Forensic Science International 200 (2010) 141–147

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Forensic Science International

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Detection of JWH-018 metabolites in smoking mixture post-administration urine

Tim Sobolevsky *, Ilya Prasolov, Grigory Rodchenkov
Moscow Antidoping Centre, Elizavetinsky per. 10, 105005 Moscow, Russia

A R T I C L E I N F O A B S T R A C T

Article history: Smoking mixtures containing the cannabimimetic indoles may still be available over-the-counter in
Received 25 January 2010 several countries. Due to the high affinity of these compounds to the cannabinoid receptors, their
Received in revised form 30 March 2010 effective dose is lower than that of the marijuana products resulting in a low concentration of the
Accepted 2 April 2010
excreted metabolites accompanied by a higher psychoactive potency. Up to now the in vivo metabolism
Available online 28 April 2010
of the cannabimimetic indoles seems to be insufficiently investigated and no data have been published
on an assay of JWH-018 in urine.
Keywords:
In this publication the urinary metabolites of JWH-018 are reported. Using gas and liquid
JWH-018
Synthetic cannabinoid
chromatography combined with tandem mass spectrometry two main monohydroxylated metabolites
Metabolism were identified in the forensic urine samples. Based on the differences in their electron ionization MS/MS
GC–MS spectra it is supposed that one is formed by hydroxylation of the indole ring whilst the other by
LC–MS hydroxylation of the N-alkyl chain. The main metabolites are almost completely glucuroconjugated,
whereas minor ones (N-despentyl hydroxy-, carboxy-, dihydroxy-, and reduced di- and trihydroxy
metabolites) were also present in the free fraction. The parent compound was not detected in urine.
ß 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction mixtures. This is not surprising as a typical marijuana abuser

Recently, aminoalkylindoles have been found to act as the
agonists of cannabinoid CB1 and CB2 receptors [1,2]. Due to their
high receptor affinity and existing potential to separate ther-
apeutic action from the psychotropic side effects, these com-
pounds are screened as pharmaceutical candidates for the
treatment of various diseases [3–5]. On the other hand, during
the last two years the synthetic cannabinoids, mostly JWH-018,
were identified as the components of smoking mixtures like
‘‘Spice’’ and its clones, openly sold as an incense [6]. They are
unofficially advertised as a semi-legal and more powerful
substitute for the marijuana products. One pack usually contains
3 g of a so-called herbal composition with approximately 0.5–1.5%
of active component impregnated on it [7,8] that corresponds to ca.
30 mg of the active component per pack, although a large variation
is expected.
Following the prohibition of JWH-018 in several countries
during 2009, the smoking mixtures containing this compound
were legal in Russia until the middle of January, 2010. Presently,
Russian drug enforcement bodies are trying to restrict their use
because of increasing number of medical reports concerning
severe complications related to the administration of the smoking
would anticipate a comparable activity of the synthetic cannabi-
noid, and this may lead to the overdose.
Up to now only the in vitro data on the metabolic behavior of
JWH-015 are available [9], whilst a number of publications deal
with the detection and structural elucidation of the synthetic
cannabinoids in herbal preparations [10–12]. To the best of our
knowledge, no reports have been published on the human
metabolism of JWH-018. Therefore, the aim of present study
was to identify the urinary markers of JWH-018 administration
and propose the analytical procedure for their assay.
2. Materials and methods
2.1. Reagents
JWH-018, or naphthalene-1-yl-(1-pentylindole-3-yl)methanone, was purchased
from Tocris Bioscience (Bristol, UK). HPLC solvents (acetonitrile, methanol, and
water) were of gradient grade and were purchased from VWR (Leuven, Belgium).
Diethyl ether was obtained from Medkhimprom (Moscow, Russia). b-Glucuroni-
dase from Escherichia coli K12 (solution in 50% glycerol) was purchased from Roche
Diagnostics (Mannheim, Germany) and used as supplied. N-methyl-N-trimethylsi-
lyltrifluoroacetamide (MSTFA) was obtained from Macherey-Nagel (Duren,
Germany). All other chemicals were obtained from Sigma–Aldrich (St. Louis, MO).
2.2. Urine samples

* Corresponding author. Tel.: +7 916 624 4389/495 258 3749;
fax: +7 499 261 9943.
E-mail address: sobolevsky@dopingtest.ru (T. Sobolevsky).
Urine samples were collected from 3 persons (2 males, 1 female, 22  1 year old)
who were seized by police in a condition of drug intoxication. As reported, the behavioral
effects were similar to those typical of the administration of the marijuana products, such
as the reddening of eyes, tachycardia, anxiety, paranoia and hallucinations accompanied
by a short-term memory defects and the impaired sense of time.

0379-0738/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2010.04.003
142 T. Sobolevsky et al. / Forensic Science International 200 (2010) 141–147

Fig. 1. EI mass spectrum and structure of JWH-018.

These persons confessed that within the last 12 h they have administered one pack
of the smoking mixture ‘‘Tropical Synergy’’. The maximal administered dose was ca. 1 g
per person. They claimed this was their first experience with smoking mixtures. These
persons were requested to provide the urine samples and upon signing the informed
consent the respective samples were collected and delivered to the laboratory. The
remainder of confiscated smoking mixture was also sent to the laboratory. As a blank
urine a certified laboratory negative control urine was used.
2.3. Testing the smoking mixture for identity
To check the composition of the smoking mixture, 5 mg of the raw material were
extracted with 1 ml of methanol in an ultrasonic bath. Following centrifugation,
5 ml of the solution was taken to dryness and treated with MSTFA/NH4I/
dithiotreitol (1000/2/1.5; v/w/w) at 70 8C for 30 min. The reaction mixture was
transferred into a 100-ml vial and 1 ml was injected into GC–MS.
2.4. Preparation of urine samples
To 3 ml of urine was added 1 ml of phosphate buffer (0.8 M, pH 6.3) and 30 ml of
b-glucuronidase. Following brief vortexing the samples were placed in an
incubator where enzymolysis was allowed to proceed at 55 8C for 60 min. After
that 1 ml of carbonate buffer (3 M, pH 10.1) was added and the samples were
extracted with 5 ml of diethyl ether by rigorous vortexing in the presence of
Na2SO4 as a salting out agent. After centrifugation at 3000 rpm for 4 min the
aqueous layer was frozen in a low-temperature bath (30 8C) and the ethereal
extract was poured out into another test tube followed by evaporation at 40 8C
under nitrogen flow. The dry residue was either dissolved in a methanol/water
mixture (60/40; v/v) for the LC–MS analysis or treated with the same MSTFA/NH4I/
dithiotreitol reagent at 70 8C for 30 min. When the urinary free fraction was
analyzed, adding b-glucuronidase and incubation at 55 8C were omitted with all
other steps being the same.

Fig. 2. RIC plotted from GC–MS fullscan data against m/z 636 (A) and m/z 270 for post-administration (B) and laboratory negative control (C) urine (14.31 min:
tetrahydrocortisol, 14.46 min: allo-tetrahydrocortisol, both as TMS artifacts).
 T. Sobolevsky et al. / Forensic Science International 200 (2010) 141–147 143

2.5. GC–MS/MS
The gas chromatographic analyses were carried out on the system comprising a
Trace GC Ultra gas chromatograph (Thermo Scientific, Rodano, Italy) coupled to a
TSQ Quantum GC triple quadrupole mass spectrometer (ThermoFisher Scientific,
San Jose, CA, USA). The separation was achieved on a Thermo TR-5MS column
(15 m  0.25 mm  0.1 mm) applying temperature programming as follows:
150 8C (1 min), heating at 10 8C/min to 300 8C (5 min). One ml injections were
done at 250 8C in the split mode (1:30) with a carrier gas flow rate set to 1.2 ml/min
(helium 99.9999%). Transfer line temperature was 300 8C, the ion source was held at
250 8C. The mass spectrometer was operated in the fullscan, MS/MS and SRM
modes. In the latter two cases, argon (99.998%) was admitted into a Q2 at the
pressure of 1.0  103 Torr and the collision energy (CE) of 10 or 20 eV.
2.6. LC–MS/MS
The HPLC analyses were performed on a TSQ Vantage AM triple quadrupole mass
spectrometer connected to an Accela liquid chromatograph (ThermoFisher
Scientific, San Jose, CA, USA). Thermo Hypersil Gold column (50 mm  2.1 mm,
particle size 1.9 mm) maintained at 40 8C was used for the separation. Injection

Fig. 3. EI mass spectra of the M1 (A), M2 (C) and MS/MS spectra of the M1 (B), M2 (D).
144 T. Sobolevsky et al. / Forensic Science International 200 (2010) 141–147
volume was 5 ml. The mobile phase flow rate was set to 250 ml/min with the
gradient elution starting at 70% of 0.1% formic acid in water and 30% of acetonitrile
followed by a linear increase to 90% of acetonitrile in 7 min, holding 90% of
acetonitrile for 1 min and then re-equilibration until the end of analysis (10 min).
The heated ESI II ion source was used for ionization. Positive and negative ions
were detected in the fullscan, MS/MS (product ion scan, parent ion scan) and SRM
modes. Argon was admitted into a Q2 at the pressure of 1.5  103 Torr resulting in
the Pirani gauge high vacuum reading of 2.0  105 Torr. Sheath gas pressure
(nitrogen from nitrogen generator, 99.9% purity) was set at 35 arbitrary units
corresponding to nitrogen consumption of ca. 10 liters per min. The vaporizer and
capillary temperatures were set at 350 8C with the spray voltage set at +3 kV (or
2.5 kV in negative ionization mode).
3. Results and discussion
3.1. Composition of smoking mixture
Based on the GC–MS analysis of plant extract, the presence of
JWH-018 in the mixture was confirmed by comparing the
retention time and electron ionization (EI) mass spectrum with
that of the reference compound. The extract was also found to
contain a C8 homolog of CP 49 497, another cannabinoid receptor
agonist occurring in herbal mixes [10], and a-tocopherol (data not
shown).
As is seen from Fig. 1, JWH-018 produces the informative mass
spectrum. Interestingly, the molecule eliminates a methyl group
and two protons resulting in the abundant ion at m/z 324 detected
as an atypical loss of 17 amu. The ions at m/z 284 and 270 are
produced by loosing the butyl and pentyl side chains, respectively.
Of structural importance are the ions with m/z 127 and 155 which
correspond to the naphthalenyl and carbonylnaphthalenyl frag-
ments and, as discussed below, are diagnostic for the metabolites
where the naphthalenyl moiety is not modified. The ion with m/z
214 is formed from the molecular ion upon the loss of the
naphthalenyl group.
3.2. Urinary metabolites of JWH-018
It is well-known that hydroxylation is a common way of the
metabolic transformation of xenobiotics as it facilitates their
elimination from an organism by glucuronidation (or sulfation) of
the hydroxylated metabolites. Based on the data reported for a
Fig. 4. SRM chromatograms for urine ca. 12 h after administration of JWH-018.
JWH analog [9] it was natural to anticipate that the in vivo
metabolism of JWH-018 should occur similarly.
3.3. GC–MS/MS
It is worthy to note that the parent compound was not found in
the post-administration urines at the detection limit of ca. 50 pg/ml
(in the SRM mode). The search for the metabolites was first
attempted using GC–MS/MS following trimethylsilylation, and in
this case hydroxylation should add 88 amu (MH+OSiMe3) to the
molecular weight of parent compound. We detected two low
abundance peaks (metabolites M1 and M2, see Fig. 6 for the
proposed structures) with [M]+ = 429 amu eluting at the very end of
the chromatograms after the distinctive doublet corresponding to
the tetrahydrocortisol and allo-tetrahydrocortisol silylation arti-
facts. The reconstructed ion chromatograms (RIC) plotted for the
tetrahydrocortisol doublet (m/z 636) and one of characteristic ions of
the metabolites M1 and M2 (m/z 270) in the post-administration and
laboratory negative control urine are presented in Fig. 2. We found
that these two metabolites are almost completely glucuroconju-
gated, so enzymolysis is a must. Other metabolites listed in Fig. 6
were not detected by GC–MS/MS.
Mass spectra of the metabolites M1 and M2 are given in Fig. 3A
and C, respectively. As is seen from these data, both metabolites
have their naphthalenyl moiety untouched, as the characteristic
ions with m/z 127 and 155 are present in the mass spectra. The loss
of trimethylsilanol (90 amu) resulting in the ion with m/z 339 is
typical when a trimethylsilylated hydroxy group is available to
cleave. The presence of ion with m/z 106 may be indicative of
hydroxylation at the indole phenyl ring.
The MS/MS spectra of the metabolites M1 and M2, both
produced at the collision energy of 10 eV (Fig. 3B and D,
respectively), clearly indicate that the metabolites have different
positions of hydroxyls as fragmentation patterns are different. The
ion with m/z 324 in the spectrum of metabolite M1 seems to be
formed upon the loss of trimethylsilanol followed by the
elimination of methyl group from the alkyl chain. This is favorable
when hydroxyl is located at the indole phenyl ring and not at the N-
alkyl side chain, therefore it is reasonable to assume that
metabolite M2 is N-alkyl hydroxylated. The loss of 17 amu from
 T. Sobolevsky et al. / Forensic Science International 200 (2010) 141–147 145

Fig. 5. RIC plotted from LC–MS fullscan data for the metabolites identified in the post-administration urine.

the molecular ion, as seen in the mass spectra of both metabolites,
is again atypical and might arise from the elimination of methyl
fragment of the trimethylsilyl group with subsequent loss of two
hydrogen atoms.
The SRM chromatogram obtained for the post-administration
urine sample is given in Fig. 4. This data clearly demonstrate that
metabolite M2 does not produce the ion with m/z 324.
At this point it may be concluded that GC–MS/MS is suitable for
detection of the monohydroxylated metabolites of JWH-018,
preferably for the M1. Nevertheless we found that the gas
chromatographic behavior of these metabolites is dependent on
the condition of column’s stationary phase, as after ca. 1000
injections the metabolite M1 and M2 peaks in the same urine
samples exhibited severe tailing, and their retention times were
shifted right by 0.3 min.
3.4. LC–MS/MS
Using liquid chromatography combined with a tandem mass
spectrometry one major peak with [M+H]+ corresponding to the
molecular weight of the monohydroxylated metabolite (m/z 358)
was observed on the chromatograms, with other related peaks

Fig. 6. Tentative structure of the identified urinary metabolites of JWH-018.

146 T. Sobolevsky et al. / Forensic Science International 200 (2010) 141–147

(RT = 3.91 min and 4.72 min, see below) being of a low abundance.
At this point it is unclear which one is the metabolite M2 detected
using GC–MS/MS.
To find more metabolites, the parent ion scan MS/MS experi-
ments were performed to detect any ions fragmenting to m/z 127 and
155, as well as to m/z 171 and 189 that should reflect hydroxylation at
the naphthalenyl moiety [9]. It allowed detecting the minor
metabolites, in addition to the data obtained by GC–MS/MS, with
the abundance being at least one order of magnitude lower than the
M1. These metabolites include: the carboxy metabolite M4 (M5
could be either carboxy or hydroxy-methoxy metabolite), dihydroxy
metabolites M6, M7, reduced trihydroxy metabolites M8, M9,
reduced dihydroxy metabolites M10, M11, and the N-despentyl
hydroxy metabolites M12, M13. Fig. 5 shows the mass chromato-
grams plotted from the fullscan data against the m/z ratio of the
[M+H]+ ions of the detected metabolites, except M12 and M13. The
ion trace for metabolites M12, M13 is not presented, as they coelute
with some endogenous compound(s) of the same molecular weight
(m/z 288) and are masked in the fullscan data. The proposed
structures of the identified metabolites are summarized in Fig. 6.
The MS/MS product ion scans were performed for each
metabolite at CE = 20 eV. It was found that ion transitions
[M+H]+ ! 127, 155 or [M+H]+ ! 171, 189 provide a high selectivity
of analysis.
The ESI(+) MS/MS spectrum for the metabolite M1 is given in
Fig. 7A. One may see that the same ions with m/z 127 and 155 as

Fig. 7. ESI(+) MS/MS spectra of the metabolites M1 (A), M4 (B), M6 (C), M7 (D), M8 (E) and M10 (F).
 T. Sobolevsky et al. / Forensic Science International 200 (2010) 141–147
observed in the electron ionization are present confirming that
naphthalenyl moiety of this metabolite is untouched.
The ESI(+) MS/MS spectrum for the metabolite M4 is shown in
Fig. 7B. It should be noted that in the ESI(+) mode the detection of
this metabolite may be difficult if the main monohydroxylated
metabolite M1 is simultaneously monitored, as the M1 and M4
elute closely and fragment to the same product ions. To
additionally support the assumption that the metabolite M4 is
carboxylated, the urine samples were analyzed with the mass
spectrometer operated in the negative ESI mode, as it is well-
known that carboxylic acids easily loose proton resulting in the
[MH] ions. Having performed the product ion scan from the ion
with m/z 370, one peak with an exceptional selectivity was
detected at the same retention time as in the positive ESI mode.
With the CE set to 20 eV, the MS/MS spectrum consisted of the only
ion with m/z 270. Structurally, this fragment may correspond to the
loss of the carboxylated N-pentyl side chain.
As our data indicate there are two closely eluting dihydroxy
metabolites M6 and M7, the first one with the monohydroxylated
naphthalenyl moiety (RT = 3.12 min, 374 ! 171, Fig. 7C) and the
second one where it seems to be untouched (RT = 3.20 min,
374 ! 155, Fig. 7D).
In addition, the trihydroxy metabolites with the reduced
naphthalenyl moiety (M8, M9) were identified. The MS/MS
spectrum for the metabolite M8 is shown in Fig. 7E. The reduced
dihydroxy metabolites (M10, M11) were also found (Fig. 7F), with
metabolite M11 being present in a negligible amount. As is seen,
the MS/MS spectrum of metabolite M10 has the same character-
istic ion transitions as the metabolite M8, fragmenting to the same
product ions m/z 171 and 189.
Since the N-despentyl hydroxy metabolites M12, M13 coelute
with some of endogenous compounds, their MS/MS spectra were
not possible to subtract and therefore are not presented. Never-
theless, the metabolite M12 may be detected by its characteristic
ion transition 288 ! 155 and 288 ! 127.
The metabolites are mostly glucuroconjugated. About 10% of
the metabolite M12 and 1% of the metabolite M10 were detected in
the free fraction. The carboxylated metabolite M4 was found only
partly conjugated.
Overall, JWH-018 metabolism proceeds in a good agreement
with the in vitro data for another aminoalkylindole [9], JWH-015,
except that the carboxylated metabolite was not reported before.
Due to their characteristic mass spectra and distinctive ion
transitions, the metabolites may be easily detected by LC–MS/
MS. At present it is not clear which of the metabolites are the most
long-term, but it may be anticipated that the monohydroxylated
and carboxylated metabolites M1 and M4 should be valuable for
147
the detection of JWH-018 in urine. Authors would also like to stress
that until confirmed by synthesis, the proposed structures of the
metabolites should be considered as tentative. In addition, since all
the data were obtained as a result of the uncontrolled administra-
tion of smoking mixture by only 3 persons, the metabolites
identified and their ratios may vary.
4. Conclusion
The metabolites of a synthetic cannabimimetic indole JWH-018
were identified in the post-administration urines. The main
monohydroxylated metabolites are excreted as conjugates with
glucuronic acid and can be reliably detected either by GC–MS/MS
or LC–MS/MS. However, LC–MS/MS seems to be a preferable
method of detection as the minor metabolites could also support
the analytical finding.
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