Studies on the metabolism of the Δ9-tetrahydrocannabinol precursor Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A) in rat using LC-MS/MS, LC-QTOF MS and GC-MS techniques

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Research ArticleReceived: 27 October 2008 Accepted: 9 July 2009 Published online in Wiley Interscience: 2 September 2009

(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 techniquesJulia Jung,a Markus R. Meyer,b Hans H. Maurer,b Christian Neusuß,c

Wolfgang Weinmanna and Volker Auwartera∗

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 detectedin serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of �9-THCA-A and toexamine particularly whether oral intake of �9-THCA-A leads to in vivo formation of �9-THC in a rat model. After oral applicationof pure �9-THCA-A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identifiedby liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) andhigh 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 identi-fied metabolites show that �9-THCA-A undergoes a hydroxylation in position 11 to 11-hydroxy-�9-tetrahydrocannabinolicacid-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 wereidentified in the rat urine as well. Furthermore, �9-THCA-A undergoes hydroxylation in position 8 to 8-alpha- and 8-beta-hydroxy-�9-tetrahydrocannabinolic acid-A, respectively, (8α-Hydroxy-�9-THCA-A and 8β-Hydroxy-�9-THCA-A, respectively)followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Severalglucuronidation conjugates of these metabolites were identified. In vivo conversion of �9-THCA-A to �9-THC was not observed.Copyright c© 2009 John Wiley & Sons, Ltd.

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 Cannabissativa, more than 60 belonging to the class of cannabinoids.[1,2]

In the plant, cannabinoids are biosynthesized and accumulated ascannabinoid acids and non-enzymatically decarboxylated intotheir neutral forms followed by degradation into secondaryproducts by temperature, auto-oxidation and light.[3 – 6] Decar-boxylation occurs slowly during storage and fermentation, andpromptly during heating or smoking. However, when smoked,�9-tetrahydrocannabinolic acid-A (�9-THCA-A) is only partiallyconverted to �9-THC. Dussy et al. investigated the temperaturedependence of the decarboxylation of �9-THCA-A under variousanalytical and smoking conditions.[7] The highest conversion rateresulting in about 70% �9-THC was observed under optimizedanalytical conditions (temperatures higher than 140 ◦C), whereasin 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 con-sumers using liquid chromatography-tandem mass spectrometry(LC-MS/MS) by our research group.[8] The highest �9-THCA-A

concentration was found, when blood sampling occurred shortlyafter consumption of cannabis resulting in a high molar ratio of�9-THCA-A/�9-THC. The inclusion of �9-THCA-A into our stan-dard procedure for the quantification of �9-THC and its metabo-lites by gas chromatography–mass spectrometry (GC-MS) re-vealed that more than 80% of the �9-THC positive serum samplesof cases from driving under the influence of drugs (DUID) werealso positive for �9-THCA-A.[9] Furthermore, �9-THCA-A was de-

∗ Correspondence to: Volker Auwarter, Institute of Forensic Medicine, ForensicToxicology, University Medical Centre Freiburg, Albertstraße 9, D-79104Freiburg, Germany. E-mail: [email protected]

a Institute of Forensic Medicine, Forensic Toxicology, University Medical CentreFreiburg, D-79104 Freiburg, Germany

b Department of Experimental and Clinical Toxicology, Institute of Experimentaland Clinical Pharmacology and Toxicology, Saarland University, D-66421Homburg (Saar), Germany

c Department of Chemistry, University of Applied Sciences, D-73430 Aalen,Germany

J. Mass. Spectrom. 2009, 44, 1423–1433 Copyright c© 2009 John Wiley & Sons, Ltd.

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

Parent compound/metabolite Transitions

DeclusteringPotential

[eV]

CollisionEnergy

[eV]

Retentiontime[min]

�9-THCA-A 357.2 → 313.2 −30 −35 15.84

357.2 → 245.2 −30 −50

357.2 → 191.1 −30 −50

�9-THCA-A glucuronide (ester) 533.2 → 357.2 −30 −35 14.74

533.2 → 313.2 −30 −50

533.2 → 245.2∗ −30 −50

�9-THCA-A glucuronide (ether) 533.2 → 357.2 −30 −35 12.40

533.2 → 313.2 −30 −50

533.2 → 245.2∗ −30 −50

11-OH-�9-THCA-A 373.2 → 311.2 −30 −35 13.54

373.2 → 268.2 −30 −50

373.2 → 173.1 −30 −50

11-OH-�9-THCA-A glucuronide 549.2 → 373.2 −30 −35 12.63

549.2 → 311.2 −30 −50

549.2 → 268.2∗ −30 −50

8β ,11-Bis-OH-�9-THCA-A 389.2 → 327.2 −30 −35 11.94

389.2 → 309.2 −30 −50

389.2 → 269.2 −30 −50

8β ,11-Bis-OH-�9-THCA-A glucuronide 565.2 → 389.2 −30 −35 11.05

565.2 → 327.2∗ −30 −50

565.2 → 309.2∗ −30 −50

8α,11-Bis-OH-�9-THCA-A 389.2 → 327.2 −30 −35 11.56

389.2 → 309.2 −30 −50

389.2 → 269.2 −30 −50

8α,11-Bis-OH-�9-THCA-A glucuronide 565.2 → 389.2 −30 −35 10.60

565.2 → 327.2 −30 −50

565.2 → 309.2∗ −30 −50

�9-THCA-A-8-one 371.2 → 327.2 −30 −35 13.79

371.2 → 284.2 −30 −35

371.2 → 189.1 −30 −50

�9-THCA-A-8-one glucuronide 547.2 → 371.2 −30 −35 12.79

547.2 → 327.2 −30 −50

547.2 → 284.2 −30 −50

�9-THCA-A-COOH 387.2 → 299.2 −30 −35 12.01

387.2 → 245.2 −30 −50

387.2 → 191.1 −30 −50

�9-THCA-A-COOH glucuronide 563.2 → 387.2 −30 −35 10.83

563.2 → 299.2 −30 −50

563.2 → 245.2 −30 −50

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

tected in oral fluid up to 8 h after marijuana smoking by Mooreet al.[10]

�9-THC is the well-known psychoactive component inCannabis sativa and the most widely used illegal drug. In addition,�9-THC shows a variety of therapeutic properties such as the reliefof nausea caused by cancer chemotherapy, the compensationof anorexia and nausea under HIV therapy and the suppressionof spasticity and neuropathic pain associated with multiplesclerosis.[11 – 16]

In the liver, �9-THC is subject to metabolic degradation.Thus, 11-hydroxy-�9-tetrahydrocannbinol (11-OH-�9-THC) is

formed by the cytochrome P450 isoenzyme CYP2C9.[17 – 21] Inrat liver microsomes, cytochrome P450 isoenzyme CYP2C11is the major enzyme responsible for the 11-hydroxylation inmicrosomes from male rats, whereas CYP2C6 plays an impor-tant role in the 11-hydroxylation in microsomes from femalerats.[22,23] Further oxidation of 11-OH-�9-THC via the intermediatealdehyde 11-oxo-�9-tetrahydrocannbinol to 11-nor-9-carboxy-�9-tetrahydrocannbinol (�9-THC-COOH) is catalyzed by a mi-crosomal oxygenase.[19,24] To a small extent, �9-THC is alsoeliminated as acetalic (ether) glucuronide.[25] After enzymaticglucuronidation of the carboxy function, 11-nor-9-carboxy-

www.interscience.wiley.com/journal/jms Copyright c© 2009 John Wiley & Sons, Ltd. J. Mass. Spectrom. 2009, 44, 1423–1433

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�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, retentiontimes, structures and predominant fragmentation patterns of �9-THCA-A and its metabolites. The numbers of the spectra correspond to those of thestructures shown in Fig. 3.

�9-tetrahydrocannbinol glucuronide (�9-THC-COOH glu-curonide) is excreted in urine.[26] Several minor metabolitescarrying hydroxyl functions at C1′ to C5′ of the side chainhave been reported, which are transformed to the corre-sponding carboxylic acids by beta-oxidation.[21] Furthermore,�9-THC undergoes hydroxylation in position eight to twodiastereomeric 8-hydroxy metabolites followed by dehydra-tion as well as formation of an epoxide at C9–10 fol-lowed by hydrolysis or glutathione conjugation.[20,21] Bothmetabolic steps are catalyzed by CYP3A4.[20] In addition,several combinations of the metabolic steps have beendescribed.[21]

Several models for estimation of the time of last cannabisconsumption and of the mental impact of �9-THC in relation tothe concentrations of �9-THC and one or two of its metaboliteshave been established.[27 – 31] Two mathematic models for theprediction of time of last cannabis use from the analysis of asingle plasma specimen were developed by Huestis et al.[29,30]

Model I is based on the �9-THC concentrations and model II isbased on the ratio of �9-THC-COOH and �9-THC concentrations.Applying these models to clinical studies, they correctly predictedthe time of cannabis use for more than 90% of the specimensevaluated, but tended to underestimate this interval at latertimes. Therefore, today, the combination of model I and II is

recommended, which increases the accuracy of the predictions.Furthermore, in clinical studies the test persons normally arecannabinoid free and cannabis use is defined regarding timeand dose. However, most cannabis consumers suspected forDUID are chronic users and take several doses within a fewhours.

The mental impact of �9-THC is estimated by the cannabisinfluence factor (CIF) introduced by Daldrup et al.[31] The CIFis based on the ratio of the sum of the molar concentra-tions of �9-THC and 11-OH-�9-THC divided by the molarconcentration of �9-THC-COOH. The calculated CIF is lowerin frequent consumers due to accumulation of �9-THC-COOH.Improved prediction and analytical evidence of the time oflast cannabis consumption would be helpful for the interpre-tation of �9-THC concentrations. Possibly, �9-THCA-A and/orits metabolite/s can provide valuable information regardingthe time between last cannabis consumption and blood sam-pling.

The aim of the presented study was to identify the metabo-lites of �9-THCA-A in rat urine, which could be used asmarkers of cannabis consumption and to examine particularlywhether oral intake of �9-THCA-A leads to in vivo formation of�9-THC.

J. Mass. Spectrom. 2009, 44, 1423–1433 Copyright c© 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jms

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Figure 1b. (Continued).

Experimental

Chemicals and materials

�9-THCA-A was isolated from Marijuana by an in-houseprocedure with a purity of more than 95%.[32] The methano-lic solutions (100 µg/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 purchasedfrom Promochem (Wesel, Germany). N-methyl-N-(trimethylsilyl)-trifluoracetamide (MSTFA) was obtained from Sigma-Aldrich(Steinheim, Germany). All other chemicals and biochemicals

were of the highest analytical grade and were provided byMerck (Darmstadt, Germany). Deionized water was prepared on acartridge deionizer from Memtech (Moorenweis, Germany). Solidphase 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 Wistarrats (Charles River, Sulzfleck, Germany) for toxicological diagnosticpurposes according to the corresponding German law. Two ratswere administered a single 15 mg/kg body mass (BM) doseof �9-THCA-A (10.1 mg �9-THCA-A/0.5 ml ethanol) by gastric


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Figure 1c. (Continued).

intubation. During the study, the rats were housed in metabolismcages. The urine was separated from the faeces by a glassconstruction designed in-house and collected over a 24 h periodin a fluoridated Erlenmeyer flask (2.5 mg potassium fluoride/1 mlurine). The urine samples were pooled and aliquots of 200 µl werestored at −20 ◦C prior to analysis. Blank rat urine samples hadbeen collected before drug administration to check whether theywere free of interfering compounds.

Sample preparation for identification of metabolites byLC-MS/MS and LC-QTOF MS

A 200 µl 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 µl of a mixture of

β-D-glucuronidase (40 U/ml) and arylsulfatase (20 U/ml) fromHelix pomatia. After addition of 500 µl refrigerated acetonitrile forprotein precipitation and centrifugation at 4000 rpm for 10 min,540 µl of the supernatant was transferred to an autosampler vial.After adding 50 µl internal standard (IS) (50 ng �9-THC-COOH-D3)to the samples, they were evaporated to dryness at 40 ◦C undera gentle stream of nitrogen. For LC-MS/MS analysis, the residuewas reconstituted in 100 µl of the LC mobile phase (solventA : B, 90 : 10, v : v) and 20 µl were injected into the LC-MS/MSsystem.

Another 100 µl aliquot of urine was prepared as describedabove, but without enzymatic cleavage of conjugates prior toprotein precipitation.


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Figure 1d. (Continued).

Figure 2. Reconstructed LC-MRM ion chromatogram of a pooled rat urinesample collected 24 h after oral intake of 15 mg/kg BM �9-THCA-A. Thenumbers correspond to those of the spectra and structures shown in Fig. 1.

Sample preparation for identification of �9-THC and itsmetabolites by GC-MS

After addition of 25 µl IS (5 ng �9-THC-D3, 5 ng 11-OH-�9-THC-D3, 25 ng �9-THC-COOH-D3) to a 1 ml aliquotof urine, the urine was adjusted to pH 5.2 withacetic acid (1 mol/l) and incubated at 50 ◦C for1.5 h with 100 µl of a mixture of β-D-glucuronidase(40 U/ml) and arylsulfatase (20 U/ml) from Helix pomatia, thendiluted with 2 ml 0.1 M acetic acid, shortly mixed and transferredto a SPE column preconditioned with 2 ml of methanol and of0.1 M acetic acid each at a flow rate of 2 ml/min. After sampleloading at a flow rate of 1 ml/min, the SPE columns were washedwith 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 ofnitrogen for 2 min. Elution was performed with 1.5 ml of acetoni-trile. The extracts were evaporated to dryness at 60 ◦C under agentle stream of nitrogen. After addition of 25 µl ethyl acetate and25 µl MSTFA, derivatization was carried out at 90 ◦C for 45 min.1 µl of the derivatized extracts were injected into the GC-MSsystem.

Another aliquot of urine (1 ml) was prepared as describedabove, but without enzymatic cleavage of conjugates, and onewith alkaline hydrolysis for cleavage of conjugates by adding250 µl of 10 M sodium hydroxide to 1 ml of urine and incubationat 60 ◦C for 45 min followed by neutralization with 2 ml of 0.1 Macetic acid.

LC-MS/MS and LC-TOF-MS apparatus for identificationof metabolites

The LC-MS/MS system consisted of a SIL-20AC Prominence au-tosampler, a CTO-20AC Prominence column oven, a CBM-20Acommunications bus module, a DGM-20A3 Prominence degasser,two LC-20AD SP Prominence liquid chromatography pumps(Shimadzu GmbH, Duisburg, Germany) combined with a QTrap3200 linear ion trap triple-quadrupole mass spectrometer (MS)equipped with a TurboIonSpray interface and Analyst softwareversion 1.4.2 (Applied Biosystems/Sciex, Darmstadt, Germany).Separation was performed at 40 ◦C with a Luna phenylhexylcolumn (50 × 2 mm, 5 µm) fitted with a phenyl propyl guardcartridge (4 × 2 mm) (Phenomenex, Torrance, CA, USA) usinggradient elution with 5 mM ammonium acetate pH 6.5 (sol-vent A), and acetonitrile (solvent B) with a total flow rate of0.2 ml/min and the following solvent gradient: 0–3 min, 10%B; 3–20 min linear from 10 to 70% B; 20–21 min linear from70 to 95% B; 21–23 min, 95% B; 23–25 min linear from 95 to10% B; 25–30 min, 10% B. With a switching valve (Rheodyne,Bensheim, Germany), the LC-effluent was admitted to the MSonly between 5 min and 21 min of the chromatographic reten-tion time (RT). Negative electrospray ionization (ESI) was usedand the ion source was operated at 400 ◦C with a needle volt-age of −4500 V and with nitrogen as curtain gas and nebulizergas. For the detection of the deprotonated molecules, a single-quadrupole mass spectrum was acquired in the Q1-scan mode(m/z 50–600 amu) and the enhanced mass spectrometer (EMS)scan mode (m/z 50–600 amu) using three different decluster-ing potentials (−30, −50 and −130 eV). For confirmation of the


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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 thoseof 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 glu-curonide: precursor of m/z 357, scanned from m/z 345 to 745 amu;11-OH-�9-THCA-A glucuronide: precursor of m/z 373, scannedfrom m/z 345 to 745 amu; �9-THCA-A-COOH glucuronide: precur-sor of m/z 387, scanned from m/z 345 to 745 amu; �9-THCA-A-8-one glucuronide: precursor of m/z 371, scanned from m/z 345 to745 amu; 8,11-Bis-OH-�9-THCA-A glucuronide: precursor of m/z389, 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 theQ1-scan mode and in the EMS scan mode as precursor ions. Themultiple reaction monitoring (MRM) method was set up using thetransitions 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 thermostattedAnalytical Sampler, a UltiMate 3000 thermostatted ColumnCompartment, two UltiMate 3000 analytical pumps (Dionex

Corporation, Sunnyvale, USA) combined with a micrOTOFQand Bruker Daltonics DataAnalysis software version 3.4 (BrukerDaltonics, Bremen, Germany). Separation was performed at 40 ◦Cwith a Luna phenylhexyl column (50 × 2 mm, 5 µm) fitted witha phenyl propyl guard cartridge (4 × 2 mm) (Phenomenex,Torrance, CA, USA) using gradient elution with 5 mM ammoniumacetate pH 6.5 (solvent A) and acetonitrile (solvent B) witha total flow rate of 0.2 ml/min and the following solventgradient: 0–3 min, 10% B; 3–20 min linear from 10 to 70%B; 20–21 min linear from 70 to 95% B; 21–23 min, 95% B;23–25 min linear from 95 to 10% B; 25–30 min, 10% B. ThemicrOTOFQ was primarily operated in ‘RF-only’ modus, i.e.MS/MS spectra were not acquired. An electrospray voltage of4500 V was applied at the inlet of the MS (negative mode).The ion transfer was optimized by means of sodium acetatecluster (5 mM NaOH in 0.2% acetic acid in isopropanol : water,1 : 1, v : v).


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B

Figure 3b. (Continued).

GC-MS apparatus for identification of �9-THC and itsmetabolites

The samples were analyzed using a 6890 Series GC sys-tem combined with a 5973 Series mass selective detector, a7683 B Series injector and a Chem Station G1701GA versionD.03.00.611 (Agilent, Waldbronn, Germany). The GC conditionswere as follows: splitless injection mode; column, Optima-5-MS capillary (30 m × 0.25 mm I.D., 0.25 µm film thickness)(Macherey-Nagel, Duren, Germany); injection port temperature,250 ◦C; carrier gas, helium; flow rate, 1.5 ml/min; oven tempera-ture, 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 for3 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-

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,

12–16 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, 16–17 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, 17–18 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 determina-

tion 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, respec-

tively and the limits of quantification were 0.21 ng/ml for �9-THC,

0.28 ng/ml for 11-OH-�9-THC and 5–7 ng/ml for �9-THC-COOH,

respectively.


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Results and Discussion

Identification of the metabolites by LC-MS/MS andLC-QTOF MS

The urinary metabolites of �9-THCA-A were separated by LC andidentified by ESI-MS after protein precipitation with and withoutenzymatic cleavage of conjugates. The analytical strategy foridentification of the metabolites was as follows: For the detectionof the deprotonated metabolites single-quadrupole mass spectrawere acquired in the Q1-scan mode and the EMS mode with thelinear ion trap applying three different declustering potentials(−30, −90, −130 V). The existence of glucuronides was confirmedusing the precursor ion scan mode. EPI spectra were recorded withcollision energies of −20, −35, and −50 eV for each metabolite.Molecular masses of the metabolites were verified by LC-QTOFMS in the MS mode (quadrupole in ‘RF-only’ modus) with a massaccuracy of better than ±10 ppm and the attribution of a correctisotopic pattern (sigma fit).

The postulated structures of the metabolites were deducedfrom the fragments detected in the EPI scan mode, which were in-terpreted in correlation with those of the parent compound (Fig. 1).The mass spectra, the structures and the predominant fragmen-tation patterns of �9-THCA-A and its main metabolites are shownin Fig. 1. The numbers of the respective mass spectra in Fig. 1 aregiven in brackets. In the rat urine samples the following metabolitesof �9-THCA-A (retention time, RT: 15.84 min) (1) could be identi-fied: �9-tetrahydrocannabinolic acid-A glucuronide (�9-THCA-Aglucuronide) (RT: 14.74 min and 12.40 min, respectively) (2),11-hydroxy-�9-tetrahydrocannabinolic acid-A (11-OH-�9-THCA-A) (RT: 13.54 min) (3), 11-hydroxy-�9-tetrahydrocannabinolic acid-A glucuronide (11-OH-�9-THCA-A glucuronide) (RT: 12.63 min)(4), 8β ,11-Bis-hydroxy-�9-THCA-A (8β ,11-Bis-OH-�9-THCA-A)(RT: 11.56 min) (5), 8β ,11-Bis-hydroxy-�9-THCA-A glucuronide(8β ,11-Bis-OH-�9-THCA-A glucuronide) (RT: 10.60 min) (6),8α,11-Bis-hydroxy-�9-THCA-A (8α,11-Bis-OH-�9-THCA-A) (RT:11.94 min) (7), 8α,11-Bis-hydroxy-�9-THCA-A glucuronide(8α,11-Bis-OH-�9-THCA-A glucuronide) (RT: 11.05 min) (8),�9-THCA-A-8-one (RT: 13.79 min) (9), �9-THCA-A-8-one glucuronide (RT: 12.79 min) (10), 11-nor-9-carboxy-�9-tetrahydrocannabinolic acid-A(�9-THCA-A-COOH) (RT: 12.01 min) (11), and11-nor-9-carboxy-�9-tetrahydrocannabinolic acid-A glucuronide(�9-THCA-A-COOH glucuronide) (RT: 10.83 min) (12).

In the following paragraph, possible fragmentation patternsof �9-THCA-A and its postulated metabolites are discussed.�9-THCA-A (1) showed a molecular ion of m/z 357. Decarboxyla-tion of the carboxy group in position two may lead to a fragmention of m/z 313. Neutral loss of C5H8 may lead to a fragment ionof m/z 245 and an additional α-cleavage of the pentyl side chaincould 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. Furtherfragmentation 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 andadditional water elimination in position 11 may lead to a fragmention of m/z 311. The fragment ions of m/z 268 and of m/z 173 couldnot be related to structures. A molecular ion of m/z 549 was shownby 11-OH-�9-THCA-A glucuronide (4). Loss of glucuronic acid(�m = 176) would result in a fragment ion of m/z 373. Further

fragmentation was observed according to that of the aglycon11-OH-�9-THCA-A.

Also 8β ,11-Bis-OH-�9-THCA-A (5) and 8α,11-Bis-OH-�9-THCA-A (7) showed molecular ions of m/z 389. Water elimina-tion could produce a fragment ion of m/z 371 and additionaldecarboxylation of the carboxy group in position two maylead to a fragment ion of m/z 327. Further water eliminationwould result in a fragment ion of m/z 309. The fragment ion ofm/z 312 could not be related to a structure. Molecular ions ofm/z 565 were shown by 8β ,11-Bis-OH-�9-THCA-A glucuronide (6)and 8α,11-Bis-OH-�9-THCA-A glucuronide (8). Loss of glucuronicacid (�m = 176) may lead to the fragment ion of m/z 389. Waterelimination and additional decarboxylation of the carboxy groupin position two would result in a fragment ion of m/z 327.

Also �9-THCA-A-8-one (9) showed molecular ions of m/z371. Decarboxylation of the carboxy group in position twomay lead to a fragment ion of m/z 327. The fragment ionsof 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/z547. Loss of glucuronic acid (�m = 176) would result in a fragmention of m/z 371. Further fragmentation was observed according tothat 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 wouldresult in a fragment ion of m/z 343 and further decarboxylationof the carboxy group in position 11 may lead to a fragment ionof m/z 299. Neutral loss of C4H6 could lead to a fragment ion ofm/z 245 and an additional α-cleavage of the pentyl side chain toa 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. Furtherfragmentation was observed according to that of the aglycon11-nor-9-carboxy-�9-THCA-A.

A MRM method was set up using the transitions obtainedfrom the EPI spectra (Fig. 1). Using the MRM mode, two�9-THCA-A glucuronides were detected (Fig. 2). Mass spectro-metric differentiation was not possible, however differences in theRT strongly suggest the compound eluting at 12.40 min to be theether glucuronide linked at the hydroxy group in position one andthe compound eluting at 14.74 min to be the ester glucuronidelinked at the carboxy group in position two. Furthermore, anothermonohydroxylated �9-THCA-A metabolite was detected in theMRM mode, but the metabolite could not be related to a structure.It can be assumed, that the metabolite is hydroxylated in the sidechain.

The molecular formulas of the metabolites were confirmed byaccurate mass measurement using a high resolution mircrOTOF Qwith a tolerance of ±10 ppm for the major and of ±20 ppm forthe minor metabolites (Table. 2).

Identification of �9-THC and metabolites by GC-MS

For the detection of �9-THC and its metabolites, the raturine samples were analyzed by GC-MS in the SIM mode afteralkaline hydrolysis, enzymatic cleavage of conjugates and withoutcleavage of conjugates, respectively, SPE and trimethylsilylation.

Neither in the native urine sample nor in the urine sampleincubated with β-D-glucuronidase/arylsulfatase, �9-THC orits metabolites 11-OH-�9-THC and �9-THC-COOH could bedetected. Although β-D-glucuronidase/arylsulfatase from Helixpomatia is not as effective as β-D-glucuronidase/arylsulfatase fromE. coli in cleavage of some glucuronides, earlier experiments in our


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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 theconfirmation of the �9-THCA-A metabolites

MetaboliteCalculated mass

[M-H]− (amu) Suggested formulaMeasured mass[M-H]− (amu) Error (ppm) Sigma fit

�9-THCA-A 357.2071 C22H29O4 357.2089 −4.9 0.0214

�9-THCA-A glucuronide 533.2390 C28H37O10 − − −11-OH-�9-THCA-A 373.2020 C22H29O5 373.2016 1.1 0.0096

11-OH-�9-THCA-A glucuronide 549.2341 C28H37O11 549.2346 −0.9 0.0232

8β ,11-Bis-OH-�9-THCA 389.1970 C22H29O6 389.1959 2.6 0.0224

8β ,11-Bis-OH-�9-THCA glucuronide 565.2284 C28H37O12 +a +a +a

8α,11-Bis-OH-�9-THCA 389.1970 C22H29O6 389.2005b −9.8b −8α,11-Bis-OH-�9-THCA glucuronide 565.2284 C28H37O12 +a +a +a

�9-THCA-A-8-one 371.1864 C22H27O5 371.1864 0.1 0.0433

�9-THCA-A-8-one glucuronide 547.2178 C28H35O11 +a +a +a

�9-THCA-A-COOH 387.1813 C22H27O6 387.1815 −0.4 −�9-THCA-A-COOH glucuronide 563.2134 C28H35O12 563.2119 2.7 0.139c

a Signal with low abundance, not sufficient for unequivocal identification.b Partly resolved interference observed.c Overlapping isotopic pattern.

research group showed that glucuronides of �9-THC and11-OH-�9-THC can be cleaved using β-D-glucuronidase/arylsulfatase from Helix pomatia. Furthermore,�9-THC-COOH, which is freed up from its glucuronide by β-D-glucuronidase/arylsulfatase from Helix pomatia, would present asthe known main metabolite in urine, if �9-THCA-A was convertedto �9-THC. Therefore, in this case it would have been possible toidentify at least �9-THC-COOH after enzymatic cleavage in therat urine, which was not the case and proves that �9-THCA-Aand/or its major oxidative metabolites are not decarboxylatedmetabolically.

However, after alkaline hydrolysis �9-THC as well as11-OH-�9-THC and �9-THC-COOH were identified. Because�9-THCA-A is readily converted into �9-THCat alkaline pH values,[33] it can be con-cluded, that �9-THCA-A and its main metabolites11-OH-�9-THCA-A and �9-THCA-A-COOH were partly de-carboxylated due to the high pH during the hydrolysisstep 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 metabolicpathway of �9-THCA-A, shown in Fig. 3 (A and B), can bepostulated: �9-THCA-A undergoes a single hydroxylation inposition 11 to 11-OH-�9-THCA-A. Via the intermediate alde-hyde 11-oxo-�9-THCA-A, 11-OH-�9-THCA-A is further oxidized to�9-THCA-A-COOH. The parent compound and both main metabo-lites were also excreted as glucuronides, i.e. �9-THCA-A glu-curonide, 11-OH-�9-THCA-A glucuronide and �9-THCA-A-COOHglucuronide.

Additionally, �9-THCA-A undergoes further single hydroxy-lation in position 8 to 8α- or 8β-OH-�9-THCA-A followed bydehydration to �9-THCA-A-8-one. Both metabolites are furtheroxidized in position 11 to their bishydroxylated forms. Also ex-creted as glucuronides are 8α,11-Bis-OH-�9-THCA, 8β ,11-Bis-OH-�9-THCA and �9-THCAA-8-one, respectively, i.e. 8α,11-Bis-OH-�9-THCA glucuronide, 8β ,11-Bis-OH-�9-THCA glucuronide and�9-THCA-A-8-one glucuronide, respectively. Although several of

the metabolites have multiple hydroxyl and carboxyl groups thatcould have multiple glucuronide moieties, di-or tri-glucuronideswere not identified. There was no evidence for the transformationof �9-THCA-A to �9-THC.

Limitations

These results provide information on the qualitative metabolismof �9-THCA-A, but their generalizability might be limited becausethey only reflect the metabolism in two rats at a single pointin time after a single dose of �9-THCA-A. The dosage wasrelatively high, which could have recruited secondary rather thanprimary metabolic pathways. Humans may metabolize�9-THCA-Adifferently; therefore, the comparability of metabolism in humansand in rats has to be checked with regard to the kind of metabolitesformed and the kinetics of their formation.

Conclusions

Twelve metabolites of �9-THCA-A were detected and iden-tified 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 presentas glucuronides. Neither �9-THC nor its metabolites weredetected, which shows, that no in vivo decarboxylation of�9-THCA-A and/or its main oxidative metabolites does occurafter oral application in rat.

The metabolism studies presented here show that the mainmetabolites of �9-THCA-A are formed in close analogy to�9-THC metabolism. It can be assumed that these metabolitescan be detected in serum and urine after cannabis consumption;therefore, kinetic studies of �9-THCA-A and its metabolites maylead to new markers for recent cannabis abuse. In future studies,the duration of detectability of THCA-A and its metabolites aftersingle and multiple consumptions of cannabis, respectively, shouldbe determined and these data should be compared with those forTHC and its metabolites.


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Acknowledgements

The authors thank Gabriela Herzog, Jurgen Kempf, Claudia Steinert,Ariane Wohlfarth, Gabriele Ulrich, Andreas H. Ewald, ChristophSauer and Svenja C. Bunz for their help.

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Documents

Studies on the metabolism of the Δ9-tetrahydrocannabinol precursor Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A) in rat using LC-MS/MS, LC-QTOF MS and GC-MS techniques