Forensic Science International 310 (2020) 110237

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Forensic Science International

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Case Report

Clonazolam a new designer benzodiazepine intoxication confirmed

by blood concentration
_ d,
Karina Sommerfeld-Klattac,* , Magdalena Łukasik-Głe˛bockaa,b , Artur Tezyk
Paweł Panien a _
 ski , Czesław Zaba , Barbara Zielin
d  ska-Psuja c

a
Department of Emergency Medicine, Poznan University of Medical Sciences, Poznan, Poland
b
Department of Toxicology, Raszeja Hospital, Poznan, Poland
c
Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland
d
Department of Forensic Sciences, Poznan University of Medical Sciences, Poznan, Poland

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

Article history: Background: Recently the number of new psychoactive substances have significantly increased, becoming
Received 1 October 2019 popular among experienced users of designer drugs. A significant group includes benzodiazepine
Received in revised form 28 February 2020 derivatives, which have not been introduced as medications but are abused by people experimenting
Accepted 4 March 2020
with new and classical psychoactive substances.
Available online 6 March 2020
Case presentation: The aim of this paper was to present the case of a clonazolam ingestion by a person who
was not habituated to benzodiazepines. The intake caused only prolonged coma, decreased muscle tone,
Keywords:
and deep tendon reflexes without any other concomitant toxicity and cardio-respiratory failure.
Clonazolam
New designer benzodiazepine
Conclusions: Clonazolam concentrations in patient’s blood, measured three times were 0.077 mg/L,
Blood confirmation 0.015 mg/L, 0.009 mg/L after 4, 8 and 12 h, respectively. Clonazolam’s human toxicity has not been well
established, so any case of poisoning should be closely monitored.
© 2020 Elsevier B.V. All rights reserved.

1. Background compound in the group of the studied benzodiazepines. It shows

Benzodiazepines are a numerous group of compounds showing
anxiolytic, myorelaxant, hypnotic, anticonvulsant, and sedative
effect. A significant number of their derivatives, as well as their
different pharmacological effects, cause that they belong to the
most often prescribed drugs in the developed countries. A wide
range of applications of this group of drugs and their properties
often lead to abusing of benzodiazepines due to prolong
application for therapeutic effect, taking them without medical
indications, to relieve the effects of other psychoactive substances
or to intoxicate [1].
Clonazolam (6-(2-chlorophenyl)-1-methyl-8-nitro-4H-s-tri-
azolo(4,3-a)-(1,4)-benzodiazepine) is a triazolo-analog of the
registered drug clonazepam. It was synthesized in the seventies
of the twentieth century, and classified as the most active
Abbreviations: GCS, Glasgow Coma Scale; HR, heart rate; BP, blood pressure; GC-
FID, gas chromatography with flame ionization detection; EMIT, Enzyme Multi
Immunoassay Technique; LC–MS/MS, liquid chromatography coupled with triple
quadrupole mass spectrometer; MRM, multiple reaction monitoring; CNS, central
nervous system.
* Corresponding author at: Poznan  University of Medical Sciences, Department of
Toxicology, Dojazd Str. 30, 60-631, Poznan , Poland.
E-mail address: ksommerfeld@ump.edu.pl (K. Sommerfeld-Klatta).
hypnotic, anxiolytic, sedative, muscle-relaxant and anticonvul-
sant effects. According to the users’ experience, it is applied in
minimal doses (orally in capsules, tablets, or in liquid form) and
can induce amnesia. The recommended doses range from 0.5 mg
(for non-addicted persons) to 4 mg (for persons who regularly
use benzodiazepines). Clonazolam is regarded by users as a
harmless substance causing pleasant impressions and eliminat-
ing fear [2–5].
2. Case presentation
A 26-year-old woman (55 kg, 176 cm) was admitted to the
toxicology department due to unintentional poisoning of clona-
zolam. She claimed that 4 h before admission, she took 10 mg
(powder) of clonazolam (0.18 mg/kg of body weight), twenty times
exceeded the recommended dose (it was impossible to analyze
ingested powder). She had been suffering from acute bronchitis for
a few days, and because of the persistent cough, she had problems
with sleeping. She was probably given clonazolam by her boyfriend
(the source of the purchase remains unknown). The patient had not
been chronically ill and had not taken any psychoactive substances
before. On admission to hospital (about four hours after the
poisoning), the patient was deeply unconscious, GCS (Glasgow

http://dx.doi.org/10.1016/j.forsciint.2020.110237
0379-0738/© 2020 Elsevier B.V. All rights reserved.
2 K. Sommerfeld-Klatta et al. / Forensic Science International 310 (2020) 110237
Coma Scale)2 100 %. On both sides over the lungs wheezing was
heard. After the next 4 h, the patient was still in a coma moreover, a
tachycardia was observed (100–110/min). After 12 h following
taking the drug (8 h after admission to hospital), the patient was
still deep asleep (GCS - 12 points) and did not make any verbal
contact (HR 110/min, BP 124/64), the pupils and reflexes were as
before. The diuresis was good. In the 24th hour after taking the
drug, tachycardia was observed (HR 135/min), possibly due to the
high temperature 38.3  C, caused by acute bronchitis, so antibiotics
(amoxicillin with clavulanic acid) were administered. The patient
was conscious however, still slowed down. In the following hours,
the condition of the patient improved. The patient left the hospital
without any consequences of acute poisoning after eight days of
treatment. Psychological and psychiatric examinations revealed
neither mental disorders nor addiction.
3. Screening toxicological analysis
Preliminary toxicological tests, made after admission to
hospital laboratory, showed the presence of benzodiazepines in
the urine (qualitative immunological test Hydrex Diagnostics, with
the sensitivity of 300 ng/mL). Gas chromatography with flame
ionization detection (GC-FID) excluded presence of ethanol in
blood, whereas the presence of psychoactive substances in the
urine (amphetamine and its derivatives, cannabinoids, opiates
(morphine), cocaine metabolite) was excluded by homogenous
enzymatic immunological test with EMIT (Enzyme Multi Immuno-
assay Technique, Syva Siemens) method.
4. LC–MS/MS analysis
In the forensic laboratory of the Department of Forensic
Sciences, the substance was identified and measured in the
patient’s blood (4, 8, and 12 h after the ingestion). The HPLC system
comprised an Agilent 1200 Infinity system consisting of a binary
pump, degasser, thermostated autosampler and column oven
compartment coupled with triple quadrupole mass spectrometer
Agilent 6410B (Agilent Technologies, PaloAlto, CA, USA). Chro-
matographic separation was performed on a Poroshell 120 EC-C18
column (2.7 mm, 3.0  75 mm; Agilent Technologies, CA, USA) with
a Poroshell 120 EC-C18 guard column (2.7 mm, 3.0 mm  5 mm;
Agilent Technologies, CA, USA). The column oven temperature was
maintained at 40  C. The mobile phase comprised 0.1 % formic acid
in distillate water (A) and 0.1 % formic acid in acetonitrile (B). The
mobile phase was delivered at 0.5 mL/min in a gradient mode
(Table 1). Analysis worked in multiple reaction monitoring (MRM)
mode with two reactions for the compound, equipped with
electrospray ionization source (Table 2). To validate the clonazolam
method, its specificity, selectivity, matrix effect, recovery, linearity,
limit of detection (LOD), limit of quantification (LOQ) and precision
were examined. The LOD was determined as the lowest
clonazolam concentration (0.01 mg/L) tested in which the peak
signal was three times higher than the chromatogram background.
The LOQ was determined as the lowest concentration with less
than 20 % coefficient of variation (CV) for precision and with less
Table 1
The HPLC gradient mode of mobile phases.
Time [min] A [%] B [%] Flow [mL/min] Pressure [bar]
1 0 90 10 0.5 400
2 6 80 20 0.5 400
3 10 50 50 0.5 400
4 16 35 65 0.5 400
5 17 90 10 0.5 400
6 20 90 10 0.5 400
Table 2
MRM transition, collision energy (CE), and fragmentor voltage of clonazolam
identification.
MRM (m/z) CE (eV) Fragmentor voltage
Clonazolam 354,1-308,1 25 147
354,1-280,1 37
Oxazepam D5 292,1-274,1 13 87
292,1-246,1 21
than 20 % accuracy for clonazolam identification (Table 3). The
matrix effect and recovery were evaluated by comparing the peak
areas of analytes for standard sample extract and blank serum
extract piked with standard after extraction (Table 4).
5. Sample preparation and clonazolam results
Extraction of clonazolam was performed using liquid-liquid
extraction method at an alkaline medium (Tris(hydroxymethyl)
aminomethane buffer pH 9.0, mixed with acetonitrile and ethyl
acetate). For the quantitative analysis of clonazolam, the blood
sample internal standard (D5-oxazepam) was added [6]. A
calibration curve for clonazolam (Cerilliant-Certified Reference
Material) was prepared in the range of 0.01-0.2 mg/L. After 4 h, the
concentration of clonazolam was 0.077 mg/L, then after 8 h, the
measured level was 0.015 mg/L and after 12 h 0.009 mg/L.
Moreover, in order to prove that the patient had not taken any
other benzodiazepines or psychoactive substances before, a
screening test of the blood was carried out, applying the LC–
MS/MS method regarding amphetamine and its derivatives,
cannabinoids, opiates and cocaine, as well as the most common
benzodiazepines (like midazolam, triazolam, alprazolam, diaze-
pam, flurazepam). The blood sample used in the screening test was
the one secured right after the patient’s admission to hospital.
6. Discussion
Recently, designer benzodiazepines are rapidly increasing as
drugs of abuse. However, they have never been applied in medical
treatment, and limited data on its human acute toxicity and toxic
levels in blood exist. Benzodiazepines, as a class of drugs are
considered safe, but their toxicity is dose-dependent and they
interact with alcohol and other drugs acting depressively on
central nervous system [1]. The paper presents documented case
report of clonazolam powder ingestion with coma and triple blood
concentration evaluation.
Clonazolam is reported by users to be highly potent, causing
strong sedation and amnesia following oral or liquid form. Slowly
increasing effects appear within 30 40 min after taking the drug,
which causes a feeling of slight relaxation, sedation and the so-
called sense of “suspension”. The peak of the effect is reached after
about 1–1.5 h. According to the users, the sedative effect with
myorelaxant action dominates [5].
Recently, due to its availability, mainly online, clonazolam sold
as a “new safe diazepine” has been arousing an increasing interest.
The annual number of benzodiazepine designers’ abuses increase
each year (from 26 in 2014 to 112 in 2017), with most common
exposures reported to etizolam and clonazolam [7]. There are also
some results of studies, which discuss the influence of a few new
benzodiazepines on drivers including clonazolam, where their
concentrations in the blood ranged from 0.0019–0.011 mg/L
(median 0.0052 mg/L). However, such concentrations were
regarded as unimportant comparing to other identified substances,
including benzodiazepine derivatives such as alprazolam, diaze-
pam as well as amphetamine [8].
 K. Sommerfeld-Klatta et al. / Forensic Science International 310 (2020) 110237
Table 3
LOD, LOQ and linearity of clonazolam identification.
LOD [mg/L] LOQ [mg/L]
Clonazolam 0.005 0.015
Table 4
The results of intra- and inter-day precision, accuracy and matrix effect.
Concentration [mg/L]
Clonazolam 0.01
0.1
1 7
The determined clonazolam concentrations were similar to the
ones described in the case of the other new designer benzodiaze-
pine, flubromazolam which caused prolonged, severe intoxication
associated with coma, hypotension and rhabdomyolysis [9].
Flubromazolam’s level detected in the patient’s serum was
0.059 mg/L and urine at 0.105 mg/L about 19 h after the ingestion
of 3 mg dose. These effects were far more severe although the
patient has experimented with new psychoactive substances for
several years and the woman poisoned with clonazolam has not
use benzodiazepines at all. This discrepancy might suggest
differences in potency and safety profile of these two drugs. There
are also some results that present the tentative identification of
clonazolam and flubromazolam in candy-like pills taken by 32-old
year man to cut down on his methadone use [10].
In the presented case of clonazolam intoxication, the first
measured concentration in the blood (0.077 mg/L), 4 h after
ingestion, was much higher than in the studies described by
Hoiseth, because it was undoubtedly due to a single high dose
(0.18 mg/b.w.). After at least 12 h, the concentration reaching
0.009 mg/L, was within the range of the compared group [8].
The mechanism of action can be significant for the evaluation of
potential toxicity of clonazolam. Benzodiazepines act as positive
allosteric modulators on the g-amino butyric acid (GABA-A)
receptor. The GABA-A receptor is a ligand-gated chloride-selective
ion channel. The primary toxicity of benzodiazepines is central
nervous system (CNS) depression which is produced by enhancing
the function of GABA-A receptors, which consist of polypeptide
subunits which variation determines the potency of its sedative,
anxiolytic, hypnotic, amnestic, and musle relaxing properties.
There is a limited information of clonazolam’s acute human
toxicity. Generally, oral overdoses of benzodiazepines may lead to
sedation, but rarely life-threatening cardio-respiratory failure.
Typically, the patient falls into a coma but maintaining normal vital
function. These drugs are not known to cause any specific organ
toxicity. Deaths resulted from isolated benzodiazepine ingestions
alone are extremely rare. It is due to special property of the GABA-A
receptors which after ingestion of benzodiazepines do not open
GABA channels even at high doses. On the other side long-term
high-dose use of benzodiazepines alone or with another psycho-
active substance (e.g. ethanol) presents an apparent decrease in
the efficacy of GABA-A receptors, presumably a mechanism of
tolerance. So the effects of benzodiazepine are minimal or none
[1,4,7].
In the reported case, there was observed a coma (with Glasgow
Coma Scale 3) with decreased muscle tone and weakened
tendinous reflex. Neither alcohol nor any other psychoactive
substances and no regular, long-term benzodiazepine usage which
could have influenced the patient’s condition, were found. Despite
high concentration of the drug in the blood (0.077 mg/L in the 4th
3
Calibration [mg/L] Calibration curve
a b R
0.01-2.00 0.00619 0.00002 0.999
Intra-day assay (n = 5) CV % Matrix effect CV % Recovery CV %
10 11 12
7 9 10
9 6
hour after ingestion), there were not noted any symptoms of
respiratory failure or circulatory disorders. The condition of the
patient did not require the administration of a specific antidote
(flumazenil). After 24 h from the moment of poisoning the patient
was conscious, however kept falling asleep when being left alone.
After 8 days the patient left the hospital. She was in good condition,
suffering no consequences of clonazolam poisoning.
7. Conclusions
In 2007 phenazepam appeared as the first one on new
psychoactive substances market and after that in the following
years more than twenty new benzodiazepine derivatives have
been identified analytically such as: mexazolam, flutoprazepam
and flunitrazolam.
Clonazolam is a newly identified psychoactive substance which
safety profile has not been well established. Despite clonazolam is
considered to be highly potent benzodiazepine and it is active in
very small doses it seems to be relatively safe in acute poisoning.
Even in the naive person, clonazolam oral dose of 0.18 mg/b.w.
resulting with high blood concentration of 0.077 mg/L in the 4th
hour after ingestion, caused only prolonged coma, decreased
muscle tone and deep tendon reflexes without any other
concomitant toxicity and in particular without cardio-respiratory
failure. That does not change the fact that its consumption with
alcohol or with other CNS depressants might be more dangerous.
Clonazolam has not been introduced into medical treatments, but
recently it has been rediscovered as a new psychoactive substance.
Therefore its toxicity should be closely monitored.
CRediT authorship contribution statement
Karina Sommerfeld-Klatta: Conceptualization, Methodology,
Investigation, Writing - original draft. Magdalena Łukasik-
Głe˛bocka: Resources, Investigation, Writing - original draft. Artur
_
Tezyk: Validation, Formal analysis, Methodology. Paweł Panien -
_
ski: Data curation. Czesław Zaba: Supervision. Barbara Zielin  ska-
Psuja: Supervision, Writing - original draft, Writing - review &
editing.
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