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Drug Metab Rev, Early Online: 1–51

! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03602532.2015.1029635

REVIEW ARTICLE

Synthetic cannabinoids pharmacokinetics and detection methods in

biological matrices
Marisol S. Castaneto1,2, Ariane Wohlfarth1, Nathalie A. Desrosiers1,2*, Rebecca L. Hartman1,2, David A. Gorelick3, and
Marilyn A. Huestis1
1
Department of Chemistry and Drug Metabolism, National Institute on Drug Abuse, NIH, Baltimore, MD, USA, 2Program in Toxicology, University of
Maryland School of Medicine, Baltimore, MD, USA, and 3Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD, USA
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Abstract Keywords
Synthetic cannabinoids (SC), originally developed as research tools, are now highly abused Analysis, GC-MS, LC-MS/MS, metabolite
novel psychoactive substances. We present a comprehensive systematic review covering in vivo profiling, methods, novel psychoactive
and in vitro animal and human pharmacokinetics and analytical methods for identifying SC and substances, pharmacokinetics, review,
their metabolites in biological matrices. Of two main phases of SC research, the first synthetic cannabinoids
investigated therapeutic applications, and the second abuse-related issues. Administration
studies showed high lipophilicity and distribution into brain and fat tissue. Metabolite profiling History
studies, mostly with human liver microsomes and human hepatocytes, structurally elucidated
metabolites and identified suitable SC markers. In general, SC underwent hydroxylation at Received 1 February 2015
various molecular sites, defluorination of fluorinated analogs and phase II metabolites were Accepted 6 March 2015
Published online 8 April 2015
For personal use only.

almost exclusively glucuronides. Analytical methods are critical for documenting intake,
with different strategies applied to adequately address the continuous emergence of new
compounds. Immunoassays have different cross-reactivities for different SC classes, but cannot
keep pace with changing analyte targets. Gas chromatography and liquid chromatography
mass spectrometry assays – first for a few, then numerous analytes – are available but
constrained by reference standard availability, and must be continuously updated and
revalidated. In blood and oral fluid, parent compounds are frequently present, albeit in low
concentrations; for urinary detection, metabolites must be identified and interpretation is
complex due to shared metabolic pathways. A new approach is non-targeted HRMS screening
that is more flexible and permits retrospective data analysis. We suggest that streamlined
assessment of new SC’s pharmacokinetics and advanced HRMS screening provide a promising
strategy to maintain relevant assays.

Introduction more pronounced cannabimimetic effects with more cognitive

Synthetic cannabinoids (SC), the largest class of novel
psychoactive substances (NPS) emerging over the last
decade, are marketed as ‘‘legal’’ alternatives to cannabis
(UNODC, 2011). SC bind to cannabinoid CB1 and/or CB2
cannabinoid receptors and were originally developed to
investigate the endogenous cannabinoid system or as potential
clinical pharmacotherapies (Castaneto et al., 2014b). Since
the mid-1960s, hundreds of SC were synthesized, but abuse
did not begin until the early 2000s (Gurney et al., 2014). Most
abused SC are CB1 receptor agonists with significantly higher
affinity than delta-9-tetrahydrocannabinol (THC), yielding
*Present address: New York State Police Forensic Investigation Center,
Albany, NY, USA
Address for correspondence: Professor Dr. (h.c.) Marilyn A. Huestis,
Chief, Chemistry and Drug Metabolism, IRP, National Institute on Drug
Abuse, National Institutes of Health, Biomedical Research Center, 251
Bayview Boulevard Suite 200 Room 05A-721, Baltimore, MD 21224,
USA. Tel: +1 443-740-2524. Fax: +1 443-740-2823. E-mail:
mhuestis@intra.nida.nih.gov
impairment, sensory perception changes and transient hallu-
cinations (Papanti et al., 2013). SC also induce adverse
physiological effects not observed with cannabis intake such
as vomiting, seizures, hyperglycemia and hypokalemia (CDC,
2013b), stroke (Freeman et al., 2013), myocardial infarction
(Mir et al., 2011) and acute kidney injury (CDC, 2013a).
Controlled SC administration studies are needed; however,
the lack of pre-clinical toxicology data makes conducting
such studies in humans currently unfeasible.
The American Association of Poison Control Centers
reported a sharp increase in SC exposure from 2096 in 2010
to 6968 calls in 2011, decreasing to 5230 in 2012, and further
declining to 2668 in 2013; however, calls are trending up
again with 3677 for 2014 (AAPCC, 2014). The reduction in
calls for 2013 was most likely a combination of SC placement
into Schedule I, reduced use due to public awareness of SC
toxicity, and physician experience in treating these exposures.
Many SC are now scheduled drugs in the USA under the
Controlled Substance Act (US DEA, 2013a,b, 2014), banned
by the World Anti-Doping Agency (WADA, 2014) and
controlled in many countries (UNODC, 2014).
 2 M. S. Castaneto et al.
SC identification in biological matrices is essential to
document intake in clinical and forensic settings and associ-
ate intake with drug toxicity; therefore, requiring laboratories
to establish reliable analytical methods and determine win-
dows of SC detection in biological samples. Notably, SC
detection is challenging in two aspects: first, doses and
concentrations in the body are low due to high potency.
Windows of detection in blood for acute intake are short
(Teske et al., 2010), although chronic use can lead to
accumulation in fatty tissues yielding longer detection
windows (Kneisel et al., 2014). Second, the best urinary
targets are metabolites, not the parent compound that is
usually extensively metabolized. Urine is the preferable
matrix to increase detection probability after SC intake.
Structurally different from THC, neither parent SC nor
metabolites are detected with standard cannabinoid immuno-
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assays. SC and their metabolites’ reference standards are
needed for method development and quantification, and
for forensically supportable identifications. Therefore, it is
critical to identify the most important analytical targets
for each SC by in vitro and in vivo studies. SC gas
chromatography mass spectrometry (GC-MS) and liquid
chromatography tandem mass spectrometry (LC-MS/MS)
methods are increasingly available, but short-lived, due to
the rapid emergence and need to constantly incorporate new
SC. High-resolution mass spectrometry (HRMS) methods,
especially when non-targeted, permit retrospective data
For personal use only.
interrogation and easier addition of new SC due to common
acquisition parameters.
We recently published a comprehensive review of SC
epidemiology, pharmacodynamics and receptor activity
(Castaneto et al., 2014b). The scope of the current review is
limited to published literature addressing in vivo and in vitro
SC pharmacokinetics and analytical methods for SC detection
and quantification in biological matrices.
Methods
We conducted a comprehensive literature search of seven
electronic databases (PubMedÕ , EmbaseÔ, Web of
ScienceÔ, ScopusÔ, Cochrane, Biological Abstracts and
Chemical Abstracts via STNÕ and SciFinderÕ platforms),
Google Scholar and Google up to 31 December 2013 except
for Biological Abstracts and Chemical abstracts (up to 30
November 2011) and also used additional search strategies
and keywords described in detail in our SC epidemiology and
pharmacodynamics review (Castaneto et al., 2014b). We
identified 3161 potentially SC-related articles, of which 881
records were considered relevant addressing SC epidemi-
ology, animal and human pharmacodynamics and receptor
interactions, animal and human pharmacokinetics, chemical
synthesis, legal status and street use and marketing. Of these,
70 articles investigated animal and human SC pharmacokin-
etics or detection and quantification in human biological
matrices and were incorporated in this review. We also
expanded our search from 1 January 2014 to 30 September
2014, focusing on these topics in EmbaseÔ, Web of
ScienceÔ, PubMed and Google Scholar using the search
filters ‘‘synthetic cannabinoids’’ plus ‘‘blood’’ or ‘‘urine’’
or ‘‘saliva’’ or ‘‘oral fluids’’ or ‘‘plasma’’ or ‘‘serum’’
Drug Metab Rev, Early Online: 1–51
or ‘‘hair’’. An additional 188 articles were identified, but only
32 were topic appropriate, yielding a total of 102 articles for
this review.
Results
Animal pharmacokinetics
We identified 19 articles (16 manuscripts, 3 abstracts) for
in vivo and in vitro SC pre-clinical pharmacokinetics studies
summarized in Table 1. This section highlights in greater
detail in vivo SC dog, guinea pig and chimeric mouse models,
administration via nose-only smoke exposure and multi-dose
studies demonstrating prolonged SC windows of detection.
In vivo SC studies included CP55,940, JWH-015, JWH-018,
AM2201, JWH-073, JWH-122, JWH-200, JWH-210, JWH-
250, JWH-398 and WIN55,212-2 administered intravenously
(IV), orally (PO), by gastric intubation (GI), smoke inhalation
(SM) or intraperitoneal injection (IP). Frequency and length
of exposures varied from once a day for 30 min to three times
a day for four weeks. SC in vitro studies with mouse S9
microsomal fractions, rat liver microsomes and slices, and
guinea pig skin are included. The majority of articles (n ¼ 12)
were published between 2011 and 2014, reported SC
distribution, and parent and/or metabolite concentrations in
biological matrices, e.g. adipose tissue, blood, brain, hair,
liver and urine.
Animal absorption and distribution studies
One of the earliest SC in vivo studies in 1987, included PO
administration of 0.6 mg/kg CP55,940 to one female dog,
with blood collected over 25 h (Fouda et al., 1987). CP55,940
plasma peak (Cmax) was about 80 mg/L 0.5 h after dosing and
the half-life (t1/2) was 8 h.
As most recreational SC intake is by smoking, pharmaco-
kinetics in mice after nose-only smoke exposure to 200 mg
‘‘Buzz’’ herbal product containing 10.8 mg (5.4% w/w)
JWH-018 were investigated (Poklis et al., 2012a). Six mice,
sacrificed 20 min post-exposure, had JWH-018 concentrations
of 82 ± 42 mg/kg in blood, 1990 ± 72 mg/kg in liver and
510 ± 166 mg/kg in brain. Later, these authors also investi-
gated blood and brain pharmacokinetics in two groups of five
mice after 10-min smoke exposure to ‘‘Magic Gold’’
containing 3.6% JWH-018, 5.7% JWH-073 and 0.1% JWH-
398 (Poklis et al., 2012b). The first group, sacrificed 20 min
post-exposure, had mean ± SD blood concentrations of
88 ± 42 mg/L JWH-018 and 134 ± 62 mg/L JWH-073 with
corresponding brain concentrations of 317 ± 81 mg/kg
JWH-018 and 584 ± 163 mg/kg JWH-073. JWH-398 was not
detected in any sample. The second group was sacrificed 20 h
post-exposure with measurable blood JWH-018 in only two
mice (3.4 and 9.4 mg/L) and JWH-073 at 4.3 mg/L in only one.
Mean brain JWH-018 concentration was 19 ± 9 ng/g, mea-
sureable in all five mice; JWH-073 was not detectable in any
20 h sample. Average brain-to-blood ratios after 20 min were
4.4 for JWH-018 and 5.2 for JWH-073.
Following 150 mg/kg IV WIN55,212-2 administration to
seven guinea pigs, plasma Cmax was 439 ± 120 mg/L and t1/2
4.9 ± 3.0 h (Valiveti et al., 2004a). Transdermal WIN55,212-2
was proposed as a route of administration to reduce the
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Table 1. Pharmacokinetic studies of synthetic cannabinoids and in vivo and in vitro biotransformation.

Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
AB-001 In vivo, human, oral Urine 7 N-dealkylation (N-pentyl) AB-001 OH (ada- Not reported Dose: 0.22–0.52 mg/kg; Grigoryev et al.
(self-experiment) Monohydroxylation (ada- mantane ring) Highest metabolite (2012)
(N ¼ 2) mantane, N-pentyl) concentration at 5–7 h;
detectable up to 160 h
DOI: 10.3109/03602532.2015.1029635

AB-FUBINACA In vitro, HLM HLM 1 Monohydroxylation AB-FUBINACA OH Not reported Only one metabolite Takayama et al.
(aminooxobutane) detected (2014)

ADB-FUBINACA In vitro, HLM HLM 1 Monohydroxylation ADB-FUBINACA ADB-FUBINACA Only one metabolite Takayama et al.
(aminooxobutane) OH OH (aminooxobu- detected (2014)
tane moiety)

AB-PINACA In vitro, HLM HLM 3 Monohydroxylation AB-PINACA-N-OH- Not reported Only two metabolites Takayama et al.
(N-pentyl, aminooxo- pentyl detected (2014)
butane moiety)
Synthetic cannabinoids
3

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4

Table 1. Continued

Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
AKB-48 In vitro, human Human 15 Mono or dihydroxylation Di-OH-AKB-48 Glucuronides No pentanoic acid Gandhi et al.
hepatocytes hepatocytes (adamantane, N- detected (2013)
pentyl) Carbonylation
(adamantane,
N-pentyl)
M. S. Castaneto et al.

5F-AKB-48 In vitro, HLM HLM 17 Defluorination 5F-AKB-48 OH or di- Glucuronides Oxidative defluorination Holm et al. (2014)
Mono, di, or trihydroxy- OH (adamantane), occurred without
lation (adamantane, 5F-AKB-48-N- NADPH
indazole, N-fluoropen- OH-fluoropentyl
tyl) or 5F-AKB-48
Carboxylation (N-pentyl) OH-indazole

AM694 In vivo, human, oral Urine 6 Defluorination AM694 N-OH-pentyl, Not specified Dose: 167 mg/kg body Grigoryev et al.
and smoke (self- Monohydroxylation AM694 N-penta- mass (PO) and (2013a)
experiment) (N-fluoropentyl, noic acid 16.7 mg/kg (smoke)
(N ¼ 1) N-pentyl)
Carboxylation (N-pentyl)

AM2201 In vitro, HLM and HLM 6 Defluorination JWH-018-N-5-OH- Glucuronides (con- Defluorination by Chimalakonda
CYP450 isoforms Monohydroxylation pentyl firmed from CYP1A2, followed by et al. (2012),
(CYP1A2, 2C9, (N-fluoropentyl, authentic human CYP2C9 and Patton et al.
-2D6, -2E1, and N-pentyl) urine samples) CYP2C19; CYP2D6 (2013a,b)
-3A4) Carboxylation (N-pentyl) catalyze OH-AM2201
In vitro, HLM and HLM 15 Defluorination AM2201-dihydrodiol Glucuronides SC isolated from herbal Sobolevsky et al.
CYP450 isoforms Epoxide hydrolysis (naphthyl) blend and incubated (2012)
(CYP3A4 and (naphthalene) with HLM
CYP2B6) Mono- or dihydroxylation
(N-fluoropentyl,
N-pentyl, naphthyl)
Carboxylation (N-pentyl)
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Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
In vivo, human, oral Serum, Urine 4 (serum) Serum: JWH-018-N-penta- Not specified Cmax ¼ 0.56 mg/L Hutter et al.
(self-experiment, 6 (urine) Defluorination noic acid for serum AM2201 at 1.5 h (2013)
N ¼ 1) Monohydroxylation and urine detected up to 150 h
DOI: 10.3109/03602532.2015.1029635

(N-fluoropentyl, post-ingestion; JWH-

indole) 018 and JWH-073
Carboxylation (N-pentyl) urinary OH and
Urine: Defluorination COOH metabolites
Monohydroxylation detected
(N-fluoropentyl,
indole, N-butyl)
Carboxylation (N-pentyl,
N-butyl)
Demethylation (N-pentyl)
In vitro, HLM HLM 4 Defluorination JWH-018-N-5-OH- Not specified Jang et al. (2014b)
Monohydroxylation pentyl
(N-fluoropentyl)
Carboxylation (N-pentyl)
In vivo, rat, IP (N ¼ 3) Urine 5 Defluorination JWH-018-N-penta- Not specified AM2201 15 mg/kg once a Jang et al. (2014a)
Monohydroxylation noic acid day  3 days; JWH-
(N-fluoropentyl, 018 and JWH-073
indole, N-pentyl) urinary metabolites
Carboxylation (N-pentyl, detected
N-butyl)
In vivo, rat, IP (N ¼ 5 Hair 3 Defluorination JWH-018-N-penta- Not specified Parent AM2201 quanti- Kim et al. (2014)
pigmented, N ¼ 5 Monohydroxylation noic acid fied 3.6 ± 0.8 pg/mg
non-pigmented (N-pentyl, indole) pigmented;
hair) Carboxylation (N-pentyl) 4.6 ± 0.9 pg/mg non-
pigmented
AM2201, JWH-018, In vivo, human, puff OF, serum None None Parent SC only Not reported Smoked 5 herbal product Kneisel et al.
JWH-019, JWH-073, (without inhal- with 12 SC; initial (2013b)
JWH-122, JWH-200, ation, N ¼ 2) concentration 7–
JWH-210, JWH-250, 577 mg/L; no parent
JWH-251, JWH-307, analyte detected after
MAM2201 and RCS-4 55 h; serum negative
ortho isomer
CP55,940 In vivo, dog, oral Plasma None None CP55,940 only Not reported Cmax ¼ 80 mg/L at Fouda et al.
(N ¼ 1) Tmax ¼ 8 h (1987)
In vitro, mouse S9 S9 microsomal 5 Monohydroxylation OH-CP55,940 Not reported Thomas & Martin
microsomal fractions (heptyl moiety) (1990)
fractions
In vitro, human skin, Human skin None None CP55,940 only Not reported Valiveti et al.
diffusion (2004b)

(continued )
Synthetic cannabinoids
5
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Table 1. Continued
6

Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
JWH-015 In vitro, RLM RLM 22 Dihydrodiol formation JWH-015 dihydrodiol Not reported Not reported Zhang et al.
(naphthyl) (2006)
Mono- or dihydroxylation
(N-propyl, indole,
naphthyl)
M. S. Castaneto et al.

Dehydrogenation
(N-propyl)
N-dealkylation
In vitro, HLM HLM 18 Dihydrodiol formation Not specified Not reported Mazzarino et al.
(naphthyl) (2014)
Mono-, di- or trihydrox-
ylation (N-propyl,
indole, naphthyl)
Dehydrogenation
(N-propyl)
N-dealkylation
In vitro, rat liver Rat liver slices 4 Monohydroxylation N-desalkyl and Glucuronides Also employed in silico Strano-Rossi et al.
slices (N-pentyl, indole) OH-indole metabolite prediction (2014)
N-dealkylation software
JWH-018 In vivo, rat, GI Urine 3 N-dealkylation OH-JWH-018- Not reported Abstract; data not Kramer et al.
desalkyl complete (2008)
In vivo, human, Blood None Parent only None Not reported Teske et al. (2010)
smoked (self-
experiment, N ¼ 2)
In vitro, HLM HLM 13 Dihydrodiol formation JWH-018-N-OH- Not reported JWH-018 dihydrodiol has Wintermeyer et al.
(naphthyl) pentyl, JWH-018- two isomers (2010)
Mono-, di- or trihydrox- OH-indole, JWH-
ylation (N-pentyl, 018-OH-naphthyl
indole, naphthyl) metabolites
N-dealkylation
Carboxylation (N-pentyl)
Dehydrogenation
(N-pentyl)
In vitro, HLM and HLM, HIM None Glucuronidation occurred Not determined Glucuronide 6 JWH-018 metabolites Chimalakonda
HIM, selected for all monohydroxy- incubated with UGT et al. (2011a)
Phase I incubated lated and carboxylated to determine phase II
with UGT1A1, metabolites metabolites; All
UGT1A10, metabolites, except
UG1A9, UGT2B7 for JWH-018-N-4-OH-
indole were
glucuronidated
In vivo, human, OF None Parent only Not reported Not reported Highest OF concentra- Coulter et al.
smoked (N ¼ 2) tions: 3–35 mg/L at (2011)
0.3 h. JWH-018 was
still present after 12 h.
In vitro, HLM HLM 4 Dihydrodiol formation OH-JWH-018- Not reported Information extracted Logan et al.
(naphthyl) desalkyl from presentation (2011)
Mono or dihydroxylation
(N-pentyl, naphthyl)
N-dealkylation
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Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
In vivo, rat, IP Blood, Urine 6 Mono-, di- trihydroxyl- OH-JWH-018- Not reported Information extracted Logan et al.
ation (not specified), desalkyl (OH pos- from presentation (2011)
N-dealkylation ition not specified)
Carboxylation (N-pentyl)
In vitro, HLM HLM 4 Monohydroxylation JWH-018-N-5-OH- Not specified ElSohly et al.
(N-pentyl, indole) pentyl (2011)
In vivo, rat, IP Hair 2 (hair) Hair: Monohydroxylation Not specified Not reported JWH-018 5 mg/kg/day Kikura-Hanajiri
DOI: 10.3109/03602532.2015.1029635

Urine 4 (urine) (indole) Carboxylation  10 d; abstract only, et al. (2011)

(N-pentyl) data not complete
Urine: Monohydoxylation
(N-pentyl, indole)
N-dealkylation
Carboxylation (N-pentyl)
In vitro, HLM and HLM 6 Monohydroxylation JWH-018-N-4- and Not reported CYP1A2 catalyzed OH Chimalakonda
CYP450 isoforms (N-pentyl, naphthyl) 5-OH-pentyl and and COOH, et al. (2012)
(CYP1A2, -2C9, Carboxylation (N-pentyl) JWH-018-N- CYP2C9 ! N-OH;
-2D6, -2E1, and pentanoic acid CYP2C19 ! N-4-OH-
-3A4) pentyl
In vivo, mouse, nose- Blood None Parent only Not reported Not reported After 30 min exposure: Poklis et al.
smoke inhalation Brain JWH-018: 82 ± 2 mg/ (2012a)
(N ¼ 6) Liver kg blood,
510 ± 166 mg/kg brain,
1990 ± 752 mg/kg liver
In vitro, HLM HLM 3 Monohydroxylation JWH-018-N-5-OH- Not detected Patton et al.
(N-pentyl) pentyl (2013a)
Carboxylation (N-pentyl)
In vitro, HLM HLM 3 Monohydroxylation JWH-018-N-4-OH- Not reported Jang et al. (2014a)
(N-pentyl) pentyl
Carboxylation (N-pentyl)
JWH-018 and In vivo, rat, IP (N ¼ 4) Urine 4 N-dealkylation OH-JWH-018- Not specified All metabolites dealky- Grigoryev et al.
JWH-073 Monohydroxylation desalkyl lated and (2011b)
(indole, naphthyl) monohydroxylated
In vivo, human, Blood None Parent, mono- or Not reported Not reported Highest concentration Kacinko et al.
smoked (controlled dihydroxylation 4.8 mg/L JWH-018 and (2011)
administration, (N-pentyl, indole, 4.2 mg/L JWH-073 at
N ¼ 6) naphthyl) 19 min from 1 of 6
N-dealkylation participants; metabol-
Carboxylation (N-pentyl) ite detected in
blood at 1 h post-
administration
In vivo, human, Urine 3 Monohydroxylation JWH-018-N-5-OH- Not specified Metabolites detected De Jager et al.
smoked (self- (N-pentyl) pentyl up to 65 h with peak (2012)
experiment, Carboxylation (N-pentyl, conc. 3–16.5 h
N ¼ 1) N-butyl)
In vivo, mouse, Blood None Parent only JWH-018 and Not reported Both SC in brain4blood Poklis et al.
smoke (inhalation Brain JWH-073 only after 20 min. After (2012b)
exposure) Liver 20 h, only JWH-018
in brain was measured.
JWH-018 and JWH-
073 blood detected
Synthetic cannabinoids

after 20 h
7

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8

Table 1. Continued

Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
M. S. Castaneto et al.

JWH-073 In vivo, rats, IP Hair 3 Monohydroxylation (N- JWH-073-N-3-OH- Not reported JWH-073 parent Kim et al. (2013)
butyl) Carboxylation butyl concentration4
(N-butyl) metabolites
In vitro, HLM HLM 5 Monohydroxylation JWH-073-N-OH- Not reported Gambaro et al.
(N-butyl, indole, butyl or OH- (2014)
naphthyl) naphthyl

JWH-073 4-methylnaphthoyl In vitro, HLM HLM 3 Monohydroxylation JWH-073-4-methyl- Not reported Gambaro et al.
analogue (N-butyl, indole, napthyoyl (2014)
naphthyl) OH-naphthyl

JWH-098 In vitro, rat liver Rat liver slices 6 Monohydroxylation JWH-098 O-demethy- Glucuronide Employed in silico Strano-Rossi et al.
slices (N-pentyl, indole, lated metabolite OH-JWH-098 metabolite prediction (2014)
naphthyl) software
O-demethylation
(naphthyl)
Carbonylation (N-pentyl)
Demethylation (naphthyl)
N-dealkylation

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Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
JWH-122 In vitro, HLM HLM 20 Mono-, di- or trihydrox- JWH-122N-OH or Not reported De Brabanter
ylation (N-pentyl, di-OH-pentyl or et al. (2013a)
indole, naphthyl) JWH-122 OH-
Dehydrogenation or di-OH naphthyl
(N-pentyl)
DOI: 10.3109/03602532.2015.1029635

Dihydrodiol formation
(naphthyl)
Carboxylation
(N-pentyl)
Demethylation
(naphthyl)
N-dealkylation
In vivo, mouse (chi- Urine 33 Mono-, di- or trihydrox- JWH-122-N-OH- Glucuronides (11) One murine-specific De Brabanter
meric and non- ylation (N-pentyl, pentyl and sulfates monohydroxylated et al. (2013a)
chimeric), PO indole, naphthyl) conjugates (6) metabolite (unspeci-
Dehydrogenation fied reaction site)
(N-pentyl) was detected in mice,
Dihydrodiol formation and not in HLM
(naphthyl)
N-dealkylation
In vitro, HLM HLM 4 Monohydroxylation JWH-122-OH- Not reported Gambaro et al.
(N-pentyl, indole, naphthyl (2014)
naphthyl)
In vitro, HLM HLM 3 Monohydroxylation JWH-122-N-4-OH- Not reported Jang et al. (2014a)
(N-pentyl) pentyl
Carboxylation
(N-pentyl)
In vivo, rats, PO Adipose tissue None Parent only JWH-122 only Not reported Single oral dose 20 mg/kg Schaefer et al.
given and JWH-122 (2014)
9 ng/g measured after
30 days
JWH-200 In vitro, HLM HLM 22 Monohydroxylation JWH-200-N-2-OH- Not reported De Brabanter
(morpholine, indole, ethyl (loss of mor- et al. (2013b)
naphthyl, N-ethyl) pholine ring)
Dehydrogenation (mor-
pholine, N-ethyl)
Dihydrodiol formation
(naphthyl) producing
2-OH-ethyl (2), mor-
pholine cleavage pro-
ducing N-2-OH-ethyl
(1), morpholine cleav-
age and carboxylation
at N-ethyl (1),
morpholine

(continued )
Synthetic cannabinoids
9
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10

Table 1. Continued

Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
In vivo, mouse (chi- Urine 23 Morpholine cleavage JWH-200-N carboxy Glucuronides (9) One monohydroxylated De Brabanter
meric, non-chi- Monohydroxylation (loss of morpho- and sulfate (2) JWH-200 detected in et al. (2013b)
meric), PO (naphthyl, morpho- line ring) conjugates mouse, but not in
line) Dihydrodiol for- HLM
mation (naphthyl)
M. S. Castaneto et al.

Carboxylation (N-ethyl,
after morpholine
cleavage), Morpholine
ring opening
Morpholine ring
opening + loss of
ethylene
JWH-210 In vitro, HLM HLM 18 Mono-, di- or trihydrox- Not specified Not reported Mazzarino et al.
ylation (indole, (2014)
N-pentyl, naphthyl)
Dihydrodiol formation
(naphthyl)
N-dealkylation
Carboxylation,
N-dealkylation
Dehydrogenation
(N-pentyl)
In vivo, rat, PO Adipose tissue None Parent only JWH-210 only Not reported Single 20 mg/kg PO Schaefer et al.
JWH-210 adminis- (2014)
tered, and 116 ng/g
JWH-122 quantified
30 days after dosing
JWH-250 In vivo, rat, GI Urine 22 Mono-, di- or tri- OH-JWH-250- Conjugated Grigoryev et al.
hydroxylation desalkyl (rats), (unspecified) (2011a)
(N-pentyl, indole, WH-250 OH-
benzyl) indole or N-pentyl
Dehydrogenation (in authentic
(N-pentyl) human urine)
N-dealkylation

JWH-251 In vitro, rat liver Rat liver slices 7 Monohydroxylation JWH-251-N-OH- Glucuronides major Employed in silico Strano-Rossi et al.
slices (N-pentyl, indole, pentyl metabolites metabolite prediction (2014)
benzyl) software
N-dealkylation
Carbonylation
(N-pentyl)
Carboxylation
(N-pentyl)
Drug Metab Rev, Early Online: 1–51

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Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
JWH-307 In vitro, rat liver Rat liver slices 3 Mono- or dihydroxylation JWH-307-OH or car- Not reported Employed in silico Strano-Rossi et al.
slices (N-pentyl, fluorophe- bonylated metabolite prediction (2014)
nyl) metabolites software
Dehydrogenation
(N-pentyl)
Carbonylation (N-pentyl)
DOI: 10.3109/03602532.2015.1029635

MAM2201 In vitro, HLM HLM 3 Defluorination JWH-122-N-5-OH- Not reported Jang et al. (2014a)
Monohydroxylation pentyl
(N-pentyl)
Carboxylation (N-pentyl)

PB-22 In vitro, HLM HLM 1 Ester hydrolysis 1-H-pentylindole-COOH Not reported Takayama et al.
(quinolinyl) + (2014)
carboxylation
In vitro, human Human 20 Ester hydrolysis Pentylindole-PB-22- Glucuronides (n ¼ 5) Wohlfarth et al.
hepatocytes hepatocytes (quinolinyl) N-pentanoic acid, and cysteine con- (2014a)
Monohydroxylation Pentylindole-PB- jugates (n ¼ 1)
(N-pentyl, indole, 22-N-4-OH-pentyl
quinolinyl)
Carbonylation (N-pentyl)
Carboxylation (N-pentyl)
Dihydrodiol
formation (quinolinyl)
5F-PB-22 In vitro, HLM HLM 1 Ester hydrolysis Fluoropentyl-1-H- Not reported Takayama et al.
(quinolinyl) + pentylindole- (2014)
carboxylation COOH
In vitro, human Human 20 Ester hydrolysis Pentylindole-PB-22- Glucuronides (n ¼ 7) Wohlfarth et al.
hepatocytes hepatocytes (quinolinyl) N-pentanoic acid and one cysteine (2014a)
Monohydroxylation conjugate
(indole, at N-pentyl,
quinolinyl)
Defluorinatiom
Carboxylation (N-pentyl)
Dihydrodiol formation
(quinolinyl)
Synthetic cannabinoids
11

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12

Table 1. Continued

Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
RCS-4 In vitro, human Human Monohydroxylation O-desmethyl-RCS-4- Glucuronides (n ¼ 11) Gandhi et al.
M. S. Castaneto et al.

hepatocytes hepatocytes 18 (indole, N-pentyl) (2) N-OH-pentyl and sulfate (n ¼ 1) (2014b)

Carboxylation (N-pentyl) conjugate
O-demethylation
(phenyl)
Demethylation (N-pentyl)

RCS-8 In vitro, human Human 32 Mono- and dihydroxyl- RCS-8-OH (OH pos- Glucuronides (n ¼ 15) Wohlfarth et al.
hepatocytes hepatocytes ation (phenyl ring, ition unspecified) (2014b)
cyclohexyl and
unspecified positions)
O-demethylation (phenyl)

STS-135 In vitro, human hep- Human 29 Mono-, di- or trihydrox- STS-135-OH (ada- Glucuronides STS-135 half-life and Gandhi et al.
atocytes and HLM hepatocytes ylation (adamantane, mantane) ring, (n ¼ 6) intrinsic clearance: (2014a)
indole, N-fluoropen- desalkyl-STS-135- t1/2 ¼ 3.1 ± 0.2 min
tyl) N-OH-pentyl and CLint ¼
Carbonylation (N-fluoro- 208.8 mlmin1kg1
pentyl, adamantane)
Carboxylation (N-pentyl)
Defluorination

(continued )
Drug Metab Rev, Early Online: 1–51
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Study type,
species, route of Total # Biotransformation, Major phase I
Compounds administration Matrix metabolites Phase I (reaction sites) metabolite(s) Phase II metabolites Other findings References
UR-144 In vitro, HLM and HLM 16 Mono- or dihydroxyl- UR-144-OH (unspeci- Glucuronides (con- Sobolevsky et al.
P450 isoforms ation (indole, fied OH position) firmed in authentic (2012)
CY3A4 and N-pentyl) human urine
CYP2B6 Dehydrogenation samples)
(N-pentyl)
N-dealkylation (1),
DOI: 10.3109/03602532.2015.1029635

Dihydrodiol formation
(indole)

WIN55,212-2 In vitro, RLM RLM 8 Dihydrodiol formation WIN55,212-2 dihy- Not reported Zhang et al.
(naphthyl, indole), drodiol at naphthyl (2002)
Monohydroxylation moiety (2 isomers)
(indole)
Dehydrogenation
(morpholine).
In vivo, hairless Plasma None Parent only WIN55,212-2 only Not reported Plasma (drug via IV Valiveti et al.
guinea pig, bolus): Cmax ¼ (2004a)
transdermal 439 ± 120 mg/L, t1/2a
¼ 0.12 ± 0.02 h, 8.1 h
In vitro, 1 cm2 human Skin None Parent only WIN55,212-2 only Not reported Not reported Valiveti et al.
and guinea pig (2004a,b)
skin
In vivo, mouse, IP Blood (trunk), None Parent only WIN55,212-2 only Not reported Detected 30% adipose Barna et al. (2009)
Adipose tissue tissue, 3% plasma and
Brain 0.3% brain total
injected (0.3–
3.0 mg/kg)
XLR-11 In vitro, human Human 30 Defluorination UR-144-N-5-OH- Glucuronides (n ¼ 16) Wohlfarth et al.
hepatocytes hepatocytes Carboxylation pentyl and UR- (2013a)
(N-pentyl, TMCP) 144-N-pentanoic
Mono- or dihydroxylation acid
(TMCP, N-pentyl)
carboxylation at N-pentyl
Aldehyde formation
(TMCP)
Hemi-acetal formation
(TMCP) carboxylation
at TMCP (1)
Internal dehydration
(TMCP) (1)
Synthetic cannabinoids

COOH: carboxy; GI: gastric intubation; HLM: human liver microsomes; HIM: human intestinal microsomes; IP: intraperitoneal injections; IV: intravenous; OH: hydroxy; PO: oral; RLM: rat liver microsomes;
TMCP: tetramethylcyclopropyl.
13
 14 M. S. Castaneto et al.
cannabinoid’s psychotropic effects. When guinea pig skin
was incubated in 5 g/L WIN55,212-2 for 48 h at 32  C,
the permeability coefficient was 4.33  104 cm/h with
777 ng/cm2h steady state flux and a 15.1 ± 2.1 h lag time.
The total skin WIN55,212-2 content was 2609 ± 93 mg/g
of skin with 26.6 mg/cm2 cumulative drug content in 48 h.
Transdermal WIN55,212-2 in guinea pig skin had 1.80 ±
0.08 mg/L solubility.
Two studies investigated SC distribution in rodent adipose
tissue after single SC doses. In three mice, euthanized 30 min
after 0.3, 1.0 or 3.0 mg/kg IP WIN55,212-2 (Barna et al.,
2009), 30% of total injected dose was found in adipose tissue,
followed by blood (3%) and brain (0.3%). Four weeks after a
single 20 mg/kg PO JWH-122 or JWH-210 dose in two rats,
adipose JWH-122 concentrations were 116 ng/g and for
JWH-210 9 ng/g (Schaefer et al., 2014).
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Animal biotransformation studies
In vivo. One hour after 10 mg/kg IP JWH-018 to rats, only
JWH-018 was detected in blood, while 3 h after dosing,
mono-, di- and trihydroxylated metabolites with unspecified
hydroxyl positions, a desalkylated hydroxy metabolite and
JWH-018-N-pentanoic acid were identified (Logan et al.,
2011). In urine 5 h after administration, the desalkyl hydroxy
metabolite was predominant followed by other hydroxylated
metabolites and only a trace amount of JWH-018-N-pentanoic
For personal use only.
acid.
Biotransformation of 2.5 mg GI JWH-122 was compared
between normal and chimeric mice transplanted with about
80% human hepatocytes (De Brabanter et al., 2013a). This
model provides an alternative approach to determining phase
I and II metabolites and their prevalence in humans. In both
animal groups, JWH-122 monohydroxylated metabolites were
the most prevalent urinary metabolites, with hydroxylation
sites at either the N-pentyl, indole or naphthalene moiety;
peak areas were higher in chimeric than normal mice. In
addition, di- and trihydroxylation at the same molecular sites,
dehydrogenation at the N-pentyl chain, dihydrodiol formation
at the naphthalene ring, N-desalkylation and reaction com-
binations occurred. No parent JWH-122 was detected in
any sample.
Using the same approach, a single 2.5 mg GI JWH-200
dose produced a predominant metabolite generated by loss of
the morpholine ring followed by N-ethyl carboxylation in the
chimeric mice (De Brabanter et al., 2013b). Other metabolites
were products of mono- or di-hydroxylation at the indole
alkyl moiety or dihydrodiol formation at the naphthalene
substructures with or without loss of the morpholine ring.
Ring opening followed by decarboxylation or loss of ethylene
also was observed. Phase II metabolites were mostly
glucuronides, although sulfates also were observed with
lower prevalence. No parent JWH-200 was detected in urine
over 24 h.
Following 15 mg/kg IP AM2201 once daily for 3 days
to three rats, 35.9–132 mg/L JWH-018-N-pentanoic acid
was the most predominant urinary metabolite, followed by
11.4–17.1 mg/L AM2201-6-hydroxyindole and 52.5 mg/L
(limits of quantification, LOQ) AM2201-N-4-hydroxypentyl,
AM2201-6-hydroxyindole, JWH-018-N-5-hydroxypentyl,
Drug Metab Rev, Early Online: 1–51
JWH-018-N-pentanoic acid and JWH-073-N-butanoic acid
(Jang et al., 2014b). AM2201 was not detected.
Five rats with and without pigmented hair received
10 mg/kg IP AM2201 once a day for 4 weeks (Kim et al.,
2014). Hair samples collected the following week had four
times higher JWH-018-N-pentanoic acid concentrations
(15.4 ± 3.7 ng/g) than AM2201 (3.6 ± 0.8 ng/g) in pigmented
and 21.5 ± 5.1 ng/g versus 4.6 ± 0.9 ng/g, respectively, in
non-pigmented hair. AM2201-6-hydroxyindole, AM2201-
N-4-hydroxypentyl and JWH-018-N-5-hydroxypentyl metab-
olite concentrations were 0.1–0.9 ng/g (LOQ 0.1 ng/g). No
significant concentration differences were observed for any
analyte between pigmented and non-pigmented hair.
In vitro. There were four in vitro metabolism studies
investigating SC with mouse S9 microsomal fractions, rat
liver microsomes (RLM) or rat liver slices. To determine
whether SC with similar chemical structures to THC undergo
similar oxidative metabolism and possibly produce pharma-
cologically active metabolites, tritium-labeled CP55,940, a
cyclohexylphenol, was incubated in mouse S9 microsomal
fractions (Thomas & Martin, 1990). Five monohydroxylated
metabolites were identified, all hydroxylated at the 10 ,10 -
dimethylheptyl side chain. Side-chain hydroxylation reactions
also occur with THC and other cannabinoids (Harvey, 1991).
Pharmacological intrinsic activity and binding studies for
CP55,490 hydroxylated metabolites were not performed;
although the authors hypothesized that these metabolites
could remain pharmacologically active and potentially cross
the blood–brain barrier, as observed with other hydroxylated
cannabinoid metabolites.
WIN55,212-2 metabolism in RLM produced two major
metabolites generated from the formation of epoxide inter-
mediates at the naphthalene moiety and subsequent hydrolysis
producing dihydrodiol metabolites (Zhang et al., 2002).
Epoxide formation can occur on different reaction sites of
the naphthalene ring yielding isomers. These major metab-
olites underwent further hydroxylation generating three tri-
hydroxylated metabolites.
The same authors also incubated JWH-015, a naphthoyl-
indole and CB2 agonist, with RLM, and although the
structures differed, the major metabolites also were dihydro-
diols (Zhang et al., 2006). Additional JWH-015 phase I
metabolites were generated by mono- and di-hydroxylation at
the indole alkyl moiety, dealkylation with and without mono-
or di-hydroxylation at the naphthalene moiety, dehydrogen-
ation and combinations of reactions.
JWH-015, JWH-098, JWH-251 and JWH-307 biotrans-
formation in rat liver slices produced hydroxylated,
N-dealkylated (JWH-015/JWH-098/JWH-251), carboxylated
(JWH-251), O-demethylated (JWH-098), carbonylated
(JWH-251/JWH307), dehydrogenated (JWH-307) and
glucuronidated (JWH-015/JWH-098/JWH-251) metabolites
(Strano-Rossi et al., 2014). This study also evaluated an in
silico metabolite prediction software for phase I metabolites
catalyzed by cytochrome P450 and flavin-containing mono-
oxygenases. The most probable predicted sites of metabolism
were on the N-alkyl, indole and benzyl or naphthalene
substructures. Products of N-dealkylation (JWH-015), aro-
matic hydroxylation (JWH-098), aliphatic carbonylation or
 DOI: 10.3109/03602532.2015.1029635
dehydrogenation (JWH-015) and aliphatic hydroxylation for
all four SC scored highest.
Human pharmacokinetics
We identified 31 manuscripts evaluating in vivo and in vitro
human SC pharmacokinetics of AB-001, AM694, AM2201,
CP55,940, JWH-015, JWH-018, JWH-019, JWH-073, JWH-
073 4-methoxynaphthoyl, JWH-098, JWH-122, JWH-200,
JWH-210, JWH-250, JWH-251, JWH-307, MAM2201, PB-
22/5F-PB-22, RCS-4, RCS-4 ortho isomer, RCS-8, STS-135,
UR-144, WIN55,212-2 and XLR-11. All were published
between 2010 and 2014, except for one CP55,940 and
WIN55,212-2 dermal absorption study published in 2004.
In vitro experiments were performed with human liver
microsomes (HLM), human hepatocytes and skin tissue. For
in vivo human studies, SC and metabolites were measured in
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blood or serum, oral fluid (OF) and urine. Results of all
studies are summarized in Table 1.
Human absorption and distribution studies
Eight manuscripts reported SC intake in humans, seven from
self-experiments and one controlled administration study
approved by the local Institutional Review Board (IRB), but
no Food and Drug Administration New Drug Application was
filed. Smoking was the most common route of administration
in 7 of 8 studies, and naphthoylindoles, adamantoylindoles
For personal use only.
and benzoylindoles were investigated. JWH-018 or mixtures
of JWH-018 and JWH-073, two naphthoylindoles, were most
commonly evaluated (6 articles).
Following 50 mg/kg smoked JWH-018, one male and one
female had 8.1 and 10.2 mg/L JWH-018 in their serum 5 min
after smoking, respectively (Teske et al., 2010), rapidly
decreasing to 4.6 and 6.1 mg/L 15 min after intake. JWH-018
was above the 0.07 mg/L limit of detection (LOD) in the male
subject for 48 h.
Data are published for only one of six subjects smoking
over 30 min 0.3 g herbal blend containing 17 mg/g JWH-018
and 22 mg/g JWH-073 (Kacinko et al., 2011). Blood JWH-
018 and JWH-073 19 min after smoking were 4.8 and
4.2 mg/L, respectively, rapidly declining to 1.5 mg/L JWH-
018 and 1.0 mg/L JWH-073 at 53 min, and 0.2 mg/L for both at
107 min (0.1 mg/L LOQ). Hydroxylated and glucuronidated
metabolites were detected in urine.
A single self-administered dose of a herbal blend contain-
ing JWH-018 and JWH-073 was smoked to obtain urine
samples to evaluate the authors’ quantitative analytical
method (De Jager et al., 2012). Peak urine concentra-
tions were 10 mg/L JWH-018-N-5-hydroxypentyl, 2 mg/L
JWH-018-N-pentanoic acid and 1 mg/L JWH-073-N-butanoic
acid.
After 5 mg PO AM2201 (N ¼ 1), serum Cmax was
0.56 mg/L at 1.5 h (Hutter et al., 2013). Serum concentrations
declined to 50.02 mg/L after 21 h, but remained detectable for
5 days. AM2201 was semi-quantified in OF at 0.02 mg/L from
only one sample collected approximately 5 h post-dose. In
urine, six metabolites quantified from 0.02 to 6.9 mg/g
(creatinine-normalized), and were undetectable after 11 days.
Oral fluid (OF) contamination was evaluated in two
volunteers following three puffs (without inhalation) of
Synthetic cannabinoids 15
herbal blends containing 12 SC (AM2201, JWH-018,
JWH-019, JWH-073, JWH-122, JWH-200, JWH-210, JWH-
250, JWH-251, JWH-307, MAM2201, RCS-4 ortho isomer)
(Kneisel et al., 2013b). One blood sample at 3 h and several
OF samples up to 90 h after inhalation were collected from
each participant. All SC were detected in OF for at least 6 h,
with initial concentrations of 7–577 mg/L, decreasing rapidly
by 70% after 30 min. Windows of detection varied by SC and
differed between subjects. The shortest OF detection window
was 6 h for JWH-200 and the RCS-4 ortho isomer, whereas
JWH-307 had the longest detection at 55 h. SC were not
detected in blood.
Oral fluid (OF) was collected 20 min to 12 h after
volunteer A smoked ‘‘Black Mamba’’ and volunteer B
smoked ‘‘Blueberry Posh’’, two herbal incenses containing
JWH-018 (Coulter et al., 2011). JWH-018 was 3 mg/L in OF at
20 min in volunteer A, falling to 50.5 mg/L LOQ at 5 h. For
volunteer B, JWH-018 OF was 35 mg/L 20 min after smoking,
rapidly declining to 10 mg/L at 40 min and detectable for 12 h.
In vitro transdermal permeation of 5 g/L WIN55,212-2 was
significantly lower in 1 cm2 human skin than guinea pig skin,
but no statistical difference in total drug accumulated over
48 h incubation at 32  C was observed (Valiveti et al., 2004a).
Transdermal permeation of human skin by CP55,940 and
WIN55,212-2, prepared in 4% bovine serum albumin and
0.5% Brij, was evaluated over 48 h (Valiveti et al., 2004b).
WIN55,212-2 skin accumulation was higher with Brij than
bovine serum albumin solution (73.8 ± 6.6 versus 16.9 ±
1.4 nmol/cm2), while no significant difference (16.8 ±
3.0 nmol/cm2 versus 15.2 ± 2.0 nmol/cm2) between the two
CP55,940 drug preparations was observed. In the same
Brij solution, the diffusion rate of 21.7 ± 3.9 mmol/g skin
WIN55,212-2 was significantly greater than that of 1.1 ±
0.4 mmol/g skin CP55,940.
Human biotransformation studies
Controlled administration biotransformation studies. In the
only IRB-approved human controlled administration study,
JWH-073 mono-, di- and trihydroxylated, desalkylated and
carboxylated metabolites were qualitatively confirmed in the
1 h blood sample of one subject who smoked an herbal blend
containing JWH-018 and JWH-073 (Kacinko et al., 2011).
In the single AM2201 oral ingestion study (Hutter et al.,
2013), serum concentrations of 0.04 mg/L AM2201-N-hydro-
xypentyl, 0.22 mg/L AM2201-6-hydroxyindole, 0.73 mg/L
JWH-018-N-pentanoic acid and 0.42 mg/L JWH-018-N-5-
hydroxypentyl were found. Creatinine-normalized concentra-
tions for six urinary metabolites’ 23 h after dosing were
0.21 mg/g AM2201-N-4-hydroxypentyl, 0.15 mg/g AM2201-6-
hydroxyindole, 1.9 mg/g JWH-018-N-5-hydroxypentyl, 1.3 mg/g
JWH-018-N-pentanoic acid, 0.06 mg/g JWH-073-N-4-hydro-
xybutyl and 0.06 mg/g JWH-073-N-butanoic acid. AM2201,
JWH-018 and JWH-073 metabolites were measurable up to
113, 238 and 68 h, respectively.
After smoking 0.15 g herbal blend containing JWH-018
and JWH-073, peak concentration of the primary urinary
metabolite, JWH-018-N-5-hydroxypentyl, was 10 mg/L, fol-
lowed by 2 mg/L JWH-018-N-pentanoic acid and 1 mg/L
JWH-073-N-butanoic acid (De Jager et al., 2012) in a single
 16 M. S. Castaneto et al.
self-experimenter. JWH-018 and JWH-073 metabolites were
detected 40.1 mg/L (LOQ) for 2–3 days.
A 47-year-old male and 43-year-old female ingested 13
and 26 mg of the adamantoylindole-type AB-001, respect-
ively, with urine collection for 1 week (Grigoryev et al.,
2012). Seven AB-001 metabolites were identified as products
of hydroxylation, N-dealkylation and a combination of these
reactions, with no unchanged AB-001 detected in urine.
The adamantane ring was the most frequent site of
biotransformation.
A 47-year-old male ingested 10 mg AM694 (a dose
equivalent to 167 mg/kg) and 2 weeks later smoked tobacco
laced with AM694 (dose equivalent to 16.7 mg/kg) (Grigoryev
et al., 2013a). Six urinary metabolites were identified
following AM694 defluorination, hydroxylation and carb-
oxylation with sites of reaction on the fluoropentylindole
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moiety. AM694 was not detected.
Biotransformation studies performed in vitro with and
without confirmation in authentic specimens
Due to insufficient SC toxicity data, which preclude IRB- and
FDA-approved human controlled drug administrations,
in vitro experiments are alternative approaches for metabolite
profiling and structure elucidation. We identified 23 articles
describing SC in vitro human studies, with the majority
(n ¼ 21) conducted between 2010 and 2014. HLM was the
For personal use only.
primary in vitro system in cited studies; fewer studies utilized
human hepatocytes. Table 1 contains data on pharmacokinetic
studies for JWH-018, the most studied SC, as well as AB- and
ABD-FUBINACA, AB-PINACA, AKB-48, AM2201, JWH-
015, JWH-073, JWH-073 4-methoxynaphthoyl, JWH-122,
JWH-200, JWH-210, PB-22, 5F-PB-22, MAM2201, RCS-4,
RCS-8, STS-135, UR-144 and XLR-11.
One of the earliest JWH-018 metabolite studies with
HLM identified 13 phase I metabolites with modifications at
three molecular sites, the N-pentyl (dehydrogenation,
hydroxylation, carboxylation), indole (N-dealkylation,
hydroxylation) and naphthyl substructure (hydroxylation
and dihydrodiol formation) (Wintermeyer et al., 2010).
Monohydroxylated and dihydrodiol metabolites were most
prevalent. Other investigators also incubated JWH-018 with
HLM and analyzed an authentic urine specimen from an
individual who admitted using ‘‘Spice’’ (ElSohly et al.,
2011). In HLM, they found two major metabolites mono-
hydroxylated: one at the pentyl chain, and one at the indole
core. The authors assigned the hydroxyl groups to the 5-
pentyl and 6-indole position, respectively; however, these
assignments are not conclusive and contradict other studies.
In urine, 4 monohydroxylated, 2 dihydroxylated, 1 trihy-
droxylated metabolites and JWH-018-N-pentanoic acid were
confirmed. Moreover, HLM produced 4 monohydroxylated
JWH-073 metabolites, one likely JWH-073-N-4-hydroxybu-
tyl. These metabolites are most likely generated by JWH-
018 N-pentanoic acid decarboxylation and further oxidation
(Hutter et al., 2012a). Dihydrodiol metabolites were not
reported, although they were major metabolites in another
study (Wintermeyer et al., 2010). Glucuronide formation was
not observed although the required co-factor uridine-
50 -diphosphoglucuronic acid was added during incubation.
Drug Metab Rev, Early Online: 1–51
JWH-073-N-butanoic acid was identified as the common
metabolite in authentic urine specimens of JWH-018 and
JWH-073 suspected users (Chimalakonda et al., 2011b). As
described above, JWH-073-N-butanoic acid is most likely
generated by JWH-018-N-pentanoic acid decarboxylation,
oxidation and carboxylation. JWH-073-N-3-hydroxybutyl was
specific to JWH-073, while JWH-018-N-pentanoic acid,
JWH-018-N-4-hydroxypentyl and JWH-018-N-5-hydroxypen-
tyl could not be generated from JWH-073.
Glucuronidation of JWH-018, JWH-073 and 12 commer-
cially available metabolites were investigated with HLM,
human intestinal microsomes and 12 different human recom-
binant uridine diphosphate-glucuronosyltransferase (UGT)
isoforms individually (Chimalakonda et al., 2011a).
UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7
isoenzymes were primarily responsible for JWH-018 and
JWH-073 metabolites’ conjugation and had high affinity for
hydroxylated metabolites (Km 12–18 mmol/L).
When JWH-018 and AM2201 were incubated with HLM,
JWH-018-N-pentanoic acid was the more prevalent metabol-
ite in AM2201- than in JWH-018-incubated samples
(Chimalakonda et al., 2012). AM2201 oxidative defluorina-
tion primarily generated JWH-018-N-5-hydroxypentyl, a
major metabolite, followed by JWH-018-N-pentanoic acid.
In contrast, JWH-018 primarily produced JWH-018-N-
4-hydroxypentyl followed by JWH-018-6-hydroxyindole.
Metabolite prevalence was verified in authentic human
urine specimens from JWH-018 and AM2201 users showing
the same results: JWH-018-N-4-hydroxypentyl was a major
metabolite for JWH-018, JWH-018-N-5-hydroxypentyl for
AM2201. In addition to metabolite profiling, cytochrome
P450 phenotyping was conducted on JWH-018 and AM2201
by incubating drugs with the selected CYP450 isoforms
CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. For
JWH-018, generation of JWH-018-N-4- and 5-hydroxypentyl
was primarily mediated by CYP2C9 followed by CYP1A2
and CYP2C19. CYP3A4 catalyzed JWH-018-N-4-hydroxy-
pentyl production but with lower activity than CYP1A2
and CP2C19. For AM2201, JWH-018-N-5-hydroxypentyl,
JWH-018-N-pentanoic acid and AM2201-N-4-hydroxypentyl
were catalyzed by CYP2C9 (highest activity), CYP1A2
and CYP2C19. The same group verified JWH-018 and
AM2201 metabolite patterns in authentic human urine
samples. Consistent with the previous study, prevalence
of metabolites after JWH-018 intake was JWH-018-N-4-
hydroxypentyl4JWH-018-N-5-hydroxypentyl4JWH-018-N-
pentanoic acid; and after AM2201 intake, JWH-018-N-5-
hydroxypentyl4JWH-018-N-pentanoic acid4AM2201-N-4-
hydroxypentyl (Patton et al., 2013b). JWH-073-N-butanoic
acid was detected in both.
Two pairs of fluorinated/non-fluorinated SC naphthoylin-
doles – AM2201/JWH-018 and MAM2201/JWH-122 – were
incubated with HLM (Jang et al., 2014a). Profiling was
limited to metabolites with commercially available standards
including JWH-018-N-4 and 5-hydroxypentyl, JWH-018-N-
pentanoic acid, AM2201-N-4-hydroxypentyl, JWH-122-N-4
and 5-hydroxypentyl, JWH-122-N-pentanoic acid (also
known as MAM2201-N-pentanoic acid) and MAM2201-
N-4-hydroxypentyl. Biotransformation of AM2201 and
MAM2201 predominantly generated JWH-018- and
 DOI: 10.3109/03602532.2015.1029635
JWH-122-N-5-hydroxypentyl metabolites, respectively, with
lower AM2201- or MAM2201-N-4-hydroxypentyl concentra-
tions. In addition, pentanoic acid metabolites were detected at
higher concentrations after consumption of the fluorinated
than non-fluorinated analog. Subsequently, authentic urine
specimens from individuals who were suspected of either
MAM2201 or JWH-122 abuse were analyzed and JWH-122-
N-5-hydroxypentyl was confirmed as the predominant metab-
olite in the MAM2201 group (1.1–84.8 mg/L), but only minor
in the JWH-122 group (0.1–2.8 mg/L). MAM2201-N-4-
hydroxypentyl also was detected, but not in all MAM2201
samples. The same group previously investigated AM2201
metabolism in rats, compared findings in authentic urine
specimens, and reported JWH-018-N-5-hydroxypentyl, the
product of oxidative defluorination, to be the major metab-
olite after suspected AM2201 intake (Jang et al., 2014b).
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
Although, this metabolite also was found in urine samples of
JWH-018 users, its concentration was lower than JWH-018-
N-4-hydroxypentyl concentration; therefore, Jang et al. sug-
gested that in the absence of fluorinated AM2201 metabolites,
a high JWH-018-N-4-hydroxypentyl to JWH-018-N-5-hydro-
xypentyl ratio indicates JWH-018 rather than AM2201 intake.
JWH-122 incubated with HLM generated 30 metabolites
as products of mono-, di- and tri-hydroxylation, dehydrogen-
ation, dihydrodiol formation, N-dealkylation, carboxylation
and combinations of these reactions (De Brabanter et al.,
2013a). Hydroxylation occurred at the indole alkyl or
For personal use only.
naphthyl substructures. In vitro findings were compared
with a chimeric mouse model, which confirmed most major
HLM metabolites and also identified one murine-specific
monohydroxylated metabolite.
In a separate study, JWH-200 incubated with HLM
produced 22 metabolites generated by the same reactions
observed for JWH-122, and also JWH-200-specific reactions
– morpholine ring cleavage or ring opening (De Brabanter
et al., 2013b). When compared to in vivo chimeric mouse
JWH-200 metabolites, a single murine-specific monohy-
droxylated JWH-200 metabolite was observed, whereas the
hydroxylated JWH-200 dihydrodiol metabolite in HLM was
absent in mouse urine.
JWH-018, JWH-073, 4-methylnaphthoyl-JWH-073 and
JWH-122 were investigated in separate HLM incubations.
The most prevalent and common oxidative reaction was
hydroxylation at the N-alkyl or indole or naphthyl substruc-
tures, carboxylation at the N-alkyl chain and N-dealkylation
were less prevalent (Gambaro et al., 2014).
JWH-015 and JWH-210 underwent mono-, di- and tri-
hydroxylation, N-dealkylation, dihydrodiol formation and
dehydrogenation and combinations of these reactions after
incubation with HLM (Mazzarino et al., 2014). In addition,
JWH-210 also underwent carboxylation.
Human liver microsomes (HLM) incubation of a UR-144
herbal extract produced 16 UR-144 phase I metabolites.
Hydroxylation at the tetracyclomethylpropyl ring generated
the most abundant metabolites (Sobolevsky et al., 2012).
Other phase I reactions included N-dealkylation plus indole
hydroxylation, dihydrodiol formation and indole dihydroxyla-
tion. Monohydroxylation also occurred at the indole alkyl
moiety and produced minor metabolites. When HLM metab-
olites were verified in five authentic urine specimens, all
Synthetic cannabinoids 17
in vitro metabolites, except for N-despentylhydroxy and
dehydrogenated hydroxy UR-144, were found. One urine
specimen contained UR-144 parent, although in low concen-
tration. Based on self-reported smoking time, the urinary
metabolites’ windows of detection were suggested to be
1 week.
Our group incubated XLR-11 with human hepatocytes,
identifying 14 phase I and 16 phase II metabolites (Wohlfarth
et al., 2013a). The XLR-11 major metabolite was 20 -carboxy-
XLR-11, a product of carboxylation at the tetramethylcyclo-
propyl substructure, followed by UR-144-N-pentanoic
acid (oxidative defluorination and carboxylation) and
UR-144-N-5-hydroxypentyl (oxidative defluorination and
hydroxylation).
Sixteen RCS-4 metabolites were identified in authentic
urine specimens from intoxication cases as products of
O-demethylation, mono- and di-hydroxylation at the
N-pentyl and/or benzoyl substructures, N-dealkylation and
reaction combinations (Kavanagh et al., 2012). In compari-
son, we incubated RCS-4 with human hepatocytes and
identified 18 metabolites, which were generated by
O-demethylation, aromatic or aliphatic monohydroxylation,
dihydroxylation, carboxylation, N-dealkylation and reaction
combinations (Gandhi et al., 2014b). All phase II metabolites,
except one sulfate conjugate, were glucuronides. We also
identified O-demethylated RCS-4 as the major metabolite, the
same major metabolite identified in authentic urine specimens
(Kavanagh et al., 2012). In addition, RCS-4-N-pentanoic
acid (with and without O-demethylation) and RCS-4-N-2-
hydroxypentyl were found as minor metabolites in vitro, but
these were not detected in authentic urine.
RCS-8 incubated with human hepatocytes generated eight
phase I and 15 phase II metabolites (Wohlfarth et al., 2014b).
The predominant metabolite, RCS-8-dihydroxy glucuronide,
was hydroxylated at the cyclohexyl and phenyl moiety and
further glucuronidated at the phenyl hydroxyl group. Five
isomers were found in total. O-demethylation with or without
hydroxylation was observed in six of 15 phase II metabolites.
PB-22 and 5F-PB-22 incubated with human hepatocytes
produced 14 PB-22 and 12 5F-PB-22 phase I metabolites
including several phase II metabolites (Wohlfarth et al.,
2014a). Ester hydrolysis was the predominant metabolic
pathway for both analytes producing 1-pentyl-1H-indole-3-
carboxylic acid (PI-COOH) and 5F-PI-COOH, which under-
went further biotransformations. Moreover, minor metabolites
with intact quinolinyl moiety were identified as products of
epoxide hydrolysis at the quinolinyl moiety, hydroxylation at
the indole alkyl or quinolinyl moiety, ketone formation and
carboxylation at the N-pentyl chain and for 5F-PB-22 only –
defluorination. Five PB-22 and seven 5F-PB-22 metabolites
were observed as glucuronides with one cysteine-conjugated
metabolite for PI-COOH and 5F-PI-COOH. Others found that
PB-22 and 5F-PB-22 incubated with HLM produced PI-
COOH or 5F-PI-COOH, generated by ester hydrolysis, but did
not observe 5F-PB-22 defluorination (Takayama et al., 2014).
AKB-48, an adamantoylindazole, generated 15 phase I and
two phase II metabolites in hepatocytes with two major metab-
olites, AKB-48-hydroxy-adamantoyl and AKB-48-dihydroxy-
adamantoyl, which also were the only phase II glucuronidated
metabolites (Gandhi et al., 2013). Ketone formation at the
 18 M. S. Castaneto et al.
N-pentyl side chain also was observed; notably, AKB48-N-
pentanoic acid was not detected.
5F-AKB-48 incubated with HLM produced 16 phase I
metabolites generated by oxidative defluorination, hydroxyl-
ation at either the adamantane or N-fluoropentyl substructures
and N-dealkylation (Holm et al., 2014). Notably, 5F-AKB-48
oxidative defluorination also occurred without adding the
co-factor NADPH indicating involvement of enzymes other
than CYP450. The most intense metabolite was AKB-
48-hydroxy-adamantoyl-N-pentanoic acid. Fifteen of 16
metabolites identified in vitro were verified in an authentic
urine sample, previously screened for 5F-AKB-48 hydroxy
by LC-QTOF. In urine, AKB-48-hydroxy-adamantoyl-N-
pentanoic acid was the most abundant metabolite.
A structurally similar SC, STS-135, a fluorinated ada-
mantoylindole, was studied with HLM and human hepato-
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
cytes to evaluate metabolite stability and its metabolic profile,
respectively (Gandhi et al., 2014a). Half-life was
3.1 ± 0.2 min and predicted intrinsic clearance was
208.8 mL/minkg, which classified STS-135 as an intermedi-
ate clearance drug. Seventeen phase I metabolites were
identified, most generated by mono-, di- and tri-hydroxylation
at the adamantane ring, and to a lesser extent oxidative
defluorination, which played a less dominant role in STS-135
metabolism than observed for other 5-fluoropentyl side chain-
containing SC.
Separate HLM incubations of AB-FUBINACA and ADB-
For personal use only.
FUBINACA, and AB-PINACA, two fluorinated and one
non-fluorinated indazole carboxamides, generated one mono-
hydroxylated metabolite at the aminooxobutane for AB- and
ADB-FUBINACA and three monohydroxylated metabolites
at either the aminooxobutane or N-pentyl substructure, for
AB-PINACA (Takayama et al., 2014).
Analytical methods to identify SC in human biological
matrices
We identified 65 articles detecting SC and metabolites in
human blood, plasma, serum, hair, OF and urine, all
published from 2010 to 2014. Method validation data are
summarized in Table 2, while metabolites identified by these
methods in corresponding in vivo or in vitro studies are listed
in Table 3. Five articles reported immunoassay method
validation targeting SC analytes in urine, the rest described
qualitative or quantitative chromatographic mass spectrom-
etry assays. Sample preparation included simple dilution,
liquid-liquid extraction (LLE), protein precipitation, solid
phase extraction (SPE), salting-out LLE (SALLE), supported
liquid extraction (SLE), acid/base/enzyme hydrolysis, wash-
ing with or without base digestion (for hair only) and extract
derivatization. Samples were analyzed by GC-MS, LC-MS/
MS and LC-HRMS for qualitative and quantitative confirm-
ation. Method validation parameters included evaluation of
LOD/LOQ, assay imprecision and bias, analyte recovery,
interference or cross-talk, matrix effects, extraction and
process efficiency, autosampler stability and short-term
analyte stability at different storage conditions. Cutoff
evaluation also was included in the immunoassay method
validation. The following subsections highlight analytical
methods applicable to clinical and forensic cases including
Drug Metab Rev, Early Online: 1–51
current knowledge on SC analyte stability in different
matrices. Validation data for all methods are provided in
Table 2. It is important to note that the extent of the
confirmation assays always depended on the commercial
availability of standards at the time of method validation.
Blood (blood/plasma/serum)
Nineteen articles described method validation for qualitative
(n ¼ 5) and quantitative (n ¼ 14) SC confirmation in blood,
serum and/or plasma. For qualitative confirmation, we chose
to highlight the most current and extensive methods identify-
ing SC in blood (Guale et al., 2013), serum (Huppertz et al.,
2014) and a method targeting SC via precursor ion scan with
common substructures (Mazzarino et al., 2014). We also
selected quantitative assays that included the most SC
analytes in blood (Ammann et al., 2012) and serum
(Kneisel & Auwärter, 2012).
Thirty SC were quantified in serum by LC-MS/MS
following LLE sample preparation, and scheduled multiple
reaction monitoring (MRM) (Kneisel & Auwärter, 2012),
achieving LOQs between 0.1 and 2.0 mg/L. The method was
applied to 833 serum samples from forensic cases and 227
were positive for at least one SC with 80% JWH-210, 63%
JWH-122, 29% AM2201, 10% JWH-018 and 56% for JWH-
019, JWH-073, JWH-081, JWH-200, JWH-203, JWH-307
and/or RCS-4.
Others quantified 25 SC in ante- and post-mortem blood
following LLE (Ammann et al., 2012). LOQ were 0.5 mg/L for
SC analyzed in positive ionization mode and 5.0 mg/L LOQ
for SC requiring negative ionization mode.
A screening method for 12 SC in blood utilized automated
SPE and LC-TOF-MS (Guale et al., 2013). Compounds
exceeding a specified intensity threshold were compared to an
in-house library and evaluated retention time, accurate mass
and isotopic pattern. For forensic purposes, fragment ions and
their ratios also must be evaluated, but this method served as a
screening tool similar to an immunoassay.
An LC-MS/MS screening method for 46 SC and 8 labeled
analogs in serum was developed with heated electrospray
ionization (ESI), which enhanced sensitivity and obtained an
LOD range from 0.1 to 0.5 mg/L (Huppertz et al., 2014). The
mass spectrometer was operated in untargeted auto MSn mode
with an inclusion list and generated MS, MS/MS and MS3
data. Candidates were evaluated based on retention time,
precursor ion and MS/MS spectra matching with an in-house
library. Thirty authentic serum samples from forensic cases
were analyzed by this screening method and another quan-
titative LC-MS/MS assay (Kneisel & Auwärter, 2012);
all samples screened and confirmed positive for one or
more SC.
A method detecting 15 structurally similar SC by LC-MS/
MS in blood, urine and OF using precursor ion scans for
characteristic fragments of naphthoylindole SC (m/z 127, 144
and/or 155) was developed (Mazzarino et al., 2014). HLM
samples incubated with JWH-015 and JWH-210 documented
that all JWH-210 and eight JWH-015 metabolites retained an
intact m/z 144 fragment and nine JWH-015 metabolites intact
m/z 127 and 155 fragments. This method was designed to
detect unknown compounds having these characteristics
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Table 2. Analytical methods for the identification of synthetic cannabinoids in biological matrices.

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
JWH-018 and QUAL Urine Hydrolysis, LLE LC-MS/MS heated- Not fully validated Authentic urine speci- No parent SC, 13 metab- Sobolevsky et al.
metabolites ESI due to the absence mens collected from olites detected: JWH- (2010)
of metabolite individuals suspected 018-N-OH-indole (2),
DOI: 10.3109/03602532.2015.1029635

standards. of SC intake N-OH-pentyl,

N-COOH, di-OH (at
indole and naphthyl)
or at indole and N-5-
OH-pentyl, dihydro-
diol (at naphthyl, 2),
dihydrodiol + OH-
indole (2),
N-dealkylated + OH-
indole (2) and
N-COOH or COOH
methoxy metabolite.
JWH-018 QUANT Serum LLE LC-MS/MS-ESI+ Validated LOD, LOQ, Authentic serum speci- JWH-018 was detected in Teske et al. (2010)
Linearity, ME mens from two volun- serum samples.
teers who smoked LOD ¼ 0.07 mg/L;
herbal product con- LOQ ¼ 0.21 mg/L
taining SC
JWH-018 metabolites: QUAL and Urine Hydrolysis, LLE LC-MS/MS-ESI+ LOD, LOQ, Authentic urine doping Validation only for JWH- Beuck et al.
N-5-OH-pentyl, 5-OH- QUANT Recovery, controls specimens 018-N-OH-pentyl and (2011)
indole, N-COOH, Linearity, Bias, (n ¼ 4) JWH-018-N-COOH,
N-dealkylated-5-OH- ME, Imprecision, synthesized in-house.
indole and 2-OH- Stability Metabolites stable for
naphthoyl 4 weeks at RT or 4  C.
LOD ¼ 0.1 mg/L;
LOQ ¼ 0.5 mg/L
JWH-018 metabolites: QUANT Urine Hydrolysis, SPE LC-MS/MS-ESI+ LOD, LOQ, Authentic urine speci- LOD/LOQ ¼ 0.1 mg/L. Chimalakonda
N-4- and 5-OH-pentyl Imprecision, Bias, mens from four indi- JWH-018-N-4-OH- et al. (2011b)
and COOH. Carryover viduals suspected of pentyl4JWH-018-
JWH-073 metabolites: SC intake N-COOH4JWH-018-
N-3- and 4-OH-butyl N-5-OH-pentyl (all
and COOH three analytes Gluc-
conjugated) in JWH-
018 users’ urine.
JWH-073-N-COOH
detected in JWH-018
suspects; but JWH-
073-N-3-OH-butyl and
N-4-OH-butyl in
JWH-073 suspected
user’ urine only

(continued )
Synthetic cannabinoids
19
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For personal use only.
20

Table 2. Continued

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
JWH-018, JWH-250, QUANT OF SPE LC-MS/MS-EIS + / LOD, LOQ, Linearity, Authentic OF specimens JWH-073, JWH-018 and Coulter et al.
JWH-073, CP47, 497 Imprecision, Bias, from two volunteers JWH-250 unstable at (2011)
(C8), CP47, 497, ME, Interference who smoked herbal RT, but remained
M. S. Castaneto et al.

HU-210 Stability product containing SC 480% at 4  C after 1

Recovery (extraction week. CP47, 497-C8
pad) and HU-210 stable at
RT & 4  C after 1
week. Extracts stable
on autosampler at 7  C
for 48 h. Authentic
samples stable for at
least 1 month.
LOQ ¼ 0.5 mg/L.
Analyte recovery from
collection pad 60%
(55–74%).
CP47, 497 QUANT Urine Dilution LC-MS/MS-ESI LOD, LOQ, Linearity, None Stability not evaluated. Dowling & Regan
Imprecision, Bias, LOD:10 mg/L, (2011)
Uncertainty LOQ:20 mg/L
JWH-015, JWH-018, QUANT and Serum LLE LC-MS/MS-ESI LOD, LOQ, Authentic serum speci- Stable for three freeze- Dresen et al.
JWH-073, JWH-081, QUAL Selectivity, mens (n ¼ 101) from thaw cycles, for 1 (2011)
JWH-200, JWH-250, Linearity, hospitals, detoxifica- week at 20  C. JWH-
WIN55,212-2, metha- Imprecision, Bias, tion and therapy cen- JWH-081 unstable at
nandamide, JWH-019, Carryover, ters, forensic RT (35 to 26%)
JWH-020 Recovery, ME, psychiatric centers after 48 h. Processed
Stability samples stable at 4 C
after 5 h.
LOD ¼ 0.1 mg/L,
LOQ ¼ 0.1–0.6 mg/L.
In authentic samples,
JWH-081 (0.11–
16.9 mg/L) most
prevalent (56/101),
then JWH-250 (47),
JWH-018 (9), JWH-
073 (6), JWH-015 (2).
JWH-018 and metabol- QUANT Urine Hydrolysis, LLE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine speci- Stability not evaluated. ElSohly et al.
ites: 6-OH-indole, Bias, Imprecision mens (n ¼ 33) col- LOD ¼ 0.01–0.1 mg/L, (2011)
N-5-OH-pentyl, lected under forensic LOQ ¼ 1 mg/L. JWH-
N-COOH and random workplace 018-N-COOH and -5-
drug testing. OH-pentyl were pre-
sent in all samples.
Highest concentration
was 27 256 mg/L JWH-
018-N-COOH.

(continued )
Drug Metab Rev, Early Online: 1–51
 Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
For personal use only.

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
 
JWH-018, JWH-073, QUANT and WB LLE LC-MS/MS-ESI + LOD, LOQ, Linearity, Authentic WB specimen Stable at 3 C and 10 C Kacinko et al.
JWH-250, JWH-019 QUAL Bias, Imprecision, from a known JWH- for at least 30 days, (2011)
(QL only) Interference, 018/JWH-073 user for three freeze-thaw
Extraction cycles, and on auto-
DOI: 10.3109/03602532.2015.1029635

Efficiency, ME, sampler at RT. JWH-

Process efficiency, 018 and JWH-073
Carryover, quantified in blood in
Dilution Integrity 19–199 min sample.
LOD ¼ 0.006–
0.016 mg/L;
LOQ ¼ 0.1 mg/L.
JWH-018 and metabolites QUAL Urine Hydrolysis, LLE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine doping Stability not evaluated. Möller et al.
(n ¼ 19) in Imprecision, control specimens LOD ¼ 0.1 mg/L. (2011)
Wintermeyer et al. Specificity, (n ¼ 7500) Predominant metabol-
(2010) Recovery ite detected was JWH-
018 N-OH-pentyl
metabolite.
JWH-018 and metabol- QUANT Urine Hydrolysis, LLE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine speci- Stability not evaluated. Moran et al.
ites: 4-, 5-, 6-, 7-OH- Bias, Imprecision mens from three sus- LOD ¼52 mg/L, (2011)
indole, N-5-OH-pentyl pected SC users. LOQ ¼ 1.8–10.8 mg/L.
and -COOH. Identified JWH-018
JWH-073 and metabol- metabolites: 5- and 6-
ites: 4-,5-,6-,7-OH- OH-indole, N-5-OH-
indole, N-4-OH-butyl pentyl, N-COOH (all
and -COOH 85–100% Gluc). Also,
identified JWH-073-3-
OH-indole (100%
Gluc) and JWH-073-
N-COOH (5% Gluc).
AM694, AM1241, QUANT WB LLE LC-MS/MS-ESI+/ LOD, LOQ, None Extracts stable for 24 h Ammann et al.
CP47, 497, CP47, 497 Selectivity, provided reconstituted (2012)
C8-homolog, HU-210, Linearity, Bias, in methanol. Analytes
JWH-007, JWH-015, Imprecision, stable after three
JWH-018, JWH-019, Stability, freeze/thaw cycles and
JWH-030, JWH-073, Extraction at 1 month stored in
JWH-081, JWH-203, Efficiency, ME 20  C. LOQ ¼ 0.5
JWH-210, JWH-250, and 5 mg/L. No
JWH-251, JWH-302, authentic specimens
JWH-398, RCS-4, analyzed.
RCS-4 2- and 3-meth-
oxy homolog, RCS-8,
RCS-4-C-4-homolog,
WIN48 098,
WIN55,212-2
mesylate

(continued )
Synthetic cannabinoids
21

QUAL
or
Analyte(s) QUANT
Metabolites only: JWH- QUANT
018-N-5-OH-pentyl
and -COOH, JWH-
019-5-OH-indole,
JWH-073-N-4-OH-
butyl and -COOH,
JWH-122-N-5-OH-
pentyl, JWH-200-5-
OH-indole, JWH-250-
5-OH-indole and
N-COOH, RCS-4 N-5-
OH-pentyl
AB-001 QUAL
JWH-018, JWH-018-2-, QUAL
4-,5-,6- and -7-OH-
indole, JWH-018-N-5-
OH-pentyl, JWH-018-
N-COOH, JWH-073,
JWH-073-4-, 5-, 6-
and -7-OH-indole,
JWH-073-N-4-OH-
butyl, JWH-073-
N-COOH
Metabolites only: JWH- QUAL
018-N-pentanoic acid,
JWH-018-N-5-OH-
pentyl, JWH-018 4-,
5-, 6-, and 7-OH-
indole, JWH-073-
N-COOH, JWH-073-
N-4-OH-butyl, JWH-
073 6-OH-indole,
JWH-122-N-5-OH-
pentyl
AM694, AM2201, JWH- QUANT
007, JWH-015, JWH-
018, JWH-019, JWH-
020, JWH-073, JWH-
081, JWH-122, JWH-
200, JWH-203, JWH-
210, JWH-250, JWH-
251, JWH-398,
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For personal use only.
22
Table 2. Continued
Matrix Sample preparation Method Validation Application Study findings References
Urine Hydrolysis, LLE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine speci- Stability not evaluated. De Jager et al.
Bias, Imprecision, mens from a volunteer LOQ ¼ 0.1 mg/L. (2012)
Carryover, ME who smoked herbal JWH-018-N-5-OH-
blend ‘‘Kronic’’ con- pentyl and -COOH
taining JWH-018 and and JWH-073-
M. S. Castaneto et al.
JWH-083 N-COOH detectable
565 h post-smoking at
LLOQ 0.1 mg/L.
Urine Hydrolysis (acid), GC-MS Not fully validated Authentic urine speci- Seven major metabolites Grigoryev et al.
LLE, TMS- due to lack of mens from two volun- and unspecified (2012)
derivatized metabolite teers (self-experiment) number of minor
standards. who ingested 0.22 and metabolites detected.
0.55 mg/kg AB-001
Urine LLE/SPE LC-MS/MS-ESI+ Interference Stability Authentic urine samples Extracts stabled at cooled Heltsley et al.
collected from US temperature for 24 h. (2012)
athletes (n ¼ 5496) LOD ¼ 1 mg/L.
Synthetic cannabinoid
metabolites were
detected in 4.5%
(n ¼ 266) samples with
50% of the samples
contained JWH-018
and JWH-073 metab-
olites while 49% con-
tained JWH-018
metabolites only.
Urine Hydrolysis, LLE LC-MS/MS-ESI+, Method not fully Authentic urine speci- Identified metabolites Hutter et al.
LC-TOF/MS-ESI+ validated; utilized mens from patient were further charac- (2012a)
for MetID with positive drug terized by LC-
test (serum) TOF-MS. Metabolites
of JWH-081, JWH-
210, JWH-250 and
RCS-4 also were
detected in urine sam-
ples by LC-TOF-MS.
Hair Washing LLE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic hair specimens Stability not evaluated. Hutter et al.
Selectivity, (n ¼ 8) from inpatient LOQ ¼ 0.5 pg/mg. (2012b)
Specificity psychiatric ward Hair samples were
patients with positive positive for 1 SC
SC in serum highest concentration
of 78 pg/mg JWH-081.
Other analytes
detected 4LLOQ
(continued )
Drug Metab Rev, Early Online: 1–51

QUAL
or
Analyte(s) QUANT
methanandamide,
RCS-4, RCS-4 ortho
isomer, RCS-8, WIN
48 098, WIN 55 212-2
RCS-4 and metabolites QUAL
AM694, AM1220, QUANT
AM2201, AM2233,
CRA-13, JWH-007,
JWH-015, JWH-018,
JWH-018 adamantyl
derivative, JWH-019,
JWH-020, JWH-073,
JWH-081, JWH-122,
JWH-200, JWH-203,
JWH-210, JWH-250,
JWH-251, JWH-307,
JWH-387, JWH-398,
MAM2201, metha-
nandamide, RCS-4,
RCS-4 ortho isomer,
RCS-8, WIN 48 098,
WIN 55 212-2
AM694, AM1220, QUANT
AM2201, AM2233,
CRA-13, JWH-007,
JWH-015, JWH-018,
JWH-018 adamantyl
derivative, JWH-019,
JWH-020, JWH-073,
JWH-081, JWH-122,
JWH-200, JWH-203,
JWH-210, JWH-250,
JWH-251, JWH-307,
JWH-387, JWH-398,
MAM2201, metha-
nandamide, RCS-4,
RCS-4 ortho isomer,
RCS-8, WIN 48 098,
WIN 55 212-2
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For personal use only.
Matrix Sample preparation Method Validation Application Study findings References
(5.0 pg/mg) were
JWH-018, JWH-073,
JWH-210 and JWH-
250. Analytes also
were detected in the
washing solvents
DOI: 10.3109/03602532.2015.1029635
(water and acetone).
Urine Hydrolysis, LLE, GC-MS Method not fully Authentic urine speci- Stability not evaluated. Kavanagh et al.
methylation, TMS- validated due to mens (n ¼ 7) collected 16 RCS-4 metabolites (2012)
derivatization, lack of standards; patients suspected of identified with RCS-4-
acylation MetID purpose SC intake N-hydroxypentyl
prevalent
OF LLE LC-MS/MS-ESI+ LOD, LOQ, Authentic OF specimens Extracts were stable up to Kneisel et al.
Selectivity, (n ¼ 264) collected 7 h in autosampler at (2012)
Linearity, between December 10  C. Instability
Imprecision, Bias, 2010 and January observed in collection
Recovery, ME, 2012 pads stored between
Process Efficiency, 22–27  C, but
Stability, improved with etha-
Carryover, nol. LOD ¼ 0.015–
Recovery (collec- 0.9 mg/L.
tion pad) LOQ ¼ 0.15–30 mg/L.
SC (n ¼ 12) were
detected in 12% of
samples.
Serum LLE LC-MS/MS-ESI+ LOD, LOQ, Authentic serum speci- Extracts stable after 9 h Kneisel &
Selectivity, mens (n ¼ 833) autosampler storage at Auwarter
Linearity, between August 2011 10  C, three freeze/ (2012)
Imprecision, Bias, and January 2012 col- thaw cycles and at
Recovery, ME, lected from forensic 14 days storage at
Process Efficiency, psychiatric and 20  C. Validated
Stability, rehabilitation clinics, method was employed
Carryover criminal investigation in the. LOD ¼ 0.01–
cases and ER visits 2.0 mg/L. LOQ ¼ 0.1–
2 mg/L. In authentic
samples, the most
prevalent analyte
identified was JWH-
210 (80%) and JWH-
122 (64%) with 11
different SC. Highest
concentration quanti-
fied was 230 mg/L for
JWH-122.
Synthetic cannabinoids
(continued )
23

QUAL
or
Analyte(s) QUANT
CBD, CBN, HU-210, QUANT
JWH-018, JWH-073,
JWH-200, JWH-250,
THC
JWH-018, JWH-073 QUANT
AM2201 and metabolites: QUAL
JWH-018-N-(5-OH-
pentyl), dihydrodiol
JWH-018, di-OH-
AM2201, dihydrodiol-
AM2201, OH-
AM2201, despentyl-
AM2201, JWH-018-
N-COOH; UR-144 and
metabolites: despen-
tyl-OH-UR-144, di-
OH-UR-144, despen-
tyl-UR-144, dehy-
drated OH-UR-144,
OH-UR-144
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24
Table 2. Continued
Matrix Sample preparation Method Validation Application Study findings References
Hair Washing, NaOH LC-MS/MS-ESI+ LOD, LOQ, Authentic hair specimens Stability not evaluated. Salomone et al.
digestion, LLE Selectivity, (n ¼ 179) from foren- LOD ¼ 0.02–0.18 pg/ (2012)
Linearity, sic cases mg; LOQ ¼ 0.07–
Imprecision, Bias, 18 pg/mg. In authentic
ME, Carryover hair samples, nine
M. S. Castaneto et al.
samples confirmed for
JWH-018, JWH-073
(n ¼ 8), and JWH-250
(n ¼ 8). Highest SC
concentration was
729.4 pg/mg for
JWH-250.
WB LLE LC-MS/MS-ESI+ LOD, LOQ, Authentic post-mortem Stability not evaluated. Shanks et al.
Selectivity, WB specimens LOD ¼ 0.01 mg/L, (2012)
Linearity, (n ¼ 18) LOQ ¼ 0.05 mg/L.
Imprecision, Bias, JWH-018 and/or
Ion suppression, JWH-073 in 40% of
ME, Interference, the cases. Highest
Carryover JWH-018 and JWH-
073 (mg/L) 199 and
68.3, respectively, in
cardiac blood.
Urine Hydrolysis, LLE LC-MS/MS-ESI+ Method not fully Authentic urine speci- Stability not evaluated. Sobolevsky et al.
validated due to mens containing Urine specimens posi- (2012)
lack of standards; UR-144 (n ¼ 5) and tive with (+) AM2201
MetID purpose AM2201 (n ¼ 6) metabolites also (+)
metabolites, suspected for JWH-018, JWH-
SC users. 073 and JWH-210
metabolites.
Defluorinated
AM2201 metabolites
were excluded.
Monohydroxylated
and dihydrodiol
AM2201 metabolites
including conjugated
metabolites were
observed in AM2201
(+) urine samples.
UR-144 parent was
detected at low levels
in 1 of 5 urine (+) for
UR-144 metabolites.
Predominant metabol-
ites identified were
conjugated mono-
and di-OH UR-144
metabolites.
(continued )
Drug Metab Rev, Early Online: 1–51
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QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
4-fluoro-AMP, 4-methyl- QUAL OF Dilution LC-MS/MS-ESI+ LOD, Specificity, None SC stable for 2 months Strano-Rossi et al.
AMP, 4-methyl- ME, Ion suppres- after eight freeze/thaw (2012)
N-ethylcathinone, sion/enhancement, cycles and at RT for 4
AM694, BZP, cloben- Linearity, Memory weeks when stored in
zorex, CP47, 497, fen- effect, Stability mobile phase except
proporex, furfenorex, for CP47, 497 and HU-
DOI: 10.3109/03602532.2015.1029635

HU-210, JWH-018, 210 52 weeks stabil-

JWH-019, JWH-073, ity. LOD ¼ 1–20 mg/L.
JWH-122, JWH-200,
JWH-250, MBDB,
MDPV, mephedrone,
mephenorex, methy-
lone, methylthioAMP,
MDAI, nabilone
JWH-018-N-COOH, QUAL and Urine Hydrolysis, LLE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine speci- Stability not evaluated. Yanes & Lovett
JWH-018-N-4-OH- QUANT Selectivity, Cross mens (n ¼ 500) LOQ ¼ 4 mg/L. JWH- (2012)
pentyl, JWH-073-N- talk, ME, random workplace 018 and JWH-073
COOH, JWH-073- Recovery, drug testing and/or metabolites were
N-3-OH-butyl Carryover forensic cases identified with con-
centrations ranging
from 5.4 to 37.8 mg/L;
although positivity
rate was low (number
not specified).
Blood: UR-144 and its QUANT WB, Urine Hydrolysis (urine LC-MS/MS-ES+ LOQ, LOQ, Authentic WB and urine Stability not evaluated. Adamowicz et al.
pyrolysis product: 1- only), LLE (blood), LC- Imprecision, Bias, specimens from a LOD ¼ 0.15 mg/L; (2013)
(1-pentyl-1H-indol-3- TOFMS-ESI+ Specificity, patient in the hospital LOQ ¼ 0.5 mg/L. UR-
yl)-3-methyl-2- (urine) Recovery, ME for SC intoxication. 144 at 6.1 mg/L was
(propan-2yl)but-3-en- quantified in blood
1-one; Urine: UR-144, and its pyrolysis prod-
despentyl-UR-144, uct (concentration not
despentyl-OH-UR- reported), while OH-
144, dehydrated-OH- UR-144, UR-144-
UR-144, OH-UR-144, N-COOH and di-OH-
UR-144-N-COOH, UR-144 detected in
di-OH-UR-144, UR- urine.
144-N-5-OH-pentyl-
b-Gluc
Metabolites for LC-MS/ QUAL Urine Hydrolysis, LLE ELISA, LC-MS/MS- Sensitivity Linearity, Authentic urine speci- ELISA designed to have Arnston et al.
MS only: JWH-018- ESI+ Interference, mens (n ¼ 63, previ- 100% CXR with JWH- (2013)
N-4- and 5-OH-pentyl, Stability, Precision ously confirmed SC 018-N-5-OH-pentyl
JWH-019-N-5- and 6- and Drift, positive) was evaluated using
OH-hexyl, JWH-073- Carryover 5 mg/L cutoff and per-
N-3- and 4-OH-butyl, formance determined
JWH-250-N-4-OH- by analyzing authentic
pentyl and AM2201- urine samples previ-
N-4-OH-pentyl ously confirmed (+)
via LC-MS/MS for
JWH-018-N-4- and
Synthetic cannabinoids

(continued )
25

QUAL
or
Analyte(s) QUANT
CP47, 497, CP47, 497- QUANT
C8, HU-211JWH-018,
JWH-073, JWH-200,
JWH-250, THC
SC: AM1220, AM2201, QUANT
AM694, JWH-015,
JWH-018, JWH-019,
JWH-073, JWH-081,
JWH-122, JWH-200,
JWH-203, JWH-210,
JWH-250, JWH-251,
JWH-307 and other
NPS: a-PPP, 4-FMA,
4-MEC, butylone,
BZP, DMA, ehthyphe-
nidate, MBZP, mCPP,
MDPV, mephedrone,
methcathinone,
methylone, naphyrone,
pentedrone, pFPP,
TFMPP
AM694, JWH-018, JWH- QUAL
073, JWH-122, JWH-
081, JWH-200, JWH-
210, JWH-250
UR-144-N-OH/-COOH, QUAL
UR-144-OH-desalkyl
and UR-144 major
pyrolysis product
metabolites formed
from hydration and
hydroxylation
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26
Table 2. Continued
Matrix Sample preparation Method Validation Application Study findings References
5-OH-pentyl (0.1 mg/L
cutoff). ELISA had
96% sensitivity, 100%
specificity and 98%
accuracy. Cross-
M. S. Castaneto et al.
reactivity (41%) 20
SC metabolites.
OF SPE LC-MS/MS-ESI+/ LOQ, LOQ, Authentic OF (n ¼ 40) Stability not evaluated. De Castro et al.
Imprecision, Bias, specimens from LOD ¼ 0.025–1.0 mg/ (2013)
Specificity, drivers L; LOQ ¼ 0.1–2.5 mg/
Recovery, ME L. No samples con-
firmed for SC while
THC was quantified in
20 samples.
Serum LLE LC-MS/MS-ESI+/ LOD, LOQ, Linearity, Authentic serum Stability experiments Dziasdosz et al.
Imprecision, Bias, specimen were performed (2013)
Stability, ME according to Ammann
et al. (2012).
LOD ¼ 0.02–0.4 mg/L;
LOQ ¼ 0.05–0.5 mg/L.
Analytes were 75–89%
stable at these condi-
tions. Authentic serum
negative for SC.
Hair Base hydrolysis, LLE LC-TOF-MS-ESI+ Extraction recovery Authentic hair (n ¼ 435) Stability not evaluated. Gottardo et al.
specimens from DUID LOD ¼ 10 pg/mg (2013)
suspects Among these, eight
were (+) for JWH-
018, JWH-073, JWH-
081, JWH-122 and
JW250 at 0.010–
1.28 ng/mg.
Urine Hydrolysis, TMS- GC-MS, LC-MS/MS Method not fully Authentic urine speci- UR-144 and metabolites Grigoryev et al.
derivatized validated due to mens (n ¼ 45) from EI spectra were (2013b)
lack of available SC suspected users included based on
standards previous studies cited
in (Sobolevsky et al.,
2012). UR-144 metab-
olites (n ¼ 16) and its
major pyrolysis prod-
uct (n ¼ 21) detected.
(continued )
Drug Metab Rev, Early Online: 1–51
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QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
SC: AM694, AM1241, QUAL WB, SPE LC-TOFMS-ESI+ Extraction recovery, Authentic WB (n ¼ 5), Method was developed Guale et al. (2013)
AM2201, JWH-007, Urine, ME serum (n ¼ 11), urine and validated to screen
JWH-015, JWH-016, Serum (n ¼ 5) specimens for wide array of sub-
JWH-018, JWH-018- confirmed other DOA; stances including anti-
DOI: 10.3109/03602532.2015.1029635

6-MeO, JWH-022, none confirmed for SC depressant, antihista-

JWH-073, JWH-081,
JWH-098, JWH-122,
JWH-210, JWH-200,
JWH-203, JWH-250,
RCS-4, RCS-8,
WIN48 098
Metabolites only: JWH- QUAL and
018-N-4- and 5-OH- QUANT
pentyl, JWH-018-
N-COOH, JWH-018-
6-OH-indole, JWH-
073-N-3- and 4-OH-
butyl, JWH-073-N-
N-COOH, JWH-073-
6-OH-indole
JWH-018 and metabol- QUANT
ites: JWH-018-N-4-
and 5-OH-pentyl,
JWH-018-N-N-
COOH; JWH-073 and
metabolites: JWH-
073-N-3- and -4-OH-
butyl, JWH-073-
N-COOH
mines, benzodiazep-
ines, hypnotics,
muscle relaxants, over-
the-counter medica-
tions and stimulants
(including synthetic
cathinones) utilizing
a personal compound
database library soft-
ware. Stability N/D.
Drugs were correctly
identified in all sam-
ples except for one
(butalbital).
Urine Dilution, Hydrolysis, ELISA, LC-MS/MS- Selectivity Authentic urine speci- Analytes stability (forti- Jang et al. (2013)
SPE ESI+ LOD, LOQ, Linearity, mens (n ¼ 52) were fied negative blanks)
Bias, Imprecision, first screened with was acceptable after
Recovery, Stability ELISA and confirmed three freeze/thaw
by LC-MS/MS. cycles, at 4  C and
20  C.
LOD ¼ 0.025–0.1 mg/
L; LOQ ¼ 2.5 mg/L. In
urine specimens, 27 of
52 confirmed positive
for 1 SC metabolite.
Hair Wash, LLE LC-MS/MS-ESI+ Selectivity Authentic human hair Analytes were stable Kim et al. (2013)
LOD, LOQ, Linearity, specimens (n ¼ 18) during sample prepar-
Bias, Imprecision, from SC suspected ation and extraction up
Recovery, Process users to 24 h. LOD/
Efficiency, LOQ ¼ 0.05 pg/mg.
Stability Only JWH-018, JWH-
018-N-5-OH-pentyl,
and JWH-073 were
detected in human
hair. No significant
difference in analyte
concentrations
between pigmented
and non-pigmented
hair.

(continued )
Synthetic cannabinoids
27

QUAL
or
Analyte(s) QUANT
AM694, AM1220, QUANT
AM2233, AM2201,
JWH-007, JWH-015,
JWH-018, JWH-019,
JWH-020, JWH-073,
JWH-081, JWH-122,
JWH-200, JWH-203,
JWH-210, JWH-250,
JWH-251, JWH-307,
JWH-387, JWH-398,
JWH-412, MAM2201,
methanandamide,
RCS-4, RCS-4 ortho
isomer, RCS-8, WIN
48 098, WIN 55 212-2
AB-001, AM694, QUANT
AM1220, AM1241,
AM2201, AM2233,
JWH-007, JWH-015,
JWH-018, JWH-019,
JWH-020, JWH-073,
JWH-073-methyl,
JWH-081, JWH-098,
JWH-122, JWH-147,
JWH-200, JWH-210,
JWH-250, JWH-251,
JWH-398, MAM2201,
RCS-4, RCS-4 ortho,
RCS-8, UR-144,
WIN55,212-2
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28
Table 2. Continued
Matrix Sample preparation Method Validation Application Study findings References
OF Protein precipitation LC-MS/MS-ESI+ Selectivity Authentic OF specimens Extracts stable in the Kneisel et al.
with ACN LOD, LOQ, Linearity, collected from 2 autosampler for 9 h at (2013b)
Bias, ME volunteers 10  C, during three
Imprecision, freeze/thaw cycles
M. S. Castaneto et al.
Recovery (extrac- (except for JWH-307
tion and collection 68–73%), and long-
device), Process term storage at 20  C
Efficiency, (except for JWH-307
Stability 70–76%). Stability
Carryover studies conducted with
authentic OF contain-
ing 11 analytes in
Kneisel et al. (2013a).
Samples were pooled,
homogenized, ali-
quoted into glass and
propylene collection
tubes and stored up to
72 h at 4 or 25  C. No
analyte instability
observed for glass.
Analytes were stable
at 4  C after 72 h
(except for JWH-251
and JWH-203 77–79%
after 72 h). All ana-
lytes except JWH-200
were unstable 563% at
25  C after 24 h and
72 h. LOD ¼ 0.015–
0.9 mg/L;
LOQ ¼ 0.15–3 mg/L.
WB LLE LC-MS/MS-ESI Selectivity Authentic WB (n ¼ 3078) Stability not evaluated. Kronstrand et al.
LOD, LOQ, Linearity, specimens collected LOD/LOQ not speci- (2013)
Bias, ME, from DUID suspects, fied. In authentic WB
Imprecision and petty drug offense (collected between
Dilution integrity October 2011 and
January 2013), 28%
WB confirmed for SC
(AB-001, AM-694,
AM2201, AM-2233,
JWH-018, JWH-019,
JWH-081, JWH-122,
JWH-203, JWH-210,
JWH-250, MAM2201,
RCS-4 and UR-144)
(continued )
Drug Metab Rev, Early Online: 1–51

QUAL
or
Analyte(s) QUANT
Metabolites only: JWH- QUANT
018-N-4- and 5-OH-
pentyl, JWH-018-
N-COOH, JWH-018
methyl ester, JWH-
072-N-COOH, JWH-
073-N-3-OH-butyl,
JWH-073-N-
N-COOH
Metabolites of AM2201 QUAL
and JWH-018 (not
specified)
AM694, AM2201, HU- QUAL
210, JWH-015, JWH-
018, JWH-019, JWH-
020, JWH-073, JWH-
081, JWH-122, JWH-
200, JWH-210, JWH-
250, JWH-251, RCS-
4, RCS-4-C4, RCS-8,
WIN55,212-2
(S)-AM2201-N-4-OH- QUANT
pentyl, (R)-AM2201-
N-4-OH-pentyl, JWH-
018-N-5-OH-pentyl,
JWH-018-N-COOH,
(S)-JWH-018-N-4-
OH-pentyl, (R)-JWH-
018-N-4-OH-pentyl
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Matrix Sample preparation Method Validation Application Study findings References
Urine Hydrolysis, SALLE, LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine speci- Stability not evaluated. Lovett et al.
repeated extraction Selectivity, Cross mens (n ¼ 30) Method validated as (2013)
talk, ME, described in Yanes &
DOI: 10.3109/03602532.2015.1029635
Recovery, Lovett (2012).
Carryover LOQ ¼ 4 mg/L. JWH-
072-N-COOH acid
was identified in sam-
ples also (+) for
AM2201, JWH-018
and JWH-073 metab-
olites, suggesting a
common biomarker for
all three SC. In this
study, JWH-072-
N-COOH was synthe-
sized in-house.
Urine Hydrolysis, LLE LC-HRMS Method not fully Authentic urine sample Stability not evaluated. Mcquade et al.
validated. from SC intoxicated AM2201-OH metabol- (2013)
patient ites were detected in
specimen.
OF LLE LC-MS/MS-ESI+ LOD, LOQ, Authentic OF specimens Stability not evaluated. Oiestad et al.
Specificity, (n ¼ 45) from drivers LOD ¼ 0.05–1.2 mg/L. (2013)
Imprecision, suspected Collection recovery
ME, Recovery was 19–61% (majority
(extraction and 30–40%). In authentic
collection device) OF specimens, 20%
were positive for
JWH-018 and/or
AM2201.
WB Hydrolysis, SPE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine speci- Stability not evaluated. Patton et al.
Imprecision, Bias, mens (n ¼ 15) from LOQ ¼ 0.7–1.53 mg/L. (2013b)
ME, Carryover, SC suspected users Method employed for
chiral metabolites dis-
tinction of AM2201
and JWH-018.
Baseline separation
was achieved. In
authentic specimens,
the majority of metab-
olites detected were
UDP-UGT conjugated,
S and R chiral metab-
olites were separated
and determined.
(continued )
Synthetic cannabinoids
29

QUAL
or
Analyte(s) QUANT
AM2201, AM2201-N-4- QUANT
OH-pentyl, JWH-018-
N-4- and 5-OH-pentyl,
JWH-018-N-COOH
QL: JWH-018, QT: QUAL,
AM2201, CP47, 497, QUANT
CP47, 497-C8, HU-
210, JWH-018, JWH-
073, JWH-081, JWH-
200, JWH-250, RCS-4
54 SC/85 NPS analytes: QUAL
AB-0101, AM694,
AM1220, AM1220
azepane isomer,
AM2201, CP47, 497,
CP47, 497-C8,
CP55,940, HU-210,
JWH-007, JWH-015,
JWH-018, JWH-018-
1-methyl-hexyl, JWH-
018-6-methoxy-indole,
JWH-018-4-, 5-, 6-
and 7-OH-indole,
JWH-018-N- 4- and 5-
OH-pentyl, JWH-018-
N- COOH, JWH-019,
JWH-073, JWH-073
2- and 3-methyl hom-
ology, JWH-073-N-3-
and 4-OH-butyl, JWH-
073-4-, 5-, 6-, and
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30
Table 2. Continued
Matrix Sample preparation Method Validation Application Study findings References
WB Protein precipitation LC-MS/MS-ESI+ Method validation Authentic post-mortem Stability not evaluated. In Patton et al.
parameters not WB SC (AM2201) blood, 2.5 mg/L (2013a)
specified intoxication AM2201, 123 mg/L
JWH-018-N-5-OH-
pentyl and 50.8 mg/L
M. S. Castaneto et al.
JWH-018-N-COOH.
Unidentified AM2201
metabolite also was
detected.
OF Dilution ELISA (QL), LC-MS/ LOD, LOQ, Authentic OF specimens ELISA performance eval- Rodrigues et al.
MS-ESI+ Selectivity, (n ¼ 32) collected for uated against LC-MS/ (2013)
Imprecision probation and parole MS results and
Stability, ME, achieved 84.0% sensi-
Recovery tivity, 100% specifi-
(collection pad) city, and 84.3%
efficiency at 0.25 mg/L
cutoffs. Cross-reactiv-
ity (410%) for JWH-
015, JWH-018, JWH-
022, JWH-073,
AM1220, AM2201,
AM2232. For LC-MS/
MS validation, ana-
lytes in OF were
60% at RT, 4  C and
20  C after 7 days.
LOD ¼ 0.1 mg/L,
LOQ ¼ 0.25 mg/L.
Urine Hydrolysis, SPE LC-TOF-MS-ESI+ Cutoff selection, ME, Authentic urine speci- Stability not evaluated. Sundström et al.
Recovery (extrac- mens (n ¼ 50) from Method utilized in- (2013)
tion), PE, Stability suspected NPS users house library with 277
compound entries (85
NPS with no available
standards).
Cutoff ¼ 0.2–30 mg/L.
In authentic speci-
mens, 26 confirmed
for SC metabolites
(primarily OH-pentyl
and COOH).
(continued )
Drug Metab Rev, Early Online: 1–51
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QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
7-OH-indole, JWH-
073-N-COOH, JWH-
081, JWH-122, JWH-
122-N-5-OH-pentyl,
JWH-147, JWH-200,
JWH-200-4-OH-
DOI: 10.3109/03602532.2015.1029635

indole, JWH-201,
JWH-210, JWH-203,
JWH-250, JWH-251,
JWH-302, JWH-398,
JWH-412, MAM2201,
RCS-4, RCS-4 ortho
isomer, RCS-4-N-5-
OH-pentyl, RCS-4-
N-COOH, RCS-8,
WIN48 098,
WIN55 212
AM2201, AM2201-6- QUAL Urine Dilution, Hydrolysis LC-MS/MS-ESI+ LOD, Linearity, Authentic urine speci- Analytes were identified (Wohlfarth et al.,
OH-indole, AM2201- Imprecision, Bias, mens (n ¼ 2501) using in-house library. 2013b, 2014c)
N-4-OH-pentyl, JWH- Interference, random workplace LOD ¼ 1–10 mg/L.
018, JWH-018 5- and Carryover drug testing Extracts stable at 4  C
6-OH-indole, JWH- ME, Ion acceptable up to 72 h.
018-N-COOH, JWH- Suppression or 290 confirmed posi-
018-N-5-OH-pentyl, Enhancement, tive for SC analytes.
JWH-073, JWH-073- Stability, Only one specimen
5- and 6-OH-indole, Carryover confirmed for
JWH-073-N-4-OH- AM2201 parent
butyl, JWH-073-N- analyte.
COOH, JWH-081,
JWH-081-N-5-OH-
pentyl, JWH-122,
JWH-122-N-5-OH-
pentyl, JWH-200-5-
and 6-OH-indole,
JWH-210, JWH-210-
N-4- and 5-OH-pentyl,
JWH-210-N-COOH,
JWH-250-5-OH-
indole, JWH-250-N-4-
and 5-OH-pentyl,
MAM2201, RCS-4,
RCS-4-N-5-OH-
pentyl, RCS-4-
N-COOH
AM2201, AM694, JWH- QUANT WB LLE LC-MS/MS-ESI+ LOD, Linearity, Authentic WB specimens Stability not evaluated. Yeakel & Logan
018, JWH-019, JWH- Imprecision, Bias, (n ¼ 12) from DUID LC-MS/MS was vali- (2013)
073, JWH-081, JWH- Interference, cases positive for SC dated in Kacinko et al.
122, JWH-175, Carryover, ME, (2011) with additional
JWH-200, JWH-210, Ion Suppression/ parent analytes.
Synthetic cannabinoids

(continued )
31
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32

Table 2. Continued

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
JWH-250, RCS-4, Enhancement LOQ ¼ 0.1 mg/L. In
RCS-8 Carryover authentic WB speci-
mens, all positive for
one or more SC
including AM2201,
M. S. Castaneto et al.

XLR11, XLR11 4-OH- QUANT
pentyl, XLR11 degra-
dant (pyrolysis prod-
uct), UR-144, UR-144
degradant (pyrolysis
product), UR-144
4-OH-pentyl
JWH-018-N-COOH (cali- QUAL,
brator); LC-MS/MS QUANT
(Wohlfarth et al.,
2013b)
JWH-018, JWH-081,
JWH-122, JWH-210
and JWH-250
OF SPE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic OF specimens Extracts stable for 24 h at (Amaratunga
Selectivity, Bias, (n ¼ 498) 15  C. Analytes also et al., 2014)
Imprecision, stable in three freeze/
Dilution Integrity, thaw cycles and long-
ME, Stability, term storage for 1
Recovery (extrac- month at 20  C. No
tion and collection pyrolysis products
pad) detected in buffer
samples fortified with
parent analyte.
LOD ¼ 0.35–
1.93 mg/L;
LOQ ¼ 5 mg/L. In
authentic OF speci-
mens, 14 confirmed
for UR-144 4-OH-
pentyl and UR-144
degradant (5–60 mg/L),
but no parent detected.
UR-144 (5–30 mg/L)
confirmed in 5 sam-
ples without metabol-
ites. XLR11 degradant
(55 to 4100 mg/L)
confirmed in 37 sam-
ples, of which 30 con-
tained XLR11
(55–100 mg/L).
Urine HEIA: none HEIA (QL), LC-MS/ LOD, Linearity, Authentic urine speci- HEIA performance eval- Barnes et al.
LC-MS/MS: dilution, MS Cutoff selection, mens (n ¼ 2443) uated by LC-MS/MS (2014)
hydrolysis, protein Interference, workplace drug testing data and achieved
precipitation Carryover 75.6% sensitivity,
99.6% specificity, and
96.8% efficiency at
10 mg/L cutoff. Cross-
reactivity (410%) for
AM1220, AM2201,
AM2201-N-4-OH-
pentyl, JWH-018-
N-OH-pentyl, JWH-
073-N-4-OH-butyl,

(continued )
Drug Metab Rev, Early Online: 1–51
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For personal use only.

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
JWH-073-N-COOH,
JWH-200, JWH-200-
6-OH-indole, JWH-
398-N-COOH,
MAM2201-N-COOH
5F-PB-22, AB-PINACA, QUANT WB LLE LC-MS/MS LOD, LOQ, Bias, Authentic ante- and post- Stability not evaluated. Behonick et al.
DOI: 10.3109/03602532.2015.1029635

ADB-PINACA, Imprecision, mortem WB speci- 5F-PB-22 was quanti- (2014)

AM2201, BB-22, Cl- Matrix selectivity, mens (n ¼ 4) fied in 2 ante- and
2201, JWH-015, JWH- Interference, 2 post-mortem blood
018, JWH-019, JWH- Carryover, Ion specimens with this
073, JWH-122, JWH- enhancement or method. Highest con-
210, JWH-250, suppression centration was
MAM2201, PB-22, 1.5 mg/L.
UR-144, XLR11
AM694, AM2201, QUANT Hair Washing, Digestion LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic hair specimens Stability not evaluated. Crimele et al.
CP47, 497, HU-210, Bias, Imprecision, (n ¼ 65) from routine LOQ ¼ 500 pg/mg. All (2014)
JWH-007, JWH-015, Carryover, Ion forensic cases authentic hair speci-
JWH-018, JWH-018- suppression or mens negative for SC.
N-COOH, JWH-019, enhancement
JWH-020, JWH-073,
JWH-081, JWH- 122,
JWH-200, JWH-203,
JWH-210, JWH-250,
WIN55,212-2
AB-FUBINACA, QUANT Urine Hydrolysis, Dilution LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine specimen Analytes stable at RT and Freijo. et al.
AM694-N-COOH, Bias, Imprecision, (n ¼ 1) collected from 4–6  C for 24 h. Long (2014)
AM1248, AM2201, Carryover, patient admitted for term storage stability
AM2201-N-4-OH- Dilution integrity, SC intoxication not evaluated. LOD/
pentyl, HU-210, JWH- ME, Interference, LOQ ¼ 1–5 mg/L. In
018-N-4-OH-pentyl, Stability authentic urine speci-
JWH-018-N-COOH, mens UR-144-
JWH-019, JWH-019- N-COOH quantified at
N-5-OH-hexyl, JWH- 1370 mg/L and XLR11
073-N-3-OH-butyl, 6-OH-indole at
JWH-073-N-COOH, 311 mg/L.
JWH-081, JWH-081-
N-5-OH-pentyl, JWH-
122, JWH-122-N-5-
OH-pentyl, JWH-200-
6-OH-indole, JWH-
120, JWH-210-N-4-
OH-pentyl, JWH-250-
N-4-OH-pentyl, PB-
22, RCS-4, RCS-4-
N-5-OH-pentyl, RCS-
8, UR-144, UR-144-
N-COOH, XLR11,
XLR11-6-OH-indole,
XLR12
Synthetic cannabinoids

(continued )
33
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Table 2. Continued
34

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
57 SC (including PB-22 QUANT Serum Not specified LC-TOFMS Method validation Authentic serum speci- Quantified PB-22 at Gugelmann et al.
and UR-144- data not available. mens from a patient 55 mg/L, UR-144- (2014)
N-COOH), not and his dog with his- N-COOH 58 mg/L and
enumerated tory of synthetic can- UR-144N-4-OH-
nabinoid (‘‘Crazy pentyl 22 mg/L in
Monkey’’) serum upon ER arri-
M. S. Castaneto et al.

val, though (-) SC in

human urine upon
arrival.
5F-AKB-48, AB-001, QUAL Serum LLE LC-TOFMS-ESI+ LOD, ME Authentic serum speci- Stability not evaluated. Huppertz et al.
AKB-48, AM694, mens (n ¼ 30) from LOD ¼ 0.1–0.25 mg/L. (2014)
AM1220, AM1220 forensic cases Only one LC-MS/MS
azepane isomer, confirmed sample
AM2201, AM2232, confirmed negative
AM2233, APICA, with LC-QTOF-MS.
Cannabipiperidiethan-
one, CRA-13, JWH-
007, JWH-015, JWH-
018, JWH-019, JWH-
020, JWH-022, JWH-
073, JWH-081, JWH-
122, JWH-182, JWH-
200, JWH-203, JWH-
210, JWH-250, JWH-
251, JWH-307, JWH-
370, JWH-387, JWH-
398, JWH-412,
MAM2201,
Methanandamide,
RCS-4, RCS-4-C4,
RCS-4 ortho isomer,
RCS-8, STS-135, UR-
144, UR-144 isomer,
WIN48 098,
WIN55,212-2,
XLR11, XLR11
isomer
AM2201-N-4-OH-pentyl, QUANT Urine Hydrolysis, SPE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic urine speci- Stability not evaluated. Jang et al. (2014b)
AM2201-6-OH- Bias, Imprecision, mens from suspected LOD ¼ 0.1 mg/L;
indole, JWH-018 -N-4 Carryover, ME, JWH-018 (n ¼ 11) and LOQ ¼ 2.5 mg/L. In
and 5-OH-pentyl, PE, Recovery, AM2201 (n ¼ 9) users JWH-018 specimens,
JWH-018-N-COOH, Selectivity, JWH-018-N-4-OH-
JWH-018-6-OH- Stability pentyl confirmed in all
indole, JWH-018-N-5- specimens while JWH-
OH-pentyl b-Gluc, 018-N-COOH present
JWH-073-N-COOH in high concentration
in AM2201 speci-
mens. JWH-073-
N-COOH confirmed

(continued )
Drug Metab Rev, Early Online: 1–51

QUAL
or
Analyte(s) QUANT
AM2201, AM2201-N-4- QUANT
OH-pentyl, AM2201-
6-OH-indole, JWH-
018, JWH-018-N-4
and -5-OH-pentyl,
JWH-018-N-COOH,
JWH-073, JWH-073-
N-3- and 4-OH-butyl,
JWH-073-N-COOH,
JWH-122, JWH-122-
N-5-OH-pentyl, JWH-
122-N-COOH,
MAM2201,
MAM2201-N-4-OH-
pentyl
AM-2201-N-4-OH- QUAL
pentyl, AM-2201-6-
and 7-OH-indole,
JWH-018-N-4- and 5-
OH-pentyl, JWH-018-
5-, 6- and 7-OH-
indole, JWH-018-
N-COOH, JWH-019-
N-5- and 6-OH-hexyl,
JWH-073-N-3- and 4-
OH-butyl, JWH-073-
5-, 6- and 7-OH-
indole, JWH-073-
N-COOH, JWH-081-
N-5-OH-pentyl, JWH-
081-N-COOH, JWH-
122-N-4-OH-pentyl,
JWH-122-N-5-OH-
pentyl, JWH-210-N-4-
and 5-OH-pentyl,
JWH-210-N-COOH,
JWH-250-N-4- and 5-
OH-pentyl, JWH-250-
N-COOH, JWH-398-
N-4- and 5-OH-pentyl,
JWH-398-N-COOH,
MAM-2201-N-4-OH-
pentyl, MAM-2201-
N-COOH, RCS-4 4-
and 5-OH-pentyl,
RCS-4-N-COOH,
UR-144 4- and 5-OH-
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Matrix Sample preparation Method Validation Application Study findings References
humans exposed to
AM2201 or JWH-018.
Hair Washing, LLE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic human hair In-process stability for all Kim et al. (2014)
Bias, Imprecision, (n ¼ 9) specimens analytes acceptable
Carryover, ME, from suspected SC after 24 h.
PE, Recovery, users LOD ¼ 0.05 pg/mg;
Selectivity, LOQ ¼ 0.1 pg/mg. In
Stability hair, 7/9 suspects (+)
DOI: 10.3109/03602532.2015.1029635
AM2201 (3.7–
918 pg/mg) with con-
centration 44 JWH-
018-N-COOH
(0.2–1.1 pg/mg).
MAM2201
(0.9–258.2 pg/mg)
and JWH-122 (0.3–
888 pg/mL) concur-
rently quantified in 4
AM2201 (+) samples.
Urine SALLE HEIA Selectivity, ME, Authentic urine speci- HEIA performance Kronstrand et al.
LC-TOF-MS-ESI+ Cutoff selection, mens (n ¼ 87) from against LC-TOF-MS (2014)
Stability forensic cases was 86.8% sensitivity,
For HEIA: 81.6% specificity,
Linearity, efficiency of 83.9% at
Cross-reactivity 5 mg/L. Subsequently,
204 authentic speci-
mens were analyzed
by LC-QTOF-MS of
which 22 were positive
for 1 SC metabolites.
Metabolites were
stable515% loss at RT
for up to 2 days
(except for JWH-073-
7-OH-indole, 25%).
At 4  C, JWH-019-6-
OH indole and RCS-4-
N-5-OH-pentyl
increased by 46 and
21%, respectively. All
analytes stable at
20  C up to
15 weeks. In-house,
spectral library was
utilized to identify 38
SC metabolites.
Cutoff ¼ 2 mg/L
Synthetic cannabinoids
(continued )
35

QUAL
or
Analyte(s) QUANT
pentyl, UR-144-
N-COOH
OF, Plasma/Serum: JWH- QUAL
007, JWH-015, JWH-
018, JWH-073, JWH-
098, JWH-122, JWH-
182, JWH-200, JWH-
210- JWH-249, JWH-
250, JWH-251, JWH-
302, JWH-
424,WIN55,212-2;
Urine: JWH-018-4-
OH-indole, JWH-018-
N-5-OH-pentyl, JWH-
018-N-COOH, JWH-
073-4-OH-indole,
JWH-073-N-COOH
UR-144-N-COOH QUAL
(calibrator)
AM694, AM1220, QUANT
AM2201, HU-210,
JWH-007, JWH-015,
JWH-018, JWH-019,
JWH-020, JWH-073,
JWH-081, JWH-122,
JWH-200, JWH-203,
JWH-210, JWH-250,
JWH-251, JWH-307,
JWH-398, RCS-4,
RCS-8, WIN48 098,
WIN55,212-2
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For personal use only.
36
Table 2. Continued
Matrix Sample preparation Method Validation Application Study findings References
OF, Urine, LLE (OF); hydrolysis LC-MS/MS-ESI+ LOD, Specificity, Ion None Stability not evaluated. Mazzarino et al.
Plasma, (urine), protein suppression or Method validated for (2014)
Serum precipitation (WB) enhancement, SC quantification in
M. S. Castaneto et al.
Retention time plasma/serum and OF
repeatability, while metabolites of
Recovery, JWH-018 and JWH-
Robustness, 073 for urine.
Carryover LOD ¼ 0.2–0.5 mg/L
OF; 0.1–0.5 mg/L
plasma/serum;
0.3–0.4 mg/L urine.
Urine None ELISA Impression, Plate Authentic urine speci- ELISA cutoff established Mohr et al. (2014)
drift, Carryover, mens (n ¼ 5989) from at 5 mg/L. Performance
Stability forensic cases evaluated by confirm-
ing all presumptive
positive by LC-MS/
MS (Arnston et al.,
2013). Based on 3.6%
positivity rate,
ELISA’s overall sensi-
tivity was 86.6%.
Cross-reactivity 50%
for XLR-11-N-4-OH-
fluropentyl and UR-
144-N-4-OH-pentyl;
100% for UR-144-
N-COOH and UR-144-
N-5-OH-pentyl.
Hair Washing, Digestion, LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic hair specimens Stability not evaluated. Salomone et al.
LLE Bias, Imprecision, (n ¼ 344, previously LOD ¼ 0.2–1.3 pg/mg; (2014a)
Carryover, ME, analyzed for drugs of LOQ ¼ 0.7–4.3 pg/mg
PE, Recovery, abuse) (HU-210 80 pg/mg
Selectivity, LOQ). Authentic sam-
Specificity ples: 15 confirmed for
SC, majority was for
JWH-073 (1.6–
50.5 pg/mg) followed
by JWH-122 (n ¼ 8,
7.4–2800 pg/mg).
Other SC identified:
JWH-250, JWH-081,
JWH-018, JWH-210,
JWH-019, and
(continued )
Drug Metab Rev, Early Online: 1–51

QUAL
or
Analyte(s) QUANT
AM694, AM2201, QUANT
AM2201-6-OH-
indole, AM2201-
N-OH-pentyl,
CP47, 497-C7,
CP47, 497-C7-OH,
CP47, 497-C8,
CP47, 497-C8-OH
dimethyloctyl, HU-
210, JWH-018, JWH-
018 5- and 6-OH-
indole, JWH-018-
N-OH-pentyl, JWH-
018-N-COOH, JWH-
019, JWH-019-5-OH-
indole, JWH-019-
N-OH-hexyl, JWH-
073, JWH-073-5- and
6-OH-indole, JWH-
073-N- OH-butyl,
JWH-073-N-COOH,
JWH-081, JWH-081-
N-OH-pentyl, JWH-
122, JWH-122-N-OH-
pentyl, JWH-200-5-
and 6-OH-indole,
JWH-203, JWH-210,
JWH-210-5-OH-
indole, JWH-210-
N-OH-pentyl, JWH-
210-N-COOH, JWH-
250-5-OH-indole,
JWH-250-N-OH-
pentyl, JWH-250-
N-COOH, JWH-398,
JWH-398-N-OH-
pentyl, JWH-398-
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For personal use only.
Matrix Sample preparation Method Validation Application Study findings References
AM2201. Method was
updated to 9 SC
metabolites (Salomone
et al., 2014b). JWH-
122-N-5-OH-pentyl
was identified in 2/15
DOI: 10.3109/03602532.2015.1029635
samples at 0.72 and
2.5 pg/mg with corres-
ponding JWH-122
concentrations of 760
and 2800 pg/mg,
respectively.
Urine Dilution, Hydrolysis, LC-MS/MS-ESI+/ LOD, LOQ, Linearity, Authentic urine speci- Analytes were stable at Scheidweiler &
SLE Bias, Imprecision, mens (n ¼ 777) work- RT up to 16 h except Huestis (2014)
Carryover, ME, place drug testing for CP47, 497-C7,
Extraction effi- CP47, 497-C8, HU-
ciency, Specificity, 210 and JWH-200, at
Dilution integrity, 4  C for 72 h. All
Stability, metabolites stable
Carryover after 3 freeze/thaw
cycles while parents
were unstable except
for AM694,
CP47, 497-C7,
CP47, 497-C8, HU-
210, JWH-200 and
JWH-398. Baseline
separations for 12
alkyl OH isomers were
not obtained, therefore
were determined semi-
quantitatively.
LOD ¼ 0.05–1 mg/L;
LOQ ¼ 0.1–1 mg/L. In
authentic specimens,
290 confirmed for 22
SC metabolites at
concentration ranges
between 0.1–2434 mg/
L. Majority confirmed
for alkyl-OH and
COOH metabolites.
Synthetic cannabinoids
(continued )
37
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38

Table 2. Continued

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
N-COOH, MAM2201,
MAM2201-N-OH-
pentyl, MAM2201-
N-COOH, RCS-4,
RCS-4-N-OH-pentyl,
M. S. Castaneto et al.

RCS-4-M9, RCS-4-
M10, RCS-4-
N-COOH, UR-144-
N-OH-pentyl, UR-
144-N-COOH
Metabolites only: 5F-AB- QUAL Urine Dilution, Hydrolysis, LC-TOF-MS-ESI+ LOD, Specificity, Authentic urine speci- Employed SWATHTM Scheidweiler et al.
PINACA-N-OH- SLE+ Extraction effi- mens (n ¼ 8) (Sequential Windowed (2014)
pentyl, 5F-AKB48- ciency, ME, Acquisition of all
N-OH-pentyl, 5F-PB- Stability, Theoretical mass
22-3-COOH-indole, Carryover spectra) for non-tar-
AB-PINACA-N-OH- geted identification of
pentyl, AB-PINACA- SC metabolites in
N-COOH, ADB- urine. Analytes forti-
PINACA-N-OH- fied in blank urine
pentyl, AKB48-N-OH- with 1–20 LOD
pentyl, AKB48- concentrations were
N-COOH, AM2201-6- stable at RT after 24 h,
OH-indole, AM2201- at 4  C for 72 h
N-OH-pentyl, JWH- (including on auto-
018-5- and 6-OH- sampler), after 3
indole, JWH-018-N- freeze/thaw cycles.
OH-pentyl, JWH-018- Baseline chromato-
N-COOH, JWH-019- graphic resolution was
5-OH-indole, JWH- not obtained for alkyl
019-OH-hexyl, JWH- OH isomers.
073-5- and 6-OH- LOD ¼ 0.25–20 mg/L.
indole, JWH-073-N- In authentic urine spe-
COOH, JWH-073- cimens, identified up
N-OH-butyl, JWH- to 12 metabolites
081-N-OH-pentyl, including UR-144
JWH-122-N-OH- degradant-COOH.
pentyl, JWH-122-
N-COOH, JWH-200-
5- and 6-OH-indole,
JWH-210-N-COOH,
JWH-210-N-OH-
pentyl, JWH-250-5-
OH-indole, JWH-250-
N-OH-pentyl, JWH-
250-N-COOH, JWH-
398-N-OH-pentyl,
JWH-398-N-COOH,
MAM2201-N-OH-
pentyl, PB-22-3-

(continued )
Drug Metab Rev, Early Online: 1–51

QUAL
or
Analyte(s) QUANT
COOH-indole, PB-22-
N-OH-pentyl-3-
COOH-indole, PB-22-
N-COOH-3-COOH-
indole, PB-22-N-OH-
pentyl, PB-22-
N-COOH, RCS-4-M9,
RCS-4-N-OH-pentyl,
RCS-4-N-COOH, UR-
144 degradant-COOH,
UR-144-N-OH-pentyl,
UR-144-N-COOH,
XLR11-6-OH-indole,
XLR11-N-OH-pentyl
5F-PB-22, AM2233, BB- QUANT
22, JWH-122, PB-22
11-OH-THC, AM2201, QUANT
HU-210, JWH-018,
JWH-018-4- and 5-
OH-indole, JWH-018-
N-4- and -5-OH-
pentyl, JWH-073,
JWH-073-N-3- and 4-
OH-butyl, JWH-250,
THC, THCCOOH,
JWH-018-N-COOH QUAL
(calibrator)
AM694, AM2201, QUANT
CP47, 497, HU-210,
JWH-015, JWH-018,
JWH-019, JWH-020,
JWH-073, JWH-081,
JWH-122, JWH-200,
JWH-210, JWH-250,
JWH-251, RCS-4,
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
For personal use only.
Matrix Sample preparation Method Validation Application Study findings References
DOI: 10.3109/03602532.2015.1029635
Plasma LLE LC-HR-MS Method validation Authentic plasma speci- Stability not evaluated. Schep et al.
parameters not mens (n ¼ 2) from Blood samples were (2014)
detailed patient experiencing drawn at 5.5 and 8.3 h
seizures after smoking after ER admission.
SC-laced cigarette. All 5 SC confirmed at
9–148 ng/L (5.5 h) and
13–125 ng/L (8.3 h).
Urine Hydrolysis, SPE LC-MS/MS-ESI+ LOD, LOQ, Linearity, Authentic samples Stability not evaluated. Simoes et al.
Imprecision, Bias, (n ¼ 80) from individ- LOD ¼ 0.01–0.5 mg/L; (2014)
Selectivity, uals admitted to the LOQ ¼ 0.05–5 mg/L.
Recovery, ME ER or involved in In authentic samples,
forensic cases 5 confirmed for JWH-
018 and/or JWH-122
metabolites, THC
and/or THCCOOH.
Urine None ELISA LOD, Linearity, Authentic urine speci- ELISA performance by Spinelli et al.
Cutoff selection, mens (n ¼ 2469) LC-MS/MS data in (2014)
Plate drift, random workplace (Wohlfarth et al.,
Interference, drug testing 2014c). Performance
Carryover was 79.9% sensitivity,
99.7% specificity and
97.4% efficiency at
5 mg/L cutoff. ELISA
cross-reacted (410%)
with 18 of 73 SC
analytes evaluated.
WB LLE LC-MS/MS-ESI+ Method validation Authentic WB specimens Stability not evaluated. Tuv et al. (2014)
parameter not (n ¼ 726) from DUID Method validated in
specified suspects (Presley et al., 2013).
In WB specimens, 16
(2.2%) confirmed for
SC: AM2201, JWH-
018, JWH-081, JWH-
Synthetic cannabinoids
(continued )
39
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For personal use only.
40

Table 2. Continued

QUAL
or
Analyte(s) QUANT Matrix Sample preparation Method Validation Application Study findings References
RCS-4-C4, RCS-8, 122, JWH-250,
WIN55,212-2 RCS-4.
JWH-018, JWH-250 QUAL Urine None Biochip array tech- LOD, Linearity, Authentic urine speci- Biochip was labeled with Castaneto et al.
(calibrators) nology Imprecision, Bias, mens (n ¼ 20 017) 4 antibodies (3 for (2014a)
immunoassay Interference, from random work- JWH-018 SCI, II, and
M. S. Castaneto et al.

Cutoff place drug testing III, 1 for JWH-250,

Optimization, SCIV). Performance
Carryover evaluated with LC-
MS/MS results yielded
98.3% sensitivity,
48.1% specificity,
53.9% efficiency at
10 mg/L SCI, 20 mg/L
SCII, 5 mg/L SCIII/
SCIV cutoffs. After
cutoff optimization,
performance improved
to 87.6% sensitivity,
85.2% specificity,
85.4% efficiency at
15 mg/L SCI, 10 mg/L
SCIII (SCII and IV no
change). Cross-
reactivity (41%) 37
SC metabolites and
varied among
antibodies.

4-FMA: 4-fluoromethamphetamine; 4-MEC: 4-methylethcathinone; AMP: amphetamine; BZP: benzylpiperazine; CBD: cannabidiol; CBN: cannabinol; COOH: carboxy; DMA: dimethoxyamphetamine; DUID:
driving under the influence; EI: Electron Impact; ELISA: enzyme-linked immunosorbent assay; ER: emergency room; ESI+/: Positive/Negative Electrospray ionization; GC: Gas Chromatography; Gluc:
glucuronide; HEIA: homogenous enzyme immunoassay; HLM: human liver microsomes; HRMS: high-resolution mass spectrometry; LLE: Liquid–Liquid Extraction; LC: Liquid Chromatography; LOD: Limit
of Detection; LOQ: Limit of Quantification; LR: Linear Range; MBDB: methylbenzodioxyolybutanamine; MDAI: 5,6-methylenedioxy-2-aminoindane; MBZP: methylbenzylpiperazines; MDPV:
methylenedioxypyrovalerone; ME: matrix effects; MS: Mass Spectrometry; MS/MS: Tandem Mass Spectrometry; NaOH: sodium hydroxide; NPS: new psychoactive substances; OH: hydroxy; OF: Oral fluid;
pFPP: para-fluorophenylpiperazine; PE: process efficiency; PNEG: presumptive negative by immunoassay; PPOS: presumptive positive by immunoassay; PPP: pyrrolidinopropriophenone; QUAL: qualitative;
QUANT: quantitative; RT: Room Temperature; SALLE: salting-out assisted liquid–liquid extraction; SC: synthetic cannabinoids; SLE+: solid–liquid support extraction; SPE: Solid-Phase Extraction; TFMPP:
3-trifluoromethyphenylpiperazone; THC: delta-9-tetrahydrocannabinol; THCCOOH: THC-carboxy; TMS: trimethylsilation; TMCP: tetramethylcyclopropyl; TOF: time-of-flight; WB: Whole blood
Drug Metab Rev, Early Online: 1–51
 Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
For personal use only.

Table 3. Biotransformation of synthetic cannabinoids in vivo and in vitro.

Phase I: site of reaction (matrix) Phase II

Dihydrodiol Oxidative
Analyte Hydroxylation Carboxylation Dehydrogenation N-dealkylation formation Ketone formation O-demethylation defluorination Ring opening Conjugates
b
AB-001 Adamantane N-pentyl (urine)
DOI: 10.3109/03602532.2015.1029635

(urine)b
N-pentyl (urine)b
AB-FUBINACA Aminooxobutane
(HLM)c
ADB-FUBINACA Aminooxobutane
(HLM)c
AB-PINACA N-Pentyl and ami-
nooxobutane
(HLM)c
AKB-48 Adamantane N-pentyl (HEP)c GLUC (HLM)c
(HEP)c
N-pentyl (HEP)c
5F-AKB-48 Adamantane N-pentyl N-pentyl (HLM)c/ N-pentyl (HLM)c N-pentyl (HLM)c/ GLUC (urine)b
(HLM)c/(urine)b (HLM)c (urine)b (urine)b
Indazole alkyl
(HLM)c/(urine)b
AM694 N-pentyl (urine)b N-pentyl N-pentyl (urine)b
(urine)b
AM2201 N-fluoropentyl N-pentyl N-pentyl (HLM)c Naphthyl (HLM) N-pentyl (HLM)c/ GLUC (urine)b
(HLM)c (HLM)c (urine)b
Indole (HLM)c/
(urine)b
Naphthyl (HLM)c
CP55,940 Heptyl side chain
(HLM)a
JWH-015 Indole alkyl N-propyl N-propyl (RLM)c Naphthyl (RLM)c
(RLM)c (RLM)c
Naphthyl (RLM)c
JWH-018 Indole alkyl N-pentyl N-pentyl N-pentyl (HLM)c/ Naphthyl (HLM)c GLUC (HLM)c/
(HLM)c/ (HLM)c/ (HLM)c/ (blood)a/(urine)a,b (urine)b
(urine)a,b (blood)a/ (blood)a
Naphthyl (urine)a,b/
(HLM)c/(urine)b (HLM)a
JWH-073 N-butyl (HLM)c/ N-butyl GLUC (urine)b
(urine)b (urine)b
Indole (HLM)c
Naphthyl (HLM)c
JWH-073 4- Indole alkyl
methylnaphthoyl (HLM)c
(HLM)c
Naphthyl (HLM)c

(continued )
Synthetic cannabinoids
41
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Table 3. Continued
42

Phase I: site of reaction (matrix) Phase II

Dihydrodiol Oxidative
Analyte Hydroxylation Carboxylation Dehydrogenation N-dealkylation formation Ketone formation O-demethylation defluorination Ring opening Conjugates
a a a a
JWH-098 Indole alkyl (RLS) N-pentyl (RLS) N -pentyl (RLS) Naphthyl (RLS) GLUC (RLS)a
Naphthyl (RLS)a
JWH-122 Indole alkyl N-pentyl N-pentyl (HLM)c/ N-pentyl (HLM)c/ Naphthyl (HLM)c/ GLUC/SUL
(HLM)c/(urine)a (HLM)c (urine)a (urine)a (urine)a (urine)a
Naphthyl (HLM)c/
M. S. Castaneto et al.

(urine)a
JWH-200 N-ethyl (HLM)c N-ethyl Morpholine Naphthyl (HLM)c/ Morpholine GLUC/SUL
Morpholine (HLM)c/ (HLM)c/ (urine)a (HLM)c/ (urine)a
(HLM)c/(urine)a (urine)a (urine)a (urine)a
Indole (HLM)c N-ethyl (HLM)c
Naphthyl (HLM)c/
(urine)a
JWH-250 N-pentyl (urine)a,b N-pentyl N-pentyl (urine)a N-pentyl (urine)a UNSPb
Phenyl (urine)a (urine)b
PB-22 Ester hydrolysis N-pentyl QUL (HEP)c N-pentyl (HEP)c GLUC/CYS
(HLM)/(HEP)c (HEP)c (HEP)c
Indole alkyl (HEP)c
QUL (HEP)c
5F-PB-22 Ester hydrolysis N-pentyl QUL (HEP)c N-pentyl (HEP)c GLUC/CYS
(HEP)c (HEP)c (HEP)c
N-pentyl (HEP)c
QUL (HEP)c
RCS-4 Indole alkyl (HEP)c N-pentyl Phenyl (HEP)c GLUC/SUL
(HEP)c (HEP)c
RCS-8 Phenyl (HEP)c Phenyl (HEP)c GLUC (HEP)c
Cyclohexyl (HEP)c
STS-135 adamantane (HEP)c N-pentyl N-pentyl (HEP)c N-pentyl (HEP)c N-pentyl (HEP)c GLUC (HEP)c
N-pentyl (HEP)c (HEP)c
UR-144 N-pentyl (HLM)c/ N-pentyl N-pentyl (HLM)c N-pentyl (HLM)c Indole (HLM)c TMCP GLUC (urine)b
(urine)b (urine)b (urine)b
indole (HLM)c
WIN55,212-2 Indole (RLM)c Morpholine NAPH (RLM)c
Naphthyl (RLM)c (RLM)c
XLR-11 N-pentyl (HEP)c N-pentyl TMCP (HEP)c TMCP-HA/HK (HEP)c N-pentyl (HEP)c GLUC (HEP)b
Indole (HEP)c (HEP)c
TMCP (HEP)c TMCP (HEP)c

CYS: cysteine; HA: hemi-acetal; HK: hemi-ketal; GLUC: glucuronide; HEP: human hepatocytes; HLM: human liver microsomes; QUL: quinolinyl; RLS: rat liver slices; RLM: rat liver microsomes; SUL:
sulfate; TMCP: tetramethylcyclopropyl; UNSP: unspecified.
a
Identified in authentic animal samples.
b
Identified in authentic human samples.
c
Identified in vitro.
Drug Metab Rev, Early Online: 1–51
 DOI: 10.3109/03602532.2015.1029635
fragment(s) even without commercial standards availability.
No authentic urine samples were tested with the method.
Urine
We identified 32 methods (five immunoassays and 27 GC-
MS, LC-HRMS and LC-MS/MS procedures) for SC detection
in urine published between 2010 and 2014. The majority of
these required sample preparation including dilution, enzym-
atic hydrolysis, LLE, SALLE, SPE or SLE+ prior to analysis
(Table 2). Almost exclusively, metabolites were detected in
urine, with low parent concentrations, if identified (Grigoryev
et al., 2013b; Hutter et al., 2012a; Wohlfarth et al., 2014c).
Constantly emerging NPS and their growing chemical
diversity compelled analytical laboratories to develop chro-
matography mass spectrometry detection strategies as the first
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approach to confirming SC in biological specimens. Initially,
GC-MS and LC-MS/MS methods focused on single or a few
SC and metabolites (Chimalakonda et al., 2011b; ElSohly
et al., 2011; Grigoryev et al., 2011a,b), but it quickly became
apparent that capability to identify multiple SC was
necessary.
The first LC-MS/MS confirmation method quantified three
JWH-018 urinary metabolites from authentic urine specimens
(ElSohly et al., 2011); another method also included JWH-
073 metabolites (Chimalakonda et al., 2011b). Covering a
wider scope, an LC-MS/MS assay was developed to detect
For personal use only.
nine hydroxypentyl, hydroxyindole and carboxylated metab-
olites from 8 parent SC with 0.1 mg/L LOQ (De Jager et al.,
2012). Sample preparation included enzyme hydrolysis and
LLE. This method was employed in analyzing urine samples
from a volunteer who smoked an herbal blend laced with
JWH-018 and JWH-073.
We developed a comprehensive targeted LC-MS/MS
qualitative confirmation method for 29 SC analytes in urine,
requiring sample enzymatic hydrolysis and acetonitrile pro-
tein precipitation, achieving 0.5–10 mg/L LOD, and confirm-
ing over 2500 presumptive positive and negative random
workplace urine specimens (Wohlfarth et al., 2013b, 2014c).
The method utilized scheduled MRM followed by data-
dependent enhanced product ion scans and identified analytes
with an in-house library. A specimen was considered positive
with 60% library match, ±0.05 min expected retention time
and the presence of three characteristic fragments.
Our group also developed one of the most comprehensive
LC-MS/MS quantitative methods for 53 SC analytes in urine
with LOQ 0.1–1 mg/L, following enzymatic hydrolysis and
SLE+ extraction, and applied the assay to 777 authentic urine
specimens (Scheidweiler & Huestis, 2014). Two separate
MRM injections – one in positive and one in negative ESI
mode – were required.
Freijo et al. (2014) published another quantitative LC-MS/
MS MRM method for 29 SC including recent analogs PB-22
and AB-FUBINACA with 5 mg/L LOQ. Samples were diluted
and subjected to enzyme hydrolysis prior to LC-MS/MS
analysis. The method was applied to one authentic urine
specimen containing UR-144-N-pentanoic acid and XLR-
11-6-hydroxyindole.
Five years after SC were first detected in herbal blends, the
first validated non-targeted LC-HRMS method for screening
Synthetic cannabinoids 43
and confirming 75 NPS including 54 SC analytes in urine
was published (Sundström et al., 2013). It achieved cutoffs
ranging from 0.2 to 60 mg/L, with the MS alternately operated
between MS and bb-CID mode, switching between low and
high collision energies. Samples were enzymatically treated
and analytes extracted by mixed-mode SPE. Analyte identi-
fication was based on reverse database searching from an
in-house library, requiring ±3 mDa molecular ion mass
accuracy, acceptable isotopic pattern, ±0.2 min retention time
and 10 000 and 41000 counts for the molecular and
qualifier ions, respectively. Fourteen urine specimens from
SC-related cases were analyzed and all contained one or more
SC metabolites.
Another non-targeted qualitative LC-HRMS confirmation
method was developed for 40 SC urinary metabolites with
2 mg/L LOD, requiring sample enzymatic hydrolysis and
SALLE (Kronstrand et al., 2014). Extracts were analyzed by
LC-QTOF-MS in auto MS/MS mode. For analyte identifica-
tion, an algorithm was employed considering retention time,
accurate mass measurement, isotopic pattern and library
matching score. Authentic forensic urine samples were
screened with the SC Immunalysis HEIA immunoassay
(Pomona, CA) and confirmed by LC-HRMS, achieving 87%
sensitivity and 82% specificity. In addition, the LC-HRMS
method identified UR-144 metabolites not detected by the
immunoassay due to lack of cross-reactivity.
We recently developed a non-targeted approach, deploying
a SWATHÔ (Sequential Windowed Acquisition of all
Theoretical mass spectra) LC-HRMS method, specifically
targeting 47 SC metabolites in urine from 21 SC families with
0.25–5 mg/L LOD and a 15 min run time (Scheidweiler et al.,
2014). This method acquired MS/MS spectra for all precursor
ions between 228 and 408 Da at 30 windows of 6 Da width,
based on SC masses generally between 232 and 406 Da.
Sample preparation included enzymatic hydrolysis and SLE+
extraction. The method was applied to analyze random
workplace drug testing urine specimens.
Traditionally, laboratories utilize immunoassay screens to
rapidly differentiate presumptive positive from negative
specimens with no sample preparation and high throughput,
(Jenkins, 2013). As SC emerge, new assays must be
developed, due to lack of cross-reactivity in standard canna-
binoid immunoassays. First generation SC immunoassays
targeted JWH-018 and JWH-250 urinary metabolites with
varying cross-reactivity to other naphthoylindoles (Arnston
et al., 2013; Barnes et al., 2014; Castaneto et al., 2014a;
Spinelli et al., 2014); subsequently, urinary UR-144 and
XLR-11 metabolites were targeted (Mohr et al., 2014).
Homogenous enzyme immunoassay (HEIA) and enzyme-
linked immunosorbent assays (ELISA) for indole-core SC and
metabolites (Table 2) in urine demonstrated 79.9–98.3%
sensitivity for SC metabolites with cutoffs as low as 5 mg/L
(Arnston et al., 2013; Barnes et al., 2014; Castaneto et al.,
2014a; Mohr et al., 2014; Spinelli et al., 2014).
Oral fluid
Eight articles published between 2011 and 2014 described
parent SC detection in OF by LC-MS/MS. OF is an
alternative matrix for blood in driving under the influence
 44 M. S. Castaneto et al.
of drugs (DUID) cases and for urine in workplace drug testing
programs. Choice of OF collection device is important due to
high SC lipophilicity. Analyte recovery from collection
devices differ, with elution buffer an important factor for
sensitivity. OF contains parent SC (Kneisel et al., 2012;
Rodrigues et al., 2013), offering a detection advantage over
urine because target metabolites might be unknown and
metabolite reference standards may not be available. The
extent of metabolite disposition into OF is unknown, but
metabolites are valuable targets to document active intake as
passive OF contamination by environmental cannabis smoke
was observed (Moore et al., 2011). An ELISA targeting JWH-
200 in OF had limited cross-reactivity to naphthoylindole SC
(AM1220, AM2201, AM2232, JWH-015, JWH-018, JWH-
022 and JWH-073), but achieved 84% sensitivity and 100%
specificity with LC-MS/MS 0.25 mg/L cutoffs for 21 indole-
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core SC (Rodrigues et al., 2013) OF was collected with the
QuantisalÔ device, and the OF buffer mixture was only
diluted with internal standard prior to LC-MS/MS analysis.
A quantitative LC-MS/MS method for 6 SC in OF utilized
MRM in ESI+ and ESI mode and achieved 0.5 mg/L LOQ
(Coulter et al., 2011). OF was also collected with the
Quantisal device. Samples were subjected to SPE before
LC-MS/MS analysis and SC recovery from the OF collection
device was 55–74%.
The most extensive LC-MS/MS method for quantification
of 30 SC in OF utilized MRM in ESI+ mode, with LOQ
For personal use only.
ranging from 0.15 to 3.0 mg/L (Kneisel et al., 2012). OF
samples collected with the Draeger DCD 5000 device
underwent LLE, with analyte recovery from 14% to 77%.
The method was applied to 264 authentic OF specimens
collected from clinical and forensic settings, finding 12 SC in
31 OF specimens. JWH-210 was the most prevalent SC
(n ¼ 31), followed by JWH-122 (n ¼ 17) JWH-081 (n ¼ 8),
JWH-018 (n ¼ 7) and AM2201 (n ¼ 6), and others with lower
prevalence.
An selective reaction monitoring (SRM) LC-MS/MS with
ESI + mode, quantified 18 SC in OF collected with the
InterceptÕ collection device (Øiestad et al., 2013). OF was
subjected to LLE, achieving 0.2–2 mg/L LOQ and 19–61%
analyte recovery. The method was applied to 45 authentic OF
specimens from suspected SC users, with 20% confirming
positive for JWH-018 and/or AM2201.
Hair
Hair is a useful matrix for documenting frequent SC intake or
sustained drug abstinence. There were six SC human hair
confirmation methods and one abstract published between
2012 and 2014. Except for one (Gottardo et al., 2013),
authentic hair specimens were washed and treated with or
without base digestion prior to LLE. SC were quantified by
LC-MS/MS or screened by LC-TOFMS (Table 2).
The first comprehensive quantitative LC-MS/MS sched-
uled MRM method for 22 indole-core SC in hair required a
washing step and LLE prior to analysis, achieving 0.5 pg/mg
LOQ, except for 5.0 pg/mg for JWH-398 (Hutter et al.,
2012b). Authentic hair specimens collected from forensic
psychiatry patients with SC-positive serum samples were
analyzed. One to five SC (JWH-018, JWH-073, JWH-081,
Drug Metab Rev, Early Online: 1–51
JWH-210 and JWJ-250) were identified with concentrations
between 0.5 and 78 pg/mg.
Gottardo et al. (2013) were the first to employ LC-HRMS
for detection of 8 SC in hair with a 10 pg/mg LOD. Data
acquisition was performed in TOF-MS and MS/MS mode
with ESI+. Hair samples underwent NaOH digestion over-
night prior to LLE. In 435 authentic hair specimens from
drivers with suspended licenses, 8 samples were positive for
JWH-018, JWH-073, JWH-081, JWH-122 and/or JWH-250,
at concentrations ranging from 0.010 to 1.28 ng/mg.
A quantitative LC-MS/MS method for 23 SC in hair
employed NaOH digestion, LLE and SRM in ESI+ mode,
achieving LOQ 0.7–4.3 pg/mg except for HU-210 at 80 pg/mg
(Salomone et al., 2014b). Authentic hair specimens from 344
individuals with suspended driving licenses or drug abuse
histories were analyzed; 15 specimens contained one or
more SC, with JWH-073 identified in 11 at 1.6–50.5 pg/mg
concentrations.
Analyte stability
Stability studies of SC were generally performed with
fortified authentic matrices rather than authentic specimens.
In fortified blood, 25 indole-core SC stored at 20  C were
stable for 1 week (Ammann et al., 2012). JWH-018, JWH-
019, JWH-073 and JWH-250 fortified in blood were stable
refrigerated and at 20  C for 30 days (Kacinko et al., 2011).
Another study observed acceptable analyte stability for JWH-
015, JWH-018, JWH-073, JWH-081, JWH-200, JWH-250
and WIN55,212-2 fortified in serum after three freeze/thaw
cycles and after 1 week at 20  C (Dresen et al., 2011).
Except for JWH-081 that decreased by 65.8%, all SC were
stable at RT after 72 h. For SC with indole-core structures, no
analyte instability was observed in processed samples stored
overnight on the autosampler at RT (Kacinko et al., 2011) or
at 10  C (Kneisel & Auwarter, 2012).
Stabilities of cyclohexylphenols, dibenzoypyrans, indole-
core SC (naphthoyl, benzoyl, phenylacetyl, adamantoyl,
quinolinyl, tetracyclomethylpropyl-type) and indazole-core
SC (adamantoyl, carboxamide) in urine also were investigated
(Beuck et al., 2011; Freijo et al., 2014; Jang et al., 2013;
Kronstrand et al., 2014; Scheidweiler & Huestis, 2014;
Scheidweiler et al., 2014; Wohlfarth et al., 2013b). SC parent
and metabolites in fortified authentic urine and stored at RT,
4 and 20  C for 24 h and after three freeze/thaw cycles, were
generally stable (Beuck et al., 2011; Freijo et al., 2014; Jang
et al., 2013; Scheidweiler & Huestis, 2014; Wohlfarth et al.,
2013b). JWH-073-7-hydroxyindole suffered a 25% analyte
loss at RT after 48 h (Kronstrand et al., 2014). This is in
contrast to our observations of parent instability (420% loss)
after 16 h at RT in 16 indole-core SC, except for JWH-200,
CP47,497-C7, CP47,497-C8 and HU-210 (Scheidweiler &
Huestis, 2014). JWH-018 and JWH-073 alkyl hydroxy,
carboxy and indole metabolites were stable under long-term
storage conditions (20  C for 2–4 weeks) (Jang et al., 2013).
Another study evaluated N-dealkylated JWH-018-5-hydro-
xyindole and 20 -hydroxynaphthoyl fortified in authentic
urine at RT and 4  C after 4 weeks and reported no
significant analyte loss after long term storage (Beuck et al.,
2011). Processed samples of SC parent and metabolites with
 DOI: 10.3109/03602532.2015.1029635
indole-core substructures fortified in authentic urine remained
stable up to 72 h (Wohlfarth et al., 2013b). Longer periods of
storage time than 4 weeks and stability in authentic urine
specimens collected from SC-exposed individuals have not
been thoroughly investigated.
Authentic OF concentrations in samples collected from
volunteers who smoked a herbal mixture containing 11
indole-core SC, were stable up to 72 h at 4  C except for
JWH-251 and JWH-203 (77–79%) (Kneisel et al., 2013a). In
volunteers smoking JWH-018, SC stability in authentic OF
was one month at 4  C (Coulter et al., 2011). In the same
study, JWH-073 and JWH-250 fortified in OF were unstable
(25%) at RT for 4 days, while JWH-018, CP47,497,
CP47,497-C8 and HU-210-fortified OF concentrations only
decreased 9 to 16%. Stability was acceptable for all
analytes at 4  C for up to 1 week. In comparison, 28 indole-
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core SC OF concentrations were stable after three freeze/thaw
cycles and after 30 days at 20  C, except for JWH-307
(Kneisel et al., 2012). Indole-core SC on the autosampler was
stable for 7–9 h at 10  C (Kneisel et al., 2012, 2013a) and
after 24 h at 15  C (Amaratunga et al., 2014).
Stability of indole-core SC in OF was evaluated in
different storage tubes. Except for JWH-200, analytes stored
in polypropylene tubes at 25  C quantified 565% target after
24 h and further degraded (9.1–54% target) after 72 h (Kneisel
et al., 2013b). Analytes stored in glass or borosilicate tubes
were stable at 4 and 25  C for up to 72 h.
For personal use only.
In hair, only in-process stability during sample preparation
was investigated. Two studies reported that extracted analytes
were within ±10% theoretical target, suggesting that extracted
SC (in solvents) remain stable during 24 h sample preparation
(Hutter et al., 2012b; Salomone et al., 2014b).
Discussion
Pharmacokinetics
SC research occurred in two phases: during the first phase, SC
was investigated as clinical therapies, e.g. to modulate
appetite (Chambers et al., 2006), or treat neurological
disorders (Beaulieu & Rice, 2002; Finn & Chapman, 2004).
Animals were dosed to determine SC pharmacodynamics and
pharmacokinetics or to identify drugs with reduced psycho-
tropic effects (Valiveti et al., 2004a,b). Publications included
receptor binding affinity and drug syntheses that later
provided guidance for clandestine chemists to select potent
SC and produce the drugs as ‘‘legal highs’’ (Presley et al.,
2013) Research for pharmacotherapies continues but at a
slower pace, partly due to the lack of success in identifying
non-psychoactive cannabinoids, new research into fatty acid
amide hydrolase (FAAH) inhibitors and other novel targets
(Uhelski et al., 2014), and scheduling of SC in many countries
(UNODC, 2014).
The second phase was initiated by the introduction of these
NPS on the designer drug market in the early 2000s,
producing increasing numbers of emergency room visits and
poison control calls attributed to acute intoxications. SC
adverse effects continue to be reported, and after a decline in
2013, the number of calls to poison control centers trended
upward again in 2014 (AAPCC, 2014). Research now focuses
on documenting pharmacodynamic effects in intoxicated drug
Synthetic cannabinoids 45
users, determining pharmacokinetic properties including the
most relevant metabolites to target and developing analytical
methods. Pharmacokinetics studies in animals or in vitro were
performed as alternative approaches to human controlled drug
administration studies and provided critical information for
toxicity evaluations and method development. The ‘‘early’’
SC, JWH-018 and AM-2201, were intensively investigated.
However, it has not been possible to perform in-depth
research for all SC due to the rapid introduction of so many
chemically diverse compounds.
Absorption/distribution studies
There were few studies on this topic, but taking into account
all results across different compounds in different animal
species (Barna et al., 2009; Schaefer et al., 2014), it is evident
that SC are very lipophilic compounds and show all charac-
teristic pharmacokinetic properties of lipophilic drugs. They
are distributed quickly into fat tissue, where they can
accumulate leading to a rapid decline of parent concentration
in blood after administration as well as long detection
windows after chronic consumption (Kneisel et al., 2014).
The compounds investigated also crossed the blood–brain
barrier and accumulated in brain tissue as demonstrated
by brain-to-blood ratios 41. Cannabinoid tetrad (analgesia,
catalepsy, hypomotility and hypothermia) effects observed in
animals corresponded well with these high brain concentra-
tions (Poklis et al., 2012a,b).
There are only two in vivo animal studies that reported
plasma Cmax and t1/2 for CP55,540 and WIN55,212-2 – one
study in a dog (Fouda et al., 1987) and one in guinea pigs
(Valiveti et al., 2004a), respectively. These data give only a
first impression of plasma concentrations and half-lives,
rather than being predictive of typical values in humans, even
more so as many different compounds are consumed, each of
them with a different individual dose.
In the human self-administration studies, acute SC smoke
exposure produces peak concentrations within minutes after
intake with low blood/serum concentrations (10 mg/L),
which then rapidly decline and are only detectable for hours
to days, e.g. JWH-018 up to 48 h (Kacinko et al., 2011; Teske
et al., 2010). In DUID cases, concentrations were similarly
low (Yeakel & Logan, 2013). In contrast, in forensic cases
from rehabilitation and psychiatric clinics or severe intoxica-
tions with unknown time of consumption, concentrations
could be much higher (JWH-122 up to 230 mg/L), probably
due to chronic use (Kneisel & Auwarter, 2012). Oral SC
intake produced much lower serum concentrations, but
detectability might be extended, e.g. AM2201 was found
after up to 5 days (Hutter et al., 2013). However, without
further information regarding frequency or last intake, SC
detection in blood might not necessarily mean recent intake as
it was shown that SC may still be detectable in chronic users’
serum 30 days after last use (Kneisel et al., 2014). In
summary, although SC compounds exhibit wide structural
diversity and major differences from THC, major pharmaco-
kinetic properties are similar; hence, we assume that similar
problems, especially when interpreting results, will arise.
How to detect recent use and assess impairment, when blood
concentrations might not reflect brain concentrations? How to
 46 M. S. Castaneto et al.
prove relapse when detection windows after chronic exposure
are long? Is it possible to find a scientifically based cut-off for
these compounds in blood?
Similar to blood, SC peak OF concentrations are low and
ranged between 3 and 35 mg/L after smoking, rapidly
declining until becoming undetectable after several hours
(Coulter et al., 2011). This might render OF a good matrix to
document recent use, if this also holds true for frequent users.
In addition, oral contamination should be closely examined as
observed by Kneisel et al. (2012) because passive environ-
mental SC smoke exposure can produce positive OF results,
similar to data for THC (Moore et al., 2011). A solution is to
measure metabolites that suggest active intake. Since no
published method has targeted metabolites in OF yet, we
consider this a valuable field of research.
Only three human self-administration studies reported
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
urine results (De Jager et al., 2012; Hutter et al., 2013; Logan
et al., 2011). Various metabolites in low mg/L concentrations
were found; after a single smoked dose, detection windows
were 2–3 days and after oral consumption 10 days. In
authentic workplace urine specimens, we observed SC
metabolites at much higher concentrations ranging from 0.1
to 2434 mg/L (Castaneto et al., 2014c). Urine offers longer
windows of SC detection than blood, with the major
disadvantage being the absence of SC parent compounds,
and the need to target (initially unknown) metabolites.
Usually it takes longer for reference standards to become
For personal use only.
available, delaying method development. An important
drawback is also the shared metabolic pathways (Figure 1)
that complicate interpretation of urine SC results.
Figure 1. Merging metabolic pathways for synthetic cannabinoids AKB-48, JWH-018, JWH-073, JWH-122, PB-22 and XLR-11 and five fluoropentyl
analogs. Ester hydrolysis of PB-22 and 5F-PB-22 produce 1-pentyl-1H-indole-3-carboxylic acid (PI-COOH) and 5F-PI-COOH, respectively.
Drug Metab Rev, Early Online: 1–51
Hair as analytical matrix usually serves to monitor long-
term consumption patterns or prove abstinence. In the few
studies that analyzed authentic hair specimens obtained from
different groups, e.g. forensic psychiatry patients, DUID
offenders or patients undergoing withdrawal (Gottardo et al.,
2013; Hutter et al., 2012b; Kim et al., 2014; Salomone et al.,
2014b), several naphthoylindole SC and metabolites were
detected covering a broad concentration range. Interestingly,
no correlation between measured concentrations and reported
drug consumption could be found. Instead, concentrations
increased from proximal to distal segments, suggesting
incorporation of SC via side-stream smoke (Hutter et al.,
2012b), another similarity with THC and cannabis smoking
(Uhl & Sachs, 2004). Hair pigmentation did not play a role in
deposition processes (Kim et al., 2013; Smeal et al., 2007).
Further investigation is required as to whether re-circulation
of SC in blood secreted from fat tissue may influence the
amount incorporated in hair, as this would affect detection of
abstinence, and how environmental contamination from SC
smoke could lead to false positive results. Moreover, it would
be helpful to know if and to which extent metabolites are
incorporated in hair, as this can assist interpretation and rule
out passive contamination.
Metabolism studies
There were few published studies on SC biotransformation
in the above-mentioned first phase of SC research, namely
on CP55,940, WIN55,212-2 and JWH-015, which
were considered for therapeutic applications at the time
 DOI: 10.3109/03602532.2015.1029635
(Thomas & Martin, 1990; Zhang et al., 2002, 2006). In the
second phase, metabolism research exploded. The main focus
now is to determine the metabolic profile of each compound,
i.e. to elucidate the structure of the major metabolites, to
identify the biotransformations involved and to check for
potentially toxic metabolites. Less often metabolic stability is
assessed, receptor affinities of metabolites are determined or
the specific CYP450 isoenzymes predominantly active
identified. Eventually, all research data served to identify
suitable urinary markers to document intake, which helped in
developing analytical methods, and clarified shared metabolic
pathways, which further assisted in results interpretation. The
time that is needed for metabolism studies is one of the
limiting factors that delays method development, especially
for urinalysis.
As controlled administration studies in humans are not
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
feasible and animal studies may not completely and reliably
predict human metabolites, in vitro methods with human
enzymes or cells were the methodology of choice in most
studies. In the first phase of SC research, in vitro metabolism
studies were conducted with RLM, but most recent studies
utilized HLM or human hepatocytes. HLM are an inexpen-
sive, easy and rapid approach, but with the disadvantages that
phase II metabolites are not identified unless additional
cofactors are added. Moreover, the prevalence of metabolites
might not predict well what is later found in authentic human
samples. Human hepatocytes generate phase I and II metab-
For personal use only.
olites usually in a similar prevalence as observed in vivo.
Therefore, they are currently considered the gold standard for
in vitro metabolism studies. However, hepatocyte incubations
are more expensive, and require special handling. Two studies
used an in vivo chimeric mouse model, which offers a third
approach for metabolite profiling, although it is the most
complicated alternative. Compared to the in vitro approaches,
the chimeric mouse offers metabolism in living human
hepatocytes in a whole organism, i.e. not only metabolism
can be investigated, but also distribution and excretion
processes. However, the mice can still produce murine-
specific metabolites not present in authentic human speci-
mens and the whole approach requires specific technical
knowledge in animal breeding and handling.
In silico metabolite prediction is a different and new
approach. Prediction software helps predict potential metab-
olites, which can then be specifically targeted in in vitro
studies or when analyzing authentic specimens. Different
algorithms were developed, some based on expansive data-
bases containing metabolic pathways for thousands of com-
pounds (training-dependent), some simulating the docking in
the catalytic cavity of different enzymes and calculating
substrate reactivity (training-independent). To date, in silico
prediction remains a supportive tool and is still under
evaluation.
In any case, metabolites proposed from in vitro studies or
predicted in silico always need to be verified in authentic
human samples. Ingested dose, intake frequency, time after
dosing, genetics and polymorphisms, analyte stability and
drug–drug interactions will influence the type and prevalence
of metabolites in biological matrices. However, in most
studies, in vitro and in vivo biotransformations and profiles
matched well. Differences were usually seen in the
Synthetic cannabinoids 47
appearance of minor metabolites or the degree of biotrans-
formation (earlier, first-generation metabolites in vitro versus
later, second- or third-generation metabolites in authentic
urine samples).
All metabolism studies demonstrated that SC undergo
extensive metabolism and follow typical xenobiotics meta-
bolic pathways (Table 3). Among the naphthoylindoles,
hydroxylation was the most common biotransformation,
predominantly occurring at the indole alkyl and to a lesser
extent at the naphthyl substructure. Dihydrodiol formation at
the naphthyl, carboxylation and N-dealkylation also were
observed. Naphthoylindoles with a particular substructure
generated compound specific biotransformations like JWH-
200 morpholine ring opening (De Brabanter et al., 2013b),
RCS-8 O-demethylation (Wohlfarth et al., 2014b) and PB-22
or 5F-PB-22 ester hydrolysis (Takayama et al., 2014;
Wohlfarth et al., 2014a). Fluorinated SC underwent oxidative
defluorination in addition or before further biotransformation.
Interestingly, when comparing fluorinated and non-fluori-
nated pentyl chain carrying SC, pentanoic acid metabolites
were usually favored by the fluorinated analogs (Jang et al.,
2014a; Holm et al., 2014; Wohlfarth et al., 2013a). Other SC
families e.g. tetramethylcyclopropylindoles, when smoked,
generated pyrolysis products further undergoing oxidative
biotransformation (Adamowicz et al., 2013).
One of many challenges in SC analysis is finding unique
biomarkers. There are two primary reasons. First, shared
metabolic pathways (Figure 1) make it difficult to distinguish
the origin of some SC, and second, some compounds lose
substantial parts of the molecule in their predominant
biotransformation(s). One possible solution to the first
problem recommended by several groups is calculating the
metabolite ratios of different major metabolites as was done
for JWH-018 and AM2201 (Chimalakonda et al., 2012; Hutter
et al., 2013; Jang et al., 2014b). Another approach, which is
also a solution to the second problem, is to target specific
metabolites, even if they are minor. These metabolites must
contain the relevant molecular features, e.g. the fluorine atom
at 50 -pentyl position or the ester linker, or generate a specific
metabolite only observed for one compound of the analog
pair, e.g. for instance, JWH-018-N-4-hydroxypentyl, which is
not produced by AM2201.
The majority of phase II SC metabolites are glucuronides;
other conjugates, i.e. sulfates, were observed to date for only
three compounds (RCS-4, JWH-122 and JWH-200) and
cysteine conjugation only for the hydrolysis products of
PB-22 and 5F-PB-22. Often major metabolites were exten-
sively glucuronidated; hence, a hydrolysis step is considered
mandatory for urine specimens analyzed by mass spectrom-
etry to detect corresponding phase I metabolites (immuno-
assays generally are not preceded by hydrolysis). Although
new compounds can always differ, the current results strongly
suggest that glucuronidation is the major phase II reaction for
SC compounds. There might be glucuronide species that are
more resistant to hydrolysis than others, but laboratories can
be confident that their common hydrolysis procedures will
work sufficiently. From an analytical point of view, special
attention should be paid to acyl glucuronides, which were
identified for some compounds (PB-22, 5F-PB-22) and can
undergo isomerization producing isomers more resistant to
 48 M. S. Castaneto et al.
enzymatic hydrolysis (Regan et al., 2010). More important,
acyl glucuronides are known to be potentially toxic due to
covalent binding to protein structures.
There is a high probability that we will continue to see new
SC emerge, probably with the majority having no available
pharmacokinetic or toxicity data. Therefore, there is an urgent
and constant need to determine the metabolism of new
compounds and to identify the appropriate analytical targets.
Ideally, a streamlined procedure covering metabolic stability
assessment, in vitro metabolic profiling and confirmation of
biomarkers in authentic specimens is in place and will quickly
enable mass spectrometry metabolite data to be incorporated
in MS libraries. Another critical component is the synthesis of
reference standards by commercial entities to enable forensic
identifications. Further characterization by enzyme phenotyp-
ing, drug–drug interactions and inhibition studies are vital as
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
well as desirable. Although human controlled administration
studies would provide valuable data to understand SC
pharmacodynamics and pharmacokinetics, these studies are
not currently feasible due to the lack of pre-clinical and
in vitro toxicity data and even if they were, it would be
impossible to test every new compound. Therefore, we
assume that the current in vitro HLM and human hepatocytes
assays will continue to provide critical identification data.
Analytical methods
For personal use only.
Documenting SC intake is important for clinical and forensic
drug testing. Whether to document recent intoxication,
identify impaired driving or monitor workplace use, SC
identification in blood, urine, OF and hair provide the
evidence. However, new compounds emerge more rapidly
than laboratories can develop and validate analytical methods.
GC-MS and LC-MS/MS are versatile instruments often
employed for confirmation methods, and toxicologists applied
this approach to analyze biological specimens for SC.
Initially, methods for one or a few analytes were developed
(ElSohly et al., 2011; Yanes & Lovett, 2012), but when more
and more compounds appeared, the scope was extended to
more than 50 SC markers in one method (Scheidweiler &
Huestis, 2014). However, developing and validating these
methods is a time-consuming, costly and labor-intensive
endeavor.
Immunoassay technology also was evaluated, offering
advantages of high throughput and no sample preparation.
However, immunoassays have limited to no cross-reactivity
with new SC and metabolites and require months to years to
be developed and validated. Moreover, immunoassays are
screening methods requiring further confirmatory testing. For
these reasons, this analytical approach has difficulty iden-
tifying new SC.
After more than 6 years of the SC phenomenon, toxicolo-
gists realize that routine analytical approaches are insuffi-
cient, promoting a recent surge of innovative strategies
applying HRMS technology to develop non-targeted methods
capable of detecting known or unknown substances at the
time of analysis. The major challenge is to achieve sufficient
selectivity to detect low SC concentrations. To date, three
non-targeted LC-HRMS screening methods for drugs of abuse
including SC in biological samples were published enabling
Drug Metab Rev, Early Online: 1–51
laboratories to acquire accurate MS and MS/MS data
without pre-selecting compounds (Kronstrand et al., 2014;
Scheidweiler et al., 2014; Sundström et al., 2013). This
methodology is more flexible and allows for retrospective
analysis. Once laboratories expand their mass spectrometric
libraries as reference standards become available, previously
collected data can be interrogated for unknown compounds.
HRMS instruments, complimented by metabolite prediction
software also are powerful tools for metabolite profiling
and identification, not easily achieved with GC-MS or
LC-MS/MS.
Conclusion
The emergence of SC will pose continuous challenges to
clinical and forensic laboratories. With each new compound,
the forensic community must address three major issues: (1)
identification of suitable biomarkers via in vitro studies to
detect intake, (2) reference standard synthesis and (3)
continuous updating and validation of analytical methods,
which is a time-consuming, cost- and labor-intensive process.
Improvements are apparent: faced with logistic and analytical
limitations, laboratories are applying unconventional
approaches with non-targeted HRMS, permitting retrospect-
ive data inquiry after library updates. SC metabolite identi-
fication is becoming faster and more comprehensive, but
should be further streamlined and complemented.
Laboratories need to respond quickly, adapt to the new
emerging drug market and be innovative with new HRMS
technology. Therefore, we expect to see additional techno-
logical and logistic advancements and improved analytical
methods for SC identification in the near future.
Declaration of interest
The authors report no declaration of interest. This research
was funded by the Chemistry and Drug Metabolism Section
of the Intramural Research Program, National Institute on
Drug Abuse, National Institutes of Health.
References
Adamowicz P, Zuba D, Sekula K. (2013). Analysis of UR-144 and its
pyrolysis product in blood and their metabolites in urine. Forensic Sci
Int 233:320–327.
Amaratunga P, Thomas C, Lemberg BL, Lemberg D. (2014).
Quantitative measurement of XLR11 and UR-144 in oral fluid by
LC-MS-MS. J Anal Toxicol 38:315–321.
American Association of Poison Control Call Centers. (2014). Synthetic
Marijuana Data: December 31, 2014. AAPCC. Available: https://
aapcc.s3.amazonaws.com/files/library/Syn_Marijuana_Web_Data_
through_12.2014.pdf [last accessed 24 Jan 2014].
Ammann J, Mclaren JM, Gerostamoulos D, Beyer J. (2012). Detection
and quantification of new designer drugs in human blood: Part 1 –
Synthetic cannabinoids. J Anal Toxicol 36:372–380.
Arnston A, Ofsa B, Lancaster D, et al. (2013). Validation of a novel
immunoassay for the detection of synthetic cannabinoids and metab-
olites in urine specimens. J Anal Toxicol 37:284–290.
Barna I, Till I, Haller J. (2009). Blood, adipose tissue and brain levels of
the cannabinoid ligands WIN -55,212 and SR-141716A after their
intraperitoneal injection in mice: Compound-specific and area-
specific distribution within the brain. Eur Neuropsychopharmacol
19:533–541.
Barnes AJ, Young S, Spinelli E, et al. (2014). Evaluation of a
homogenous enzyme immunoassay for the detection of synthetic
cannabinoids in urine. Forensic Sci Int 241:27–34.
 DOI: 10.3109/03602532.2015.1029635
Beaulieu P, Rice ASC. (2002). The pharmacology of cannabinoid
derivatives: Are there applications to treatment of pain? Ann Fr
Anesth Reanim 21:493–508.
Behonick G, Shanks KG, Firchau DJ, et al. (2014). Four postmortem
case reports with quantitative detection of the synthetic cannabinoid,
5F-PB-22. J Anal Toxicol 38:559–562.
Beuck S, Moeller I, Thomas A, et al. (2011). Structure characterisation
of urinary metabolites of the cannabimimetic JWH-018 using
chemically synthesised reference material for the support of
LC-MS/MS-based drug testing. Anal Bioanal Chem 401:493–505.
Castaneto MS, Desrosiers ND, Ellefsen K, et al. (2014a). Method
validation of the biochip array technology for synthetic cannabinoids
detection in urine. Bioanalysis 6:2917–2928.
Castaneto MS, Gorelick DA, Desrosiers NA, et al. (2014b). Synthetic
cannabinoids: Epidemiology, pharmacodynamics, and clinical impli-
cations. Drug Alcohol Depend 144:12–41.
Castaneto MS, Scheidweiler KB, Gandhi A, et al. (2014c). Quantitative
urine confirmatory testing for synthetic cannabinoids in randomly
collected urine specimens. Drug Test Anal doi:10.1002/dta.1709.
Centers for Disease Control and Prevention. (2013a). Acute kidney
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
injury associated with synthetic cannabinoid use. Morb Mortal Wkly
Rep 62:93–98.
Centers for Disease Control and Prevention. (2013b). Notes from the
field: Severe illness associated with synthetic cannabinoid use –
Brunswick, georgia, 2013. Morb Mortal Wkly Rep 62:939.
Chambers AP, Koopmans HS, Pittman QJ, Sharkey KA. (2006). AM 251
produces sustained reductions in food intake and body weight that are
resistant to tolerance and conditioned taste aversion. Br J Pharmacol
147:109–116.
Chimalakonda KC, Bratton SM, Le VH, et al. (2011a). Conjugation
of synthetic cannabinoids JWH-018 and JWH-073, metabolites by
human UDP-glucuronosyltransferases. Drug Metab Dispos 39:
1967–1976.
Chimalakonda KC, Moran CL, Kennedy PD, et al. (2011b). Solid-phase
For personal use only.
extraction and quantitative measurement of omega and omega-1
metabolites of JWH-018 and JWH-073 in human urine. Anal Chem
83:6381–6388.
Chimalakonda KC, Seely KA, Bratton SM, et al. (2012). Cytochrome
P450-mediated oxidative metabolism of abused synthetic cannabin-
oids found in K2/Spice: Identification of novel cannabinoid receptor
ligands. Drug Metab Dispos 40:2174–2184.
Coulter C, Garnier M, Moore C. (2011). Synthetic cannabinoids in oral
fluid. J Anal Toxicol 35:424–430.
Crimele V, Klinger N, Etter M, et al. (2014). O21:Testing for 18
synthetic cannabinoid in hair using HPLC-MS/MS: Method develop-
ment and validation, its application to authentic samples and
preliminary results. Toxicol Anal Clin 26:S13.
De Brabanter N, Esposito S, Geldof L, et al. (2013a). In vitro and in vivo
metabolisms of 1-pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122).
Forensic Toxicol 31:212–222.
De Brabanter N, Esposito S, Tudela E, et al. (2013b). In vivo and in vitro
metabolism of the synthetic cannabinoid JWH-200. Rapid Commun
Mass Spectrom 27:2115–2126.
De Castro A, Piñeiro B, Lendoiro E, et al. (2013). Quantification of
selected synthetic cannabinoids and delta-9-tetrahydrocannabinol
in oral fluid by liquid chromatrography-tandem mass spectrometry.
J Chromatogr A 1295:99–106.
De Jager AD, Warner JV, Henman M, et al. (2012). LC-MS/MS method
for the quantitation of metabolites of eight commonly-used synthetic
cannabinoids in human urine–an Australian perspective. J Chromatogr
B Analyt Technol Biomed Life Sci 897:22–31.
Dowling G, Regan L. (2011). A method for CP74, 497 a synthetic non-
traditional cannabinoid in human urine using liquid chromatography
tandem mass spectrometry. J Chrom B Analyt Technol Biomed Life
Sci 879:253–259.
Dresen S, Kneisel S, Weinmann W, et al. (2011). Development and
validation of a liquid chromatography-tandem mass spectrometry
method for the quantitation of synthetic cannabinoids of the
aminoalkylindole type and methanandamide in serum and its appli-
cation to forensic samples. J Mass Spectrom 46:163–171.
Dziasdosz M, Weller JP, Klintschar M, et al. (2013). Scheduled multiple
reaction monitoring algorithm as a way to analyse new designer drugs
combined with synthetic cannabinoids in human serum with liquid
chromatography-tandem mass spectrometry. J Chromatogr B Analyt
Technol Biomed Life Sci 929:84–89.
Synthetic cannabinoids 49
ElSohly MA, Gul W, ElSohly KM, et al. (2011). Liquid chromatography-
tandem mass spectrometry analysis of urine specimens for K2
(JWH-018) metabolites. J Anal Toxicol 35:487–495.
Finn DP, Chapman V. (2004). Cannabinoids as analgesic agents:
Evidence from in vivo studies. Curr Neuropharmacol 2:75–89.
Fouda HG, Lukaszewicz J, Luther EW. (1987). Selected ion monitoring
analysis of CP -55,940, a cannabinoid derived analgetic agent. Biomed
Environ Mass Spectrom 14:599–602.
Freeman MJ, Rose DZ, Myers MA, et al. (2013). Ischemic stroke after
use of the synthetic marijuana ‘‘spice’’. Neurology 81:2090–2093.
Freijo Jr TD, Harris SE, Kala SV. (2014). A rapid quantitative method for
the analysis of synthetic cannabinoids by liquid chromatography-
tandem mass spectrometry. J Anal Toxicol 38:466–478.
Gambaro V, Arnoldi S, Bellucci S, et al. (2014). Characterization of
in vitro metabolites of JWH-018, JWH-073 and their 4-methyl
derivatives, markers of the abuse of these synthetic cannabinoids.
J Chromatogr B Analyt Technol Biomed Life Sci 957:68–76.
Gandhi AS, Wohlfarth A, Zhu M, et al. (2014a). High-resolution mass
spectrometric metabolite profiling of a novel synthetic designer drug,
N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide
(STS-135), using cryopreserved human hepatocytes and assessment
of metabolic stability with human liver microsomes. Drug Test Anal
7:187–198.
Gandhi AS, Zhu M, Pang S, et al. (2013). First characterization of AKB-
48 metabolism, a novel synthetic cannabinoid, using human hepato-
cytes and high-resolution mass spectrometry. AAPS J 15:1091–1098.
Gandhi AS, Zhu M, Pang S, et al. (2014b). Metabolite profiling of
RCS-4, a novel synthetic cannabinoid designer drug, using human
hepatocyte metabolism and TOF-MS. Bioanalysis 6:1471–1485.
Gottardo R, Sorio D, Musile G, et al. (2013). Screening for synthetic
cannabinoids in hair by using LC-QTOF MS: A new and powerful
approach to study the penetration of these new psychoactive
substances in the population. Med Sci Law 54:22–27.
Grigoryev A, Kavanagh P, Melnik A, et al. (2013b). Gas and liquid
chromatography-mass spectrometry detection of the urinary metabol-
ites of UR-144 and its major pyrolysis product. J Anal Toxicol 37:
265–276.
Grigoryev A, Kavanagh P, Melnik A. (2012). The detection of the
urinary metabolites of 3-[(adamantan-1-yl)carbonyl]-1-pentylindole
(AB-001), a novel cannabimimetic, by gas chromatography-mass
spectrometry. Drug Test Anal 4:519–524.
Grigoryev A, Kavanagh P, Melnik A. (2013a). The detection of
the urinary metabolites of 1-[(5-fluoropentyl)-1H-indol-3-yl]-(2-
iodophenyl)methanone (AM-694), a high affinity cannabimimetic,
by gas chromatography–mass spectrometry. Drug Test Anal 5:
110–115.
Grigoryev A, Melnik A, Savchuk S, et al. (2011a). Gas and liquid
chromatography-mass spectrometry studies on the metabolism of the
synthetic phenylacetylindole cannabimimetic JWH-250, the psycho-
active component of smoking mixtures. J Chromatogr B Analyt
Technol Biomed Life Sci 879:2519–2526.
Grigoryev A, Saychuk S, Melnik A, et al. (2011b). Chromatography-
mass spectrometry studies on the metabolism of synthetic cannabin-
oids JWH-018 and JWH-073, psychoactive components of smoking
mixtures. J Chromatogr B Analyt Technol Biomed Life Sci 879:
1126–1136.
Guale F, Shahreza S, Walterscheid JP, et al. (2013). Validation of LC-
TOF-MS screening for drugs, metabolites, and collateral compounds
in forensic toxicology specimens. J Anal Toxicol 37:17–24.
Gugelmann H, Gerona R, Li C, et al. (2014). ‘‘Crazy Monkey’’ poisons
man and dog: Human and canine seizures due to PB-22, a novel
synthetic cannabinoid. Clin Toxicol (Phila) 52:635–638.
Gurney SMR, Scott KS, Kacinko SL, et al. (2014). Pharmacology,
toxicology, and adverse effects of synthetic cannabinoid drugs.
Forensic Sci Rev 26:53–78.
Harvey DJ. (1991). Metabolism and pharmacokinetics of the cannabin-
oids. Biochem Physiol Subst Abuse 3:279–365.
Heltsley R, Shelby MK, Crouch DJ, et al. (2012). Prevalence of synthetic
cannabinoids in U.S. athletes: Initial findings. J Anal Toxicol 36:
588–593.
Holm NB, Pedersen AJ, Dalsgaard PW, Linnet K. (2014). Metabolites
of 5F-AKB-48, a synthetic cannabinoid receptor agonist, identified
in human urine and liver microsomal preparations using liquid
chromatography high-resolution mass spectrometry. Drug Test Anal
7:199–206.
 50 M. S. Castaneto et al.
Huppertz LM, Kneisel S, Auwärter V, Kempf J. (2014). A comprehen-
sive library-based, automated screening procedure for 46 synthetic
cannabinoids in serum employing liquid chromatography-quadrupole
ion trap mass spectrometry with high-temperature electrospray
ionization. J Mass Spectrom 49:117–127.
Hutter M, Broecker S, Kneisel S, Auwärter V. (2012a). Identification of
the major urinary metabolites in man of seven synthetic cannabinoids
of the aminoalkylindole type present as adulterants in ‘herbal
mixtures’ using LC-MS/MS techniques. J Mass Spectrom 47:54–65.
Hutter M, Kneisel S, Auwärter V, Neukamm MA. (2012b).
Determination of 22 synthetic cannabinoids in human hair by liquid
chromatography-tandem mass spectrometry. J Chromatogr B Analyt
Technol Biomed Life Sci 903:95–101.
Hutter M, Moosmann B, Kneisel S, Auwärter V. (2013). Characteristics
of the designer drug and synthetic cannabinoid receptor agonist
AM-2201 regarding its chemistry and metabolism. J Mass Spectrom
48:885–894.
Jang M, Shin I, Yang W, et al. (2014a). Determination of major
metabolites of MAM-2201 and JWH-122 in in vitro and in vivo
studies to distinguish their intake. Forensic Sci Int 244:85–91.
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
Jang M, Shin I, Yang W, et al. (2014b). Determination of AM-2201
metabolites in urine and comparison with JWH-018 abuse. Int J Legal
Med 128:285–294.
Jang M, Yang W, Choi H, et al. (2013). Monitoring of urinary
metabolites of JWH-018 and JWH-073 in legal cases. Forensic Sci Int
231:13–19.
Jenkins A. (2013). Forensic drug testing. In: Levine B (ed.) Principles of
forensic toxicology. 4th ed. Washington (DC): AACC Press, 31–47.
Kacinko SL, Xu A, Homan JW, et al. (2011). Development and
validation of a liquid chromatography tandem mass spectrometry
method for the identification and quantification of JWH-018, JWH-
073, JWH-019, and JWH-250 in human whole blood. J Anal Toxicol
35:386–393.
Kavanagh P, Grigoryev A, Melnik A, Simonov A. (2012). The
For personal use only.
identification of the urinary metabolites of 3-(4-methoxybenzoyl)-1-
pentylindole (RCS-4), a novel cannabimimetic, by gas chromatog-
raphy-mass spectrometry. J Anal Toxicol 36:303–311.
Kikura-Hanajiri R, Kawamura M, Shoda T, et al. (2011). Determination
of synthetic cannabinoid JWH-018 and its metabolites in rat urine and
hair samples using UPLC-MS/MS. Joint Meeting of the Society of
Forensic Toxicologist and The International Association of Forensic
Toxicologists, 25 September 2011, San Franscisco, CA.
Kim J, In S, Park Y, et al. (2013). Deposition of JWH-018, JWH-073 and
their metabolites in hair and effect of hair pigmentation. Anal Bioanal
Chem 405:9769–9778.
Kim J, Park Y, Park M, et al. (2014). Simultaneous determination of five
naphthoylindole-based synthetic cannabinoids and metabolites and
their deposition in human and rat hair. J Pharm Biomed Anal 102:
162–175.
Kneisel S, Auwärter V, Kempf J. (2012). Analysis of 30 synthetic
cannabinoids in oral fluid using liquid chromatography-electrospray
ionization tandem mass spectrometry. Drug Test Anal 5:657–669.
Kneisel S, Auwärter V. (2012). Analysis of 30 synthetic cannabinoids in
serum by liquid chromatography-electrospray ionization tandem mass
spectrometry after liquid-liquid extraction. J Mass Spectrom 47:
825–835.
Kneisel S, Speck M, Moosmann B, Auwärter V. (2013a). Stability of 11
prevalent synthetic cannabinoids in authentic neat oral fluid samples:
Glass versus polypropylene containers at different temperatures. Drug
Test Anal 5:602–606.
Kneisel S, Speck M, Moosmann B, et al. (2013b). LC/ESI-MS/MS
method for quantification of 28 synthetic cannabinoids in neat oral
fluid and its application to preliminary studies on their detection
windows. Anal Bioanal Chem 405:4691–4706.
Kneisel S, Teske J, Auwärter V. (2014). Analysis of synthetic canna-
binoids in abstinence control: Long drug detection windows in serum
and implications for practitioners. Drug Test Anal 6:135–136.
Kramer T, Meyer M, Wissenbach D, et al. (2008) Studies on the
metabolism of JWH-018, the pharmacologically active ingredient of
different misused incenses (V10). Society of Toxiocological and
Forensic Chemistry Conference, Germany.
Kronstrand R, Brinkhagen L, Birath-Karlsson C, et al. (2014). LC-
QTOF-MS as a superior strategy to immunoassay for the compre-
hensive analysis of synthetic cannabinoids in urine. Anal Bioanal
Chem 405:3599–3609.
Drug Metab Rev, Early Online: 1–51
Kronstrand R, Roman M, Andersson M, et al. (2013). Toxicological
findings of synthetic cannabinoids in recreational users. J Anal
Toxicol 37:534–541.
Logan BK, Reinhold LE, Kacinko SL. (2011). K2 and Beyond:
A Synthetic Cannabinoid Primer (K43). American Academy of
Forensic Sciences, 2011 Feb 21–26, Chicago, IL.
Lovett DP, Yanes EG, Herbelin TW, et al. (2013). Structure elucidation
and identification of a common metabolite for napthoylindole-based
synthetic cannabinoids using LC-TOF and comparison to a synthetic
reference standard. Forens Sci Int 226:81–87.
Mazzarino M, De La Torre X, Botre F. (2014). A liquid chromatography-
mass spectrometry method based on class characteristic fragmentation
pathways to detect the class of indole-derivative synthetic cannabin-
oids in biological samples. Anal Chim Acta 837:70–82.
McQuade D, Hudson S, Dargan PI, et al. (2013). First European case of
convulsions related to analytically confirmed use of the synthetic
cannabinoid receptor agonist AM-2201. Eur J Clin Pharmacol 69:
373–376.
Mir A, Obafemi A, Young A, Kane C. (2011). Myocardial infarction
associated with use of the synthetic cannabinoid K2. Pediatrics 128:
e1622–e1627.
Mohr AL, Ofsa B, Keil AM, et al. (2014). Enzyme-linked immunosorb-
ent assay (ELISA) for the detection of use of the synthetic
cannabinoid agonists UR-144 and XLR-11 in human urine. J Anal
Toxicol 38:427–431.
Möller I, Wintermeyer A, Bender K, et al. (2011). Screening for the
synthetic cannabinoid JWH-018 and its major metabolites in human
doping controls. Drug Test Anal 9:606–620.
Moore C, Coulter C, Uges D, et al. (2011). Cannabinoids in oral fluid
following passive exposure to marijuana smoke. Forensic Sci Int 212:
227–230.
Moran CL, Le VH, Chimalakonda KC, et al. (2011). Quantitative
measurement of JWH-018 and JWH-073 metabolites excreted in
human urine. Anal Chem 83:4228–4236.
Øiestad E, Johansen U, Christophersen A, Karinen R. (2013). Screening
of synthetic cannabinoids in preserved oral fluid by UPLC-MS/MS.
Bioanalysis 5:2257–2268.
Papanti D, Schifano F, Botteon G, et al. (2013). ‘‘Spiceophrenia’’:
A systematic overview of ‘‘Spice’’-related psychopathological issues
and a case report. Hum Psychopharmacol 28:379–389.
Patton AL, Chimalakonda KC, Moran CL, et al. (2013a). K2 toxicity:
Fatal case of psychiatric complications following AM2201 exposure.
J Forensic Sci 58:1676–1680.
Patton AL, Seely KA, Chimalakonda KC, et al. (2013b). Targeted
metabolomic approach for assessing human synthetic cannabinoid
exposure and pharmacology. Anal Chem 85:9390–9399.
Poklis JL, Amira D, Wise LE, et al. (2012a). Determination of
naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in mouse
blood and tissue after inhalation exposure to ‘buzz’ smoke by HPLC/
MS/MS. Biomed Chromatogr 26:1393–1398.
Poklis JL, Amira D, Wise LE, et al. (2012b). Detection and disposition
of JWH-018 and JWH-073 in mice after exposure to ‘‘Magic Gold’’
smoke. Forensic Sci Int 220:91–96.
Presley BC, Jansen-Varnum SA, Logan BK. (2013). Analysis of synthetic
cannabinoids in botanical material: A review of analytical methods
and findings. Forensic Sci Rev 25:27–46.
Regan SL, Maggs JL, Hammond TG, et al. (2010). Acyl glucuronides:
The good, the bad and the ugly. Biopharm Drug Dispos 31:367–395.
Rodrigues WC, Catbagan P, Rana S, et al. (2013). Detection of synthetic
cannabinoids in oral fluid using ELISA and LC-MS-MS. J Anal
Toxicol 37:526–533.
Salomone A, Gerace E, D’Urso F, et al. (2012). Simultaneous analysis of
several synthetic cannabinoids, THC, CBD and CBN, in hair by ultra-
high performance liquid chromatography tandem mass spectrometry:
Method validation and application to real samples. J Mass Spectrom
47:604–610.
Salomone A, Gerace E, Luciano C, et al. (2014a). O22:Quantification of
22 synthetic cannabinoids and 10 metabolites in human hair. Toxicol
Analyt Clin 26:S14.
Salomone A, Luciano C, Di Corcia D, et al. (2014b). Hair analysis as a
tool to evaluate the prevalence of synthetic cannabinoids in different
populations of drug consumers. Drug Test Anal 6:126–134.
Schaefer N, Peters B, Bregel D, et al. (2014). Can JWH-210 and JWH-
122 be detected in adipose tissue four weeks after single oral drug
administration to rats? Biomed Chromatogr 28:1043–1047.
 DOI: 10.3109/03602532.2015.1029635
Scheidweiler KB, Huestis, MA. (2014). Simultaneous quantification of
20 synthetic cannabinoids and 21 metabolites, and semi-quantification
of 12 alkyl hydroxy metabolites in human urine by liquid chroma-
tography-tandem mass spectrometry. J Chromatogr A 1327:105–117.
Scheidweiler KB, Jarvis MJ, Huestis, MA. (2014). Nontargeted SWATH
acquisition for identifying 47 synthetic cannabinoid metabolites in
human urine by liquid chromatography-high-resolution tandem mass
spectrometry. Anal Bioanal Chem 407:883–897.
Schep L, Slaughter R, Hudson S, et al. (2014). Delayed seizure-like
activity following analytically confirmed use of previously unreported
synthetic cannabinoids analogues. Hum Exp Toxicol. doi:10.1177/
0960327114550886.
Shanks KG, Dahn T, Terrell AR. (2012). Detection of JWH-018 and
JWH-073 by UPLC-MS-MS in postmortem whole blood casework.
J Anal Toxicol 36:145–152.
Simoes SS, Silva I, Ajenjo AC, et al. (2014). Validation and application
of an UPLC-MS/MS method for the quantification of synthetic
cannabinoids in urine samples and analysis of seized materials from
the Portugues market. Forensic Sci Int 243:117–125.
Smeal SJ (2007). The incorporation of delta-9-tetrahydrocannabinol and
Drug Metabolism Reviews Downloaded from informahealthcare.com by Kainan University on 04/22/15
its metabolite, 11-nor-carboxy-delta-9-tetrahydrocannabinol, into hair.
PhD Thesis. Salt Lake City (UT): University of Utah.
Sobolevsky T, Prasolov I, Rodchenkov G. (2012). Detection of urinary
metabolites of AM-2201 and UR-144, two novel synthetic cannabin-
oids. Drug Test Anal 4:745–753.
Sobolevsky T, Prasolov I, Rodchenvkov G. (2010). Detection of JWH-
018 metabolites in smoking mixture post-administration urine.
Forensic Sci Int 200:141–147.
Spinelli E, Barnes AJ, Young S, et al. (2014). Performance character-
istics of an ELISA screening assay for urinary synthetic cannabinoids.
Drug Test Anal. doi:10.1002/dta.1702.
Strano-Rossi S, Anzillotti L, Castrignano E, et al. (2012). Ultra high per-
formance liquid chromatography-electrospray ionization-tandem mass
spectrometry screening method for direct analysis of designer drugs,
For personal use only.
‘‘spice’’ and stimulants in oral fluid. J Chromatogr A 1258:37–42.
Strano-Rossi S, Anzillotti L, Dragoni S, et al. (2014). Metabolism of
JWH-015, JWH-098, JWH-251, and JWH-307 in silico and in vitro:
A pilot study for the detection of unknown synthetic cannabinoids
metabolites. Anal Bioanal Chem 406:3621–3636.
Sundström M, Pelander A, Angerer V, et al. (2013). A high-sensitivity
ultra-high performance liquid chromatography/high-resolution time-
of-flight mass spectrometry (UHPLC-HR-TOFMS) method for
screening synthetic cannabinoids and other drugs of abuse in urine.
Anal Bioanal Chem 405:8463–8474.
Takayama T, Suzuki M, Todoroki K, et al. (2014). UPLC/ESI-MS/
MS-based determination of metabolism of several new illicit drugs,
ADB-FUBINACA, AB-FUBINACA, AB-PINACA, QUPIC, 5F-
QUPIC and alpha-PVT, by human liver microsome. Biomed
Chromatogr 28:831–838.
Teske J, Weller J-P, Fieguth A, et al. (2010). Sensitive and rapid
quantification of the cannabinoid receptor agonist naphthalen-1-yl-(1-
pentylindol-3-yl)methanone (JWH -018) in human serum by liquid
chromatography-tandem mass spectrometry. J Chromatog B Analyt
Technol Biomed Life Sci 878:2659–2663.
Thomas BF, Martin BR. (1990). In vitro metabolism of (-)-cis-3-
[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)
cyclohexanol, a synthetic bicyclic cannabinoid analog. Drug Metab
Dispos 18:1046–1054.
Tuv SS, Krabseth H, Karinen R, et al. (2014). Prevalence of synthetic
cannabinoids in blood samples from Norwegian drivers suspected of
impaired driving during a seven weeks period. Accid Anal Prev 62:
26–31.
Uhelski ML, Khasabova IA, Simone DA. (2014). Inhibition of
anandamide hydrolysis attenuates nociceptor sensitization in a
murine model of chemotherapy-induced peripheral neuropathy.
J Neurophysiol 113:1501–1510.
Synthetic cannabinoids 51
Uhl M, Sachs H. (2004). Cannabinoids in hair: Strategy to prove
marijuana/hashish consumption. Forensic Sci Int 145:143–147.
United Nations Office on Drugs and Crime. (2011). Synthetic
Cannabinoids in Herbal Products. Available from: http://www.unodc.
org/unodc/en/scientists/synthetic-cannabinoids-in-herbal-products.html
[last accessed 15 Sept 2014].
United Nations on Drugs and Crime. (2014). World Drug Report 2014.
Available at: http://www.unodc.org/wdr2014/ [last accessed 15 Jan
2015].
US Drug Enforcement Administration. (2013a). Establishment of drug
codes for 26 substances. Final rule. Fed Regist 78:664–666.
US Drug Enforcement Administration. (2013b). Schedules of controlled
substances: Temporary placement of three synthetic cannabinoids into
Schedule I. Final order. Fed Regist 78:28735–28739.
US Drug Enforcement Administration. (2014). Schedules of Controlled
Substances: Temporary Placement of Four Synthetic Cannabinoids
Into Schedule I: Notice of Intent. Fed Regist 79:7577–7582.
Valiveti S, Hammell DC, Earles DC, Stinchcomb AL. (2004a).
Transdermal delivery of the synthetic cannabinoid WIN 55,212-2:
In vitro/in vivo correlation. Pharm Res 21:1137–1145.
Valiveti S, Kiptoo PK, Hammell DC, Stinchcomb AL. (2004b).
Transdermal permeation of WIN 55,212-2 and CP 55,940 in human
skin in vitro. Int J Pharm 278:173–180.
Wintermeyer A, Moller I, Thevis M, et al. (2010). In vitro phase I
metabolism of the synthetic cannabimimetic JWH-018. Anal Bioanal
Chem 398:2141–2153.
Wohlfarth A, Gandhi AS, Pang S, et al. (2014a). Metabolism of synthetic
cannabinoids PB-22 and its 5-fluoro analog, 5F-PB-22, by human
hepatocyte incubation and high-resolution mass spectrometry. Anal
Bioanal Chem 406:1763–1780.
Wohlfarth A, Pang S, Zhu M, et al. (2013a). First metabolic profile of
XLR-11, a novel synthetic cannabinoid, obtained by using human
hepatocytes and high-resolution mass spectrometry. Clin Chem 59:
1638–1648.
Wohlfarth A, Pang S, Zhu M, et al. (2014b). Metabolism of RCS-8,
a synthetic cannabinoid with cyclohexyl structure, in human hepato-
cytes by high-resolution MS. Bioanalysis 6:1187–1200.
Wohlfarth A, Scheidweiler KB, Castaneto M, et al. (2014c). Urinary
prevalence, metabolite detection rates, temporal patterns and evalu-
ation of suitable LC-MS/MS targets to document synthetic cannabin-
oid intake in US military urine specimens. Clin Chem Lab Med
53:423–434.
Wohlfarth A, Scheidweiler KB, Chen X, et al. (2013b). Qualitative
confirmation of 9 synthetic cannabinoids and 20 metabolites in
human urine using LC–MS/MS and library search. Anal Chem 85:
3730–3738.
World Anti-Doping Agency. (2014). The 2014 Prohibited List
International Standard. Available from: http://www.wada-ama.org/
Documents/World_Anti-Doping_Program/WADP-Prohibited-list/2014/
WADA-prohibited-list-2014-EN.pdf [last accessed 15 Jan 2014].
Yanes EG, Lovett DP. (2012). High-throughput bioanalytical method for
analysis of synthetic cannabinoid metabolites in urine using salting-
out sample preparation and LC-MS/MS. J Chromatogr B Analyt
Technol Biomed Life Sci 909:42–50.
Yeakel JK, Logan BK. (2013). Blood synthetic cannabinoid concentra-
tions in cases of suspected impaired driving. J Anal Toxicol 37:
547–551.
Zhang Q, Ma P, Cole RB, Wang, G. (2006). Identification of in vitro
metabolites of JWH-015, an aminoalkylindole agonist for the
peripheral cannabinoid receptor (CB2) by HPLC-MS/MS. Anal
Bioanal Chem 386:1345–1355.
Zhang Q, Ma P, Iszard M, et al. (2002). In vitro metabolism of R(+)-
[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo [1,2,3-de]1,4-
benzoxazinyl]-(1-naphthalenyl) methanone mesylate, a cannabinoid
receptor agonist. Drug Metab Dispos 30:1077–1086.