Forensic Toxicology最新文献

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.

Purpose: Regional traditional plant khat, which have been recreationally used world-wide recently, has been proven to be a mixture of several biologically active ingredients. Herein, a chosen specimen, vitreous humor (VH) and a novel pretreatment, electromembrane extraction (EME), are applied for forensic investigations of such abused plant.

Methods: VH, as an alternative matrix, is being used for evaluating possible compounds more and more; EME, a novel and efficient pretreatment method, is applied to detect the ingredients from natural complex matrices with advantages of a more sustainable microextraction technique. This study aims to analyze the ingredients of khat, norephedrine (NE), norpseudoephedrine (NPE) and cathinone (CTN), as well as their concentrations in VH of khat-treated mice applying EME.

Results: After optimization, 2-ethylnitrobenzene (ENB)/undecanol was used as the support liquid membrane (SLM), HCl (pH = 2) as the acceptor solution, extraction voltage at 60 V, and extraction time for 30 min. The established EME was combined with liquid chromatography-ultraviolet spectrometry (LC-UV) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to evaluate spiked VH. The LOD of NE, NPE, and CTN were 0.40-1.90 µg/mL with linearity (R² > 0.9624) and repeatability (< 13%).

Conclusions: By this method, NE, NPE, and CTN were detected to be 14.4 ± 0.54 µg/mL, 8.50 ± 0.69 µg/mL, and 90.5 ± 7.88 µg/mL in VH of mice administrated with khat for 28 days.

Purpose: Identification and quantification of sulfide ion in biological samples are required in forensic purpose. Gas chromatography-mass spectrometry (GC/MS) has been used for the analysis of sulfide ion by using derivatization reagents. However, conventional derivatization reagents require special attention for derivatization. To simplify the derivatization protocol, we examined ethenesulfonyl fluoride (ESF) as a derivatizing reagent of sulfide ion.

Methods: To 100 μL of whole blood sample containing sulfide ion, 100 μL of boric acid buffer (pH 8.0), 100 μL of acetone solution containing internal standard, 100 μL of acetone solution containing 600 mM concentration of ESF, and 100 μL of hexane were added in a 1.5-mL plastic tube. The mixture was vortexed at room temperature, the tubes were centrifuged, and the organic layer was injected into the GC/MS.

Results: ESF exhibited higher reactivity toward sulfide ion than interfering compounds present in whole blood, allowing for selective derivatization. With the optimized protocol, the detection limit for sulfide ion was 0.01 μg/mL. The calibration curve showed good linearity (R² = 0.9999) in the range of 0.05-10.0 μg/mL, and the precision (% relative standard deviation) and the accuracy (% bias) were within ± 10% (intra- and inter-day).

Conclusion: This GC/MS-based method is a valuable tool for forensic investigations and various analytical fields, offering reliable quantification of sulfide ion in whole blood.

Purpose: Diphenhydramine is an antihistaminic agent available in numerous over-the-counter preparations, while modafinil is a wakefulness-promoting agent, available only by prescription, but also used recreationally, when purchased from the black market. Structurally, both substances belong to the class of so-called benzhydryl compounds, which can complicate their proper differentiation. The authors point out the possibility of misattributing modafinil in diphenhydramine-positive cases due to the likely coelution of nordiphenhydramine and modafinil.

Methods: Post-mortem blood and vitreous humor samples were subjected to liquid-liquid extraction using ethyl acetate in an alkaline environment (pH = 9), followed by a detailed toxicological analysis utilizing ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry.

Results: Through the application of full scan mode, multiple reaction monitoring (MRM), and product ion scan mode, the presence of modafinil was excluded in diphenhydramine-positive biological matrices (blood and vitreous humor).

Conclusions: In the analysis of benzhydryl compounds, particular caution should be exercised, with each case verified by comparison with a certified analytical standard, and, where possible, by detecting the metabolites of these compounds.

Purpose: A principal objective of forensic entomotoxicology is to apply insect specimens for post-mortem toxicological analysis. Successful identification of drugs in necrophagous insects may depend on pharmacokinetic processes occurring in larvae. We thus applied a model system involving Lucilia sericata (Meigen, 1826) (Diptera, Calliphoridae) to investigate pharmacokinetics of diazepam in larvae in vitro, followed by a field experiment with Göttingen Minipigs.

Methods: Lucilia sericata larvae were fed one of four diazepam concentrations at constant temperature, sampled regularly, and analysed for diazepam and metabolites by liquid chromatography tandem mass spectrometry (LC-MS/MS). Two Göttingen Minipigs of 60 kg each were euthanised one hour after oral administration of 25 mg/kg diazepam and placed outdoors. While available, samples of peripheral blood, cardiac blood, liver, and fly larvae were collected over 70 days. Extracts from porcine samples and larvae were analysed by LC-MS/MS. Some larvae were bred to adulthood and identified morphologically together with 718 larvae.

Results: Oxazepam was a primary metabolite of diazepam in L. sericata larvae. The most prevalent fly species on minipig carcasses were Lucilia caesar (Linnaeus, 1758) (Diptera, Calliphoridae) and Lucilia illustris (Meigen, 1826) (Diptera, Calliphoridae). Diazepam and metabolites were detected in all larval samples, even weeks after porcine samples were unacquirable due to post-mortem decomposition. Ratios of oxazepam and nordazepam to diazepam concentrations in larvae were significantly higher than in associated porcine samples, confirming metabolism in larvae.

Conclusion: These findings are relevant to forensic casework, as there is potential for misinterpreting that the deceased consumed oxazepam or nordazepam rather than diazepam. This caution may also apply to other drugs that can form through metabolism in larvae.