http://informahealthcare.com/dmrISSN: 0360-2532 (print), 1097-9883 (electronic)

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 inbiological matrices

Marisol S. Castaneto1,2, Ariane Wohlfarth1, Nathalie A. Desrosiers1,2*, Rebecca L. Hartman1,2, David A. Gorelick3, andMarilyn A. Huestis1

1Department 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

Abstract

Synthetic cannabinoids (SC), originally developed as research tools, are now highly abusednovel psychoactive substances. We present a comprehensive systematic review covering in vivoand in vitro animal and human pharmacokinetics and analytical methods for identifying SC andtheir metabolites in biological matrices. Of two main phases of SC research, the firstinvestigated therapeutic applications, and the second abuse-related issues. Administrationstudies showed high lipophilicity and distribution into brain and fat tissue. Metabolite profilingstudies, mostly with human liver microsomes and human hepatocytes, structurally elucidatedmetabolites and identified suitable SC markers. In general, SC underwent hydroxylation atvarious molecular sites, defluorination of fluorinated analogs and phase II metabolites werealmost exclusively glucuronides. Analytical methods are critical for documenting intake,with different strategies applied to adequately address the continuous emergence of newcompounds. Immunoassays have different cross-reactivities for different SC classes, but cannotkeep pace with changing analyte targets. Gas chromatography and liquid chromatographymass spectrometry assays – first for a few, then numerous analytes – are available butconstrained by reference standard availability, and must be continuously updated andrevalidated. In blood and oral fluid, parent compounds are frequently present, albeit in lowconcentrations; for urinary detection, metabolites must be identified and interpretation iscomplex due to shared metabolic pathways. A new approach is non-targeted HRMS screeningthat is more flexible and permits retrospective data analysis. We suggest that streamlinedassessment of new SC’s pharmacokinetics and advanced HRMS screening provide a promisingstrategy to maintain relevant assays.

Keywords

Analysis, GC-MS, LC-MS/MS, metaboliteprofiling, methods, novel psychoactivesubstances, pharmacokinetics, review,synthetic cannabinoids

History

Received 1 February 2015Accepted 6 March 2015Published online 8 April 2015

Introduction

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

more pronounced cannabimimetic effects with more cognitive

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).

*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 DrugAbuse, National Institutes of Health, Biomedical Research Center, 251Bayview 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

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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-

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

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 (PubMed�, Embase�, Web of

Science�, Scopus�, Cochrane, Biological Abstracts and

Chemical Abstracts via STN� and SciFinder� 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 Embase�, Web of

Science�, PubMed and Google Scholar using the search

filters ‘‘synthetic cannabinoids’’ plus ‘‘blood’’ or ‘‘urine’’

or ‘‘saliva’’ or ‘‘oral fluids’’ or ‘‘plasma’’ or ‘‘serum’’

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 (n¼ 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

2 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

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DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 3

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73

,JW

H-1

22

,JW

H-2

00

,JW

H-2

10

,JW

H-2

50

,JW

H-2

51

,JW

H-3

07

,M

AM

22

01

and

RC

S-4

ort

ho

iso

mer

Invi

vo,

hu

man

,p

uff

(wit

ho

ut

inh

al-

atio

n,

N¼

2)

OF

,se

rum

No

ne

No

ne

Par

ent

SC

on

lyN

ot

rep

ort

edS

mo

ked

5h

erb

alp

rod

uct

wit

h1

2S

C;

init

ial

con

cen

trat

ion

7–

57

7mg

/L;

no

par

ent

anal

yte

det

ecte

daf

ter

55

h;

seru

mn

egat

ive

Kn

eise

let

al.

(20

13

b)

CP

55

,94

0In

vivo

,d

og

,o

ral

(N¼

1)

Pla

sma

No

ne

No

ne

CP

55

,94

0o

nly

No

tre

po

rted

Cm

ax¼

80mg

/Lat

Tm

ax¼

8h

Fo

ud

aet

al.

(19

87

)

Invi

tro,

mo

use

S9

mic

roso

mal

frac

tio

ns

S9

mic

roso

mal

frac

tio

ns

5M

on

ohy

dro

xyla

tio

n(h

epty

lm

oie

ty)

OH

-CP

55

,94

0N

ot

rep

ort

edT

ho

mas

&M

arti

n(1

99

0)

Invi

tro,

hu

man

skin

,d

iffu

sio

nH

um

ansk

inN

on

eN

on

eC

P5

5,9

40

on

lyN

ot

rep

ort

edV

aliv

eti

etal

.(2

00

4b

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 5

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le1

.C

on

tin

ued

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

JWH

-01

5In

vitr

o,

RL

MR

LM

22

Dih

yd

rod

iol

form

atio

n(n

aph

thyl)

Mo

no

-o

rd

ihy

dro

xyla

tio

n(N

-pro

py

l,in

do

le,

nap

hth

yl)

Deh

yd

rogen

atio

n(N

-pro

py

l)N

-dea

lkyla

tio

n

JWH

-01

5d

ihy

dro

dio

lN

ot

rep

ort

edN

ot

rep

ort

edZ

han

get

al.

(20

06

)

Invi

tro,

HL

MH

LM

18

Dih

yd

rod

iol

form

atio

n(n

aph

thyl)

Mo

no

-,d

i-o

rtr

ihy

dro

x-

yla

tio

n(N

-pro

pyl,

ind

ole

,n

aph

thyl)

Deh

yd

rogen

atio

n(N

-pro

py

l)N

-dea

lkyla

tio

n

No

tsp

ecif

ied

No

tre

po

rted

Maz

zari

no

etal

.(2

01

4)

Invi

tro,

rat

liver

slic

esR

atli

ver

slic

es4

Mo

no

hy

dro

xyla

tio

n(N

-pen

tyl,

ind

ole

)N

-dea

lkyla

tio

n

N-d

esal

kyl

and

OH

-in

do

leG

lucu

ron

ides

Als

oem

plo

yed

insi

lico

met

abo

lite

pre

dic

tio

nso

ftw

are

Str

ano

-Ro

ssi

etal

.(2

01

4)

JWH

-01

8In

vivo

,ra

t,G

IU

rin

e3

N-d

ealk

yla

tio

nO

H-J

WH

-01

8-

des

alk

yl

No

tre

po

rted

Ab

stra

ct;

dat

an

ot

com

ple

teK

ram

eret

al.

(20

08

)In

vivo

,h

um

an,

smo

ked

(sel

f-ex

per

imen

t,N¼

2)

Blo

od

No

ne

Par

ent

on

lyN

on

eN

ot

rep

ort

edT

eske

etal

.(2

01

0)

Invi

tro,

HL

MH

LM

13

Dih

yd

rod

iol

form

atio

n(n

aph

thyl)

Mo

no

-,d

i-o

rtr

ihy

dro

x-

yla

tio

n(N

-pen

tyl,

ind

ole

,n

aph

thyl)

N-d

ealk

yla

tio

nC

arb

ox

yla

tio

n(N

-pen

tyl)

Deh

yd

rogen

atio

n(N

-pen

tyl)

JWH

-01

8-N

-OH

-p

enty

l,JW

H-0

18

-O

H-i

nd

ole

,JW

H-

01

8-O

H-n

aph

thyl

met

abo

lite

s

No

tre

po

rted

JWH

-01

8d

ihy

dro

dio

lh

astw

ois

om

ers

Win

term

eyer

etal

.(2

01

0)

Invi

tro,

HL

Man

dH

IM,

sele

cted

Ph

ase

Iin

cub

ated

wit

hU

GT

1A

1,

UG

T1

A1

0,

UG

1A

9,

UG

T2

B7

HL

M,

HIM

No

ne

Glu

curo

nid

atio

no

ccu

rred

for

all

mo

no

hy

dro

xy-

late

dan

dca

rbox

yla

ted

met

abo

lite

s

No

td

eter

min

edG

lucu

ron

ide

6JW

H-0

18

met

abo

lite

sin

cub

ated

wit

hU

GT

tod

eter

min

ep

has

eII

met

abo

lite

s;A

llm

etab

oli

tes,

exce

pt

for

JWH

-01

8-N

-4-O

H-

ind

ole

wer

eg

lucu

ron

idat

ed

Ch

imal

ako

nd

aet

al.

(20

11

a)

Invi

vo,

hu

man

,sm

oked

(N¼

2)

OF

No

ne

Par

ent

on

lyN

ot

rep

ort

edN

ot

rep

ort

edH

igh

est

OF

con

cen

tra-

tio

ns:

3–

35mg

/Lat

0.3

h.

JWH

-01

8w

asst

ill

pre

sen

taf

ter

12

h.

Co

ult

eret

al.

(20

11

)

Invi

tro,

HL

MH

LM

4D

ihy

dro

dio

lfo

rmat

ion

(nap

hth

yl)

Mo

no

or

dih

yd

rox

yla

tio

n(N

-pen

tyl,

nap

hth

yl)

N-d

ealk

yla

tio

n

OH

-JW

H-0

18

-d

esal

ky

lN

ot

rep

ort

edIn

form

atio

nex

trac

ted

fro

mp

rese

nta

tio

nL

ogan

etal

.(2

01

1)

(co

nti

nu

ed)

6 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

Invi

vo,

rat,

IPB

loo

d,

Uri

ne

�6

Mo

no

-,d

i-tr

ihy

dro

xyl-

atio

n(n

ot

spec

ifie

d),

N-d

ealk

yla

tio

nC

arb

ox

yla

tio

n(N

-pen

tyl)

OH

-JW

H-0

18

-d

esal

kyl

(OH

po

s-it

ion

no

tsp

ecif

ied

)

No

tre

po

rted

Info

rmat

ion

extr

acte

dfr

om

pre

sen

tati

on

Lo

gan

etal

.(2

01

1)

Invi

tro,

HL

MH

LM

4M

on

ohy

dro

xyla

tio

n(N

-pen

tyl,

ind

ole

)JW

H-0

18

-N-5

-OH

-p

enty

lN

ot

spec

ifie

dE

lSo

hly

etal

.(2

01

1)

Invi

vo,

rat,

IPH

air

Uri

ne

2(h

air)

4(u

rin

e)H

air:

Mo

no

hy

dro

xyla

tio

n(i

nd

ole

)C

arb

ox

yla

tio

n(N

-pen

tyl)

Uri

ne:

Mo

no

hy

dox

yla

tio

n(N

-pen

tyl,

ind

ole

)N

-dea

lkyla

tio

nC

arb

ox

yla

tio

n(N

-pen

tyl)

No

tsp

ecif

ied

No

tre

po

rted

JWH

-01

85

mg

/kg

/day

�1

0d

;ab

stra

cto

nly

,d

ata

no

tco

mp

lete

Kik

ura

-Han

ajir

iet

al.

(20

11

)

Invi

tro,

HL

Man

dC

YP

45

0is

ofo

rms

(CY

P1

A2

,-2

C9

,-2

D6

,-2

E1

,an

d-3

A4

)

HL

M6

Mo

no

hy

dro

xyla

tio

n(N

-pen

tyl,

nap

hth

yl)

Car

box

yla

tio

n(N

-pen

tyl)

JWH

-01

8-N

-4-

and

5-O

H-p

enty

lan

dJW

H-0

18

-N-

pen

tan

oic

acid

No

tre

po

rted

CY

P1

A2

cata

lyze

dO

Han

dC

OO

H,

CY

P2

C9!

N-O

H;

CY

P2

C1

9!

N-4

-OH

-p

enty

l

Ch

imal

ako

nd

aet

al.

(20

12

)

Invi

vo,

mo

use

,n

ose

-sm

ok

ein

hal

atio

n(N¼

6)

Blo

od

Bra

inL

iver

No

ne

Par

ent

on

lyN

ot

rep

ort

edN

ot

rep

ort

edA

fter

30

min

exp

osu

re:

JWH

-01

8:

82

±2mg

/k

gb

loo

d,

51

0±

16

6mg

/kg

bra

in,

19

90

±7

52mg

/kg

liver

Po

kli

set

al.

(20

12

a)

Invi

tro,

HL

MH

LM

3M

on

ohy

dro

xyla

tio

n(N

-pen

tyl)

Car

box

yla

tio

n(N

-pen

tyl)

JWH

-01

8-N

-5-O

H-

pen

tyl

No

td

etec

ted

Pat

ton

etal

.(2

01

3a)

Invi

tro,

HL

MH

LM

3M

on

ohy

dro

xyla

tio

n(N

-pen

tyl)

Car

box

yla

tio

n(N

-pen

tyl)

JWH

-01

8-N

-4-O

H-

pen

tyl

No

tre

po

rted

Jan

get

al.

(20

14

a)

JWH

-01

8an

dJW

H-0

73

Invi

vo,

rat,

IP(N¼

4)

Uri

ne

4N

-dea

lkyla

tio

nM

on

ohy

dro

xyla

tio

n(i

nd

ole

,n

aph

thyl)

OH

-JW

H-0

18

-d

esal

kyl

No

tsp

ecif

ied

All

met

abo

lite

sd

ealk

y-

late

dan

dm

on

ohy

dro

xyla

ted

Gri

go

ryev

etal

.(2

01

1b

)

Invi

vo,

hu

man

,sm

ok

ed(c

on

tro

lled

adm

inis

trat

ion

,N¼

6)

Blo

od

No

ne

Par

ent,

mo

no

-o

rd

ihy

dro

xyla

tio

n(N

-pen

tyl,

ind

ole

,n

aph

thyl)

N-d

ealk

yla

tio

nC

arb

ox

yla

tio

n(N

-pen

tyl)

No

tre

po

rted

No

tre

po

rted

Hig

hes

tco

nce

ntr

atio

n4

.8mg

/LJW

H-0

18

and

4.2mg

/LJW

H-0

73

at1

9m

infr

om

1o

f6

par

tici

pan

ts;

met

abo

l-it

ed

etec

ted

inb

loo

dat

1h

po

st-

adm

inis

trat

ion

Kac

inko

etal

.(2

01

1)

Invi

vo,

hu

man

,sm

ok

ed(s

elf-

exp

erim

ent,

N¼

1)

Uri

ne

3M

on

ohy

dro

xyla

tio

n(N

-pen

tyl)

Car

box

yla

tio

n(N

-pen

tyl,

N-b

uty

l)

JWH

-01

8-N

-5-O

H-

pen

tyl

No

tsp

ecif

ied

Met

abo

lite

sd

etec

ted

up

to6

5h

wit

hp

eak

con

c.3

–1

6.5

h

De

Jag

eret

al.

(20

12

)

Invi

vo,

mo

use

,sm

ok

e(i

nh

alat

ion

exp

osu

re)

Blo

od

Bra

inL

iver

No

ne

Par

ent

on

lyJW

H-0

18

and

JWH

-07

3o

nly

No

tre

po

rted

Bo

thS

Cin

bra

in4

blo

od

afte

r2

0m

in.

Aft

er2

0h

,o

nly

JWH

-01

8in

bra

inw

asm

easu

red

.JW

H-0

18

and

JWH

-0

73

blo

od

det

ecte

daf

ter

20

h

Po

kli

set

al.

(20

12

b)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 7

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le1

.C

on

tin

ued

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

JWH

-07

3In

vivo

,ra

ts,

IPH

air

3M

on

ohy

dro

xyla

tio

n(N

-b

uty

l)C

arb

ox

yla

tio

n(N

-bu

tyl)

JWH

-07

3-N

-3-O

H-

bu

tyl

No

tre

po

rted

JWH

-07

3p

aren

tco

nce

ntr

atio

n4

met

abo

lite

s

Kim

etal

.(2

01

3)

Invi

tro,

HL

MH

LM

5M

on

ohy

dro

xyla

tio

n(N

-bu

tyl,

ind

ole

,n

aph

thyl)

JWH

-07

3-N

-OH

-b

uty

lo

rO

H-

nap

hth

yl

No

tre

po

rted

Gam

bar

oet

al.

(20

14

)

JWH

-07

34

-met

hyln

aph

thoyl

anal

og

ue

Invi

tro,

HL

MH

LM

3M

on

ohy

dro

xyla

tio

n(N

-bu

tyl,

ind

ole

,n

aph

thyl)

JWH

-07

3-4

-met

hyl-

nap

thyoyl

OH

-nap

hth

yl

No

tre

po

rted

Gam

bar

oet

al.

(20

14

)

JWH

-09

8In

vitr

o,

rat

liver

slic

esR

atli

ver

slic

es6

Mo

no

hy

dro

xyla

tio

n(N

-pen

tyl,

ind

ole

,n

aph

thyl)

O-d

emet

hyla

tio

n(n

aph

thyl)

Car

bo

nyla

tio

n(N

-pen

tyl)

Dem

ethyla

tio

n(n

aph

thyl)

N-d

ealk

yla

tio

n

JWH

-09

8O

-dem

eth

y-

late

dm

etab

oli

teG

lucu

ron

ide

OH

-JW

H-0

98

Em

plo

yed

insi

lico

met

abo

lite

pre

dic

tio

nso

ftw

are

Str

ano

-Ro

ssi

etal

.(2

01

4)

(co

nti

nu

ed)

8 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

JWH

-12

2In

vitr

o,

HL

MH

LM

20

Mo

no

-,d

i-o

rtr

ihy

dro

x-

yla

tio

n(N

-pen

tyl,

ind

ole

,n

aph

thyl)

Deh

yd

rogen

atio

n(N

-pen

tyl)

Dih

yd

rod

iol

form

atio

n(n

aph

thyl)

Car

box

yla

tio

n(N

-pen

tyl)

Dem

ethyla

tio

n(n

aph

thyl)

N-d

ealk

yla

tio

n

JWH

-12

2N

-OH

or

di-

OH

-pen

tyl

or

JWH

-12

2O

H-

or

di-

OH

nap

hth

yl

No

tre

po

rted

De

Bra

ban

ter

etal

.(2

01

3a)

Invi

vo,

mo

use

(chi-

mer

ican

dn

on

-ch

imer

ic),

PO

Uri

ne

33

Mo

no

-,d

i-o

rtr

ihy

dro

x-

yla

tio

n(N

-pen

tyl,

ind

ole

,n

aph

thyl)

Deh

yd

rogen

atio

n(N

-pen

tyl)

Dih

yd

rod

iol

form

atio

n(n

aph

thyl)

N-d

ealk

yla

tio

n

JWH

-12

2-N

-OH

-p

enty

lG

lucu

ron

ides

(11

)an

dsu

lfat

esco

nju

gat

es(6

)

On

em

uri

ne-

spec

ific

mo

no

hy

dro

xyla

ted

met

abo

lite

(un

spec

i-fi

edre

acti

on

site

)w

asd

etec

ted

inm

ice,

and

no

tin

HL

M

De

Bra

ban

ter

etal

.(2

01

3a)

Invi

tro,

HL

MH

LM

4M

on

ohy

dro

xyla

tio

n(N

-pen

tyl,

ind

ole

,n

aph

thyl)

JWH

-12

2-O

H-

nap

hth

yl

No

tre

po

rted

Gam

bar

oet

al.

(20

14

)

Invi

tro,

HL

MH

LM

3M

on

ohy

dro

xyla

tio

n(N

-pen

tyl)

Car

box

yla

tio

n(N

-pen

tyl)

JWH

-12

2-N

-4-O

H-

pen

tyl

No

tre

po

rted

Jan

get

al.

(20

14

a)

Invi

vo,

rats

,P

OA

dip

ose

tiss

ue

No

ne

Par

ent

on

lyJW

H-1

22

on

lyN

ot

rep

ort

edS

ing

leo

ral

do

se2

0m

g/k

gg

iven

and

JWH

-12

29

ng

/gm

easu

red

afte

r3

0d

ays

Sch

aefe

ret

al.

(20

14

)

JWH

-20

0In

vitr

o,

HL

MH

LM

22

Mo

no

hy

dro

xyla

tio

n(m

orp

ho

lin

e,in

do

le,

nap

hth

yl,

N-e

thyl)

Deh

yd

rogen

atio

n(m

or-

ph

oli

ne,

N-e

thyl)

Dih

yd

rod

iol

form

atio

n(n

aph

thyl)

pro

du

cin

g2

-OH

-eth

yl

(2),

mo

r-p

ho

lin

ecl

eavag

ep

ro-

du

cin

gN

-2-O

H-e

thyl

(1),

mo

rph

oli

ne

clea

v-

age

and

carb

ox

yla

tio

nat

N-e

thyl

(1),

mo

rph

oli

ne

JWH

-20

0-N

-2-O

H-

ethyl

(lo

sso

fm

or-

ph

oli

ne

rin

g)

No

tre

po

rted

De

Bra

ban

ter

etal

.(2

01

3b

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 9

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

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Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le1

.C

on

tin

ued

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

Invi

vo,

mo

use

(ch

i-m

eric

,n

on

-chi-

mer

ic),

PO

Uri

ne

23

Mo

rph

oli

ne

clea

vag

eM

on

ohy

dro

xyla

tio

n(n

aph

thyl,

mo

rph

o-

lin

e)D

ihyd

rod

iol

for-

mat

ion

(nap

hth

yl)

Car

box

yla

tio

n(N

-eth

yl,

afte

rm

orp

ho

lin

ecl

eavag

e),

Mo

rph

oli

ne

rin

go

pen

ing

Mo

rph

oli

ne

rin

go

pen

ing

+lo

sso

fet

hyle

ne

JWH

-20

0-N

carb

ox

y(l

oss

of

mo

rph

o-

lin

eri

ng

)

Glu

curo

nid

es(9

)an

dsu

lfat

e(2

)co

nju

gat

es

On

em

on

ohy

dro

xyla

ted

JWH

-20

0d

etec

ted

inm

ou

se,

bu

tn

ot

inH

LM

De

Bra

ban

ter

etal

.(2

01

3b

)

JWH

-21

0In

vitr

o,

HL

MH

LM

18

Mo

no

-,d

i-o

rtr

ihy

dro

x-

yla

tio

n(i

nd

ole

,N

-pen

tyl,

nap

hth

yl)

Dih

yd

rod

iol

form

atio

n(n

aph

thyl)

N-d

ealk

yla

tio

nC

arb

ox

yla

tio

n,

N-d

ealk

yla

tio

nD

ehy

dro

gen

atio

n(N

-pen

tyl)

No

tsp

ecif

ied

No

tre

po

rted

Maz

zari

no

etal

.(2

01

4)

Invi

vo,

rat,

PO

Ad

ipo

seti

ssu

eN

on

eP

aren

to

nly

JWH

-21

0o

nly

No

tre

po

rted

Sin

gle

20

mg

/kg

PO

JWH

-21

0ad

min

is-

tere

d,

and

11

6n

g/g

JWH

-12

2qu

anti

fied

30

day

saf

ter

do

sin

g

Sch

aefe

ret

al.

(20

14

)

JWH

-25

0In

vivo

,ra

t,G

IU

rin

e2

2M

on

o-,

di-

or

tri-

hy

dro

xyla

tio

n(N

-pen

tyl,

ind

ole

,b

enzy

l)D

ehy

dro

gen

atio

n(N

-pen

tyl)

N-d

ealk

yla

tio

n

OH

-JW

H-2

50

-d

esal

kyl

(rat

s),

WH

-25

0O

H-

ind

ole

or

N-p

enty

l(i

nau

then

tic

hu

man

uri

ne)

Co

nju

gat

ed(u

nsp

ecif

ied

)G

rig

ory

evet

al.

(20

11

a)

JWH

-25

1In

vitr

o,

rat

liver

slic

esR

atli

ver

slic

es7

Mo

no

hy

dro

xy

lati

on

(N-p

enty

l,in

do

le,

ben

zyl)

N-d

ealk

yla

tio

nC

arb

onyla

tio

n(N

-pen

tyl)

Car

box

yla

tio

n(N

-pen

tyl)

JWH

-25

1-N

-OH

-p

enty

lG

lucu

ron

ides

maj

or

met

abo

lite

sE

mp

loyed

insi

lico

met

abo

lite

pre

dic

tio

nso

ftw

are

Str

ano

-Ro

ssi

etal

.(2

01

4)

(co

nti

nu

ed)

10 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

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Pat

holo

gy L

ab o

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/09/

15Fo

r pe

rson

al u

se o

nly.

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

JWH

-30

7In

vitr

o,

rat

liver

slic

esR

atli

ver

slic

es3

Mo

no

-o

rd

ihy

dro

xy

lati

on

(N-p

enty

l,fl

uo

rop

he-

nyl)

Deh

yd

rogen

atio

n(N

-pen

tyl)

Car

bo

nyla

tio

n(N

-pen

tyl)

JWH

-30

7-O

Ho

rca

r-b

onyla

ted

met

abo

lite

s

No

tre

po

rted

Em

plo

yed

insi

lico

met

abo

lite

pre

dic

tio

nso

ftw

are

Str

ano

-Ro

ssi

etal

.(2

01

4)

MA

M2

20

1In

vitr

o,

HL

MH

LM

3D

eflu

ori

nat

ion

Mo

no

hy

dro

xyla

tio

n(N

-pen

tyl)

Car

box

yla

tio

n(N

-pen

tyl)

JWH

-12

2-N

-5-O

H-

pen

tyl

No

tre

po

rted

Jan

get

al.

(20

14

a)

PB

-22

Invi

tro

,H

LM

HL

M1

Est

erhy

dro

lysi

s(q

uin

oli

nyl)

+ca

rbox

yla

tio

n

1-H

-pen

tyli

nd

ole

-CO

OH

No

tre

po

rted

Tak

ayam

aet

al.

(20

14

)

Invi

tro

,h

um

anh

epat

ocy

tes

Hu

man

hep

ato

cyte

s2

0E

ster

hy

dro

lysi

s(q

uin

oli

nyl)

Mo

no

hy

dro

xyla

tio

n(N

-pen

tyl,

ind

ole

,qu

ino

linyl)

Car

bo

nyla

tio

n(N

-pen

tyl)

Car

box

yla

tio

n(N

-pen

tyl)

Dih

yd

rod

iol

form

atio

n(q

uin

oli

nyl)

Pen

tyli

nd

ole

-PB

-22

-N

-pen

tan

oic

acid

,P

enty

lin

do

le-P

B-

22

-N-4

-OH

-pen

tyl

Glu

curo

nid

es(n¼

5)

and

cyst

ein

eco

n-

jugat

es(n¼

1)

Wo

hlf

arth

etal

.(2

01

4a)

5F

-PB

-22

Invi

tro

,H

LM

HL

M1

Est

erhy

dro

lysi

s(q

uin

oli

nyl)

+ca

rbox

yla

tio

n

Flu

oro

pen

tyl-

1-H

-p

enty

lin

do

le-

CO

OH

No

tre

po

rted

Tak

ayam

aet

al.

(20

14

)

Invi

tro

,h

um

anh

epat

ocy

tes

Hu

man

hep

ato

cyte

s2

0E

ster

hy

dro

lysi

s(q

uin

oli

nyl)

Mo

no

hy

dro

xyla

tio

n(i

nd

ole

,at

N-p

enty

l,qu

ino

linyl)

Def

luo

rin

atio

mC

arb

ox

yla

tio

n(N

-pen

tyl)

Dih

yd

rod

iol

form

atio

n(q

uin

oli

nyl)

Pen

tyli

nd

ole

-PB

-22

-N

-pen

tan

oic

acid

Glu

curo

nid

es(n¼

7)

and

on

ecy

stei

ne

con

jug

ate

Wo

hlf

arth

etal

.(2

01

4a)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 11

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

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info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le1

.C

on

tin

ued

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

RC

S-4

Invi

tro

,h

um

anh

epat

ocy

tes

Hu

man

hep

ato

cyte

s1

8M

on

oh

ydro

xyla

tio

n(i

nd

ole

,N

-pen

tyl)

(2)

Car

box

yla

tio

n(N

-pen

tyl)

O-d

emet

hyla

tio

n(p

hen

yl)

Dem

eth

yla

tio

n(N

-pen

tyl)

O-d

esm

eth

yl-

RC

S-4

-N

-OH

-pen

tyl

Glu

curo

nid

es(n¼

11

)an

dsu

lfat

e(n¼

1)

con

jugat

e

Gan

dh

iet

al.

(20

14

b)

RC

S-8

Invi

tro

,h

um

anh

epat

ocy

tes

Hu

man

hep

ato

cyte

s3

2M

on

o-

and

dih

yd

rox

yl-

atio

n(p

hen

yl

rin

g,

cycl

oh

exyl

and

un

spec

ifie

dp

osi

tio

ns)

O-d

emet

hyla

tio

n(p

hen

yl)

RC

S-8

-OH

(OH

po

s-it

ion

un

spec

ifie

d)

Glu

curo

nid

es(n¼

15

)W

oh

lfar

thet

al.

(20

14

b)

ST

S-1

35

Invi

tro

,h

um

anh

ep-

ato

cyte

san

dH

LM

Hu

man

hep

ato

cyte

s2

9M

on

o-,

di-

or

trih

yd

rox

-yla

tio

n(a

dam

anta

ne,

ind

ole

,N

-flu

oro

pen

-ty

l)C

arb

onyla

tio

n(N

-flu

oro

-p

enty

l,ad

aman

tan

e)C

arb

ox

yla

tio

n(N

-pen

tyl)

Def

luo

rin

atio

n

ST

S-1

35

-OH

(ad

a-m

anta

ne)

rin

g,

des

alk

yl-

ST

S-1

35

-N

-OH

-pen

tyl

Glu

curo

nid

es(n¼

6)

ST

S-1

35

hal

f-li

fean

din

trin

sic

clea

ran

ce:

t 1/2¼

3.1

±0

.2m

inan

dC

Lin

t¼

20

8.8

ml�m

in�

1�k

g�

1

Gan

dh

iet

al.

(20

14

a)

(co

nti

nu

ed)

12 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

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Dow

nloa

ded

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info

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care

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Pat

holo

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/09/

15Fo

r pe

rson

al u

se o

nly.

Co

mp

ou

nd

s

Stu

dy

typ

e,sp

ecie

s,ro

ute

of

adm

inis

trat

ion

Mat

rix

To

tal

#m

etab

oli

tes

Bio

tran

sfo

rmat

ion

,P

has

eI

(rea

ctio

nsi

tes)

Maj

or

ph

ase

Im

etab

oli

te(s

)P

has

eII

met

abo

lite

sO

ther

fin

din

gs

Ref

eren

ces

UR

-14

4In

vitr

o,

HL

Man

dP

45

0is

ofo

rms

CY

3A

4an

dC

YP

2B

6

HL

M1

6M

on

o-

or

dih

yd

rox

yl-

atio

n(i

nd

ole

,N

-pen

tyl)

Deh

yd

rogen

atio

n(N

-pen

tyl)

N-d

ealk

yla

tio

n(1

),D

ihyd

rod

iol

form

atio

n(i

nd

ole

)

UR

-14

4-O

H(u

nsp

eci-

fied

OH

po

siti

on

)G

lucu

ron

ides

(co

n-

firm

edin

auth

enti

ch

um

anu

rin

esa

mp

les)

So

bo

levsk

yet

al.

(20

12

)

WIN

55

,21

2-2

Invi

tro

,R

LM

RL

M8

Dih

yd

rod

iol

form

atio

n(n

aph

thyl,

ind

ole

),M

on

ohy

dro

xyla

tio

n(i

nd

ole

)D

ehy

dro

gen

atio

n(m

orp

ho

lin

e).

WIN

55

,21

2-2

dih

y-

dro

dio

lat

nap

hth

yl

mo

iety

(2is

om

ers)

No

tre

po

rted

Zh

ang

etal

.(2

00

2)

Invi

vo,

hai

rles

sg

uin

eap

ig,

tran

sder

mal

Pla

sma

No

ne

Par

ent

on

lyW

IN5

5,2

12

-2o

nly

No

tre

po

rted

Pla

sma

(dru

gv

iaIV

bo

lus)

:C

max¼

43

9±

12

0mg

/L,

t 1/2a

¼0

.12

±0

.02

h,

8.1

h

Val

ivet

iet

al.

(20

04

a)

Invi

tro

,1

cm2

hu

man

and

gu

inea

pig

skin

Sk

inN

on

eP

aren

to

nly

WIN

55

,21

2-2

on

lyN

ot

rep

ort

edN

ot

rep

ort

edV

aliv

eti

etal

.(2

00

4a,

b)

Invi

vo,

mo

use

,IP

Blo

od

(tru

nk

),A

dip

ose

tiss

ue

Bra

in

No

ne

Par

ent

on

lyW

IN5

5,2

12

-2o

nly

No

tre

po

rted

Det

ecte

d3

0%

adip

ose

tiss

ue,

3%

pla

sma

and

0.3

%b

rain

tota

lin

ject

ed(0

.3–

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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� 10�4 cm/h with

777 ng/cm2�h 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 ±

0.08mg/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).

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

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

11.4–17.1mg/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,

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

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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

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

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

4.2mg/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 (0.1mg/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 (N¼ 1), serum Cmax was

0.56mg/L at 1.5 h (Hutter et al., 2013). Serum concentrations

declined to50.02 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

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, 0.15mg/g AM2201-6-

hydroxyindole, 1.9mg/g JWH-018-N-5-hydroxypentyl, 1.3mg/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

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self-experimenter. JWH-018 and JWH-073 metabolites were

detected40.1 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

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

(n¼ 21) conducted between 2010 and 2014. HLM was the

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.

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

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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).

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

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

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

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 17

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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-

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/min�kg, 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-

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 biologicalmatrices

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

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

(n¼ 5) and quantitative (n¼ 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 & Auwarter, 2012).

Thirty SC were quantified in serum by LC-MS/MS

following LLE sample preparation, and scheduled multiple

reaction monitoring (MRM) (Kneisel & Auwarter, 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 and56% 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 & Auwarter, 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

18 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

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Tab

le2

.A

nal

yti

cal

met

ho

ds

for

the

iden

tifi

cati

on

of

syn

thet

icca

nn

abin

oid

sin

bio

log

ical

mat

rice

s.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

JWH

-01

8an

dm

etab

oli

tes

QU

AL

Uri

ne

Hy

dro

lysi

s,L

LE

LC

-MS

/MS

hea

ted

-E

SI

No

tfu

lly

val

idat

edd

ue

toth

eab

sen

ceo

fm

etab

oli

test

and

ard

s.

Au

then

tic

uri

ne

spec

i-m

ens

coll

ecte

dfr

om

ind

ivid

ual

ssu

spec

ted

of

SC

inta

ke

No

par

ent

SC

,1

3m

etab

-o

lite

sd

etec

ted

:JW

H-

01

8-N

-OH

-in

do

le(2

),N

-OH

-pen

tyl,

N-C

OO

H,

di-

OH

(at

ind

ole

and

nap

hth

yl)

or

atin

do

lean

dN

-5-

OH

-pen

tyl,

dih

yd

ro-

dio

l(a

tn

aph

thyl,

2),

dih

yd

rod

iol+

OH

-in

do

le(2

),N

-dea

lkyla

ted

+O

H-

ind

ole

(2)

and

N-C

OO

Ho

rC

OO

Hm

eth

ox

ym

etab

oli

te.

So

bo

levsk

yet

al.

(20

10

)

JWH

-01

8Q

UA

NT

Ser

um

LL

EL

C-M

S/M

S-E

SI+

Val

idat

edL

OD

,L

OQ

,L

inea

rity

,M

EA

uth

enti

cse

rum

spec

i-m

ens

fro

mtw

ovo

lun

-te

ers

wh

osm

oked

her

bal

pro

du

ctco

n-

tain

ing

SC

JWH

-01

8w

asd

etec

ted

inse

rum

sam

ple

s.L

OD¼

0.0

7mg

/L;

LO

Q¼

0.2

1mg

/L

Tes

ke

etal

.(2

01

0)

JWH

-01

8m

etab

oli

tes:

N-5

-OH

-pen

tyl,

5-O

H-

ind

ole

,N

-CO

OH

,N

-dea

lkyla

ted

-5-O

H-

ind

ole

and

2-O

H-

nap

hth

oyl

QU

AL

and

QU

AN

TU

rin

eH

yd

roly

sis,

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,

Rec

over

y,L

inea

rity

,B

ias,

ME

,Im

pre

cisi

on

,S

tab

ilit

y

Au

then

tic

uri

ne

do

pin

gco

ntr

ols

spec

imen

s(n¼

4)

Val

idat

ion

on

lyfo

rJW

H-

01

8-N

-OH

-pen

tyl

and

JWH

-01

8-N

-CO

OH

,sy

nth

esiz

edin

-ho

use

.M

etab

oli

tes

stab

lefo

r4

wee

ks

atR

To

r4� C

.L

OD¼

0.1mg

/L;

LO

Q¼

0.5mg

/L

Beu

cket

al.

(20

11

)

JWH

-01

8m

etab

oli

tes:

N-4

-an

d5

-OH

-pen

tyl

and

CO

OH

.JW

H-0

73

met

abo

lite

s:N

-3-

and

4-O

H-b

uty

lan

dC

OO

H

QU

AN

TU

rin

eH

yd

roly

sis,

SP

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,

Imp

reci

sio

n,

Bia

s,C

arry

over

Au

then

tic

uri

ne

spec

i-m

ens

fro

mfo

ur

ind

i-v

idu

als

susp

ecte

do

fS

Cin

take

LO

D/L

OQ¼

0.1mg

/L.

JWH

-01

8-N

-4-O

H-

pen

tyl4

JWH

-01

8-

N-C

OO

H4

JWH

-01

8-

N-5

-OH

-pen

tyl

(all

thre

ean

aly

tes

Glu

c-co

nju

gat

ed)

inJW

H-

01

8u

sers

’u

rin

e.JW

H-0

73

-N-C

OO

Hd

etec

ted

inJW

H-0

18

susp

ects

;b

ut

JWH

-0

73

-N-3

-OH

-bu

tyl

and

N-4

-OH

-bu

tyl

inJW

H-0

73

susp

ecte

du

ser’

uri

ne

on

ly

Ch

imal

ako

nd

aet

al.

(20

11

b)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 19

Dru

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from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

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al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

JWH

-01

8,

JWH

-25

0,

JWH

-07

3,

CP

47

,49

7(C

8),

CP

47

,49

7,

HU

-21

0

QU

AN

TO

FS

PE

LC

-MS

/MS

-EIS

+/�

LO

D,

LO

Q,L

inea

rity

,Im

pre

cisi

on

,B

ias,

ME

,In

terf

eren

ceS

tab

ilit

yR

ecover

y(e

xtr

acti

on

pad

)

Au

then

tic

OF

spec

imen

sfr

om

two

vo

lun

teer

sw

ho

smo

ked

her

bal

pro

du

ctco

nta

inin

gS

C

JWH

-07

3,

JWH

-01

8an

dJW

H-2

50

un

stab

leat

RT

,b

ut

rem

ain

ed4

80

%at

4� C

afte

r1

wee

k.

CP

47

,49

7-C

8an

dH

U-2

10

stab

leat

RT

&4� C

afte

r1

wee

k.

Ex

trac

tsst

able

on

auto

sam

ple

rat

7� C

for

48

h.

Au

then

tic

sam

ple

sst

able

for

atle

ast

1m

on

th.

LO

Q¼

0.5mg

/L.

An

aly

tere

cover

yfr

om

coll

ecti

on

pad

60

%(5

5–

74

%).

Co

ult

eret

al.

(20

11

)

CP

47

,49

7Q

UA

NT

Uri

ne

Dil

uti

on

LC

-MS

/MS

-ES

I�L

OD

,L

OQ

,L

inea

rity

,Im

pre

cisi

on

,B

ias,

Un

cert

ain

ty

No

ne

Sta

bil

ity

no

tev

alu

ated

.L

OD

:10mg

/L,

LO

Q:2

0mg

/L

Dow

lin

g&

Reg

an(2

01

1)

JWH

-01

5,

JWH

-01

8,

JWH

-07

3,

JWH

-08

1,

JWH

-20

0,

JWH

-25

0,

WIN

55

,21

2-2

,m

eth

a-n

and

amid

e,JW

H-0

19

,JW

H-0

20

QU

AN

Tan

dQ

UA

LS

eru

mL

LE

LC

-MS

/MS

-ES

IL

OD

,L

OQ

,S

elec

tiv

ity,

Lin

eari

ty,

Imp

reci

sio

n,

Bia

s,C

arry

over

,R

ecover

y,M

E,

Sta

bil

ity

Au

then

tic

seru

msp

eci-

men

s(n¼

10

1)

fro

mh

osp

ital

s,d

etox

ific

a-ti

on

and

ther

apy

cen

-te

rs,

fore

nsi

cp

sych

iatr

icce

nte

rs

Sta

ble

for

thre

efr

eeze

-th

awcy

cles

,fo

r1

wee

kat�

20� C

.JW

H-

JWH

-08

1u

nst

able

atR

T(�

35

to�

26

%)

afte

r4

8h

.P

roce

ssed

sam

ple

sst

able

at4� C

afte

r5

h.

LO

D¼

0.1mg

/L,

LO

Q¼

0.1

–0

.6mg

/L.

Inau

then

tic

sam

ple

s,JW

H-0

81

(0.1

1–

16

.9mg

/L)

mo

stp

reval

ent

(56

/10

1),

then

JWH

-25

0(4

7),

JWH

-01

8(9

),JW

H-

07

3(6

),JW

H-0

15

(2).

Dre

sen

etal

.(2

01

1)

JWH

-01

8an

dm

etab

ol-

ites

:6

-OH

-in

do

le,

N-5

-OH

-pen

tyl,

N-C

OO

H

QU

AN

TU

rin

eH

yd

roly

sis,

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

nA

uth

enti

cu

rin

esp

eci-

men

s(n¼

33

)co

l-le

cted

un

der

fore

nsi

can

dra

nd

om

wo

rkp

lace

dru

gte

stin

g.

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.0

1–

0.1mg

/L,

LO

Q¼

1mg

/L.

JWH

-0

18

-N-C

OO

Han

d-5

-O

H-p

enty

lw

ere

pre

-se

nt

inal

lsa

mp

les.

Hig

hes

tco

nce

ntr

atio

nw

as2

72

56mg

/LJW

H-

01

8-N

-CO

OH

.

ElS

oh

lyet

al.

(20

11

)

(co

nti

nu

ed)

20 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

JWH

-01

8,

JWH

-07

3,

JWH

-25

0,

JWH

-01

9(Q

Lo

nly

)

QU

AN

Tan

dQ

UA

LW

BL

LE

LC

-MS

/MS

-ES

I+

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Inte

rfer

ence

,E

xtr

acti

on

Eff

icie

ncy

,M

E,

Pro

cess

effi

cien

cy,

Car

ryover

,D

ilu

tio

nIn

teg

rity

Au

then

tic

WB

spec

imen

fro

ma

kn

ow

nJW

H-

01

8/J

WH

-07

3u

ser

Sta

ble

at3� C

and�

10� C

for

atle

ast

30

day

s,fo

rth

ree

free

ze-t

haw

cycl

es,

and

on

auto

-sa

mp

ler

atR

T.

JWH

-0

18

and

JWH

-07

3qu

anti

fied

inb

loo

din

19

–1

99

min

sam

ple

.L

OD¼

0.0

06

–0

.01

6mg

/L;

LO

Q¼

0.1mg

/L.

Kac

inko

etal

.(2

01

1)

JWH

-01

8an

dm

etab

oli

tes

(n¼

19

)in

Win

term

eyer

etal

.(2

01

0)

QU

AL

Uri

ne

Hy

dro

lysi

s,L

LE

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,L

inea

rity

,Im

pre

cisi

on

,S

pec

ific

ity,

Rec

over

y

Au

then

tic

uri

ne

do

pin

gco

ntr

ol

spec

imen

s(n¼

75

00

)

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.1mg

/L.

Pre

do

min

ant

met

abo

l-it

ed

etec

ted

was

JWH

-0

18

N-O

H-p

enty

lm

etab

oli

te.

Mo

ller

etal

.(2

01

1)

JWH

-01

8an

dm

etab

ol-

ites

:4

-,5

-,6

-,7

-OH

-in

do

le,

N-5

-OH

-pen

tyl

and

-CO

OH

.JW

H-0

73

and

met

abo

l-it

es:

4-,

5-,

6-,

7-O

H-

ind

ole

,N

-4-O

H-b

uty

lan

d-C

OO

H

QU

AN

TU

rin

eH

yd

roly

sis,

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

nA

uth

enti

cu

rin

esp

eci-

men

sfr

om

thre

esu

s-p

ecte

dS

Cu

sers

.

Sta

bil

ity

no

tev

alu

ated

.L

OD¼5

2mg

/L,

LO

Q¼

1.8

–1

0.8mg

/L.

Iden

tifi

edJW

H-0

18

met

abo

lite

s:5

-an

d6

-O

H-i

nd

ole

,N

-5-O

H-

pen

tyl,

N-C

OO

H(a

ll8

5–

10

0%

Glu

c).

Als

o,

iden

tifi

edJW

H-0

73

-3-

OH

-in

do

le(1

00

%G

luc)

and

JWH

-07

3-

N-C

OO

H(5

%G

luc)

.

Mo

ran

etal

.(2

01

1)

AM

69

4,

AM

12

41

,C

P4

7,4

97

,C

P4

7,4

97

C8

-ho

mo

log

,H

U-2

10

,JW

H-0

07

,JW

H-0

15

,JW

H-0

18

,JW

H-0

19

,JW

H-0

30

,JW

H-0

73

,JW

H-0

81

,JW

H-2

03

,JW

H-2

10

,JW

H-2

50

,JW

H-2

51

,JW

H-3

02

,JW

H-3

98

,R

CS

-4,

RC

S-4

2-

and

3-m

eth

-ox

yh

om

olo

g,

RC

S-8

,R

CS

-4-C

-4-h

om

olo

g,

WIN

48

09

8,

WIN

55

,21

2-2

mes

yla

te

QU

AN

TW

BL

LE

LC

-MS

/MS

-ES

I+/�

LO

D,

LO

Q,

Sel

ecti

vit

y,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Sta

bil

ity,

Ex

trac

tio

nE

ffic

ien

cy,

ME

No

ne

Ex

trac

tsst

able

for

24

hp

rov

ided

reco

nst

itu

ted

inm

eth

ano

l.A

nal

yte

sst

able

afte

rth

ree

free

ze/t

haw

cycl

esan

dat

1m

on

thst

ore

din

�2

0� C

.L

OQ¼

0.5

and

5mg

/L.

No

auth

enti

csp

ecim

ens

anal

yze

d.

Am

man

net

al.

(20

12

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 21

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

Met

abo

lite

so

nly

:JW

H-

01

8-N

-5-O

H-p

enty

lan

d-C

OO

H,

JWH

-0

19

-5-O

H-i

nd

ole

,JW

H-0

73

-N-4

-OH

-b

uty

lan

d-C

OO

H,

JWH

-12

2-N

-5-O

H-

pen

tyl,

JWH

-20

0-5

-O

H-i

nd

ole

,JW

H-2

50

-5

-OH

-in

do

lean

dN

-CO

OH

,R

CS

-4N

-5-

OH

-pen

tyl

QU

AN

TU

rin

eH

yd

roly

sis,

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Car

ryover

,M

E

Au

then

tic

uri

ne

spec

i-m

ens

fro

ma

vo

lun

teer

wh

osm

oked

her

bal

ble

nd

‘‘K

ron

ic’’

con

-ta

inin

gJW

H-0

18

and

JWH

-08

3

Sta

bil

ity

no

tev

alu

ated

.L

OQ¼

0.1mg

/L.

JWH

-01

8-N

-5-O

H-

pen

tyl

and

-CO

OH

and

JWH

-07

3-

N-C

OO

Hd

etec

tab

le5

65

hp

ost

-sm

ok

ing

atL

LO

Q0

.1mg

/L.

De

Jager

etal

.(2

01

2)

AB

-00

1Q

UA

LU

rin

eH

yd

roly

sis

(aci

d),

LL

E,

TM

S-

der

ivat

ized

GC

-MS

No

tfu

lly

val

idat

edd

ue

tola

cko

fm

etab

oli

test

and

ard

s.

Au

then

tic

uri

ne

spec

i-m

ens

fro

mtw

ovo

lun

-te

ers

(sel

f-ex

per

imen

t)w

ho

inges

ted

0.2

2an

d0

.55

mg

/kg

AB

-00

1

Sev

enm

ajo

rm

etab

oli

tes

and

un

spec

ifie

dn

um

ber

of

min

or

met

abo

lite

sd

etec

ted

.

Gri

go

ryev

etal

.(2

01

2)

JWH

-01

8,

JWH

-01

8-2

-,4

-,5

-,6

-an

d-7

-OH

-in

do

le,

JWH

-01

8-N

-5-

OH

-pen

tyl,

JWH

-01

8-

N-C

OO

H,

JWH

-07

3,

JWH

-07

3-4

-,5

-,6

-an

d-7

-OH

-in

do

le,

JWH

-07

3-N

-4-O

H-

bu

tyl,

JWH

-07

3-

N-C

OO

H

QU

AL

Uri

ne

LL

E/S

PE

LC

-MS

/MS

-ES

I+In

terf

eren

ceS

tab

ilit

yA

uth

enti

cu

rin

esa

mp

les

coll

ecte

dfr

om

US

ath

lete

s(n¼

54

96

)

Ex

trac

tsst

able

dat

coo

led

tem

per

atu

refo

r2

4h

.L

OD¼

1mg

/L.

Sy

nth

etic

can

nab

ino

idm

etab

oli

tes

wer

ed

etec

ted

in4

.5%

(n¼

26

6)

sam

ple

sw

ith

50

%o

fth

esa

mp

les

con

tain

edJW

H-0

18

and

JWH

-07

3m

etab

-o

lite

sw

hil

e4

9%

con

-ta

ined

JWH

-01

8m

etab

oli

tes

on

ly.

Hel

tsle

yet

al.

(20

12

)

Met

abo

lite

so

nly

:JW

H-

01

8-N

-pen

tan

oic

acid

,JW

H-0

18

-N-5

-OH

-p

enty

l,JW

H-0

18

4-,

5-,

6-,

and

7-O

H-

ind

ole

,JW

H-0

73

-N

-CO

OH

,JW

H-0

73

-N

-4-O

H-b

uty

l,JW

H-

07

36

-OH

-in

do

le,

JWH

-12

2-N

-5-O

H-

pen

tyl

QU

AL

Uri

ne

Hy

dro

lysi

s,L

LE

LC

-MS

/MS

-ES

I+,

LC

-TO

F/M

S-E

SI+

Met

ho

dn

ot

full

yval

idat

ed;

uti

lize

dfo

rM

etID

Au

then

tic

uri

ne

spec

i-m

ens

fro

mp

atie

nt

wit

hp

osi

tive

dru

gte

st(s

eru

m)

Iden

tifi

edm

etab

oli

tes

wer

efu

rth

erch

arac

-te

rize

dby

LC

-T

OF

-MS

.M

etab

oli

tes

of

JWH

-08

1,

JWH

-2

10

,JW

H-2

50

and

RC

S-4

also

wer

ed

etec

ted

inu

rin

esa

m-

ple

sby

LC

-TO

F-M

S.

Hu

tter

etal

.(2

01

2a)

AM

69

4,

AM

22

01

,JW

H-

00

7,

JWH

-01

5,

JWH

-0

18

,JW

H-0

19

,JW

H-

02

0,

JWH

-07

3,

JWH

-0

81

,JW

H-1

22

,JW

H-

20

0,

JWH

-20

3,

JWH

-2

10

,JW

H-2

50

,JW

H-

25

1,

JWH

-39

8,

QU

AN

TH

air

Was

hin

gL

LE

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,L

inea

rity

,S

elec

tiv

ity,

Sp

ecif

icit

y

Au

then

tic

hai

rsp

ecim

ens

(n¼

8)

fro

min

pat

ien

tp

sych

iatr

icw

ard

pat

ien

tsw

ith

po

siti

ve

SC

inse

rum

Sta

bil

ity

no

tev

alu

ated

.L

OQ¼

0.5

pg

/mg

.H

air

sam

ple

sw

ere

po

siti

ve

for�

1S

Ch

igh

est

con

cen

trat

ion

of

78

pg

/mg

JWH

-08

1.

Oth

eran

aly

tes

det

ecte

d4

LL

OQ

Hu

tter

etal

.(2

01

2b

)

(co

nti

nu

ed)

22 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

met

han

and

amid

e,R

CS

-4,

RC

S-4

ort

ho

iso

mer

,R

CS

-8,

WIN

48

09

8,

WIN

55

21

2-2

(5.0

pg

/mg

)w

ere

JWH

-01

8,

JWH

-07

3,

JWH

-21

0an

dJW

H-

25

0.

An

aly

tes

also

wer

ed

etec

ted

inth

ew

ash

ing

solv

ents

(wat

eran

dac

eto

ne)

.R

CS

-4an

dm

etab

oli

tes

QU

AL

Uri

ne

Hy

dro

lysi

s,L

LE

,m

ethyla

tio

n,

TM

S-

der

ivat

izat

ion

,ac

yla

tio

n

GC

-MS

Met

ho

dn

ot

full

yval

idat

edd

ue

tola

cko

fst

and

ard

s;M

etID

pu

rpo

se

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

7)

coll

ecte

dp

atie

nts

susp

ecte

do

fS

Cin

take

Sta

bil

ity

no

tev

alu

ated

.1

6R

CS

-4m

etab

oli

tes

iden

tifi

edw

ith

RC

S-4

-N

-hy

dro

xy

pen

tyl

pre

val

ent

Kav

anag

het

al.

(20

12

)

AM

69

4,

AM

12

20

,A

M2

20

1,

AM

22

33

,C

RA

-13

,JW

H-0

07

,JW

H-0

15

,JW

H-0

18

,JW

H-0

18

adam

anty

ld

eriv

ativ

e,JW

H-0

19

,JW

H-0

20

,JW

H-0

73

,JW

H-0

81

,JW

H-1

22

,JW

H-2

00

,JW

H-2

03

,JW

H-2

10

,JW

H-2

50

,JW

H-2

51

,JW

H-3

07

,JW

H-3

87

,JW

H-3

98

,M

AM

22

01

,m

eth

a-n

and

amid

e,R

CS

-4,

RC

S-4

ort

ho

iso

mer

,R

CS

-8,

WIN

48

09

8,

WIN

55

21

2-2

QU

AN

TO

FL

LE

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,S

elec

tiv

ity,

Lin

eari

ty,

Imp

reci

sio

n,

Bia

s,R

ecover

y,M

E,

Pro

cess

Eff

icie

ncy

,S

tab

ilit

y,C

arry

over

,R

ecover

y(c

oll

ec-

tio

np

ad)

Au

then

tic

OF

spec

imen

s(n¼

26

4)

coll

ecte

db

etw

een

Dec

emb

er2

01

0an

dJa

nu

ary

20

12

Ex

trac

tsw

ere

stab

leu

pto

7h

inau

tosa

mp

ler

at1

0� C

.In

stab

ilit

yo

bse

rved

inco

llec

tio

np

ads

sto

red

bet

wee

n2

2–

27� C

,b

ut

imp

roved

wit

het

ha-

no

l.L

OD¼

0.0

15

–0

.9mg

/L.

LO

Q¼

0.1

5–

30mg

/L.

SC

(n¼

12

)w

ere

det

ecte

din

12

%o

fsa

mp

les.

Kn

eise

let

al.

(20

12

)

AM

69

4,

AM

12

20

,A

M2

20

1,

AM

22

33

,C

RA

-13

,JW

H-0

07

,JW

H-0

15

,JW

H-0

18

,JW

H-0

18

adam

anty

ld

eriv

ativ

e,JW

H-0

19

,JW

H-0

20

,JW

H-0

73

,JW

H-0

81

,JW

H-1

22

,JW

H-2

00

,JW

H-2

03

,JW

H-2

10

,JW

H-2

50

,JW

H-2

51

,JW

H-3

07

,JW

H-3

87

,JW

H-3

98

,M

AM

22

01

,m

eth

a-n

and

amid

e,R

CS

-4,

RC

S-4

ort

ho

iso

mer

,R

CS

-8,

WIN

48

09

8,

WIN

55

21

2-2

QU

AN

TS

eru

mL

LE

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,S

elec

tiv

ity,

Lin

eari

ty,

Imp

reci

sio

n,

Bia

s,R

ecover

y,M

E,

Pro

cess

Eff

icie

ncy

,S

tab

ilit

y,C

arry

over

Au

then

tic

seru

msp

eci-

men

s(n¼

83

3)

bet

wee

nA

ug

ust

20

11

and

Jan

uar

y2

01

2co

l-le

cted

fro

mfo

ren

sic

psy

chia

tric

and

reh

abil

itat

ion

clin

ics,

crim

inal

inves

tigat

ion

case

san

dE

Rv

isit

s

Ex

trac

tsst

able

afte

r9

hau

tosa

mp

ler

sto

rage

at1

0� C

,th

ree

free

ze/

thaw

cycl

esan

dat

14

day

sst

ora

ge

at�

20� C

.V

alid

ated

met

ho

dw

asem

plo

yed

inth

e.L

OD¼

0.0

1–

2.0mg

/L.

LO

Q¼

0.1

–2mg

/L.

Inau

then

tic

sam

ple

s,th

em

ost

pre

val

ent

anal

yte

iden

tifi

edw

asJW

H-

21

0(8

0%

)an

dJW

H-

12

2(6

4%

)w

ith

11

dif

fere

nt

SC

.H

igh

est

con

cen

trat

ion

qu

anti

-fi

edw

as2

30mg

/Lfo

rJW

H-1

22

.

Kn

eise

l&

Au

war

ter

(20

12

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 23

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

CB

D,

CB

N,

HU

-21

0,

JWH

-01

8,

JWH

-07

3,

JWH

-20

0,

JWH

-25

0,

TH

C

QU

AN

TH

air

Was

hin

g,

NaO

Hd

iges

tio

n,

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,

Sel

ecti

vit

y,L

inea

rity

,Im

pre

cisi

on

,B

ias,

ME

,C

arry

over

Au

then

tic

hai

rsp

ecim

ens

(n¼

17

9)

fro

mfo

ren

-si

cca

ses

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.0

2–

0.1

8p

g/

mg

;L

OQ¼

0.0

7–

18

pg

/mg

.In

auth

enti

ch

air

sam

ple

s,n

ine

sam

ple

sco

nfi

rmed

for

JWH

-01

8,

JWH

-07

3(n¼

8),

and

JWH

-25

0(n¼

8).

Hig

hes

tS

Cco

nce

ntr

atio

nw

as7

29

.4p

g/m

gfo

rJW

H-2

50

.

Sal

om

on

eet

al.

(20

12

)

JWH

-01

8,

JWH

-07

3Q

UA

NT

WB

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,

Sel

ecti

vit

y,L

inea

rity

,Im

pre

cisi

on

,B

ias,

Ion

sup

pre

ssio

n,

ME

,In

terf

eren

ce,

Car

ryover

Au

then

tic

po

st-m

ort

emW

Bsp

ecim

ens

(n¼

18

)

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.0

1mg

/L,

LO

Q¼

0.0

5mg

/L.

JWH

-01

8an

d/o

rJW

H-0

73

in4

0%

of

the

case

s.H

igh

est

JWH

-01

8an

dJW

H-

07

3(m

g/L

)1

99

and

68

.3,

resp

ecti

vel

y,in

card

iac

blo

od

.

Sh

ank

set

al.

(20

12

)

AM

22

01

and

met

abo

lite

s:JW

H-0

18

-N-(

5-O

H-

pen

tyl)

,d

ihy

dro

dio

lJW

H-0

18

,d

i-O

H-

AM

22

01

,d

ihy

dro

dio

l-A

M2

20

1,

OH

-A

M2

20

1,

des

pen

tyl-

AM

22

01

,JW

H-0

18

-N

-CO

OH

;U

R-1

44

and

met

abo

lite

s:d

esp

en-

tyl-

OH

-UR

-14

4,

di-

OH

-UR

-14

4,

des

pen

-ty

l-U

R-1

44

,d

ehy-

dra

ted

OH

-UR

-14

4,

OH

-UR

-14

4

QU

AL

Uri

ne

Hy

dro

lysi

s,L

LE

LC

-MS

/MS

-ES

I+M

eth

od

no

tfu

lly

val

idat

edd

ue

tola

cko

fst

and

ard

s;M

etID

pu

rpo

se

Au

then

tic

uri

ne

spec

i-m

ens

con

tain

ing

UR

-14

4(n¼

5)

and

AM

22

01

(n¼

6)

met

abo

lite

s,su

spec

ted

SC

use

rs.

Sta

bil

ity

no

tev

alu

ated

.U

rin

esp

ecim

ens

po

si-

tive

wit

h(+

)A

M2

20

1m

etab

oli

tes

also

(+)

for

JWH

-01

8,

JWH

-0

73

and

JWH

-21

0m

etab

oli

tes.

Def

luo

rin

ated

AM

22

01

met

abo

lite

sw

ere

excl

ud

ed.

Mo

no

hy

dro

xy

late

dan

dd

ihy

dro

dio

lA

M2

20

1m

etab

oli

tes

incl

ud

ing

con

jug

ated

met

abo

lite

sw

ere

ob

serv

edin

AM

22

01

(+)

uri

ne

sam

ple

s.U

R-1

44

par

ent

was

det

ecte

dat

low

level

sin

1o

f5

uri

ne

(+)

for

UR

-14

4m

etab

oli

tes.

Pre

do

min

ant

met

abo

l-it

esid

enti

fied

wer

eco

nju

gat

edm

on

o-

and

di-

OH

UR

-14

4m

etab

oli

tes.

So

bo

levsk

yet

al.

(20

12

)

(co

nti

nu

ed)

24 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

4-f

luo

ro-A

MP

,4

-met

hyl-

AM

P,

4-m

eth

yl-

N-e

thylc

ath

ino

ne,

AM

69

4,

BZ

P,

clo

ben

-zo

rex

,C

P4

7,4

97

,fe

n-

pro

po

rex

,fu

rfen

ore

x,

HU

-21

0,

JWH

-01

8,

JWH

-01

9,

JWH

-07

3,

JWH

-12

2,

JWH

-20

0,

JWH

-25

0,

MB

DB

,M

DP

V,

mep

hed

ron

e,m

eph

eno

rex

,m

ethy-

lon

e,m

ethylt

hio

AM

P,

MD

AI,

nab

ilo

ne

QU

AL

OF

Dil

uti

on

LC

-MS

/MS

-ES

I+L

OD

,S

pec

ific

ity,

ME

,Io

nsu

pp

res-

sio

n/e

nh

ance

men

t,L

inea

rity

,M

emo

ryef

fect

,S

tab

ilit

y

No

ne

SC

stab

lefo

r2

mo

nth

saf

ter

eig

ht

free

ze/t

haw

cycl

esan

dat

RT

for

4w

eek

sw

hen

sto

red

inm

ob

ile

ph

ase

exce

pt

for

CP

47

,49

7an

dH

U-

21

05

2w

eek

sst

abil

-it

y.L

OD¼

1–

20mg

/L.

Str

ano

-Ro

ssi

etal

.(2

01

2)

JWH

-01

8-N

-CO

OH

,JW

H-0

18

-N-4

-OH

-p

enty

l,JW

H-0

73

-N-

CO

OH

,JW

H-0

73

-N

-3-O

H-b

uty

l

QU

AL

and

QU

AN

TU

rin

eH

yd

roly

sis,

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,S

elec

tiv

ity,

Cro

ssta

lk,

ME

,R

ecover

y,C

arry

over

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

50

0)

ran

do

mw

ork

pla

ced

rug

test

ing

and

/or

fore

nsi

cca

ses

Sta

bil

ity

no

tev

alu

ated

.L

OQ¼

4mg

/L.

JWH

-0

18

and

JWH

-07

3m

etab

oli

tes

wer

eid

enti

fied

wit

hco

n-

cen

trat

ion

sra

ng

ing

fro

m5

.4to

37

.8mg

/L;

alth

ou

gh

po

siti

vit

yra

tew

aslo

w(n

um

ber

no

tsp

ecif

ied

).

Yan

es&

Lovet

t(2

01

2)

Blo

od

:U

R-1

44

and

its

py

roly

sis

pro

du

ct:

1-

(1-p

enty

l-1

H-i

nd

ol-

3-

yl)

-3-m

ethyl-

2-

(pro

pan

-2yl)

bu

t-3

-en

-1

-on

e;U

rin

e:U

R-1

44

,d

esp

enty

l-U

R-1

44

,d

esp

enty

l-O

H-U

R-

14

4,

deh

ydra

ted

-OH

-U

R-1

44

,O

H-U

R-1

44

,U

R-1

44

-N-C

OO

H,

di-

OH

-UR

-14

4,

UR

-1

44

-N-5

-OH

-pen

tyl-

b-G

luc

QU

AN

TW

B,

Uri

ne

Hy

dro

lysi

s(u

rin

eo

nly

),L

LE

LC

-MS

/MS

-ES

+(b

loo

d),

LC

-T

OF

MS

-ES

I+(u

rin

e)

LO

Q,

LO

Q,

Imp

reci

sio

n,

Bia

s,S

pec

ific

ity,

Rec

over

y,M

E

Au

then

tic

WB

and

uri

ne

spec

imen

sfr

om

ap

atie

nt

inth

eh

osp

ital

for

SC

into

xic

atio

n.

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.1

5mg

/L;

LO

Q¼

0.5mg

/L.

UR

-1

44

at6

.1mg

/Lw

asqu

anti

fied

inb

loo

dan

dit

sp

yro

lysi

sp

rod

-u

ct(c

on

cen

trat

ion

no

tre

po

rted

),w

hil

eO

H-

UR

-14

4,

UR

-14

4-

N-C

OO

Han

dd

i-O

H-

UR

-14

4d

etec

ted

inu

rin

e.

Ad

amow

icz

etal

.(2

01

3)

Met

abo

lite

sfo

rL

C-M

S/

MS

on

ly:

JWH

-01

8-

N-4

-an

d5

-OH

-pen

tyl,

JWH

-01

9-N

-5-

and

6-

OH

-hex

yl,

JWH

-07

3-

N-3

-an

d4

-OH

-bu

tyl,

JWH

-25

0-N

-4-O

H-

pen

tyl

and

AM

22

01

-N

-4-O

H-p

enty

l

QU

AL

Uri

ne

Hy

dro

lysi

s,L

LE

EL

ISA

,L

C-M

S/M

S-

ES

I+S

ensi

tiv

ity

Lin

eari

ty,

Inte

rfer

ence

,S

tab

ilit

y,P

reci

sio

nan

dD

rift

,C

arry

over

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

63

,p

rev

i-o

usl

yco

nfi

rmed

SC

po

siti

ve)

EL

ISA

des

ign

edto

hav

e1

00

%C

XR

wit

hJW

H-

01

8-N

-5-O

H-p

enty

lw

asev

alu

ated

usi

ng

5mg

/Lcu

toff

and

per

-fo

rman

ced

eter

min

edby

anal

yzi

ng

auth

enti

cu

rin

esa

mp

les

pre

vi-

ou

sly

con

firm

ed(+

)v

iaL

C-M

S/M

Sfo

rJW

H-0

18

-N-4

-an

d

Arn

sto

net

al.

(20

13

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 25

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

5-O

H-p

enty

l(�

0.1mg

/Lcu

toff

).E

LIS

Ah

ad9

6%

sen

siti

vit

y,1

00

%sp

ecif

icit

yan

d9

8%

accu

racy

.C

ross

-re

acti

vit

y(4

1%

)2

0S

Cm

etab

oli

tes.

CP

47

,49

7,

CP

47

,49

7-

C8

,H

U-2

11

JWH

-01

8,

JWH

-07

3,

JWH

-20

0,

JWH

-25

0,

TH

C

QU

AN

TO

FS

PE

LC

-MS

/MS

-ES

I+/�

LO

Q,

LO

Q,

Imp

reci

sio

n,

Bia

s,S

pec

ific

ity,

Rec

over

y,M

E

Au

then

tic

OF

(n¼

40

)sp

ecim

ens

fro

md

riv

ers

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.0

25

–1

.0mg

/L

;L

OQ¼

0.1

–2

.5mg

/L

.N

osa

mp

les

con

-fi

rmed

for

SC

wh

ile

TH

Cw

asqu

anti

fied

in2

0sa

mp

les.

De

Cas

tro

etal

.(2

01

3)

SC

:A

M1

22

0,

AM

22

01

,A

M6

94

,JW

H-0

15

,JW

H-0

18

,JW

H-0

19

,JW

H-0

73

,JW

H-0

81

,JW

H-1

22

,JW

H-2

00

,JW

H-2

03

,JW

H-2

10

,JW

H-2

50

,JW

H-2

51

,JW

H-3

07

and

oth

erN

PS

:a-

PP

P,

4-F

MA

,4

-ME

C,

bu

tylo

ne,

BZ

P,

DM

A,

ehth

yp

he-

nid

ate,

MB

ZP

,m

CP

P,

MD

PV

,m

eph

edro

ne,

met

hca

thin

on

e,m

ethylo

ne,

nap

hy

ron

e,p

ente

dro

ne,

pF

PP

,T

FM

PP

QU

AN

TS

eru

mL

LE

LC

-MS

/MS

-ES

I+/�

LO

D,

LO

Q,L

inea

rity

,Im

pre

cisi

on

,B

ias,

Sta

bil

ity,

ME

Au

then

tic

seru

msp

ecim

enS

tab

ilit

yex

per

imen

tsw

ere

per

form

edac

cord

ing

toA

mm

ann

etal

.(2

01

2).

LO

D¼

0.0

2–

0.4mg

/L;

LO

Q¼

0.0

5–

0.5mg

/L.

An

aly

tes

wer

e7

5–

89

%st

able

atth

ese

con

di-

tio

ns.

Au

then

tic

seru

mn

egat

ive

for

SC

.

Dzi

asd

osz

etal

.(2

01

3)

AM

69

4,

JWH

-01

8,

JWH

-0

73

,JW

H-1

22

,JW

H-

08

1,

JWH

-20

0,

JWH

-2

10

,JW

H-2

50

QU

AL

Hai

rB

ase

hy

dro

lysi

s,L

LE

LC

-TO

F-M

S-E

SI+

Ex

trac

tio

nre

cover

yA

uth

enti

ch

air

(n¼

43

5)

spec

imen

sfr

om

DU

IDsu

spec

ts

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

10

pg

/mg

Am

on

gth

ese,

eig

ht

wer

e(+

)fo

rJW

H-

01

8,

JWH

-07

3,

JWH

-0

81

,JW

H-1

22

and

JW2

50

at0

.01

0–

1.2

8n

g/m

g.

Go

ttar

do

etal

.(2

01

3)

UR

-14

4-N

-OH

/-C

OO

H,

UR

-144-O

H-d

esal

kyl

and

UR

-14

4m

ajo

rp

yro

lysi

sp

rod

uct

met

abo

lite

sfo

rmed

fro

mhy

dra

tio

nan

dhy

dro

xyla

tio

n

QU

AL

Uri

ne

Hy

dro

lysi

s,T

MS

-d

eriv

atiz

edG

C-M

S,

LC

-MS

/MS

Met

ho

dn

ot

full

yval

idat

edd

ue

tola

cko

fav

aila

ble

stan

dar

ds

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

45

)fr

om

SC

susp

ecte

du

sers

UR

-14

4an

dm

etab

oli

tes

EI

spec

tra

wer

ein

clu

ded

bas

edo

np

rev

iou

sst

ud

ies

cite

din

(So

bo

levsk

yet

al.,

20

12

).U

R-1

44

met

ab-

oli

tes

(n¼

16

)an

dit

sm

ajo

rp

yro

lysi

sp

rod

-u

ct(n¼

21

)d

etec

ted

.

Gri

go

ryev

etal

.(2

01

3b

)

(co

nti

nu

ed)

26 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

SC

:A

M6

94

,A

M1

24

1,

AM

22

01

,JW

H-0

07

,JW

H-0

15

,JW

H-0

16

,JW

H-0

18

,JW

H-0

18

-6

-MeO

,JW

H-0

22

,JW

H-0

73

,JW

H-0

81

,JW

H-0

98

,JW

H-1

22

,JW

H-2

10

,JW

H-2

00

,JW

H-2

03

,JW

H-2

50

,R

CS

-4,

RC

S-8

,W

IN4

80

98

QU

AL

WB

, Uri

ne,

Ser

um

SP

EL

C-T

OF

MS

-ES

I+E

xtr

acti

on

reco

ver

y,M

EA

uth

enti

cW

B(n¼

5),

seru

m(n¼

11

),u

rin

e(n¼

5)

spec

imen

sco

nfi

rmed

oth

erD

OA

;n

on

eco

nfi

rmed

for

SC

Met

ho

dw

asd

evel

op

edan

dval

idat

edto

scre

enfo

rw

ide

arra

yo

fsu

b-

stan

ces

incl

ud

ing

anti

-d

epre

ssan

t,an

tih

ista

-m

ines

,b

enzo

dia

zep

-in

es,

hy

pn

oti

cs,

mu

scle

rela

xan

ts,over

-th

e-co

un

ter

med

ica-

tio

ns

and

stim

ula

nts

(in

clu

din

gsy

nth

etic

cath

ino

nes

)u

tili

zin

ga

per

son

alco

mp

ou

nd

dat

abas

eli

bra

ryso

ft-

war

e.S

tab

ilit

yN

/D.

Dru

gs

wer

eco

rrec

tly

iden

tifi

edin

all

sam

-p

les

exce

pt

for

on

e(b

uta

lbit

al).

Gu

ale

etal

.(2

01

3)

Met

abo

lite

so

nly

:JW

H-

01

8-N

-4-

and

5-O

H-

pen

tyl,

JWH

-01

8-

N-C

OO

H,

JWH

-01

8-

6-O

H-i

nd

ole

,JW

H-

07

3-N

-3-

and

4-O

H-

bu

tyl,

JWH

-07

3-N

-N

-CO

OH

,JW

H-0

73

-6

-OH

-in

do

le

QU

AL

and

QU

AN

TU

rin

eD

ilu

tio

n,

Hy

dro

lysi

s,S

PE

EL

ISA

,L

C-M

S/M

S-

ES

I+S

elec

tiv

ity

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Rec

over

y,S

tab

ilit

y

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

52

)w

ere

firs

tsc

reen

edw

ith

EL

ISA

and

con

firm

edby

LC

-MS

/MS

.

An

aly

tes

stab

ilit

y(f

ort

i-fi

edn

egat

ive

bla

nk

s)w

asac

cep

tab

leaf

ter

thre

efr

eeze

/th

awcy

cles

,at

4� C

and

�2

0� C

.L

OD¼

0.0

25

–0

.1mg

/L

;L

OQ¼

2.5mg

/L.

Inu

rin

esp

ecim

ens,

27

of

52

con

firm

edp

osi

tive

for�

1S

Cm

etab

oli

te.

Jan

get

al.

(20

13

)

JWH

-01

8an

dm

etab

ol-

ites

:JW

H-0

18

-N-4

-an

d5

-OH

-pen

tyl,

JWH

-01

8-N

-N-

CO

OH

;JW

H-0

73

and

met

abo

lite

s:JW

H-

07

3-N

-3-

and

-4-O

H-

bu

tyl,

JWH

-07

3-

N-C

OO

H

QU

AN

TH

air

Was

h,

LL

EL

C-M

S/M

S-E

SI+

Sel

ecti

vit

yL

OD

,L

OQ

,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Rec

over

y,P

roce

ssE

ffic

ien

cy,

Sta

bil

ity

Au

then

tic

hu

man

hai

rsp

ecim

ens

(n¼

18

)fr

om

SC

susp

ecte

du

sers

An

aly

tes

wer

est

able

du

rin

gsa

mp

lep

rep

ar-

atio

nan

dex

trac

tio

nu

pto

24

h.

LO

D/

LO

Q¼

0.0

5p

g/m

g.

On

lyJW

H-0

18

,JW

H-

01

8-N

-5-O

H-p

enty

l,an

dJW

H-0

73

wer

ed

etec

ted

inh

um

anh

air.

No

sig

nif

ican

td

iffe

ren

cein

anal

yte

con

cen

trat

ion

sb

etw

een

pig

men

ted

and

no

n-p

igm

ente

dh

air.

Kim

etal

.(2

01

3)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 27

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

AM

69

4,

AM

12

20

,A

M2

23

3,

AM

22

01

,JW

H-0

07

,JW

H-0

15

,JW

H-0

18

,JW

H-0

19

,JW

H-0

20

,JW

H-0

73

,JW

H-0

81

,JW

H-1

22

,JW

H-2

00

,JW

H-2

03

,JW

H-2

10

,JW

H-2

50

,JW

H-2

51

,JW

H-3

07

,JW

H-3

87

,JW

H-3

98

,JW

H-4

12

,M

AM

22

01

,m

eth

anan

dam

ide,

RC

S-4

,R

CS

-4o

rth

ois

om

er,

RC

S-8

,W

IN4

80

98

,W

IN5

52

12

-2

QU

AN

TO

FP

rote

inp

reci

pit

atio

nw

ith

AC

NL

C-M

S/M

S-E

SI+

Sel

ecti

vit

yL

OD

,L

OQ

,L

inea

rity

,B

ias,

ME

Imp

reci

sio

n,

Rec

over

y(e

xtr

ac-

tio

nan

dco

llec

tio

nd

evic

e),

Pro

cess

Eff

icie

ncy

,S

tab

ilit

yC

arry

over

Au

then

tic

OF

spec

imen

sco

llec

ted

fro

m2

vo

lun

teer

s

Ex

trac

tsst

able

inth

eau

tosa

mp

ler

for

9h

at1

0� C

,d

uri

ng

thre

efr

eeze

/th

awcy

cles

(exce

pt

for

JWH

-30

76

8–

73

%),

and

lon

g-

term

sto

rage

at�

20� C

(exce

pt

for

JWH

-30

77

0–

76

%).

Sta

bil

ity

stu

die

sco

nd

uct

edw

ith

auth

enti

cO

Fco

nta

in-

ing

11

anal

yte

sin

Kn

eise

let

al.

(20

13

a).

Sam

ple

sw

ere

po

ole

d,

ho

mo

gen

ized

,al

i-qu

ote

din

tog

lass

and

pro

pyle

ne

coll

ecti

on

tub

esan

dst

ore

du

pto

72

hat

4o

r2

5� C

.N

oan

aly

tein

stab

ilit

yo

bse

rved

for

gla

ss.

An

aly

tes

wer

est

able

at4� C

afte

r7

2h

(exce

pt

for

JWH

-25

1an

dJW

H-2

03

77

–7

9%

afte

r7

2h

).A

llan

a-ly

tes

exce

pt

JWH

-20

0w

ere

un

stab

le5

63

%at

25� C

afte

r2

4h

and

72

h.

LO

D¼

0.0

15

–0

.9mg

/L;

LO

Q¼

0.1

5–

3mg

/L.

Kn

eise

let

al.

(20

13

b)

AB

-00

1,

AM

69

4,

AM

12

20

,A

M1

24

1,

AM

22

01

,A

M2

23

3,

JWH

-00

7,

JWH

-01

5,

JWH

-01

8,

JWH

-01

9,

JWH

-02

0,

JWH

-07

3,

JWH

-07

3-m

ethyl,

JWH

-08

1,

JWH

-09

8,

JWH

-12

2,

JWH

-14

7,

JWH

-20

0,

JWH

-21

0,

JWH

-25

0,

JWH

-25

1,

JWH

-39

8,

MA

M2

20

1,

RC

S-4

,R

CS

-4o

rth

o,

RC

S-8

,U

R-1

44

,W

IN5

5,2

12

-2

QU

AN

TW

BL

LE

LC

-MS

/MS

-ES

IS

elec

tiv

ity

LO

D,

LO

Q,L

inea

rity

,B

ias,

ME

,Im

pre

cisi

on

Dil

uti

on

inte

gri

ty

Au

then

tic

WB

(n¼

30

78

)sp

ecim

ens

coll

ecte

dfr

om

DU

IDsu

spec

ts,

and

pet

tyd

rug

off

ense

Sta

bil

ity

no

tev

alu

ated

.L

OD

/LO

Qn

ot

spec

i-fi

ed.

Inau

then

tic

WB

(co

llec

ted

bet

wee

nO

cto

ber

20

11

and

Jan

uar

y2

01

3),

28

%W

Bco

nfi

rmed

for

SC

(AB

-00

1,

AM

-69

4,

AM

22

01

,A

M-2

23

3,

JWH

-01

8,

JWH

-01

9,

JWH

-08

1,

JWH

-12

2,

JWH

-20

3,

JWH

-21

0,

JWH

-25

0,

MA

M2

20

1,

RC

S-4

and

UR

-14

4)

Kro

nst

ran

det

al.

(20

13

)

(co

nti

nu

ed)

28 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

Met

abo

lite

so

nly

:JW

H-

01

8-N

-4-

and

5-O

H-

pen

tyl,

JWH

-01

8-

N-C

OO

H,

JWH

-01

8m

ethyl

este

r,JW

H-

07

2-N

-CO

OH

,JW

H-

07

3-N

-3-O

H-b

uty

l,JW

H-0

73

-N-

N-C

OO

H

QU

AN

TU

rin

eH

yd

roly

sis,

SA

LL

E,

rep

eate

dex

trac

tio

nL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,S

elec

tiv

ity,

Cro

ssta

lk,

ME

,R

ecover

y,C

arry

over

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

30

)S

tab

ilit

yn

ot

eval

uat

ed.

Met

ho

dval

idat

edas

des

crib

edin

Yan

es&

Lovet

t(2

01

2).

LO

Q¼

4mg

/L.

JWH

-0

72

-N-C

OO

Hac

idw

asid

enti

fied

insa

m-

ple

sal

so(+

)fo

rA

M2

20

1,

JWH

-01

8an

dJW

H-0

73

met

ab-

oli

tes,

sug

ges

tin

ga

com

mo

nb

iom

arker

for

all

thre

eS

C.

Inth

isst

ud

y,JW

H-0

72

-N

-CO

OH

was

syn

the-

size

din

-ho

use

.

Lovet

tet

al.

(20

13

)

Met

abo

lite

so

fA

M2

20

1an

dJW

H-0

18

(no

tsp

ecif

ied

)

QU

AL

Uri

ne

Hy

dro

lysi

s,L

LE

LC

-HR

MS

Met

ho

dn

ot

full

yval

idat

ed.

Au

then

tic

uri

ne

sam

ple

fro

mS

Cin

tox

icat

edp

atie

nt

Sta

bil

ity

no

tev

alu

ated

.A

M2

20

1-O

Hm

etab

ol-

ites

wer

ed

etec

ted

insp

ecim

en.

Mcq

uad

eet

al.

(20

13

)

AM

69

4,

AM

22

01

,H

U-

21

0,

JWH

-01

5,

JWH

-0

18

,JW

H-0

19

,JW

H-

02

0,

JWH

-07

3,

JWH

-0

81

,JW

H-1

22

,JW

H-

20

0,

JWH

-21

0,

JWH

-2

50

,JW

H-2

51

,R

CS

-4

,R

CS

-4-C

4,

RC

S-8

,W

IN5

5,2

12

-2

QU

AL

OF

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,

Sp

ecif

icit

y,Im

pre

cisi

on

,M

E,

Rec

over

y(e

xtr

acti

on

and

coll

ecti

on

dev

ice)

Au

then

tic

OF

spec

imen

s(n¼

45

)fr

om

dri

ver

ssu

spec

ted

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.0

5–

1.2mg

/L.

Co

llec

tio

nre

cover

yw

as1

9–

61

%(m

ajo

rity

30

–4

0%

).In

auth

enti

cO

Fsp

ecim

ens,

20

%w

ere

po

siti

ve

for

JWH

-01

8an

d/o

rA

M2

20

1.

Oie

stad

etal

.(2

01

3)

(S)-

AM

22

01

-N-4

-OH

-p

enty

l,(R

)-A

M2

20

1-

N-4

-OH

-pen

tyl,

JWH

-0

18

-N-5

-OH

-pen

tyl,

JWH

-01

8-N

-CO

OH

,(S

)-JW

H-0

18

-N-4

-O

H-p

enty

l,(R

)-JW

H-

01

8-N

-4-O

H-p

enty

l

QU

AN

TW

BH

yd

roly

sis,

SP

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,Im

pre

cisi

on

,B

ias,

ME

,C

arry

over

,

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

15

)fr

om

SC

susp

ecte

du

sers

Sta

bil

ity

no

tev

alu

ated

.L

OQ¼

0.7

–1

.53mg

/L.

Met

ho

dem

plo

yed

for

chir

alm

etab

oli

tes

dis

-ti

nct

ion

of

AM

22

01

and

JWH

-01

8.

Bas

elin

ese

par

atio

nw

asac

hie

ved

.In

auth

enti

csp

ecim

ens,

the

maj

ori

tyo

fm

etab

-o

lite

sd

etec

ted

wer

eU

DP

-UG

Tco

nju

gat

ed,

San

dR

chir

alm

etab

-o

lite

sw

ere

sep

arat

edan

dd

eter

min

ed.

Pat

ton

etal

.(2

01

3b

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 29

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

AM

22

01

,A

M2

20

1-N

-4-

OH

-pen

tyl,

JWH

-01

8-

N-4

-an

d5

-OH

-pen

tyl,

JWH

-01

8-N

-CO

OH

QU

AN

TW

BP

rote

inp

reci

pit

atio

nL

C-M

S/M

S-E

SI+

Met

ho

dval

idat

ion

par

amet

ers

no

tsp

ecif

ied

Au

then

tic

po

st-m

ort

emW

BS

C(A

M2

20

1)

into

xic

atio

n

Sta

bil

ity

no

tev

alu

ated

.In

blo

od

,2

.5mg

/LA

M2

20

1,

12

3mg

/LJW

H-0

18

-N-5

-OH

-p

enty

lan

d5

0.8mg

/LJW

H-0

18

-N-C

OO

H.

Un

iden

tifi

edA

M2

20

1m

etab

oli

teal

sow

asd

etec

ted

.

Pat

ton

etal

.(2

01

3a)

QL

:JW

H-0

18

,Q

T:

AM

22

01

,C

P4

7,4

97

,C

P4

7,4

97

-C8

,H

U-

21

0,

JWH

-01

8,

JWH

-0

73

,JW

H-0

81

,JW

H-

20

0,

JWH

-25

0,

RC

S-4

QU

AL

,Q

UA

NT

OF

Dil

uti

on

EL

ISA

(QL

),L

C-M

S/

MS

-ES

I+L

OD

,L

OQ

,S

elec

tiv

ity,

Imp

reci

sio

nS

tab

ilit

y,M

E,

Rec

over

y(c

oll

ecti

on

pad

)

Au

then

tic

OF

spec

imen

s(n¼

32

)co

llec

ted

for

pro

bat

ion

and

par

ole

EL

ISA

per

form

ance

eval

-u

ated

agai

nst

LC

-MS

/M

Sre

sult

san

dac

hie

ved

84

.0%

sen

si-

tiv

ity,

10

0%

spec

ifi-

city

,an

d8

4.3

%ef

fici

ency

at0

.25mg

/Lcu

toff

s.C

ross

-rea

ctiv

-it

y(4

10

%)

for

JWH

-0

15

,JW

H-0

18

,JW

H-

02

2,

JWH

-07

3,

AM

12

20

,A

M2

20

1,

AM

22

32

.F

or

LC

-MS

/M

Sval

idat

ion

,an

a-ly

tes

inO

Fw

ere

�6

0%

atR

T,

4� C

and

�2

0� C

afte

r7

day

s.L

OD¼

0.1mg

/L,

LO

Q¼

0.2

5mg

/L.

Ro

dri

gu

eset

al.

(20

13

)

54

SC

/85

NP

San

aly

tes:

AB

-01

01

,A

M6

94

,A

M1

22

0,

AM

12

20

azep

ane

iso

mer

,A

M2

20

1,

CP

47

,49

7,

CP

47

,49

7-C

8,

CP

55

,94

0,

HU

-21

0,

JWH

-00

7,

JWH

-01

5,

JWH

-01

8,

JWH

-01

8-

1-m

ethyl-

hex

yl,

JWH

-0

18

-6-m

eth

ox

y-i

nd

ole

,JW

H-0

18

-4-,

5-,

6-

and

7-O

H-i

nd

ole

,JW

H-0

18

-N-

4-

and

5-

OH

-pen

tyl,

JWH

-01

8-

N-

CO

OH

,JW

H-0

19

,JW

H-0

73

,JW

H-0

73

2-

and

3-m

ethyl

ho

m-

olo

gy,

JWH

-07

3-N

-3-

and

4-O

H-b

uty

l,JW

H-

07

3-4

-,5

-,6

-,an

d

QU

AL

Uri

ne

Hy

dro

lysi

s,S

PE

LC

-TO

F-M

S-E

SI+

Cu

toff

sele

ctio

n,

ME

,R

ecover

y(e

xtr

ac-

tio

n),

PE

,S

tab

ilit

y

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

50

)fr

om

susp

ecte

dN

PS

use

rs

Sta

bil

ity

no

tev

alu

ated

.M

eth

od

uti

lize

din

-h

ou

seli

bra

ryw

ith

27

7co

mp

ou

nd

entr

ies

(85

NP

Sw

ith

no

avai

lab

lest

and

ard

s).

Cu

toff¼

0.2

–3

0mg

/L.

Inau

then

tic

spec

i-m

ens,

26

con

firm

edfo

rS

Cm

etab

oli

tes

(pri

mar

ily

OH

-pen

tyl

and

CO

OH

).

Su

nd

stro

met

al.

(20

13

)

(co

nti

nu

ed)

30 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

7-O

H-i

nd

ole

,JW

H-

07

3-N

-CO

OH

,JW

H-

08

1,

JWH

-12

2,

JWH

-1

22

-N-5

-OH

-pen

tyl,

JWH

-14

7,

JWH

-20

0,

JWH

-20

0-4

-OH

-in

do

le,

JWH

-20

1,

JWH

-21

0,

JWH

-20

3,

JWH

-25

0,

JWH

-25

1,

JWH

-30

2,

JWH

-39

8,

JWH

-41

2,

MA

M2

20

1,

RC

S-4

,R

CS

-4o

rth

ois

om

er,

RC

S-4

-N-5

-O

H-p

enty

l,R

CS

-4-

N-C

OO

H,

RC

S-8

,W

IN4

80

98

,W

IN5

52

12

AM

22

01

,A

M2

20

1-6

-O

H-i

nd

ole

,A

M2

20

1-

N-4

-OH

-pen

tyl,

JWH

-0

18

,JW

H-0

18

5-

and

6-O

H-i

nd

ole

,JW

H-

01

8-N

-CO

OH

,JW

H-

01

8-N

-5-O

H-p

enty

l,JW

H-0

73

,JW

H-0

73

-5

-an

d6

-OH

-in

do

le,

JWH

-07

3-N

-4-O

H-

bu

tyl,

JWH

-07

3-N

-C

OO

H,

JWH

-08

1,

JWH

-08

1-N

-5-O

H-

pen

tyl,

JWH

-12

2,

JWH

-12

2-N

-5-O

H-

pen

tyl,

JWH

-20

0-5

-an

d6

-OH

-in

do

le,

JWH

-21

0,

JWH

-21

0-

N-4

-an

d5

-OH

-pen

tyl,

JWH

-21

0-N

-CO

OH

,JW

H-2

50

-5-O

H-

ind

ole

,JW

H-2

50

-N-4

-an

d5

-OH

-pen

tyl,

MA

M2

20

1,

RC

S-4

,R

CS

-4-N

-5-O

H-

pen

tyl,

RC

S-4

-N

-CO

OH

QU

AL

Uri

ne

Dil

uti

on

,H

yd

roly

sis

LC

-MS

/MS

-ES

I+L

OD

,L

inea

rity

,Im

pre

cisi

on

,B

ias,

Inte

rfer

ence

,C

arry

over

ME

,Io

nS

up

pre

ssio

no

rE

nh

ance

men

t,S

tab

ilit

y,C

arry

over

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

25

01

)ra

nd

om

wo

rkp

lace

dru

gte

stin

g

An

aly

tes

wer

eid

enti

fied

usi

ng

in-h

ou

seli

bra

ry.

LO

D¼

1–

10mg

/L.

Ex

trac

tsst

able

at4� C

acce

pta

ble

up

to7

2h

.2

90

con

firm

edp

osi

-ti

ve

for

SC

anal

yte

s.O

nly

on

esp

ecim

enco

nfi

rmed

for

AM

22

01

par

ent

anal

yte

.

(Wo

hlf

arth

etal

.,2

01

3b

,2

01

4c)

AM

22

01

,A

M6

94

,JW

H-

01

8,

JWH

-01

9,

JWH

-0

73

,JW

H-0

81

,JW

H-

12

2,

JWH

-17

5,

JWH

-20

0,

JWH

-21

0,

QU

AN

TW

BL

LE

LC

-MS

/MS

-ES

I+L

OD

,L

inea

rity

,Im

pre

cisi

on

,B

ias,

Inte

rfer

ence

,C

arry

over

,M

E,

Ion

Su

pp

ress

ion

/

Au

then

tic

WB

spec

imen

s(n¼

12

)fr

om

DU

IDca

ses

po

siti

ve

for

SC

Sta

bil

ity

no

tev

alu

ated

.L

C-M

S/M

Sw

asval

i-d

ated

inK

acin

ko

etal

.(2

01

1)

wit

had

dit

ion

alp

aren

tan

aly

tes.

Yea

kel

&L

og

an(2

01

3)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 31

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

JWH

-25

0,

RC

S-4

,R

CS

-8E

nh

ance

men

tC

arry

over

LO

Q¼

0.1mg

/L.

Inau

then

tic

WB

spec

i-m

ens,

all

po

siti

ve

for

on

eo

rm

ore

SC

incl

ud

ing

AM

22

01

,JW

H-0

18

,JW

H-0

81

,JW

H-1

22

,JW

H-2

10

and

JWH

-25

0X

LR

11

,X

LR

11

4-O

H-

pen

tyl,

XL

R1

1d

egra

-d

ant

(py

roly

sis

pro

d-

uct

),U

R-1

44

,U

R-1

44

deg

rad

ant

(py

roly

sis

pro

du

ct),

UR

-14

44

-OH

-pen

tyl

QU

AN

TO

FS

PE

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,L

inea

rity

,S

elec

tiv

ity,

Bia

s,Im

pre

cisi

on

,D

ilu

tio

nIn

teg

rity

,M

E,

Sta

bil

ity,

Rec

over

y(e

xtr

ac-

tio

nan

dco

llec

tio

np

ad)

Au

then

tic

OF

spec

imen

s(n¼

49

8)

Ex

trac

tsst

able

for

24

hat

15� C

.A

nal

yte

sal

sost

able

inth

ree

free

ze/

thaw

cycl

esan

dlo

ng

-te

rmst

ora

ge

for

1m

on

that�

20� C

.N

op

yro

lysi

sp

rod

uct

sd

etec

ted

inb

uff

ersa

mp

les

fort

ifie

dw

ith

par

ent

anal

yte

.L

OD¼

0.3

5–

1.9

3mg

/L;

LO

Q¼

5mg

/L.

Inau

then

tic

OF

spec

i-m

ens,

14

con

firm

edfo

rU

R-1

44

4-O

H-

pen

tyl

and

UR

-14

4d

egra

dan

t(5

–6

0mg

/L),

bu

tn

op

aren

td

etec

ted

.U

R-1

44

(5–

30mg

/L)

con

firm

edin

5sa

m-

ple

sw

ith

ou

tm

etab

ol-

ites

.X

LR

11

deg

rad

ant

(55

to4

10

0mg

/L)

con

firm

edin

37

sam

-p

les,

of

wh

ich

30

con

-ta

ined

XL

R1

1(5

5–

10

0mg

/L).

(Am

arat

un

ga

etal

.,2

01

4)

JWH

-01

8-N

-CO

OH

(cal

i-b

rato

r);

LC

-MS

/MS

(Wo

hlf

arth

etal

.,2

01

3b

)

QU

AL

,Q

UA

NT

Uri

ne

HE

IA:

no

ne

LC

-MS

/MS

:d

ilu

tio

n,

hy

dro

lysi

s,p

rote

inp

reci

pit

atio

n

HE

IA(Q

L),

LC

-MS

/M

SL

OD

,L

inea

rity

,C

uto

ffse

lect

ion

,In

terf

eren

ce,

Car

ryover

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

24

43

)w

ork

pla

ced

rug

test

ing

HE

IAp

erfo

rman

ceev

al-

uat

edby

LC

-MS

/MS

dat

aan

dac

hie

ved

75

.6%

sen

siti

vit

y,9

9.6

%sp

ecif

icit

y,an

d9

6.8

%ef

fici

ency

at1

0mg

/Lcu

toff

.C

ross

-re

acti

vit

y(4

10

%)

for

AM

12

20

,A

M2

20

1,

AM

22

01

-N-4

-OH

-p

enty

l,JW

H-0

18

-N

-OH

-pen

tyl,

JWH

-0

73

-N-4

-OH

-bu

tyl,

Bar

nes

etal

.(2

01

4)

(co

nti

nu

ed)

32 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

JWH

-07

3-N

-CO

OH

,JW

H-2

00

,JW

H-2

00

-6

-OH

-in

do

le,

JWH

-3

98

-N-C

OO

H,

MA

M2

20

1-N

-CO

OH

5F

-PB

-22

,A

B-P

INA

CA

,A

DB

-PIN

AC

A,

AM

22

01

,B

B-2

2,

Cl-

22

01

,JW

H-0

15

,JW

H-

01

8,

JWH

-01

9,

JWH

-0

73

,JW

H-1

22

,JW

H-

21

0,

JWH

-25

0,

MA

M2

20

1,

PB

-22

,U

R-1

44

,X

LR

11

QU

AN

TW

BL

LE

LC

-MS

/MS

LO

D,

LO

Q,

Bia

s,Im

pre

cisi

on

,M

atri

xse

lect

ivit

y,In

terf

eren

ce,

Car

ryover

,Io

nen

han

cem

ent

or

sup

pre

ssio

n

Au

then

tic

ante

-an

dp

ost

-m

ort

emW

Bsp

eci-

men

s(n¼

4)

Sta

bil

ity

no

tev

alu

ated

.5

F-P

B-2

2w

asqu

anti

-fi

edin

2an

te-

and

2p

ost

-mo

rtem

blo

od

spec

imen

sw

ith

this

met

ho

d.

Hig

hes

tco

n-

cen

trat

ion

was

1.5mg

/L.

Beh

on

ick

etal

.(2

01

4)

AM

69

4,

AM

22

01

,C

P4

7,4

97

,H

U-2

10

,JW

H-0

07

,JW

H-0

15

,JW

H-0

18

,JW

H-0

18

-N

-CO

OH

,JW

H-0

19

,JW

H-0

20

,JW

H-0

73

,JW

H-0

81

,JW

H-

12

2,

JWH

-20

0,

JWH

-20

3,

JWH

-21

0,

JWH

-25

0,

WIN

55

,21

2-2

QU

AN

TH

air

Was

hin

g,

Dig

esti

on

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Car

ryover

,Io

nsu

pp

ress

ion

or

enh

ance

men

t

Au

then

tic

hai

rsp

ecim

ens

(n¼

65

)fr

om

rou

tin

efo

ren

sic

case

s

Sta

bil

ity

no

tev

alu

ated

.L

OQ¼

50

0p

g/m

g.

All

auth

enti

ch

air

spec

i-m

ens

neg

ativ

efo

rS

C.

Cri

mel

eet

al.

(20

14

)

AB

-FU

BIN

AC

A,

AM

69

4-N

-CO

OH

,A

M1

24

8,

AM

22

01

,A

M2

20

1-N

-4-O

H-

pen

tyl,

HU

-21

0,

JWH

-0

18

-N-4

-OH

-pen

tyl,

JWH

-01

8-N

-CO

OH

,JW

H-0

19

,JW

H-0

19

-N

-5-O

H-h

exyl,

JWH

-0

73

-N-3

-OH

-bu

tyl,

JWH

-07

3-N

-CO

OH

,JW

H-0

81

,JW

H-0

81

-N

-5-O

H-p

enty

l,JW

H-

12

2,

JWH

-12

2-N

-5-

OH

-pen

tyl,

JWH

-20

0-

6-O

H-i

nd

ole

,JW

H-

12

0,

JWH

-21

0-N

-4-

OH

-pen

tyl,

JWH

-25

0-

N-4

-OH

-pen

tyl,

PB

-2

2,

RC

S-4

,R

CS

-4-

N-5

-OH

-pen

tyl,

RC

S-

8,

UR

-14

4,

UR

-14

4-

N-C

OO

H,

XL

R1

1,

XL

R1

1-6

-OH

-in

do

le,

XL

R1

2

QU

AN

TU

rin

eH

yd

roly

sis,

Dil

uti

on

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Car

ryover

,D

ilu

tio

nin

teg

rity

,M

E,

Inte

rfer

ence

,S

tab

ilit

y

Au

then

tic

uri

ne

spec

imen

(n¼

1)

coll

ecte

dfr

om

pat

ien

tad

mit

ted

for

SC

into

xic

atio

n

An

aly

tes

stab

leat

RT

and

4–

6� C

for

24

h.

Lo

ng

term

sto

rage

stab

ilit

yn

ot

eval

uat

ed.

LO

D/

LO

Q¼

1–

5mg

/L.

Inau

then

tic

uri

ne

spec

i-m

ens

UR

-14

4-

N-C

OO

Hqu

anti

fied

at1

37

0mg

/Lan

dX

LR

11

6-O

H-i

nd

ole

at3

11mg

/L.

Fre

ijo

.et

al.

(20

14

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 33

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

57

SC

(in

clu

din

gP

B-2

2an

dU

R-1

44

-N

-CO

OH

),n

ot

enu

mer

ated

QU

AN

TS

eru

mN

ot

spec

ifie

dL

C-T

OF

MS

Met

ho

dval

idat

ion

dat

an

ot

avai

lab

le.

Au

then

tic

seru

msp

eci-

men

sfr

om

ap

atie

nt

and

his

do

gw

ith

his

-to

ryo

fsy

nth

etic

can

-n

abin

oid

(‘‘C

razy

Mo

nkey

’’)

Qu

anti

fied

PB

-22

at5

5mg

/L,

UR

-14

4-

N-C

OO

H5

8mg

/Lan

dU

R-1

44

N-4

-OH

-p

enty

l2

2mg

/Lin

seru

mu

po

nE

Rar

ri-

val

,th

ou

gh

(-)

SC

inh

um

anu

rin

eu

po

nar

riv

al.

Gu

gel

man

net

al.

(20

14

)

5F

-AK

B-4

8,

AB

-00

1,

AK

B-4

8,

AM

69

4,

AM

12

20

,A

M1

22

0az

epan

eis

om

er,

AM

22

01

,A

M2

23

2,

AM

22

33

,A

PIC

A,

Can

nab

ipip

erid

ieth

an-

on

e,C

RA

-13

,JW

H-

00

7,

JWH

-01

5,

JWH

-0

18

,JW

H-0

19

,JW

H-

02

0,

JWH

-02

2,

JWH

-0

73

,JW

H-0

81

,JW

H-

12

2,

JWH

-18

2,

JWH

-2

00

,JW

H-2

03

,JW

H-

21

0,

JWH

-25

0,

JWH

-2

51

,JW

H-3

07

,JW

H-

37

0,

JWH

-38

7,

JWH

-3

98

,JW

H-4

12

,M

AM

22

01

,M

eth

anan

dam

ide,

RC

S-4

,R

CS

-4-C

4,

RC

S-4

ort

ho

iso

mer

,R

CS

-8,

ST

S-1

35

,U

R-

14

4,

UR

-14

4is

om

er,

WIN

48

09

8,

WIN

55

,21

2-2

,X

LR

11

,X

LR

11

iso

mer

QU

AL

Ser

um

LL

EL

C-T

OF

MS

-ES

I+L

OD

,M

EA

uth

enti

cse

rum

spec

i-m

ens

(n¼

30

)fr

om

fore

nsi

cca

ses

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.1

–0

.25mg

/L.

On

lyo

ne

LC

-MS

/MS

con

firm

edsa

mp

leco

nfi

rmed

neg

ativ

ew

ith

LC

-QT

OF

-MS

.

Hu

pp

ertz

etal

.(2

01

4)

AM

22

01

-N-4

-OH

-pen

tyl,

AM

22

01

-6-O

H-

ind

ole

,JW

H-0

18

-N-4

and

5-O

H-p

enty

l,JW

H-0

18

-N-C

OO

H,

JWH

-01

8-6

-OH

-in

do

le,

JWH

-01

8-N

-5-

OH

-pen

tylb-

Glu

c,JW

H-0

73

-N-C

OO

H

QU

AN

TU

rin

eH

yd

roly

sis,

SP

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Car

ryover

,M

E,

PE

,R

ecover

y,S

elec

tiv

ity,

Sta

bil

ity

Au

then

tic

uri

ne

spec

i-m

ens

fro

msu

spec

ted

JWH

-01

8(n¼

11

)an

dA

M2

20

1(n¼

9)

use

rs

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.1mg

/L;

LO

Q¼

2.5mg

/L.

InJW

H-0

18

spec

imen

s,JW

H-0

18

-N-4

-OH

-p

enty

lco

nfi

rmed

inal

lsp

ecim

ens

wh

ile

JWH

-0

18

-N-C

OO

Hp

rese

nt

inh

igh

con

cen

trat

ion

inA

M2

20

1sp

eci-

men

s.JW

H-0

73

-N

-CO

OH

con

firm

ed

Jan

get

al.

(20

14

b)

(co

nti

nu

ed)

34 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

hu

man

sex

po

sed

toA

M2

20

1o

rJW

H-0

18

.A

M2

20

1,

AM

22

01

-N-4

-O

H-p

enty

l,A

M2

20

1-

6-O

H-i

nd

ole

,JW

H-

01

8,

JWH

-01

8-N

-4an

d-5

-OH

-pen

tyl,

JWH

-01

8-N

-CO

OH

,JW

H-0

73

,JW

H-0

73

-N

-3-

and

4-O

H-b

uty

l,JW

H-0

73

-N-C

OO

H,

JWH

-12

2,

JWH

-12

2-

N-5

-OH

-pen

tyl,

JWH

-1

22

-N-C

OO

H,

MA

M2

20

1,

MA

M2

20

1-N

-4-O

H-

pen

tyl

QU

AN

TH

air

Was

hin

g,

LL

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Car

ryover

,M

E,

PE

,R

ecover

y,S

elec

tiv

ity,

Sta

bil

ity

Au

then

tic

hu

man

hai

r(n¼

9)

spec

imen

sfr

om

susp

ecte

dS

Cu

sers

In-p

roce

ssst

abil

ity

for

all

anal

yte

sac

cep

tab

leaf

ter

24

h.

LO

D¼

0.0

5p

g/m

g;

LO

Q¼

0.1

pg

/mg

.In

hai

r,7

/9su

spec

ts(+

)A

M2

20

1(3

.7–

91

8p

g/m

g)

wit

hco

n-

cen

trat

ion44

JWH

-0

18

-N-C

OO

H(0

.2–

1.1

pg

/mg

).M

AM

22

01

(0.9

–2

58

.2p

g/m

g)

and

JWH

-12

2(0

.3–

88

8p

g/m

L)

con

cur-

ren

tly

qu

anti

fied

in4

AM

22

01

(+)

sam

ple

s.

Kim

etal

.(2

01

4)

AM

-22

01-N

-4-O

H-

pen

tyl,

AM

-22

01

-6-

and

7-O

H-i

nd

ole

,JW

H-0

18

-N-4

-an

d5

-O

H-p

enty

l,JW

H-0

18

-5

-,6

-an

d7

-OH

-in

do

le,

JWH

-01

8-

N-C

OO

H,

JWH

-01

9-

N-5

-an

d6

-OH

-hex

yl,

JWH

-07

3-N

-3-

and

4-

OH

-bu

tyl,

JWH

-07

3-

5-,

6-

and

7-O

H-

ind

ole

,JW

H-0

73

-N

-CO

OH

,JW

H-0

81

-N

-5-O

H-p

enty

l,JW

H-

08

1-N

-CO

OH

,JW

H-

12

2-N

-4-O

H-p

enty

l,JW

H-1

22

-N-5

-OH

-p

enty

l,JW

H-2

10

-N-4

-an

d5

-OH

-pen

tyl,

JWH

-21

0-N

-CO

OH

,JW

H-2

50

-N-4

-an

d5

-O

H-p

enty

l,JW

H-2

50

-N

-CO

OH

,JW

H-3

98

-N

-4-

and

5-O

H-p

enty

l,JW

H-3

98

-N-C

OO

H,

MA

M-2

20

1-N

-4-O

H-

pen

tyl,

MA

M-2

20

1-

N-C

OO

H,

RC

S-4

4-

and

5-O

H-p

enty

l,R

CS

-4-N

-CO

OH

,U

R-1

44

4-

and

5-O

H-

QU

AL

Uri

ne

SA

LL

EH

EIA

LC

-TO

F-M

S-E

SI+

Sel

ecti

vit

y,M

E,

Cu

toff

sele

ctio

n,

Sta

bil

ity

Fo

rH

EIA

:L

inea

rity

,C

ross

-rea

ctiv

ity

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

87

)fr

om

fore

nsi

cca

ses

HE

IAp

erfo

rman

ceag

ain

stL

C-T

OF

-MS

was

86

.8%

sen

siti

vit

y,8

1.6

%sp

ecif

icit

y,ef

fici

ency

of

83

.9%

at5mg

/L.

Su

bse

qu

entl

y,2

04

auth

enti

csp

eci-

men

sw

ere

anal

yze

dby

LC

-QT

OF

-MS

of

wh

ich

22

wer

ep

osi

tive

for�

1S

Cm

etab

oli

tes.

Met

abo

lite

sw

ere

stab

le5

15

%lo

ssat

RT

for

up

to2

day

s(e

xce

pt

for

JWH

-07

3-

7-O

H-i

nd

ole

,�

25

%).

At

4� C

,JW

H-0

19

-6-

OH

ind

ole

and

RC

S-4

-N

-5-O

H-p

enty

lin

crea

sed

by

46

and

21

%,

resp

ecti

vel

y.A

llan

aly

tes

stab

leat

�2

0� C

up

to1

5w

eek

s.In

-ho

use

,sp

ectr

alli

bra

ryw

asu

tili

zed

toid

enti

fy3

8S

Cm

etab

oli

tes.

Cu

toff¼

2mg

/L

Kro

nst

ran

det

al.

(20

14

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 35

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

pen

tyl,

UR

-14

4-

N-C

OO

HO

F,

Pla

sma/

Ser

um

:JW

H-

00

7,

JWH

-01

5,

JWH

-0

18

,JW

H-0

73

,JW

H-

09

8,

JWH

-12

2,

JWH

-1

82

,JW

H-2

00

,JW

H-

21

0-

JWH

-24

9,

JWH

-2

50

,JW

H-2

51

,JW

H-

30

2,

JWH

-4

24

,WIN

55

,21

2-2

;U

rin

e:JW

H-0

18

-4-

OH

-in

do

le,

JWH

-01

8-

N-5

-OH

-pen

tyl,

JWH

-0

18

-N-C

OO

H,

JWH

-0

73

-4-O

H-i

nd

ole

,JW

H-0

73

-N-C

OO

H

QU

AL

OF

,U

rin

e,P

lasm

a,S

eru

m

LL

E(O

F);

hy

dro

lysi

s(u

rin

e),

pro

tein

pre

cip

itat

ion

(WB

)

LC

-MS

/MS

-ES

I+L

OD

,S

pec

ific

ity,

Ion

sup

pre

ssio

no

ren

han

cem

ent,

Ret

enti

on

tim

ere

pea

tab

ilit

y,R

ecover

y,R

ob

ust

nes

s,C

arry

over

No

ne

Sta

bil

ity

no

tev

alu

ated

.M

eth

od

val

idat

edfo

rS

Cqu

anti

fica

tio

nin

pla

sma/

seru

man

dO

Fw

hil

em

etab

oli

tes

of

JWH

-01

8an

dJW

H-

07

3fo

ru

rin

e.L

OD¼

0.2

–0

.5mg

/LO

F;

0.1

–0

.5mg

/Lp

lasm

a/se

rum

;0

.3–

0.4mg

/Lu

rin

e.

Maz

zari

no

etal

.(2

01

4)

UR

-14

4-N

-CO

OH

(cal

ibra

tor)

QU

AL

Uri

ne

No

ne

EL

ISA

Imp

ress

ion

,P

late

dri

ft,

Car

ryover

,S

tab

ilit

y

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

59

89

)fr

om

fore

nsi

cca

ses

EL

ISA

cuto

ffes

tab

lish

edat

5mg

/L.P

erfo

rman

ceev

alu

ated

by

con

firm

-in

gal

lp

resu

mp

tive

po

siti

ve

by

LC

-MS

/M

S(A

rnst

on

etal

.,2

01

3).

Bas

edo

n3

.6%

po

siti

vit

yra

te,

EL

ISA

’sover

all

sen

si-

tiv

ity

was

86

.6%

.C

ross

-rea

ctiv

ity

50

%fo

rX

LR

-11

-N-4

-OH

-fl

uro

pen

tyl

and

UR

-1

44

-N-4

-OH

-pen

tyl;

10

0%

for

UR

-14

4-

N-C

OO

Han

dU

R-1

44

-N

-5-O

H-p

enty

l.

Mo

hr

etal

.(2

01

4)

AM

69

4,

AM

12

20

,A

M2

20

1,

HU

-21

0,

JWH

-00

7,

JWH

-01

5,

JWH

-01

8,

JWH

-01

9,

JWH

-02

0,

JWH

-07

3,

JWH

-08

1,

JWH

-12

2,

JWH

-20

0,

JWH

-20

3,

JWH

-21

0,

JWH

-25

0,

JWH

-25

1,

JWH

-30

7,

JWH

-39

8,

RC

S-4

,R

CS

-8,

WIN

48

09

8,

WIN

55

,21

2-2

QU

AN

TH

air

Was

hin

g,

Dig

esti

on

,L

LE

LC

-MS

/MS

-ES

I+L

OD

,L

OQ

,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Car

ryover

,M

E,

PE

,R

ecover

y,S

elec

tiv

ity,

Sp

ecif

icit

y

Au

then

tic

hai

rsp

ecim

ens

(n¼

34

4,

pre

vio

usl

yan

aly

zed

for

dru

gs

of

abu

se)

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.2

–1

.3p

g/m

g;

LO

Q¼

0.7

–4

.3p

g/m

g(H

U-2

10

80

pg

/mg

LO

Q).

Au

then

tic

sam

-p

les:

15

con

firm

edfo

rS

C,

maj

ori

tyw

asfo

rJW

H-0

73

(1.6

–5

0.5

pg

/mg

)fo

llow

edby

JWH

-12

2(n¼

8,

7.4

–2

80

0p

g/m

g).

Oth

erS

Cid

enti

fied

:JW

H-2

50

,JW

H-0

81

,JW

H-0

18

,JW

H-2

10

,JW

H-0

19

,an

d

Sal

om

on

eet

al.

(20

14

a)

(co

nti

nu

ed)

36 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

AM

22

01

.M

eth

od

was

up

dat

edto

9S

Cm

etab

oli

tes

(Sal

om

on

eet

al.,

20

14

b).

JWH

-1

22

-N-5

-OH

-pen

tyl

was

iden

tifi

edin

2/1

5sa

mp

les

at0

.72

and

2.5

pg

/mg

wit

hco

rres

-p

on

din

gJW

H-1

22

con

cen

trat

ion

so

f7

60

and

28

00

pg

/mg

,re

spec

tivel

y.A

M6

94

,A

M2

20

1,

AM

22

01

-6-O

H-

ind

ole

,A

M2

20

1-

N-O

H-p

enty

l,C

P4

7,4

97

-C7

,C

P4

7,4

97

-C7

-OH

,C

P4

7,4

97

-C8

,C

P4

7,4

97

-C8

-OH

dim

ethylo

cty

l,H

U-

21

0,

JWH

-01

8,

JWH

-0

18

5-

and

6-O

H-

ind

ole

,JW

H-0

18

-N

-OH

-pen

tyl,

JWH

-0

18

-N-C

OO

H,

JWH

-0

19

,JW

H-0

19

-5-O

H-

ind

ole

,JW

H-0

19

-N

-OH

-hex

yl,

JWH

-0

73

,JW

H-0

73

-5-

and

6-O

H-i

nd

ole

,JW

H-

07

3-N

-O

H-b

uty

l,JW

H-0

73

-N-C

OO

H,

JWH

-08

1,

JWH

-08

1-

N-O

H-p

enty

l,JW

H-

12

2,

JWH

-12

2-N

-OH

-p

enty

l,JW

H-2

00

-5-

and

6-O

H-i

nd

ole

,JW

H-2

03

,JW

H-2

10

,JW

H-2

10

-5-O

H-

ind

ole

,JW

H-2

10

-N

-OH

-pen

tyl,

JWH

-2

10

-N-C

OO

H,

JWH

-2

50

-5-O

H-i

nd

ole

,JW

H-2

50

-N-O

H-

pen

tyl,

JWH

-25

0-

N-C

OO

H,

JWH

-39

8,

JWH

-39

8-N

-OH

-p

enty

l,JW

H-3

98

-

QU

AN

TU

rin

eD

ilu

tio

n,

Hy

dro

lysi

s,S

LE

LC

-MS

/MS

-ES

I+/�

LO

D,

LO

Q,L

inea

rity

,B

ias,

Imp

reci

sio

n,

Car

ryover

,M

E,

Ex

trac

tio

nef

fi-

cien

cy,

Sp

ecif

icit

y,D

ilu

tio

nin

teg

rity

,S

tab

ilit

y,C

arry

over

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

77

7)

wo

rk-

pla

ced

rug

test

ing

An

aly

tes

wer

est

able

atR

Tu

pto

16

hex

cep

tfo

rC

P4

7,4

97

-C7

,C

P4

7,4

97

-C8

,H

U-

21

0an

dJW

H-2

00

,at

4� C

for

72

h.

All

met

abo

lite

sst

able

afte

r3

free

ze/t

haw

cycl

esw

hil

ep

aren

tsw

ere

un

stab

leex

cep

tfo

rA

M6

94

,C

P4

7,4

97

-C7

,C

P4

7,4

97

-C8

,H

U-

21

0,

JWH

-20

0an

dJW

H-3

98

.B

asel

ine

sep

arat

ion

sfo

r1

2al

kyl

OH

iso

mer

sw

ere

no

to

bta

ined

,th

eref

ore

wer

ed

eter

min

edse

mi-

qu

anti

tati

vel

y.L

OD¼

0.0

5–

1mg

/L;

LO

Q¼

0.1

–1mg

/L.

Inau

then

tic

spec

imen

s,2

90

con

firm

edfo

r2

2S

Cm

etab

oli

tes

atco

nce

ntr

atio

nra

nges

bet

wee

n0

.1–

24

34mg

/L

.M

ajo

rity

con

firm

edfo

ral

kyl-

OH

and

CO

OH

met

abo

lite

s.

Sch

eid

wei

ler

&H

ues

tis

(20

14

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 37

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

N-C

OO

H,

MA

M2

20

1,

MA

M2

20

1-N

-OH

-p

enty

l,M

AM

22

01

-N

-CO

OH

,R

CS

-4,

RC

S-4

-N-O

H-p

enty

l,R

CS

-4-M

9,

RC

S-4

-M

10

,R

CS

-4-

N-C

OO

H,

UR

-14

4-

N-O

H-p

enty

l,U

R-

14

4-N

-CO

OH

Met

abo

lite

so

nly

:5

F-A

B-

PIN

AC

A-N

-OH

-p

enty

l,5

F-A

KB

48

-N

-OH

-pen

tyl,

5F

-PB

-2

2-3

-CO

OH

-in

do

le,

AB

-PIN

AC

A-N

-OH

-p

enty

l,A

B-P

INA

CA

-N

-CO

OH

,A

DB

-P

INA

CA

-N-O

H-

pen

tyl,

AK

B4

8-N

-OH

-p

enty

l,A

KB

48

-N

-CO

OH

,A

M2

20

1-6

-O

H-i

nd

ole

,A

M2

20

1-

N-O

H-p

enty

l,JW

H-

01

8-5

-an

d6

-OH

-in

do

le,

JWH

-01

8-N

-O

H-p

enty

l,JW

H-0

18

-N

-CO

OH

,JW

H-0

19

-5

-OH

-in

do

le,

JWH

-0

19

-OH

-hex

yl,

JWH

-0

73

-5-

and

6-O

H-

ind

ole

,JW

H-0

73

-N-

CO

OH

,JW

H-0

73

-N

-OH

-bu

tyl,

JWH

-0

81

-N-O

H-p

enty

l,JW

H-1

22

-N-O

H-

pen

tyl,

JWH

-12

2-

N-C

OO

H,

JWH

-20

0-

5-

and

6-O

H-i

nd

ole

,JW

H-2

10

-N-C

OO

H,

JWH

-21

0-N

-OH

-p

enty

l,JW

H-2

50

-5-

OH

-in

do

le,

JWH

-25

0-

N-O

H-p

enty

l,JW

H-

25

0-N

-CO

OH

,JW

H-

39

8-N

-OH

-pen

tyl,

JWH

-39

8-N

-CO

OH

,M

AM

22

01

-N-O

H-

pen

tyl,

PB

-22

-3-

QU

AL

Uri

ne

Dil

uti

on

,H

yd

roly

sis,

SL

E+

LC

-TO

F-M

S-E

SI+

LO

D,

Sp

ecif

icit

y,E

xtr

acti

on

effi

-ci

ency

,M

E,

Sta

bil

ity,

Car

ryover

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

8)

Em

plo

yed

SW

AT

HT

M

(Seq

uen

tial

Win

dow

edA

cqu

isit

ion

of

all

Th

eore

tica

lm

ass

spec

tra)

for

no

n-t

ar-

get

edid

enti

fica

tio

no

fS

Cm

etab

oli

tes

inu

rin

e.A

nal

yte

sfo

rti-

fied

inb

lan

ku

rin

ew

ith

1–

20�

LO

Dco

nce

ntr

atio

ns

wer

est

able

atR

Taf

ter

24

h,

at4� C

for

72

h(i

ncl

ud

ing

on

auto

-sa

mp

ler)

,af

ter

3fr

eeze

/th

awcy

cles

.B

asel

ine

chro

mat

o-

gra

ph

icre

solu

tio

nw

asn

ot

ob

tain

edfo

ral

kyl

OH

iso

mer

s.L

OD¼

0.2

5–

20mg

/L.

Inau

then

tic

uri

ne

spe-

cim

ens,

iden

tifi

edu

pto

12

met

abo

lite

sin

clu

din

gU

R-1

44

deg

rad

ant-

CO

OH

.

Sch

eid

wei

ler

etal

.(2

01

4)

(co

nti

nu

ed)

38 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

CO

OH

-in

do

le,

PB

-22

-N

-OH

-pen

tyl-

3-

CO

OH

-in

do

le,

PB

-22

-N

-CO

OH

-3-C

OO

H-

ind

ole

,P

B-2

2-N

-OH

-p

enty

l,P

B-2

2-

N-C

OO

H,

RC

S-4

-M9

,R

CS

-4-N

-OH

-pen

tyl,

RC

S-4

-N-C

OO

H,

UR

-1

44

deg

rad

ant-

CO

OH

,U

R-1

44

-N-O

H-p

enty

l,U

R-1

44

-N-C

OO

H,

XL

R1

1-6

-OH

-in

do

le,

XL

R1

1-N

-OH

-pen

tyl

5F

-PB

-22

,A

M2

23

3,

BB

-2

2,

JWH

-12

2,

PB

-22

QU

AN

TP

lasm

aL

LE

LC

-HR

-MS

Met

ho

dval

idat

ion

par

amet

ers

no

td

etai

led

Au

then

tic

pla

sma

spec

i-m

ens

(n¼

2)

fro

mp

atie

nt

exp

erie

nci

ng

seiz

ure

saf

ter

smo

kin

gS

C-l

aced

cigar

ette

.

Sta

bil

ity

no

tev

alu

ated

.B

loo

dsa

mp

les

wer

ed

raw

nat

5.5

and

8.3

haf

ter

ER

adm

issi

on

.A

ll5

SC

con

firm

edat

9–

14

8n

g/L

(5.5

h)

and

13

–1

25

ng

/L(8

.3h

).

Sch

epet

al.

(20

14

)

11

-OH

-TH

C,

AM

22

01

,H

U-2

10

,JW

H-0

18

,JW

H-0

18

-4-

and

5-

OH

-in

do

le,

JWH

-01

8-

N-4

-an

d-5

-OH

-p

enty

l,JW

H-0

73

,JW

H-0

73

-N-3

-an

d4

-O

H-b

uty

l,JW

H-2

50

,T

HC

,T

HC

CO

OH

,

QU

AN

TU

rin

eH

yd

roly

sis,

SP

EL

C-M

S/M

S-E

SI+

LO

D,

LO

Q,L

inea

rity

,Im

pre

cisi

on

,B

ias,

Sel

ecti

vit

y,R

ecover

y,M

E

Au

then

tic

sam

ple

s(n¼

80

)fr

om

ind

ivid

-u

als

adm

itte

dto

the

ER

or

invo

lved

info

ren

sic

case

s

Sta

bil

ity

no

tev

alu

ated

.L

OD¼

0.0

1–

0.5mg

/L;

LO

Q¼

0.0

5–

5mg

/L.

Inau

then

tic

sam

ple

s,5

con

firm

edfo

rJW

H-

01

8an

d/o

rJW

H-1

22

met

abo

lite

s,T

HC

and

/or

TH

CC

OO

H.

Sim

oes

etal

.(2

01

4)

JWH

-01

8-N

-CO

OH

(cal

ibra

tor)

QU

AL

Uri

ne

No

ne

EL

ISA

LO

D,

Lin

eari

ty,

Cu

toff

sele

ctio

n,

Pla

ted

rift

,In

terf

eren

ce,

Car

ryover

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

24

69

)ra

nd

om

wo

rkp

lace

dru

gte

stin

g

EL

ISA

per

form

ance

by

LC

-MS

/MS

dat

ain

(Wo

hlf

arth

etal

.,2

01

4c)

.P

erfo

rman

cew

as7

9.9

%se

nsi

tiv

ity,

99

.7%

spec

ific

ity

and

97

.4%

effi

cien

cyat

5mg

/Lcu

toff

.E

LIS

Acr

oss

-rea

cted

(41

0%

)w

ith

18

of

73

SC

anal

yte

sev

alu

ated

.

Sp

inel

liet

al.

(20

14

)

AM

69

4,

AM

22

01

,C

P4

7,4

97

,H

U-2

10

,JW

H-0

15

,JW

H-0

18

,JW

H-0

19

,JW

H-0

20

,JW

H-0

73

,JW

H-0

81

,JW

H-1

22

,JW

H-2

00

,JW

H-2

10

,JW

H-2

50

,JW

H-2

51

,R

CS

-4,

QU

AN

TW

BL

LE

LC

-MS

/MS

-ES

I+M

eth

od

val

idat

ion

par

amet

ern

ot

spec

ifie

d

Au

then

tic

WB

spec

imen

s(n¼

72

6)

fro

mD

UID

susp

ects

Sta

bil

ity

no

tev

alu

ated

.M

eth

od

val

idat

edin

(Pre

sley

etal

.,2

01

3).

InW

Bsp

ecim

ens,

16

(2.2

%)

con

firm

edfo

rS

C:

AM

22

01

,JW

H-

01

8,

JWH

-08

1,

JWH

-

Tu

vet

al.

(20

14

)

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 39

Dru

g M

etab

olis

m R

evie

ws

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

NIH

Pat

holo

gy L

ab o

n 04

/09/

15Fo

r pe

rson

al u

se o

nly.

Tab

le2

.C

on

tin

ued

An

aly

te(s

)

QU

AL

or

QU

AN

TM

atri

xS

amp

lep

rep

arat

ion

Met

ho

dV

alid

atio

nA

pp

lica

tio

nS

tud

yfi

nd

ing

sR

efer

ence

s

RC

S-4

-C4

,R

CS

-8,

WIN

55

,21

2-2

12

2,

JWH

-25

0,

RC

S-4

.JW

H-0

18

,JW

H-2

50

(cal

ibra

tors

)Q

UA

LU

rin

eN

on

eB

ioch

ipar

ray

tech

-n

olo

gy

imm

un

oas

say

LO

D,

Lin

eari

ty,

Imp

reci

sio

n,

Bia

s,In

terf

eren

ce,

Cu

toff

Op

tim

izat

ion

,C

arry

over

Au

then

tic

uri

ne

spec

i-m

ens

(n¼

20

01

7)

fro

mra

nd

om

wo

rk-

pla

ced

rug

test

ing

Bio

chip

was

lab

eled

wit

h4

anti

bo

die

s(3

for

JWH

-01

8S

CI,

II,

and

III,

1fo

rJW

H-2

50

,S

CIV

).P

erfo

rman

ceev

alu

ated

wit

hL

C-

MS

/MS

resu

lts

yie

lded

98

.3%

sen

siti

vit

y,4

8.1

%sp

ecif

icit

y,5

3.9

%ef

fici

ency

at1

0mg

/LS

CI,

20mg

/LS

CII

,5mg

/LS

CII

I/S

CIV

cuto

ffs.

Aft

ercu

toff

op

tim

izat

ion

,p

erfo

rman

ceim

pro

ved

to8

7.6

%se

nsi

tiv

ity,

85

.2%

spec

ific

ity,

85

.4%

effi

cien

cyat

15mg

/LS

CI,

10mg

/LS

CII

I(S

CII

and

IVn

och

ange)

.C

ross

-re

acti

vit

y(4

1%

)3

7S

Cm

etab

oli

tes

and

var

ied

amo

ng

anti

bo

die

s.

Cas

tan

eto

etal

.(2

01

4a)

4-F

MA

:4

-flu

oro

met

ham

ph

etam

ine;

4-M

EC

:4

-met

hyle

thca

thin

on

e;A

MP

:am

ph

etam

ine;

BZ

P:

ben

zylp

iper

azin

e;C

BD

:ca

nn

abid

iol;

CB

N:

can

nab

ino

l;C

OO

H:

carb

ox

y;

DM

A:

dim

eth

ox

yam

ph

etam

ine;

DU

ID:

dri

vin

gu

nd

erth

ein

flu

ence

;E

I:E

lect

ron

Imp

act;

EL

ISA

:en

zym

e-li

nked

imm

un

oso

rben

tas

say;

ER

:em

ergen

cyro

om

;E

SI+

/�:

Po

siti

ve/

Neg

ativ

eE

lect

rosp

ray

ion

izat

ion

;G

C:

Gas

Ch

rom

ato

gra

ph

y;

Glu

c:g

lucu

ron

ide;

HE

IA:

ho

mo

gen

ou

sen

zym

eim

mu

no

assa

y;

HL

M:

hu

man

liver

mic

roso

mes

;H

RM

S:

hig

h-r

eso

luti

on

mas

ssp

ectr

om

etry

;L

LE

:L

iqu

id–

Liq

uid

Ex

trac

tio

n;

LC

:L

iqu

idC

hro

mat

og

rap

hy;

LO

D:

Lim

ito

fD

etec

tio

n;

LO

Q:

Lim

ito

fQ

uan

tifi

cati

on

;L

R:

Lin

ear

Ran

ge;

MB

DB

:m

ethylb

enzo

dio

xyo

lyb

uta

nam

ine;

MD

AI:

5,6

-met

hyle

ned

iox

y-2

-am

ino

ind

ane;

MB

ZP

:m

ethylb

enzy

lpip

eraz

ines

;M

DP

V:

met

hyle

ned

iox

yp

yro

val

ero

ne;

ME

:m

atri

xef

fect

s;M

S:

Mas

sS

pec

tro

met

ry;

MS

/MS

:T

and

emM

ass

Sp

ectr

om

etry

;N

aOH

:so

diu

mhy

dro

xid

e;N

PS

:n

ewp

sych

oac

tive

sub

stan

ces;

OH

:hy

dro

xy;

OF

:O

ral

flu

id;

pF

PP

:p

ara-

flu

oro

ph

enylp

iper

azin

e;P

E:

pro

cess

effi

cien

cy;

PN

EG

:p

resu

mp

tive

neg

ativ

eb

yim

mu

no

assa

y;

PP

OS

:p

resu

mp

tive

po

siti

ve

by

imm

un

oas

say;

PP

P:

py

rro

lid

ino

pro

pri

op

hen

on

e;Q

UA

L:

qu

alit

ativ

e;Q

UA

NT

:q

uan

tita

tive;

RT

:R

oo

mT

emp

erat

ure

;S

AL

LE

:sa

ltin

g-o

ut

assi

sted

liqu

id–

liqu

idex

trac

tio

n;

SC

:sy

nth

etic

can

nab

ino

ids;

SL

E+

:so

lid

–li

qu

idsu

pp

ort

extr

acti

on

;S

PE

:S

oli

d-P

has

eE

xtr

acti

on

;T

FM

PP

:3

-tri

flu

oro

met

hy

ph

enylp

iper

azo

ne;

TH

C:

del

ta-9

-tet

rahy

dro

can

nab

ino

l;T

HC

CO

OH

:T

HC

-car

box

y;

TM

S:

trim

eth

yls

ilat

ion

;T

MC

P:

tetr

amet

hylc

ycl

op

rop

yl;

TO

F:

tim

e-o

f-fl

igh

t;W

B:

Wh

ole

blo

od

40 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

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Tab

le3

.B

iotr

ansf

orm

atio

no

fsy

nth

etic

can

nab

ino

ids

invi

voan

din

vitr

o.

Ph

ase

I:si

teo

fre

acti

on

(mat

rix

)P

has

eII

An

aly

teH

yd

rox

yla

tio

nC

arb

ox

yla

tio

nD

ehy

dro

gen

atio

nN

-dea

lkyla

tio

nD

ihyd

rod

iol

form

atio

nK

eto

ne

form

atio

nO

-dem

ethyla

tio

nO

xid

ativ

ed

eflu

ori

nat

ion

Rin

go

pen

ing

Co

nju

gat

es

AB

-00

1A

dam

anta

ne

(uri

ne)

b

N-p

enty

l(u

rin

e)b

N-p

enty

l(u

rin

e)b

AB

-FU

BIN

AC

AA

min

ooxo

bu

tan

e(H

LM

)c

AD

B-F

UB

INA

CA

Am

ino

oxo

bu

tan

e(H

LM

)c

AB

-PIN

AC

AN

-Pen

tyl

and

ami-

no

oxo

bu

tan

e(H

LM

)c

AK

B-4

8A

dam

anta

ne

(HE

P)c

N-p

enty

l(H

EP

)c

N-p

enty

l(H

EP

)cG

LU

C(H

LM

)c

5F

-AK

B-4

8A

dam

anta

ne

(HL

M)c

/(u

rin

e)b

Ind

azo

leal

kyl

(HL

M)c

/(u

rin

e)b

N-p

enty

l(H

LM

)cN

-pen

tyl

(HL

M)c

/(u

rin

e)b

N-p

enty

l(H

LM

)cN

-pen

tyl

(HL

M)c

/(u

rin

e)b

GL

UC

(uri

ne)

b

AM

69

4N

-pen

tyl

(uri

ne)

bN

-pen

tyl

(uri

ne)

bN

-pen

tyl

(uri

ne)

b

AM

22

01

N-f

luo

rop

enty

l(H

LM

)c

Ind

ole

(HL

M)c

/(u

rin

e)b

Nap

hth

yl

(HL

M)c

N-p

enty

l(H

LM

)cN

-pen

tyl

(HL

M)c

Nap

hth

yl

(HL

M)

N-p

enty

l(H

LM

)c/

(uri

ne)

bG

LU

C(u

rin

e)b

CP

55

,94

0H

epty

lsi

de

chai

n(H

LM

)a

JWH

-01

5In

do

leal

kyl

(RL

M)c

Nap

hth

yl

(RL

M)c

N-p

rop

yl

(RL

M)c

N-p

rop

yl

(RL

M)c

Nap

hth

yl

(RL

M)c

JWH

-01

8In

do

leal

kyl

(HL

M)c

/(u

rin

e)a,b

Nap

hth

yl

(HL

M)c

/(u

rin

e)b

N-p

enty

l(H

LM

)c/

(blo

od

)a/

(uri

ne)

a,b

/(H

LM

)a

N-p

enty

l(H

LM

)c/

(blo

od

)a

N-p

enty

l(H

LM

)c/

(blo

od

)a/(

uri

ne)

a,b

Nap

hth

yl

(HL

M)c

GL

UC

(HL

M)c

/(u

rin

e)b

JWH

-07

3N

-bu

tyl

(HL

M)c

/(u

rin

e)b

Ind

ole

(HL

M)c

Nap

hth

yl

(HL

M)c

N-b

uty

l(u

rin

e)b

GL

UC

(uri

ne)

b

JWH

-07

34

-m

ethyln

aph

thoyl

Ind

ole

alk

yl

(HL

M)c

(HL

M)c

Nap

hth

yl

(HL

M)c

(co

nti

nu

ed)

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 41

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Tab

le3

.C

on

tin

ued

Ph

ase

I:si

teo

fre

acti

on

(mat

rix

)P

has

eII

An

aly

teH

yd

rox

yla

tio

nC

arb

ox

yla

tio

nD

ehy

dro

gen

atio

nN

-dea

lkyla

tio

nD

ihyd

rod

iol

form

atio

nK

eto

ne

form

atio

nO

-dem

ethyla

tio

nO

xid

ativ

ed

eflu

ori

nat

ion

Rin

go

pen

ing

Co

nju

gat

es

JWH

-09

8In

do

leal

kyl

(RL

S)a

Nap

hth

yl

(RL

S)a

N-p

enty

l(R

LS

)aN

-pen

tyl

(RL

S)a

Nap

hth

yl

(RL

S)a

GL

UC

(RL

S)a

JWH

-12

2In

do

leal

kyl

(HL

M)c

/(u

rin

e)a

Nap

hth

yl

(HL

M)c

/(u

rin

e)a

N-p

enty

l(H

LM

)cN

-pen

tyl

(HL

M)c

/(u

rin

e)a

N-p

enty

l(H

LM

)c/

(uri

ne)

aN

aph

thyl

(HL

M)c

/(u

rin

e)a

GL

UC

/SU

L(u

rin

e)a

JWH

-20

0N

-eth

yl

(HL

M)c

Mo

rph

oli

ne

(HL

M)c

/(u

rin

e)a

Ind

ole

(HL

M)c

Nap

hth

yl

(HL

M)c

/(u

rin

e)a

N-e

thyl

(HL

M)c

/(u

rin

e)a

Mo

rph

oli

ne

(HL

M)c

/(u

rin

e)a

N-e

thyl

(HL

M)c

Nap

hth

yl

(HL

M)c

/(u

rin

e)a

Mo

rph

oli

ne

(HL

M)c

/(u

rin

e)a

GL

UC

/SU

L(u

rin

e)a

JWH

-25

0N

-pen

tyl

(uri

ne)

a,b

Ph

enyl

(uri

ne)

aN

-pen

tyl

(uri

ne)

bN

-pen

tyl

(uri

ne)

aN

-pen

tyl

(uri

ne)

aU

NS

Pb

PB

-22

Est

erhy

dro

lysi

s(H

LM

)/(H

EP

)c

Ind

ole

alk

yl

(HE

P)c

QU

L(H

EP

)c

N-p

enty

l(H

EP

)cQ

UL

(HE

P)c

N-p

enty

l(H

EP

)cG

LU

C/C

YS

(HE

P)c

5F

-PB

-22

Est

erhy

dro

lysi

s(H

EP

)c

N-p

enty

l(H

EP

)c

QU

L(H

EP

)c

N-p

enty

l(H

EP

)cQ

UL

(HE

P)c

N-p

enty

l(H

EP

)cG

LU

C/C

YS

(HE

P)c

RC

S-4

Ind

ole

alk

yl

(HE

P)c

N-p

enty

l(H

EP

)cP

hen

yl

(HE

P)c

GL

UC

/SU

L(H

EP

)c

RC

S-8

Ph

enyl

(HE

P)c

Cycl

oh

exyl

(HE

P)c

Ph

enyl

(HE

P)c

GL

UC

(HE

P)c

ST

S-1

35

adam

anta

ne

(HE

P)c

N-p

enty

l(H

EP

)cN

-pen

tyl

(HE

P)c

N-p

enty

l(H

EP

)cN

-pen

tyl

(HE

P)c

N-p

enty

l(H

EP

)cG

LU

C(H

EP

)c

UR

-14

4N

-pen

tyl

(HL

M)c

/(u

rin

e)b

ind

ole

(HL

M)c

N-p

enty

l(u

rin

e)b

N-p

enty

l(H

LM

)cN

-pen

tyl

(HL

M)c

Ind

ole

(HL

M)c

TM

CP

(uri

ne)

bG

LU

C(u

rin

e)b

WIN

55

,21

2-2

Ind

ole

(RL

M)c

Nap

hth

yl

(RL

M)c

Mo

rph

oli

ne

(RL

M)c

NA

PH

(RL

M)c

XL

R-1

1N

-pen

tyl

(HE

P)c

Ind

ole

(HE

P)c

TM

CP

(HE

P)c

N-p

enty

l(H

EP

)c

TM

CP

(HE

P)c

TM

CP

(HE

P)c

TM

CP

-HA

/HK

(HE

P)c

N-p

enty

l(H

EP

)cG

LU

C(H

EP

)b

CY

S:

cyst

ein

e;H

A:

hem

i-ac

etal

;H

K:

hem

i-ket

al;

GL

UC

:g

lucu

ron

ide;

HE

P:

hu

man

hep

ato

cyte

s;H

LM

:h

um

anli

ver

mic

roso

mes

;Q

UL

:qu

ino

linyl;

RL

S:

rat

liver

slic

es;

RL

M:

rat

liver

mic

roso

mes

;S

UL

:su

lfat

e;T

MC

P:

tetr

amet

hylc

ycl

op

rop

yl;

UN

SP

:u

nsp

ecif

ied

.aId

enti

fied

inau

then

tic

anim

alsa

mp

les.

bId

enti

fied

inau

then

tic

hu

man

sam

ple

s.cId

enti

fied

invi

tro

.

42 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

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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

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

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 0.5–10mg/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

and confirming 75 NPS including 54 SC analytes in urine

was published (Sundstrom 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 SWATH� (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

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 43

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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 0.25mg/L cutoffs for 21 indole-

core SC (Rodrigues et al., 2013) OF was collected with the

Quantisal� 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

ranging from 0.15 to 3.0mg/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

(n¼ 31), followed by JWH-122 (n¼ 17) JWH-081 (n¼ 8),

JWH-018 (n¼ 7) and AM2201 (n¼ 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

Intercept� 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,

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

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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-

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 quantified565% 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.

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

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 230mg/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

DOI: 10.3109/03602532.2015.1029635 Synthetic cannabinoids 45

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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

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

available, delaying method development. An important

drawback is also the shared metabolic pathways (Figure 1)

that complicate interpretation of urine SC results.

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

Figure 1. Merging metabolic pathways for synthetic cannabinoids AKB-48, JWH-018, JWH-073, JWH-122, PB-22 and XLR-11 and five fluoropentylanalogs. Ester hydrolysis of PB-22 and 5F-PB-22 produce 1-pentyl-1H-indole-3-carboxylic acid (PI-COOH) and 5F-PI-COOH, respectively.

46 M. S. Castaneto et al. Drug Metab Rev, Early Online: 1–51

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(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

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-

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

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

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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

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

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

laboratories to acquire accurate MS and MS/MS data

without pre-selecting compounds (Kronstrand et al., 2014;

Scheidweiler et al., 2014; Sundstrom 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.

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