Journal of Analytical Toxicology, Vol. 23, May/June 1999
[ Letter to the Editor
Ag-Tetrahydrocannabivarin (Ag-THCV) as a Marker for
the Ingestion of Cannabis versus Marinol |
To the Editor:
A9-THC (Figure 1, 1), the active ingredient in marijuana, has been approved by the FDA as an anti-emetic for cancer patients
receiving chemotherapy and as an appetite stimulant for AIDS patients suffering from the wasting syndrome.
The procedures used in workplace drug testing and criminal justice system drug testing rely on the identification of the
major A9-THCmetabolite (11-nor-A9-THC-9-COOH,Figure 1, 2) in biological fluids, mainly in urine, to identify marijuana
users.
Although the prescription drug Marinol is approved only for the two indications listed previously, off-label prescription
could make it available to others, and the drug could be claimed as the reason for a positive test for marijuana. Therefore,
the question is, could one determine if the Ag-THC metabolite found in the urine of a given individual is the result of
Marinol use only? That is, could one determine if marijuana (or other cannabis-related products) was used, with or without
Marinol? This letter offers a scientific basis to the answer for this the question based on the identification of the metabolite of
the C3 homologue of A9-THC in the urine of cannabis users.
The bulk active material used in the manufacture of Marinol is produced by a synthetic process that leads only to A9-THC
with its C5 side chain. Other side products of the synthetic process such as AS-THC and CBN also contain a C5 side chain.
On the other hand, although A9-THC is the major cannabinoid in drug-type cannabis, other cannabinoids do exist at
different levels (1). One such cannabinoid closely related to A9-THC that is found in most drug-type samples is the C3
homologue of Ag-THCknown as Ag-tetrahydrocannabivarin (A9-THCV,Figure 1, 3_).Being a homologue, this cannabinoid is
expected to behave as Ag-THCin every respect with differences related only to the side chain. Gill (3) described the isolation
and characterization of A9-THCVand reported its mass spectral fragmentation to be parallel to that of A9-THC.The mass
spectrum of Ag-THCVfrom a cannabis sample was compared with that of Ag-THC.In addition, to prove that these
cannabinoid homologues would have similar fragmentation and ion ratios that differ only in masses, the mass spectrum of
the C4 homologue of A9-THC (known also to be component of the cannabis plant [4]) was recorded and compared with the
spectra of the C3 and C5 homologues. Figure 2 shows the mass spectra of the three homologues, which are essentially
identical except for the differences in ion masses.
Now that the similarity in the mass spectral
fragmentation of the parent cannbinoids is established, it
R 1
is believed that A9-THCVwould be metabolized in much
the same way as A9-THC, resulting in 11-nor-A9-THCV-9COOH (Figure 1, _4) that could be detected in the urine of
subjects ingesting cannabis products containing Ag-THCV
OH~R
(5). Again, being a lower homologue, the retention time of
its metabolite derviative would be lower than that of the
C5 homologue, but the ion ratios of the C3 homologue
should be similar to the corresponding ion ratios of the
C5 homologue. For example, ions at m/z 313, 357, and
1 R 1 = CH3; R = C5Hu = Ag-THC
372 are monitored for the methyl derivative of 11-nor-A92 R1 = COOH; R = CsH u = ll-Nor-Ag-THC-9-COOH
THC-9-COOH with ion ratios of m/z 357/313 and 372/313
3_ R] = CH3; R = C3H7 = Ag-THCV
monitored for identification. The corresponding ions for
4 R 1 = COOH; R = C3H/ = ll-Nor-Ag-THCV-9-COOH
the same derivative of the C3 homologue would be at m/z
285, 329, and 344, with the ion ratios 329/285 and 344/285
Figure I. Chemical structures of Ag-THC ~ , 11-nor-Ag-THC-9being in the same range as 357/313 and 372/313,
COOH ~), Ag-THCV (3), and 11-nor-Ag-THCV-9-COOH (4).
respectively.
Figure 3 shows the ion chromatograms from the analysis
222
Reproduction(photocopying)of editorialcontentof this journal is prohibitedwithout publisher'spermission.
Journal of Analytical Toxicology, Vol. 23, May/June 1999
of a urine specimen collected from a cannabis user. Table I shows the ions monitored, the expected ion ratios, and the ion
ratio found. The peak at the shorter retention time relative to 11-nor-Ag-THC-9-COOH(Figure 1, ~) with the ion masses 28
units less than 11-nor-A9-THC-9-COOHwas determined to be that of 11-nor-Ag-THCV-9-COOH(Figure 1, 41).
This approach was used to analyze a urine specimen, the donor of which had indicated on his consent form that he was
using Marinol prescribed to him by his physician for back pain. The specimen showed high levels of both 11-nor-Ag-THC-9COOH and 11-nor-A~-THCV-9-COOH.Upon confronting the individual with the test results, he admitted use of marijuana.
Subsequently, we have synthesized the carboxy metabolite of THCV (Figure 1, 4) and established that the mass
fragmentation of its derivative is identical to that of the C5 homologue as previously anticipated. Figure 4 shows the mass
spectra of the methyl derivative of both metabolites. In addition, incubation of THCVwith human hepatocytes resulted in the
formation of the acid metabolite found to be identical to the synthetic standard.
It is therefore concluded that the detection of the Ag-THCVmetabolite in the urine of an individual would be a positive
indication that marijuana (or a related product) had been ingested sometime (within days) prior to collection of the urine
~r
aO000 *
3~000 ]
2~,~ ~.,)"~ rain,| TM,:V.c.
2
80000 2
30~":,)
2S,',90 ]
60000 -
:O;,,:.) ]
I ~O@O
l 0,000
271
40000 S
1
20000
5,)00 .] ~11'~5 117
oi[
m/z 285
A
'
9 't.~,) . . . .
.~-t3
16]1~74 ,"~r'
"'
:go . . . .
l
0
~6,:," "
92 ~ 0
....
.~,:~,:,'
"
~,~
,j
''l''--
7.I
"
7.2
7.3
7.5
7.6
7.7
7.8
7.0
THC:V.
2iS
90000 1
80000
B
300
70000 1
60~00
40000
7.4
Time(min)
Scan 255 (4.~B6 min)l
SOOOO
, ~ ~ ,
l""l"'l' '7i ~''
;I'''I"'I"
mlz
==
9
,~
I'
rn/z 329
60000
217
50000
.1
1
40000
257
30000
20000
I0000
0
9 't6o . . . .
~o ....
~6o"
" "~o"
"
'''l'''l'''l'''l'''l'''l
"~6o" "
7.1
7.2
7.3
rn/z
sin)l
203
9 "1do . . . .
147
Id@
1~,>"
7.9
m/z 344
20000
I0000
224 I
"
t
"'.'do"
m/z
Figure 2. Mass spectra of THC (A), C-4 THC (B), and C-3 THC (THCV)
(c).
''I '''
30000
I
115
7.8
24d
4000,~)
2000t~J
7.7
C
120t~)O0
600000
7.6
THCV.O
211
286
1000000
'''I'''I'
7.5
Time (min)
Scan 225 (4.370
tSO0000
ts
1400000
7.4
0
"
7.I
7.2
7.3
7.4
7.5
7,6
7,7
7,8
7,9
Time (rain)
Figure 3. GC-MS ion chromalograms for T H C V C O O H methyl derivative
from the urine of a cannabis user.
223
Journal of Analytical Toxicology, Vol. 23, May/June 1999
285
A
1800000"
1600000"
1400000
1200000
1000000
800000
600000
400000
217
260
161~
",9
91
115
~11~
,241
200000
0 .....,. . . . ._i.~..~,_........ C...... .- ,.,-~L,~&-~,-~ ...... L.,.~,.. . . . .
80
120
160
200
240
280
Mass/charge
329
,~.
. . . . . . .
320
360
313
900000
800000
700000
600000
500000
400000.
300000~
200O0O
1000000
B
and that the presence of 11-nor-A9THCV-9-COOH is a marker for such
ingestion with or without Marinol.
This is particularly true because
analysis of Marinol showed no THCV,
and analysis of plasma samples from
subjects ingesting Marinol showed no
A9-THCV-9-COOH. Details of the
studies outlined in this letter will be
the subject of upcoming
communications.
357
I 372
91
119
161
1~o
oo 207
L
245
271
'
'
341
/
/
1
....... L _ b ....
100
200
300
Mass/charge
Figure4. Massspectraof the methyl derivativesof THCVCOOH(A) and THCCOOH (B).
Table I. Ions Monitored and Ion Ratios Found in the Analysis of a Urine
Specimen of a Cannabis User
Compound
11-nor-Ag-THC-9-COOH
Retentiontime I
(min)
9.13
Ions monitored*
313,357,372
Mahmoud A. EISohly 1,~,Shixia Feng 1,
Timothy P. Murphyl, Samir A. Ross2,
Alison Nimrod2, Zlatko Mehmedic2,
and Neil Fortner3
1ElSohly Laboratories, Inc., 5
Industrial Park Drive, Oxford,
Mississippi 38655; 2National Center
for the Development of Natural
Products, University of Mississippi,
University, Mississippi 38677; and
3pharmChem Laboratories, 1505
O'Brien Drive, Menlo Park, California
94025
Ion ratios
expected
found
(357/313)*69.3%
(372/313)* 38.9%
69.8%
39.7%
References
1. R. Brenneisen and M. EISohly. Chromatographic and spectroscopic profiles of
cannabis of different origins: part 1.
11-nor-Ag-THCV-9-COOH
7.208
285,329,344
(329/285)*69.3% 74.0%
J. Forensic Sci. 33:1385-1404 (1988).
(344/285)* 38.9% 44.8%
2. C.E. Turner, K. Hadley, and P.S.Fetterman.
Constituents of Cannabis sativa L., VI:
* Methyl derivative.
* Based onstandard 11-nor-Ag-THC-9-COOH at 50 ng/mL.
propyl homologs in samples of known
* In comparison with the respective ions of the C5 homologue.
geographical origin. J. Pharm. Sci. 62:
1739-1741 (1973).
3. E.W. Gill. Propyl homologue of tetrahydrocannabinol: its isolation from cannabis, properties and synthesis. J. Chem. Soc. (C) 579-582 (1971).
4. D.J. Harvey. Characterization of the butyl homologues of delta-l-tetrahydrocannabinol, cannbinol, and cannabidiol in samples of cannabis
by combined gas chromatography and mass spectrometry. J. Pharm. Pharmacol. 28:280 (1976).
5. R. Foltz. Analysis of urine specimens for cannabinoids to distinguish the ingestion of Marinol | from smoked THC. Proceedings of the California
Association of Toxicologists, November 1996, pp 28-31.
224