Neurotoxicology 73 (2019) 8–16

Contents lists available at ScienceDirect

Neurotoxicology
journal homepage: www.elsevier.com/locate/neuro

Review

An expanding world of new psychoactive substances—designer T

benzodiazepines
⁎
Jolanta B. Zawilska , Jakub Wojcieszak
Department of Pharmacodynamics, Medical University of Łódź, 90-151 Łódź, Muszynskiego 1, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: The abuse of new psychoactive substances (NPS) has been increasing dramatically since the late 2000s world-
Benzodiazepines wide. Between 2009 and 2017, a total of 803 individual NPS were reported to the United Nations Office of Drugs
New psychoactive substances and Crime by 111 countries and territories. Although the most popular compounds are synthetic cannabino-
Toxicity mimetics and psychostimulatory derivatives of cathinone (so-called β-keto-amphetamines), novel benzodiaze-
Metabolism
pines have recently emerged on the recreational drug market. The misuse/abuse of “designer benzodiazepines”
Impaired driving
(DBZD), a common name for the benzodiazepine class NPS, has become an increasing problem in many coun-
tries. The DBZD group includes pharmaceutical drug candidates that have never been approved for medical use,
compounds that were synthesized by a simple structural modification of a registered drug, and some active
metabolites of registered benzodiazepines. This survey presents members of the DBZD group, describes the
epidemiological trends and clinical effects associated with DBZD use, and discusses available data on their
metabolism. Special emphasis is given to cases of intoxications involving these compounds.

1. Introduction
Benzodiazepines were introduced as therapeutic drugs in the early
1960s. They act as positive allosteric modulators of γ-aminobutyric acid
(GABA)-A receptors, which are composed of heteropentameric multi-
subunit proteins. The drugs bind to a specific high affinity binding site
of GABA-A receptors, located at the α/γ subunit interface. The phar-
macological activity of the benzodiazepines is determined by the type
of GABA-A receptor α subunit to which they bind. Thus, the sedative,
anterograde amnesic and anticonvulsant actions, as well as the addic-
tive potential of these drugs, require the presence of α1-containing
GABA-A receptors, while the anxiolytic effects are mediated by GABA-A
receptors containing α2 subunits, and the myorelaxant actions by
GABA-A receptors containing α2, α3, and α5 subunits (Tan et al.,
2011). Benzodiazepines are nowadays widely applied in the therapy of
psychiatric and neurological disorders, including anxiety and panic
attacks, insomnia, muscle spasms, epilepsy and alcohol withdrawal.
Some are also used as a premedication prior to surgery and intra-op-
erative medications. In addition, benzodiazepines are commonly used
in self-medication, and misused in combination with other psychoactive
substances, namely opioids, psychostimulants and alcohol (EMCDDA,
2018a; Griffin et al., 2013; UNODC, 2017a). There are also reports on
the use of benzodiazepines in drug-facilitated crime and driving under
the influence of drugs (e.g., Bertol et al., 2018; Drummer et al., 2012;
Kriikku et al., 2012; Valen et al., 2017; Xiang et al., 2015).
Over the last decade, an increasing number of new benzodiazepines
that are not registered as medical products have been introduced into
the recreational drug market (see Table 1). Currently, new psychoactive
substances (NPS) belonging to the benzodiazepine class are commonly
referred to as “designer benzodiazepines” (DBZD), and their misuse or
abuse has become an increasing problem in many countries. This survey
presents members of the DBZD group, describes the epidemiological
trends and clinical effects associated with DBZD use, and discusses
available data on their metabolism.
2. Methodology
Review of the literature was based on the exhaustive search carried
out in PubMed (U.S. National Library of Medicine), using “designer
benzodiazepines” and each of the compound name listed in Table 1 as
keywords. Only papers written in English and with full texts available
by December 2018 were included. Additionally, official reports pub-
lished by the United Nations Office on Drugs and Crime (UNODOC),
European Monitoring Centre for Drugs and Drug Addiction (EMCDDA)
and World Health Organization (WHO), and governmental acts were
reviewed. Furthermore, in each article and report retrived, references

⁎
Corresponding author.
E-mail address: jolanta.zawilska@umed.lodz.pl (J.B. Zawilska).

https://doi.org/10.1016/j.neuro.2019.02.015
Received 4 January 2019; Received in revised form 12 February 2019; Accepted 21 February 2019
Available online 23 February 2019
0161-813X/ © 2019 Elsevier B.V. All rights reserved.
J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16

Table 1 Table 1 (continued)

Chemical structures and names of designer benzodiazepines.
Chemical structure Names
Chemical structure Names

1,4-BENZODIAZEPINES
Cloniprazepam
A likely prodrug of clonazepam.
IUPAC name:
5-(2-Chlorophenyl)-1-(cyclopropylmethyl)-7-
nitro-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-
one

Phenazepam1
IUPAC name:
7-Bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-
Desmethylflunitrazepam (also known as 1,4-benzodiazepin-2-one
norflunitrazepam, Ro-4435 and fonazepam) Trade names: BD98, Elzepam, Fenazepam,
The active metabolite of the potent drug Phenazef, Phenazepam, Phezipam,
flunitrazepam. Phenorelaxan, Trankvezipam
IUPAC name: Street names: Bonsai, Bonsai Supersleep, Fenaz,
5-(2-fluorophenyl)-7-nitro-1,3-dihydro-2H-1,4- Soviet Benzo, Panda
benzodiazepin-2-one
3-Hydroxyphenazepam
The active metabolite of phenazepam.
IUPAC name:
Diclazepam (2-Chlorodiazepam, Ro5-3448) 7-Bromo-5-(2-chlorophenyl)-3-hydroxy-1,3-
IUPAC name: dihydro-2H-1,4-benzodiazepin-2-one
7-Chloro-5-(2-chlorophenyl)-1-methyl-1,3-
dihydro-2H-1,4-benzodiazepin-2-one

TRIAZOLOBENZODIAZEPINES
Adinazolam (Deracyn®, Adinazolamum)
IUPAC name:
1-(8-Chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a]
[1,4]benzodiazepin-1-yl)-N,N-
4’-Chlorodiazepam (Ro5-4864)
dimethylmethanamine
IUPAC name:
7-Chloro-5-(4-chlorophenyl)-1-methyl-3H-1,4-
benzodiazepin-2-one

Bromazolam
IUPAC name:
8-Bromo-1-methyl-6-phenyl-4H-[1,2,4]triazolo
[4,3-a][1,4]benzodiazepine
Flubromazepam
IUPAC name:
7-Bromo-5-(2-fluorophenyl)-1,3-dihydro-2H-
1,4-benzodiazepin-2-one

Clonazolam (Clonitrazolam)
IUPAC name:
Meclonazepam [(S)-3-methylclonazepam] 6-(2-Chlorophenyl)-1-methyl-8-nitro-4H-[1,2,4]
IUPAC name: triazolo[4,3-a][1,4]benzodiazepine
(3S)-5-(2-Chlorophenyl)-3-methyl-7-nitro-1,3-
dihydro-2H-1,4-benzodiazepin-2-one

Flualprazolam
Nifoxipam (3- IUPAC name:
hydroxydesmethylflunitrazepam, DP 370) 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-benzo
IUPAC name: [f][1,2,4]triazolo[4,3-a][1,4]diazepine
5-(2-Fluorophenyl)-3-hydroxy-7-nitro-2,3-
dihydro-1H-1,4-benzodiazepin-2-one

Nitemazepam Flubromazolam
IUPAC name: IUPAC name:
3-Hydroxy-1-methyl-7-nitro-5-phenyl-2,3- 8-Bromo-6-(2-fluorophenyl)-1-methyl-4H-
dihydro-1H-1,4-benzodiazepin-2-one
(continued on next page)

9
J.B. Zawilska and J. Wojcieszak
Table 1 (continued)
Chemical structure Names
[1,2,4]triazolo[4,3-a][1,4]benzodiazepine
Street name: liquid Xanax
Flunitrazolam
IUPAC name:
6-(2-Fluorophenyl)-1-methyl-8-nitro-4H-[1,2,4]
triazolo[4,3-a][1,4]benzodiazepine
Nitrazolam (NitrazolaM)
IUPAC name:
1-Methyl-8-nitro-6-phenyl-4H-[1,2,4]triazolo
[4,3-a][1,4]benzodiazepine
Pyrazolam
IUPAC name:
8-Bromo-1-methyl-6-(pyridin-2-yl)-4H-[1,2,4]
triazolo[4,3-a][1,4]benzodiazepine
Zapizolam
IUPAC name:
8-Chloro-6-(2-chlorophenyl)-4H-pyrido[2,3-f]
[1,2,4]triazolo[4,3-a][1,4]diazepine
THIENOTRIAZOLOBENZODIAZEPINES
Etizolam2
IUPAC name:
4-(2-Chlorophenyl)-2-ethyl-9-methyl-6H-thieno
[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine
International trade names: Arophalm, Capsafe,
Depas, Dezolam, Eticalm, Etidrale, Etisedan,
Etizolan, Guperies, Medipeace, Mozun,
Nonnerv, Palgin, Pasaden, Sedekopan,
Sylazepam
Street names: Etiz, Eitizzy, Etizest
Deschloroetizolam
IUPAC name:
4-Phenyl-2-ethyl-9-methyl-6H-thieno[3,2-f]
[1,2,4]triazolo[4,3-a][1,4]diazepine
Street names: Etizolam-2, Etiz-dechlorinate,
Legal-Eti
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Neurotoxicology 73 (2019) 8–16
Table 1 (continued)
Chemical structure Names
Metizolam (desmethyletizolam)
A metabolite of etizolam.
IUPAC name:
4-(2-Chlorophenyl)-2-ethyl-6H-thieno[3,2-f]
[1,2,4]triazolo[4,3-a][1,4]diazepine
Fluclotizolam
IUPAC name:
2-Chloro-4-(2-fluorophenyl)-9-methyl-6H-
thieno[3,2-f][1,2,4]triazolo[4,3-a]diazepine
1
Phenazepam is used as a prescription medicine in the Russian Federation,
Estonia, Latvia, Lithuania and Belarus (WHO, 2015).
2
Etizolam is used as a prescription medicine in Japan, India and Italy (WHO,
2017).
were checked carefully in order to find possible additional publications,
missed during the initial search.
3. Legal status of designer benzodiazepines
In Canada, all benzodiazepines are classified as schedule IV drugs
(Ministry of Justice, Canada, 2018). In the United Kingdom, broma-
zolam, 4-chlorodiazepam, clonazolam, deschloroetizolam, diclazepam,
etizolam, flubromazepam, flubromazolam, fonezepam, 3-hydro-
xyphenazepam, meclonazepam, metizolam, nifoxipam, nitrazolam and
pyrazolam have been classified as Class C drugs by the May 2017
amendment to The Misuse of Drugs Act 1971 (Advice Council on Misuse
of Drugs, 2017). Several DBZD have been placed under national control
in other countries (UNODC, 2017c; WHO, 2015, 2017):
• China: phenazepam;
• Denmark: clonazolam, deschloroetizolam, diclazepam, flu-
bromazepam, flubromazolam, metizolam, nifoxipam, phenazepam
and pyrazolam;
• Finland: clonazolam, deschloroetizolam, diclazepam, etizolam, flu-
bromazepam, flubromazolam, meclonazepam, nifoxipam, phena-
zepam and pyrazolam;
•
Germany: etizolam and phenazepam;
• Ireland: phenazepam;
• Netherlands: phenazepam;
• Poland: etizolam;
• The Republic of Korea: diclazepam;
• Sweden: adinazolam, cloniprazepam, desmethylflunitrazepam,
J.B. Zawilska and J. Wojcieszak
flubromazepam, flunitrazolam, phenazepam, pyrazolam and zapi-
zolam;
• Switzerland: clonazolam, deschloroetizolam, diclazepam, etizolam,
flubromazepam, flubromazolam, nifoxipam and pyrazolam;
• Turkey: adinazolam, deschloroetizolam, diclazepam, flu-
bromazepam, flubromazolam, meclonazepam and pyrazolam;
• The United Arab Emirates: diclazepam, etizolam, flubromazepam
and pyrazolam;
• The United States: etizolam is not currently listed under the
Controlled Substances Act, but has been declared a controlled sub-
stance in some of the states, i.e., Alabama, Arkansas, Arizona,
Florida, Georgia, Indiana, Mississippi, and Virginia.
4. Designer benzodiazepines – historical background, appearance
of products, patterns of consumption
The first recreationally-used benzodiazepine was phenazepam
(2007) followed by etizolam (2011) (EMCDDA, 2018a). Phenazepam
was originally developed in the Soviet Union in the 1970s, and pre-
scribed to treat anxiety and alcohol withdrawal (WHO, 2015). Etizolam
is an anxiolytic medicine, originally developed in Japan, where it was
introduced under the brand name of Depas in 1984. Both compounds
are used as prescribed medicines in a few countries (see Table 1).
Since 2012, new benzodiazepines have been introduced on the re-
creational drug market and distributed by online retailers mostly as
“research chemicals”. Between 2011 and 2016, the United Nations
Office on Drugs and Crime (UNODC) Early Warning Advisory (EWA)
received a total of 209 reports of DBZD, most of them from European
countries (UNODC, 2017a). In Europe, the number of countries re-
porting the emergence of new benzodiazepines as NPS has increased
from three in 2011 to 21 in 2015. The European Monitoring Centre for
Drugs and Drug Addiction (EMCDDA) is currently monitoring 23 DBZD,
with more than half having been detected since 2015 (EMCDDA, 2018a,
2018b). The most frequently reported drugs from this group are eti-
zolam, diclazepam, flubromazolam, phenazepam and pyrazolam
(EMCDDA, 2018a; UNODC, 2017a). Notably, etizolam, diclazepam,
flubromazolam and phenazepam account for more than 80% of all ta-
blets containing DBZD that have been seized in Europe since 2005
(EMCDDA, 2018a). DBZD, mostly clonazolam, flubromazepam, nifox-
ipam, nimetazepam, phenazepam and pyrazolam were also identified in
South-East Asia (UNODC, 2017a, 2017b).
Most of DBZD either derive from pharmaceutical drug candidates
(e.g., clonazolam, deschloroetizolam, diclazepam, flubromazepam,
meclonazepam and pyrazolam) that have never been approved for
medical use, or are synthesized by a simple structural modification of a
registered drug (e.g., flubromazolam). Some active metabolites of re-
gistered benzodiazepines are also sold as NPS. For example, des-
methylflunitrazepam and 3-hydroxydesmethylflunitrazepam, the active
metabolites of the potent drug flunitrazepam, are marketed under the
names of fonazepam and nifoxipam, respectively (Katselou et al.,
2017).
DBZD are sold as tablets, pills, capsules, pellets, powders, blotters,
and recently liquids under their own names (Abouchedid et al., 2018;
Corkery et al., 2012; Pope et al., 2018; US Department of Justice,
2009). However, some of them are also sold on the illicit drug market as
counterfeit forms of diazepam and alprazolam, commonly prescribed
anxiolytic medicines, which increases the risk of unintentional overdose
and intoxication. For instance, tablets sold on the illicit drug market in
Europe as alprazolam were found to contain flubromazolam, and those
sold as diazepam contained various other DBZD: phenazepam, eti-
zolam, diclazepam and flubromazepam (EMCDDA, 2016; UNODC,
2017a). In California, etizolam, together with fentanyl, was found in
serum samples of intoxicated subjects who took counterfeit alprazolam
tablets (Arens et al., 2016). Phenazepam, together with synthetic can-
nabinomimetics, was identified in smokable products called “Pineapple
Express” and “Purple Haze”, marketed in New Zealand as containing
11
Neurotoxicology 73 (2019) 8–16
natural extracts (Couch and Madhavaram, 2012). Additionally, there
are reports of phenazepam being sold in combination with stimulant
recreational drugs, such as dimethocaine (distributed under the brand
name of “Dimethocaine Phenazepam Legal Powder”) (Advisory Council
on the Misuse of Drugs, 2011).
At present, limited information is available regarding the pre-
valence and pattern of DBZD use, and existing studies should be re-
garded with caution as many are based on online surveys and self-re-
ports on user websites. The common route of administration for DBZD
is oral consumption, such as by pills, or powder inserted into gel cap-
sules or dissolved in ethanol or propylene glycol. Alternatively, rectal,
sublingual, intravenous or intranasal routes can be used, as well as
inhalation (smoking and vaporization). Intravenous administration was
reported to give a “rush” (Anderson and Kjeligren, 2017; Carpenter
et al., 2018).
5. Biological effects
Information on the effects of DBZD are largely limited to self-re-
ported experiences from user websites and case reports/series
(Anderson and Kjeligren, 2017; Benerjee, 2018; Benesch and Iqbal,
2018; Corkery et al., 2012; Dargan et al., 2013; Ghazi and Mohmand,
2017; Gupta and Garg, 2014; Manchester et al., 2018; O’Connell et al.,
2015).
By analogy to pharmaceutical benzodiazepines, the desired actions
of DBZD include increased sociability, muscle relaxation, feelings of
well-being or even euphoria. The sleep-inducing effects, and the control
of anxiety, nervousness, panic attacks and chronic pain are used for self-
medication. The drugs are often taken by opioid users in order to in-
crease the opioid high and to help to sedate the descent phase, as well
as by users of psychostimulants to facilitate a come-down after an ex-
tensive dose, and by individuals addicted to alcohol to augment its
effect synergistically. In individuals addicted to benzodiazepines,
opioids, stimulants and alcohol DBZD are used to alleviate withdrawal
or abstinence syndromes (Abouchedid et al., 2018; Anderson and
Kjeligren, 2017; EMCDDA, 2018a; Jones et al., 2012; WHO, 2015,
2017).
The common adverse effects of DBZD include somnolence, impaired
balance, ataxia, loss of coordination, impaired thinking and self-as-
sessment capability, muscle weakness, confusion, slurred speech,
blurred vision, amnesia, dizziness, drowsiness, lethargy, fatigue and
palpitations. At high doses, they could induce delirium, auditory and
visual hallucinations, seizures, deep sleep and coma (Ali et al., 2015;
Anderson and Kjeligren, 2017; Benerjee, 2018; Benesch and Iqbal,
2018; Carpenter et al., 2018; Corkery et al., 2012; Dargan et al., 2013;
Huppertz et al., 2018; Łukasik-Głębocka et al., 2016; Manchester et al.,
2018; O’Connell et al., 2015; WHO, 2015, 2017). There are also reports
of paradoxical excitement and increase in energy (Anderson and
Kjeligren, 2017). Users of long-acting flubromazolam have described
unpleasant night dreams, sleeping paralysis, and somnambulistic states
that persisted for several days (Anderson and Kjeligren, 2017). Sus-
tained involuntary closing of the eyelids (blepharospasms) were spor-
adically observed in patients who had used etizolam for at least one
month (Wakakura et al., 2004). The toxic effects of phenazepam and, in
particular, flubromazolam may last for several days (e.g., Anderson and
Kjeligren, 2017; Dargan et al., 2013). Like pharmaceutical benzodia-
zepines, DBZD can have severe toxicity when concomitantly used with
other CNS depressant drugs, especially opioids and alcohol, which in-
creases the risk of respiratory depression and death (EMCDDA, 2018a).
Chronic use of DBZD results in the development of tolerance, as well
as psychological and physical dependence. Abrupt cessation may lead
to withdrawal symptoms such as anxiety, insomnia, restlessness, sei-
zures and life-threatening convulsions (Anderson and Kjeligren, 2017;
Corkery et al., 2012; Gupta and Garg, 2014; Nishi et al., 2014; WHO,
2015, 2017). Analysis of anonymous self-reports published on a
Swedish drug forum revealed that flubromazolam withdrawal
J.B. Zawilska and J. Wojcieszak
symptoms are worse and longer than those produced by any benzo-
diazepine, and include muscle aches, sleeping disorders, severe anxiety
and panic attacks, dissociative symptoms, perceptual distortions,
cramping, chills, seizures, and vomiting (Anderson and Kjeligren,
2017).
6. Acute intoxication cases involving designer benzodiazepines
Bäckberg et al. (2018) published data on acute intoxication cases
presenting for emergency care in Sweden during 2012–2016 and col-
lected within the STRIDA program. DBZD were detected in 217 (11.3%)
out of 1913 urine samples analyzed. The mean age of patients was 28
years (range 15–66; median 26), and 81% were men. The first analy-
tically confirmed intoxication involving DBZD (etizolam) was found in
January 2012. The frequency of samples tested positive for DBZD in-
creased from 4% in 2012, 8% in 2013, 11% in 2014, to 19% in 2015,
and then declined to 6% in 2016. Altogether 14 DBZD were found:
flubromazolam (92 cases), pyrazolam (33), flubromazepam (33), me-
clonazepam (26), etizolam (20), clonazolam (16), 3-hydro-
xyphenazepam (eight), nifoxipam (five), diclazepam (four), metizolam
(four), bromazepam (one), deschloroetizolam (one), ketazolam (one),
and phenazepam (one). Twenty four patients (11%) tested positive only
for DBZD. In the remaining cases other psychoactive drugs were de-
tected, including standard prescription benzodiazepines (49%), other
depressants such as opioids, ethanol and cannabis (29%), stimulants
including phenethylamines and synthetic cathinones (11%); in half of
the samples a mixture of drugs was identified. Medical records and
clinical data obtained for 23 patients who were intoxicated only with
DBZD show that the most prominent clinical signs were CNS depression
with a sedative-hypnotic toxidrome, tachycardia, dilated pupils, and
slurred speech (Bäckberg et al., 2018). Carpenter et al. (2018) analyzed
cases of intoxication related to DBZD and reported by 40 states to the
U.S. National Poison Data System during January 1, 2014 – December
31, 2017. Among 474 cases, 234 were classified as single compound
exposures, and their number steadily increased from 26 in 2014, 30 in
2015, 66 in 2016 to 122 in 2017. Most of the cases (77%) represented
acute intoxication. The median age of patients was 25 years (range
13–63), and 86% were men. The most common benzodiazepine found
in these single-agent exposures was etizolam (162 cases; 69%), fol-
lowed by clonazolam (50 cases; 21%). Other identified compounds
include flubromazolam (13 cases), diclazepam (three), flubromazepam
(one), meclonazepam (one), and norflurazepam (one). The most
common clinical signs of intoxications were drowsiness/lethargy
(65%), slurred speech (17%), and confusion (14%). Less frequently
observed symptoms were agitation/irritability, ataxia and tachycardia
(each 9%), hypotension (8%), coma (4%), and bradycardia (4%). There
was one death among the cluster with a reportedly intentional ingestion
of etizolam. In 85% of the subjects, the duration of clinical symptoms
was shorter than 24 h. In one case, intoxication with etizolam, the
symptoms persisted for longer than one month (Carpenter et al., 2018).
7. Fatal intoxications related to the use of designer
benzodiazepines
By analogy to pharmaceutical benzodiazepines, DBZD are involved
mostly in polydrug deaths. In South Scotland during 2010–2014, there
were 228 cases where phenazepam was detected in post-mortem fe-
moral blood (Shearer et al., 2015). In two of these cases, the cause of
death was solely attributed to phenazepam (blood concentration of
1.2 mg/L and 1.6 mg/L). In 54 cases, the cause of death was related to
phenazepam along with one or more other drugs, mainly methadone,
morphine and alcohol. The blood concentration of phenazepam in these
cases ranged from < 0.005 to 0.9 mg/L (median 0.10 mg/L). Of the 83
cases where phenazepam was present but not included in the cause of
death, its concentration in blood ranged from 0.005 to 0.46 mg/L
(median 0.05 mg/L). In these cases methadone, morphine and ethanol
12
Neurotoxicology 73 (2019) 8–16
were found (Shearer et al., 2015). Another report presents tissue dis-
tribution and concentration of phenazepam and its metabolite, 3-hy-
droxyphenazepam, in biological fluids and tissues from 29 post-mortem
cases from Scotland, investigated between January 2011 and April
2013 (Crichton et al., 2015). In 27 cases, where the cause of death was
not directly related to phenazepam, its median concentration in fe-
moral, cardiac and subclavian blood was 0.097 mg/L (0.007–0.360 mg/
L), 0.086 mg/L (0.014–0.310 mg/L) and 0.039 mg/L (0.016–0.270 mg/
L), respectively. Apart from blood, phenazepam was also detected in
vitreous humour: 0.013 mg/L (0.007–0.054 mg/L), urine: 0.016 mg/L
(0.007–0.049 mg/L), thalamus: 0.325 mg/kg (0.065–1.013 mg/kg),
liver: 0.584 mg/kg (0.099–2.125 mg/kg) and psoas muscle: 0.166 mg/
kg (0.034–0.469 mg/kg). In the remaining two cases phenazepam was
either the certified cause of death or a contributing factor. In the former
one, concentrations of phenazepam and 3-hydroxyphenazepam in fe-
moral blood were 1.64 mg/L and 0.43 mg/L, and in urine 0.08 mg/L
and 0.19 mg/L. In the latter case (multidrug fatal intoxication) con-
centrations of phenazepam and 3-hydroxyphenazepam in femoral
blood were 0.97 mg/L and 0.17 mg/L, and in urine 0.55 mg/L and
3.52 mg/L (Crichton et al., 2015). The authors of this report suggest
that phenazepam may undergo post-mortem redistribution (Crichton
et al., 2015). Whether post-mortem distribution might occur in the case
of other DBZD remains to be elucidated.
Maskell et al. (2011) reported nine cases of death in the United
Kingdom where phenazepam was detected in post-mortem toxicology
but not implicated in the cause of death. Death was attributed to
opioids in seven cases, but other drugs in two cases. The deceased were
men and women aged 31–45 years. Phenazepam was found in 17 au-
topsy cases in Finland between July 1, 2010 and June 30, 2011. Most of
the deaths were accidental overdoses. All of the deceased were male
with a mean age of 35 ± 7 years. The median phenazepam blood
concentration was 0.048 mg/L (range 0.007–1.600 mg/L). One or sev-
eral opioids were detected in blood/urine in 12 of these cases, alcohol
in nine and amphetamine in four (Krrikku et al., 2012).
In Scotland, etizolam was the third most frequently detected sub-
stance reported among all drug-related deaths in 2016, after heroin/
morphine and methadone. The number of deaths related to etizolam
increased from 58 in 2015 to 270 in 2016, with more women than men
being among the deceased (UNODC, 2018). In addition, 134 deaths due
to intoxication with diclazepam were reported in Scotland in 2016
(UNODC, 2018).
A case of a 42-year old Caucasian male who died as the result of
multidrug intoxication was described by Bailey et al. (2010). Near the
body, a vial of white powder labeled "Phenazepam, Purity 99%, CAS
No. 51753-57-2, Research Sample", was found. Phenazepam, morphine,
codeine, and thebaine were present in his femoral blood at concentra-
tions of 386, 116, 85 and 72 ng/mL, respectively. Elsewhere, a number
of substances were detected in femoral blood of a 22-year old man,
whose death was determined to be the aspiration of chyme, possibly
due to a loss of consciousness: mitragynine (790 μg/L), etizolam
(280 μg/L), pregabaline (3 mg/L), pipamperon (7.4 μg/L), lorazepam
(6.9 μg/L), triazolam (1.1 μg/L), fluoxetine (89 μg/L), quetiapine
(18 μg/L), olanzapine (5.9 μg/L), and (likely) 2-MMC (∼5.2 μg/L)
(Domingo et al., 2017). Another report describes the case of a 42-year
old man who died in the course of myocardial infarction due to a
multidrug intoxication. Toxicological analysis revealed the presence of
etizolam (0.3 mg/L) in his blood, as well as the synthetic cathinones
3,4-methylenedioxypyrovalerone (0.046 mg/L) and pentedrone
(0.16 mg/L), together with ephedrine (0.068 mg/L), olanzapine
(4.2 mg/L) and mitrazapine (0.57 mg/L) (Liveri et al., 2016). Other
fatal poisoning cases were described by Tanaka and coworkers (Tanaka
et al., 2011a, 2011b). In one, toxicological analysis revealed the pre-
sence of etizolam, phenobarbital, promethazine and chlorpromazine in
femoral blood at concentrations of 86 ng/mL, 5082 μg/mL, 0.107 μg/
mL and 0.144 μg/mL, respectively (Tanaka et al., 2011a). In another
case, brotizolam, 7-aminoflunitrazepam (a metabolite of flunitrazepam)
J.B. Zawilska and J. Wojcieszak
and ethanol were found to be present in femoral blood at concentra-
tions of 0.025 μg/mL, 0.094 μg/L and 0.29 mg/mL, respectively
(Tanaka et al., 2011b). The drugs were also detected in the stomach
contents of these two deceased (Tanaka et al., 2011a, 2011b).
There are four case reports of deaths related to intoxication with
DBZD in combination with synthetic opioids (MT-45, U-47700, AH-
7921) used as NPS. Papsun et al. (2016) describe the case of a 35-year
old man with a known history of drug abuse who was found dead. MT-
45 and etizolam were detected in his femoral blood at concentrations of
520 ng/mL and 35 ng/mL, respectively. Similarly, Partridge et al.
(2018) present a case of a 28-year-old man with a history of illicit drug
use. U-47700 (330 μg/L), diclazepam (70 μg/L), flubromazepam
(10 μg/L), methylamphetamine (290 μg/L) and amphetamine (150 μg/
L) were found in post-mortem peripheral blood.
In another study, mixed intoxication by U-47700 and flu-
bromazepam was found to have a lethal outcome in a 24‐year‐old man
admitted to a hospital after suffering apnoea associated with their
consumption. The concentrations of U‐47700 and flubromazepam in a
blood sample collected after admission were 370 μg/L and 830 μg/L,
respectively. The man died six days later (Koch et al., 2018). Finally,
intoxication with AH-7921 in combination with other psychoactive
drugs, including DBZD, was the cause of death of a young woman. The
concentrations of drugs in her blood were as follows: AH-7921 –
0.33 mg/L, methoxetamine – 0.064 mg/L, etizolam – 0.27 mg/L, phe-
nazepam – 1.33 mg/L, 7-aminonitrazepam – 0.043 mg/L, diazepam –
0.046 mg/L, nordiazepam – 0.073 mg/L, and oxazepam – 0.018 mg/L
(Karinen et al., 2014).
8. Designer benzodiazepines and impaired driving
Over the last decade, an increasing number of apprehended drivers
have been found to test positive for DBZD. The subjects commonly
exhibited slurred speech, lack of balance, slow reactions, drowsiness
and confusion (Kerrigan et al., 2013; Stephenson et al., 2013). Phena-
zepam was detected in 0.5% of 166 individuals driving under the in-
fluence of drugs (DUID) that were killed in road traffic accidents in
Norway during 2006–2008 (Gjerde et al., 2011). Also in Norway, DBZD
were detected in 77 out of 22,022 samples collected during the period
July 1, 2013 – May 31, 2016. Sixty-nine were found to be under the
influence of drugs, and 14 of them were involved in a traffic accident
(Høiseth et al., 2016). The following median (range) blood concentra-
tions of DBZD were recorded: flubromazolam – 0.012 mg/L
(0.00048–0.10; n = 25), flubromazepam – 0.055 mg/L (0.0047–1.2;
n = 24), diclazepam – 0.013 mg/L (0.0021–0.057; n = 15), etizolam –
0.050 mg/L (0.019–0.17; n = 14), clonazolam – 0.0053 mg/L
(0.0019–0.011; n = 7), and pyrazolam – 0.074 mg/L (n = 1). In six
cases, DBZD were the only drugs detected in blood samples. The other
most frequently detected drugs were Δ9-THC (51% of the cases), am-
phetamine (44% of the cases) and ethanol (14% of the cases). In three
cases, the DBZD were detected together with other NPS: cannabino-
mimetics - 5F-APINACA (n = 2) and APINACA (n = 1); psychostimu-
lants - methiopropamine (n = 1) and α-PVP (n = 1) (Høiseth et al.,
2016).
In Finland, 141 out of 4007 blood samples collected from appre-
hended drivers during the period between July 1, 2010 and June 30,
2011 tested positive for phenazepam. The majority of the drivers, 88%,
were male. The mean age of the men was 34 ± 2 years and of women
27 ± 5 years. The median phenazepam concentration in blood was
0.061 mg/L (range 0.004–3.600 mg/L). In seven cases phenazepam was
the only psychoactive substance present (median concentration
0.270 mg/L). Other substances identified together with phenazepam
were amphetamine and various pharmaceutical benzodiazepines
(Kriikku et al., 2012).
In the state of Georgia (U.S.) phenazepam was found in blood
samples taken from 11 impaired drivers between March, 2010 and
August, 2011. Concentrations of the drug ranged from 0.04 to 3.2 mg/L,
13
Neurotoxicology 73 (2019) 8–16
with a median of 0.17 mg/L. In five cases phenazepam was the only
drug detected. The observed effects where symptomatic of CNS de-
pression, with slurred speech, lack of balance, slow reactions, drowsi-
ness and confusion (Stephenson et al., 2013).
9. Metabolism
Unlike pharmaceutical benzodiazepines, the vast majority of DBZD
have not undergone clinical trials, and our knowledge of their phar-
macokinetic properties and biotransformation is very limited. Assuming
that these substances could be extensively metabolized, there is a high
need for effective analytical methods to detect their biotransformation
products in biological samples, particularly urine.
Three main strategies are used to examine the metabolism of DBZD:
(1) incubation of substances with human liver microsomes (HLM) fol-
lowed by analysis of the produced metabolites, (2) analysis of urine
samples of a large cohort of NPS users, and in rare cases (3) analysis of
urine samples in controlled self-administration studies.
The two main biotransformation pathways for benzodiazepines are
generally oxidation and glucuronidation; for an excellent review see
Moosmann and Auwäter (2018). Phase I metabolism of DBZD involves
hydroxylation, N-dealkylation, and in the case of nitrobenzodiazepines
– nitro reduction to the 7-amino compound; compounds containing
bromine atom in their structure can also undergo debromination. The
phase II reaction involves glucuronidation of the parent compound
and/or its phase I metabolites, and acetylation of 7-amino metabolites
of nitrobenzodiazepines (see Table 2). It should emphasized that not
only some active metabolites of registered benzodiazepines are sold as
NPS, but also some metabolites of DBZD (i.e., 3-hydroxyphenazepam,
deschloroetizolam and desmethyletizolam; delorazepam, lorazepam
and lormetazepam formed from diclazepam; clonazepam produced
from cloniprazepam) can be responsible for the pharmacological ac-
tivity and toxic effects of the substance.
DBZD vary significantly in terms of effective doses and duration of
action (Table 3), from short-acting drugs, e.g., etizolam and adinazolam
(elimination half-life of approximately three hours) to long-acting ones,
e.g., diclazepam (elimination half-life of around 42 h), phenazepam
(elimination half-lives of around 15 h after intravenous injection and
103 h after oral administration), and flubromazepam (elimination half-
life of around 106 h) (Lomas and Maskell, 2015; Moosmann et al., 2013,
2014). The delayed onset of action may lead users to re-dose the drug
before the effects of the first dose have been experienced, thus poten-
tially leading to overdose (Corkery et al., 2012). Drugs with a long
elimination half-life are likely to accumulate in an organism, increasing
the risk of intoxication.
10. Analytical detection of designer benzodiazepines and their
metabolites
The identification and quantification of DBZD, both parent com-
pounds and their metabolites, is an important task in the field of clinical
and forensic toxicology. A liquid-liquid extraction and solid-phase ex-
traction are used for sample clean-up and extraction of DBZD. Urine
samples should be hydrolyzed using for example β-glucoronidase (Kintz
et al., 2017a; Pettersson Bergstrand et al., 2016). Some DBZD have been
detected in blood and urine using immunochemical assays with high
cross-reactivity, such as enzyme multiplied immunoassay technique
(EMIT), cloned enzyme donor immunoassay (CEDIA), enzyme-linked
immunosorbent assay (ELISA), and kinetic interaction of microparticles
in solution (KIMS) technique (O’Connor et al., 2016; Pettersson
Bergstrand et al., 2017). However, immunochemical screening of bio-
logical specimens for DBZD has two important drawbacks: (1) the
drugs, especially the newest ones, may be missed when screened by
immunoassay if they are not in the scope of the confirmation panel of
benzodiazepines; (2) blood/serum levels of DBZD can be too low to be
detected by immunoassays (see Moosmann and Auwäter, 2018;
J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16

Table 2
Metabolism of designer benzodiazepines.
Compound Metabolites Reference

Adinazolam In vitro (HLM): N‐desmethyladinazolam, N, N‐didesmethyladinazolam Moosmann et al., 2016

Diclazepam In vitro (HLM): monohydroxylation → delorazepam, desmetylation → lormetazepam El Balkhi et al., 2017
Human urine: delorazepam, lorazepam, lormetazepam El Balkhi et al., 2017; Moosmann et al., 2014
Human serum: delorazepam
Etizolam In vitro (HLM): three monohydroxylated metabolites, keto-metabolite, etizolam glucuronide El Balkhi et al., 2017; Pettersson Bergstrand
Post-mortem blood: α-hydroxyetizolam, 8-hydroxyetizolam et al., 2019
Nakame et al., 2008
Deschloroetizolam In vitro (HLM): hydroxydeschloroetizolam, dihydroxydeschloroetizolam, El Balkhi et al., 2017; Pettersson Bergstrand
deschloroetizolam glucuronide et al., 2019
Flubromazolam In vitro (HLM, human hepatocytes): 4-hydroxyflubromazolam, α-hydroxyflubromazolam, El Balkhi et al., 2017; Noble et al., 2017;
dihydroxyflubromazolam, flubromazolam glucuronide Pettersson Bergstrand et al., 2019; Wohlfarth
Mice urine: α-hydroxyflubromazolam, 4‐hydroxyflubromazolam, α‐hydroxyflubromazolam et al., 2017
glucuronide, α,4‐dihydroxyflubromazolam, flubromazolam glucuronide Wohlfarth et al., 2017
Human urine: α-hydroxyflubromazolam, 4‐hydroxyflubromazolam, flubromazolam Huppertz et al., 2018; Noble et al., 2017;
glucuronide, α‐hydroxyflubromazolam glucuronide, 4‐hydroxyflubromazolam glucuronide, Wohlfarth et al., 2017; Pettersson Bergstrand
dihydroxyflubromazolam et al., 2018
Metizolam In vitro (HLM): 2-hydroxymetizolam, N-hydroxymetizolam, metizolam glucuronide Kintz et al., 2017a; Moosmann et al., 2016;
Human urine: 2-hydroxymetizolam, N-hydroxymetizolam, 2-hydroxymetizolam glucuronide Pettersson Bergstrand et al., 2019
Kintz et al., 2017a
Norflurazepam In vitro (HLM): hydroxynorflurazepam, dihydroxynorflurazepam. Moosmann et al., 2018
Phenazepam Human urine: 3-hydroxyphenazepam, 5-bromo-(2-chlorophenyl)-2-aminobenzophenone WHO, 2015
(ABPH), 6-bromo-(2-chlorophenyl) quinazoline-2-one (QNZ)
Pyrazolam Human urine: pyrazolam glucuronide Pettersson Bergstrand et al., 2018
COMPOUNDS WITH NITRO GROUP
Clonazolam In vitro (HLM): aminoclonazolam, desmethylclonazolam, hydroxyclonazolam El Balkhi et al., 2017
Human urine: 7-aminoclonazolam, 7-acetaminoclonazolam, hydroxyclonazolam, 7- Meyer et al., 2016
aminoclonazolam glucuronide, 7-acetaminoclonazolam glucuronide, hydroxyclonazolam
glucuronide
Cloniprazepam In vitro (HLM): 7-aminocloniprazepam, hydroxycloniprazepam, dihydroxycloniprazepam, 3- Moosmann et al., 2016; Mortelé et al., 2018
ketocloniprazepam, clonazepam, 7‐aminoclonazepam, hydroxyclonazepam, 3-hydroxy-7-
aminoclonazepam, hydroxycloniprazepam glucuronide
Flunitrazolam In vitro (HLM): hydroxyflunitrazolam, dihydroxyflunitrazolam, aminoflunitrazolam, Moosmann et al., 2018; Petterson Bergstrand
flunitrazolam glucuronide et al., 2019
Human urine: desnitroflunitrazolam, 7-aminoflunitrazolam, 7-acetamidoflunitrazolam, Ameline et al., 2018
hydroxyflunitrazolam
Fonazepam (norflunitrazepam) In vitro (HLM): 7-aminofonazepam (7-aminonorflunitrazepam), 3-hydroxyfonazepam (3- Moosmann et al., 2016
hydroxynorflunitrazepam; nifoxipam)
Meclonazepam In vitro (HLM): aminomeclonazepam, hydroxymeclonazepam. El Balkhi et al., 2017
Human urine: 7-aminomeclonazepam, 7-acetaminomeclonazepam Meyer et al., 2016; Vikingsson et al., 2017
Nifoxipam In vitro (HLM):7-aminonifoxipam, denitro-nifoxipam, nifoxipam glucuronide El Balkhi et al., 2017; Pettersson Bergstrand
Human urine: 7-aminonifoxipam, 7-acetaminonifoxipam, nifoxipam glucuronide et al., 2019
Meyer et al., 2016
Nitrazolam In vitro (HLM): 8‐aminonitrazolam, 4-hydroxynitrazolam/α-hydroxynitrazolam Moosmann et al., 2016

HLM – human liver microsomes. Main metabolites are written in bold.

Pettersson Bergstrand et al., 2017). Thus, results of the initial im-
munoassay screening need confirmation using chromatographic
methods, in particular based on mass spectrometry (MS) with up to date
library of references. To fulfill this requirement various methods of
DBZD and their metabolites detection in bodily fluids and tissue sam-
ples (blood, serum, urine, vitreous humour, hair, liver, brain, muscle)
have been published. They are based on gas chromatography (GC), e.g.,
with flame-ionization detection (GC–FID) or coupled to MS (GC–MS),
e.g., with electron ionization (GC–El–MS) or with negative–ion che-
mical ionization (GC–NCI–MS), as well as liquid chromatography (LC),
e.g., with diode-array detection (HPLC–DAD) or coupled to MS (LC–MS
or LC–MS/MS), e.g., liquid chromatography–triple quadrupole–mass
spectrometry (LC–QqQ–MS/MS) or liquid chromatography–quadrupole
time of flight–mass spectrometry (LC–QTOF–MS). (Crichton et al.,
2015; Kintz et al., 2017b; reviewed by Moosmann and Auwäter, 2018).
Recently, an ultra-assisted low-density solvent dispersive liquid-liquid
microextraction (US-LDS-DLLME) plus gas chromatography–triple
quadrupole mass spectrometry (GC-QQQ-MS) (Meng et al., 2017), and a
non–aqueous capillary electrophoresis (NACE)–tandem mass spectro-
metry (Švidrnoch et al., 2018) have been developed and used for the
identification of DBZD in urine and serum, respectively.
11. Conclusions
Novel benzodiazepines used as NPS are an example of designer
drugs consisting of derivatives of prescription medications or their
metabolites legally used in psychiatry and neurology. Although these
drugs produce effects similar to registered benzodiazepines, they could
be very harmful, as they can produce marked cognitive and motor
impairment after acute use, which may be a causal factor of traffic
accidents. Secondly, as in the case of medical benzodiazepines, chronic
use of DBZD may lead to the development of tolerance and drug de-
pendence. Finally, the pattern of abuse involving the use of DBZDs
poses a threat of serious interaction with alcohol and other CNS de-
pressants, leading to the hyper-additive accumulation of their effects,
and a greater risk of death due to respiratory depression.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
Supported by grants from the National Science Centre (NCN,
Cracow, Poland (2014/13/B/NZ7/02237)) and Medical University of
Łódź, Łódź, Poland (503/3-011-01/503-31-002).

14
J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16

Table 3 publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/
Oral doses and duration of action of designer benzodiazepines (TripSit file/616781/misuse_of_drugs_act_circular_008_2017.pdf.
Advisory Council on the Misuse of Drugs, 2011. Phenazepam Advice. Available from:
FactSheets). http://www.homeoffice.gov.uk/publications/agencies-public-bodies/acmd1/acmd-
Compound Dose (oral) Duration of action advice-phenazepam?view=Binary. Accessed October 12, 2018.
Ali, A., Jerry, J.M., Khawam, E.A., 2015. Delirium induced by a new synthetic legal in-
toxicating drug: phenazepam. Psychosomatics 56 (4), 414–418. https://doi.org/10.
Adinazolam Light 5-15 mg Onset 10-25 min 1016/j.psym.2014.05.018.
Common 15-30 mg Duration 2-5 h Ameline, A., Richeval, C., Gaulier, J.-M., Raul, J.-S., Kintz, P., 2018. Detection of the
Strong 30-50 mg+ After effects 1-16 h designer benzodiazepine flunitrazolam in urine and preliminary data on its meta-
Bromazolam Light 0.5-1 mg Onset 15-45 min bolism. Drug Test. Anal. https://doi.org/10.1002/dta.2480.
Common 1-3 mg Duration 5-8 h Anderson, M., Kjeligren, A., 2017. The slippery slope of flubromazolam: experiences of a
Strong 3-5 mg+ After effects 1-12 h novel psychoactive benzodiazepine as discussed on a Swedish online forum. Nordic
Studies Alc. Drugs 34 (3), 217–229. https://doi.org/10.1177/1455072517706304.
Clonazolam Threshold 50-75 μg Onset 10-30 min
Arens, A.M., van Wijk, X.M., Vo, K.T., Lynch, K.L., Wu, A.H., Smollin, C.G., 2016. Adverse
Light 75-200 μg Duration 6-10 h effects from counterfeit alprazolam tablets. JAMA Intern. Med. 176 (10), 1554–1555.
Common 200-400 μg After effects 1-12 h https://doi.org/10.1001/jamainternmed.2016.4306.
Heavy 500-1000 μg Bäckberg, M., Pettersson Bergstrand, M., Beck, O., Helander, A., 2018. Occurrence and
Desmethyl- Light 0.5-1 mg Onset 40-60 min time course of NPS benzodiazepines in Sweden - results from intoxication cases in the
flunitrazepam Common 1-2 mg Duration 5-10 h STRIDA project. Clin. Toxicol. Phila. (Phila) https://doi.org/ 10.1080/
Strong 2-4 mg+ After effects 2-12 h 15563650.2018.1506130.
Diclazepam Light 0.25-1 mg Onset 15-90 min Bailey, K., Richards-Waugh, L., Clay, D., Gebhardt, M., Mahmoud, H., Kraner, J.C., 2010.
Fatality involving the ingestion of phenazepam and poppy seed tea. J. Anal. Toxicol.
Common 1-2 mg Duration 8-12 h
34 (8), 527–532.
Strong > 2 mg After effects 1-24 h Benerjee, D., 2018. Etizolam withdrawal catatonia: the first case report. Asian J.
Etizolam Light 0.5-1 mg Onset 10-40 min Psychiatr. 37, 32–33. https://doi.org/10.1016/j.ajp.2018.07.019.
Common 1-2 mg Duration 5-8 h Benesch, M.G.K., Iqbal, S.J., 2018. Novel psychoactive substances: overdose of 3-fluor-
Strong 2-4 mg+ After effects 6-24 h ophenmetrazine (3-FPM) and etizolam in a 33-year-old man. BMJ Case Rep.
Deschloroetizolam Light 2-4 mg Onset 1-5 min 2018https://doi.org/10.1136/bcr-2018-224995. pii: bcr-2018-224995.
Common 4-6 mg Duration 8-10 h Bertol, E., Di Milia, M.G., Fioravanti, A., Mari, F., Palumbo, D., Pascali, J.P., Vaiano, F.,
2018. Proactive drugs in DFSA cases: toxicological findings in an eight-years study.
Strong 6-12 mg After effects 1-8 h
Forensic Sci. Int. 291, 207–215. https://doi.org/10.1016/j.forsciint.2018.08.032.
Flualprazolam Light 0.125-0.25 mg Onset 10-30 min
Carpenter, J.E., Murray, B.P., Dunkley, C., Kazzi, Z.N., Gittinger, M.H., 2018. Designer
Common 0.25-0.5 mg Duration 6-14 h benzodiazepines: a report of exposures recorded in the National Poison Data System,
Strong 0.5-1 mg After effects 1-36 h 2014–2017. Clin. Toxicol. Phila. (Phila). https://doi.org/10.1080/15563650.2018.
Heavy 1-2 mg 1510502.
Flubromazepam Light 2-4 mg Onset 15-90 min Corkery, J.M., Schifano, F., Ghodse, A.H., 2012. Phenazepam abuse in the UK: an
Common 4-8 mg Duration 12-18 h emerging problem causing serious adverse health problems, including death. Hum.
Strong 8-12 mg+ After effects > 36 h Psychopharmacol. 27 (3), 254–261. https://doi.org/10.1002/hup.2222.
Couch, R.A., Madhavaram, H., 2012. Phenazepam and cannabinomimetics sold as herbal
Flubromazolam Threshold 80 μg Onset 20-45 min
highs in New Zealand. Drug Test. Anal. 4 (6), 409–414.
Light 100-200 μg Duration 3-6 h Crichton, M.L., Shenton, C.F., Drummond, G., Beer, L.J., Seetohul, L.N., Maskell, P.D.,
Common 200-400 μg After effects 1-14 h 2015. Analysis of phenazepam and 3-hydroxyphenazepam in post-mortem fluids and
Strong 400-600 μg+ tissues. Drug Test. Anal. 7 (10), 926–936. https://doi.org/10.1002/dta.1790.
Fluclotizolam Light 0.25 mg Onset 10-30 min Dargan, P.I., Davies, S., Puchnarewicz, M., Johnston, A., Wood, D.M., 2013. First reported
Common 0.25-0.5 mg Duration 6-14 h case in the UK of acute prolonged neuropsychiatric toxicity associated with analy-
Strong 0.5-0.75 mg After effects 1-36 h tically confirmed recreational use of phenazepam. Eur. J. Clin. Pharmacol. 69 (3),
Heavy 0.75 mg+ 361–363. https://doi.org/10.1007/s00228-012-1361-z.
Domingo, O., Roider, G., Stöver, A., Graw, M., Musshoff, F., Sachs, H., Bicker, W., 2017.
Flunitrazolam Threshold 30-40 μg Onset 10-30 min
Mitragynine concentrations in two fatalities. Forensic Sci. Int. 271, e1–e7. https://
Light 40-80 μg Duration 4-5 h doi.org/10.1016/j.forsciint. 2016.12.020.
Common 80-150 μg After effects 1-16 h Drummer, O.H., Kourtis, I., Beyer, J., Tayler, P., Boorman, M., Gerostamoulos, D., 2012.
Strong 150-300 μg The prevalence of drugs in injured drivers. Forensic Sci. Int. 215 (1–3), 14–17.
Metizolam Light 1-2 mg Onset 30-90 min https://doi.org/10.1016/j.forsciint.2011.01.040.
Common 2-4 mg Duration 5-8 h El Balkhi, S., Chaslot, M., Picard, N., Dulaurent, S., Delage, M., Mathieu, O., Saint-
Strong 4-6 mg After effects 10-30 h Marcoux, F., 2017. Characterization and identification of eight designer benzodia-
zepine metabolites by incubation with human liver microsomes and analysis by a
Heavy > 6 mg
triple quadrupole mass spectrometer. Int. J. Legal Med. 131 (4), 979–988. https://
Meclonazepam Common 3-6 mg Onset 20-60 min
doi.org/10.1007/s00414-017-1541-6.
Heavy 6-12 mg Duration 9-15 hours EMCDDA, 2016. European Drug Report. Trends and Developments. Accessed 29
After effects 1-6 hrs November 2018. http://www.emcdda.europa.eu/system/files/publications/2637/
Nimetazepam Light 2.5-5 mg Onset 15-30 min TDAT16001ENN.pdf.
Common 5-10 mg Duration 5-8 h EMCDDA, 2018a. Perspective on Drugs. The Misuse of Benzodiazepines Among High-risk
Strong 10-20 mg+ After effects 12-24 h Opioid Users in Europe. Accessed October 9, 2018. http://www.emcdda.europa.eu/
Nitrazolam Light 0.5-1 mg Onset 15-30 min system/files/publications/2733/Misuse%20of%20benzos_POD2015.pdf.
EMCDDA, 2018b. European Drug Report. Trends and Developments, 2018. Accessed
Common 1-2 mg Duration 5-10 h
October 9, 2018. http://www.emcdda.europa.eu/system/files/publications/8585/
Strong 2-3 mg+ After effects 2-24 h 20181816_TDAT18001ENN_PDF.pdf.
Phenazepam Light 0.5-1 mg Onset 15-60 min Ghazi, M.A., Mohmand, M., 2017. Co-occurring addition of synthetic benzodiazepine
Common 1-2 mg Duration > 18 h clonazolam and propylhexedrine presenting as acute brief psychosis. J. Addict. Med.
Heavy 2-4 mg After effects > 36 h Ther. Sci. 5 (2), 1037.
3-Hydroxyphenazepam Light 0.5-1 mg Onset 30-90 min Gjerde, H., Christophersen, A.S., Normann, P.T., Mørland, J., 2011. Toxicological in-
Common 1-2 mg Duration 10-24 h vestigations of drivers killed in road traffic accidents in Norway during 2006-2008.
Forensic Sci. Int. 212 (1–3), 102–109. https://doi.org/10.1016/j.forsciint.2011.05.
Heavy 2-4 mg After effects 2-24 h
021.
Pyrazolam Light 1-2 mg Onset 10-15 min
Griffin 3rd, C.E., Kaye, A.M., Bueno, F.R., Kaye, A.D., 2013. Benzodiazepine pharma-
Common 2-3 mg Duration 5-8 h cology and central nervous system-mediated effects. Ochsner J. 13 (2), 214–223.
Strong 3-4 mg After effects 1-12 h Gupta, S., Garg, B., 2014. A case of etizolam dependence. Indian J. Pharmacol. 46 (6),
655–656. https://doi.org/10.4103/0253-7613.144943.
Høiseth, G., Tuv, S.S., Karinen, R., 2016. Blood concentrations of new designer benzo-
diazepines in forensic cases. Forensic Sci. Int. 268, 35–38. https://doi.org/10.1016/j.
References forsciint.2016.09.006.
Huppertz, L.M., Moosmann, B., Auwärter, V., 2018. Flubromazolam - Basic pharmaco-
Abouchedid, R., Gilks, T., Dargan, P.I., Archer, J.R.H., Wood, D.M., 2018. Assessment of kinetic evaluation of a highly potent designer benzodiazepine. Drug Test. Anal. 10
the availability, cost, and motivations for use over time of the new psychoactive (1), 206–211. https://doi.org/10.1002/dta.2203.
substances – benzodiazepines diclazepam, flubromazepam, and pyrazolam – in the Jones, J.D., Mogali, S., Comer, S.D., 2012. Polydrug abuse: a review of opioid and ben-
UK. J. Med. Toxicol. 14 (2), 134–143. https://doi.org/10.1007/s13181-018-0659-3. zodiazepine combination use. Drug Alcohol Depend. 125 (1–2), 8–18. https://doi.
Advice Council on Misuse of Drugs, 2017. Circular 008/2017: a Change to the Misuse of org/10.1016/j.drugalcdep. 2012.07.004.
Drugs Act 1971 to Control U-47,700, Twelve Methylphenidate Related Substances Karinen, R., Tuv, S.S., Rogde, S., Peres, M.D., Johansen, U., Frost, J., Vindenes, V.,
and Sixteen ‘designer’ Benzodiazepines. Accessed 29 November 2018. https://assets. Øiestad, Å.M., 2014. Lethal poisonings with AH-7921 in combination with other

15
J.B. Zawilska and J. Wojcieszak
substances. Forensic Sci. Int. 244, e21–24. https://doi.org/10.1016/j.forsciint.2014.
08.013.
Katselou, M., Papoutsis, I., Nikolaou, P., Spiliopoulou, C., Athanaselis, S., 2017.
Metabolites replace the parent drug in the drug arena. The cases of fonazepam and
nifoxipam. Forensic Toxicol. 35 (1), 1–10. https://doi.org/10.1007/s11419-016-
0338-5.
Kerrigan, S., Mellon, M.B., Hinners, P., 2013. Detection of phenazepam in impaired
driving. J. Anal. Toxicol. 37 (8), 605–610. https://doi.org/10.1093/jat/bkt075.
Kintz, P., Richeval, C., Jamey, C., Ameline, A., Allorge, D., Gaulier, J.M., Raul, J.S.,
2017a. Detection of the designer benzodiazepine metizolam in urine and preliminary
data on its metabolism. Drug Test. Anal. 9 (7), 1026–1033. https://doi.org/10.1002/
dta.2099.
Kintz, P., Jamey, C., Ameline, A., Richeval, C., Raul, J.S., 2017b. Characterization of
metizolam, a designer benzodiazepine, in alternative biological specimens. Toxicol.
Anal. Clin. 29, 57–63 https://dx.doi.org/1016/j.toxac.2016.09.04.
Koch, K., Auwärter, V., Hermanns‐Clausen, M., Wilde, M., Neukamm, M.A., 2018. Mixed
intoxication by the synthetic opioid U‐47700 and the benzodiazepine flubromazepam
with lethal outcome: pharmacokinetic data. Drug Test. Anal https://doi.org/
10.1002/dta.2391.
Kriikku, P., Wilhelm, L., Rintatalo, J., Hurme, J., Kramer, J., Ojanperä, I., 2012.
Phenazepam abuse in Finland: findings from apprehended drivers, post-mortem cases
and police confiscations. Forensic Sci. Int. 220 (1–3), 111–117. https://doi.org/10.
1016/j.forsciint.2012.02.006.
Liveri, K., Constantinou, M.A., Afxentiou, M., Kanari, P., 2016. A fatal intoxication related
to MDPV and pentedrone combined with antipsychotic and antidepressant substances
in Cyprus. Forensic Sci. Int. 265, 160–165. https://doi.org/10.1016/j.forsciint.2016.
02.017.
Lomas, E.C., Maskell, P.D., 2015. Phenazepam: more information coming in from the
cold. J. Forensic Leg. Med. 36, 61–62. https://doi.org/10.1016/j.jflm.2015.08.017.
Łukasik-Głębocka, M., Sommerfeld, K., Teżyk, A., Zielińska-Psuja, B., Panieński, P., Żaba,
C., 2016. Flubromazolam – A new life-threatening designer benzodiazepine. Clin.
Toxicol. Phila. (Phila) 54 (1), 66–68. https://doi.org/10.3109/15563650.2015.
1112907.
Manchester, K.R., Lomas, E.C., Waters, L., Dempsey, F.C., Maskell, P.D., 2018. The
emergence of new psychoactive substance (NPS) benzodiazepines: a review. Drug
Test. Anal. 10 (2), 392–393. https://doi.org/10.1002/dta.2349.
Maskell, P.D., Paoli, G.D., Seetohul, L.N., Pounder, D.J., 2011. Phenazepam is currently
being misused in the UK. BMJ 343, d4207. https://doi.org/10.1136/bmj.d4207.
Meng, L., Zhu, B., Zheng, K., Fu, S., 2017. Ultrasound-assisted low-density solvent dis-
persive liquid-liquid microextraction for the determination of 4 designer benzodia-
zepines in urine samples by gas chromatography-triple quadrupole mass spectro-
metry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1053, 9–15. https://doi.
org/10.1016/j.jchromb.2017.04.008.
Meyer, M.R., Pettersson Bergstrand, M., Helander, A., Beck, O., 2016. Identification of
main human urinary metabolites of the designer nitrobenzodiazepines clonazolam,
meclonazepam, and nifoxipam by nano-liquid chromatography-high-resolution mass
spectrometry for drug testing purposes. Anal. Bioanal. Chem. 408 (13), 3571–3591.
https://doi.org/10.1007/s00216-016-9439-6.
Ministry of Justice, Canada, 2018. Benzodiazepines and Other Targeted Substances
Regulations. Accessed January 3, 2019. https://laws-lois.justice.gc.ca/PDF/SOR-
2000-217.pdf.
Moosmann, B., Auwäter, V., 2018. Designer benzodiazepines: another class of new psy-
choactive substances. Handb. Exp. Pharmacol. https://doi.org/10.1007/164_2018_
154.
Moosmann, B., Huppertz, L.M., Hutter, M., Buchwald, A., Ferlaino, S., Auwärter, V.,
2013. Detection and identification of the designer benzodiazepine flubromazepam
and preliminary data on its metabolism and pharmacokinetics. J. Mass Spectrom. 48
(11), 1150–1159. https://doi.org/10.1002/jms.3279.
Moosmann, B., Bisel, P., Auwärter, V., 2014. Characterization of the designer benzodia-
zepine diclazepam and preliminary data on its metabolism and pharmacokinetics.
Drug Test. Anal. 6 (7–8), 757–763. https://doi.org/10.1002/dta.1628.
Moosmann, B., Bisel, P., Franz, F., Huppertz, L.M., Auwärter, V., 2016. Characterization
and in vitro phase I microsomal metabolism of designer benzodiazepines - an update
comprising adinazolam, cloniprazepam, fonazepam, 3-hydroxyphenazepam, meti-
zolam and nitrazolam. J. Mass Spectrom. 51 (11), 1080–1089. https://doi.org/10.
1002/jms.3840.
Moosmann, B., Bisel, P., Westphal, F., Wilde, M., Kempf, J., Angerer, V., Auwärter, V.,
2018. Characterization and in vitro phase I microsomal metabolism of designer
benzodiazepines - an update comprising flunitrazolam, norflurazepam and 4’-chlor-
odiazepam (Ro5-4864). Drug Test. Anal https://doi.org. 10.1002/dta.2561.
Mortelé, O., Vervliet, P., Gys, C., Degreef, M., Cuykx, M., Maudens, K., Covaci, A., van
Nuijs, A.L.N., Lai, F.Y., 2018. In vitro Phase I and Phase II metabolism of the new
designer benzodiazepine cloniprazepam using liquid chromatography coupled to
quadrupole time-of-flight mass spectrometry. J. Pharm. Biomed. Anal. 153, 158–167.
https://doi.org/10.1016/j.jpba.2018.02.032.
Noble, C., Mardal, M., Bjerre Holm, N., Stybe Johansen, S., Linnet, K., 2017. In vitro
studies on flubromazolam metabolism and detection of its metabolites in authentic
forensic samples. Drug Test. Anal. 9 (8), 1182–1191 https://doi.org/ 10.1002/
dta.2146.
O’Connell, C.W., Sadler, C.A., Tolia, V.M., Ly, B.T., Saitman, A.M., Fitzgerald, R.L., 2015.
Overdose of etizolam: the abuse and rise of a benzodiazepine analog. Ann. Emerg.
Med. 65 (4), 465–466. https://doi.org/10.1016/j.annemergmed.2014.12.019.
Papsun, D., Krywanczyk, A., Vose, J.C., Bundock, E.A., Logan, B.K., 2016. Analysis of MT-
45, a novel synthetic opioid, in human whole blood by LC-MS-MS and its identifi-
cation in a drug-related death. J. Anal. Toxicol. 40 (4), 313–317. https://doi.org/10.
16
Neurotoxicology 73 (2019) 8–16
1093/jat/bkw012.
Partridge, E., Trobbiani, S., Stockham, P., Charlwood, C., Kostakis, C., 2018. A case study
involving U-47700, diclazepam and flubromazepam–application of retrospective
analysis of HRMS data. J. Anal. Toxicol. 42 (9), 655–660. https://doi.org/10.1093/
jat/bky039.
Pettersson Bergstrand, M., Helander, A., Beck, O., 2016. Development and application of
a multi-component LC-MS/MS method for determination of designer benzodiazepines
in urine. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1035, 104–110. https://
doi.org/10.1016/j.jchromb.2016.08.047.
Pettersson Bergstrand, M., Helander, A., Hansson, T., Beck, O., 2017. Detectability of
designer benzodiazepines in CEDIA, EMIT II Plus, HEIA, and KIMS II im-
munochemical screening assays. Drug Test. Anal. 9 (4), 640–645. https://doi.org/10.
1002/dta.2003.
Pettersson Bergstrand, M., Meyer, M.R., Beck, O., Helander, A., 2018. Human urinary
metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam
studied by liquid chromatography-high resolution mass spectrometry. Drug Test.
Anal. 10 (3), 496–506. https://doi.org/10.1002/dta.2243.
Pettersson Bergstrand, M., Richter, L.H.J., Maurer, H.H., Wagmann, L., Meyer, M.R.,
2019. In vitro glucuronidation of designer benzodiazepines by human UDP-glucur-
onyltransferases. Drug Test. Anal. 11 (1), 45–50. https://doi.org/10.1002/dta.2463.
Pope, J.D., Choy, K.W., Drummer, O.H., Schneider, H.G., 2018. Novel benzodiazepines
(clonazolam and flubromazolam) identified in candy-like pills. J. Appl. Lab. Med. 3
(1), 48–55. https://doi.org/10.1373/jalm.2017.025387.
Shearer, K., Bryce, C., Parsons, M., Torrance, H., 2015. Phenazepam: a review of medico-
legal deaths in South Scotland between 2010 and 2014. Forensic Sci. Int. 254,
197–204. https://doi.org/10.1016/j.forsciint.2015.07.033.
Stephenson, J.B., Golz, D.E., Brasher, M.J., 2013. Phenazepam and its effects on driving.
J. Anal. Toxicol. 37 (1), 25–29. https://doi.org/10.1093/jat/bks080.
Švidrnoch, M., Boráňová, B., Tomková, J., Ondra, P., Maier, V., 2018. Simultaneous de-
termination of designer benzodiazepines in human serum using non-aqueous capil-
lary electrophoresis - Tandem mass spectrometry with successive multiple ionic -
Polymer layer coated capillary. Talanta 176, 69–76 https://doi.org/ 10.1016/j.ta-
lanta.2017.08.010.
Tan, K.R., Rudolph, U., Lüscher, C., 2011. Hooked on benzodiazepines: GABAA receptor
subtypes and addiction. Trends Neurosci. 34 (4), 188–197 https://doi.org/ 10.1016/
j.tins.2011.01.004.
Tanaka, N., Kinoshita, H., Nishiguchi, M., Jamal, M., Kumihashi, M., Takahashi, M.,
Nishio, H., Ameno, K., 2011a. An autopsy case of multiple psychotropic drug poi-
soning. Soud. Lek. 56 (3), 38–39.
Tanaka, N., Kinoshita, H., Jamal, M., Ohkubo, E., Kumihashi, M., Ameno, K., 2011b. A
case of drowning whilst under the influence of brotizolam, flunitrazepam and
ethanol. Soud. Lek. 56 (1), 5–6.
TripSit FactSheets, http://drugs.tripsit.me. Accessed 11 October 2018.
UNODC, 2017a. Non-medical Use of Benzodiazepines: a Growing Threat to Public
Health? Global Smart Update. pp. 18. https://www.unodc.org/documents/scientific/
Global_SMART_Update_2017_Vol_18.pdf.
UNODC, 2017b. The Challenge of Synthetic Drugs in East and South-East Asia. Trends and
Patterns of Amphetamine-type Stimulants and New Psychoactive Substances.
Accessed 9 October 2018.. https://www.unodc.org/documents/scientific/Trends_
and_Patterns_of_ATS_and_NPS_2017.pdf.
UNODC, 2017c. April 2017 – UNODC: Several Countries Place Benzodiazepine
Derivatives Under National Control. Accessed 9 October 2018.. https://www.unodc.
org/LSS/Announcement/Details/065118d5-b238-48d8-9d7f-b95fb5d11411.
UNODC, 2018. June 2018 – Scotland: Number of Deaths Involving Benzodiazepine-type
NPS Etizolam and Diclazepam Increase Sharply in 2016. Accessed October 9, 2018.
https://www.unodc.org/LSS/Announcement/Details/0ec8745d-da7e-4594-a47f-
c47005a8ae96.
US Department of Justice Drug Enforcement Administration, 2009. Blotter acid mimic
(actually containing phenazepam) in North Carolina. Microgram Bulletin 42, 94.
Valen, A., Bogstrand, S.T., Vindenes, V., Gjerde, H., 2017. Toxicological findings in sus-
pected drug-impaired drivers in Norway - Trends during 1990-2015. Forensic Sci. Int.
280, 15–24. https://doi.org/10.1016/j.forsciint.2017.09.010.
Vikingsson, S., Wohlfarth, A., Andersson, M., Gréen, H., Roman, M., Josefsson, M.,
Kugelberg, F.C., Kronstrand, R., 2017. Identifying metabolites of meclonazepam by
high-resolution mass spectrometry using human liver microsomes, hepatocytes, a
mouse model, and authentic urine samples. AAPS J. 19 (3), 736–742. https://doi.
org/10.1208/s12248-016-0040-x.
Wakakura, M., Tsubouchi, T., Inouye, J., 2004. Etizolam and benzodiazepine induced
blepharospasm. J. Neurol. Neurosurg. Psychiatry. 75 (3), 506–507.
WHO, 2015. Phenazepam. Pre-review Report. Agenda Item 5.8. Expert Committee on
Drug Dependence Thirty-seventh Meeting. Geneva, 16-20 November 2015. . http://
www.who.int/medicines/access/controlled-substances/5.8_Phenazepam_PreRev.pdf.
WHO, 2017. ETIZOLAM Critical Review Report. Agenda Item 4.13. Expert Committee on
Drug DependenceThirty-ninth Meeting Geneva, 6-10 November. 2017. http://www.
who.int/medicines/access/controlled-substances/CriticalReview_Etizolam.pdf.
Wohlfarth, A., Vikingsson, S., Roman, M., Andersson, M., Kugelberg, F.C., Green, H.,
Kronstrand, R., 2017. Looking at flubromazolam metabolism from four different
angles: metabolite profiling in human liver microsomes, human hepatocytes, mice
and authentic human urine samples with liquid chromatography high-resolution
mass spectrometry. Forensic Sci. Int. 274, 55–63. https://doi.org/10.1016/j.forsciint.
2016.10.021.
Xiang, P., Shen, M., Drummer, O.H., 2015. Review: drug concentrations in hair and their
relevance in drug facilitated crimes. J. Forensic Leg. Med. 36, 126–135. https://doi.
org/10.1016/j.jflm.2015.09.009.