Alpha-methyltryptamine and 5-(2-methylaminopropyl)-benzofuran (5-MAPB) fatal co-intoxication: case report and review of literature.

The scientific literature contains little reliable data regarding new psychoactive substances and designer drugs, making it difficult to assess toxic blood levels and potentially lethal threshold. Here, we report a fatal co-intoxication involving two uncommon drugs ‒ alpha-methyltryptamine (AMT) and 5-(2-methylaminopropyl)-benzofuran (5-MAPB) ‒ combined with exposure to benzodiazepines, ephedrine, and norephedrine. AMT and 5-MAPB were quantified using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC/MS-MS), revealing concentrations of AMT 4690 ng/mL and 5-MAPB 101 ng/mL in postmortem peripheral blood. We additionally reviewed the literature to help interpret the likely roles of these molecules in the occurrence of death.

α-Methyltryptamine (α-MT) Metabolite Profiling in Human Hepatocyte Incubations and Postmortem Urine and Blood.

α-MT is a hallucinogenic and stimulant tryptamine that was involved in several overdose fatalities in the United States and Europe. Analytical toxicology, and particularly the identification of metabolite biomarkers in biological samples, often is the only way to prove tryptamine use in clinical and forensic caseworks. We aimed to identify optimal α-MT metabolite biomarkers of consumption in humans. We identified α-MT metabolites in 10-donor-pooled human hepatocyte incubations and postmortem urine and blood from an α-MT overdose case using in silico metabolite predictions, liquid chromatography high-resolution-tandem mass spectrometry (LC-HRMS/MS), and software-assisted data mining. Nine metabolites were identified in vitro and eight additional metabolites were found in urine; five metabolites were found in blood. Metabolic transformations were hydroxylation, O-sulfation, O-glucuronidation, N-glucuronidation, and N-acetylation, consistent with the metabolism of structural analogues. The findings in hepatocyte incubations and postmortem samples were consistent, proving the in vitro model suitability. We suggest α-MT, hydroxy-α-MT glucuronide, and two hydroxy-α-MT sulfates as biomarkers of α-MT use in non-hydrolyzed urine; we suggest α-MT, two hydroxy-α-MT sulfates and N-acetyl-α-MT as biomarkers of α-MT use in blood. Further studies on α-MT clinical and forensic caseworks with different doses and routes of administration are necessary to better explore α-MT metabolism.

Toxicological detection of pholcodine in blood, urine and hair in three cases of fatal intoxication.

Pholcodine is an opioid antitussive reputed for its low toxicity and absence of addictive effect. We report three cases of pholcodine intoxication with fatal outcome. Large concentrations of pholcodine were quantified by gas chromatography coupled to mass spectrometry (GC/MS) in peripheral postmortem blood (respectively 2890 ng/mL, 979 ng/mL and 12,280 ng/mL). Segmental hair analyses by GC/MS and detected pholcodine in three 1.5-2 cm segments (38-161 ng/mg, 8.54-41.6 ng/mg, and 0.26-2.66 ng/mg, respectively). These findings underline that pholcodine can be involved in fatal poisoning and raise the question of misuse or abuse and of taking account of this drug in opioid overdose prevention policies.

Detection of insulins in postmortem tissues: an optimized workflow based on immunopurification and LC-MS/HRMS detection.

Diabetes is a worldwide disease in perpetual expansion. Type 1 and sometimes type 2 diabetic patients require daily human insulin (HI) or analog administration. Easy access to insulins for insulin-treated diabetics, their relatives, and medical professionals can enable abuse for suicidal or homicidal purpose. However, demonstrating insulin overdose in postmortem blood is challenging. Tissue analyses are contributive, as insulins can accumulate before death or undergo only limited degradation. The present study describes an assay for HI and synthetic analogs (lispro, aspart, glulisine, detemir and degludec, glargine and its main metabolite (M1)) in liver, kidney, muscle, and injection site samples. It is based on a 5-step sample preparation (reduction of tissue sample size, homogenization, extraction, concentration, and immunopurification) associated with liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/HRMS). Selectivity and limit of detection (LOD) for all target analogs were assessed in the above matrices. LOD was determined at 25 ng/g for HI and for analogs except detemir and degludec, where LOD was 50 ng/g in kidney and injection site samples and 80 ng/g in the liver and muscle. The method was applied to13 forensic cases in which insulin use was suspected.

Fatal 4-MEC Intoxication: Case Report and Review of Literature.

ABSTRACT: Synthetic cathinones are one of the major pharmacological families of new psychoactive substances and 4-methylethcathinone (4-MEC) has emerged in recent years as a recreational psychostimulant. We report a case of a 35-year-old man found dead and naked at home by his friend. Although no anatomic cause of death was observed at autopsy, toxicological analysis identified 4-MEC and hydroxyzine at therapeutic level (160 ng/mL). 4-Methylethcathinone was quantified in autopsy samples by a validated method consisting in liquid-liquid extraction and gas chromatography coupled to tandem mass spectrometry: peripheral blood, 14.6 µg/mL; cardiac blood, 43.4 µg/mL; urine, 619 µg/mL; vitreous humor, right 2.9 µg/mL and left 4.4 µg/mL; bile, 43.5 µg/mL; and gastric content, 28.2 µg/mL. The cause of death was 4-MEC intoxication and the manner of death could be either accidental or suicidal. The literature concerning 4-MEC was reviewed, focusing on distribution in classical postmortem matrices and 4-MEC metabolism and postmortem redistribution and stability.

Fatal intoxication involving 4-methylpentedrone (4-MPD) in a context of chemsex.

4-methylpentedrone (4-MPD) is a new psychoactive substance (NPS) belonging to the cathinone class. We report an original case of death mainly involving 4-MPD, along with cocaine, sildenafil, bromazepam and nevirapine. The investigation data and the autopsy findings indicated fatal intoxication in a chemsex context (drug use during sex). 4-MPD concentrations were determined in peripheral blood (1285 ng/mL), cardiac blood (1128 ng/mL), urine (>10,000 ng/mL), bile (1187 ng/mL) and vitreous humor (734 and 875 ng/mL in left and right samples, respectively) by gas chromatography (GC) coupled to tandem mass spectrometry. 4-MPD metabolites were explored by GC coupled to high resolution mass spectrometry. Due to the paucity of data concerning 4-MPD, its use and effects were gathered from online user testimonies. This case illustrates the toxicity of this infrequent pentedrone derivate and confirms the significant overdose risk associated with chemsex.

Is insulin intoxication still the perfect crime? Analysis and interpretation of postmortem insulin: review and perspectives in forensic toxicology.

Insulin is an anabolic hormone essential to glucose homeostasis. Insulin therapy, comprising human insulin (HI) or biosynthetic analogs, is critical for the management of type-1 diabetes and many of type-2 diabetes. However, medication error including non-adapted dose and confusion of insulin type, and misuse, such as massive self-administration or with criminal intent, can have lethal consequences. The aim of this paper is to review the state of knowledge of insulin analysis in biological samples and of the interpretation of insulin concentrations in the situation of insulin-related death investigations. Analytic aspects are considered, as quantification can be strongly impacted by methodology. Immunoanalysis, the historical technique, has a prominent role due to its sensitivity and ease of implementation. Recently, liquid chromatography coupled to mass spectrometry has provided indispensable selectivity in forensic contexts, distinguishing HI, analogs, and degradation products. We review the numerous antemortem (dose, associated pathology, injection-to-death interval, etc.) and postmortem parameters (in corpore degradation, in vitro degradation related to hemolysis, etc.) involved in the interpretation of insulin concentration. The interest and limitations of various alternative matrices providing a valuable complement to blood analysis are discussed. Vitreous humor is one of the most interesting, but the low diffusion of insulin in this matrix entails very low concentrations. Injection site analysis is relevant for identifying which type of insulin was administered. Muscle and renal cortex are matrices of particular interest, although additional studies are required. A table containing most case reports of fatal insulin poisoning published, with analytical data, completes this review. A logic diagram is proposed to highlight analytical issues and the main parameters to be considered for the interpretation of blood concentrations. Finally, it remains a challenge to provide reliable biological data and solid interpretation in the context of death related to insulin overdose. However, the progress of analytical tools is making the "perfect crime" ever more difficult to commit.

Unpredictable Behavior Under the Influence of "Magic Mushrooms": A Case Report and Review of the Literature.

Fatalities implicating psychedelic mushrooms are not a common clinical situation in everyday forensic medicine. Despite classification as an illegal drug in many countries, psilocybin mushrooms have the reputation of being safe. We report the case of a young man who jumped from a second story balcony under the influence of psilocybin mushrooms. The psilocin assay was performed by gas chromatography coupled to an electron-impact ionization time-of-flight detector (GC-EI-TOF) after solid-phase extraction. Total psilocin was quantified in peripheral and cardiac blood as 60 and 67 ng/mL, respectively, and in urine (2230 ng/mL), bile (3102 ng/mL), and vitreous humor (57 ng/mL). This case report and review of literature highlights the danger of psilocybin mushrooms. Isolated use of psilocybin mushrooms by a regular consumer without psychiatric history, even under "safe" circumstances, can lead to a fatal outcome.

Vitreous humor analysis for the detection of xenobiotics in forensic toxicology: a review.

Vitreous humor (VH) is a gelatinous substance contained in the posterior chamber of the eye, playing a mechanical role in the eyeball. It has been the subject of numerous studies in various forensic applications, primarily for the assessment of postmortem interval and for postmortem chemical analysis. Since most of the xenobiotics present in the bloodstream are detected in VH after crossing the selective blood-retinal barrier, VH is an alternative matrix useful for forensic toxicology. VH analysis offers particular advantages over other biological matrices: it is less prone to postmortem redistribution, is easy to collect, has relatively few interfering compounds for the analytical process, and shows sample stability over time after death. The present study is an overview of VH physiology, drug transport and elimination. Collection, storage, analytical techniques and interpretation of results from qualitative and quantitative points of view are dealt with. The distribution of xenobiotics in VH samples is thus discussed and illustrated by a table reporting the concentrations of 106 drugs from more than 300 case reports. For this purpose, a survey was conducted of publications found in the MEDLINE database from 1969 through April 30, 2015.

Quantification of five compounds with heterogeneous physicochemical properties (morphine, 6-monoacetylmorphine, cyamemazine, meprobamate and caffeine) in 11 fluids and tissues, using automated solid-phase extraction and gas chromatography-tandem mass spectrometry.

An automated solid-phase extraction (SPE) protocol followed by gas chromatography coupled with tandem mass spectrometry was developed for quantification of caffeine, cyamemazine, meprobamate, morphine and 6-monoacetylmorphine (6-MAM) in 11 biological matrices [blood, urine, bile, vitreous humor, liver, kidney, lung and skeletal muscle, brain, adipose tissue and bone marrow (BM)]. The assay was validated for linearity, within- and between-day precision and accuracy, limits of quantification, selectivity, extraction recovery (ER), sample dilution and autosampler stability on BM. For the other matrices, partial validation was performed (limits of quantification, linearity, within-day precision, accuracy, selectivity and ER). The lower limits of quantification were 12.5 ng/mL(ng/g) for 6-MAM, morphine and cyamemazine, 100 ng/mL(ng/g) for meprobamate and 50 ng/mL(ng/g) for caffeine. Analysis of real-case samples demonstrated the performance of the assay in forensic toxicology to investigate challenging cases in which, for example, blood is not available or in which analysis in alternative matrices could be relevant. The SPE protocol was also assessed as an extraction procedure that could target other relevant analytes of interest. The extraction procedure was applied to 12 molecules of forensic interest with various physicochemical properties (alimemazine, alprazolam, amitriptyline, citalopram, cocaine, diazepam, levomepromazine, nordazepam, tramadol, venlafaxine, pentobarbital and phenobarbital). All drugs were able to be detected at therapeutic concentrations in blood and in the alternate matrices.

Structural elucidation of two photolytic degradation products of tetrabenazine.

During solution formulation study of tetrabenazine (TBZ), a dopamine depleting agent, used in chorea associated with Huntington's disease and symptomatic treatment of hyperkinetic movement disorder it was observed a strong discoloration upon storage. We investigated this physico-chemical behavior by implementing forced degradation studies. It was observed yellowing only under Suntest(®) light exposure of TBZ solution. LC-MS (liquid chromatography coupled to mass spectrometer detection) analysis of light exposed TBZ samples allowed us to propose 1,11b-dedihydrotetrabenazine (DTBZ) and 1,3,4,11b-detetrahydrotetrabenazine (TTBZ) as the main TBZ impurities. Synthesis and complete structural determination of DTBZ and TTBZ·HCl by NMR and X-ray crystallography were carried out. They were identical in LC-MS with polar impurities found in light exposed TBZ samples. However, even if these TBZ degradation products are correlated with discoloration of TBZ solution there is no evidence they are directly responsible of it.

A safe and practical method for the preparation of 7α-thioether and thioester derivatives of spironolactone.

Spironolactone is a renal competitive aldosterone antagonist. One of its most important metabolite is the 7α-methylthio spironolactone: thus it is very important to have an efficient and safe access to this compound, for pharmacokinetic studies. In this context, we synthesized this metabolite by thioalkylation of 7α-thio spironolactone using Hünig's base with a very good yield. We also used our procedure to prepare, with an easy work-up and high yields, 7α-thioether and thioester derivatives of spironolactone, that could be useful for further Structure-Activity Relationships studies.

Interpretation of drug concentrations in an alternative matrix: the case of meprobamate in vitreous humor.

The use of vitreous humor (VH) as an alternative matrix to blood in the field of forensic toxicology has been described for numerous drugs. Interpretation of drug concentrations measured in VH, as in other matrices, requires statistical analysis of a data set obtained on a significant series. In the present study, two diagnostic tests interpreting postmortem VH concentrations of meprobamate in 117 sets of autopsy data are reported. (1) A VH meprobamate concentration threshold of 28 mg/l was statistically equivalent to that of blood meprobamate concentration threshold of 50 mg/l distinguishing overdose from therapeutic use in blood. The intrinsic qualities of the test were good, with sensitivity of 0.95 and absolute specificity of 1. (2) A novel interpretation tool is proposed, allowing blood concentration range to be evaluated, with a known probability, from VH concentration.

State-of-the-art of bone marrow analysis in forensic toxicology: a review.

Although blood is the reference medium in the field of forensic toxicology, alternative matrices are required in case of limited, unavailable or unusable blood samples. The present review investigated the suitability of bone marrow (BM) as an alternative matrix to characterize xenobiotic consumption and its influence on the occurrence of death. Basic data on BM physiology are reported in order to highlight the specificities of this matrix and their analytical and toxicokinetic consequences. A review of case reports, animal and human studies involving BM sample analysis focuses on the various parameters of interpretation of toxicological results: analytic limits, sampling location, pharmacokinetics, blood/BM concentration correlation, stability and postmortem redistribution. Tables summarizing the analytical conditions and quantification of 45 compounds from BM samples provide a useful tool for toxicologists. A specific section devoted to ethanol shows that, despite successful quantification, interpretation is highly dependent on postmortem interval. In conclusion, BM is an interesting alternative matrix, and further experimental data and validated assays are required to confirm its great potential relevance in forensic toxicology.

Postmortem measurement of human chorionic gonadotropin in vitreous humor and bile.

Postmortem human chorionic gonadotrophin (HCG) blood assay can confirm postmortem diagnosis of pregnancy or document situations in which HCG levels are elevated. In some cases, however, blood sampling is not possible at autopsy. In this study, HCG was quantified by enzyme-linked fluorescent assay (ELFA) in the bile (n = 5), vitreous humor (n = 4), and postmortem blood (n = 4) of five pregnant women. There were no false negatives in the pregnant subjects (n = 5) or false positives in controls (n = 34), enabling this test to be recommended for routine use in forensic contexts in which the detection of elevated HCG levels could be of interest.