BIOCHEMICAL MEDICINE 8, 341-344 ( 1973)

Marihuana Extraction and Purification for

Oral Administration of Known Amounts
of Ag-Tetrahydrocannabinol (THC)

R. STRAIGHT, A. W. WAYNE, E. G. LEWIS,

AND E. C. BECK
Biochemisty and, Neuropsychology Laboratories,
Veterans Administration Hospital and Unitiersity of Utah,
Salt Lake City, Utah 84113

Received September 15, 1972

We have developed a simple method for the extraction, purification,

estimation, and oral administration of as-THC from a standardized batch
of cannabis plant material for physiological and psychological studies
of the effect of marihuana on man and primates. The technique of oral
administration is applicable also to synthetic a”-THC recently made avail-
able by the National Institute of Mental Health.

MATERIALS AND METHODS

Cannabis s&vu, Mexican, female, 1969 crop, U. S. grown, batch, Me

ll( 2-PF-126) was obtained from the Center for Studies of Narcotic and
Drug Abuse, NIMH, PHS, U. S. Department of Health, Education and
Welfare, The material was in the form of dried, chopped stems and
leaves in sealed brown bottles and was stored at -5°C in the dark until
ready for use. The NIMH assay by glc reported 1.5% a”-THC, 0.09%
cannabidiol (CBD), 0.05% cannabinol (CBN) and trace amounts of
A’-THC. Approximately 90%of AS-THC was in the acid form.
Synthetic as-THC for use as a standard was obtained from NIMH,
batch No. ADL-16122-28, C,,H,,,,O, MW i314.5. This material assayed
93% As-THC, 3.4% A”-THC, and 3.4% exocyclic THC.
As is frequently the case ( 1, 2)) most of the A”-THC was in the tetra-
hydrocannabinolic acid (A!‘-THCA) form as determined by tic. We
found that in order to obtain adequate yields of J,“-THC it was necessary
to decarboxylate the THCA. This was done by heating Cannabis material
(2-4). Our conditions were a thin layer (5-6 mm) of Cannabis material
placed in a 200°C oven for 9 min. This converts about 95%of the THCA
341
Copyright @ 1973 by Academic Press, Inc.
All rights of reproduction in any form reserved.
342 STRAJ.GHT ET AL.

to THC. It is important not to overheat the material or the THC will be

converted to CBN. During the heating, the material turned brown and
there was a 10% weight loss presumably from loss of moisture and volatile
oils.
The heated material, ground to a fine powder, was extracted over-
night in a Soxhlet extractor with petroleum ether (37”49”C). This
extract was concentrated to a tar on a flash exaporator at 40°C. The
tar was dissolved in ethanol and an equal volume of water was added.
This solution was extracted three times with hexane. The hexane extracts
were pooled and then evaporated on a flash evaporator at 40°C. The
residue obtained represents from 6 to 10% weight of the original material.
The residue was chromatographed on a Sephadex LH 20 column
(2.5 X 100 cm) and eluted by upward flow with 5% ethyl acetate in
chloroform at 1.3 ml/min. The LH 20 column is best prepared by pour-
ing the gel in a slurry of methanol and reequilibrating with 5% ethyl
acetate-chloroform after the column is assembled. Three to five grams
of residue per 5 ml of solvent were applied to the column. This amount
of material overloads the column but gives good separation of the peak
containing A”-THC. The chromatography was monitored visually by
watching the pigmented compounds separate on the column and by
recording the optical density of the eluate at 280 nm. The identity of
A”-THC in peak 4 was confirmed by TLC on Eastman 6060 silica gel,
developed with hexane : acetone, 3: 1. A”-Tetrahydrocannibinol and other
cannabinoids were detected by spraying the chromatogram with 0.1%
Fast Blue B (Napthanil Diazo Blue B) in 70% ethanol.
The A”-THC fraction was concentrated and rechromatographed on the
same column. The purified A”-THC fraction gave a single spot by two
tic systems, NJ-dimethylformamide, cyclohexane (5)) and hexane :
acetone (3:l) (6) on Eastman 6060 silica gel tic plates. The purified
A”-THC fraction was concentrated by flash evaporation at 40°C weighed
for recovery, and diluted to 25 ml with ethanol for preparation of oral
doses.
The amount of A”-THC in an extract was also determined by com-
parison of TLC spot size with synthetic reference A”-THC in the range
of OS-3 pg.
The oral dose prepared in amounts of 10-55 mg by applying measured
volumes of ethanolic solution of A”-THC to white sugar cubes. The cubes
containing AS-THC and the blanks were then colored golden yellow with
an ethanol extract of red food coloring and each cube was flavored
with three drops of oil of peppermint to further disguise the cubes. Each
cube was wrapped in aluminum foil, numbered, and stored in a freezer
until used.
 EXTRACXOh- AKD ORAL USE OF 4’ THC 343

FIG. 1. Elution of various cannabinoids on a Sephadex LH 20 column equilibrated

with chloroform, 5% ethyl acetate. Compounds eluted from column: ( 1) brown pig-
ment; ( 2) dirty red pigment band; (3) CBN-C, (cannabivarin); (4 ) THC-C,
( tetrahydrocannabirol; ( 5 ) CBN-C, ( cannabinol); ( 6) CBD-C, ( cannabidiol ); ( 7 )
CBD-C,, ( cannabidvarin ) ; ( 8) THC-C, ( tetrahydrocannabivarin) ; ( 9 ) THCA and
unknown peak stripped with methanol.

RESULTS AND DISCUSSION

Figure 1 shows the elution pattern of the cannabinoids chromato-

graphed on the Sephadex LH 20 column. Thin-layer chromatography of
the eluted peaks showed compounds that had the characteristic colors
of the three main cannabinoids: THC Red, CBN Violet, and CBD
Orange, but were present in smalIer quantities and had different Rr
values. These spots have been tentatively identified as the propal
homologs of the main cannabinoids (7, 8).
Some THCA (45%) which was not decarboxylated and some other
unidentified compounds are retained on the column, but can be eluted
with methanol.
Increasing the ethyl acetate concentration in the eluent decreases peak
retention times but with a loss of separation. Possibly reduced column
running time could be achieved, with good separation, by running a
gradient. Starting with chloroform until the first pigment band emerges
and then running a gradient increasing to 100% of ethyI acetate.
The total amount of THC recovered from NIMH Cannabis material
was 1.9% by weight, which agrees with their analysis by gIc.

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344 STRAIGHT ET AL.

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