Abstract
Synthetic cathinones, frequently used in chemsex, are difficult to identify in postmortem blood due to their short half-life and in vitro degradation. This unusual case of chemsex involving 3-CMC highlights the importance of freezing a blood aliquot and testing for metabolites. A 62-year-old man was found dead with chemsex paraphernalia beside him. Peripheral blood with sodium fluoride (PB), cardiac blood (CB) and urine were collected at autopsy. A PB sample was frozen at −20 °C for cathinone testing. In PB at +4 °C, alcohols were measured by HS-GC/FID, drugs and narcotics by LC-HRMS, GHB by GC-MS and cannabinoids by LC-MS/MS. Analysis of PB at +4 °C revealed a toxic concentration of GHB (330 µg/mL). Determination of cathinones in frozen PB revealed the presence of 3-CMC (17 ng/mL), dihydro-3-CMC and N-desmethyldihydro-3-CMC, while no cathinones or metabolites were identified in samples stored at +4 °C. Only metabolites were detected in urine. This case confirms the need to systematically screen for cathinones in suspected chemsex cases and, due to their instability, to screen for different metabolites and to freeze a blood sample after autopsy.
Keywords: 3-CMC, chemsex, postmortem blood, instability, metabolites
1. Introduction
3-CMC, or 1-(3-chlorophenyl)-2-(methylamino)-propan-1-one or clophedrone, is a synthetic halogenated, N-alkylated cathinone substituted on the ring with a chiral centre. It has two enantiomers ((R)-3-CMC and (S)-3-CMC) and two positional isomers, 2-chloromethcathinone (2-CMC) and 4-chloromethcathinone (clephedrone, 4-CMC). This New Psychoactive Substance (NPS) was first identified in a police seizure of 0.72 g of powder in Sweden in 2014 [1]. The spread of this molecule appears to have remained marginal until 2019, particularly compared with 4-CMC, but then accelerated, accounting for almost a quarter of the quantity of cathinones seized in Europe in 2020 [2]. 2-CMC (1-(2-chlorophenyl)-2-(methylamino)propan-1-one) appears to be much less widespread, and although it has been seized in at least two European countries since 2016, it has not been officially reported to the European Union Drug Agency (EUDA) [3]. Until 2022, 3-CMC appears to be manufactured, imported, distributed, sold and used as a “legal” substitute for 4-CMC as well as other controlled psychostimulants. In 2022, the European Union adopted a proposal to control this NPS, based on risk assessments [1]. According to information provided by seizures, 3-CMC is generally available on the drug market as a white to light yellow powder, but also as “crystals” or “stones”. More rarely, tablets, capsules, liquids or blotter papers have been reported. Adulterants and/or diluents typical of the stimulant market have also occasionally been reported, such as caffeine, benzocaine, paracetamol and mannitol [1]. It can be absorbed orally, by intravenous injection (slam), intra-rectally (plug) or snorted, but its corrosive nature makes this route not recommended on user forums. According to the latter, the suggested oral intake is 75 to 100 mg. Intravenous doses of 40 to 70 mg produce comparable effects [4].
Cathinones, particularly the isomers of MMC and CMC, are very frequently involved in the practice of chemsex [5]. Substances known as “Chems”, GHB, cathinones, cocaine, certain amphetamines (methamphetamine/MDMA) and ketamine are used to enhance sexual experience. Other molecules, such as poppers and PDE-5 inhibitors, are also considered as adjuvants to facilitate sexual intercourse [6]. In this context, cathinones are used for their psychostimulant and euphoric effects, improving alertness, motor excitement, empathy, entactogenic effect, increased stamina and sexual appetite [7]. They are often absorbed intravenously (slam) in order to obtain a stronger effect [8]. They are generally associated with GHB. This one, generally absorbed in the form of a precursor, mainly GBL, is highly popular in chemsex for its disinhibiting, euphoric and relaxing effects. It also increases stamina, quality of sexual performance and promotes relaxation of the anal sphincter. Unfortunately, overdoses are common and GHB is often the cause of acute or fatal poisoning observed in the context of chemsex, by respiratory depression and impaired consciousness [9].
Although 3-CMC is very often identified in drug crises, there is little pharmacological data on this molecule. Substituted methcathinones, such as 3-CMC and 4-CMC, are structurally like methamphetamine and MDMA and increase non-selective dopamine and serotonin release in the synaptic cleft [10,11,12]. They exhibit empathogenic properties and appear to have a lower abuse potential than pyrrolidinophenones [13,14]. In addition, limited animal studies suggest that 3-CMC can induce a cocaine-like discriminative stimulus and increase spontaneous locomotor activity in a dose-dependent manner [1]. It is recognized that 3-CMC is used recreationally, particularly in the context of chemsex, due to its clinical effects, which are considered similar to those of 4-CMC (euphoria, improved mood, increased energy). However, the possibility that some clinical effects may differ between positional isomers has been raised [12]. Although there is no conclusive data in the literature, it therefore seems important to be able to differentiate positional isomers. Finally, their instability in postmortem blood complicates their identification and quantification [15]. This case of chemsex with 3-CMC underlines the importance not only of early freezing of a blood sample, but also of assaying the various metabolites and determining the positional isomers.
2. Case Report
A 62-year-old male was found dead at home, naked, face down. At his side were found three sachets of white powder, two syringes, one full, the other almost empty with a residue of around 20 µL, an already opened bottle labelled “Iron Fist”, dildos, and two computer screens, suggesting a case of chemsex. According to investigators, the victim was alone at the time of death because all exits were locked from the inside and there was no evidence to suggest that anyone else was present. Powder sachets, the bottle and syringes were sealed and stored at room temperature. The autopsy, performed 4 days later, revealed cadaveric changes (green abdominal skin discoloration, corneal opacification, areas of epidermolysis), three recent punctures opposite a venous line in the left elbow, diffuse polyvisceral congestion, cardiomegaly with healthy coronary arteries, congestive and edematous lungs, signs of inhalation of gastric contents with fluid in the respiratory tract, and nail cyanosis. There were no signs of violence. Usual anatomopathological samples (whole heart, lung, kidney, liver fragments, one brain slice) and toxicological samples (peripheral blood with 1% sodium fluoride (NaF) (PB), cardiac blood (CB), gastric contents (GC) and urine (UR)) were collected. Hair, vitreous humour and bile were not collected. Toxicological samples were frozen at −20 °C for 10 days. They were then transported to the laboratory at +4 °C and received 48 h later. Upon arrival at the laboratory, given the probable context of chemsex and use of cathinones, an aliquot of PB was refrozen at −20 °C in a tube with NaF for specific NPS testing. All other samples were stored at +4 °C. Analyses were carried out within two months of death.
3. Materials and Methods
3.1. Chemical and Reagents
Water purity was 18.2 mΩ/cm (Millipore, Guyancourt, France). Whole blood was obtained from the Etablissement Français du Sang (EFS, Marseille, France). Acetonitrile, formic acid, ammonia and methanol were obtained from VWR International (Radnor, PA, USA), ammonium formate from Sigma Aldrich (Munich, Germany). GHB, GHB-D6 and mephedrone-D3 were purchased from LGC Standard (Molsheim, France), 3-CMC and 4-CMC hydrochloride salts from Cayman chemical (Ann Arbor, MI, USA), isoamyl nitrite and isoamyl alcohol from Toronto Research Chemicals (TRC, Toronto, ON, Canada). QuEChERS salts (Phenomenex, Le Pecq, France) were constituted by magnesium sulfate (MgSO4; 1.0 g), sodium chloride (NaCl; 1.0 g), sodium citrate tribasic dihydrate (SCTD; 0.5 g), and sodium citrate dibasic sesquihydrate (SCDS; 0.5 g). BSTFA + 1% TMCS (N,O-bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane) was obtained from Fluka (Saint-Quentin Fallavier, France).
3.2. Screening in Biological Fluids by High Resolution–Mass Spectrometry (LC-HRMS)
Non-targeted screening of biological fluids for active drug and narcotics principles and metabolites, including New Psychoactive Substances (NPS), was carried out using LC-HRMS method adapted from a previously published technique [16]. A total of 25 μL of internal standard (IS) and 40 mg of QuEChERS salts were added to 200 μL of the sample, followed by 750 μL of acetonitrile at −20 °C. The mixture was vortexed and centrifuged, the organic layer was evaporated and reconstituted in a mobile phase solution. A 5 μL sample was injected into a Vanquish liquid chromatographic system using a Luna® Omega Polar C18 column (Phenomenex, Le Pecq, France). The mobile phase consisted of water with 0.1% formic acid and methanol with 0.1% formic acid, with a 14 min total run time. MS analysis was performed using an Exploris 120 mass spectrometer (ThermoFisherScientific, Les Ullis, France) with heated electrospray ionization (HESI), operating in data-dependent analysis mode. Full scan analysis was conducted at 60,000 with a resolution of FWHM, followed by DDA at 16,000 FWHM. Data were processed using TraceFinder® v5.2 software for quantitative analysis of 3-CMC. For untargeted screening, Compound Discoverer® v3.3 (ThermoFisherScientific) software was used, cross-referencing spectra with databases including HighResNPS (https://highresnps.com/) and MzCloud™ (ThermoFisherScientific). Mean limit of identification (LOI) was 8.8 ng/mL (min = 0.05 ng/mL, max = 500 ng/mL) and mean limit of detection (LOD) was 0.25 ng/mL (min = 0.05 ng/mL, max = 5 ng/mL). The method was linear in the 5 to 500 ng/mL range (0.5 to 50 ng/mL for cannabinoids, 6-acetylmorphine and buprenorphine) with correlation coefficients >0.99, intra- and inter-day accuracy and precision were <15% for all compounds. The method was successfully applied to 31 routine samples. Additionally, the presence of 3-CMC metabolites was confirmed using fragmentation spectra provided in the study by Berardinelli et al. [17].
3.3. GHB Quantification in Biological Fluids by Gas Chromatography-Mass Spectrometry (GC/MS)
Twenty μL of blood or urine was treated with 45 μL of acetonitrile, in the presence of 20 μL of GHB-d6 (40 µg/mL) used as internal standard. After agitation and centrifugation (13,500 rpm for 5 min), the supernatant was collected and evaporated to dryness under nitrogen flow. The residue was derivatized by adding 35 μL BSTFA + 1% TMCS, then incubated for 25 min at 70 °C. A 1-μL aliquot of the derivatized extract was injected into the column of a GC system 7890A from Agilent (Santa Clara, CA, USA). The flow of carrier gas (helium) through the column (HP5-MS capillary column, 30 m × 0.25 mm I.D.× 0.25 µm film thickness) was 1.0 mL/min. The injector temperature was 270 °C and spitless injection was employed. The column oven temperature was programmed to rise from an initial temperature of 100 °C, maintained for 2 min, to 290 °C at 30 °C/min, maintained for the final 4 min. The MS system was a simple quadrupole coupled with an electronic impact ionization source (Agilent 5975C). Data were recorded in full scan and ions monitored were: 233 m/z (quantification) and 204 m/z (qualification) for GHB and 239 m/z for GHB-d6. Quantitative data processing: quantification for GHB was performed using the Mass Hunter Quantitative Analysis v10.1 software (Agilent). The method has been validated according to ICH guidelines.
3.4. Further Analyses in Biological Fluids
Alcohols were determined by headspace-gas chromatography with flame ionization detector (HS-GC/FID, Agilent, Les Ullis, France). Blood carboxyhaemoglobin level was determined spectrophotometrically using a CO-oxymeter (ABL 80, Radiometer SAS, Neuilly-Plaisance, France). Cannabinoids were measured with a specific LC-MS/MS technique using a published method [18]. An assay for antiretrovirals was carried out using LC-MS/MS in PB at +4 °C using a previously validated technique [19]. Finally, poppers are not routinely tested.
3.5. LC-MS/MS Screening on Seized Products
This method was adapted from routine procedure for screening biological matrices. Two hundred µL of a cold 95/5 acetonitrile–methanol was added to samples (100 µL). The mixture was vortexed, centrifuged, and the supernatant diluted 1:10. Chromatographic separation was achieved on a Waters H-Class system using a BEH C18 column (Waters, Guyancourt, France). Mobile phases consisted of acidified water with ammonia (A) and acidified acetonitrile (B). A 10 min gradient at 0.5 mL/min was applied with the column at 40 °C and a 1 µL injection volume. Mass spectrometric detection was performed on a Waters TQ-XS® triple quadrupole (Waters, Guyancourt, France) using ESI in MRM mode. Each compound was monitored with at least two transitions, and identification required correct retention time and ion ratios. Reference transitions and spectral parameters were obtained from in-house mixtures covering a wide range of therapeutics drugs, drugs of abuse, NPS, and specific targets including GHB. During cathinone screening, detection of transitions compatible with 4-CMC but with a shifted retention time suggested an isomer, later confirmed as 3-CMC using a certified reference material (CRM). Product ion confirmation was added by acquiring full-scan spectra at peak apex. Quantification of 3-CMC used a shortened C18 column and a faster gradient. The method was linear from 5 to 500 ng/mL with acceptable precision and bias across calibration levels and quality controls.
3.6. Gas Chromatography Coupled with Electronic Impact (GC-MS/EI) Screening on Seized Products
To complete the LC-MS/MS targeted screening, a non-targeted screening procedure was conducted with Gas Chromatography coupled with Electronic Impact (GC-MS/EI). The method was derived from our routine method for biological matrix screening. A total of 100 μL of sample was mixed with 200 µL of a cold 95/5 (v/v) mixture of acetonitrile and methanol, vortexed and centrifuged 10 min at 14,500 rpm. Two hundred microliters of supernatant were evaporated to dryness under nitrogen flow. The residue was derivatized by adding 25 μL BSTFA + 1% TCMS, then incubated for 30 min at 70 °C. A 1-μL aliquot of the derivatized extract was injected into the column of a GC system Trace 1300 (Thermo Fisher Scientific, Les Ulis, France). The flow of carrier gas (helium) through the column (capillary column Zebron ZB-Semivolatiles, 30 m × 0.25 mm I.D. × 0.25 µm film thickness, Phenomenex, Torrance, CA, USA) was 1.0 mL/min. Splitless injection was employed. The column oven temperature was programmed to rise from an initial temperature of 40 °C, maintained for 2 min, to 300 °C at 10 °C/min, maintained for the final 5 min. The MS system was a simple quadrupole coupled with an electronic impact ionization source (ISQ 7000, Thermo). Data were recorded in full scan and processed with Chromeleon v7.2.10. (Thermo). Databases used for compound identification were swdrugs 3.1 and NIST MS Search.
3.7. Poppers Analysis by HS-GC/FID
Poppers were analyzed by HS-GC/FID using a Trace 1300 chromatograph with a TriPlus 500 headspace unit (Thermo). Samples (50 µL) were mixed with methylene chloride and ethyl acetate (internal standard). Ten µL of this mixture was placed in a headspace vial. After 10 min at 120 °C, 1 µL of headspace gas was injected (1:120 split) into a ZB-624 Plus column (Phenomenex, Le Pecq, France). Oven temperature was increased from 50 °C to 100 °C. Helium was used as a carrier gas at 10 mL/min, and detection was performed by FID at 300 °C. Isoamyl nitrite and isoamyl alcohol were quantified with Chromeleon (Thermo). The method was linear between 5 and 100% purity, with CV and bias below 15%.
4. Results
4.1. Biological Fluids
Carboxyhaemoglobin levels in CB were physiological at 2%. Ethanol concentration was 0.06 g/L in PB at +4 °C, 0.06 g/L in GC and 0.2 g/L in UR, suggesting that ethanol had been absorbed several hours before death, although postmortem neoformation cannot be formally excluded in the absence of vitreous humour [20]. GHB measurement showed a concentration of 330 µg/mL in PB at +4 °C, and 921 µg/mL in UR, indicating severe intoxication [21]. Testing for cathinones in PB at −20 °C revealed the presence of 3-CMC at 17 ng/mL. In addition, four metabolites were detected: dihydro-3-CMC, N-desmethyldihydro-3-CMC, N-desmethyl-ω-carboxy-3-CMC and N-desmethyl-3-CMC, they were not quantified due to the absence of CRM. The same analysis in PB stored at +4 °C, CB, UR and GC did not reveal 3-CMC, but confirmed the presence of dihydro-3-CMC, N-desmethyldihydro-3-CMC, N-desmethyl-ω-carboxy-3-CMC and N-desmethyl-3-CMC (chemical structure of each metabolite in Figure 1).
Figure 1.
Chemical structure of 3-CMC and its four metabolites.
Extracted-ion chromatograms of 3-CMC and its four metabolites for the five matrices are presented in Figure 2. Finally, drug assays showed concentrations within the therapeutic range expected in the residual period for dolutegravir (623 ng/mL) and rilpivirine (74 ng/mL).
Figure 2.
Extracted-ion chromatograms of 3-CMC and its four metabolites for the five matrices.
4.2. Seized Products
LC-MS/MS analysis of the powder with standards of 3- and 4-CMC confirmed the presence of 3-CMC with a content close to 100% (range 87.9–117.0%, mean 104.0%) in the three sachets. The full syringe also contained 3-CMC (241 mg/mL for a volume of 600 µL). Analysis of the vial labelled “Iron Fist” showed the presence of amyl nitrite (82.9%) and isoamyl alcohol (14.9%). Neither GHB nor any of its precursors were detected in the seized products. However, the very small volume of liquid contained in the second syringe prevented an exhaustive search.
4.3. Pathological Examination
Pathological examination revealed mitral valve disease, micro-infarct scars and acute emphysema lesions, suggesting a mechanism of asphyxia with probable positional involvement, since the victim was found on the floor in a prone position.
5. Discussion
Taking into account toxicological and anatomopathological findings, the death was attributed to positional asphyxia, probably due to GHB overdose. The pre-existing heart disease probably accelerated the fatal process. The urine/blood ratio of GHB concentrations suggests a survival period of a few hours [21]. The circumstances surrounding the discovery of the body and the association of GHB, cathinone and poppers are strongly suggestive of chemsex [22,23] and the traces of venipuncture on the left elbow probably correspond to slamming [8].
On the other hand, there were no psychostimulants such as cocaine or amphetamines, generally found in this context [24,25]. Dolutegravir and rilpivirine do not correspond to pre-exposure (PrEP) or post-exposure prophylaxis (PEP), but probably to the victim’s long-term antiretroviral treatment, since these two molecules are combined in the specialty Juluca® [26,27], indicated for the treatment of human immunodeficiency virus type 1 (HIV-1) infection in virologically controlled adults on stable antiretroviral therapy. The risk of pharmacokinetic interactions between cathinones and certain antiretrovirals is regularly raised, as some antiretrovirals are inhibitors of CYP3A4 (ritonavir, cobicistat) [28]. In this case, dolutegravir is metabolized mainly by UGT1A1 and marginally by CYP3A4. It has no major inhibitory or inductive effect [29]. Rilpivirine, metabolized by the CYP3A family, does not appear to have an inhibitory or inductive effect on therapeutic doses [30]. It is therefore likely that these two antiretrovirals do not interact with cathinones, although metabolic pathways of 3-CMC are not yet fully described. GHB concentrations are comparable to those found in the literature in cases of chemsex [21,24,25]. The involvement of GHB in the determinism of death seems clear, since at high doses it induces bradycardia, convulsions and cardiorespiratory collapse [31]. However, in the majority of cases of death from acute intoxication in a chemsex context, it is the combination of GHB and cathinones that is highlighted [32]. In this case, given the delay between death and autopsy (4 days), the time taken to transport samples (48 h), variations in storage temperature and the instability of the molecule, it is possible that the initial concentration of 3-CMC was higher and may have played a role in the cause of death. Finally, the possible involvement of poppers, although not confirmed by blood tests, must also be considered, especially as the bottle containing the amyl nitrite and isoamyl alcohol had already been opened when the body was discovered. Finally, it should be noted that chemsex online, particularly since the lockdowns related to the COVID pandemic, is an important practice in France [33]. This type of activity has also increased, particularly among users living in rural areas, or who do not necessarily want social interaction. These chemsex sessions via screens are generally carried out via videoconferencing platforms (Zoom, Skype, etc.) [34]. Unfortunately, in this case, we did not have access to the results of the computer forensic expertise. To the author’s knowledge, this is the first documented case of death following virtual chemsex.
Pharmacokinetic data on 3-CMC are scarce. Lopes et al., in a recent study based on the use of human liver microsomes, determined that the half-life of 3-CMC is around 54 min and the intrinsic clearance around 15 mL/min/kg [35]. Plasma protein binding, particularly to albumin, is around 80% [36]. According to a recent publication, there are five phase I and 7 phase II metabolites of 3-CMC, the main reactions being ketoreduction and N-demethylation followed by O-glucuronidation [17]. In this study, the four most abundant phase I metabolites correspond to those found in the five biological matrices analyzed, with the highest concentrations found in urine. The ω-hydroxylated metabolite and glucuronoconjugated phase II metabolites were not detected. In the publication of Berardinelli et al., the detection was performed in negative ionization mode, the absence of detection is probably due to the analysis being carried out in positive mode in our case [17]. The N-acetylated metabolite was also not detected, but this was only detected in vitro in the study by Berardinelli et al. [17]. As presented in previous studies, reduction in the ketone in the beta position creates a chiral carbon, giving rise to two new diastereoisomers [17,37]. This explains the split appearance of the chromatographic peak of dihydro-3-CMC, as well as the shoulder of the N-desmethyl-dihydro-3-CMC peak.
In this case, 3-CMC was only detected in the peripheral blood sample stored at −20 °C. Interestingly, this sample had undergone a brief freeze–thaw–refreeze cycle, with a 48 h thawing period, unlike the other peripheral blood sample, which was not refrozen on arrival at the laboratory. The instability of cathinones is a phenomenon widely described in the scientific literature. According to Glicksberg and Kerrigan, unsubstituted and ring-substituted secondary amines are the least stable, while pyrrolidine analogues and methylenedioxy derivatives are the most stable [38]. Romanczuk et al. tested the stability of 3-CMC and 3-dihydro-CMC for 12 months in blood stored at +4 °C, in blood acidified with 1 M HCl at +4 °C, and finally in blood at −30 °C [3]. Their results clearly show that 3-CMC rapidly disappears in samples at +4 °C, and that acidification brings no major improvement, unlike freezing, which significantly delays 3-CMC degradation. The recommended solution is therefore to freeze the biological sample in the event of suspicion with NPS or in a chemsex context [39]. However, this information is often delayed. This underlines the importance of looking for metabolites, particularly dihydro-3-CMC, which is stable at +4 °C for 12 months [3]. The stability of other metabolites has not yet been tested.
6. Conclusions
In conclusion, this unusual case of death during an online chemsex session confirms the major role played by GHB and highlights the difficulties in interpreting post-mortem concentrations of CMC derivatives, due to their short detection window and high instability. Given the current state of knowledge regarding the relationship between the concentration and toxicity of CMC analogues, determining their involvement in the lethal mechanism is difficult, particularly since these are often polyintoxications involving multiple toxic mechanisms, as is the case here. The first step, in order to determine more precisely the concentrations of these molecules and then to understand the concentration/toxicity relationship, would be to freeze a sample of peripheral blood and systematically search for metabolites, notably dihydro-3-CMC, in the various samples. In this case, even though the concentration of 3-CMC is low, we must consider the possibility that this molecule may have contributed to the lethal process.
Abbreviations
The following abbreviations are used in this manuscript:
| 2-CMC | 2-chloromethcathinone |
| 3-CMC | 3-chloromethcathinone |
| 4-CMC | 4-chloromethcathinone |
| CB | Cardiac blood |
| EUDA | European Union Drug Agency |
| GBL | Gamma butyrolactone |
| GC | Gastric content |
| GC-MS/EI | Gas Chromatography coupled with Electronic Impact |
| GC-MS | Gas chromatography–mass spectrometry |
| GHB | Gamma hydroxybutyrate |
| HS-GC/FID | Headspace gas chromatography with flame ionization detector |
| LC-HRMS | Liquid chromatography-high resolution mass spectrometry |
| LC-MS/MS | Liquid chromatography-mass tandem spectrometry |
| MDMA | Methylenedioxymethamphetamine |
| MMC | Methylmethcathinone |
| NPS | New Psychoactive Substances |
| PB | Peripheral blood |
| QuEChERS | Quick, Easy, Cheap, Effective, Rugged and Safe |
| UR | Urine |
Author Contributions
Conceptualization, A.-L.P.-A. and N.F.; investigation: A.P., C.D., R.C., V.B.-C. and C.S.; supervision: A.-L.P.-A. and N.F.; writing—original draft: A.-L.P.-A., N.F., A.P., R.C. and M.-D.P.; writing—review and editing: A.-L.P.-A., N.F., M.-D.P. and G.L. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
In this case, neither the ethics committee nor the consent of legal representatives is required, as this is an expert assessment ordered by an investigating magistrate as part of an investigation into the causes of death to determine whether a third party was involved. The investigation is now closed.
Informed Consent Statement
In this case, neither the ethics committee nor the consent of legal representatives is required, as this is an expert assessment ordered by an investigating magistrate as part of an investigation into the causes of death to determine whether a third party was involved. The investigation is now closed.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author, upon reasonable request.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research received no external funding.
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author, upon reasonable request.




