First evidence in an oversea French department of the deadly risk of protonitazene use: about 5 post mortem cases.

Protonitazene is a synthetic benzoimidazole opioid of the nitazenes class, developed in the 1950s as an effective analgesic, but never released in the market due to severe side effects and major risk of dependence. The laboratory was involved in the determination of the cause of death for 5 subjects deceased in a French department of the Indian Ocean. The 5 victims were male, aged between 20 and 35 years. The first 2 victims were found dead in their prison cell and the 3 other victims were found dead in a squat. Therefore, we have developed and validated a specific procedure to identify and quantify the drug in post mortem specimens using LC-MS/MS. The procedure involves extraction of 0.5 mL fluid at pH 9.5 with a mixture of organic solvents in presence of 20 ng fentanyl-d5 used as internal standard. Linearity of the method was verified from 0.1 to 20 ng/mL in both whole blood and urine (r2 = 0.9983 and 0.9993, respectively). The limit of detection was estimated at 0.05 ng/mL in each matrix. Protonitazene was identified at < LOQ to 0.8 ng/mL, 0.4 to 2.9 ng/mL and 3.0 to 8.0 ng/mL in femoral blood, urine and bile, respectively. Post mortem concentrations were very low, which is consistent with reported high toxicity of protonitazene. As nitazenes represent a growing threat to public health in various parts of the world, this method seems to be a good response to the challenges posed by the identification of this class of substances.

Anabolic steroids and extreme violence: a case of murder after chronic intake and under acute influence of metandienone and trenbolone.

A 32-year-old male went to the police to claim he just killed his girlfriend by inflicting several stabs with a kitchen knife. He was very nervous and particularly aggressive. About 90 min after the assault, a blood specimen was collected with natrium fluoride as preservative. The blood was free of alcohol, pharmaceuticals and drugs of abuse, but tested positive by LC-MS/MS for metandienone (32 ng/mL) and trenbolone (9 ng/mL). The perpetrator admitted regular consumption of anabolic steroids to enhance his muscular mass, as he was a professional security agent. To document long-term steroid abuse, a hair specimen was collected 3 weeks after the assault, which tested positive for both drugs. Segmental analyses revealed in the proximal 1.5 cm segment, corresponding to the period of the assault, the simultaneous presence of metandienone (11 pg/mg) and trenbolone (14 pg/mg), while only metandienone (3 pg/mg) was identified in the distal 1.5 cm segment. As aggressiveness and violence can be associated with abuse of anabolic steroids, the aetiology of this domestic crime was listed to be due impulsive behaviour in a context of antisocial lifestyle.

Perspectives in Evaluating Selective Androgen Receptor Modulators in Human Hair: A Short Communication.

BACKGROUND: As hair testing increases the window of drug detection and permits the differentiation of long-term use from a single exposure when performing segmental analyses (which also allows establishing the pattern of use), this matrix should be considered as a suitable complement to standard investigations in clinical, forensic, and sport toxicology. The authors were recently involved in 3 cases where hair analysis was used to demonstrate the use of selective androgen receptor modulators (SARMs), including ligandrol (LGD-4033), andarine (S-4), and ostarine (S-22). SARMs are increasingly being abused as "safe" alternatives to steroids. METHODS: After decontamination using dichloromethane, hair specimens were segmented, cut into very small segments (<1 mm), incubated overnight in a buffer, and extracted using a mixture of organic solvents. Drugs were tested using liquid chromatography-tandem mass spectrometry and confirmed using liquid chromatography/HRMS. RESULTS: The determined concentrations were as follows: ligandrol, 14-42 pg/mg; andarine, 0.1-0.7 pg/mg; and ostarine, 3-21 pg/mg. CONCLUSIONS: To enhance performance, SARMs must be used on a long-term basis, which can have serious clinical consequences, including liver damage, myocardial infarction, and blood clots. Hair testing for SARMs has additional benefits versus urine analysis as it can detect the parent compound and numerous metabolites.

Evidence of ostarine cross-contamination via sweat in 2 athletes sharing the same neoprene hamstring sleeves. An original situation of drug transfer where the anti-doping rule violation was suspended by the sport authorities.

The presence of ostarine, a selective androgen receptor modulator (SARM) in an athlete's sample constitutes one of the most frequent anti-doping rules violation. It is possible to challenge this violation but it is the athlete who has to demonstrate he / she is innocent. The conditions to evidence no fault or negligence are mostly based on 2 points: 1. the athlete or his/her legal representative must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete about the fact that he / she did not knowingly take the prohibited substance, i.e. that the violation was not intentional. During a 2-weeks period, a male athlete tested two times positive for ostarine in urine (<0.1 ng/ml) and he challenged these results. His hair and nail tests returned negative (LOQ at 0.5 pg/mg). He admitted using two neoprene hamstring sleeves of another athlete who confessed abusing ostarine. This was confirmed in his hair (190 pg/mg), his fingernail clippings (780 pg/mg) and his toenail clippings (45 pg/mg). To document the presence of ostarine in the hamstring sleeves and therefore possible drug transfer, the hamstring sleeves were analysed. Ostarine was identified in 12 different selected pieces (about 1 g) of the sleeves at concentrations ranging from 3 to 142 pg/g. Sport authorities (USADA) agreed that the most likely source of contamination was the hamstring sleeves, thus confirming the scenario of drug transfer and gave the athlete a no fault.

Interest of hair tests to discriminate a tail end of a doping regimen from a possible contamination in case of challenging an anti-doping rule violation. V. Case reports involving trimetazidine, a drug where the concentration after a single 20 mg dose has been established.

The identification of trimetazidine, a medicine used for treating stable angina pectoris and for preventing angina attacks, has been recently observed in doping cases involving high profile athletes from various countries over the world. In all the files where the authors have been involved, the urine concentration of trimetazidine was low (<2 ng/mL), and the athletes argued that contamination was the source of their adverse analytical finding. It is possible to challenge imposed sanctions in relation to an adverse analytical finding, but it is the responsibility of the athlete to demonstrate he/she is innocent and can qualify for no fault or negligence. When the delay between the urine collection and the notification of the violation was not too long (less than 6 months), these athletes requested a head hair test. Trimetazidine was analyzed by an original LC-MS/MS method involving pH 9.5 borate buffer overnight incubation of 20 mg and subsequent solvents extraction in presence of trimetazidine-D8 used as internal standard. Linearity was verified from 1 to 200 pg/mg (R2 = 0.9987). Limit of detection of the method was 0.1 pg/mg. The hair specimen of a male subject, collected 4 weeks after single oral ingestion of 20 mg trimetazidine, tested positive at 146 pg/mg in the corresponding segment. Concentrations of trimetazidine measured in several hair specimens (n = 5) collected from athletes challenging their anti-doping rule violation were below 1 pg/mg, which is consistent with incidental exposure due to contamination. This is the first evidence that trimetazidine is incorporated in human hair after a single therapeutic dose administration.

Evidence of ostarine excretion in oral fluid after a single controlled oral administration.

The presence of ostarine, a selective androgen receptor modulator (SARM) in an athlete's urine specimen constitutes one of the most frequent anti-doping rules violation as the drug is listed as a member of the S1.2 class "other anabolic agents" of the World Anti-doping Agency Prohibited List, forbidden in- and out-competition. It is possible to challenge this violation but it is at the charge of the athlete to prove innocence. The conditions to evidence no fault or negligence are mostly based on 2 points: 1. the athlete must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete that the violation was not intentional. Some months before the Olympic games, a female athlete was suspended by a national anti-doping agency because of an adverse analytical finding for ostarine. She claimed that her violation was due to drug transfer when kissing her boyfriend, who did not inform her about his ostarine daily intake. To document this claim (excretion of ostarine in oral fluid in sufficient amounts), a male volunteer ingested 17.3 mg of ostarine (dose verified by 1H NMR). Oral fluid was collected over 8 h using the NeoSal™ collection device and was tested by liquid chromatography coupled to tandem mass spectrometry. Maximal ostarine concentration was 468 ng/mL at T + 15 min, which can also be partially attributed to mouth contamination. Ostarine was detectable during the whole period of test, with concentrations at 1-2 ng/mL after T + 4 h. These results support drug transfer during kissing and subsequent possible contamination of the partner.

Hair and dietary supplements testing to identify contamination with roxadustat in an adverse analytical finding.

Roxadustat is an oral inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylase, which increases endogenous erythropoiesis. WADA has included roxadustat and other HIF stabilizers on its list of prohibited substances. We describe here the case of an elite athlete (female, 31 years old, 168 cm and 53 kg) with an adverse analytical finding (AAF) with concentration of roxadustat in her urine at 0.289 ng/mL in the A sample and 0.529 ng/mL in the B sample (83% higher than A). A stability study was carried out, showing total stability of roxadustat at this concentration in urine exposed to light for 50 h, so photoisomerization degradation cannot explain the difference in concentration. Her urine had been completely negative in a control test carried out three days previously, while roxadustat had been shown to be present in urine for at least 20 days after administration of pharmacologically effective doses to an athlete. Hair concentration was 0.39 and 0.35 pg/mg in the segments corresponding to the presumed period of intake, with few adjacent segments also positive (0.29-0.33 pg/mg), likely explained by cosmetic treatments. Concentrations found in a patient treated with a pharmacologically active dose (between 100 and 120 mg 3 days a week) were more than 100 times higher (between 41 and 57 pg/mg). Numerous supplements and pharmaceuticals taken by the athlete were analyzed. Only collagen powder showed the presence of roxadustat, at a very low but highly variable concentration (100 pg/g-1000 pg/g). A female volunteer (58 years old, 169 cm and 65 kg), taking this powder at the same doses as the athlete (10 g of powder 5 times for 6 days) presented 7 roxadustat-positive urine samples (although lower than those observed in the athlete) out of 34 sampled over 7 days, the difference in powder sampling location, age, weight, height, pharmacokinetic parameters variability and level of sporting activity between the athlete and the volunteer probably explaining the difference in concentrations observed. All these results could be consistent with an AAF due to contamination by dietary supplements, which are becoming increasingly common due to the current exposome of athletes in our society.

Simultaneous detection of three hypoxia-inducible factor stabilizers-molidustat, roxadustat, and vadadustat-in multiple keratinized matrices and its application in a doping context.

In a doping case, a top athlete challenged an anti-doping rule violation, involving molidustat. Molidustat is a stabilizing agent of the hypoxia-inducible factor (HIF) recently developed. It is currently undergoing clinical trials for anemia associated with chronic kidney disease. HIF stabilizers are banned at all times by the World Anti-Doping Agency (class S2). Because of their pharmacological proprieties, these new drugs can enhance athletic performance. The athlete's defense wanted to analyze multiple keratinized matrices as they allow long-term investigations. Requests concerning HIF stabilizers are constantly growing. We have therefore developed a liquid chromatography coupled with tandem mass spectrometry method to identify and quantify three molecules of this class: molidustat, vadadustat, and roxadustat. Thirty milligrams of keratinized matrices were incubated in 1 mL of pH 8.4 diammonium hydrogen phosphate buffer for 16 h at 40°C with 1 ng of testosterone-D3, used as internal standard. After extraction with ethyl acetate/diethyl ether (80/20), the organic phase was evaporated, and the dry residue was reconstituted in 30 µL of initial phase. The method was linear from 5 to 1000 pg/mg for the three analytes. Limits of quantification were 2, 0.5, and 5 pg/mg for molidustat, roxadustat, and vadadustat, respectively. The analysis of the athlete's head hair (collected 1 month after the urine test) showed a concentration of molidustat of 135 pg/mg, and his beard hair and his fingernails clippings contained 55 and 40 pg/mg, respectively.

In vitro characterization of protonitazene metabolites, using human liver microsomes, and first application to two urines collected from death cases.

Protonitazene, or N,N-diethyl-5-nitro-2-[(4-propoxyphenyl)methyl]-1H-benzimidazole-1-ethanamine, is a novel synthetic opioid, which belongs to the nitazene family. Over the last four years, nitazenes have re-emerged on the new psychoactive substances market and have been reported in several fatal intoxication cases. The metabolism of several nitazene analogues have already been studied, but to date, no data exists regarding protonitazene. The aim of the study was the detection of protonitazene and its metabolites in authentic human urine collected in two fatal intoxication cases, comparing the data after in vitro incubation with human liver microsomes, and subsequent analysis by ultra-performance liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-high-resolution mass spectrometry. Protonitazene metabolites, including N-desethyl-protonitazene, 5-amino-protonitazene and 4-hydroxy-nitazene, were characterized in vitro and were identified in the urine of both cases. The ratios between metabolites and parent protonitazene, higher than 1, were calculated to estimate the proportionality of metabolites. The results suggest that testing protonitazene metabolites should increase the window detection of exposure to protonitazene.

Is it really possible to kill with insulin without leaving traces? From lifesaver to killer, the issues surrounding the analytical characterization of postmortem insulin illustrated by an exemplary case.

Evidence of an insulin overdose is very complicated in the medico-legal field. The analysis and subsequent interpretation of results is complex, especially when treating postmortem blood samples. The instability of insulin, the special pre-analytical conditions and the absence of specific analytical methods has led most laboratories not to analyze insulin in their routine with a consequent underestimation of cases. This paper aims to assess the difficulties associated with the analytical characterization of insulin by describing a case that typically represents most of the inconveniences encountered following a suspected insulin overdose. The case concerns a man found dead at home by his brother. After an external examination, which did not reveal a specific cause of death, toxicological analysis was requested which did not reveal any substance of toxicological interest. Only 9 months later, it was reported to the toxicologist that the subject was diabetic, on insulin lispro treatment and that three empty syringes were found next to his body. Following analysis by LC-high-resolution mass spectrometry, the presence of insulin lispro at a concentration of 1.1 ng/mL, a therapeutic concentration, was evidenced. Despite the low concentration found, overdose cannot be excluded and this paper will describe the criteria evaluated to reach this conclusion. This case highlights that the interpretation of a postmortem insulin concentration is very complex and requires the evaluation of various elements including the circumstances of death, the subject's medical history, the interval between death and sampling and the sample storage.