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• Antidiabetic intoxications can be fatal. • Hair analysis is important to analyze the historical consumption of antidiabetics in cas of factitious hypoglycaemias. • A method for identifying and quantifying antidiabetic agents in hair is needed. • In the method developed by the authors, it is possible to simultaneously identify and quantify 13 different antidiabetics. Oral antidiabetics are the drugs used to control blood sugar in diabetic subjects. The greatest risk of using these drugs is hypoglycaemia, which can be fatal if managed inappropriately. The diagnosis of hypoglycemia may be simple in diabetic subjects but can become a challenge in subjects with no history of exposure to these drugs. The major interest of testing for these compounds in hair is in the case of unexpected hypoglycaemias, as it enables discrimination between hypoglycaemias caused by antidiabetics and other reasons (e.g. insulinoma). Therefore it is important for a toxicology laboratory to screen for antidiabetics in hair due to the large window of detection this matrix allows associated to its long stability over time. In this study, a method has been developed and validated using liquid-chromatography coupled to tandem mass spectrometry for the analysis of 13 oral antidiabetics in hair. After addition of three different internal standards (hydroxy-tolbutamide-d9 for sulfonylureas, repaglinide-ethyl-d5 for glinides and vildagliptin-d3 for gliptins) and incubation in an ultrasonic bath in methanol, the hair was dissolved in NaOH and then subjected to liquid–liquid extraction. The validation procedure demonstrated an acceptable linearity for all compounds between 1 and 50,000 pg/mg. LOD and LOQ were between 0.5 and 5 pg/mg and 1–10 pg/mg respectively. Repeatability and reproducibility were below 20 % at two concentrations for all the analytes. The method was successfully applied to the hair of 18 diabetic patients under treatment of oral antidiabetics. The hair tested positive for gliclazide (3–21,400 pg/mg), sitagliptin (1.4–1.8 pg/mg), vildagliptin (3.3 – 1,740 pg/mg) and repaglinide (14.1 pg/mg). [ABSTRACT FROM AUTHOR]

• Development and validation of a rapid method for SARMs in hair using UHPLC-MS/MS. • Simultaneous analysis of 9 SARMs and 2 metabolic modulators in hair. • First identification of S23 in authentic human hair in a doping case. Selective Androgen Receptors Modulators (SARMs) are a new class of doping drugs that emerged in sport since 2008. Easy access on the Internet also leads to their misuse by amateurs. It seems important for a laboratory of toxicology to develop a targeted screening of SARMs, given their health risks. A method has been developed and validated for the analysis in hair of 9 SARMs (AC262536, ACP-105, andarine, LGD-4033, MK-0773, MK 677, ostarine, RAD 410 and S23) and 2 other metabolic modulators (GW501516, SR9009), using liquid chromatography coupled to tandem mass spectrometry. After addition of bicalutamide-D 4 used as internal standard and incubation in phosphate buffer pH = 9.5, 20 mg of hair samples were extracted with liquid/liquid extraction. Linearity was verified for all compounds between 0.5 and 50 and 2000 pg/mg. LOD and LOQ were determined between 0.1–20 and 0.5–50 pg/mg respectively, according to the various analytes. Intra- and inter-day precision (CV < 20%), matrix effects and recovery were evaluated for all compounds with CVs < 20%. The application and the interest of SARMs screening was demonstrated in a doping case. Three SARMs were detected namely andarine (120–1644 pg/mg), ostarine (1–9 pg/mg) and S23 (0.6–16 pg/mg) in 6x1 cm segments of the subject. [ABSTRACT FROM AUTHOR]

During the Belle Epoque, the use of cocaine, well known to the South American populations, spread among the European elite in the form of a tonic drink created from coca leaves macerated in Bordeaux wine, the Mariani wine. Nowadays, bottles in circulation are extremely rare but the investigations of a Parisian historian led him to the discovery of a bottle of wine that belonged to Angelo Mariani's estate at the end of the 19th century. Our laboratory was asked to analyse the contents of the bottle in order to search for the main active compounds and to estimate the state of preservation. The analysis of the centenarian product collected was aimed at identifying the alcohols and solvents by HS-GC/FID and HS-GC/MS after dilution in water, and the main alkaloids contained in all the samples by LC-HRMS screening and LC-MS/MS quantification. The Mariani wine presented an alcohol content of 15.7°. The screening and the subsequent dosage revealed the following substances: cocaine (86 ng/mL), benzoylecgonine (383 ng/mL), cocaethylene (17 ng/mL), ecgonine methyl ester (130 ng/mL), caffeine, cuscohygrine, cinnamoylcocaine and synephrine. The alkaloids found in the Mariani wine confirm the real presence of coca leaves in this drink. Their low concentrations may indicate drug instability. The presence of cocaethylene demonstrates that it can be formed without passing through the liver, which was supposed to be the site of production when cocaine and ethanol are present in the body at the same time. These appear to be the first report in the literature presenting the analysis of Mariani wine with the dosage of cocaine derivatives. • First analysis of a 19th century bottle of Mariani wine using MS technologies. • Determination of the real presence of cocaine and metabolites in Mariani wine. • Evidence of the presence of cocaethylene formed outside the liver system. [ABSTRACT FROM AUTHOR]

• Protonitazene is a novel synthetic opioid, which belongs to the nitazene family. • Nitazenes have re-emerged on the new psychoactive substances market and have been reported in several fatal intoxication cases. • Protonitazene metabolism was studied in vitro after drug incubation with human liver microsomes. • The identified metabolites were compared to the responses obtained in two authentic human urine specimens.. 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. [ABSTRACT FROM AUTHOR]

The selective androgen receptor modulators are a recent class of anabolic agents, used to improve athletic performance. Among these molecules, there is (2 S)-N-(4-cyano-3-trifluoromethylphenyl)− 3-(3-fluoro-4-chlorophenoxyl)2-hydroxy-2-methyl-propanamide, commonly known as S-23. This molecule appeared very recently on the doping market. As a result, very few data are available in the literature, and nothing has been published about long-term effects of S-23. The authors focused on the detection of S-23 and its metabolites in human urine, following a single oral administration of approx. 8 mg to a volunteer, using standard ultra-performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-MS/MS), and ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-Q-TOF-MS). To the best of the authors knowledge, this seems to be the first study ever achieved on S-23. In vitro experiment was performed, using human liver microsomes, in order to investigate the potential CYP- and UGT-dependent S-23 metabolites. Four metabolites were produced, which were identified as hydroxy-S-23 (C 18 H 12 O 4 N 2 ClF 4 : m/z [M-H-] 431.0423); O -dephenylate-S-23 (C 12 H 10 O 3 N 2 F 3 : m/z [M-H-] 287.0647); S-23-glucuronide (C 24 H 20 O 9 N 2 ClF 4 : m/z [M-H-] 591.0794) and hydroxy-S-23-glucuronide (C 24 H 20 O 10 N 2 ClF 4 : m/z [M-H-] 607.0743). After consumption of S-23, the parent drug was detectable in hydrolyzed urine from 2 h post administration up to 28 days, with concentrations ranging between 0.5 and 93 ng/mL. In the urine, only one of the four metabolites identified in vitro was detected, hydroxy-S-23. This metabolite was detected up to 28 days. It does not seem to increase the window of detection of S-23 as the ratio between hydroxy-S-23 and the parent drug was always lower than 1. Another metabolite, dihydroxy-S-23, not identified in vitro, was identified in the urine of the volunteer. Hair sample, collected one month after the consumption of a single tablet, was negative for S-23 and hydroxy-S-23, with a LOQ at 0.1 pg/mg. [Display omitted] • The authors focused on the detection of S-23 and its metabolites in human urine, following a oral administration of 8 mg. • Four metabolites were produced in vitro, using human liver microsomes. • Parent S-23 was detectable in hydrolyzed urine up to 28 days. • Hydroxy-S-23 was detected up to 28 days, in urine. Dihydroxy-S-23 was detected from + 8 hours up to 5 days. • Hair sample, collected one month after the exposure, was negative for S-23 and metabolites, with a LOQ at 0.1 pg/mg. [ABSTRACT FROM AUTHOR]

Clomiphene is a selective estrogen receptor modulator. It is indicated for the treatment of female infertility issues but in sport, it can be misused to stimulate endogenous testosterone secretion in men. Therefore, it has been prohibited at all times by the World Anti-doping Agency. The aim of this study was to get data to be able to interpret concentrations in athletes. A healthy volunteer (male, 62 years-old) ingested a single therapeutic dose of clomiphene (Clomid™, 50 mg). Strands of hair (blond, 4 cm) were collected one month after the ingestion. Body hair (beard, axillary, pubic and chest hair), and finger and toenails were collected over 4–5 months. A previous method was modified to identify and quantify clomiphene in keratinous matrices. 30 mg of specimen were sonicated and incubated in 1 mL of methanol, in presence of 200 pg of clomiphene-D 5 (internal standard). After centrifugation and evaporation of the organic phase, the samples were analyzed using LC-MS/MS. Linearity was verified in hair and nail clippings between 1 and 500 pg/mg. The limits of detection and quantification were determined at 0.3 and 1 pg/mg respectively. The study demonstrated that clomiphene tested positive in all the analyzed specimens at 9 pg/mg in head hair, from 28 to 486 pg/mg (body hair) and from 4 to 57 pg/mg (nails). Clomiphene was identified for the first time in multiple keratinous matrices. This study demonstrated that a single oral therapeutic dose is detectable in keratinous matrices over a long period of time. • A single dose of clomiphene (50 mg) is detectable in head hair, body hair and nail clippings. • A single therapeutic dose is detectable for a long period in keratinous matrices. • Higher concentrations of clomiphene are identified in body hair than in head hair. [ABSTRACT FROM AUTHOR]

• Letrozole can be diverted from its therapeutic use. • Letrozole counteracts feminizing effects due to anabolic steroids abuse. • Hair is the specimen of choice to document long-term drug exposure. • Two independent methods (LC-MS/MS and LC-HRMS) are essential for legal rules. Letrozole is a reversible aromatase inhibitor, used in the treatment of hormone-dependent woman cancer. No indication for medical use is available for men. In recent years, several cases of doping with letrozole have been observed, especially among high level athletes. Aromatase inhibitors reverse the harmful effects (feminizing) of the abuse of anabolic androgenic steroids. Letrozole is included on the list of products prohibited in- and out-competition by the World Anti-Doping Agency, under section S4.1. The aim of the present work was to develop a specific method to identify letrozole in human hair of a male amateur athlete by LC-MS/MS and confirmation by LC-HRMS, after incubation of 20 mg of matrix in 1 mL of methanol. The chromatographic separation was performed using a reverse phase column HSS C18 with a gradient elution of 15 min (from 87% to 5% of formate buffer adjusted to pH 3). Linearity was observed from 1 to 1000 pg/mg (r2 = 0.9999), after spiking blank hair with the corresponding amounts of letrozole. The limit of detection was estimated at 0.5 pg/mg and the lower limit of quantification was the first point of the calibration curve, i.e. 1 pg/mg. The precision was lower than 20% and there was no interference with the analytes by chemicals or any extractable endogenous materials present in hair. Letrozole was identified in the male amateur athlete hair at 310 pg/mg (segment 0–2 cm) and 245 pg/mg (segment 2–4 cm). [ABSTRACT FROM AUTHOR]

• Girls are better able to delay gratification than boys. • Boys with higher prenatal testosterone levels are less able to delay gratification. • Boys with higher prenatal testosterone levels show more attentions problems/overactive behavior. • Boys with more attention problems/overactive behavior are less able to delay gratification. Sex differences in self-control become apparent during preschool years. Girls are better able to delay their gratification and show less attention problems and overactive behavior than boys. In this context, organizational effects of gonadal steroids affecting the neural circuitry underlying self-control could be responsible for these early sex differences. In the present study testosterone levels measured in amniotic fluid (via ultra performance liquid chromatography and tandem mass spectrometry) were used to examine the role of organizational sex hormones on self-control. One hundred and twenty-two 40-month-old children participated in a delay of gratification task (DoG task) and their parents reported on their attention problems and overactive behavior. Girls waited significantly longer for their preferred reward than boys, and significantly more girls than boys waited the maximum period of time, providing evidence for sex differences in delay of gratification. Boys that were rated as suffering from more attention problems and overactive behavior waited significantly shorter in the DoG task. Amniotic testosterone measures were reliable in boys only. Most importantly, boys who waited shorter in the DoG task and boys who were reported to suffer from more attention problems and overactive behavior had higher prenatal testosterone levels. These findings extend our knowledge concerning organizational effects of testosterone on the brain circuitry underlying self-control in boys, and are of relevance for understanding how sex differences in behavioral disorders are connected with a lack of self-control. [ABSTRACT FROM AUTHOR]

The authors report the case of a 41-year-old man known as a polydrug addict and found dead at home. The initial toxicological screening was inconclusive for a toxic death. However, 7 small bags of unknown powders and crystals, supposed to be NPS, were found near the body and sent to the laboratory 3 weeks later. This article focuses on the interest of nuclear magnetic resonance (NMR) spectroscopy to solve this fatal case. The 7 items were identified by 1H NMR and their purities were measured by the quantitative NMR (qNMR) method using maleic acid as internal standard. The compounds were 4-chloro-α-pyrrolidinopentiophenone, dibutylone, 4-chloroethcathinone, 4-α-pyrrolidinohexuiphenone, 3,4-methylenedioxy-N-benzylcathinone, 3-methoxy-phencyclidine and α-pyrrolidinopropiophenone, with purities in the range 51-89%. In the biological specimens of the victim, only 3-methoxy-phencyclidine (3-MeO-PCP) was identified in cardiac blood, peripheral blood and urine at 743, 489 and 16,700 ng/mL. Hair (6 cm) concentration of the drug was 15,600 pg/mg. The other NPS were not detected. The peripheral blood concentration was higher than the values reported in previous intoxicated and fatal cases. The toxicological significance of the measured concentration in hair was difficult to establish because this is only the second case describing a test result for 3-MeO-PCP. [ABSTRACT FROM AUTHOR]

Objective: Nowadays, post-mortem computed tomography (PMCT) has become an integral part of Forensic practice. The purpose of the study was to determine PMCT impact on diagnosis of the cause of death within the context of the external examination of the body, when autopsy has, at first, not been requested.Material and Methods: We reviewed the records of 145 cases for which unenhanced PMCT was performed in addition to the external examination of the body from January 2014 to July 2015 at the Institute of Forensic medicine in Strasbourg (France). We confronted final reports from forensic pathologist to the corresponding PMCT reports. Data were collected in a contingency table and the impact of PMCT on the final conclusions of the forensic pathologist was evaluated via a Chi2 test.Results: PMCT results significantly impact the final conclusions of forensic pathologist (p<0,001). In some cases, PMCT permits etiological diagnosis by revealing a cause of death hidden from external examination (mainly natural death) or by supporting the clinical findings of the forensic pathologist. In other cases (traumatic death), PMCT enables fast and exhaustive lesion assessment. Lastly, there are situations where PMCT may be ineffective (intoxication, hanging or some natural deaths).Conclusion: Performing PMCT within the context of the external examination of the body when autopsy has, at first, not been requested could bring significant benefits in diagnosing the cause of death. The impact of PMCT varies depending on the circumstances of death. [ABSTRACT FROM AUTHOR]

The major problem after testing the hair of a child for drugs is the interpretation of the findings. In 2016, the laboratory received several hair specimens with the request to verify if there was any evidence of previous methadone exposure by the donor of the sample. Case 1 was a child admitted to the Emergency Unit for intense sedation and breathing difficulties. Cases 2-4 involved children found dead at home. In all cases, methadone and EDDP, its metabolite, were found in blood. After decontamination, the hair was analysed with LC-MS/MS for methadone and EDDP. The LOQ for both drugs was 10pg/mg. Concentrations were in the range 60-1590 and <10-220pg/mg for methadone and EDDP, respectively. In all of the cases, segmental analysis revealed approximately the same amount of drug along the hair lock. As a consequence, contamination was considered as an issue and interpretation of the results was a challenge that deserves particular attention. It must be considered that the amount of hair from children, available for analysis, can be low, particularly when several drugs have to be tested. This has consequences on the limit of quantitation and the identification of the metabolite(s). It must be also noted that hair from children is finer and more porous in comparison with adult (risk of higher contamination by sweat versus adults). It is very difficult to put any window of detection when testing for drugs in young children as hair growing is asynchronous. It is even more complicated as it has been demonstrated that drugs can be incorporated during pregnancy in the hair of the foetus, which will contribute to the positive findings after delivery. Several weeks or months after delivery, identification of a drug in hair can indicate: 1, in-utero exposure, or 2, exposure after delivery, or 3, a mix of both situations. Whereas the detection of drugs in a child's hair unambiguously shows drug handling in the environment of the child, it is difficult to distinguish between systemic incorporation into hair after ingestion or inhalation and external deposition into hair from smoke, dust, or contaminated surfaces. However, the interpretation of hair results with respect to systemic or only external exposure is particularly important in case of children for a realistic assessment of the toxic health risk. Practising scientists have the responsibility to inform the child protection authorities, courts, etc about these limitations. [ABSTRACT FROM AUTHOR]

Determining fetal death causes is a complex problem for the forensic pathologist. Beyond the medico-legal context, the expert must be able to evaluate the viability of the fetus at the time of death, to eliminate in-utero fetal death and to determine if the death is related to a fetal, a maternal, a placental cause, or simply related to obstetrical complications. The authors present the case of a 21-year-old woman who unexpectedly gave birth to a fetus during a party. As pregnancy was not acknowledged by the mother (regular menstrual cycles and use of hormonal contraception), no obstetrical check-up had been performed. She would have presented violent abdominal pain and expelled a mass in the toilet. The fetus body, enclosed in the amniotic pouch, and the placenta were found in the toilet. A forensic autopsy was performed jointly by a forensic pathologist and a specialist in fetal pathology. Histological, toxicological and genetic samples were collected. Body morphometry and bone maturation indicated a gestational age of 31–32 weeks of amenorrhea. A significant asphyxia syndrome and non-specific multi-visceral congestion were noted at autopsy. Histological analysis of the fetal tissues revealed a lung and skeletal muscle maturation in accordance with the estimated term. At the brain level, there were signs of anoxia and abnormal cortical development with periventricular nodular heterotopia areas. The placenta microscopic analysis revealed acute chorioamniotitis, the probable cause of the premature fetal expulsion. Toxicological analyses revealed the presence of ecstasy (48 ng/mL) and its metabolite MDA (2 ng/mL) in fetal blood. Although negative in blood, THC-COOH tested positive in urine (9 ng/mL). The fetus was repetitively exposed to cannabis, as Δ9-THC tested positive in hair (51 pg/mg). Maternal hair analysis on 4 × 3 cm evidenced a long-term use of cannabis, while results support single massive exposure to ecstasy. In this article, the authors try to explain the reflexive pathway carried out to establish death causes and the maternal toxic consumption imputability on the cerebral malformations and fetal death. This case illustrates both the interest of toxicological analyses in cases of fetal death and the importance of a collaborative work between forensic and fetal pathologists and toxicologists, which appeared critical to answer in the best conditions to the magistrates questions, as well as to the bereaved families. [ABSTRACT FROM AUTHOR]

A case of fatal left iliac penetrating wound in a thirty-year-old man, during a fight, was presented. The medical file described a transfixing five-centimeter large wound on the left iliac vein and a puncture wound on the left iliac artery. After performing an autopsy, we concluded that the death was secondary to multivisceral failure, secondary to hemorrhagic shock. The judge investigating the case requested a detailed forensic medical reconstructive opinion focusing on the sequence of events leading to the fatal blow. Our having access to the videorecording and the photographs of the crime scene, added to the testimonies of witnesses proved to be a great help to the investigation. The data from the crime scene video recordings allowed us to estimate the victim's physical activity after the fatal stabbing (running between 20 and 140 meters) and the time when he collapsed (between 32s and 1 min) after the stabbing which is rarely described with methods using Closed-Circuit Television material, especially in cases of fatal vascular injuries. Those data are usually estimated from eyewitnesses' accounts, which remain an unreliable method. • Using CTTV cameras allow documenting physical activity after fatal vascular injury. • In vascular injuries the vital prognosis depends largely on the venous lesions. • Further importance of the blood traces photography and vascular lesions description. [ABSTRACT FROM AUTHOR]

• Metformin intoxication can be fatal. • Hair analysis is important to analyze the historical consumption of metformin in fatalities. • Metformin hair concentration could be altered by sweat contamination due to fatal hypoglycaemia during the agony period. • Metformin hair concentration after acute poisoning are 10 times more elevated when compared to therapeutic users. Hair analysis is very useful for toxicological investigations since, by providing a wider detection window, it gives the possibility to perform a retrospective study on the historical consumption of a substance. Unfortunately, there are no data available for hair concentrations in metformin-related deaths. In this study, the authors present 2 cases of fatal metformin intoxication in which, for the first time, hair analysis was performed using a specific GC–MS/MS method. Metformin was tested positive in femoral blood (112.3 mg/L and 64.7 mg/L respectively) and cardiac blood (226.9 and 203.2 mg/L) of the two subjects. For case 1, other samples were also tested positive, including vitreous humor (31.1 mg/L) and gastric contents (773.5 mg/L). In case 2, metformin was measured at 844.9 mg/L in urine. Metformin hair concentrations were 28.3–44.8 and 22.5 ng/mg for both cases, respectively. The concentrations found in the 2 fatal cases are clearly higher than those obtained in a previous study with subjects under treatment (0.3–3.8 ng/mg) or those found in 3 post-mortem cases where metformin death was excluded (0.6–1.4 ng/mg). Excessive sweating during the agonal phase due to fatal hypoglycemia could explain these elevated concentrations as sweat can have contaminated the hair. [ABSTRACT FROM AUTHOR]

• Testosterone and estradiol from amniotic fluid were correlated with infants' performance on a mental rotation task and an index of vigilance. • For 6-months-old boys (N = 104) mental rotation but not vigilance was correlated with prenatal testosterone but not with estradiol. • For 6-months-old girls (N = 104) mental rotation but not vigilance was correlated with prenatal estradiol but not with testosterone. • In both cases, only about 5% of the within-sex variance in mental rotation performance was due to prenatal sex hormones indicating small effects. Sex differences in mental rotation, robust in adults, have recently been reported for infants' looking times although the pattern of results is not completely conclusive. In this context, organizational effects of gonadal steroids affecting the neural circuitry underlying spatial cognition could be (partly) responsible for the early sex difference. In the present study testosterone and estradiol levels measured in amniotic fluid via ultra performance liquid chromatography and tandem mass spectrometry were used to examine the role of prenatal sex hormones on infants' looking times during mental rotation. N = 208 six-month-old infants participated in an expectation of violation task with 3D cube figures. Mental rotation was defined as the difference in looking times for familiar versus mirrored cube figures whereas vigilance was defined as the sum of both looking times. Sex differences were absent for mental rotation as well as for vigilance. Most importantly, however, for boys mental rotation but not vigilance was correlated with prenatal testosterone but not with estradiol. For girls mental rotation but not vigilance was correlated with prenatal estradiol but not with testosterone although it has to be noted that the testosterone values for girls suffered from a floor effect. Only 5% of the within-sex variance was due to prenatal sex hormones indicating small effects. These findings extend our knowledge concerning organizational effects of prenatal sex hormones on the brain circuitry underlying spatial cognition. [ABSTRACT FROM AUTHOR]