Forensic Toxicology最新文献

{"title":"Effects of 4-position substitutions of diphenidine on blood-brain barrier penetration and dopamine release in the nucleus accumbens of rats.","authors":"Yuta Takahashi, Katsuhiro Okuda, Kazuo Matsubara, Yoshikazu Tasaki, Masaru Asari, Kanae Mori, Ryo Namba, Keiko Shimizu","doi":"10.1007/s11419-026-00771-6","DOIUrl":"10.1007/s11419-026-00771-6","url":null,"abstract":"<p><strong>Purpose: </strong>Diphenidine (DPD) is a dissociative novel psychoactive substance (NPS) structurally related to phencyclidine and ketamine. Although DPD is legally regulated in Japan and several other countries, analogues sharing the core scaffold are not comprehensively regulated. Therefore, it is possible that analogues with minor scaffold modifications may continue to emerge. This study examined the effects of methoxy or hydroxy substitution at the 4-position of DPD on its blood-brain barrier (BBB) penetration and dopamine release in the synaptic cleft.</p><p><strong>Methods: </strong>Using in vivo brain microdialysis in freely moving unanesthetized rats, DPD, 4-methoxydiphenidine (4MeO-DPD), and 4-hydroxydiphenidine (4OH-DPD) (20 mg/kg, i.p. each) were administered, and concentrations in the nucleus accumbens and plasma were quantified by liquid chromatography-mass spectrometry. Extracellular dopamine levels were determined by high-performance liquid chromatography with electrochemical detection. To investigate carrier-mediated BBB transport, verapamil (P-glycoprotein, P-gp, inhibitor) or diphenhydramine (organic cation transporter, OCT, inhibitor) was administered 1 h prior to each compound.</p><p><strong>Results: </strong>DPD and its analogues showed distinct BBB penetration profiles, among which 4OH-DPD showed the highest brain concentrations and dopamine release. Verapamil but not diphenhydramine pretreatment significantly increased brain extracellular concentrations and prolonged elimination half-lives of all compounds, particularly 4MeO-DPD. P-gp inhibition elevated brain-to-plasma concentration ratios, indicating restricted BBB penetration by P-gp. The dopamine concentration profile was consistent with those observed for DPD and its analogues.</p><p><strong>Conclusions: </strong>This study demonstrates that 4MeO-DPD and 4OH-DPD strongly elicit dopamine release compared with DPD. These findings show that P-gp regulates BBB penetration, offering important insights for the toxicological risk assessment for newly emerging NPS.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"395-404"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147927972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput measurement for micro-segmental hair analysis using parallel column regeneration in liquid chromatography/tandem mass spectrometry.","authors":"Kenji Kuwayama, Hajime Miyaguchi, Tatsuyuki Kanamori, Kenji Tsujikawa, Tadashi Yamamuro, Yuki Okada, Yuko T Iwata","doi":"10.1007/s11419-026-00764-5","DOIUrl":"10.1007/s11419-026-00764-5","url":null,"abstract":"<p><p>PURPOSE: We previously developed micro-segmental analysis (MSA), which measured detailed distributions of drugs in hair by segmenting a single hair strand at a 0.4-mm interval. MSA is effective in estimating the day of ingestion of hypnotics in drug-facilitated crimes. However, the disadvantage is that it needs considerable time to provide analytical results because hundreds of segments are analyzed using liquid chromatography (LC)-tandem mass spectrometry. In this study, the LC conditions were modified to improve the throughput of measurements. METHODS: Instrumental parts and parameters for LC were optimized to shorten the cycle time while maintaining separation between etizolam and triazolam, which have relatively similar retention times and the same nominal mass. Additionally, parallel column regeneration (PCR) available in two-dimensional LC system, which can switch channels through two identical columns, was used. RESULTS: The cycle time was reduced from 429 s to 235 s by changing the sample needle volume (from 15 to 30 µL), column length (from 150 to 50 mm) and injection volume (from 50 to 30 µL), and steepening the mobile phase gradient. Moreover, the use of PCR reduced the cycle time to 194 s by paralleling sample measurements with washing and equilibration of the lines. The effectiveness of the LC method modified using PCR was confirmed by the MSA of a long hair (978 segments) collected from a participant who had ingested hypnotics. CONCLUSIONS: This high-throughput measurement greatly contributed to saving machine time and early providing evidence of drug-facilitated crimes, showing detailed distributions of drugs in hair.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"419-428"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147354472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Use of nitrogen as an alternative to helium in gas chromatography/mass spectrometry for forensic toxicological analysis.","authors":"Kazumichi Kakazu, Kenji Ninomiya, Chiaki Fuke, Natsuki Ikematsu, Maki Fukasawa, Mio Takayama","doi":"10.1007/s11419-025-00744-1","DOIUrl":"10.1007/s11419-025-00744-1","url":null,"abstract":"<p><strong>Purpose: </strong>Although helium is commonly used as a carrier gas for gas chromatography/mass spectrometry (GC-MS), this practice is unsustainable owing to the unstable supply and rising prices of helium. Therefore, this study investigated nitrogen as an alternative carrier gas in forensic toxicology applications to suggest a simple transfer of analytical conditions.</p><p><strong>Methods: </strong>GC-MS using nitrogen was compared with the conventional helium-based method for 34 compounds, including illegal drugs, medications, and pesticides. The targets were analyzed in a 10 µg/mL solution under constant linear velocity and pulsed splitless modes. Three analytical conditions were evaluated by varying the linear velocity and ionization voltage.</p><p><strong>Results: </strong>With nitrogen as the carrier gas, the average peak height was 10% compared to that with helium. The similarity index of the obtained mass spectra and that acquired with helium averaged at 84. However, under conditions at which the velocity decreased, the peak height was approximately the same, whereas the spectral similarity index increased to an average of 91. Furthermore, under conditions in which the ionization voltage was changed, the spectral similarity index slightly decreased to an average of 81, whereas the peak height significantly increased to an average of 87%.</p><p><strong>Conclusions: </strong>Although analysis using nitrogen with the same level of detection sensitivity as that used with helium was challenging, qualitative analysis was considered sufficient for samples with high concentrations, such as powders, tablets, liquids, and plants. Thus, nitrogen can serve as a viable alternative carrier gas to helium in GC-MS analysis.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"281-295"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147282993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Artificial intelligence assisted behavioral profiling of synthetic cannabinoids in planarians.","authors":"Jay R Vargas, Laura L Hernandez, Leo H Lai, Helen H Chang","doi":"10.1007/s11419-026-00769-0","DOIUrl":"10.1007/s11419-026-00769-0","url":null,"abstract":"<p><p>PURPOSE: Freshwater planarians provide a rapid and scalable biological model for detecting drug-induced neurobehavioral effects. This study evaluated whether automated behavioral profiling in Dugesia dorotocephala could distinguish phytocannabinoid and synthetic cannabinoid exposure based on organism-level motor responses. METHODS: Planarians were acutely exposed to Δ⁹-tetrahydrocannabinol (Δ⁹-THC), AB-PINACA, MA-CHMINACA, A-796,260, or JWH-412 at concentrations of 5–60 µg/mL in artificial spring water containing PEG-400. Locomotion and posture were recorded for 5 min and analyzed using LabGym, a supervised deep-learning–based behavioral classification system that quantified gliding, headshake activity, and sustained C-shaped postures. RESULTS: Distinct compound-associated behavioral profiles were observed. Δ⁹-THC and JWH-412 produced marked suppression of total locomotion relative to pooled controls. AB-PINACA and MA-CHMINACA preserved overall movement volume but produced severe disruption of coordinated gliding accompanied by frequent abnormal postural states. A-796,260 produced comparatively mild effects on locomotor organization. These findings revealed separable behavioral patterns that were further resolved in a two-dimensional state space by PCA (91.4% variance captured). These results demonstrate that cannabinoid exposures differing in pharmacological class produce separable behavioral patterns defined by both movement magnitude and organization. CONCLUSIONS: Automated planarian behavioral profiling provides a biologically grounded functional assay capable of distinguishing synthetic cannabinoids based on integrated motor signatures. This scalable non-vertebrate platform may support forensic toxicology by enabling early functional characterization of emerging synthetic cannabinoids and other novel psychoactive substances relevant to regulatory monitoring and public health surveillance.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"385-394"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147716443","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and quantification of two etomidate analogues ABP-700 and 2,6-dichloro-3-fluoro-etomidate in e-cigarette liquid and hair by GC-MS and LC-MS/MS.","authors":"Yinyin Dai, Jian Jiang, Amin Wurita, Koutaro Hasegawa","doi":"10.1007/s11419-026-00755-6","DOIUrl":"10.1007/s11419-026-00755-6","url":null,"abstract":"<p><strong>Purpose: </strong>In response to the Chinese government's regulation of etomidate and some of its analogs, illicit manufacturers have turned to novel structural analogs of etomidate to circumvent legal restrictions. This study aims to establish a qualitative and quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection of two emerging etomidate analogs-ABP-700 and 2,6-dichloro-3-fluoro-etomidate-and apply it to the analysis of hair samples from actual abusers.</p><p><strong>Methods: </strong>Hair samples were washed, segmented, cryogenically ground, and extracted with methanol. For extraction, precisely 20 mg of homogenized hair sample was spiked with deuterated internal standard and subjected to methanol extraction with vortex mixing followed by centrifugation. The resulting supernatant was filtered through a 0.22 μm membrane filter prior to UPLC-MS/MS analysis.</p><p><strong>Results: </strong>The method showed excellent quantitative linearity (r² > 0.99) over the range of 0.01-1 ng/mg. The limits of detection and quantification were 0.005 ng/mg and 0.01 ng/mg for both analytes. In authentic hair samples from suspected abusers, ABP-700 was detected at 0.11 ng/mg and 0.02 ng/mg, while 2,6-dichloro-3-fluoro-etomidate was quantified at 0.13 ng/mg. Concentrations in seized e-cigarette liquids were 22.6 µg/mg and 48.6 µg/mg, respectively.</p><p><strong>Conclusions: </strong>This study establishes a validated analytical method for the simultaneous determination of ABP-700 and 2,6-dichloro-3-fluoro-etomidate in human hair matrices. To our knowledge, this is the first reported methodology enabling both identification and quantification of these novel etomidate analogs in hair samples, with successful application to forensic case samples.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"327-340"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145997716","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Study on the expression changes of hepatic Gluconeogenesis-related proteins induced by insulin Overdose-related liver injury.","authors":"Yuhao Yuan, Yiling Li, Jiaxin Zhang, Xiangting Gao, Chen Chen, Zhonghao Yu, Yiwu Zhou","doi":"10.1007/s11419-025-00754-z","DOIUrl":"10.1007/s11419-025-00754-z","url":null,"abstract":"<p><p>PURPOSE: Insulin overdose severely disrupts hepatic glucose metabolism, leading to toxic changes in the liver that may play an important role in the diagnosis and identification of insulin overdose. METHODS: A rat model of insulin overdose was established in this study. ALT and AST levels were measured to assess liver function. Hematoxylin and eosin, oil red O, and periodic acid-Schiff staining were used to evaluate pathological liver injury. Ultrastructural changes in hepatocyte organelles were observed under a transmission electron microscope. The expression levels of liver gluconeogenesis-related proteins, including forkhead box protein O1 (FOXO1) and phosphorylated FOXO1 (p-FOXO1), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase catalytic subunit (G6PC), chromosome region maintenance 1 (CRM1), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3), were detected in the rat model and human autopsy liver tissues using immunohistochemistry and immunofluorescence. RESULTS: The insulin overdose group showed elevated ALT/AST, hepatocyte fatty degeneration, glycogen depletion, mitochondrial swelling, and cristae loss. The recovery group exhibited milder fatty changes, glycogen accumulation, and restored mitochondria. Insulin overdose increased hepatic p-FOXO1, CRM1, and 14-3-3 expression but decreased PEPCK and G6PC. FOXO1 shifted from nuclear to cytoplasmic localization in overdose groups, with partial nuclear recovery post-recovery. CONCLUSION: Insulin overdose leads to hypoglycemia, which in turn causes hepatocyte damage. Insulin overdose promotes the nuclear translocation of FOXO1 protein in hepatocytes, and this process shows high reproducibility in human cases of fatal insulin overdose. The coordinated changes in these indicators have significant value in detecting and diagnosing fatal cases of insulin overdose.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"296-312"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145849080","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative analysis and method validation of mirogabalin in postmortem blood and urine using liquid chromatography-tandem mass spectrometry.","authors":"Kenta Yuyama, Masako Suzuki, Itaru Yamagishi, Hideki Nozawa, Kayoko Minakata, Koutaro Hasegawa","doi":"10.1007/s11419-026-00766-3","DOIUrl":"10.1007/s11419-026-00766-3","url":null,"abstract":"<p><p>PURPOSE: Mirogabalin is a gabapentinoid used to treat peripheral neuropathic pain. While its adverse effect profile is generally mild, postmortem toxicological data are currently unavailable. This study aimed to develop and validate a quantitative method for determining mirogabalin concentrations in postmortem blood and urine using liquid chromatography–tandem mass spectrometry (LC-MS/MS), and to evaluate its concentrations in a fatal multiple drug intoxication case involving mirogabalin. METHODS: Postmortem blood and urine specimens were collected during a forensic autopsy following a mirogabalin overdose. For method development and validation, blank blood and urine samples were spiked with mirogabalin standard solutions. Mirogabalin was extracted using the QuEChERS method, further purified using Captiva ND Lipids cartridges, and analyzed by LC-MS/MS. The method was validated according to the U.S. Food and Drug Administration bioanalytical guideline. RESULTS: The method demonstrated good linearity over the range of 0.2–100 µg/mL (r > 0.99), with an LLOQ of 0.1 µg/mL. Mirogabalin concentrations in postmortem blood and urine were 1.99 µg/mL and 3.56 µg/mL, respectively. Accuracy and precision were within acceptable limits. Matrix effects were observed, indicating ion suppression. Recovery rates were generally within the recommended range. Mirogabalin also showed sufficient stability. CONCLUSIONS: This is the first study to establish and validate an LC-MS/MS method for quantifying mirogabalin in postmortem blood and urine. The findings provide valuable data for forensic toxicological interpretation and evaluation of fatal intoxication cases associated with mirogabalin.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"363-371"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147503514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of dithiocarbamate fungicides by hydrophilic interaction liquid chromatography-mass spectrometry.","authors":"Daisuke Watanabe, Shuhei Sonoda","doi":"10.1007/s11419-026-00761-8","DOIUrl":"10.1007/s11419-026-00761-8","url":null,"abstract":"<p><p>PURPOSE: Many dithiocarbamate (DTC) fungicides are complexes between organic anions and metal cations. They are difficult to be analyzed, and no simple methods have been established to directly determine their components. In this study, we developed a simple method to detect organic anions and metal cations simultaneously without derivatization. METHODS: DTC compounds were dissolved in alkaline ethylenediaminetetraacetic acid (EDTA) solution to generate organic anions and metal-EDTA complexes. They were analyzed simultaneously by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS). In addition to commercially available DTC formulations, soil samples stained with the formulations were analyzed by simulating illegal dumping cases onto the ground. RESULTS: All DTC standards and formulations were dissolved in alkaline EDTA solution, and the corresponding organic anions and metal-EDTA complexes were detected. DTC components were also detected in the soil samples without significant interference from the soil, and the DTC formulations were also detectable. CONCLUSIONS: Conventional detection methods require derivatization and only provide the classification of organic anions; however, our method enables complete discrimination of DTCs, including the metal cations, without any derivatization. Our method enables the forensic analysis of DTCs in not only poisoning cases, but also illegal dumping cases.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"411-418"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147354489","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"'3-Trifluoromethylphenylpiperazine (TFMPP)' misidentification in aripiprazole-positive cases: a cautionary note.","authors":"Karolina Nowak, Marcin Zawadzki, Paweł Szpot","doi":"10.1007/s11419-026-00767-2","DOIUrl":"10.1007/s11419-026-00767-2","url":null,"abstract":"<p><p>PURPOSE: Aripiprazole is an atypical antipsychotic agent with antidepressant properties, indicated for the treatment of schizophrenia, bipolar I disorder, and major depressive disorder, available only by prescription. The authors note that 3-trifluoromethylphenylpiperazine (TFMPP) may be misattributed in aripiprazole-positive cases, due to its likely coelution with 1-(2,3-dichlorophenyl)piperazine (DCPP), one of aripiprazole’s metabolites. METHODS: Biological matrices collected post-mortem (blood and vitreous humor) underwent liquid-liquid extraction with ethyl acetate in an alkaline medium (pH 9), followed by in-depth toxicological assessment via ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. RESULTS: Comprehensive analysis using multiple reaction monitoring (MRM) and product ion scan modes confirmed the absence of TFMPP in biological samples positive for aripiprazole. CONCLUSIONS: In toxicological analyses, particularly in forensic toxicology, utmost caution must be exercised when reporting and interpreting results, both in cases involving living subjects and post-mortem materials. It is recommended to monitor not only major metabolites but also minor ones. Analytical methods should be developed using certified reference standards, and in disputed cases, methodologies should be established that allow for verification of the contested results.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":"429-436"},"PeriodicalIF":2.1,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147573002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Post-mortem degradation of flunitrazepam by aldehyde oxidase 1 and hemoglobin in blood.","authors":"Yoshikazu Yamagishi, Hiroyuki Inoue, Sayaka Nagasawa, Hirotaro Iwase, Yasumitsu Ogra","doi":"10.1007/s11419-026-00777-0","DOIUrl":"https://doi.org/10.1007/s11419-026-00777-0","url":null,"abstract":"<p><strong>Purpose: </strong>Flunitrazepam is a widely used hypnotic agent detected in many fatal intoxication and suicide cases. However, its concentration in blood during autopsy differs from that at the time of death; it is either undetected or detected at lower concentrations than expected in post-mortem blood. In this study, we evaluated interactions between flunitrazepam and human blood using liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS).</p><p><strong>Methods: </strong>We measured flunitrazepam, 7-aminoflunitrazepam via reduction by aldehyde oxidase 1 (AOX1), and the degradation product of 7-aminoflunitrazepam via the Fenton reaction by hemoglobin (Hb) in human blood. In addition, we also detected flunitrazepam, 7-aminoflunitrazepam, and their degradation products in the blood of individuals who died from intentional ingestion of flunitrazepam.</p><p><strong>Results: </strong>We found that flunitrazepam was reduced to 7-aminoflunitrazepam by AOX1 in blood, and 7-aminoflunitrazepam was oxidized by Hb via the Fenton reaction. The mass spectra obtained by LC-Q-Orbitrap-MS revealed the formation of 7-amino-5-(2-fluorocyclohexa-1,3-dien-1-yl)-3-hydroxy-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (7-amino-3-hydroxyflunitrazepam) in the Hb/H₂O₂ reaction mixture containing 7-aminoflunitrazepam, as well as in the blood of some individuals who died from intentional ingestion of flunitrazepam.</p><p><strong>Conclusion: </strong>7-Amino-3-hydroxyflunitrazepam is the degradation product of flunitrazepam at various temperatures. The degradation is brought about by post-mortem changes via AOX1-mediated reduction and Hb-mediated Fenton reaction. Thus, 7-amino-3-hydroxyflunitrazepam may be a valuable biomarker for correcting blood flunitrazepam concentration at the time of death in clinical cases.</p>","PeriodicalId":12329,"journal":{"name":"Forensic Toxicology","volume":" ","pages":""},"PeriodicalIF":2.1,"publicationDate":"2026-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148293935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}