Identification of clobromazolam in Australian emergency department intoxications using data-independent high-resolution mass spectrometry and the HighResNPS.com database.

The proliferation of novel psychoactive substances (NPSs) continues to challenge toxicology laboratories. In particular, the United Nations Office on Drugs and Crime considers designer benzodiazepines to be a current primary threat among all NPSs. Herein, we report detection of a new emerging designer benzodiazepine, clobromazolam, using high-resolution mass spectrometry and untargeted data acquisition in combination with a "suspect screening" method built from the crowd-sourced HighResNPS.com database. Our laboratory first detected clobromazolam in emergency department presenting intoxications included within the Emerging Drugs Network of Australia-Victoria project in the state of Victoria, Australia, from April 2022 to March 2023. Clobromazolam was the most frequent designer benzodiazepine detected in this cohort (100/993 cases, 10%). No patients reported intentional administration of clobromazolam, although over half reported exposure to alprazolam, which was detected in only 7% of cases. Polydrug use was prevalent (98%), with phenazepam (45%), methylamphetamine (71%) and other benzodiazepines (60%) most frequently co-detected. This is the first case series published in the literature concerning clobromazolam in clinical patients. The identification of clobromazolam in patients presenting to emergency departments in Victoria demonstrates how high-resolution mass spectrometry coupled with the HighResNPS.com database can be a valuable tool to assist toxicology laboratories in keeping abreast of emerging psychoactive drug use.

Non-fatal intoxications involving the novel benzodiazepine clonazolam: case series from the Emerging Drugs Network of Australia - Victoria project.

INTRODUCTION: Clonazolam is an unregistered novel benzodiazepine which emerged in global illicit drug markets in 2014. We describe the clinical features of four cases of non-fatal clonazolam mono-intoxications from patients presenting to emergency departments in Australia. CASES: Four patients aged between 16 and 19 years presented to hospital with a sedative toxidrome (Glasgow Coma Scale range 8-13) and elevated heart rate (median heart rate 100 beats per minute, range 92-105) following reported benzodiazepine exposure. Three patients reported the use of a large quantity (7-20 tablets) of Xanax®, a brand of alprazolam not commercially available in Australia. Two patients required nasopharyngeal airway insertion following the development of airway obstruction. The median time to return of a normal conscious state (Glasgow Coma Scale 15) was 23 h (range 5-30 h). Clonazolam (range 0.2-2.1 µg/L) and its main metabolite 8-aminoclonazolam (range 5.9-19.1 µg/L) were the only substances detected by liquid chromatography-tandem mass spectrometry in blood samples of all patients. CONCLUSION: Clonazolam intoxication resulted in sedation with mild sinus tachycardia. Three patients who reported multiple tablet exposures experienced prolonged sedation, and two of these patients developed airway obstruction. In this series, clonazolam was unknowingly ingested through possible illicit substitution within an unregulated counterfeit benzodiazepine product.

Microbial degradation products of lurasidone and their significance in postmortem toxicology.

Recent research reported that lurasidone degrades in unpreserved ante-mortem human whole blood inoculated with microorganisms known to dominate postmortem blood specimens. In vitro degradation occurred at a similar rate to risperidone, known to degrade in authentic postmortem specimens until below analytical detection limits. To identify the lurasidone degradation products formed, an Agilent 6520 liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) operating in auto-MS/MS mode was used. Numerous degradation products not previously reported in prior in vitro or in vivo pharmacokinetic studies or forced degradation studies were detected. Accurate mass data, mass fragmentation data, acetylation experiments, and a proposed mechanism of degradation analogous to risperidone supports initial identification of the major degradation product as N-debenzisothiazole-lurasidone (calculated m/z [M + H]+ = 360.2646). A standard was unavailable to conclusively confirm this identification. Retrospective data analysis of postmortem cases involving lurasidone identified the presence of the major degradation product in four of six cases where lurasidone was also detected. This finding is significant for toxicology laboratories screening for this drug in postmortem casework. The major postmortem lurasidone degradation product has consequently been added to the LC-QTOF-MS drug screen at Forensic Science SA (FSSA) to indicate postmortem lurasidone degradation in authentic postmortem blood specimens and as a marker of lurasidone administration in the event lurasidone is degraded to concentrations below detection limits.

In vitro degradation of ziprasidone in human whole blood.

A systematic study was performed into the degradation of ziprasidone in simulated postmortem blood. Fifteen potential degradation products not previously reported in the literature were observed. Four resulted from degradation in human blood, whereas the remaining products resulted from reaction with solvents: four from alkaline degradation, four from reaction with acetaldehyde, and three from reaction with acetone. To identify possible degradation products, a liquid chromatograph-diode array detector (LC-DAD) and liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) operating in auto-MS/MS mode were used. It was indicated from red-shifted UV-Vis spectra, accurate mass data, mass fragmentation data, and a deuteration experiment that the site of ziprasidone degradation, in the in vitro blood experiments, was the methylene carbon of the oxindole moiety. The major in vitro blood degradation products were proposed to be E/Z isomers of 3-ethylidene-ziprasidone. Further, another in vitro degradation product in microbially inoculated blood specimens was proposed to be 3-ethyl-ziprasidone. 3-Ethylidene-ziprasidone was hypothesized to form from the reaction of ziprasidone with acetaldehyde derived from the ethanol used to spike ziprasidone into the in vitro blood experiments. Data from two postmortem investigations were available for retrospective reanalysis. Attempts were made to detect degradation products of ziprasidone, but none were found.

Intoxications in an Australian Emergency Department Involving 'Nitazene' Benzylbenzimidazole Synthetic Opioids (Etodesnitazene, Butonitazene and Protonitazene).

Benzimidazole synthetic opioids are highly potent µ-opioid receptor agonists with heroin-like effects, including dose-dependent respiratory depression and a high risk of abuse and toxicity. Benzimidazoles were first detected in 2019 in Europe and Canada, with analytical confirmation of etodesnitazene, protonitazene and butonitazene in 2021. We report the first detections of these compounds in Australia, in two patients presenting with drug toxicity to Emergency Departments (EDs) in the state of Victoria. Case 1 was a female in her 20s who rectally administered etodesnitazene and was found unconscious with respiratory depression and hypotension. Case 2 was a female in her 30s who presented to the ED in a sedated state after taking a formulation of protonitazene that also contained butonitazene, in addition to methylamphetamine. She responded positively to naloxone. Novel synthetic opioids were used with prior experience of the formulations purchased; however, the unpredictability of their effects was demonstrated by the acute toxicity experienced with this occasion of use. Toxicosurveillance of ED presentations with analytical confirmation of drugs is crucial in identifying emerging drugs in the community and informing harm reduction strategies.

Investigations into the stability of 17 psychoactive drugs in a "simulated postmortem blood" model.

In the postmortem environment, some drugs and metabolites may degrade due to microbial activity, even forming degradation products that are not produced in humans. Consequently, underestimation or overestimation of perimortem drug concentrations or even false negatives are possible when analyzing postmortem specimens. Therefore, understanding whether medications may be susceptible to microbial degradation is critical in order to ensure that reliable detection and quantitation of drugs and their degradation products is achieved in toxicology screening methods. In this study, a "simulated postmortem blood" model constructed of antemortem human whole blood inoculated with a broad population of human fecal microorganisms was used to investigate the stability of 17 antidepressant and antipsychotic drugs. Microbial communities present in the experiments were determined to be relevant to postmortem blood microorganisms by 16S rRNA sequencing analyses. After 7 days of exposure to the community at 37°C, drug stability was evaluated using liquid chromatography coupled with diode array detection (LC-DAD) and with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Most of the investigated drugs were found to be stable in inoculated samples and noninoculated controls. However, the 1,2-benzisothiazole antipsychotics, ziprasidone and lurasidone, were found to degrade at a rate comparable with the known labile control, risperidone. In longer experiments (7 to 12 months), where specimens were stored at -20°C, 4°C, and ambient temperature, N-dealkylation degradation products were detected for many of the drugs, with greater formation in specimens stored at -20°C than at 4°C.

Harnessing Thin-Film Continuous-Flow Assembly Lines.

Inspired by nature's ability to construct complex molecules through sequential synthetic transformations, an assembly line synthesis of α-aminophosphonates has been developed. In this approach, simple starting materials are continuously fed through a thin-film reactor where the intermediates accrue molecular complexity as they progress through the flow system. Flow chemistry allows rapid multistep transformations to occur via reaction compartmentalization, an approach not amenable to using conventional flasks. Thin film processing can also access facile in situ solvent exchange to drive reaction efficiency, and through this method, α-aminophosphonate synthesis requires only 443 s residence time to produce 3.22 g h(-1) . Assembly-line synthesis allows unprecedented reaction flexibility and processing efficiency.