Research Article

Received: 27 October 2008

Accepted: 9 July 2009

Published online in Wiley Interscience:

(www.interscience.com) DOI 10.1002/jms.1624

Studies on the metabolism of the

9-tetrahydrocannabinol precursor

9-tetrahydrocannabinolic acid A
(
9-THCA-A) in rat using LC-MS/MS,
LC-QTOF MS and GC-MS techniques
Julia Jung,a Markus R. Meyer,b Hans H. Maurer,b Christian Neusu,
c
a

Wolfgang Weinmanna and Volker Auwarter

In Cannabis sativa,
9-Tetrahydrocannabinolic acid-A (
9-THCA-A) is the non-psychoactive precursor of

9-tetrahydrocannabinol (
9-THC). In fresh plant material, about 90% of the total
9-THC is available as
9-THCA-A.
When heated (smoked or baked),
9-THCA-A is only partially converted to
9-THC and therefore,
9-THCA-A can be detected
in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of
9-THCA-A and to
examine particularly whether oral intake of
9-THCA-A leads to in vivo formation of
9-THC in a rat model. After oral application
of pure
9-THCA-A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified
by liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and
high resolution LC-MS using time of flight-mass spectrometry (TOF-MS) for accurate mass measurement. For detection of

9-THC and its metabolites, urine extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). The identified metabolites show that
9-THCA-A undergoes a hydroxylation in position 11 to 11-hydroxy-
9-tetrahydrocannabinolic
acid-A (11-OH-
9-THCA-A), which is further oxidized via the intermediate aldehyde 11-oxo-
9-THCA-A to 11-nor-9-carboxy
9-tetrahydrocannabinolic acid-A (
9-THCA-A-COOH). Glucuronides of the parent compound and both main metabolites were
identified in the rat urine as well. Furthermore,
9-THCA-A undergoes hydroxylation in position 8 to 8-alpha- and 8-betahydroxy-
9-tetrahydrocannabinolic acid-A, respectively, (8-Hydroxy-
9-THCA-A and 8-Hydroxy-
9-THCA-A, respectively)
followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several
glucuronidation conjugates of these metabolites were identified. In vivo conversion of
9-THCA-A to
9-THC was not observed.
c 2009 John Wiley & Sons, Ltd.
Copyright

Keywords:
9-Tetrahydrocannabinolic acid-A (
9-THCA-A); metabolism; LC-MS/MS; LC-QTOF MS; GC-MS

Introduction
More than 400 compounds have been identified in Cannabis
sativa, more than 60 belonging to the class of cannabinoids.[1,2]
In the plant, cannabinoids are biosynthesized and accumulated as
cannabinoid acids and non-enzymatically decarboxylated into
their neutral forms followed by degradation into secondary
products by temperature, auto-oxidation and light.[3 6] Decarboxylation occurs slowly during storage and fermentation, and
promptly during heating or smoking. However, when smoked,

9-tetrahydrocannabinolic acid-A (
9-THCA-A) is only partially
converted to
9-THC. Dussy et al. investigated the temperature
dependence of the decarboxylation of
9-THCA-A under various
analytical and smoking conditions.[7] The highest conversion rate
resulting in about 70%
9-THC was observed under optimized
analytical conditions (temperatures higher than 140 C), whereas
in a simulated smoking process only about 30% of the spiked

9-THCA-A was recovered as
9-THC.

9-THCA-A was detected in serum and urine of cannabis consumers using liquid chromatography-tandem mass spectrometry
(LC-MS/MS) by our research group.[8] The highest
9-THCA-A

J. Mass. Spectrom. (2009)

concentration was found, when blood sampling occurred shortly

after consumption of cannabis resulting in a high molar ratio of

9-THCA-A/
9-THC. The inclusion of
9-THCA-A into our standard procedure for the quantification of
9-THC and its metabolites by gas chromatographymass spectrometry (GC-MS) revealed that more than 80% of the
9-THC positive serum samples
of cases from driving under the influence of drugs (DUID) were
also positive for
9-THCA-A.[9] Furthermore,
9-THCA-A was de-

Correspondence to: Volker Auwarter, Institute of Forensic Medicine, Forensic

Toxicology, University Medical Centre Freiburg, Albertstrae 9, D-79104
Freiburg, Germany. E-mail: volker.auwaerter@uniklinik-freiburg.de

a Institute of Forensic Medicine, Forensic Toxicology, University Medical Centre

Freiburg, D-79104 Freiburg, Germany
b Department of Experimental and Clinical Toxicology, Institute of Experimental
and Clinical Pharmacology and Toxicology, Saarland University, D-66421
Homburg (Saar), Germany
c Department of Chemistry, University of Applied Sciences, D-73430 Aalen,
Germany

c 2009 John Wiley & Sons, Ltd.

Copyright

J. Jung et al.

Table 1. Transitions obtained from the EPI spectra of pooled rat urine samples, which were used for MRM method set up

Transitions

Declustering
Potential
[eV]

Collision
Energy
[eV]

Retention
time
[min]

9-THCA-A

357.2 313.2
357.2 245.2
357.2 191.1

30
30
30

35
50
50

15.84

9-THCA-A glucuronide (ester)

533.2 357.2
533.2 313.2
533.2 245.2

30
30
30

35
50
50

14.74

9-THCA-A glucuronide (ether)

533.2 357.2
533.2 313.2
533.2 245.2

30
30
30

35
50
50

12.40

11-OH-
9-THCA-A

373.2 311.2
373.2 268.2
373.2 173.1

30
30
30

35
50
50

13.54

11-OH-
9-THCA-A glucuronide

549.2 373.2
549.2 311.2
549.2 268.2

30
30
30

35
50
50

12.63

8,11-Bis-OH-
9-THCA-A

389.2 327.2
389.2 309.2
389.2 269.2

30
30
30

35
50
50

11.94

8,11-Bis-OH-
9-THCA-A glucuronide

565.2 389.2
565.2 327.2
565.2 309.2

30
30
30

35
50
50

11.05

8,11-Bis-OH-
9-THCA-A

389.2 327.2
389.2 309.2
389.2 269.2

30
30
30

35
50
50

11.56

8,11-Bis-OH-
9-THCA-A glucuronide

565.2 389.2
565.2 327.2
565.2 309.2

30
30
30

35
50
50

10.60

9-THCA-A-8-one

371.2 327.2
371.2 284.2
371.2 189.1

30
30
30

35
35
50

13.79

9-THCA-A-8-one glucuronide

547.2 371.2
547.2 327.2
547.2 284.2

30
30
30

35
50
50

12.79

9-THCA-A-COOH

387.2 299.2
387.2 245.2
387.2 191.1

30
30
30

35
50
50

12.01

9-THCA-A-COOH glucuronide

563.2 387.2
563.2 299.2
563.2 245.2

30
30
30

35
50
50

10.83

Parent compound/metabolite

calculated m/z values from known fragmentation patterns of the aglycon

tected in oral fluid up to 8 h after marijuana smoking by Moore

et al.[10]

9-THC is the well-known psychoactive component in
Cannabis sativa and the most widely used illegal drug. In addition,

9-THC shows a variety of therapeutic properties such as the relief
of nausea caused by cancer chemotherapy, the compensation
of anorexia and nausea under HIV therapy and the suppression
of spasticity and neuropathic pain associated with multiple
sclerosis.[11 16]
In the liver,
9-THC is subject to metabolic degradation.
Thus, 11-hydroxy-
9-tetrahydrocannbinol (11-OH-
9-THC) is

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formed by the cytochrome P450 isoenzyme CYP2C9.[17 21] In

rat liver microsomes, cytochrome P450 isoenzyme CYP2C11
is the major enzyme responsible for the 11-hydroxylation in
microsomes from male rats, whereas CYP2C6 plays an important role in the 11-hydroxylation in microsomes from female
rats.[22,23] Further oxidation of 11-OH-
9-THC via the intermediate
aldehyde 11-oxo-
9-tetrahydrocannbinol to 11-nor-9-carboxy
9-tetrahydrocannbinol (
9-THC-COOH) is catalyzed by a microsomal oxygenase.[19,24] To a small extent,
9-THC is also
eliminated as acetalic (ether) glucuronide.[25] After enzymatic
glucuronidation of the carboxy function, 11-nor-9-carboxy-

c 2009 John Wiley & Sons, Ltd.

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J. Mass. Spectrom. (2009)


9-THCA-A metabolism in rats

Figure 1a. Negative LC-ESI-MS/MS EPI spectra (CE 20, 35, 50eV, summed) and reconstructed ion chromatograms (RIC), respectively, retention
times, structures and predominant fragmentation patterns of
9-THCA-A and its metabolites. The numbers of the spectra correspond to those of the
structures shown in Fig. 3.

9-tetrahydrocannbinol glucuronide (
9-THC-COOH glucuronide) is excreted in urine.[26] Several minor metabolites

carrying hydroxyl functions at C1 to C5 of the side chain
have been reported, which are transformed to the corresponding carboxylic acids by beta-oxidation.[21] Furthermore,

9-THC undergoes hydroxylation in position eight to two
diastereomeric 8-hydroxy metabolites followed by dehydration as well as formation of an epoxide at C910 followed by hydrolysis or glutathione conjugation.[20,21] Both
metabolic steps are catalyzed by CYP3A4.[20] In addition,
several combinations of the metabolic steps have been
described.[21]
Several models for estimation of the time of last cannabis
consumption and of the mental impact of
9-THC in relation to
the concentrations of
9-THC and one or two of its metabolites
have been established.[27 31] Two mathematic models for the
prediction of time of last cannabis use from the analysis of a
single plasma specimen were developed by Huestis et al.[29,30]
Model I is based on the
9-THC concentrations and model II is
based on the ratio of
9-THC-COOH and
9-THC concentrations.
Applying these models to clinical studies, they correctly predicted
the time of cannabis use for more than 90% of the specimens
evaluated, but tended to underestimate this interval at later
times. Therefore, today, the combination of model I and II is

J. Mass. Spectrom. (2009)

recommended, which increases the accuracy of the predictions.

Furthermore, in clinical studies the test persons normally are
cannabinoid free and cannabis use is defined regarding time
and dose. However, most cannabis consumers suspected for
DUID are chronic users and take several doses within a few
hours.
The mental impact of
9-THC is estimated by the cannabis
influence factor (CIF) introduced by Daldrup et al.[31] The CIF
is based on the ratio of the sum of the molar concentrations of
9-THC and 11-OH-
9-THC divided by the molar
concentration of
9-THC-COOH. The calculated CIF is lower
in frequent consumers due to accumulation of
9-THC-COOH.
Improved prediction and analytical evidence of the time of
last cannabis consumption would be helpful for the interpretation of
9-THC concentrations. Possibly,
9-THCA-A and/or
its metabolite/s can provide valuable information regarding
the time between last cannabis consumption and blood sampling.
The aim of the presented study was to identify the metabolites of
9-THCA-A in rat urine, which could be used as
markers of cannabis consumption and to examine particularly
whether oral intake of
9-THCA-A leads to in vivo formation of

9-THC.

c 2009 John Wiley & Sons, Ltd.

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J. Jung et al.

Figure 1b. (Continued).

Experimental
Chemicals and materials

9-THCA-A was isolated from Marijuana by an in-house
procedure with a purity of more than 95%.[32] The methanolic solutions (100 g/ml) of ()-
9-tetrahydrocannabinol-D3
(
9-THC-D3 ),
()-11-hydroxy-
9-tetrahydrocannabinol-D3
(11-OH-
9-THC-D3 )
and
()-11-nor-9-carboxy
9-tetrahydrocannabinol-D3 (
9-THC-COOH-D3 ) were purchased
from Promochem (Wesel, Germany). N-methyl-N-(trimethylsilyl)trifluoracetamide (MSTFA) was obtained from Sigma-Aldrich
(Steinheim, Germany). All other chemicals and biochemicals

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were of the highest analytical grade and were provided by

Merck (Darmstadt, Germany). Deionized water was prepared on a
cartridge deionizer from Memtech (Moorenweis, Germany). Solid
phase extraction (SPE) columns (Chromabond C18 , 3 ml, 500 mg)
were supplied by Macherey-Nagel (Duren,
Germany).

Urine samples
Investigations were performed using the urine of male Wistar
rats (Charles River, Sulzfleck, Germany) for toxicological diagnostic
purposes according to the corresponding German law. Two rats
were administered a single 15 mg/kg body mass (BM) dose
of
9-THCA-A (10.1 mg
9-THCA-A/0.5 ml ethanol) by gastric

c 2009 John Wiley & Sons, Ltd.

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J. Mass. Spectrom. (2009)


9-THCA-A metabolism in rats

Figure 1c. (Continued).

intubation. During the study, the rats were housed in metabolism

cages. The urine was separated from the faeces by a glass
construction designed in-house and collected over a 24 h period
in a fluoridated Erlenmeyer flask (2.5 mg potassium fluoride/1 ml
urine). The urine samples were pooled and aliquots of 200 l were
stored at 20 C prior to analysis. Blank rat urine samples had
been collected before drug administration to check whether they
were free of interfering compounds.
Sample preparation for identification of metabolites by
LC-MS/MS and LC-QTOF MS
A 200 l aliquot of urine was adjusted to pH 5.2 with acetic acid
(1 mol/l) and incubated at 50 C for 1.5 h with 20 l of a mixture of

J. Mass. Spectrom. (2009)

-D-glucuronidase (40 U/ml) and arylsulfatase (20 U/ml) from

Helix pomatia. After addition of 500 l refrigerated acetonitrile for
protein precipitation and centrifugation at 4000 rpm for 10 min,
540 l of the supernatant was transferred to an autosampler vial.
After adding 50 l internal standard (IS) (50 ng
9-THC-COOH-D3 )
to the samples, they were evaporated to dryness at 40 C under
a gentle stream of nitrogen. For LC-MS/MS analysis, the residue
was reconstituted in 100 l of the LC mobile phase (solvent
A : B, 90 : 10, v : v) and 20 l were injected into the LC-MS/MS
system.
Another 100 l aliquot of urine was prepared as described
above, but without enzymatic cleavage of conjugates prior to
protein precipitation.

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J. Jung et al.

Figure 1d. (Continued).

Another aliquot of urine (1 ml) was prepared as described

above, but without enzymatic cleavage of conjugates, and one
with alkaline hydrolysis for cleavage of conjugates by adding
250 l of 10 M sodium hydroxide to 1 ml of urine and incubation
at 60 C for 45 min followed by neutralization with 2 ml of 0.1 M
acetic acid.
LC-MS/MS and LC-TOF-MS apparatus for identification
of metabolites

Figure 2. Reconstructed LC-MRM ion chromatogram of a pooled rat urine

sample collected 24 h after oral intake of 15 mg/kg BM
9-THCA-A. The
numbers correspond to those of the spectra and structures shown in Fig. 1.

Sample preparation for identification of
9-THC and its

metabolites by GC-MS
After addition of 25 l IS (5 ng
9-THC-D3 , 5 ng 11-OH
9-THC-D3 , 25 ng
9-THC-COOH-D3 ) to a 1 ml aliquot
of urine, the urine was adjusted to pH 5.2 with
acetic acid (1 mol/l) and incubated at 50 C for
1.5 h with 100 l of a mixture of -D-glucuronidase
(40 U/ml) and arylsulfatase (20 U/ml) from Helix pomatia, then
diluted with 2 ml 0.1 M acetic acid, shortly mixed and transferred
to a SPE column preconditioned with 2 ml of methanol and of
0.1 M acetic acid each at a flow rate of 2 ml/min. After sample
loading at a flow rate of 1 ml/min, the SPE columns were washed
with 1 ml of 0.1 M acetic acid and of acetonitrile/water (70 : 30,
v : v) (flow rate: 1 ml/min) and dried under a gentle stream of
nitrogen for 2 min. Elution was performed with 1.5 ml of acetonitrile. The extracts were evaporated to dryness at 60 C under a
gentle stream of nitrogen. After addition of 25 l ethyl acetate and
25 l MSTFA, derivatization was carried out at 90 C for 45 min.
1 l of the derivatized extracts were injected into the GC-MS
system.

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The LC-MS/MS system consisted of a SIL-20AC Prominence autosampler, a CTO-20AC Prominence column oven, a CBM-20A
communications bus module, a DGM-20A3 Prominence degasser,
two LC-20AD SP Prominence liquid chromatography pumps
(Shimadzu GmbH, Duisburg, Germany) combined with a QTrap
3200 linear ion trap triple-quadrupole mass spectrometer (MS)
equipped with a TurboIonSpray interface and Analyst software
version 1.4.2 (Applied Biosystems/Sciex, Darmstadt, Germany).
Separation was performed at 40 C with a Luna phenylhexyl
column (50 2 mm, 5 m) fitted with a phenyl propyl guard
cartridge (4 2 mm) (Phenomenex, Torrance, CA, USA) using
gradient elution with 5 mM ammonium acetate pH 6.5 (solvent A), and acetonitrile (solvent B) with a total flow rate of
0.2 ml/min and the following solvent gradient: 03 min, 10%
B; 320 min linear from 10 to 70% B; 2021 min linear from
70 to 95% B; 2123 min, 95% B; 2325 min linear from 95 to
10% B; 2530 min, 10% B. With a switching valve (Rheodyne,
Bensheim, Germany), the LC-effluent was admitted to the MS
only between 5 min and 21 min of the chromatographic retention time (RT). Negative electrospray ionization (ESI) was used
and the ion source was operated at 400 C with a needle voltage of 4500 V and with nitrogen as curtain gas and nebulizer
gas. For the detection of the deprotonated molecules, a singlequadrupole mass spectrum was acquired in the Q1-scan mode
(m/z 50600 amu) and the enhanced mass spectrometer (EMS)
scan mode (m/z 50600 amu) using three different declustering potentials (30, 50 and 130 eV). For confirmation of the

c 2009 John Wiley & Sons, Ltd.

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J. Mass. Spectrom. (2009)


9-THCA-A metabolism in rats

A

Figure 3a. Proposed metabolic pathway of
9-THCA-A in rats: phase I metabolism (A) and phase II metabolism (B). The numbers correspond to those
of the spectra, structures and peaks shown in Fig. 1 and 2. The compounds in brackets are assumed intermediates.

glucuronides, precursor ion scan mode was used (
9-THCA-A glucuronide: precursor of m/z 357, scanned from m/z 345 to 745 amu;
11-OH-
9-THCA-A glucuronide: precursor of m/z 373, scanned
from m/z 345 to 745 amu;
9-THCA-A-COOH glucuronide: precursor of m/z 387, scanned from m/z 345 to 745 amu;
9-THCA-A-8one glucuronide: precursor of m/z 371, scanned from m/z 345 to
745 amu; 8,11-Bis-OH-
9-THCA-A glucuronide: precursor of m/z
389, scanned from m/z 345 to 745 amu). Enhanced product ion
(EPI) spectra were recorded at three different collision energies
(20, 35, and 50 eV) using the molecular ions found in the
Q1-scan mode and in the EMS scan mode as precursor ions. The
multiple reaction monitoring (MRM) method was set up using the
transitions obtained from the EPI spectra (Table 1).
For further confirmation the LC-Quadrupole Time-of-Flight MS
(LC-QTOF MS) system consisted of a UltiMate 3000 thermostatted
Analytical Sampler, a UltiMate 3000 thermostatted Column
Compartment, two UltiMate 3000 analytical pumps (Dionex

J. Mass. Spectrom. (2009)

Corporation, Sunnyvale, USA) combined with a micrOTOFQ

and Bruker Daltonics DataAnalysis software version 3.4 (Bruker
Daltonics, Bremen, Germany). Separation was performed at 40 C
with a Luna phenylhexyl column (50 2 mm, 5 m) fitted with
a phenyl propyl guard cartridge (4 2 mm) (Phenomenex,
Torrance, CA, USA) using gradient elution with 5 mM ammonium
acetate pH 6.5 (solvent A) and acetonitrile (solvent B) with
a total flow rate of 0.2 ml/min and the following solvent
gradient: 03 min, 10% B; 320 min linear from 10 to 70%
B; 2021 min linear from 70 to 95% B; 2123 min, 95% B;
2325 min linear from 95 to 10% B; 2530 min, 10% B. The
micrOTOFQ was primarily operated in RF-only modus, i.e.
MS/MS spectra were not acquired. An electrospray voltage of
4500 V was applied at the inlet of the MS (negative mode).
The ion transfer was optimized by means of sodium acetate
cluster (5 mM NaOH in 0.2% acetic acid in isopropanol : water,
1 : 1, v : v).

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J. Jung et al.
B

Figure 3b. (Continued).

GC-MS apparatus for identification of
9-THC and its

metabolites
The samples were analyzed using a 6890 Series GC system combined with a 5973 Series mass selective detector, a
7683 B Series injector and a Chem Station G1701GA version
D.03.00.611 (Agilent, Waldbronn, Germany). The GC conditions
were as follows: splitless injection mode; column, Optima-5MS capillary (30 m 0.25 mm I.D., 0.25 m film thickness)
(Macherey-Nagel, Duren,
Germany); injection port temperature,

250 C; carrier gas, helium; flow rate, 1.5 ml/min; oven temperature, initially 140 C for 2 min, increased to 200 C at 60 C/min,
to 230 C at 2.5 C/min, to 310 C at 60 C/min, 310 C for
3 min.
The MS conditions were as follows: transfer line heater, 280 C;
ion source temperature, 230 C; electron impact ionization (EI)
mode; ionization energy, 70 eV; electron multiplier voltage
(EMV), 400 V. Analysis was performed in selected-ion moni-

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toring (SIM) mode using a fully validated standard procedure

for the detection of the trimethylsilylated (TMS) derivatives
of
9-THC, 11-OH-
9-THC and
9-THC-COOH with the
following program: solvent delay, 12 min; time window I,
1216 min, m/z 306.2, 374.3, 389.3 (target ion, t) for

9-THC-D3 and m/z 303.2, 371.3, 386.3 (t) for
9-THC;
time window II, 1617 min, m/z, 374.3 (t), 462.4, 477.4 for 11-OH
9-THC-D3 and m/z, 371.3 (t), 459.4, 474.4 for 11-OH-
9-THC;
time window III, 1718 min, m/z, 374.3 (t), 476.3, 491.3 for

9-THC-COOH-D3
and
m/z,
371.3
(t),
473.3,
488.3 for
9-THC-COOH. The limits of determination were 0.21 ng/ml for
9-THC, 0.08 ng/ml for
11-OH-
9-THC and 2.0 ng/ml for
9-THC-COOH, respectively and the limits of quantification were 0.21 ng/ml for
9-THC,
0.28 ng/ml for 11-OH-
9-THC and 57 ng/ml for
9-THC-COOH,
respectively.

c 2009 John Wiley & Sons, Ltd.

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J. Mass. Spectrom. (2009)


9-THCA-A metabolism in rats

Results and Discussion

Identification of the metabolites by LC-MS/MS and
LC-QTOF MS
The urinary metabolites of
9-THCA-A were separated by LC and
identified by ESI-MS after protein precipitation with and without
enzymatic cleavage of conjugates. The analytical strategy for
identification of the metabolites was as follows: For the detection
of the deprotonated metabolites single-quadrupole mass spectra
were acquired in the Q1-scan mode and the EMS mode with the
linear ion trap applying three different declustering potentials
(30, 90, 130 V). The existence of glucuronides was confirmed
using the precursor ion scan mode. EPI spectra were recorded with
collision energies of 20, 35, and 50 eV for each metabolite.
Molecular masses of the metabolites were verified by LC-QTOF
MS in the MS mode (quadrupole in RF-only modus) with a mass
accuracy of better than 10 ppm and the attribution of a correct
isotopic pattern (sigma fit).
The postulated structures of the metabolites were deduced
from the fragments detected in the EPI scan mode, which were interpreted in correlation with those of the parent compound (Fig. 1).
The mass spectra, the structures and the predominant fragmentation patterns of
9-THCA-A and its main metabolites are shown
in Fig. 1. The numbers of the respective mass spectra in Fig. 1 are
given in brackets. In the rat urine samples the following metabolites
of
9-THCA-A (retention time, RT: 15.84 min) (1) could be identified:
9-tetrahydrocannabinolic acid-A glucuronide (
9-THCA-A
glucuronide) (RT: 14.74 min and 12.40 min, respectively) (2),
11-hydroxy-
9-tetrahydrocannabinolic acid-A (11-OH-
9-THCAA) (RT: 13.54 min) (3), 11-hydroxy-
9-tetrahydrocannabinolic acidA glucuronide (11-OH-
9-THCA-A glucuronide) (RT: 12.63 min)
(4), 8,11-Bis-hydroxy-
9-THCA-A (8,11-Bis-OH-
9-THCA-A)
(RT: 11.56 min) (5), 8,11-Bis-hydroxy-
9-THCA-A glucuronide
(8,11-Bis-OH-
9-THCA-A glucuronide) (RT: 10.60 min) (6),
8,11-Bis-hydroxy-
9-THCA-A (8,11-Bis-OH-
9-THCA-A) (RT:
11.94 min) (7), 8,11-Bis-hydroxy-
9-THCA-A glucuronide
(8,11-Bis-OH-
9-THCA-A glucuronide) (RT: 11.05 min) (8),

9-THCA-A-8-one
(RT:
13.79 min)
(9),

9-THCAA-8-one
glucuronide
(RT:
12.79 min)
(10),
11nor-9-carboxy-
9-tetrahydrocannabinolic
acid-A
(
9-THCA-A-COOH)
(RT:
12.01 min)
(11),
and
11-nor-9-carboxy-
9-tetrahydrocannabinolic acid-A glucuronide
(
9-THCA-A-COOH glucuronide) (RT: 10.83 min) (12).
In the following paragraph, possible fragmentation patterns
of
9-THCA-A and its postulated metabolites are discussed.

9-THCA-A (1) showed a molecular ion of m/z 357. Decarboxylation of the carboxy group in position two may lead to a fragment
ion of m/z 313. Neutral loss of C5 H8 may lead to a fragment ion
of m/z 245 and an additional -cleavage of the pentyl side chain
could produce a fragment ion of m/z 191.
9-THCA-A glucuronide
(2) showed a molecular ion of m/z 533. Loss of glucuronic acid
(
m = 176) would result in a fragment ion of m/z 357. Further
fragmentation was observed according to that of the aglycon

9-THCA-A.
A molecular ion of m/z 373 was shown by 11-OH-
9-THCA-A
(3). Decarboxylation of the carboxy group in position two and
additional water elimination in position 11 may lead to a fragment
ion of m/z 311. The fragment ions of m/z 268 and of m/z 173 could
not be related to structures. A molecular ion of m/z 549 was shown
by 11-OH-
9-THCA-A glucuronide (4). Loss of glucuronic acid
(
m = 176) would result in a fragment ion of m/z 373. Further

J. Mass. Spectrom. (2009)

fragmentation was observed according to that of the aglycon

11-OH-
9-THCA-A.
Also 8,11-Bis-OH-
9-THCA-A (5) and 8,11-Bis-OH
9-THCA-A (7) showed molecular ions of m/z 389. Water elimination could produce a fragment ion of m/z 371 and additional
decarboxylation of the carboxy group in position two may
lead to a fragment ion of m/z 327. Further water elimination
would result in a fragment ion of m/z 309. The fragment ion of
m/z 312 could not be related to a structure. Molecular ions of
m/z 565 were shown by 8,11-Bis-OH-
9-THCA-A glucuronide (6)
and 8,11-Bis-OH-
9-THCA-A glucuronide (8). Loss of glucuronic
acid (
m = 176) may lead to the fragment ion of m/z 389. Water
elimination and additional decarboxylation of the carboxy group
in position two would result in a fragment ion of m/z 327.
Also
9-THCA-A-8-one (9) showed molecular ions of m/z
371. Decarboxylation of the carboxy group in position two
may lead to a fragment ion of m/z 327. The fragment ions
of m/z 284 and m/z 189 could not be related to structures.

9-THCA-A-8-one glucuronide (10) showed molecular ions of m/z
547. Loss of glucuronic acid (
m = 176) would result in a fragment
ion of m/z 371. Further fragmentation was observed according to
that of the aglycon
9-THCA-A-8-one.
A molecular ion of m/z 387 was shown by
9-THCA-A-COOH
(11). Decarboxylation of the carboxy group in position two would
result in a fragment ion of m/z 343 and further decarboxylation
of the carboxy group in position 11 may lead to a fragment ion
of m/z 299. Neutral loss of C4 H6 could lead to a fragment ion of
m/z 245 and an additional -cleavage of the pentyl side chain to
a fragment ion of m/z 191. The
9-THCA-A-COOH glucuronide
(12) showed a molecular ion of m/z 563. Loss of glucuronic acid
(
m = 176) may lead to the fragment ion of m/z 387. Further
fragmentation was observed according to that of the aglycon
11-nor-9-carboxy-
9-THCA-A.
A MRM method was set up using the transitions obtained
from the EPI spectra (Fig. 1). Using the MRM mode, two

9-THCA-A glucuronides were detected (Fig. 2). Mass spectrometric differentiation was not possible, however differences in the
RT strongly suggest the compound eluting at 12.40 min to be the
ether glucuronide linked at the hydroxy group in position one and
the compound eluting at 14.74 min to be the ester glucuronide
linked at the carboxy group in position two. Furthermore, another
monohydroxylated
9-THCA-A metabolite was detected in the
MRM mode, but the metabolite could not be related to a structure.
It can be assumed, that the metabolite is hydroxylated in the side
chain.
The molecular formulas of the metabolites were confirmed by
accurate mass measurement using a high resolution mircrOTOF Q
with a tolerance of 10 ppm for the major and of 20 ppm for
the minor metabolites (Table. 2).
Identification of
9-THC and metabolites by GC-MS
For the detection of
9-THC and its metabolites, the rat
urine samples were analyzed by GC-MS in the SIM mode after
alkaline hydrolysis, enzymatic cleavage of conjugates and without
cleavage of conjugates, respectively, SPE and trimethylsilylation.
Neither in the native urine sample nor in the urine sample
incubated with -D-glucuronidase/arylsulfatase,
9-THC or
its metabolites 11-OH-
9-THC and
9-THC-COOH could be
detected. Although -D-glucuronidase/arylsulfatase from Helix
pomatia is not as effective as -D-glucuronidase/arylsulfatase from
E. coli in cleavage of some glucuronides, earlier experiments in our

c 2009 John Wiley & Sons, Ltd.

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www.interscience.wiley.com/journal/jms

J. Jung et al.

Table 2. Results of LC-QTOF MS screening of a pooled rat urine sample collected 24 h after oral intake of 15 mg/kg BM
9-THCA-A for the
confirmation of the
9-THCA-A metabolites
Metabolite

Calculated mass
[M-H] (amu)

9-THCA-A

9-THCA-A glucuronide
11-OH-
9-THCA-A
11-OH-
9-THCA-A glucuronide
8,11-Bis-OH-
9-THCA
8,11-Bis-OH-
9-THCA glucuronide
8,11-Bis-OH-
9-THCA
8,11-Bis-OH-
9-THCA glucuronide

9-THCA-A-8-one

9-THCA-A-8-one glucuronide

9-THCA-A-COOH

9-THCA-A-COOH glucuronide

357.2071
533.2390
373.2020
549.2341
389.1970
565.2284
389.1970
565.2284
371.1864
547.2178
387.1813
563.2134

Suggested formula
C22 H29 O4
C28 H37 O10
C22 H29 O5
C28 H37 O11
C22 H29 O6
C28 H37 O12
C22 H29 O6
C28 H37 O12
C22 H27 O5
C28 H35 O11
C22 H27 O6
C28 H35 O12

Measured mass
[M-H] (amu)

Error (ppm)

Sigma fit

357.2089

373.2016
549.2346
389.1959
+a
389.2005b
+a
371.1864
+a
387.1815
563.2119

4.9

1.1
0.9
2.6
+a
9.8b
+a
0.1
+a
0.4
2.7

0.0214

0.0096
0.0232
0.0224
+a

+a
0.0433
+a

0.139c

Signal with low abundance, not sufficient for unequivocal identification.

Partly resolved interference observed.
c Overlapping isotopic pattern.
b

research group showed that glucuronides of
9-THC and

11-OH-
9-THC
can
be
cleaved
using
-Dglucuronidase/arylsulfatase from Helix pomatia. Furthermore,

9-THC-COOH, which is freed up from its glucuronide by -Dglucuronidase/arylsulfatase from Helix pomatia, would present as
the known main metabolite in urine, if
9-THCA-A was converted
to
9-THC. Therefore, in this case it would have been possible to
identify at least
9-THC-COOH after enzymatic cleavage in the
rat urine, which was not the case and proves that
9-THCA-A
and/or its major oxidative metabolites are not decarboxylated
metabolically.
However, after alkaline hydrolysis
9-THC as well as
11-OH-
9-THC and
9-THC-COOH were identified. Because

9-THCA-A
is
readily
converted
into

9-THC
it
can
be
conat
alkaline
pH
values,[33]
cluded, that
9-THCA-A and its main metabolites
11-OH-
9-THCA-A and
9-THCA-A-COOH were partly decarboxylated due to the high pH during the hydrolysis
step resulting in detection of
9-THC, 11-OH-
9-THC and

9-THC-COOH.
Proposed metabolic pathway
On the basis of the identified metabolites, the following metabolic
pathway of
9-THCA-A, shown in Fig. 3 (A and B), can be
postulated:
9-THCA-A undergoes a single hydroxylation in
position 11 to 11-OH-
9-THCA-A. Via the intermediate aldehyde 11-oxo-
9-THCA-A, 11-OH-
9-THCA-A is further oxidized to

9-THCA-A-COOH. The parent compound and both main metabolites were also excreted as glucuronides, i.e.
9-THCA-A glucuronide, 11-OH-
9-THCA-A glucuronide and
9-THCA-A-COOH
glucuronide.
Additionally,
9-THCA-A undergoes further single hydroxylation in position 8 to 8- or 8-OH-
9-THCA-A followed by
dehydration to
9-THCA-A-8-one. Both metabolites are further
oxidized in position 11 to their bishydroxylated forms. Also excreted as glucuronides are 8,11-Bis-OH-
9-THCA, 8,11-Bis-OH
9-THCA and
9-THCAA-8-one, respectively, i.e. 8,11-Bis-OH
9-THCA glucuronide, 8,11-Bis-OH-
9-THCA glucuronide and

9-THCA-A-8-one glucuronide, respectively. Although several of

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the metabolites have multiple hydroxyl and carboxyl groups that

could have multiple glucuronide moieties, di-or tri-glucuronides
were not identified. There was no evidence for the transformation
of
9-THCA-A to
9-THC.
Limitations
These results provide information on the qualitative metabolism
of
9-THCA-A, but their generalizability might be limited because
they only reflect the metabolism in two rats at a single point
in time after a single dose of
9-THCA-A. The dosage was
relatively high, which could have recruited secondary rather than
primary metabolic pathways. Humans may metabolize
9-THCA-A
differently; therefore, the comparability of metabolism in humans
and in rats has to be checked with regard to the kind of metabolites
formed and the kinetics of their formation.

Conclusions
Twelve metabolites of
9-THCA-A were detected and identified in rat urine after a single oral dose. Hydroxylation of

9-THCA-A
in
position
11
to
11-OH
9-THCA-A
and
further
oxidation
to

9-THCA-A-COOH are the main phase I steps. The

9-THCA-A and both main metabolites were also present
as glucuronides. Neither
9-THC nor its metabolites were
detected, which shows, that no in vivo decarboxylation of

9-THCA-A and/or its main oxidative metabolites does occur
after oral application in rat.
The metabolism studies presented here show that the main
metabolites of
9-THCA-A are formed in close analogy to

9-THC metabolism. It can be assumed that these metabolites
can be detected in serum and urine after cannabis consumption;
therefore, kinetic studies of
9-THCA-A and its metabolites may
lead to new markers for recent cannabis abuse. In future studies,
the duration of detectability of THCA-A and its metabolites after
single and multiple consumptions of cannabis, respectively, should
be determined and these data should be compared with those for
THC and its metabolites.

c 2009 John Wiley & Sons, Ltd.

Copyright

J. Mass. Spectrom. (2009)


9-THCA-A metabolism in rats

Acknowledgements
The authors thank Gabriela Herzog, Jurgen
Kempf, Claudia Steinert,

Ariane Wohlfarth, Gabriele Ulrich, Andreas H. Ewald, Christoph

Sauer and Svenja C. Bunz for their help.

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