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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. 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
533.2 357.2
533.2 313.2
533.2 245.2
30
30
30
35
50
50
14.74
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
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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.
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J. Jung et al.
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-carboxy9-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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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
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J. Jung et al.
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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
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
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J. Jung et al.
B
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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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
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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-OH9-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.
References
[1] C. E. Turner, M. A. Elsohly, E. G. Boeren. Constituents of Cannabis
Sativa L. XVII. A review of natural constituents. Journal of Natural
Products 1980, 43, 169.
[2] R. Mechoulam, S. Ben-Shabat. From gan-zi-gun-nu to anandamide
and 2-arachidonoylglycerol: the ongoing story of cannabis. Natural
Product Reports 1999, 16, 131.
[3] T. Yamauchi, Y. Shoyama, H. Aramaki, T. Azuma, I. Nishioka.
Tetrahydrocannabinolic acid, a genuine substance of
tetrahydrocannabinol. Chemical and Pharmaceutical Bulletin 1967,
15, 1075.
[4] Y. Shoyama, I. Yamauchi, V. Nishioka. Cannabis, Cannabigerolic
acid monomethyl ether and cannabinolic acid. Chemical and
Pharmaceutical Bulletin 1970, 18, 1327.
[5] G. S. Lewis, C. E. Turner. Constituents of Cannabis sativa L. XIIIStability of dosage form prepared by impregnating synthetic
()-9-trans-tetrahydrocannabinol on placebo Cannabis plant
material. Journal of Pharmaceutical Sciences 1978, 67, 876.
[6] R. K. Razdan, A. J. Puttick, B. A. Zitko, G. R. Handrick. Hashish
VI: conversion of ()-1(6)-tetrahydrocannabinol to ()1(7)-tetrahydrocannabinol. Stability of ()-1- and ()-1(6)tetrahydrocannabinols. Experientia 1972, 28, 121.
[7] F. E. Dussy,
C. Hamberg,
M. Luginbuhl,
T. Schwerzmann,
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