Authentic Novichok (A-series) nerve agents are highly restricted, making the rapid preparation of positive controls and reference materials challenging in forensic toxicology. This study aimed to evaluate p-nitrophenoxy analogs as surrogate compounds for Novichok-related biomarker verification by assessing their synthetic accessibility, ability to generate diagnostically relevant butyrylcholinesterase (BuChE) nonapeptide biomarkers, and functional relevance in cholinesterase inhibition and reactivation assays. Six surrogate compounds representing phosphate- and phosphonate-type analogs were prepared using a revised synthetic sequence with scaffold-dependent optimization of the reaction and purification conditions. Their utility was evaluated using plasma BuChE inhibition assays, 2-pyridine aldoxime methochloride (2-PAM)-mediated reactivation studies, inhibition kinetics with horse BuChE and human acetylcholinesterase, and liquid chromatography–tandem mass spectrometry analysis of BuChE-derived nonapeptide adducts in plasma. Operational applicability was examined using the OPCW biomedical proficiency test (BioPT) evaluation criteria. All six surrogate compounds were obtained by structural class-dependent optimizations of their synthesis and purification. The plasma BuChE IC50 values of authentic A230, A232, A234, A242, A262, and A2xx were 12.3, 9.6, 8.6, 10.1, 10.6, and 24.5 ng/mL, respectively, whereas those of the corresponding surrogates were 58.5, 38.6, 55.2, 3942, 3829, and 2575 ng/mL. 2-PAM-mediated BuChE reactivation showed similar qualitative patterns within each authentic/surrogate pair. All surrogates generated analytically equivalent BuChE nonapeptides and could be used to prepare qualitative reference materials for the OPCW BioPT-related workflow. p-Nitrophenoxy Novichok-related analogs provide a practical surrogate platform for preparing diagnostically relevant BuChE nonapeptide reference materials without using authentic A-series nerve agents.
Quantitative analysis of volatile hydrocarbons (VHCs) in postmortem blood provides insight into the circumstances of fire-related deaths. Detection of specific VHCs can distinguish construction fires from those involving gasoline or kerosene. Although VHC concentrations have been examined in heart blood, their concentrations in other specimens remain unclear. We evaluated the correlations of carboxyhemoglobin and VHC concentrations among heart blood and femoral vein blood or various organ tissues to investigate the viability of alternative specimens for cases where heart blood cannot be collected. Heart blood, femoral vein blood, brain, liver, and muscle samples were collected from victims of fire-related death cases processed at our department. Seventeen VHCs were analyzed using headspace gas chromatography–mass spectrometry. In construction fire cases, carboxyhemoglobin concentrations in heart blood were positively correlated with those in femoral vein blood. Benzene, toluene, and styrene are commonly detected in fire-related deaths, and the concentrations of these compounds in heart blood were positively correlated with the corresponding concentrations in femoral vein blood. The concentrations in heart blood were significantly higher than those in femoral vein blood. Tissue concentrations were also positively correlated with those in heart blood. Among the samples, brain tissue had the highest concentrations. In gasoline- and kerosene-related fire cases, the VHC concentration patterns in the brain were similar to those observed in heart blood. These results suggest that brain tissue is an appropriate specimen for VHCs analysis and can be used when VHCs are undetectable in heart blood or heart blood is not available.
Hexahydrocannabinol (HHC) is an emerging semi-synthetic cannabinoid, but analytical data for all structurally confirmed stereoisomers of its hydroxylated metabolites remain limited. In this study, we synthesized all four stereoisomers of 8-hydroxyhexahydrocannabinol (8-OH-HHC) and comparatively characterized them, together with their trimethylsilyl (TMS) derivatives, by gas chromatography mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A particular analytical advantage of the present work is that it includes LC-MS/MS data for (8S,9R)-8-OH-HHC, for which no standard-based analytical data was previously available. Four synthetic 8-OH-HHC stereoisomers were analyzed by GC-MS and LC-MS/MS. For GC-MS, both underivatized compounds and their TMS derivatives were examined. Retention behavior, electron ionization mass spectra, and LC-MS/MS transitions were compared among the stereoisomers. Under the tested GC conditions, the four stereoisomers and their TMS derivatives showed different retention times, although some differences were small and should be interpreted cautiously. Two pairs of stereoisomers exhibited similar electron ionization fragmentation patterns, whereas one pair showed differences in the relative abundances of selected ions. In addition to providing GC-MS data for all four underivatized stereoisomers, this study also provides GC retention and EI mass spectral data for all four TMS derivatives and a comparative evaluation of relative ion abundances among these closely related isomers. In LC-MS/MS, the stereoisomers showed partially overlapping transition patterns and closely eluting peaks under the tested conditions. These data provide a comparative analytical reference dataset for four structurally confirmed 8-OH-HHC stereoisomers and their TMS derivatives and may support future forensic studies of HHC-related compounds.
Xylazine, an α2-adrenergic agonist, is widely used in veterinary medicine for anesthesia, sedation, and analgesia. However, non-therapeutic use, overdose, and deliberate administration may pose serious health risks to animals, necessitating reliable detection methods for both clinical and forensic purposes. In this study, a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed and fully validated for the determination of xylazine and its metabolites in canine urine. The method exhibited excellent selectivity and linearity over the range of 0.05–100 ng/mL, with coefficients of determination (R²) ≥ 0.9989. Recovery values ranged from 70.3
Hair toxicological analysis enables the verification of long-term drug use monitoring and supports epidemiological studies. In recreational contexts, multi-analyte methods are crucial for accurately characterizing consumption patterns. The present work aimed to develop and validate an LC-MS/MS method for the analysis of 47 drugs of abuse and related compounds in hair samples. A 20 mg hair aliquot was incubated with methanol at 40 °C for 16 h. The supernatant was dried and injected into the LC-MS/MS system. The monitored substances were amphetamines, cocaine and related compounds, cannabinoids, opioids, hallucinogens, synthetic cathinones, synthetic phenethylamines, and synthetic cannabinoids. The method was validated in accordance with international recommendations and subsequently applied to 81 hair samples collected at Brazilian electronic dance music festivals. The method had limits of detection of 0.5-5 pg/mg and limits of quantification of 5–50 pg/mg. All compounds showed suitable linearity (R² > 0.9903). The precision assays showed CV
