-blue        visible         lavender                                      
 0.40           not visible      visible         aquamarine blue
 0.36            sky-blue        visible         darker purple
 0.31           not visible      visible         light blue
 0.28           not visible      visible         brownish orange becoming pink                                     
 0.16            sky-blue        visible         pinkish brown becoming brownish purple       
 0.13           not visible      visible         light brown
 
Rf  0.16: Psilocybin
Rf  0.36: Psilocin

When developed the same way but dipped in 20% pTSA, only psilocybin, 
psilocin and three other spots (Rf 0.13, 0.34 and 0.76) were visible.
Cellulose plates developed in butanol-acetic acid-water (12:3:5) also 
showed ample separation of psilocybin and psilocin, but only resolved four 
of the other spots. Psilocybin had an Rf of 0.48, and psilocin migrated to
Rf  0.78.
Alumina plates developed in butanol-acetic acid-water (12:3:5) did not 
separate psilocybin from psilocin.
Solvent butanol-acetic acid-water (2:1:1), when used with 20 x 5 cm silica
gel plates, also effectively separated psilocybin (Rf 0.21) from psilocin 
(Rf 0.46), as well as yielding several other spots, but gave less effective
separation of other peaks than did butanol-acetic acid-water (12:3:5). 
Similarly, good separation of psilocybin (Rf 0.14) from psilocin (Rf 0.45) 
was obtained with 1.5% ammonia solution in methanol, but only four other 
spots were observed. Conc. ammonia-water-n-propanol (12:188:500) resolved 
nine spots in addition to psilocybin (Rf 0.11) and psilocin (Rf 0.58). 
However, the spots were streaked and were not as cleanly resolved as in 
butanol-acetic acid-water (12:3:5). Ammonia-water-n-propanol (10:50:150) 
resolved eight spots in addition to psilocybin (Rf 0.16) and psilocin 
(Rf 0.82), but also displayed considerable streaking. Methanol, n-propanol,
water-saturated n-butanol and methanol saturated with conc. aqueous 
ammonia in chloroform (5:95) did not adequately resolve psilocybin and 
psilocin.

The only solvent system/plate type even close to what you are using is 
possibly the 1.5% ammonia solution in methanol. My guess is that your 
spot #4 is psilocybin Rf: ~0.2 (using your picture of the developed plate 
and assuming that each hash-mark is .1 RF), spot #3 is psilocin Rf: ~0.5. 
I have no idea what spots 1 & 2 are and the researchers didn't mention what
the other four spots were when 1.5% ammonia solution in methanol was used
as the solvent. Spot #2 Rf: ~0.8,  Spot #1 Rf: ~1.0 (if it moved with the
solvent front)
It also appears that you have some streaking between spot #3 and spot #2. 
It could be possible that the plate needs to be developed for longer than 
12 minutes.

---

> So, who's grading this thing anyway?  .. and how'd I do? ;)


the grader is "the honorable gene poole - entheogenic research scientist"

poole says you got an A.  hee also looked at the reference you posted
earlier, and concluded that sally's #4 was psilocybine and #3 was
psilocine with #1, and #2 remaining unidentified.

the answer to the extra credit portion is - reduce the amount of methanol
to slow the dot run.


> BTW Sally, why are you using ethyl acetate-methanol-ammonia hydroxide for
> the solvent? Do you have a ref for this or did you make this one up off the
> top of your head? Also, I would suggest using a published solvent system
> such as butanol-acetic acid-water (12:3:5) with a 20 x 5 cm silica gel 
> plate like the one in the above experiment... but that's just my opinion.

the 17/2/6drop solvent was recommended by sasha as little as two months
ago.  this for isolating psilocybine/psilocine and mescaline... and a
chloroform/methanol mixture for lsa.  he mentioned it is a good gp indole
seperating solvent for liquid column chromotography (and could be used
for TLC also).

***

based on the pop quiz results, and further data from later experiments by
sally, gene poole would currently recommend...

 - keep the 17/2/6drop solvent for column seperation and TLC work if
   sally is only interested in seperating out psilocybine/psilocine
   because it develops quickly, low solvent cost, and easily resolves
   the two desired compounds. (use ehrlich's reagent in addition too)

 - use the (12:3:5 - butanol-acetic acid-water) solvent for exacting
   laboratory research and analysis of fungal material.

 - dot #1 is probably not a compound from the extraction at all, but
   some artifact of the process (sally has a learning curve to overcome),
   because nothing came out before dot #2 via column chromotograophy.

 - dot #2 was a compound that crystallized into beautiful clear white
   thin needles, but was not entheogenic in quantities up to 100 mg
   (according to sally).


***

the next quiz will cover liquid column chromotography, so start studying!

**********

>  - dot #2 was a compound that crystallized into beautiful clear white
>    thin needles, but was not entheogenic in quantities up to 100 mg
>    (according to sally).


sally continued her experiments, and reported the following :

 - with more extacting TLC techniques, two smaller dots were found
   between the original dot#3 and dot#4.

 - dot#3 (sky blue under u.v. light) is not psilocine, since it
   proved to be inactive in quantities up to 70 mg.


*******

pop quiz - TLC of ps. cubensis.

sally extracts fungal material using methanol, and evaporates off
the methanol.  the resulting goo is dissolved in a solvent mixture
of ethyl acetate/methanol/amonium hydroxide (17ml/2ml/6drops) and
TLCed.  below is what sally's plate looked like :

|
--(*) - dot #1---- <- solvent run after 12 minutes
| (*) - dot #2
|
|
| (*) - dot #3
|
|
| (*) - dot #4
|
---X - sample----- <- baseline

dot #1 moves at the speed of the solvent run, so it is actually
distributed evenly along the entire top edge of the solvent run.
in normal light it is a light yellowish colour parcipitate, just
visible against the white silica background.  under shortwave
light, it appears very dark.

dot#2 is of less quantity than dot #1, is not visible in normal
light, and appears as a fairly dark spot under shortwave light.
it almost looks as if a light blue flourescence is occuring
just below the dark spot when illuminated with shortwave light.
this blueing dissappears within an hour or two.

dot #3 is the smallest dot, much less than the others, is not
visible in normal light, and appears as a light blue flourensing
spot under shortwave light.  the blue disappears within an hour
or two, but a faint dark spot is still visible under shortwave
light for several days.

dot #4 is the largest dot, roughtly the quantitly of dot #1.  on
the dry plate, it appears as a dark blue/green to brown spot
easily visible in normal light.  under shortwave light it appears
as a very dark spot.


quiz question -

sally believe that dot #4 is psilocybine.  is she correct, and what
are dots #1, #2, and #3?


extra credit -

how should sally adjust the solvent mixture so that all four dots
appear below the top edge of the solvent run?

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Extraction and analysis of indole derivatives from fungal biomass. 
                             Journal of Basic Microbiology, 1994, 34(1):17-22. 
                             (UI: 94267730) 

                             Abstract: The occurrence 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 simple one-step 
                             procedure with methanol for the quantitative extraction is still the safest 
                             method to obtain the genuine alkaloids from fungal biomass. 

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This is the procedure Hoffman used. I found it over on hyperreal. I had a dream a while back that this worked real well. 

                             Dry the mushrooms. 
                             - This important step is most likely to cause the greatest 
                             loss of yield depending on how it is done. 
                             Crush or grind the dried carpophores or mycelium to a powder. 

                             Shake and allow to stand (e.g. 30 mins) in chloroform. Use maybe 
                             twice the dry weight in solvents at every step, or enough to 
                             well cover the powder. 

                             Filter and discard the chloroform. 

                             SHake the reidue and allow to stand with acetone. 

                             Filter and discard the acetone. 

                             Shake residue and allow to stand with methanol. 
                             Filter. 
                             Shake residue and allow to stand with methanol. 
                             Filter. 
                             Shake residue and allow to stand with methanol. 
                             Filter. 

                             Discard residue. 
                             Combine methanol extracts. 

                             Evaporate methanol to dryness, preferably in a vacuum, although 
                             low heat will do. 
                             This will yield a crude extract containing the active tryptamines, 
                             suitable for most purposes. 
                             This can be further chromatographed on cellulose etc. to give pure 
                             psilocin and psilocybin. The recommended solvents are 
                             n-Butanol saturated with water, and n-butanol:acetic acid:water 
                             (24:10:10). 

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