Jacob Bell
New Member
Confirmation of the Presence of
11-Hydroxy-r 9-Tetrahydrocannabinol
in the Urine of Marijuana Smokers
Edward J. Woodhouse, Ph.D.
The widespread use of marijuana has stimulated interest
in the metabolic fate of its constituents, both physiologically
active and inactive. Previous studies have shown
that the physiologically active constituents, A 9-
tetrahydrocannabinol (A9-THC) and a metabolite, 11-
hydroxy-,A9-tetrahydrocannabinol remain in the plasma of
human subjects for several days and are excreted in the
urine and feces for more than 8 days.1'2 The subjects of
those studies were injected with [14 C] (A9-THC), the metabolism
and disposition of which was followed by
radiochemical methods. This report presents the first confirmatory
evidence for 1 -hydroxy-A9-tetrahydrocannabinol
in the urine of human marijuana smokers.
Four male subjects, ranging in age from 21 to 28
years, who were experienced marijuana smokers, volunteered
for this investigation. The subjects were both responsible
and concerned that the investigation be conducted in a
scientific manner. The majority of the subjects also consumed
alcohol and smoked tobacco. No other drugs were
consumed during this investigation. The subjects were
requested to convene for a 12-hour period during which
they could smoke as much or as little marijuana as they
desired. Seven grades of marijuana were provided, differing
only in THC and cannabinol content, ranging from a reliable
Mexican variety (0.6% THC), through spiked samples
up to 5.0% THC. Spiked samples were prepared by adding
diethyl ether extracts of Mexican marijuana to the original
marijuana. After evaporation of the ether, all marijuana
samples were assayed for THC, cannabinol (CBN) and
cannabidiol (CBD) using thin-layer chromatography3 with
Fast Blue B visualization reagent. The samples all contained
approximately equal quantities of THC and CBN with no
detectable level of CBD. Approximately 20 g of marijuana
were consumed by the subjects in the 12-hour session. The
bulk of the marijuana consumed was the variety lowest in
THC content. No record was kept of how much marijuana
each individual smoked since this was a preliminary experiment
designed just to establish whether or not marijuana
constituents or their metabolites appeared in the urines of
smokers and to establish an analytical method for this purpose.
All urine specimens voided by the four subjects
during the 12-hour period were collected in glass containers
and refrigerated until analyzed. Urine specimens were also
collected from three control subjects, none of whom had
ever smoked marijuana. The control subjects did not consume
any drugs, other than alcohol and tobacco during the
12-hour period of this investigation.
In preparation for the confirmation of the presence
of cannabinoid metabolites in the urine, an in vitro preparation4
of the major metabolites ofA9-tetrahydrocannabinol
and cannabinol was undertaken to provide reference standards
of pure metabolites. The metabolites, 11 -hydroxy-A9-
tetrahydrocannabinol (1 I-OH-A9-THC) and 1 1-hydroxycannabinol
( 1-OH-CBN), as shown in Figure 1, were
purified by extracting the metabolism media with chloroform,
evaporating to dryness and extracting the dry residue
with diethyl ether. Preparative thin-layer chromatography5
further purified the metabolism mixtures and provided the
isolated metabolites. The identity of these metabolites as the
I 1-hydroxy derivatives was confirmed by mass spectrometry
and by gas chromatography/mass spectrometry6 of
the trimethylsilylether derivatives. The fragmentation patterns
as shown in Table I agree with those of Wall, et al.7
who have also studied the in vitro metabolism of cannabinoids.
Both marijuana smokers' and control subjects' urine
specimens were analyzed for cannabinoid content by a combination
of liquid extraction, thin-layer chromatography,
and mass spectrometry. Urine samples were pooled and half
the pool was extracted at the natural pH with an equal volume
of ethyl acetate. The rest of the pool was hydrolyzed8
to free cannabinoids from glucuronides and then extracted
with an equal volume of ethyl acetate. The extracts were
evaporated to dryness at ambient temperature, and then extracted
with diethyl ether to effect further cleanup. The
ether extracts were evaporated to dryness and redissolved in
0.5 ml methanol. Thin-layer chromatography of the
marijuana smokers' urine residues under the same conditions
as in Note 5 yielded positives at the same Rf value
(0.65) as the I 1-hydroxy-cannabinoid standards only when
the urine had been hydrolyzed. Unhydrolyzed urine and the
control subjects' urine residues showed no positives at the
above Rf value.
The area of the TLC plate at Rf 0.65 was eluted
with hot methanol, filtered and evaporated to dryness. Mass
spectrometry of this residue yielded the fragmentation pattern
shown in Table 2 when introduced to the mass spectrometer
by direct-inlet at 2500 C. This is interpreted as
confirmatory evidence for the presence of I 1-hydroxy-A9-
tetrahydrocannabinol in the urine of marijuana smokers.
Eluates of the areas adjacent to the I I-OH-A9-THC
spot indicated the presence of I I-OH-CBN by the virtue of
the fact that mass spectrometric fragments of mass 326 and
295 were present. These fractions were not clean enough,
however, for any confirmatory evidence.
The conclusion drawn from this investigation is that
I 1-hydroxy-A9-tetrahydrocannabinol is present in the
hydrolyzed urine of marijuana smokers. Other cannabinoids,
especially I 1-hydroxy-cannabinol are indicated.
The results are qualitative as yet, but do indicate that similar
metabolic processes are occurring when marijuana is
smoked as occur when tetrahydrocannabinol is injected. Further
studies with quantitative assays are underway to determine
the sensitivity of these and other techniques in an effort
to understand more of the metabolism and disposition of
marijuana when consumed by inhalation. The present results
indicate a method for detecting smokers of marijuana may
now be feasible, although the detection limits and time
factors involved in the metabolism require further study.
References and Notes
1. Lemberger, L.; Tamarkin, N. R.; Axelrod, J.; and Kopin, I. J. Science,
173: 72, 1971.
2. Lemberger, L.; Silberstein, S. D.; Axelrod, J.; and Kopin, I. J. Science,
170: 1320, 1970.
3. Thin-layer chromatographic conditions were: Silica Gel G on Glass
Plates Developed in Dry Benzene. Location Reagent: 0.5% Fast Blue B in
0.1 N HCI followed by 0.2 N sodium hydroxide.
4.The in vitro preparation was as follows: 50 g of liver from rats pretreated
with phenobarbital prior to sacrifice was homogenized using 500
ml. 0.1 M potassium phosphate buffer containing 0.013 M magnesium
chloride. The ice cold homogenate was centrifuged at 10,OOOG, and to the
supernatant was added 100 mg of A9-THC or CBN plus 0.65 g NADP, 1.4
g G6P, and 100 units of G6P-dehydrogenase. The mixture was incubated
aerobically for 2 hr at 370 C.
5. The TLC conditions were: Silica Gel G on glass (500, ); developing solvents
and R, values; (a) 10% tert-butanol in chloroform, R, for both metabolites,
0.65, and (b) 5% acetone in chloroform, R, for both metabolites,
0.18. Visualization reagent was 0.5% Fast Blue B in 0.1 N HCI followed
by 0.2 N sodium hydroxide. THC metabolite produced a red spot, CBN
metabolite produced a violet spot.
6. Mass spectrometry conducted on an Atlas CH-4 model (Varian) with a 70
ev ion beam. Samples of extract (5p 1) were introduced by direct inlet. Gas
chromatograph was a Varian Model 1200 connected to the mass spectrometer
through a Watson-Biemann helium separator. A 5 ft column, 5
mm ID, of 3% OV1 on Gas Chrom Q was employed at 230' with He flow
of 20 ml/min. Retention times were: 11-OH-THC, 5.2 min; 11-OH-CBN,
6.8 min for the trimethyl silyl derivatives.
7. These findings are in accord with the results of M. E. Wall, et al., at
Research Triangle Institute, North Carolina, who found the same metabolites
in liver homogenates of pure A 9-THC and CBN. These results
were presented by M. E. Wall at the New York Academy of Sciences Conference
on Marijuana, New York, May 20-21, 1971. Details are presented
in Ann. New York Acad. Sciences, 191: 23, 1971. Evidence for in vitro
and in vivo formation of l1-hydroxy-THC has also been presented by the
following workers:
Nilsson, I.M.; Agurell, S.; Nilsson. J.L.G.; Ohlsson, A.; Sandberg, F.;
Wahlqvist, M.: Science, 168; 1228, 1970.
Ben-Zvi, Z.; Mechoulam, R.; and Burstein, S.; J. Amer. Chem. Soc., 92
(11), 3468, 1970.
8. Hydrolysis conditions: 18 hr at 370 C; pH 5.2; 4,000 Fishmann Units of 13-
glucuronidase added per urine specimen.
9. A-9THC is indicated since it is the major form of THC present in
marijuana. A-8-THC, a minor constituent also yields a similar mass spectrum
and also forms an lI-hydroxy metabolite in vitro; see: Folta, R. L.;
Fentiman, A.F., Jr.; Leighty, E.G.; Walter, J.L.; Drewes, H.R.;
Schwartz, W.E.; Page, T.F., Jr.; and Truitt, E.B., Jr., Science, 168: 844
1970.
ACKNOWLEDGMENT
The studies reported were conducted with the support of a contract from the
Insurance Institute for Highway Safety, Watergate 600, Washington, D.C.
Dr. Woodhouse is Senior Chemist, Midwest Research Institute, 425 Volker
Boulevard, Kansas City, Missouri 64110. This paper was submitted for
publication in November, 1971.
Source: Confirmation of the presence of 11-hydroxy- 9-tetrahydrocannabinol in the urine of marijuana smokers
11-Hydroxy-r 9-Tetrahydrocannabinol
in the Urine of Marijuana Smokers
Edward J. Woodhouse, Ph.D.
The widespread use of marijuana has stimulated interest
in the metabolic fate of its constituents, both physiologically
active and inactive. Previous studies have shown
that the physiologically active constituents, A 9-
tetrahydrocannabinol (A9-THC) and a metabolite, 11-
hydroxy-,A9-tetrahydrocannabinol remain in the plasma of
human subjects for several days and are excreted in the
urine and feces for more than 8 days.1'2 The subjects of
those studies were injected with [14 C] (A9-THC), the metabolism
and disposition of which was followed by
radiochemical methods. This report presents the first confirmatory
evidence for 1 -hydroxy-A9-tetrahydrocannabinol
in the urine of human marijuana smokers.
Four male subjects, ranging in age from 21 to 28
years, who were experienced marijuana smokers, volunteered
for this investigation. The subjects were both responsible
and concerned that the investigation be conducted in a
scientific manner. The majority of the subjects also consumed
alcohol and smoked tobacco. No other drugs were
consumed during this investigation. The subjects were
requested to convene for a 12-hour period during which
they could smoke as much or as little marijuana as they
desired. Seven grades of marijuana were provided, differing
only in THC and cannabinol content, ranging from a reliable
Mexican variety (0.6% THC), through spiked samples
up to 5.0% THC. Spiked samples were prepared by adding
diethyl ether extracts of Mexican marijuana to the original
marijuana. After evaporation of the ether, all marijuana
samples were assayed for THC, cannabinol (CBN) and
cannabidiol (CBD) using thin-layer chromatography3 with
Fast Blue B visualization reagent. The samples all contained
approximately equal quantities of THC and CBN with no
detectable level of CBD. Approximately 20 g of marijuana
were consumed by the subjects in the 12-hour session. The
bulk of the marijuana consumed was the variety lowest in
THC content. No record was kept of how much marijuana
each individual smoked since this was a preliminary experiment
designed just to establish whether or not marijuana
constituents or their metabolites appeared in the urines of
smokers and to establish an analytical method for this purpose.
All urine specimens voided by the four subjects
during the 12-hour period were collected in glass containers
and refrigerated until analyzed. Urine specimens were also
collected from three control subjects, none of whom had
ever smoked marijuana. The control subjects did not consume
any drugs, other than alcohol and tobacco during the
12-hour period of this investigation.
In preparation for the confirmation of the presence
of cannabinoid metabolites in the urine, an in vitro preparation4
of the major metabolites ofA9-tetrahydrocannabinol
and cannabinol was undertaken to provide reference standards
of pure metabolites. The metabolites, 11 -hydroxy-A9-
tetrahydrocannabinol (1 I-OH-A9-THC) and 1 1-hydroxycannabinol
( 1-OH-CBN), as shown in Figure 1, were
purified by extracting the metabolism media with chloroform,
evaporating to dryness and extracting the dry residue
with diethyl ether. Preparative thin-layer chromatography5
further purified the metabolism mixtures and provided the
isolated metabolites. The identity of these metabolites as the
I 1-hydroxy derivatives was confirmed by mass spectrometry
and by gas chromatography/mass spectrometry6 of
the trimethylsilylether derivatives. The fragmentation patterns
as shown in Table I agree with those of Wall, et al.7
who have also studied the in vitro metabolism of cannabinoids.
Both marijuana smokers' and control subjects' urine
specimens were analyzed for cannabinoid content by a combination
of liquid extraction, thin-layer chromatography,
and mass spectrometry. Urine samples were pooled and half
the pool was extracted at the natural pH with an equal volume
of ethyl acetate. The rest of the pool was hydrolyzed8
to free cannabinoids from glucuronides and then extracted
with an equal volume of ethyl acetate. The extracts were
evaporated to dryness at ambient temperature, and then extracted
with diethyl ether to effect further cleanup. The
ether extracts were evaporated to dryness and redissolved in
0.5 ml methanol. Thin-layer chromatography of the
marijuana smokers' urine residues under the same conditions
as in Note 5 yielded positives at the same Rf value
(0.65) as the I 1-hydroxy-cannabinoid standards only when
the urine had been hydrolyzed. Unhydrolyzed urine and the
control subjects' urine residues showed no positives at the
above Rf value.
The area of the TLC plate at Rf 0.65 was eluted
with hot methanol, filtered and evaporated to dryness. Mass
spectrometry of this residue yielded the fragmentation pattern
shown in Table 2 when introduced to the mass spectrometer
by direct-inlet at 2500 C. This is interpreted as
confirmatory evidence for the presence of I 1-hydroxy-A9-
tetrahydrocannabinol in the urine of marijuana smokers.
Eluates of the areas adjacent to the I I-OH-A9-THC
spot indicated the presence of I I-OH-CBN by the virtue of
the fact that mass spectrometric fragments of mass 326 and
295 were present. These fractions were not clean enough,
however, for any confirmatory evidence.
The conclusion drawn from this investigation is that
I 1-hydroxy-A9-tetrahydrocannabinol is present in the
hydrolyzed urine of marijuana smokers. Other cannabinoids,
especially I 1-hydroxy-cannabinol are indicated.
The results are qualitative as yet, but do indicate that similar
metabolic processes are occurring when marijuana is
smoked as occur when tetrahydrocannabinol is injected. Further
studies with quantitative assays are underway to determine
the sensitivity of these and other techniques in an effort
to understand more of the metabolism and disposition of
marijuana when consumed by inhalation. The present results
indicate a method for detecting smokers of marijuana may
now be feasible, although the detection limits and time
factors involved in the metabolism require further study.
References and Notes
1. Lemberger, L.; Tamarkin, N. R.; Axelrod, J.; and Kopin, I. J. Science,
173: 72, 1971.
2. Lemberger, L.; Silberstein, S. D.; Axelrod, J.; and Kopin, I. J. Science,
170: 1320, 1970.
3. Thin-layer chromatographic conditions were: Silica Gel G on Glass
Plates Developed in Dry Benzene. Location Reagent: 0.5% Fast Blue B in
0.1 N HCI followed by 0.2 N sodium hydroxide.
4.The in vitro preparation was as follows: 50 g of liver from rats pretreated
with phenobarbital prior to sacrifice was homogenized using 500
ml. 0.1 M potassium phosphate buffer containing 0.013 M magnesium
chloride. The ice cold homogenate was centrifuged at 10,OOOG, and to the
supernatant was added 100 mg of A9-THC or CBN plus 0.65 g NADP, 1.4
g G6P, and 100 units of G6P-dehydrogenase. The mixture was incubated
aerobically for 2 hr at 370 C.
5. The TLC conditions were: Silica Gel G on glass (500, ); developing solvents
and R, values; (a) 10% tert-butanol in chloroform, R, for both metabolites,
0.65, and (b) 5% acetone in chloroform, R, for both metabolites,
0.18. Visualization reagent was 0.5% Fast Blue B in 0.1 N HCI followed
by 0.2 N sodium hydroxide. THC metabolite produced a red spot, CBN
metabolite produced a violet spot.
6. Mass spectrometry conducted on an Atlas CH-4 model (Varian) with a 70
ev ion beam. Samples of extract (5p 1) were introduced by direct inlet. Gas
chromatograph was a Varian Model 1200 connected to the mass spectrometer
through a Watson-Biemann helium separator. A 5 ft column, 5
mm ID, of 3% OV1 on Gas Chrom Q was employed at 230' with He flow
of 20 ml/min. Retention times were: 11-OH-THC, 5.2 min; 11-OH-CBN,
6.8 min for the trimethyl silyl derivatives.
7. These findings are in accord with the results of M. E. Wall, et al., at
Research Triangle Institute, North Carolina, who found the same metabolites
in liver homogenates of pure A 9-THC and CBN. These results
were presented by M. E. Wall at the New York Academy of Sciences Conference
on Marijuana, New York, May 20-21, 1971. Details are presented
in Ann. New York Acad. Sciences, 191: 23, 1971. Evidence for in vitro
and in vivo formation of l1-hydroxy-THC has also been presented by the
following workers:
Nilsson, I.M.; Agurell, S.; Nilsson. J.L.G.; Ohlsson, A.; Sandberg, F.;
Wahlqvist, M.: Science, 168; 1228, 1970.
Ben-Zvi, Z.; Mechoulam, R.; and Burstein, S.; J. Amer. Chem. Soc., 92
(11), 3468, 1970.
8. Hydrolysis conditions: 18 hr at 370 C; pH 5.2; 4,000 Fishmann Units of 13-
glucuronidase added per urine specimen.
9. A-9THC is indicated since it is the major form of THC present in
marijuana. A-8-THC, a minor constituent also yields a similar mass spectrum
and also forms an lI-hydroxy metabolite in vitro; see: Folta, R. L.;
Fentiman, A.F., Jr.; Leighty, E.G.; Walter, J.L.; Drewes, H.R.;
Schwartz, W.E.; Page, T.F., Jr.; and Truitt, E.B., Jr., Science, 168: 844
1970.
ACKNOWLEDGMENT
The studies reported were conducted with the support of a contract from the
Insurance Institute for Highway Safety, Watergate 600, Washington, D.C.
Dr. Woodhouse is Senior Chemist, Midwest Research Institute, 425 Volker
Boulevard, Kansas City, Missouri 64110. This paper was submitted for
publication in November, 1971.
Source: Confirmation of the presence of 11-hydroxy- 9-tetrahydrocannabinol in the urine of marijuana smokers
