Forensic Science International 349 (2023) 111775

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Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

Review article

Non-fentanyl new synthetic opioids – An update

Jolanta B. Zawilska a, *, Piotr Adamowicz b, Marta Kurpeta a, Jakub Wojcieszak a
a
Department of Pharmacodynamics, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland
b
Department of Forensic Toxicology, Institute of Forensic Research, Westerplatte 9, 31-033 Krakow, Poland

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

Keywords: Background: New synthetic opioids (NSO) constitute one of the fastest-growing group of New Psychoactive
New synthetic opioids (NSO) Substances, which emerged on the illicit drug marker in the second half of 2000’s. The most popular and the
Non-fentanyl opioids largest NSO subgroup are high potency fentanyl and its analogs. Subsequent to core-structure scheduling of
U-compounds
fentanyl-related substances many opioids with different chemical structures are now emerging on the illicit drug
MT-45
market, rendering the landscape highly complex and dynamic.
Analytical Toxicology, Treatment of opioid
overdose Methods: PubMed, Scopus and Google Scholar were searched for appropriate articles up to December 2022.
2-Benzylbenzimidazoles (nitazenes) Moreover, a search for reports was conducted on Institutional websites to identify documentation published by
Brorphine World Health Organization, United Nations Office on Drugs and Crime, United States Drug Enforcement
Administration, and European Monitoring Centre for Drugs and Drug Addiction. Only articles or reports written
in English were selected.
Results: Non-fentanyl derived synthetic opioids, i.e., 2-benzylbenzimidazoles (nitazenes), brorphine, U-com­
pounds, AH-7921, MT-45 and related compounds are characterized, describing them in terms of available forms,
pharmacology, metabolism as well as their toxic effects. Sample procedures and analytical techniques available
for detection and quantification of these compounds in biological matrices are also presented. Finally, as
overdoses involving highly potent NSO may be difficult to reverse, the effectiveness of naloxone as a rescue agent
in NSO overdose is discussed.
Conclusions: Current review presents key information on non-fentanyl derived NSO. Access to upto-date data on
substances of abuse is of great importance for clinicians, public health authorities and professionals performing
analyses of biological samples.

1. Introduction semisynthetic substances in pure form, mixture or preparation that can

be categorized using at least one of the following criteria:
Over the last two decades we have witnessed a major transformation
of the illicit drug market induced by a rapid increase in appearance and
use of New Psychoactive Substances (NPS) with chemical structures or
pharmacological profiles that are similar to traditional drugs of abuse.
Generally, NPS are designed to be perceived as legal substitutes of
scheduled compounds. The illicit drug market is highly dynamic. Once a
compound becomes prohibited, new unscheduled molecules quickly
appear, enlarging the library of psychoactive compounds [1]. NPS are
considered as a global phenomenon, with 1127 substances reported to
the United Nations Office on Drugs and Crime (UNODC) Early Warning
Advisory between 2009 and 2021 [2].
Very recently, due to the constantly growing number of NPS and
their diversity, the definition of this group of psychoactive compounds
has been extended [3]. NPS are defined “as natural, synthetic or
• A substance that has been discovered or synthesized for the first time
since the mid-2000 s and is being ingested, regardless of degree of
psychoactive effect (e.g., MDMB-4en-PINACA and N-pyrrolidino
etonitazene);
• A substance that was previously discovered, synthesized or reported
(e.g., patents, literature and publications) but has been observed in
the current illicit drug supply or identified in toxicological samples
for the first time in more than 10 years (e.g., 2-methyl AP-237 and
isotonitazene);
• A substance that since the mid-2000 s has been used in a novel way
or differing manner from its originally intended use (i.e., different
dosage form or amount to produce effects and different preparation)
(e.g., loperamide and xylazine);

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

https://doi.org/10.1016/j.forsciint.2023.111775
Received 31 December 2022; Received in revised form 22 June 2023; Accepted 26 June 2023
Available online 28 June 2023
0379-0738/© 2023 Elsevier B.V. All rights reserved.
J.B. Zawilska et al.
• A substance that previously was not well described or studied but
now presents significant challenges or threats due to an altered
toxicological effect profile as a result of increased use or popularity
(e.g., mitragynine) and/or
• A substance that is not controlled by the United Nations drug con­
ventions (1961 Single Convention on Narcotic Drugs or the 1971
Convention on Psychotropic Substances) but that may pose a public
health threat comparable to that posed by substances listed in these
conventions (e.g., quetiapine and O-desmethyl tramadol)”.
In general, NPS are categorized according to three basic criteria: (1)
their origin (plant-based, semi synthetic, synthetic), (2) chemical
structure, and (3) psychotropic effects [2]. Based on exerted psycho­
tropic effects and behavioral changes in individuals being under influ­
ence of specific substances, NPS can be divided into the following main
categories [4,5]:
• Synthetic cannabinoids – synthetic agonists of cannabinoid receptors
mimicking effects of Δ9-tetrahydrocannabinol (Δ9-THC);
• Psychostimulants – substitutes for amphetamine, cocaine or 3,4-
methylenodioxymethamphetamine (MDMA);
• Hallucinogens – exerting similar effects to lysergic acid diethylamide
(LSD) or phencyclidine;
• Depressants – synthetic opioids (copying effects of morphine/heroin)
and designer, non-pharmaceutical benzodiazepines.
Of these, the group of new synthetic opioids (NSO) has been one of
the fastest growing NPS subclasses in recent years, with 73 compounds
detected in Europe between 2009 and 2021 [6]. As emphasized in the
recent World Drug Report “Opioid NPS are the potentially most harmful
group of NPS and, in contrast to the general decline in the number of
NPS, the number of opioid NPS has continued to grow. The number of
opioid NPS found on markets worldwide grew from just one substance in
2009, to 14 in 2015, 56 in 2019 and 87 in 2020, by which time synthetic
opioids had become the third most numerous group of NPS….” [2].
NSO are basically categorized into fentanyl and its analogs (fentan­
yls) and non-fentanyl structured compounds, i.e., diphenylethylpiper­
azines (e.g., MT-45), cinnamylpiperazines (e.g., 2-methyl AP-237),
cyclohexylbenzamides (U-47700 and its analogs, AH-7921), 2-benzyl­
benzimidazoles (nitazenes), benzimidazolones (e.g., brorphine), atyp­
ical opioid agonists (e.g., mitragynine) and others [3]. By analogy to
traditional μ-opioid receptors (MOR) agonists, the use of NSO can result
in sedation, euphoria, and respiratory depression that can progress to
coma and death.
Most NSO are not “new” compounds. In fact, plenty of them were
synthesized several decades ago by medical chemistry researches and
pharmaceutical industry in their search for novel non-addictive opioid
analgesics with reduced ability to evoke harmful side-effects; some of
them were created to serve for research purposes exclusively. However,
due to undesirable profile of pharmacotoxicological actions these drugs
were never advanced to clinical trials. By analogy to other groups of
NPS, data on NSO have been mined from scientific literature or old
patents. Notwithstanding, due to the low cost of starting materials and
equipment required for clandestine laboratory production, relatively
simple three-four step synthesis and enormous profit potential, NSO are
establishing a strong position on the illegal drug market as stand-alone
products, adulterants in heroin (‘fake heroin’), or constituents of coun­
terfeit prescription medications, e.g., hydromorphone, oxycodone, or
falsified Xanax [7–9]. The most popular, particularly between 2012 and
2018, and the largest NSO group are high potency fentanyls. They are
considered by many the greatest concern due to their prevalence, po­
tency and diversity [10]. Fentanyls were mainly synthesized in China
and shipped all over the word, but most of all to the US. After
core-structure scheduling of fentanyls in the US in 2018, and a similar
action combined with a ban on fentanyl precursors N-phenethyl-4-pi­
peridinone and 4-anilino-N-phenethylpiperidine taken by China in 2019
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Forensic Science International 349 (2023) 111775
[11,12], the number of new fentanyl analogs started to decrease dras­
tically from 2018 onwards [13,14]. NSO with chemical structures
different from fentanyl quickly replaced the scheduled ones. Among the
first representatives of illicit non-fentantyl opioids were AH-7921 in
2012 and MT-45 in 2013. From 2014, several compounds of the ‘U se­
ries’ became increasingly detected, with U-47700 being one of the most
prevalent drug. Few years later, new potent opioids, dominated by
2-benzylbenzimidazoles and benzimidazolones, appeared on the recre­
ational drug market [15].
As fentanyl and its analogs have already been the subject of a number
of detailed reviews (e.g., [9,16–18]), these compounds are not discussed
in the present paper. Instead, we focused on a new ‘wave’ of synthetic
opioids with a special emphasis given to the recent ones, nitazenes and
brorphine.
2. Methodology
PubMed, Scopus, Web of Science and Google Scholar data bases were
searched for relevant articles (up to December 2022). The following
keywords were used alone or in combination: ‘new psychoactive sub­
stances’, ‘new synthetic opioids’, ‘fentanyl’, ‘fentanyl analogs’, ‘non-
fentanyl synthetic opioids’, ‘MT-45’, ‘AH-7921’, ‘U-compounds”, ‘2-
benzylbenzimidazoles’, ‘nitazenes’, ‘brorphine’, ‘intoxication’, ‘death’,
‘fatal poisoning’, ‘analytical methods’, ‘naloxone’, ‘opioid overdose
treatment’. Further studies were retrieved by a hand search through the
reference lists of the selected articles. A search for reports was also
conducted on institutional websites to identify documentation published
by international agencies or institutions such as World Health Organi­
zation (WHO), United Nations Office on Drugs and Crime (UNODC),
United States Drug Enforcement Administration (DEA), and European
Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Only ar­
ticles or reports written in English were selected.
3. Characterization of the main classes of non-fentanyl NSO
3.1. Diphenylethylpiperazines
MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) (Fig. 1), also
known as IC-6, CDEP and NSC299236, was originally developed in the
1970’s by the Dainippon Pharmaceutical Co, Ltd. in Japan as a potential
analgesic substitute for morphine. Racemic MT-45 and its (S)-isomer are
morphine-like opioid analgesics with the (S)-isomer being more potent
than morphine in most rodent assays [19]. The compound was first
detected in seized samples in Sweden and Japan [20,21]. Administration
routes include nasal insufflation (‘snorting’), oral intake, smoking,
intravenous and intramuscular injection, and rectal administration [9,
20]. Clinical data from Sweden suggest MT-45 may exert ototoxic ac­
tivity [22]. Blood and serum concentration found in non-fatal cases was
6.0–157 ng/mL and 112–280 ng/mL, respectively [22]. MT-45 caused a
total of 28 deaths in Sweden within a short, nine-month period between
November 2013 and July 2014 [20]. The concentration of MT-45 in
postmortem femoral blood ranged from 6 to 1900 ng/g. In 24 of them,
MT-45 was found in combination with at least one other psychoactive
substance, including benzodiazepines, antidepressants (fluoxetine, ser­
traline, mirtazapine, and venlafaxine), antipsychotic drugs (olanzapine,
quetiapine, and levomepromazine), antiepileptic drugs (gabapentin,
lamotrigine, and carbamazepine), opioids (morphine, codeine, trama­
dol, fentanyl, and hydrocodone), ethylphenidate, methiopropamine,
2-aminoindane, amphetamine, Δ9-THC, and ethanol [20,23]. MT-45 is
metabolized by N-dealkylation, mono- and dihydroxylation, with 1,
2-diphenylethylpiperazine and 1-cyclohexylpiperazine being the main
metabolites [20]. Dainippon Pharmaceutical also synthesized MT-45
analogs: AD-1211 [24] and diphenpipenol [25].
J.B. Zawilska et al.
Fig. 1. Chemical structures of popular piperazine- and cyclohexylbenzamide-derived new synthetic opioids.
3.2. Cinnamylpiperazines
AP-237 (1-butyryl-4-cinnamylpiperazine, also known as bucinna­
zine), 2-methyl AP-237 and AP-238 (Fig. 1) are examples of
cinnamylpiperazine-type drugs with MOR agonistic activity. They
appeared on the European and American market in 2019–2020 [26].
AP-237 and AP-238 have been used in China as analgesics in cancer
patients [27]. 2-Methyl AP-237 was found to bind to MOR with appre­
ciable affinity (Ki = 12.9 nM). In in vitro [35S]GTPγS binding assay,
2-methyl AP-237 activated MOR with low efficacy and potency (EC50 =
620 nM); fentanyl and morphine were 22 and 15 times more potent,
respectively [28,29]. Information obtained from Internet forums sug­
gests that individuals abusing 2-methyl AP-237 are likely to abuse pre­
scription opioid analgesics, heroin and other NSO. 2-Methyl AP-237 is
typically administered orally and by snorting. The ‘caustic’ properties of
2-methyl AP-237 are mentioned frequently, indicating unpleasant sen­
sations associated with certain routes of administration (e.g., gastric
problems after oral ingestion or burning sensations after snorting). In the
US, at least 17 confirmed cases of fatal poisonings and several reports of
non-fatal intoxication associated with 2-methyl AP-237 were reported
[10,26,28]. AP-237 and 2-methyl AP-237 are metabolized by hydrox­
ylation, N-dealkylation and oxidation [27].
3.3. Cyclohexylbenzamides
3.3.1. AH-7921
AH-7921 (3,4-dichloro-N-(1-(dimethylamino)cyclohexylmethyl)
benzamide) (Fig. 1), known also as ‘doxylam’, ‘doxylan’ and ‘CN 2924
29 98’, was developed in mid-1970’s by Allen & Hanburys Ltd., the UK,
as a potent opioid analgesic agent. AH-7921 is an agonist of μ and κ
opioid receptors, MOR and KOR, respectively, with a moderate prefer­
ence toward MOR, and a narrow therapeutic window [9,30]. AH-7921
was firstly identified in July 2012 in the UK in samples of a product
known Doxylam. Importantly, the name Doxylam, used by Internet
suppliers/retailers selling the substance and user websites, can be easily
confused with the name of doxylamine, an over-the-counter antihista­
minic with sedative properties. Doxylamine is used in a short-term
treatment of insomnia, and in combination with decongestants and
other medications to relieve rhinitis and nasal congestion caused by a
common cold or allergy. An accidental use of AH-7921 by people who
intend to purchase medications containing doxylamine from online
vendors, might lead to serious health problems [9,31]. AH-7921 was
often used as a substitute for heroin, and also combined with psycho­
active controlled drugs, medicines, NPS (cannabinoids, synthetic cath­
inones) and alcohol [9,31]. The substance is sold in the form of capsules
and tablets. Routes of administration include nasal insufflation, sublin­
gual application, intravenous injection, a combination of insufflation
and oral consumption, or rectal administration; available doses range
from 10 to 150 mg [30]. Fifteen deaths associated with AH-7921 have
been reported to the European Early Warning System, all of them
occurred within a 10-month period between December 2012 and
September 2013 [31]. Analysis of blood samples from nine decedents in
Sweden revealed AH-7921 concentrations ranged from 0.03 to
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0.99 µg/g blood [32]. In most cases, other psychoactive compounds,
mainly benzodiazepines but also amphetamines, synthetic cathinones,
codeine, acetaminophen, and methoxetamine were detected in post­
mortem samples [9]. The two most dominant metabolites in vitro and
also identified in the urine case specimen were desmethyl- and
di-desmethyl-AH-7921 [33].
3.3.2. U-compounds/U-drugs
These substances were originally synthesized in the 1970’s and
1980’s by a pharmaceutical company Upjohn in a search for non-
addicting analgesics. Based on the absence or presence of the methy­
lene group in their chemical structure, U-compounds can be broadly
categorized into two major groups (Fig. 2):
(1) U-47700 series M compounds with high affinity for MOR and low
for KOR: U-47700, N-ethyl-U-47700, isopropyl-U-47700, 3,4-
difluoro-U-47700, U-47109, U-77891, U-47931A (bromado­
line), N-methyl-U-47931E, U-48520 and U-49900;
(2) U-50488 series K compounds with high affinity for KOR and low
for MOR: U-504884, U-51574, U-48800, U-69593 and U-62066
(spiradoline). These compounds are important research tools for
studying the role of KOR in animal models (for an excellent re­
view see [34])
U-47700 (3,4-dichloro-N-[2-(dimethyloamino)cyclohexyl]-N-meth­
ylbenzamide) was the first of the U-series that appeared on the recrea­
tional drug market beginning in 2014. U-47700 replaced scheduled AH-
7921, its structural isomer. The popularity of U-47700 increased in 2015
and 2016. Clandestine U-47700 has been advertised as heroin, an oxy­
codone substitute, and sold under street names of ’Pink’, Pinky’ or ‘pink
heroin’ (owing to synthesis impurities that color the drug powder in
light pink), ‘U4‘ or ‘Fake morphine’, and sometimes referred to as
‘synthetic cocaine’ [34–36]. More recently this drug was identified as a
constituent in a potent opioid cocktail ‘gray death’ (cited in [34]).
U-47700 is typically sold as powder, but also as tablets, liquid to use in
inhalers, nasal spray and ‘spice like’ herbal incense. Reported routes of
administration involve oral, insufflation, intravenous, rectal and inha­
lation of aerosol [36]. Accumulated pharmacological data indicate that
U-47700 is a selective MOR agonist. Although its in vitro activity is
lower than that of morphine, preclinical studies found U-47700 to be
7.5–10-fold more efficacious than morphine and about 10 times less
potent than fentanyl [37,38]. The subjective effects of U-47700, in
particular its potent euphoric effect, combined with a low price (a
common insufflation dose, between 6 and 8 mg, costs less than 0.5 USD)
and easy access, are very attractive to potential users [36,39]. The
short-lived euphoric effect of this drug creates a desire to re-dose [14].
U-47700 has been reported in case reports and/or seized materials in
several countries, including the US, the UK, Sweden, Belgium, Italy,
Czech Republic, Finland, Brazil and Australia [36,40]. There are several
reports on U-47700-related intoxications, including fatal ones. Most of
the cases involved poly-drug use; they are nicely summarized by Bau­
mann and co-workers [34]. Only a few cases have reported U-47700 as a
sole substance involved in both non-fatal intoxications and fatal drug
J.B. Zawilska et al.
Fig. 2. Chemical structures of U-compounds.
overdoses. In the middle of 2017, there was a series of opioid overdoses
in the state of Georgia, the US, evoked by use of counterfeit Percocept®
pills containing U-47700 and cyclopropylfentanyl [41]. U-47700 blood
concentrations in non-fatal intoxications ranged from 3.4 to
282.4 ng/mL and serum concentrations from 7.2 to 394 ng/mL [42]. In
non-fatal intoxications involving only U-47700, blood or serum con­
centrations ranged from 18 to 294 ng/mL [34]. In a case series of 26
deaths associated with U-47700 exposure reported from Germany the
median femoral blood concentration was 610 ng/mL (range
27–2200 ng/mL) [43]. The femoral blood concentrations for the
U-47700 fatalities, summarized by Adamowicz & Nowak [42], were in
the range 0.4 ̶ 3040 ng/mL. Studies on distribution of U-47700 in
samples taken from 58 independent overdose victims revealed high
concentration of this compound in the blood from different regions:
femoral (0.4–492 ng/mL), cardiac (>500 ng/mL), iliac (>100 ng/mL),
and subclavian (68 ng/mL). High concentrations of U-47700 were also
found in the vitreous humor (0.1–328 ng/mL), and in the brain (1 to
>600 ng/mL) [44].
U-49900 (3,4-dichloro-N-[2-(diethylamino)cyclohexyl]-N-methyl­
benzamide) is the diethyl analog of U-47700. In 2016, after U-47700
scheduling in the US, U-49900 was identified through online drug fo­
rums and NPS vendors [45]. In a short time, other U-compounds
emerged on the market, such as U-51754 in 2016, U-48800, U-47931E
(bromadoline) and U-77891 in 2017, U-504883, 3,4-methylene­
dioxy-U-47700 and isopropyl-U-47700 between 2017 and 2018. While
U-49900 succeeded U-47700, it did not replace U-47700 on the market,
possibly due to rumors stating that it failed to induce satisfactory level of
euphoria and analgesia [7].
Using [35S]GTPγS assay, potencies of eight different U-compounds:
U-47700, isopropyl-U-47700, U-49900, U-47931E, N-methyl-U-47931E,
U-51754, U-48520, and U-48800 at MOR and KOR were examined in
vitro. U-47700 showed the highest potency at MOR (EC50 = 111 nM),
whereas U-51754 was the most potent compound at KOR (EC50 =
120 nM) [46]. N-Dealkylation, hydroxylation, and their combinations
are the predominant metabolic pathways for U-compounds [47–52].
N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 were identified as
major metabolites of U-47700 in urine samples obtained from human
casework. Furthermore, the presence of these compounds was found in
blood samples obtained from human subjects exposed to U-47700, an
observation indicating that these metabolites are the major species
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formed [47–52]. N,N-didesethyl-N-desmethyl-U-49900 was identified
as the primary metabolite of U-49900 in human urine samples [49].
3.4. 2-Benzylbenzimidazoles (nitazenes)
The most recent NSO subclass to emerge and proliferate on the illicit
drug market are 2-benzylbenzimidazoles (nitazenes). First compounds
from this group were invented by the Swiss pharmaceutical company
CIBA in the mid-1950’s.
[53]. Currently, no drugs in this class are approved for medicinal use.
Between 1966 and 2003, the only 2-benzylbenzimidazole opioid that
was sporadically identified in the illicit drug supply was etonitazene, a
harmful opioid described to be 1000 times more potent than morphine
in a rodent model of antinociception [53]. However, it was the
appearance of isotonitazene in 2019 that triggered a rapid expansion of
2-benzylbenzimidazole opioids on the recreational drug market. This
drug dominated the NSO market in the first half of 2020 [54]. By now,
isotonitazene has decreased in popularity, following its international
scheduling in June 2021 [55]. After isotonitazene began to wane, other
nitazene analogs appeared worldwide (Fig. 3).
Papsun and co-workers examined 384 casework blood samples
collected between January 2020 and December 2021 in the US for the
presence of nitrazenes and a benzimidazolone compound – brorphine.
The mean concentration (in ng/mL) of isotonitazene was 2.8 ± 9.2
(range 0.11–99), metonitazene 5.1 ± 7.0 (range 0.5–50), brorphine 3.9
± 12 (range (0.27–111). 224 blood samples also contained fentanyl.
Methamphetamine and/or amphetamine, cocaine, and/or benzoy­
lecgonine, cannabinoids and designer benzodiazepines (etizolam, clo­
nazolam, bromazolam, phenazepam, and flualprazolam) were also
identified [10]. During 2020 and 2021, a total of 52 nitazene-involved
fatal drug overdoses were identified in Tennessee, the US, using State
Unintentional Drug Overdose Reporting System data; 10 in 2020, and 42
in 2021. In 2020, nine nitazene-involved deaths were attributed to
isotonitazene, and one to metonitazene. In 2021, 36 deaths involved
metonitazene, five – etonitazene, two – protonitazene and one – iso­
tonitazene. Other psychoactive substances, such as fentanyl, metham­
phetamine, amphetamine and flualprazolam, were also identified [56].
Walton and co-workers [57] analyzed 171 biological samples from 114
individual cases (47 postmortem and 67 from
driving-under-the-influence-of-drugs (DUID) subjects for the presence of
J.B. Zawilska et al.
Fig. 3. Chemical structures of 2-benzylbenzimidazoles.
nitazene analogs. Isonitazene (n = 25), N-desethylisotonitazene
(n = 13), metonitazene (n = 35), protonitazene (n = 4), butonitazene
(n = 3), etodesnitazene (n = 11), flunitazene (n = 4) and 4′-hydrox­
ynitazene (n = 10) were confirmed quantitatively in postmortem sam­
ples. Only isotonitazene (n = 50) and N-desethylisotonitazene (n = 12)
were confirmed in DUID samples. These newly emerging 2-benzylbenzi­
midazole analogs were commonly found in combination with flualpra­
zolam and fentanyl. During 2021–2022, EMCDDA received reports from
Ireland, Slovenia, and Norway of fake oxycodone tablets containing
metonitazene and etonitazepyne, as well as brorphine. The tablets
looked similar to commercially produced oxycodone tablets. They are
round, blue, half-scored, and debossed with ‘M’ on the unscored side and
‘30’ on the scored side oxycodone [1]. Pyrrolidino etonitazene, eto­
desnitazene and isotonitazene were also identified in tablets seized at an
International Mail Facility in the US between December 2021 and May
2022. They were labeled to contain health supplements but were found
to contain tablets looking like these described above [58].
The extensive chemical and pharmacological characterization of 14
nitazenes (both parent compounds and metabolites) was performed by
Vanderputte and co-workers [59]. MOR activity of all compounds was
evaluated using two in vitro recruitment assays, MOR-β-arrestin2 and
MOR-mini-Gi. N-desethylisotonitazene and etonitazene had the highest
potencies in both assays. These compounds were also the most effica­
cious in terms of both β-arrestin2 and mini-Gi recruitment. Metodesni­
tazene and 4′-hydroxynitazene were the least efficacious compounds.
Except for N-desethylisotonitazene, the evaluated metabolites, i.e.,
4’-hydroxynitazene, 5-aminoisotonitazene and N-desethyletonitazene
were less active than their parent compounds. Comparison of potency of
each compound versus that of fentanyl led to classification of nitazenes
into four categories: (a) those with a potency around 20 times higher
(etonitazene, N-desethylisotonitazene), (b) 1.5 − 10 times higher (iso­
tonitazene, metonitazene, N-desethyletonitazene, protonitazene), (c)
2–10 times lower (butonitazene, clonitazene, isotodesnitazene, eto­
desnitazene), and (d) 12–50 times lower (4’-hydroxynitazene, 5-amino­
isotonitazene, flunitazene, metodesnitazene) than that of fentanyl [59].
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3.4.1. Etonitazene
Etonitazene or etodesnitazene (2-{2-[(4-ethoxyphenyl)methyl]− 5-
nitro-1H-benzimidazol-1-yl}-N,N-diethylethan-1-amine) was the first
compound from the nitazene group encountered on the illicit drug
market. In MOR activation assays monitoring either β-arrestin2 or mini-
Gi recruitment etonitazene was 22-times (β-arrestin2) and 20-times
(mini-Gi) more potent than fentanyl [60].
The presence of etonitazene in a form of a “brownish looking pow­
der” was reported in Italy in the late 1960 s, in Germany in 1987, in
Russia in 1998, and in the US in 2003. Etonitazene was not only a potent
but also a highly selective MOR ligand (reviewed in [8]). N- and
O-deethylation were identified as the predominant metabolism routes of
etonitazene, resulting in O-deethylated etazene, N-deethylated etazene
and N,O-dideethylated etazene. Less abundant hydroxylated products of
the listed above deethylated metabolites were also found [61].
3.4.2. Isotonitazene
Isotonitazene (N,N-diethyl-2-(2-(4-isopropoxybenzyl)− 5-nitro-1H-
benzo[d]imidazol-1-yl)ethan-1-amine), known under street names ‘Iso’,
‘Nitazene’ and ‘Toni’, is a structural analog of etonitazene. Isotonitazene
is a high affinity and potent MOR agonist in vitro with Ki and EC50 values
in subnanomolar range [13,59,62]. As an analgesic, isotonitazene was
500-times more potent than morphine in mice [8], and about 3.7- and
1500-times more potent than fentanyl and morphine, respectively, in
rats [62]. In in vivo microdialysis studies isotonitazene increased
extracellular dopamine levels in rat nucleus accumbens (NAc) shell, an
observation indicating its potential reward activity [62].
A report from BlueLight described the drug as having the “potency of
fentanyl + duration of heroin, with actual euphoria to go with it” [63].
Isotonitazene has been identified in postmortem forensic toxicology
reports and in drug seizures since April 2019 [53,63]. In January 2020,
isotonitazene was analytically confirmed in falsified tablets with
‘Dilaudid’ logo circulating in Québec, Canada. These tablets also con­
tained benzocaine, benzoylecgonine, levamisole, MDMA, and metham­
phetamine (cited in [8]). Isotonitazene can be administered orally as a
powder, tablets, or solution; intranasally, sublingually; inhaled by
J.B. Zawilska et al.
vaporizing e-liquid solutions or by smoking, and injected [53]. Doses
reported on online forums are quite variable, ranging from 1 to 10 mg
[10,64]. Beside the US [10,64], deaths involving isotonitazene were also
reported in Canada, Germany and the UK [53,63]. In a series of 18
isotonitazene-related deaths from the Midwestern United States, this
drug was the only opioid detected in half of the samples; in another half
fentanyl, heroin, tramadol, acetylfentanyl and U-47700 were also
identified. Beside opioids, some of the samples contained other psy­
choactive substances, namely benzodiazepines (etizolam, flualprazo­
lam, diazepam and oxazepam), psychostimulants (cocaine,
amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine
and MDMA), diphenhydramine and Δ9-THC. The mean concentration
of isotonitazene in central or peripheral blood samples was 2.2
± 2.1 ng/mL (median 1.75 ng/mL, range 0.4–9.5 ng/mL). The lowest
concentration of isotonitazene in the blood, 0.4 ng/mL, was found in
two cases in which it was the only opioid identified. The mean con­
centration of isotonitazene in urine samples was 2.4 ± 1.4 ng/mL (me­
dian 2.7 ng/mL, range 0.6–4.0 ng/mL). The concentration of
isotonitazene in a single sample of vitreous fluid was 0.1 ng/mL [64].
Isonitazene was also identified in 144 postmortem blood samples ([10],
see above). Isotonitazene was found to undergo N- and O-dealkylation to
form prominent urinary metabolites N-desethylisotonitazene and
N-desethyl-O-desalkylisotonitazene; 5-aminoisotonitazene was identi­
fied in most blood samples [64].
3.4.3. Metonitazene
Metonitazene (N,N-diethyl-2-(2-(4-methoxybenzyl)− 5-nitro-1H-
benzo[d]imidazol-1- yl)ethan-1-amine), also known as ‘NIH 7606’, is
one of the newest non-fentanyl-related NSO. It first appeared in the
recreational drug supply in mid-2020 s’ [65]. [3H]DAMGO binding and
[35S]GTPγS functional assays demonstrated that metonitazene is a
highly potent agonist of MOR in both rat cortical and CHO-MOR
membranes, with EC50 values of 19.1 and 10.4 nM, respectively [62].
By analogy to isotonitazene, metonitazene increased extracellular
dopamine levels in rat NAc shell [62]. In early 2021 it was reported in
the US together with butonitazene, and together with flunitazene and
clonazolam (cited in [57]). Reports from discussion forums suggest that
the drug can be used intranasally, intravenously or by vaporization [65].
Presence of metonitazene was confirmed in 20 authentic forensic post­
mortem cases [66]; most decedents had a history of opioid use disorder
and were using/seeking heroin or fentanyl at the time of death. An
average concentration in the blood was 6.3 ± 7.5 ng/mL (median
3.8 ng/mL, range 0.5–33 ng/mL) and in urine 15 ± 13 ng/mL (median
11 ng/mL, range 0.6–46 ng/mL). Metonitazene was the only opioid
identified in 30% of cases; in the rest it was detected together with other
opioids, benzodiazepines, and hallucinogens from NSP [66]. Later on,
metonitazene was identified in 122 postmortem blood samples ([10],
see above). Metonitazene is thought to undergo N-dealkylation and
O-dealkylation. N-desethylmetonitazene was found to be a prominent
metabolite in the urine and vitreous samples tested [66].
3.4.4. Protonitazene
Protonitazene (N,N-diethyl-2-(5-nitro-2-(4-propoxybenzyl)− 1H-
benzo[d]imidazol-1-yl)ethan-1-amine) is known also as propox­
ynitazene. In in vitro recruitment assays protonitazene was highly active
in MOR stimulation, with a potency and efficacy slightly greater than
those of fentanyl (β-arrestin2: 107%; mini-Gi: 129%), and significantly
greater than those of hydromorphone (β-arrestin2: 174%; mini-Gi:
365%) [59]. Protonitazene was first detected during forensic sampling
in Canada and the US in 2020, and more recently in Australia [67]. In an
alert issued in June 2022, a “yellow powder” containing protonitazene
was reported being sold as ketamine in Melbourne [68]. Protonitazene
was analytically confirmed in nine fatal poisonings in the US; the
average blood concentration was 286 ± 556 ng/mL [67]. Presence of
protonitazene, together with butonitazene and methylamphetamine, in
a blood sample from an intoxicated women, was reported in Australia
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Forensic Science International 349 (2023) 111775
[69].
3.4.5. Etonitazepyne
Etonitazepyne (2-[(4-ethoxyphenyl)methyl]− 5-nitro-1-(2-pyrroli­
din-1-ylethyl)− 1H-benzoimidazole), also known as N-piperidinyl eto­
nitazene or ‘etonitazepipne’, is the newest representative of nitazenes. In
vitro radioligand binding assays performed on rat brain tissue showed
greater affinity of etonitazepyne for MOR (Ki = 4.09 nM) than for KOR
(Ki = 959 nM) and DOR (Ki = 980 nM). Etonitazepyne also showed high
potency (EC50 = 0.348 nM) in the MOR-β-arrestin2 activation assay.
These values exceeded the potencies of both fentanyl (EC50 = 14.9 nM)
and morphine (EC50 = 290 nM) [60,70].
Etonitazepyne was reported in Europe in mid-February 2021. Later
on, it appeared on the illicit drug market in the US and New Zealand
[71]. In Wales, the presence of etonitazepyne in ‘M30‘ blue tablets sold
as oxycodone was reported [71]. Snorting and oral administration have
been described as the main routes for taking etonitazepyne. The first
identification and chemical characterization of etonitazepyne was re­
ported by Vandeputte and co-workers [70]. The detected analytical
concentration of this drug was 1.21 ng/mL and 0.51 ng/mL in serum
and urine, respectively. In New Jersey, the US, three patients with acute
opioid overdose tested positive for etonitazepyne in July 2021. Two
admitted the use of cocaine, one the use of heroin and alprazolam [72].
A severe toxicity involving etonitazepyne was reported in the UK [73].
Presence of etonitazepyne has been analytically confirmed in 21 blood
samples and one urine sample from 21 post-mortem cases, of which four
were in Canada and 17 in the US. Etonitazepyne was detected with
fentanyl in 12 cases, with methamphetamine in 12 and with benzodi­
azepines (flualprazolam, etizolam, flubromazepam, clonazolam and
desalkylflurazepam) in 11 cases. Etonitazepyne was the sole substance
found in seven cases [71].
3.5. Benzimidazolones
Brorphine (1-[1-[1-(4-bromophenyl)ethyl]-piperidin-4-yl]− 1,3-
dihydro-2H-benzo[d]imidazol-2-one) (Fig. 4) was first synthesized in
2018 as a member of a series of MOR agonists. The chemical structure of
brorphine contains similarities to both fentanyl and isotonitazene. In
vitro pharmacological studies using [35S]GTPγS binding, mini-Gi, and
β-arrestin2 recruitment assays revealed brorphine to be a potent, full
MOR agonist [74,75]. The detection of brorphine was first reported to
European Monitoring Centre for Drugs and Drug Addiction (EMCCDA)
in June 2020. Later on, fake oxycodone tablets containing brorphine
were seized in Slovenia [1]. The rise of brorphine in the US can be
directly linked to decline of isotonitazene popularity after its scheduling
mid-2020’s. Brorphine was sometimes sold under the street name
‘purple heroin’, and found in falsified oxycodone ‘A/215’ tablets [76]. In
a case of non-fatal intoxication serum concentration of brorphine was
Fig. 4. Chemical structure of brorphine.
J.B. Zawilska et al.
69.4 ng/mL [77]. Since June 2020 brorphine has been increasingly
detected in postmortem samples in the US. The first case of a fatal
intoxication with brorphine was presented by Vohra and co-workers; the
blood concentration of brorphine was 2 ng/mL. In addition to brorphine
other substances, such as ethanol, fentanyl, dipropyl-fentanyl, gaba­
pentin and chloropromazine, were detected [78]. The presence of
brorphine was confirmed in 20 authentic fatal cases; the drug was
commonly found in combination with fentanyl and flualprazolam. The
average concentration of brorphine in the blood was 2.5 ± 3.1 ng/mL
(median 1.1 ng/mL, range 0.1–10 ng/mL), and in urine 4.6
± 7.6 ng/mL (median 1.6 ng/mL, range 0.2–23 ng/mL) [79]. Brorphine
was also identified in 91 postmortem blood samples ([10], see above).
The drug is metabolized by N-dealkylation and hydroxylation [79].
4. Methods of detection
Due to the high potency and low dose required to produce desired
effects, NSO concentrations in biological materials are also low. Thus,
detection and determination of these drugs in biological matrices
require high sensitivity. For some high potent compounds the limit of
detection should be at least 0.01 ng/mL in order to detect their use [42].
At the same time, methods must be effective for identification of com­
pounds having similar chemical structures. Moreover, the rapid emer­
gence of new compounds makes analytical methods to be updated to
new trends.
The comprehensive reviews of methods used in the detection and
determination of NOS, especially fentanyl-like drugs, were presented
elsewhere [7,80–83]. Therefore, only a brief description of the methods
used in the analysis of the latest synthetic opioids in biological material
is presented below.
Most standard immunoassay drug screens are limited to a set number
of drugs. These immunoassays, routinely used in clinical and forensic
laboratories to detect traditional opioids, are often not effective for the
majority of new non-fentanyl opioids. Importantly, immunoassays
cannot distinguish between structural analogs [7,84].
Popular analytical methods, such as high performance liquid chro­
matography with diode array detection (HPLC-DAD) or gas chroma­
tography coupled with mass spectrometry (GC-MS) without prior time
consuming derivatization often lack the necessary sensitivity needed to
detect low concentrations of more potent opioids. GC-MS has the
advantage of untargeted data acquisition. However, if novel opioids are
not included in library they cannot be identified. Therefore, GC-MS
methods have been used less frequently. A GC-MS analysis was used
to detect and quantify better known compounds like U-47700 and AH-
7921 in postmortem matrices [85–87]. HPLC-DAD was used to differ­
entiate these compounds as their retention times and UV spectra differed
when analyzed by this method, despite similar structures and the same
molecular mass [88].
Considering the above difficulties, more specific and sensitive
analytical methods are needed to detect and distinguish the growing
number of NSO. Therefore, liquid chromatography-tandem mass spec­
trometry (LC-MS/MS) or ultra-high performance liquid
chromatography-tandem mass spectrometry (UHPLC-MS/MS) based
methods were the most commonly used as they are characterized by the
lowest limits of detection. Most of the methods are triple quadrupole
based and use positive-ion electrospray ionization (ESI) and a targeted
multiple monitoring (MRM) mode. The reversed phase C18 chromato­
graphic columns were mostly used. Mobile phases commonly contain
acetonitrile or methanol with water or ammonium formate (or ammo­
nium acetate), often with addition of formic acid [7,82,83]. An example
of such a method for the detection and quantitation of 12 drugs and 2
metabolites from U-series drugs in human whole blood can be the pro­
cedure reported by Fogarty and co-workers [28]. Moody and co-workers
described a method for the analysis of 19 NOS (including AH-7921,
U-47700 and MT-45) in whole blood and serum, and 17 analytes in
urine using LC-MS/MS [89]. The same technique was utilized for
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Forensic Science International 349 (2023) 111775
quantitative analysis of brorphine in the blood, serum and urine [75,79].
LC-MS/MS was also used for the quantification of nine nitazene analogs
and/or metabolites: isotonitazene, metonitazene, protonitazene, etoni­
tazene, clonitazene, flunitazene, N-desethylisotonitazene, 5-aminoisoto­
nitazene and 4′-hydroxynitazene in human whole blood, urine, and
tissues [57]. However, LC-MS/MS targeted procedures have a few
drawbacks. These methods detect only compounds for which the assay
was designed, while NSO continue to appear on the market, and they are
not detected if previously not included in the method. Such methods do
not also allow for the retrospective evaluation to identify formerly un­
expected compounds. Therefore, liquid chromatography-high resolution
mass spectrometry (LC-HRMS) methods have emerged as the most
suitable for analysis of NSO. LC-HRMS technique (using quadrupole
time-of-flight or orbitrap technology) allows the tentative identification
of an unknown compound without the availability of a reference stan­
dard or a library spectrum by deducing the molecular formula from
accurate mass. Data are acquired in an untargeted manner, and there is a
possibility of retrospective analysis to screen for new, emerging and
previously undetected compounds. It has also been used for elucidation
of the metabolic pathways of emerging synthetic opioids [7,18,28,51,
61,75].
The most commonly biological matrices used to detect and quantify
NSO are blood (whole blood, plasma, serum), urine, and post-mortem
tissues [7,82,83]. NSO were also analysed in other biological matrices,
including hair and oral fluid [90,91]. Different protocols for both single
and multiple (screening) compound extraction were described. The most
commonly used isolation techniques for NSO in biological matrices
include liquid-liquid extraction (LLE), solid phase extraction (SPE) as
well as simple protein precipitation (PP) or dilution (mostly for urine).
LLE methods are mostly performed under alkaline conditions. This
extraction commonly uses 1-chlorobutane and butyl acetate, and less
frequently other solvents, sometimes in mixtures [7,82]. Both extraction
and extraction with subsequent acid back-extraction were evaluated [7].
PP was used on its own or coupled with other extraction techniques (LLE
or SPE) for pre-treatment of blood and urine. Acetonitrile is the most
popular solvent used for this purpose, other water-miscible solvents
were also used. SPE is also a popular technique of samples pre-treatment
with the use of different columns, e.g., Clean Screen DAU, PolyChrom
ClinII or Agilent Plexa PCX [89,91,92]. Enzymatic hydrolysis with
β-glucuronidase was sometimes employed before extraction of urine
[82].
5. Treatment of NSO overdose
Naloxone is a high affinity, competitive and non-selective opioid
receptors antagonist that reverses acute opioid-induced effects. The drug
has been the ‘gold standard’ for the treatment of opioid overdose for
decades. The US Food and Drug Administration (FDA) approved the first
naloxone solution for intravenous, intramuscular and subcutaneous in­
jection in 1971 for partial or complete reversal of opioid overdose. WHO
added naloxone to its model list of essential medicines in 1983, and
injectable naloxone formulations have been off-patent since 1985. Since
1996, community-based harm reduction programs in the US have
distributed naloxone directly to opioid drug users, their family members
or friends, and service providers. A so-called ‘take-home naloxone’
(THN) is a life-saving intervention in which the drug is made available to
non-medically trained people for administration to other people expe­
riencing the opioid overdose [93,94]. The first community-based
naloxone projects in the US and Europe started in the 1990’s [94–96].
Nowadays, such projects are also used in Canada, Australia and Central
Asia. Injectable naloxone hydrochloride solution is available in two
different strengths: 0.4 mg/mL and 1 mg/mL. In 2015, the first nasal
naloxone spray, Narcan®, delivering 4 mg of naloxone per dose
(0.1 mL) became available in the US. In November 2017, the European
Commission authorized a nasal spray Nyxoid (naloxone dehydrate
1.8 mg/0.1 mL; two atomizers/package). Another nasal spray currently
J.B. Zawilska et al.
available is Nalscue (naloxone hydrochloride 0.9 mg/0.1 mL; four
atomizers/package). It should be emphasized that naloxone nasal sprays
improve safety by avoiding potential needle-stick injuries when treating
a patient population at high risk of blood-borne illnesses [95].
Facing a trend of the marked rise in opioid overdose deaths driven by
the use of highly potent NSO, often combined with several psychoactive
compounds, as well as their use as adulterants in other abused sub­
stances, it is necessary to reconsider naloxone effectiveness in these
cases. It is postulated that higher doses of naloxone may be needed to
effect a successful rescue than those typically used. In addition, the short
half-life of naloxone relative to many NSO may lead to the recurrence of
respiratory depression during an overdose event and require multiple
doses of naloxone to be administered. This is particularly important in
cases of intranasal administration, commonly used by first responders.
While naloxone is generally considered safe and effective, one of the
most common risks regarding increasing dosing of this drug is precipi­
tation of abrupt onset opioid withdrawal in those who are physically
dependent on opioids [96–98].
Several MOR antagonists with higher affinities and longer half-life
than naloxone, including nalmefene, naltrexone and most recently
samidorphan, have been studied as potential effective rescue agents in
opioid overdose. Among them, nalmefene is considered as a promising
drug due to its higher affinity to MOR and longer half-life time com­
parable to those of NSO [96,98]. Moreover, it is well absorbed through
the nasal cavity when combined with dodecyl maltodise [99].
6. Conclusions
The global occurrence of NPS poses major concerns and challenges
for law enforcement agencies, but also for public health, due to constant
diversification and rise in numbers. As soon as one substance is sched­
uled, a new analog(s) occurs on the market. NPS have gained
outstanding popularity due to their wide availability, relatively low
prices, impressive marketing strategies, and popularization via the
Internet. NSO constitute one of the fastest-growing group of NPS. Due to
high potency and propensity for adverse health effects, with respiratory
depression being the most notable cause of overdose, even low doses of
NSO can lead to severe toxicity or even death. Importantly, any newly
emerging opioid has the potential of being even more dangerous than
the last one. There are increasing reports that higher naloxone doses or
repeat administration might be required for overdose victims involving
NSO.
At present little is known on pharmacotoxicology of the newly
appearing NSO, e.g., 2-benzylbenzimidazoles and benzimidazolones. As
the presence of NSO on the illicit drug market continues to rise, it will
become increasingly important to rapidly identify and characterize new
compounds as they emerge. Along this line, analytical methods have to
be updated to new trends. Due to the high potency and low dose
required to produce desired effects, NSO concentrations in biological
materials are also low. Thus, detection and determination of these drugs
in biological matrices require high sensitivity. At the same time,
methods must be effective for identification of compounds having
similar chemical structure. Finally, multidisciplinary research in areas of
epidemiology, prevention, and public health should allow the develop­
ment of adequate measures to monitor consumption and implement
public health education policies.
CRediT authorship contribution statement
JBZ conducted the systematic review and drafted the manuscript. PA
conducted the systematic review on analytical methods, drafted the
manuscript and prepared figures. JW led on title, abstract, highlights
and provided substantial comments and revisions. MK drafted the
manuscript full text screening. All authors have read the final version of
the manuscript and approved it for publication.
8
Forensic Science International 349 (2023) 111775
Declaration of Competing Interest
Hereby, we confirm that: (1) it is original work and it has not been
published in whole or in part elsewhere and is not under consideration
by any other journal; (2) all persons named as authors have made a
major contribution to the work reported and approved the submitted
version of the manuscript, and are prepared to take public responsibility
for its contents, (3) if accepted, it will not be published elsewhere in the
same form, in English or in any other language, including electronically
without the written consent of the copyright-holder.
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