Fatal intoxication involving amphetamine, clonazafone, a benzodiazepine prodrug, and fluoro-etonitazene, a new synthetic opioid

Abstract

Intoxication cases involving new psychoactive substances (NPS) are known to provide various challenges for forensic toxicological case interpretation, starting with the identification of previously unknown substances. Furthermore, the pharmacological characteristics of these substances, including potency and metabolic processes, remain largely unstudied. In this particular medico-legal case, a 20-year-old man consumed clonazafone and fluoro-etonitazene, which were examined in blood by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, a urine screening was conducted using LC-high-resolution mass spectrometry (HRMS) to investigate the metabolism of these substances, particularly clonazafone. Clonazafone was (semi-)quantified in urine (39 ng/mL), muscle tissue (3.0 ng/g), and stomach content (76’000 ng/mL), but could not be detected in peripheral blood, heart blood, and vitreous humor (lower limit of quantification: 0.1 ng/mL). Additionally, clonazepam (1.5 ng/mL) and its metabolite 7-amino-clonazepam (140 ng/mL), as well as amphetamine (110 ng/mL) and the designer-opioid fluoro-etonitazene (3.3 ng/mL) were found in blood. Within the HR screening, desglycylclonazafone, the intermediate of clonazafone that can be further converted into clonazepam, was detected in the stomach content and urine. Screening in urine has also revealed several metabolites of clonazafone. The cause of death was assumed to be a mixed drug intoxication with fluoro-etonitazene, clonazepam, and amphetamine.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00414-025-03627-7.

Introduction

New psychoactive substances (NPS) are designed to mimic the pharmacological effects of psychoactive drugs while circumventing legal restrictions [1]. There is no general agreement, but these substances can be divided into four main groups based on their effect: synthetic stimulants, synthetic cannabinoids, synthetic hallucinogens, and synthetic depressants [2]. The focus of this case report is on synthetic depressants, specifically designer benzodiazepines (DBZDs) and new synthetic opioids (NSOs).

The increasing demand for benzodiazepines (BZDs) has also led to their popularity in the illicit drug market. In 2012, Pyrazolam was the first DBZD, which was never approved by the industry, to be seized [3]. In the following years, to circumvent continuously updated legislations, a variety of easily accessible DBZDs occurred on the illicit drug market (e.g., diclazepam, clonazolam, fluclotizolam, bromazolam, etc.) [4, 5]. The latest group to emerge is BZD prodrugs, some of which are glycine-substituted and are only converted into the active agent in the human body. These substances are usually not novel, as rilmazafone, a representative example, was approved for medical use in Japan several decades ago. It has since been subjected to comprehensive analysis, including its metabolism [6]. The first described cases of ingestion of rilmazafone were reported in 2023, in which a mislabeling has led to fatal intoxications [7]. In the meantime, the range of available BZD prodrugs is expanding, and most of the available prodrugs are metabolized to therapeutically used BZDs. Clonazafone, for example, is metabolized to clonazepam (Fig. 1). While the analytical detection of the pharmacologically active BZDs in biological matrices is routinely done in forensic toxicology laboratories (usually covered by routine quantitative methods targeting legally available BZDs), the distinction between the consumption of a therapeutically used substance and a BZD prodrug sold as NPS might be challenging. This, however, can be crucial, particularly for forensic investigations, where the legal status of a substance is of paramount importance.

Fig. 1.

Transformation and activation of clonazafone (A) in clonazepam (C) via the intermediate desglycylclonazafone (B)

NSOs are another rapidly evolving class of NPS. Starting with the recording of the first NSO in 2009 by the United Nations Office on Drugs and Crime, more and more derivatives of various substance classes have entered the illicit drug market over the years, including cyclohexylbenzamides such as U-47,700, phenethylpiperidines such as isobutylfentanyl, nitrobenzimidazoles such as isotonitazene, etc [8]. The first reported appearance of fentanyl derivatives in the European Union (EU) was in 2012, making a highly potent class of opioids available on the NPS market [9]. The emergence of fentanyl derivatives has coincided with a significant escalation in the prevalence of fatal overdoses in North America, which can be attributed to their high potency [10]. After introducing stricter legislations to ban the available fentanyl derivatives, the first representatives of the class of nitazenes occurred in the EU starting in 2019 [8]. Since then, the number of available nitazenes grew continuously –in the year 2023 alone, six new nitazenes were reported through the EU Early Warning System, and in 2024, the number of newly reported analogues even increased to seven [11]. Nitazenes were originally developed by the pharmaceutical industry in the late 1950 s, but have never gained marked access [12]. The pharmacological activity of numerous nitazenes is similar to that of fentanyl; for some derivatives, it is even higher [13].

Opioids, in general, also possess a high potential for abuse and dependency [14, 15]. Furthermore, they carry a significant risk of fatal overdose [16–18]. Moreover, the risk of a deadly intoxication is further elevated when combined with BZD use [19, 20]. This case report presents an additional example that demonstrates the significantly increased risk associated with the concurrent administration of opioids and BZDs.

Case history

According to an involved witness, a twenty-year-old male consumed BZDs, alcohol, and cannabis together with his friends. After he gradually lost consciousness, the emergency services were called, who, in turn, noted asystole and immediately began with cardiopulmonary resuscitation (CPR) and administered, amongst other drugs, naloxone. The resuscitation was terminated approximately thirty minutes later due to the development of livores. On external inspection of the corpse, the body presented no CPR-independent injuries. The only remarkable finding was a white, powdery substance in the nostrils. After about six hours of death, the body was cooled to 5 °C for 14 h at the Institute of Forensic Medicine until the autopsy was conducted. The autopsy of the 185 cm tall and 53 kg heavy (body mass index 15.5 kg/m²) male corpse revealed cerebral oedema (brain weight 1400 g), hemorrhagic pulmonary oedema (lung weight 1920 g), aspiration of gastric contents, petechial hemorrhages of the gastric mucosa, and a full urinary bladder (500 mL urine). The internal organs showed no further abnormalities, and no non-CPR-related injuries were found.

Seizures from the site of death – a small bottle filled with white powder labeled as clonazafone and a non-labeled bottle filled with a liquid – were analyzed at the Forensic Science Institute Zurich (FOR-ZH; Switzerland; data not shown). Gas chromatography-mass spectrometry (GC-MS) and infrared spectroscopy (IR) analysis confirmed the powder’s identity as clonazafone and revealed that the liquid was fluoro-etonitazene in propylene glycol, which was most likely intended for use as a vaping liquid.

Materials and methods

Chemicals and reagents

Reference substances for clonazafone and fluoro-etonitazene were not available from an official vendor. Therefore, seized clonazafone powder was obtained from FOR-ZH. Since GC-MS and IR analysis found no impurities, the powder was used as a relatively pure reference material suitable for semi-quantification. Fluoro-etonitazene was available from the test purchase of a vaping liquid at the Institute of Forensic Medicine, Freiburg, Germany. The main compound was confirmed to be N,N-diethyl-2-(2-(4-(2-fluoroethoxy)benzyl)−5-nitro-1H-benzo[d]imidazol-1-yl)ethan-1-amine (fluoro-etonitazene) via GC-MS and nuclear magnetic resonance (NMR) spectroscopy as part of the ADEBAR project [21] and isolated from the propylene-glycol matrix by employing preparative HPLC. The isolation procedure is provided in the supplementary information. The purity of the isolate was 99% as determined via high-performance liquid chromatography with diode-array detection (HPLC-DAD). The pH meter 827 pH lab from Metrohm AG (Herisau, Switzerland) was used for pH measurements. Methanolic solutions of the deuterated internal standards (IS) clonazepam-d4 (0.1 mg/mL) and fentanyl-d5 (0.1 mg/mL) were obtained from Lipomed AG (Arlesheim, Switzerland) and LGC Standards GmbH (Wesel, Germany), respectively. Water was purified with a Purelab Ultra millipore filtration unit (Labtech, Villmergen, Switzerland), acetonitrile and methanol of HPLC grade were obtained from Fluka (Buchs, Switzerland), formic acid was obtained from Biosolve (Valkenswaard, Netherlands), and ammonium formate from Merck KGaA (Darmstadt, Germany). Cloned enzyme donor immunoassay (CEDIA) kits were purchased from Thermo Scientific (Zug, Switzerland). Glucuronidase G7017 from Helix pomatia (EC 3.2.1.31), purchased from Sigma-Aldrich (Buchs, Switzerland), was used for clonazafone analysis. β-Glucuronidase/arylsulfatase from Helix pomatia was used for flouro-etonitazene analysis, as were sodium acetate trihydrate, ammonium formate, and glacial acetic acid, all of which were purchased from Merck KGaA (Darmstadt, Germany). All other reagents and chemicals were from Sigma-Aldrich (Steinheim, Germany) and of the highest grade available, but at least p.a. (pro analysi) quality.

Biological samples

Postmortem samples of the deceased were collected during the medico-legal autopsy, approximately 20 h after death. They included femoral blood, urine, heart blood, vitreous humor, stomach content, and muscle tissue, taken from the thigh. Additionally, urine samples from three anonymized ante mortem routine clonazepam cases were taken for comparison of metabolites.

Routine systematic toxicological analysis

Routine toxicological analysis was performed on femoral blood and urine. Urine was screened by CEDIA using an Indiko Plus device (Thermo Scientific, Braunschweig, Germany) for common drugs of abuse (opiates: cut-off 300 ng/mL, cocaine: cut-off 300 ng/mL, cannabis: cut-off 50 ng/mL, amphetamines: cut-off 500 ng/mL, methadone: cut-off 300 ng/mL, barbiturates: cut-off 200 ng/mL, benzodiazepines: cut-off 200 ng/mL, buprenorphine: cut-off 5 ng/mL, lysergic acid diethylamide (LSD): cut-off 0.5 ng/mL, and γ-Hydroxybutyric acid (GHB, by Bühlmann Laboratories AG): cut-off 40 µg/mL), followed by an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) ion trap screening after simple dilution with mobile phase 1:1 and addition of IS (Bruker amaZon^®; Maurer/Wissenbach/Weber database [22]). Femoral blood and urine were screened for ethanol and other volatile compounds by headspace GC flame ionization detection (HS-GC-FID). Quantification of drugs (including amphetamine and clonazepam) in femoral blood was performed by LC-MS/MS using a fully validated scheduled multiple reaction monitoring (MRM) method [23].

LC-HRMS screening

Identification of fluoro-etonitazene, clonazafone, as well as potential metabolites and intermediates, was done in femoral blood, heart blood, stomach content, and urine using LC-HRMS analysis. The samples were prepared in duplicate by conducting protein precipitation and reconstitution of the evaporated supernatant, according to Steuer et al. [23]. Data was acquired on the ZenoTOF™ 7600 system (Sciex, Darmstadt, Germany) in positive-ionization mode, coupled with a Shimadzu Nexera Series 40D X3 (Shimadzu, Kyoto, Japan). Chromatography was performed on a Phenomenex (Aschaffenburg, Germany) Kinetex PS C18 column (100 × 2.1 mm, 2.6 μm) using a gradient elution with 10 mM ammonium formate buffer in water containing 0.1% (v/v) formic acid (pH 3.5, eluent A) and acetonitrile containing 0.1% (v/v) formic acid (eluent B). The gradient was adopted from Steuer et al. [23]. Injection volume was 10 µL. MS parameters were set as follows: Source voltage at 5500 V, source temperature at 450 °C, curtain gas at 35 psi, collision gas at 7 psi, and ion source gases 1 and 2 at 50 psi. The measurement was conducted using an untargeted method in data-dependent acquisition (DDA) mode, including a maximum of 10 candidate ions above an intensity threshold of 10 counts per second (cps). The MS scan was conducted with a DP of 60 V, a CE of 5 V, and an accumulation time of 0.1 s, while the observed mass range reached from 100 to 1000 Da. At the MS/MS level, the scan range was 50–1000 Da with a DP of 60 V, a CE of 35 ± 15 V, and an accumulation time of 0.05 s. Fragmentation with electron-activated dissociation mode was conducted by applying an energy of 10 eV and a current of 3500 nA. To increase the intensity of the fragment ions, zeno-pulsing was turned on. The DDA settings included dynamic background subtraction and exclusion of former candidate ions for 3 s after 3 occurrences. Every three injections, an automatic mass calibration with the calibrant delivery system was conducted.

The targeted data processing method included clonazafone (m/z 391.0804), desglycylclonazafone (m/z 334.0589), clonazepam (m/z 316.0484), and fluoro-etonitazene (m/z 415.2140). In addition, the deglycinylated variant of desglycylclonazafone (2-Amino-2’-chlor-5-nitrobenzophenon) and the respective hydroxy derivatives and glucuronides of clonazafone and its deglycinylated derivatives were included (Table 1). The identity of clonazepam, clonazafone, and fluoro-etonitazene was verified by comparing the MS/MS spectra with MS/MS spectra generated by analyzing the pure substance in solution. Potential metabolites were tentatively identified through their fragmentation pattern on the MS and MS/MS level, including the presence of a characteristic chlorine cluster for metabolites of clonazafone. The mass error had to be below 2 ppm on MS-level as a further identification criterion.

Table 1.

Parent substances and potential metabolites in the targeted processing method, including molecular formula, theoretical mass to charge ratio (m/z), detection in urine with mass error below 2 ppm, confirmed by isotope pattern in MS and specific fragments in MS/MS, peak area, and retention time in urine

Compound	Molecular formula	m/z	Detected in urine	Mass error [ppm]	Peak area [cps]	Retention time [min]
Clonazafone (C2)	C17H15ClN4O5	391.0804	yes	0.59	9.97*103	7.12
Hydroxy-clonazafone	C17H15ClN4O6	407.0753	no	/	/	/
Hydroxy-clonazafone-glucuronide	C23H23ClN4O12	583.1074	no	/	/	/
Reduced clonazafone	C17H18ClN4O3	361.1062	no	/	/	/
Desglycylclonazafone (C1)	C15H12ClN3O4	334.0590	yes	0.63	5.92*103	6.88
Hydroxy-desglycylclonazafone	C15H12ClN3O5	350.0544	no	/	/	/
Hydroxy-desglycylclonazafone-glucuronide	C21H20ClN3O11	526.0865	no	/	/	/
2-amino-2’-chloro-5-nitrobenzophenone (C6)	C13H9ClN2O3	277.0380	yes	1.05	1.74*105	14.65
Hydroxy-2-amino-2’-chloro-5-nitrobenzophenone (C5)	C13H9ClN2O4	293.0324	yes	0.20	3.66*105	13.68
Hydroxy-2-amino-2’-chloro-5-nitrobenzophenone-glucuronide (C3)	C19H17ClN2O10	469.0645	yes	0.11	2.76*106	8.80
Clonazepam (C4)	C15H10ClN3O3	316.0484	yes	0.47	6.20*104	10.22
Fluoro-etonitazene	C22H27FN4O3	415.2140	no	/	/	/
4’-hydroxy-nitazene	C20H24N4O3	388.1905	no	/	/	/
4’-hydroxy-nitazene-O-glucuronide	C26H32N4O9	564.2226	no	/	/	/
N-desmethyl-fluoro-etonitazene	C20H23FN4O3	387.1827	no	/	/	/
N, N-didesmethyl-fluoro-etonitazene	C18H19FN4O3	359.1514	no	/	/	/
Reduced fluoro-etonitazene	C22H29FN4O	385.2398	no	/	/	/

Quantitation of clonazafone

Extraction of body fluid samples was conducted according to Steuer et al. [23], with reconstitution in 100 µL instead of 300 µL to increase sensitivity. In brief, 50 µL of IS mixture containing clonazepam-d4 and 50 µL of methanol were added to 200 µL of the liquid biological sample, followed by protein precipitation with 400 µL acetonitrile. After shaking for 10 min at 1400 rpm, the samples were centrifuged at 10’000 rpm for 10 min, and 250 µL of the supernatant was mixed with 20 µL of 20% formic acid and evaporated to dryness afterwards. 200 mg of muscle tissue was combined with 50 µL IS mixture, 50 µL methanol, and 400 µL acetonitrile and homogenized using BeadBug^TM−6 (Benchmark Scientific, Sayreville, USA) and further processed like body fluids. Urine was analyzed with and without enzymatic hydrolysis by glucuronidase. For the enzymatic hydrolysis, a safe-lock tube was filled with 50 µL of IS mixture, 50 µL methanol, 200 µL of urine, and 25 µL of 1 M sodium acetate buffer pH 5. 25 µL of glucuronidase was added, and the sample was incubated for 1 h at 60 °C in an Eppendorf ThermoMixer^® C (Eppendorf, Hamburg, Germany).

The quantitative analysis was performed using a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fisher, San Jose, California, USA) coupled with a Sciex 5500 QTrap linear ion trap quadrupole mass spectrometer (Sciex). The measurements were carried out in duplicates, in the unscheduled MRM, positive-ionization mode using the transition m/z 391.0 to 86.9 as quantifier (MRM 1) and m/z 391.0 to 115.1 as qualifier (MRM 2) with the following potentials: declustering potential (DP) 56 V, entrance potential (EP) 10 V, collision energy (CE) 22 V (MRM1) and 16 V (MRM2), and cell exit potential (CXP) 12 V (MRM1) and 8 V (MRM2). Clonazepam-d4 was used as IS. An LC gradient elution was performed using a Phenomenex (Aschaffenburg, Germany) Kinetex^® C18 column (50 × 2.1 mm, 2.6 μm) with water containing 0.1% (v/v) formic acid (eluent A) and acetonitrile containing 0.1% (v/v) formic acid (eluent B). Injection volume was 10 µL. Starting conditions were 5% eluent B until 0.5 min, then the percentage increased continuously to 40% until min 3. Between 3 and 4 min, the percentage of B increased further to 90% and remained at this level until min 4.5. Between min 4.5 and 4.6, the proportion of B decreased again to 5%, where it remained until the end of the method at min 5.5. The flow rate was set to 0.5 mL/min throughout the run. The MS was controlled by Analyst^® 1.7.2 software (Sciex), and quantitation was performed with MultiQuant^® 3.0.3 software (Sciex).

This LC-MS/MS method for the identification and quantification of clonazafone was developed for femoral blood, and was also used to quantify clonazafone in heart blood, vitreous humor, and muscle tissue. To ensure the absence of interfering signals with clonazafone or clonazepam-d4, ten blank blood samples from different sources were examined. Further, two blank blood samples spiked with IS (zero samples) were examined. The calibration range reached from 0.1 to 100 ng/mL, and the quality control QC levels were at 0.5, 16, 40, and 80 ng/mL. Additionally, an external calibration for urine was conducted, with a calibration range between 50 and 1200 ng/mL. The QC levels were at 150, 400, and 1000 ng/mL. A dilution of the stomach content (1:100) was also quantified by applying the calibration range of urine.

Quantitation of fluoro-etonitazene

The blood samples were processed into serum prior to analysis. Blood serum samples were prepared as follows: To 100 µL of serum, 10 µL of the IS mixture containing fentanyl-d5, 100 µL of 10 M ammonium formate solution, and 1 mL of ice-cold acetonitrile (−20 °C) were added. The samples were then shaken overhead for 5 min and subsequently centrifuged at 4000 rpm for 10 min. Next, the clear organic supernatant (approximately 1 mL) was transferred to a 2 mL clear glass Q-Trap vial and evaporated under nitrogen at 40 °C. For analysis, the dried residue was reconstituted in 100 µL of mobile phase (eluent A/B (95:5, v/v)). The gastric content and the muscle tissue were analyzed like the blood serum samples.

The urine sample was prepared as follows: To a test tube with 100 µL of phosphate buffer pH 6, 10 µL of the internal standard mixture, and 100 µL of the urine sample were added. An additional 10 µL of β-glucuronidase/arylsulfatase was added for the measurements, including enzymatic hydrolysis. The test tube was then gently shaken and incubated for 2 h at 45 °C. After enzymatic hydrolysis, 200 µL of 10 M ammonium formate solution and 1.5 mL of ice-cold acetonitrile (−20 °C) were added to the urine sample. Next, the sample was shaken overhead for 5 min and centrifuged at 4000 rpm for 10 min. The clear organic supernatant was transferred to a 2 mL clear glass Q-Trap vial and evaporated under nitrogen at 40 °C. For analysis, the dried residue was reconstituted in 100 µL of mobile phase (mobile phase A/B (95:5, v/v)).

Analyses were carried out using a QTrap 5500 mass spectrometer (Sciex) coupled to a Nexera 2 HPLC system (Shimadzu). Fluoro-etonitazene was detected using an unscheduled MRM approach utilizing the following ion transitions in positive mode: m/z 415.0 to 153.1 (qualifier 1), m/z 415.0 to 100.1 (quantifier), and m/z 415.0 to 72.0 (qualifier 2). MS parameters were set as follows: Source voltage at 4500 V, source temperature at 500 °C, curtain gas at 40 psi, collision gas at 9 psi, ion source gas 1 and 2 at 60 and 70 psi, respectively. MRM parameters were set as follows: DP at 60 V, EP at 10 V, CE at 45 V, and CXP at 13 V. Fentanyl-d5 was used as IS. The chromatographic separation was performed employing a Phenomenex Kinetex F5 analytical column (100 × 2.1 mm; 2.6 μm) fitted to a F5 pre-column (2.1 mm). Injection volume was 10 µL. Mobile phase A was 1% acetonitrile, 0.1% formic acid, and 2 mM ammonium formate in water; mobile phase B was acetonitrile with 0.1% formic acid and 2 mM ammonium formate. The gradient profile was as follows: Starting with 5% B at a flow rate of 0.5 mL/min, at 1 min, the concentration of B was increased linearly to 22.5% over 3.5 min, followed by another linear increase to 32.5% B over 6.25 min. Subsequently, the concentration of B was rapidly increased to 95% over 2.75 min and held at this composition for an additional 2 min. Finally, the system was returned to initial conditions (5% B) and equilibrated for 3.5 min. The total flow rate was maintained at 0.5 mL/min throughout the gradient, except for the final equilibration step, where it was reduced to 0.25 mL/min. The total run time was 19.5 min. The MS was controlled by Analyst^® 1.7.2 software (Sciex), and quantitation was performed with MultiQuant^® 3.0.3 software (Sciex).

This LC-MS/MS method was used to identify and quantify fluoro-etonitazene in femoral blood, urine, heart blood, muscle tissue, and stomach contents. To ensure the absence of interfering signals with fluoro-etonitazene or fentanyl-d5, seven blank blood serum samples from different sources were examined. The calibration range reached from 1 to 200 ng/mL.

Results and discussion

Systematic toxicological analysis

In the CEDIA^® immunoassays of urine the defined cut-off values for amphetamines and benzodiazepines were exceeded. The LC-ion trap MS screening in urine revealed amphetamine, metabolites of ibuprofen, ritalinic acid, clonazepam, and 7-amino-clonazepam. Ethanol analysis was negative in femoral blood; a trace (< 0.1 g/kg) was detected in urine. Quantitative analysis of drugs in femoral blood revealed concentrations of 110 ng/mL amphetamine, 1.5 ng/mL clonazepam, and 140 ng/mL 7-amino-clonazepam.

Analysis of clonazafone, fluoro-etonitazene, and metabolites.

In LC-HRMS analysis, unchanged clonazafone was only detected in urine and gastric content. The mass spectrum and proposed fragmentation are given in Fig. 2. The two aromatic fragments, m/z 260 and 277, and the two fragments of the side chain, m/z 87 and 115, could be clearly assigned. Since the aromatic system is chlorine-substituted and the side chain is not, metabolic changes at these sites, such as hydroxylation, could also be assigned accordingly due to the isotope pattern (see supplementary Figure S2).

Fig. 2.

LC-HRMS/MS spectrum of clonazafone and fragment assignment

An extracted ion chromatogram of the parent compound clonazafone and a few preliminary major metabolites in urine is shown in Fig. 3. Clonazafone (C2) is not only activated to clonazepam (C4), but is also metabolized by several pathways. Next to the intermediate desglycylclonazafone (C1), the two clonazafone-related analytes C5 and C6 were identified in urine. The clonazafone metabolite C3 was abundant in both blood and urine. 2-Amino-2’-chlor-5-nitrobenzophenone (C6, the deglycinylated variant of desglycylclonazafone) also appears to be hydroxylated at the aromatic system (C5) during metabolism and finally partially glucuronidated (C3) before excretion (Fig. 4). The identified glucuronidated metabolite C2 was present at a relatively high abundance, with a peak area of approximately 300 times greater than the peak area of clonazafone itself (see Table 1). The corresponding MS/MS spectra can be found in the SI (see Figure S3-8).

Fig. 3.

Chromatogramm of clonazafone and its metabolites desglycylclonazafone (C1), clonazafone (C2), hydroxy-2-amino-2’-chloro-5-nitrobenzophenone-glucuronide (C3), clonazepam (C4), hydroxy-2-amino-2’-chloro-5-nitrobenzophenone (C5), and 2-amino-2’-chloro-5-nitrobenzophenone (C6) in urine

Fig. 4.

Proposed metabolic pathway of clonazafone to metabolite C3 (hydroxy-2-amino-2’-chloro-5-nitrobenzophenone-glucuronide)

However, in three routine urine samples with declared clonazepam intake, the analytes C3, C5, and C6 were present as well. Therefore, C3, C5, and C6 do not seem to be specific metabolites of clonazafone and cannot serve as markers for clonazafone intake. Phase II conjugates of clonazafone could not be detected using the presented method, and enzymatic hydrolysis did not lead to a significant increase in the concentration of clonazafone.

Clonazafone itself could not be detected in blood or vitreous humor. In urine and muscle tissue, concentrations of 39 ng/mL and 3.0 ng/g were detected, respectively. The highest concentration of clonazafone was identified in stomach content at approximately 76’000 ng/mL. Taking into account the total volume of the stomach content of 150 mL, a quantity of approx. 11 mg clonazafone was estimated to be present in the stomach. The elevated concentration of clonazafone in the stomach content could be attributed to either oral ingestion of the substance or a trapping mechanism within the acidic stomach environment, owing to its alkaline properties and the ensuing protonation within the stomach. The fact that clonazafone is abundant in urine leads to the assumption that clonazafone was partly excreted unmetabolized. Therefore, the detection of clonazafone seems to be an obvious key distinction between the intake of the pharmaceutical drug clonazepam and the drug of abuse clonazafone. The distinction between the two substances may be of forensic interest, as the legal regulation of the two substances differs. The distinction is not trivial, as the metabolism of each substance overlaps significantly with the other. After ingestion, clonazafone is activated to clonazepam via the intermediate desglycylclonazafone, which was detected in gastric content and urine as well (together with clonazepam). However, desglycylclonazafone and clonazepam are subject to a chemical equilibrium and exhibit a preference for desglycylclonazafone at an acidic pH value, as shown for structural equivalent benzodiazepines [24]. Therefore, the presence of the intermediate might rather be an indication of the presence of clonazepam than the presence of clonazafone.

Fluoro-etonitazene was only detected in stomach content by LC-HRMS, and metabolites of fluoro-etonitazene were not detected in any matrix - most likely due to lacking sensitivity of the untargeted LC-HRMS approach. The quantification of fluoro-etonitazene by LC-MS/MS in femoral blood of the deceased revealed a concentration of 3.3 ng/mL. The concentration in urine increased from 1.7 ng/mL to 2.5 ng/mL after enzymatic hydrolysis, indicating a glucuronidation of the parent compound. Other opioids, opiates, or NSOs were not detected in femoral blood or urine. The opioid receptor antagonist naloxone was detected in femoral blood (4.9 ng/mL), resulting from the administration by the first responders. The muscle tissue, gastric content, and heart blood of the deceased also revealed the presence of fluoro-etonitazene, with the highest concentration in the stomach content at 1’300 ng/mL, resulting in a total amount of 0.2 mg in the stomach. In muscle tissue and heart blood, concentrations of 6.2 ng/g and 16 ng/mL were found, respectively. All results are listed in Table 2. The large difference in concentration between heart blood and peripheral blood, with a ratio of approximately 4.8, suggests an extensive potential for postmortem redistribution [25].

Table 2.

Average concentrations of Clonazafone and fluoro-etonitazene measured in duplicates by LC-MS/MS

Matrix	Clonazafone conc. [ng/mL]/[ng/g] (postmortem samples)	Fluoro-etonitazene conc. [ng/mL]/[ng/g] (postmortem samples)
Femoral blood	not detected	3.3
Urine	39	1.7
Urine (after glucuronidase digestion)	50	2.5
Heart blood	not detected	16
Vitreous humor	not detected	not analyzed
Muscle tissue	3.0	6.2
Stomach content	76’000	1’300

Due to the limited material of the biological case specimen, it was not possible to conduct quantification through a standard addition method, which would have been more accurate. Also, full validation of the used methods was not conducted, since the analytes of interest were rare analytes without certified reference material or reference values concerning their efficacy available. However, the selectivity was examined and no interfering signals with clonazafone, fluoro-etonitazene and their internal standards were detected in the blank blood samples or zero samples.

Cause of death and toxicological significance score (TSS)

No data is yet available on the pharmacological activity of fluoro-etonitazene. However, according to data generated in vitro, etonitazene leads to one of the strongest activations of the µ-receptor compared to fentanyl and some other representatives of the nitazenes, which is why it can be assumed that fluoro-etonitazene is also highly potent [26]. Clonazafone, once metabolically transformed to clonazepam, along with the (less) active metabolite 7-amino-clonazepam, enhances the respiratory depressant effect of the NSO fluoro-etonitazene. Considering the analysis results of clonazepam and 7-amino-clonazepam, it should also be taken into account that the metabolism of clonazepam to 7-amino-clonazepam by bacteria continues even after death [27]. Therefore, the results in this regard probably do not reflect the concentrations at the time of death. There are no data on postmortem stability of clonazafone. However, degradation of clonazafone to desglycylclonazafone may continue after death or even after blood sampling. Also, the pH of the blood at 6.8 could have caused postmortem changes, for example, concerning the equilibrium reaction between clonazepam and desglycylclonazafone. Furthermore, it cannot be ruled out that the ongoing metabolism of the active substances was facilitated by a prolonged agonal phase, which could potentially also have influenced the fluoro-etonitazene concentration. Ethanol could not be detected in blood, whereas in urine, a trace was detected. The presence of the detected trace of ethanol in urine could either be a result of postmortem microbiological processes, or derive from an ethanol consumption. Therefore, a certain degree of alcoholization cannot be ruled out, especially as a consumption of ethanol was declared, and the deceased had probably been in an agonal phase for up to several hours. Amphetamine was detected in a pharmacologically active concentration and might have contributed to death, e.g., by masking the depressive effects of the other analytes. In conclusion, the analytical results together with the autopsy findings showing cerebral and pulmonary oedema strongly indicate a mixed intoxication leading to respiratory depression as a plausible cause of death.

The toxicological significance score (TSS) was assessed according to the method described by Elliott et al. for each of the substances involved [28]. Fluoro-etonitazene, especially in combination with the benzodiazepine clonazepam, probably had a significant impact on respiration, as is the case with most opioids in combination with benzodiazepines [19, 20]; therefore, fluoro-etonitazene was estimated to have a TSS of 3. Clonazafone was probably primarily involved as a prodrug and was given a TSS of 2.

Conclusion

The cause of death was reported as a combined drug intoxication involving fluoro-etonitazene, clonazepam, and amphetamine. Clonazafone was detected in the urine, which suggests that the clonazepam detected probably resulted (at least partially) from clonazafone intake. Several metabolites resulting from phase I and phase II metabolism were also detected. However, the metabolites detected could not be conclusively attributed to clonazafone or clonazepam intake. For example, the intermediate desglycylclonazafone can also be formed in an equilibrium reaction from clonazepam at acidic pH; thus, it cannot serve as clear evidence of clonazafone ingestion. To improve the ability to prove clonazafone intake, more samples need to be investigated.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the conception and design. Material preparation, data collection and analysis were performed by Jonas Malzacher and Benedikt Pulver. The first draft of the manuscript was written by Jonas Malzacher and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

Open access funding provided by University of Zurich.

Declarations

Competing interests

The authors have no relevant financial or non-financial interests to disclose.