Testing for protonitazene in human hair using LC-MS/MS.

Protonitazene is a synthetic benzoimidazole opioid of the nitazenes class, developed in the 1950s as an effective analgesic, but never released on the market due to severe side effects and possible dependence. Despite its increasing use as a new psychoactive substance starting in 2019, its detection in human hair of intoxicated and deceased consumers has never been reported. We present the development and validation of a specific procedure to identify protonitazene in hair by LC-MS-MS. Drugs were incubated overnight at 40°C in 1 mL borate buffer, pH 9.5 with 20 mg pulverized hair and 1 ng/mg fentanyl-d5 used as internal standard. Drugs were then extracted with a mixture of organic solvents. The chromatographic separation was performed using a HSS C18 column with a 15 min gradient elution. Linearity was verified from 1 to 100 pg/mg. The limit of detection was estimated at 0.1 pg/mg. No interference was noted from a large panel of natural and synthetic opioids, fentanyl derivatives or other new synthetic opioids. Protonitazene was identified at 70 and at > 7600 pg/mg in the whole head hair specimens of two male subjects deceased from acute drug overdose in jail. Protonitazene was also identified at 14 and 54 pg/mg in two living co-prisoners. As nitazenes represent a growing threat to public health in various parts of the world, this method was developed in response to the challenges posed by the identification of this class of substances.

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.

Window of detection of cocaine-related alkaloids in oral fluid collected with the FloqSwab™ after coca tea consumption.

Coca tea is a popular drink in some countries of South America, where it is presented as a safe energy preparation, based on a limited total content of cocaine of ∼3-5 mg. Tea bags can be bought with no legal considerations in these countries both by locals and tourists, but its consumption can have consequences when consumed overseas. Driving under the influence of cocaine is banned in most of the places in the world and can be documented by oral fluid testing. A study was implemented with coca tea bags (Coca & Muna) purchased in Peru, after a French attorney-at-law contacted the laboratory to assess the involvement of coca tea in the positive oral fluid results of a driver. Ten healthy volunteers consumed 250 mL of coca tea containing 4.5 mg of cocaine. No volunteer reported any change in behavioral effects after consumption of the coca tea. Oral fluid was collected with a swab (FloqSwab™, Copan) over 8 h to follow the elimination of cocaine and its major metabolites (benzoylecgonine and ecgonine methylester). This is the procedure used by the French police. All samples were analyzed by UHPLC-MS-MS after Quantisal™ buffer desorption. As the device does not allow measurement of the amount of collected fluid, the results are qualitative. This is in accordance with the French law that requires a yes or no response about the presence of cocaine, with a minimum required performance level of 10 ng/mL of cocaine or benzoylecgonine. Parent cocaine was identified for 30-120 min. Benzoylecgonine and ecgonine methylester were identified between 1 and 8 h, with a large inter-individual variation. Although it is generally accepted that a 4-5 mg cocaine dose has no significant pharmacological effect, the consumption of coca tea can lead to the suspension of a person's driving license due to a positive oral fluid test.

Hidden administration of 5-APB in a dancing club of New Caledonia documented by urine analysis: about 3 cases.

1-Benzofuran-5-ylpropan-2-amine or 5-APB is a new psychoactive substance (NPS) with empathic effects close to ecstasy (MDMA). Although 5-APB has been observed in fatality cases, the drug has not yet been reported in the context of hidden administration for behaviour impairment, also known as drug-facilitated crime. Such a situation was recently observed on 3 separate occasions in the same dancing club of New Caledonia. It involves 3 women, aged 27, 29, and 33 years who presented, after having drunk a cocktail, anxiety, abnormal movements of the inferior jaw, and aggressiveness. No memory loss was noticed. About 12 h after the event, a urine specimen was collected in the 3 cases. Comprehensive toxicology was requested and only 5-APB was identified, at 6, 8, and 14 ng/mL. Urine ethanol tested negative, which is consistent with the limited intake before the event occurred. These results have demonstrated that NPS are circulating in New Caledonia, which was not previously reported, and that 5-APB, like ecstasy, can be used to modify the behaviour of a subject, as it can be done by a chemical weapon.

Fatal Rectal Injection of 3-MMC in a Sexual Context: Toxicological Investigations Including Metabolites Identification Using LC-HRMS.

The dead body of a 59-year-old man was found at his home by his father. The subject was naked in the corridor, wearing a black hood and a collar around the neck where a dog leash was attached. An empty syringe was discovered in the decedent's rectal vein. The autopsy revealed marked asphyxia signs with no indication of violence or trauma. Femoral blood, urine and hair (4 cm, brown) were collected and submitted for comprehensive toxicological investigation. Initial screening did not indicate the presence of ethanol or any other over-the-counter or prescription pharmaceuticals. Routine toxicology screening by liquid chromatography-tandem mass spectrometry (LC-MS-MS) tentatively identified only the cathinone stereoisomer(s), 3-methylmethcathinone (3-MMC) or mephedrone. Analysis by gas chromatography-MS to distinguish between the isomers revealed the presence of 3-MMC, which was subsequently quantified by LC-MS-MS. Femoral blood and urine concentrations were 1,437 and 16,733 ng/mL, respectively. In 4 × 1-cm hair segments, 3-MMC was detected at <10 pg/mg (limit of quantification). Further analysis by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) allowed the identification of two metabolites in both blood and urine: desmethyl-3-MMC and hydroxyl-3-MMC. The pathologist established the cause of death in this case as acute 3-MMC poisoning in the context of ChemSex.

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.

Toxicological Investigations in a Death Involving 2,5-Dimethoxy-4-Chloamphetamine (DOC) Performed on an Exhumed Body.

During a party in another country, several adults sniffed a powder presented as being lysergic acid diethylamide (LSD). The next morning, two subjects, including a French citizen, were found dead. After a body examination that concluded that the death was due to respiratory and cardiac collapses, the French citizen's corpse was returned to France and buried. Four years later, the body was exhumed, and an autopsy that did not reveal traumatic injury was performed. During the autopsy, biological specimens were collected. A comprehensive toxicological screening, followed by confirmation using ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS-MS) revealed the presence of 2,5-dimethoxy-4-chloamphetamine (DOC) in all specimens: liver (99 ng/g), spleen (28 ng/g), bone (14 ng/g), lung (15 ng/g) and pubic hair (32 pg/mg). No other drug, including pharmaceuticals and drugs of abuse were identified, but the circumstances of specimen collection can influence drug stability. Literature survey about DOC stability in biological material did not contribute in interpretation as there is no data dealing with storage for about 4 years before quantitative analysis. A stability study was performed at the laboratory. Blank blood was spiked with DOC at 1 mg/L, stored at + 4°C and -20°C and regularly tested over 6 months. The percentages of concentration remaining from the initial concentration of DOC stored for 6 months at + 4°C and -20°C were 53% and 59%, respectively. To characterize the metabolite(s) of DOC, the drug was incubated with a pool of human hepatic microsomes and the cofactors required to ensure the functioning of the main phase I enzymes. The incubation media were analyzed by liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS), and the results showed hydroxy-DOC. However, the hydroxy-metabolite was not identified in the liver or spleen of the subject. Although the French pathologist considered that it was more likely than not a toxic death, it is difficult to attribute the death to DOC alone, as it was impossible to test for ethanol and other chemically instable drugs. This case presents original data, which can be useful to increase the knowledge in designer drug toxicity.

Major discrepancy between factual antibiotic resistance and consumption in South of France: analysis of 539,037 bacterial strains.

The burden of antibiotic resistance is currently estimated by mathematical modeling, without real count of resistance to key antibiotics. Here we report the real rate of resistance to key antibiotics in bacteria isolated from humans during a 5 years period in a large area in southeast in France. We conducted a retrospective study on antibiotic susceptibility of 539,107 clinical strains isolated from hospital and private laboratories in south of France area from January 2014 to January 2019. The resistance rate to key antibiotics as well as the proportion of bacteria classified as Difficult-to-Treat (DTR) were determined and compared with the Mann-Whitney U test, the χ2 test or the Fisher's exact test. Among 539,037 isolates, we did not observe any significant increase or decrease in resistance to key antibiotics for 5 years, (oxacillin resistance in Staphylococcus aureus, carbapenem resistance in enterobacteria and Pseudomonas aeruginosa and 3rd generation cephalosporin resistance in Escherichia coli and Klebsiella pneumoniae). However, we observed a significant decrease in imipenem resistance for Acinetobacter baumannii from 2014 to 2018 (24.19-12.27%; p = 0.005) and a significant increase of ceftriaxone resistance in Klebsiella pneumoniae (9.9-24.03%; p = 0.001) and Enterobacter cloacae (24.05-42.05%; p = 0.004). Of these 539,037 isolates, 1604 (0.3%) had a DTR phenotype. Over a 5-year period, we did not observe a burden of AR in our region despite a high rate of antibiotic consumption in our country. These results highlight the need for implementation of real-time AR surveillance systems which use factual data.

Testing for SGT-151 (CUMYL-PEGACLONE) and its Metabolites in Blood and Urine after Surreptitious Administration.

The authors aim to report a case of surreptitious administration of a synthetic cannabinoid (SC) and the subsequent toxicological investigations to be able to document accurately the case for submission at a hearing. A dealer gave surreptitiously a substance to two juvenile migrants who experienced shakings and faintness. The laboratory received a blood sample from each of the two victims, who, according to the investigators, were probably exposed to SGT-151, a SC, also known as CUMYL-PEGACLONE. Blood and urine specimens from the dealer, who claimed being a user of SGT-151 were received at the same time. To characterize the metabolites of SGT-151, the drug was incubated with a pool of human hepatic microsomes and the cofactors required to ensure the functioning of the main Phase I and Phase II enzymes. The incubation media were analyzed by liquid chromatography coupled to high-resolution mass spectrometry. The metabolites identified following transformation by hepatic microsomes were mostly N-dealkylated SGT-151, mono-hydroxylated SGT-151 and di-hydroxylated SGT-151. The presence of SGT-151 (5.4 ng/mL) and its metabolite, N-dealkyl SGT-151, was confirmed in the dealer's blood. Two metabolites of SGT-151 (OH-SGT-151, diOH-SGT-151) were detected in the dealer's urine. SGT-151 (~1 ng/mL) and its metabolite N-dealkyl SGT-151 were detected in the blood samples of the two victims.

Managing ESBL-producing Enterobacteriaceae-related urinary tract infection in primary care: a tool kit for general practitioners.

In Southern France, approximately 4% of E. coli isolates from community-acquired urinary tract infections are extended spectrum beta-lactamase producers, while carriage rates for enterobacteriaceae (ESBL-E) range from 3 to 6%. General practitioners (GP) are unfamiliar with the management of patients harboring ESBL-E. Providing them with a specific tool kit should assist in their therapeutic approach and optimize antimicrobial prescription an ESBL-E tool kit was developed by a multidisciplinary team: infectious diseases (ID) specialists, microbiologists, pharmacologists, and nursing home staff. This tool kit includes treatment protocols, GP and patient information leaflets, a list of infection control measures, and contact details of ID physicians for specialized advice. A community-based (including nursing homes) prospective study was conducted in 2012 in Southeastern France to test the tool kit in the context of ESBL-E-related urinary tract infections (UTI). ESBL-E-related UTI were identified in 88 patients, 66 GPs were contacted by the microbiology laboratory, 56 stated they were offered the tool kit, 48 said they had received it, and 41 stated they had read its contents. Use of the tool kit was significantly correlated with appropriate antibiotic prescription, which concerned 36/39 tool kit users versus 13/20 non-users (p = 0.0125) and 40 GPs expressed an average satisfaction rate of 4.2 on a scale of 0 to 5. Availability of a specific tool for managing patients harboring ESBL-E, now completed with a website, can assist community-based GPs and improve antimicrobial prescription.