Forensic Toxicology最新文献

Purpose: Existing on-site color tests for cannabis (e.g., rapid Duquénois-Levine reagent, 4-aminophenol reagent) have insufficient specificity, especially in the point of distinguishing between Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and the other cannabinoids. We developed a novel Δ⁹-THC-specific color test reagent.

Methods: Cannabinoid standards were dissolved in 0.5 mL of a 2-hydroxybenzaldehyde solution in acetonitrile (2 g/100 mL) in a glass test tube (solution A). Herbal samples (approximately 10 mg) were extracted using 0.5 mL of the 2-hydroxybenzaldehyde solution in a plastic homogenization vessel. The extract was poured into a glass test tube through a filter cap (solution B). Solutions A and B were mixed with hydrochloric acid (0.5 mL) and allowed to react for 30 s, after which the mixture was extracted with chloroform (1 mL).

Results: When observing the lower layer after the chloroform extraction, Δ⁹-THC and cannabidiol (CBD) exhibited bluish and orangish colors, respectively. In contrast, cannabinol (CBN) did not show any color. The performance of the reagent was evaluated using dried herbal cannabis (n = 58) and other herbal materials (n = 13), including CBD- and CBN-supplemented herbal materials (each n = 2). Only cannabis samples with Δ⁹-THC levels ≥ 0.14% exhibited a bluish or greenish-blue color.

Conclusions: The newly developed reagent shows good specificity for Δ⁹-THC. The new reagent has the potential to replace existing on-site color tests.

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: Fluvoxamine (FLV) has been used widely as an antidepressant agent belonging to the group of second-generation selective serotonin reuptake inhibitors. However, only one work on the human metabolism of FLV was reported in 1983, examining a human urine specimen, and tentatively identified nine metabolites. Therefore, in the present work, the metabolites of FLV were examined in the liver, bile, and urine from a human cadaver, and the metabolites produced in the human liver microsomes (HLMs) in vitro were also examined.

Methods: Metabolites in each matrix were treated altogether in a tube where impurities had been precipitated using acetonitrile. The identification and tentative quantification of metabolites in human specimens and HLMs were performed using liquid chromatography (LC)-high resolution mass spectrometry (MS), LC-tandem mass spectrometry (MS/MS) and LC-QTRAP- MS/MS.

Results: Eleven new metabolites designated as M1 to M11 were detected from human cadaver specimens and HLMs. M1 was produced after acetylation at the terminal NH₂ of FLV and was the most abundant metabolite in the liver and bile, but was the third abundant one in urine. M4 was produced after demethylation at the methoxy moiety of FLV, and was the most abundant metabolite in HLMs.

Conclusions: To our knowledge, this is the first report on the existence of eleven new metabolites (M1-M11) of FLV in HLMs, human liver, bile and urine. The present eleven metabolites may be useful for the identification of FLV in human samples both antemortem and postmortem.

Purpose: Recently, numerous lysergic acid diethylamide (LSD) analogs have emerged as designer drugs globally. These compounds are mainly distributed as sheet products. In this study, two new LSD analogs were identified from sheet products.

Methods: The structures of the compounds were determined by gas chromatography-mass spectrometry, liquid chromatography-photodiode array-mass spectrometry, liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance (NMR) measurement.

Results: From the NMR analysis, two compounds in the products were identified as N,N-diethyl-7-methyl-4-(3-(trimethylsilyl)propanoyl)-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1S-LSD) and 7-allyl-N,N-diethyl-4-(thiophene-2-carbonyl)-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1T-AL-LAD). In the product where 1S-LSD was detected, the presence of a trace amount of iso-1S-LSD, a C8-epimerization product of 1S-LSD, was suggested.

Conclusions: This paper is the first to report the detection of 1S-LSD and 1T-AL-LAD in sheet products in Japan. Notably, the metabolic pathways and biological activities of 1S-LSD and 1T-AL-LAD are not explored. The possibility of the in vivo deacylation and conversion of the compoundsinto LSD or AL-LAD should be further investigated.

Purpose: Detecting hypnotics in victim urine samples collected several days after drug-facilitated crime (DFC) is challenging because most of the drugs have already been excreted. In this study, a sample preparation method was developed for extracting trace amounts of hypnotics using most of the urine excreted at one sampling time (100 mL), and large amounts of matrices were efficiently removed.

Methods: Etizolam, midazolam, ramelteon, and their metabolites were used as the target compounds. As the first step in decreasing the sample volume, solid-phase extraction using various sorbents was examined. The effects of additional clean-up columns (alumina, graphite, anion exchanger, etc.) on the removal of urine matrices were also examined. The pretreatment of 0.1-mL urine using a simple extraction column, specialized for small-scale urinalysis (Isolute Hydro DME +), was used as the reference method. The feasibility of drug detection in 100-mL urine was evaluated by comparison with a reference method.

Results: All analytes in 100-mL urine were most effectively adsorbed on a sorbent with octadecyl-bonded polymer and eluted with less than 2 mL of acetonitrile. A multilayer clean-up column consisting of alumina, octadecyl-bonded silica, and anion exchangers was effective in removing the matrices. α-Hydroxymidazolam was detected in 100 mL of urine that was collected 5 days after midazolam administration, but was undetected using the reference method.

Conclusions: This preparation method for 100-mL urine is useful as the first extraction step in detecting trace amounts of hypnotics in victim urine collected late after DFC.

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: Fire victims often inhale hydrogen cyanide (HCN) gas in addition to carbon monoxide. This study aimed to investigate the current prevalence of HCN inhalation among fire victims and assess the contribution of HCN as a toxic factor in fire-related deaths.

Methods: The study included 29 cases of fire-related deaths, where autopsies were conducted at the Department of Legal Medicine, Osaka University, from April 2014 to March 2020. No resuscitation was performed before death was confirmed and blood samples were obtained from both the left and right cardiac chambers. Blood cyanide concentrations were measured. Additionally, a physiologically based pharmacokinetic model, as described by Stamyr et al. (Arch Toxicol 89:1287-1296, 2015), was used to simulate the time course of blood concentration changes for different inhaled HCN concentrations. The inhaled HCN concentration and inhalation time that minimized the difference between the measured and simulated blood concentrations were calculated.

Results: Cyanide was detected in the cardiac blood of 76.3% of cases. In all instances, left cardiac blood concentrations were higher than those in the right cardiac blood. The simulations using the physiologically based pharmacokinetic model revealed eight cases where the inhaled HCN concentration exceeded 5000 ppm, with an inhalation time of less than 0.5 min.

Conclusions: Many fire victims inhaled HCN gas, and in a few cases, it appears that death occurred rapidly after inhalation of high HCN concentrations. These findings suggest that the contribution of cyanide gas to fire-related deaths warrants closer examination.

Purpose: To achieve the rapid analysis of drug metabolites in urine, we examined the differences in the hydrolysis efficiencies against O-glucuronide and N-glucuronide by two commercially available glucuronidases and three commercially available recombinant ones.

Methods: The metabolites analyzed included oxazepam-O-glucuronide, amitriptyline-N-glucuronide, and diphenhydramine-N-glucuronide. Hydrolysis was performed using commercially available five enzymes at two different temperatures, and the reaction progress was monitored for up to 360 min. The amount of hydrolyzed product was quantified using liquid chromatography-tandem mass spectrometry.

Results: Although no enzyme selectivity was observed for the hydrolysis of O-glucuronide, the hydrolysis efficiency against N-glucuronide varied significantly, depending on the enzyme and reaction temperature. Among the enzymes evaluated, IMCSzyme 3S and the enzyme derived from E. coli demonstrated superior hydrolysis of N-glucuronides under optimal conditions. For IMCS RT, good results were also obtained by adding twice the amount of enzyme specified.

Conclusions: Suitable enzymes and hydrolysis conditions were determined for the rapid and systematic screening of drug metabolites in human urine. These findings are expected to streamline the analytical workflow and reduce the need for tedious sample preprocessing.

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: The increasing prevalence of methamphetamine and cocaine in postmortem toxicology casework has placed significant demands on forensic laboratories. This study introduces and validates a streamlined method using salt assisted liquid-liquid extraction (SALLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to improve the efficiency and reliability of detecting amphetamine-type stimulants (ATS) and cocaine metabolites in forensic toxicology.

Methods: A new SALLE method was developed to analyze a panel of drugs, including amphetamine, methamphetamine, phentermine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), pseudoephedrine, cocaine, cocaethylene, and benzoylecgonine (BZE). Calibration models, bias, precision, recovery, matrix effects, interferences, limits of detection (LOD) and quantitation (LOQ), dilution integrity, carryover, and sample stability were evaluated following AAFS standard 036 guidelines. The method was applied to over 150 postmortem and human performance toxicology cases and compared with the traditional gas chromatography-mass spectrometry (GC/MS) approach.

Results: The SALLE-LC-MS/MS method exhibited high accuracy, with all analytes meeting bias and precision criteria (< 20%). Percent recovery values exceeded 80%, while matrix effect values (ion suppression/enhancement) remained below 20%. LODs ranged from 5-25 µg/L, and LOQs ranged from 10-50 µg/L across analytes. Processed samples were stable for up to 8 days. Analysis of 150 cases showed strong agreement with the GC/MS method, with average percent differences ranging from 5.4 to 19.4% for most analytes. The new method reduced sample preparation time by 67% and data-processing time by 80%, resulting in overall time savings of 8 h per batch.

Conclusions: The resulting validated SALLE procedure represents a significant advancement in the analysis of stimulant drugs within forensic toxicology. Its adoption at the Georgia Bureau of Investigation not only addresses current analytical challenges but also sets a precedent for the development of more efficient and reliable methods in the field.