Forensic Toxicology最新文献

Purpose: The most commonly associated substance found in Ecstasy tablets is MDMA (3,4-methylenedioxymethamphetamine). In our study, we showed how the composition of psychoactive ingredients in Ecstasy tablets seized on the drug market in Poland has changed in the years 2005-2020.

Methods: The study material consisted of nearly 20,000 single Ecstasy tablets seized by representatives of law enforcement (the police, prosecutors) from 2005 to 2020 and analysed by the Institute of Forensic Research, Krakow, Poland. The analysis of the tablets was carried out by gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography with diode array detection (HPLC-DAD) and ultra-high-performance liquid chromatography with photodiode array detection (UHPLC-PDA).

Results: Currently, new types of MDMA tablets are introduced onto the market, available in various colours and shapes. Our study showed that tablets sold on the street as Ecstasy have variable purity and sometimes contain little or no MDMA. The mean content of MDMA in one tablet seized in 2005-2011 decreased from 90 to 50 mg. In 2013, Ecstasy tablets with a very high MDMA content (average 195 mg per tablet) appeared on the market, but in the next 2 years, the MDMA content decreased again. From 2016, the average MDMA content began to rise again, ranging from 60 to 280 mg.

Conclusion: Tablets sold as Ecstasy also contained completely different psychoactive substances, including new psychoactive substances (NPS) (found in almost 20% of all examined tablets sold as Ecstasy) belonging to different chemical groups or their dangerous combinations (i.e. phenylethylamines, piperazines, tryptamines, cathinones, arylalkylamines, arylcyclohexylamines and piperidines). Such a large variety of psychoactive substances in Ecstasy tablets is associated with a high risk for users unaware of their composition.

Purpose: Diagnosis of drug intoxication in the medico-legal autopsy is challenging due to many factors such as non-specific clinical features and non-specific, inconclusive autopsy findings, etc. Thus, deaths due to drug intoxication can be misclassified in a low-resource setting where post-mortem toxicology testing is selective. The paper presents a fatal case of unrecognized nifedipine intoxication in an adult where the manner of death was undetermined after extensive investigation.

Method: The liquid-liquid extraction using chloroform was carried out on a blood sample spiked with nifedipine. Subsequently, the post-mortem blood sample was analyzed and quantified using gas chromatography-mass spectrometry with electron ionization technique.

Results: The patient before death had symptoms, such as trismus, vomiting, and dizziness. The initial blood pressure and pulse rate were 94/56 mm Hg and 110 beats per minute, respectively. The respiratory rate was 20 breaths per minute. The post-mortem examination revealed no pathological changes or injuries in any organs. Upon histopathological examination, no significant findings that could have led to death were observed in any of the organs. The level of nifedipine in the peripheral blood, 0.645 μg/ml was determined to be either  close to or exceeding the reported fatal dose. The cause of death was ascertained as acute nifedipine intoxication.

Conclusion: It is crucial to accurately determine the cause of death in cases that pose a significant threat to public health. This case highlights the challenges faced by forensic pathologists in scientifically ascertaining the cause of death accurately, especially in intoxication deaths, and the importance of comprehensive toxicology testing services including analytical toxicology for the integrity of the medico-legal death investigation system.

Purpose: The goal of the current study was to clarify the potential molecular mechanism underlying the protective effects of silymarin (SIL) administration against diazinon-induced subacute nephrotoxicity, with a special emphasis on the role of the Kelch-like-associated protein-1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway in minimizing the oxidative stress induced by diazinon (DZN).

Methods: Five equal groups of thirty adult male Wistar rats were created at random. Group 1 (G1) was maintained under typical control conditions and administered saline intragastrically (I/G) once daily for 4 weeks; G2 was administered olive oil I/G for 4 weeks; G3 was I/G administered silymarin daily for 4 weeks; G4 was I/G administered diazinon daily for 4 weeks. G5 was I/G administered silymarin daily 1 h before the I/G administration of the diazinon for 4 weeks. Blood samples were collected at the end of the experiment for the determination of complete blood cell count, and kidney function tests. Kidney specimens were collected for the evaluation of the oxidative markers, mRNA gene expression, protein markers, and histopathological examination.

Results: SIL reduced the renal dysfunction caused by DZN by restoring urea and creatinine levels, as well as oxidative indicators. Although the expression of Keap-1 was also elevated, overexpression of Nrf2 also enhanced the expression of HO-1, a crucial target enzyme of Nrf2.

Conclusions: SIL is hypothesized to potentially aid in the prevention and management of nephrotoxicity caused by DZN.

Purpose: The metabolic pathways of APP-CHMINACA were characterized to select the markers of intake for implementation into analytical assays used by the clinical and forensic communities. We have combined the evidences obtained by both in vitro experiments and administration studies on mice.

Methods: APP-CHMINACA was incubated with either human or mouse liver microsomes. Urine and blood samples were collected at different time points from mice after injection of a 3 mg/kg dose of the test compound. Samples were analyzed using liquid chromatography-tandem mass spectrometry.

Results: The in vitro studies allowed to isolate eight different metabolic reactions, formed by two metabolic routes, with no differences between human and mouse liver microsomes. The main biotransformation route involved the hydrolysis of the distal amide group and the subsequent hydroxylation on the cyclohexyl-methyl ring. The second route involved multiple hydroxylation of the parent compound, followed by reduction to generate minor metabolites. In blood samples, the most abundant substances identified were APP-CHMINACA unchanged and the metabolites formed by the hydrolysis of the distal amide together with its hydroxylated products. In urine samples, four metabolites formed following the hydroxylation of the distal amide hydrolysis metabolite were detected as the most abundant and long-term metabolites.

Conclusions: The outcomes of our study showed that the most suitable markers to detect the intake of APP-CHMINACA in blood and urine samples in the framework of toxicological, clinical and forensic investigations were the metabolite formed by the hydrolysis of the distal amide and its hydroxylated products.

Purpose: This study examines the interaction between benzoylmesaconine (BMA) and hen egg white lysozyme (HEWL) under various physiological conditions, aiming to determine how BMA affects the HEWL's structure and function.

Methods: Several analytical techniques were used, including tryptophan assay, light scattering, thioflavin T (ThT)-binding assay, dynamic light scattering, 8-anilino-1-naphthalenesulfonic acid (ANS)-binding assay, circular dichroism (CD) spectroscopy, enzyme activity assay, and molecular docking.

Results: The tryptophan assay displayed a concentration-dependent decrease in tryptophan fluorescence, showing an interaction between BMA and HEWL. Light scattering and ThT-binding assays confirmed increased protein aggregation and amyloid fibril formation, while the ANS-binding assay demonstrated altered exposed hydrophobic regions, implying structural changes. CD spectroscopy showed a reduction in α-helix content, indicating conformational alterations, and enzyme activity assays showed a loss of lytic function due to structural distortion. Finally, molecular docking identified significant bonds and hydrophobic interactions between BMA and HEWL residues.

Conclusions: BMA binding induces structural changes in proteins, forming small oligomers and amyloid fibrils that decrease HEWL enzymatic activity and disrupt functional integrity.

Purpose: Cytisine is the active ingredient in preparations used for smoking cessation. Its popularity is attributed to its low cost, efficacy, and low incidence of adverse effects. Additionally, its easy over-the-counter availability is also significant. This accessibility makes it a potential substance for use in suicidal attempts. The aim of this study was to develop a method for the determination of cytisine in biological material for use in clinical and forensic toxicology, and to apply this method in authentic cases.

Methods: Biological samples were subjected to liquid-liquid extraction using cytisine-d₄ as an internal standard. Analyses were performed using a Hydrophilic Interaction Liquid Chromatography (HILIC) column with the technique of ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry.

Results: For both matrices (blood and urine), the linear concentration range was 5-1000 ng/mL. The method met all validation requirements. The concentration of cytisine in a man taking it for smoking cessation in post-mortem materials was 21.4 ng/mL in blood, 958.9 ng/mL in urine, ca. 30 ng/mL in vitreous humor, and ca. 40 ng/mL in bile. In contrast, for a man with cytisine intoxication, the concentration was 174.6 ng/mL in blood and > 10,000 ng/mL in urine. In both cases, no N-methylcytisine was detected.

Conclusions: The developed method can be used for the determination of cytisine in post-mortem biological matrices as well as for clinical purpose. We presented the concentrations of cytisine in the post-mortem biological samples of a man taking cytisine for smoking cessation and of a man with suicidal cytisine poisoning.

Purpose: Suvorexant is an orexin receptor antagonist used in the treatment of insomnia. In this study, we investigated the urinary excretion profiles of suvorexant and its major metabolites, including conjugates, to obtain fundamental information for proving exposure to suvorexant in criminal cases.

Methods: Urine specimens were collected from three subjects for maximum 168 h after a single oral ingestion of suvorexant (10 mg), and suvorexant and its metabolites in urine were determined using liquid chromatography-tandem mass spectrometry with a C18 semi-micro column.

Results: The carboxylic and hydroxy metabolites (M4 and M9) were identified with authentic standards synthesized in our laboratory, and their glucuronides and other hydroxy metabolites (M8 and M10) were tentatively detected based on measured exact masses and product ion spectra of them. Suvorexant, M4 and M9 would be detectable for 20-34 h, 6-7 days and 42-61 h after intake, respectively. The quantitative results demonstrated that the molar ratios of accumulated amounts of M4 and M9 including their glucuronides excreted in urine to dose ranged about 2.6-6.2% and 0.37-0.51%, respectively, while that of the unchanged parent was much lower (0.011-0.013%). The ratios of the amount of glucuronide to the total amount of M4 and M9 excreted in urine was less than 10% and approximately 90%, respectively.

Conclusions: The urinary excretion profiles indicated that M4 and M9 would be effective indicators for proving suvorexant intake, and M4 could be detected until one week after intake even without enzymatic hydrolysis (limit of detection: 0.05 ng/mL).

Purpose: Distribution and abuse of imidazole-derived γ-aminobutyric acid (GABA) agonists, such as etomidate and metomidate, and their analogs have been encountered frequently especially in China. The aim of this study was to identify etomidate, metomidate, propoxate, and isopropoxate more accurately by establishing a gas chromatography-mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) method and applying it to real forensic cases.

Methods: One mg of the seized powder was dissolved in 1 mL of methanol, and subjected to GC-MS and LC-MS/MS. Hair samples were washed and cut into approximately 2 mm sections, then ground to powder by a low-temperature grinder. Twenty mg of the hair powder was extracted with 1 mL of methanol, and the supernatant was subjected to LC-MS/MS.

Results: Etomidate, metomidate, propoxate, and isopropoxate were chromatographically separated and each mass spectrum was obtained by GC-MS. For LC-MS/MS, tested validation data were all satisfactory. The seized powder samples contained isopropoxate, with an approximate content of 30.9%. Etomidate, etomidate acid, metomidate, and isopropoxate could be determined in the submitted hairs, ranging from 2.89 to 8.09 ng/mg, 0.0591-0.177 ng/mg, 0.342-2.77 ng/mg, and 33.2-130 ng/mg, respectively.

Conclusions: Mass spectra and ion chromatograms of etomidate, metomidate, isopropoxate, and propoxate were obtained by GC-MS. We have also established a simultaneous and reliable analytical method for etomidate, etomidate acid, metomidate, and isopropoxate in human hair by LC-MS/MS. This is the first report to present analytical results of a novel imidazole-derived GABA agonist isopropoxate in drug abuse cases.

Purpose

We aimed to explore the metabolite products of 1,2-diacetylbenzene (DAB) and investigate their harmful effects, physicochemical properties, and biological activities, along with those of DAB itself.

Methods

Key approaches included MetaTox, PASS online, ADMESWISS, ADMETlab 2.0, molecular docking, and molecular dynamic simulation to identify metabolites, toxic effects, Lipinski’s rule criteria, absorption, distribution, metabolism, and excretion properties, interactions with cytochrome (CYP) 450 isoforms, and the stability of the DAB-cytochrome complex.

Results

A total of 13 metabolite products from DAB were identified, involving Phase I reactions (aliphatic hydroxylation, epoxidation, oxidative dehydrogenation, and hydrogenation) and Phase II reactions (oxidative sulfation and methylation). Molecular dynamics and modeling revealed a stable interaction between CYP1A2 and DAB, suggesting the involvement of CYP1A2 in DAB metabolism. All studied compounds adhered to Lipinski’s rule, indicating their potential as inducers or activators of toxic mechanisms. The physicochemical parameters and pharmacokinetics of the investigated compounds were consistent with their harmful effects, which included neurotoxic, nephrotoxic, endocrine disruptor, and hepatotoxic consequences due to their high gastrointestinal absorption and ability to cross the blood–brain barrier. Various CYP450 isoforms exhibited different functions, and the compounds were found to act as superoxide dismutase inhibitors, neuropeptide Y2 antagonists, glutaminase inhibitors, and activators of caspases 3 and 8. DAB and its metabolites were also associated with apoptosis, oxidative stress, and neuroendocrine disruption.

Conclusion

The toxic effects of DAB and its metabolites were predicted in this study. Further research is warranted to explore their effects on other organs, such as the liver and kidneys, and to validate our findings.

Purpose

Amphetamine-type stimulants are very common, and their usage is becoming a very big social problem all over the world. Thousands of addicts encounter several health problems including mental, metabolic, behavioral and neurological disorders. In addition to these, there are several reports about the elevated risk of tendency on committing criminal cases by addicted persons. Hence, methamphetamine addiction is not only an individual health problem but also a social problem. In our study, we aimed to investigate the pathogenesis of chronic usage of methamphetamine via untargeted metabolomics approach.

Methods

38 plasma samples were carefully collected and extracted for untargeted metabolomics assay. A liquid–liquid extraction was performed to get as much metabolite as possible from the samples. After the extraction procedure, samples were transferred into vials and they were evaluated via time of flight mass spectrometry instrument.

Results

Significantly, altered metabolites were identified by the fold analysis and Welch’s test between the groups. 42 different compounds were annotated regarding to data-dependent acquisition method. Pathway analysis were also performed to understand the hazardous effect of methamphetamine on human body.

Conclusion

It has been reported that drug exposure may affect several metabolic pathways for amino acids, fats, energy metabolism and vitamins. An alternative bioinformatic model was also developed and validated in order to predict the chronic methamphetamine drug users in any criminal cases. This generated model passes the ROC curve analysis and permutation test and classify the controls and drug users correctly by evaluating the metabolic alterations between the groups.