G. TRYPTAMINES
Hallucinogenic simple indole alkaloids such as tryptamine and its anlogs were
found in many species of sacred mushrooms in Mexico and elsewhere. Some of
the plants in South America that contain harmala alkaloids also contain tryp-
tamines (see harmala alkaloids). Two active principal ingredients isolated fmm
the sacred mushroom “teonanacatl” (Psilocybe mexicana) from Mexico were
found to be 4-hydroxydimethyltryptamine (psilocin) and its corresponding 4-
phosphate psilocybin (Figure 15). Other Psilocybe species belonging to the
teonanacatl group containing psilocybin, usually together with a small amount of
psilocin, are P. caerulescens var. mazatecorum, P. zapotecorum, P. aztecor-
mum, P. semperviva, P. bonettii and P. candipedes. North American and Euro-
pean mushrooms in which psilocin and psilocybin are found are: P. pelliculosa,
P. cyanescens, P. baeocystis, P. quebecensis, P. stuntzii, and Conocybe
cyanopus in North America; and P. semilanceata, P. coprinifacies, Panaeolus
sphinctrinus, P. foenisecii, and P. subbalteatus in Europe (255). Baeocystin and
norbaeocystin (the N-demethylated derivatives of psilocybin) were first isolated
from Psilocybe baeocystis (256, 257). Subsequently, baeocystin was also iso-
lated from Psilocybe semilanceata (with psilocybin), Paneolus renenosus, P.
subbaltealus, and Copelandia chlorocystis (258,259). h u n g et al. (256) applied
TLC to a separation and identification of these tryptarnine analogs, using
Ehrlich’s reagent (5% p-dimethylaminobenzaldehydein concentrated HC1) as a
spray for color visualization of the alkaloids. This reagent reacts with active
hydrogens present in compounds to give a blue-violet color.
Dragendorff‘s re-
agent, as well as Ehrlich’s reagent, is often used for the,detection of tryptamine
alkaloids not substituted on the C-2 position of the indol ring present in plant
extracts (236).A quantitative analysis of alkaloids in Psilocybe was examined by
PC using n-BuOH saturated with water on Whatman paper and detection with p-
dimethylaminobenzaldehyde in benzene saturated with HC1 gas (Rf : psilocin
0.5, psilocybin 0.1) (260).Psilocybin and psilocin present in submerged cultures
of P . cubensis were examined on PC by a two-dimensional procedure: n-
BuOH/AcOH/H,O (4 : 1 : 5) in the first direction and n-BuOH/lN NH,OH
(5 : 1) in the second direction (261). Psilocybin and psilocin in an extract of P .
cubensis were detected by the combined technique of GC-MS (OV-101 on a Gas
Chrome Q column), after TMS derivatization (262). The retention times for bis
(TMS) psilocin and tris (TMS) psilocybin were 8.45 and 13.10 min, respec-
tively. They were clearly separated from other components in the natural extract.
HPLC (column, Partisil, with MeOH/H,O/lN NH,NO, = 240: 50: 10 and
buffered to pH 9.7 with NH,OH, detection 254 nm) was used for a convenient
and speedy analysis of psilocin, psilocybin, and baeocystin in P . semilanceata
(263). Psilocybin and baeocystin gave very similar retention times under these
conditions. Greater resolution could be obtained by increasing the ammonia
content of the solvent (264). Normal-phase HPLC has disadvantages since the
column is susceptible to contamination by polar materials and solvents contain-
ing water, which shorten column life. Thomson used reversed-phase HPLC for
quantification of these alkaloids of mushrooms in New Zealand (264), which
proved more versatile and less restrictive in terms of requirements for sample
size and solvent purity. Two different extracts were obtained from mushroom
materials dried, ground, and preserved, without drying, in sugar by a roller-
mixed extraction with MeOH for 24 hr. The mushrooms used were botanically
not identified beyond the genus Psilocybe. Psilocybin and psilocin exist as zwit-
tenons. There was no significant change in the retention volume for the former
with either cationic or anionic reagents; ,but a marked increase was seen in that
for the latter. It is suggested that either a complex was formed or that the
complex was not partitioning between the mobile and the C18 column phases.
The retention volumes of psilocybin and psilocin varied with the pH of the
mobile phase, showing that solvent pH and phosphate concentration were less
important and that the presence of phosphate was essential to delay the elution of
psilocin. For analysis of psilocybin in MeOH extracts of dried ground mush-
rooms, fluorescence spectroscopy was investigated as a method for the selective
detection and quantification on an ion-exchange column by HPLC (265).
Psilocybin fluoresces strongly at 335 nm, with excitation at 267 nm, whereas
psilocin displays weak fluorescence at 312 nm, with excitation at 260 nm. N,N-
Dimethyltryptamin (DMT) was used as the internal standard. The minimum
detectable quantities of psilocybin and psilocin are 250 pg and 30 ng, respec-
tively, with fluorescence detection at the optimum wavelengths for each compo-
nent, and 7 ng and 150 ng, respectively, by using UV absorption at 267 nm.
Norwegian P. semilanceata was extracted with MeOH containing 10% 1N
NH4N03 to examine HPLC for the quantification of psilocybin (266). Psilocybin
was detected, in addition to, three unknown compounds by UV absorption at 254
nm and fluorescence at 335 nm (excitation at 267 nm). The results obtained by
both UV absorption and fluorescence detection of psilocybin in dried mushrooms
correlated well. C18 columns and the more polar Spherisorb Phenyl columns
were used for separation of the components, but the very polar and soluble
psilocybin did not seem to be sufficiently retarded.
Abuse of mushrooms has increased in Western countries. There have been two
reports of psilocybin and Psylocibe cubensis found in chocolate cookies (267,
26
. One report identified psilocybin in chocolate by TLC, HPLC, and GC
(TMS derivatized). The other report isolated fruit bodies of P . cubensis appear-
ing on chocolate grain cookies after 30 days cultivation.
Tryptamine derivatives are also found in species of South American halluci-
nogenic plants in which harmala alkaloids were found, for example, Banisteriop-
sis rusbyana, Virola spp. and Anadenanthera spp. (see harmala alkaloids). N,N-
dimethyltryptamine (DMT), N-monomethyltryptamine (MMT), 5-methoxy-
DMT (5-MeO-DMT), and 5-hydroxy DMT (bufotenine) were found in B . rus-
byana leaves by GC and GC-MS. Contents of DMT h e r e 98% of the total
amounts of the alkaloids (233). The only plant species of Banisteriopsis where
tryptamines were detected is B . rusbyana. Agurell et al. (235) detected DMT,
MMT, 5-MeO-DMT, and 5-methoxy MMT (5-MeO-MMT) in various Virola
species by GC and GC-MS. Corothie and Nakano detected DMT in Virola
sebifera on TLC using benzene/CHCl,/Et,NH (25 :23 :2) (269). From the same
species, the authors have isolated the amides of tryptamine base, N-formyl-N-
methyl-tryptamine and N-acetyl-N-methyl tryptamine (236). Each of them was
detected as two-spot and two-peak rotamers on TLC and HPLC, respectively. No
attempt was made to isolate them preparatively by HPLC because their rate of
isomerization was expected to be too fast to allow isolation at room temperature.
Twenty tryptamine derivatives were separated by GC (two columns, F60-2 and
NGS) (270). Bufotenine, DMT, and 5-MeO-DMT were determined from the
South American snuff, epeni (V. Aublet), by a comparison of their retention
times. Agurell et al. (235) also examined Anadenanthera peregrina (Legumino-
sae) (barks) to find DMT, MMT, 5-MeO-DMT, and 5-MeO-MMT. Legler and
Tschesche isolated MMT, 5-MeO-MMT, and 5-MeO-DMT by using TLC (silica
gel G, 5% MeOH saturated with NH, in CHCl,, or AcOEt/ MeOH/ 25%
NH40H, 88 : 10 : 2 using p-dimethylaminobenzaldehydefor visualization (271).
Four indole bases were found to occur in the seeds and pods of A . peregrina by
PC by using four different solvent systems (272). Our study on alkaloids in
Anadenanthera (Piptadenia) spp showed four unknown compounds which re-
acted positively with Dragendorff's reagent in addition to bufotenine and DMT,
on paper electrophoresis (Table V) (273).
In searching for a biosynthetic scheme for bufotenine in possible precursors
labeled with 14C or 3H were incubated with tissues of A. peregrina. Products
were detected by Rf values on PC, ionic mobilities on high-voltage elec-
trophoresis, and color reaction with Ehrlich’s reagent (Table I), K2S04, and
Pauly’s reagent (274). Benington et al. (275) showed the presence of trace levels
of N- and/or 0-methylated derivatives of tryptamine and 5hydroxytryptamine
(serotonin) in the cerebrospinal fluid of schizophrenic patients. Indole al-
kylamines were converted to HFB derivatives by acyl transfer using N-hep-
taflurobutyrylimidazole (HFBI) as a derivatizing reagent for detection on GC.
Metabolism of DMT in man was evaluated to explain its psychotic effect
(276). The urine samples collected before and after injections were analyzed for
indolic compounds by PC and colorimetry. The extracts were analyzed by two-
dimensional PC (ascending) with n-BuOH saturated with 10% NH40H in the
fist, and n-BuOHIAcOHIH,O (4 : 1 : 5) in the second, dimension. Chro-
matograms were sprayed with 2% p-dimethylaminobenzaldehyde in 1.2N HCl.
5-Hydroxyindole acetic acid was found 2-4 times more often in the extracted
urine after the administration of DMT than before. @,a,
P,P-Tetradeutero-DMT
was incubated with rat brain homogenates and the metabolites identified as
indole acetic acid, tryptamine, DMT-N-oxide, MMT, MTHC, and THC by TLC
and GC-MS (Chromasorb W-HP) (277). DMT and its metabolites were convert-
ed to their corresponding HFB derivatives for GC analysis