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.

Quantitative analysis of tetrahydrocannabinol isomers and other toxicologically relevant drugs in blood.

A multi-analyte liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described, involving the separation of delta-9-tetrahydrocannabinol (delta-9-THC) and delta-8-THC in addition to other commonly encountered drugs and metabolites. Briefly, sample preparation involved an alkaline liquid-liquid extraction (methyl tert-butyl ether) of blood (100 µl). The solvent layer was transferred, evaporated to dryness, reconstituted, and samples then separated on an Agilent Poroshell 120 EC-C18 100 Å (50 mm × 3.0 mm, 2.7 µm) analytical column using a multi-step gradient elution of 50 mM ammonium formate in water (pH 3.5) and 0.1% formic acid in methanol over 14 min. A SCIEX Triple Quad 6500+ system operating in scheduled multiple reaction monitoring and positive electrospray ionization was used for detection. There were no interferences, and matrix effects were generally acceptable (±20% of neat response). Linearity was achieved within the calibration range, including methylamphetamine (MA) (10-1000 ng/ml), 3,4-methylenedioxy-N-methylamphetamine (MDMA) (10-1,000 ng/ml), cocaine (10-1000 ng/ml), and two THC isomers (1-100 ng/ml). Accuracies of MA, MDMA, cocaine, and two THC isomers were 3.6 to 8.9%, -1.2 to 4%, -5.3 to 5.8%, and -11 to 14%, respectively; while precision estimates of the same were 1.6 to 5.4%, 1.7 to 5.3%, 1.2 to 4.5%, and 2 to 10%, respectively. Autosampler stability and dilution integrity were within acceptable limits, and no carryover was detected at the limit of detection. This validated LC-MS/MS method made the routine identification of both delta-9-THC and delta-8-THC in blood possible.

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.

Fatal Intoxications from a Combination of 4-Fluoroamphetamine and 25C-NBOMe.

Six fatalities have occurred from the ingestion of a combination of new psychoactive substances (NPSs), 4-fluoroamphetamine (4FA) and 2-(4-chloro-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) over a 9-month period. Four of these fatalities (one older female and three young males) were from direct adverse effects of drugs, and one each from a fall while being intoxicated and during restraint. All cases were subject to full postmortem examinations that included collection of femoral blood. The four drug-caused fatalities had postmortem blood concentrations for 4FA and 25C-NBOMe of 330-682 ng/L (median 417) and 1.4-12 ng/mL (median 4.3), respectively. The other two cases (both young males) where death was considered to have been caused indirectly by drug intoxication had 4FA and 25C-NBOMe postmortem concentrations of 21 and 123 ng/mL, and 1.8 and 4.5 ng/mL, respectively. None of these cases showed concentrations of drugs that suggested use of high recreational doses. In one drug-caused death, capsules and a brown powder obtained from the scene were found to contain a mixture of these two NPSs. With the exception of one drug-caused death, other drugs were detected; however, the effects of the two NPSs together were regarded as the primary triggers for the deaths. There were no consistent symptoms or pathology in these cases; however, agitation/aggression was observed in two cases prior to their collapse, with seizures in possibly three cases. Pulmonary and/or cerebral edema was noted in three cases. Potentially significant natural disease (a mildly enlarged heart) was only observed in one drug-caused case. These cases illustrate a possible increased risk of sudden death with this combination of drugs, both of which can elevate serotonin concentrations as well as act as strong stimulants. These cases also illustrate the difficulty in detecting NPS in cases where no prior information is available that might suggest their use.

Postmortem Drug Redistribution: A Compilation of Postmortem/Antemortem Drug Concentration Ratios.

Postmortem drug redistribution (PMR) is a well-known phenomenon in forensic toxicology with implications for medico-legal death investigations. Paired antemortem (AM) specimen and postmortem (PM) mortuary admission femoral blood drug concentrations from 811 coronial cases were used to construct a retrospective compilation of PM/AM drug concentration ratios for 42 parent drugs and metabolites. The median PM/AM ratios for all antidepressants were > 1 and consistent with PMR In contrast, the median PM/AM ratios of most benzodiazepines were < 1. The antipsychotics were varied (0.63-3.3) and suggest the mixed effects of PMR and drug instability. Amphetamines exhibited no trends (0.90-0.95) and are likely confounded by many factors. The PM/AM ratios of cardiovascular drugs, opioids and other drugs are also reported. This research represents an expansive retrospective compilation of paired AM and PM drug concentrations for many toxicologically relevant drugs. While the median PM/AM ratios demonstrate some drug-dependent trends, there was no obvious relationship between AM specimens and PM femoral blood taken at mortuary admission.

High Throughput Detection of 327 Drugs in Blood by LC-MS-MS with Automated Data Processing.

The described procedure provides a rapid technique for the detection and semi-quantitation of a large number of drugs in blood. This procedure uses a minimal sample volume and employs a one-step liquid extraction and automated data processing to yield rapid turnaround times. A total of 327 of the most commonly used medicinal and illicit drugs in Australia were selected including various amphetamines, anesthetics, antidepressants, antipsychotics, anticonvulsants, benzodiazepines, beta blockers, opioid and nonopioid analgesics, stimulants, THC and a large number of synthetic cannabinoids and other novel psychoactive substances. The extracts were subject to 5-minute chromatography using a Kinetex C18 50 × 4.6 mm 2.6 µm solid-core analytical column and analyzed using a Sciex 3200 Q-TRAP MS-MS (+ ESI, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Matrix effects and extraction efficiencies were acceptable with most analytes showing > 80% response and low variation (within 25%RSD). Cannabinoids were most affected by the matrix and yielded poorest recovery values but were still detectable. Precision, accuracy, repeatability and multipoint linearity were assessed for all analytes. The method has been used in routine practice in the forensic toxicology service at the Victorian Institute of Forensic Medicine in over 6000 coronial investigations using both postmortem and clinical blood specimens. This technique has greatly increased throughput, reduced turnaround times and allowed for rapid same-day analysis of results when needed. The method is routinely used in routine overnight testing with results reported to pathologists within 4 h of data acquisition. This rapid toxicological technique is used in conjunction with other investigative processes such as full-body CT imaging, review of case circumstances and medical histories to provide an efficient death investigation process.

Odds of culpability associated with use of impairing drugs in injured drivers in Victoria, Australia.

Culpability analysis was conducted on 5000 drivers injured as a result of a vehicular collision and in whom comprehensive toxicology testing in blood was conducted. The sample included 1000 drivers for each of 5 years from approximately 5000-6000 drivers injured and taken to hospital in the State of Victoria. Logistic regression was used to investigate differences in the odds of culpability associated with alcohol and drug use and other selected crash attributes using the drug-free driver as the reference group. Adjusted odds ratios were obtained from multivariable logistic regression models in which other potentially explanatory driver and crash attributes were included. Drivers with alcohol present showed large increases in the odds of culpability similar to that seen in other studies investigating associations between blood alcohol concentration and crash risk. Methylamphetamine also showed a large increase in the odds of culpability (OR 19) compared to the reference group at both below and above 0.1 mg/L, whereas those drivers with Δ9-tetrahydrocannabinol (THC) present showed only modest increase in odds when all concentrations were assessed (OR 1.9, 95 %CI 1.2-3.1). Benzodiazepines in drivers also gave an increase in odds (3.2, 95 %CI 1.6-6.1), but not other medicinal drugs such as antidepressants, antipsychotics and opioids. Drivers that had combinations of impairing drugs generally gave a large increase in odds, particularly combinations of alcohol with THC or benzodiazepines, and those drivers using both THC and methamphetamine.

A systematic investigation of forensic hair decontamination procedures and their limitations.

The effectiveness of decontamination procedures used for the removal of external drug contamination in forensic hair analysis is an ongoing debate. This investigation evaluated wash methods complying with Society of Hair Testing (SoHT) guidelines and their capacity to remove cocaine (COC) and methamphetamine (MA) from artificially contaminated hair. The most effective decontamination method was determined using a systematic approach, involving (1) an initial washing solvent screen, (2) optimization of wash duration, (3) comparison of sequential wash methods, and (4) reanalysis of clinical hair samples. For analysis, hair was subjected to micro-pulverized methanolic extraction prior to quantitation by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Methanol (MeOH) and 0.1 M phosphate buffer (pH 6) were the most effective organic and aqueous solvents, respectively, removing 28%-38% of COC and 16%-31% of MA. Wash durations longer than 30-60 minutes did not remove additional amounts, and a more efficient sequential wash method was subsequently developed. Despite this, the interpretation of reportable results relative to the SoHT cut-off levels was unchanged for most clinical hair samples reanalyzed after washing by agitation for 30 minutes with MeOH. These findings highlight the inability of decontamination solvents to completely remove external COC and MA contamination from hair, including wash methods adhering to SoHT guidelines.

Analysis of Benzodiazepines for Drug-Facilitated Assaults and Abuse Settings (Urine).

An overview of the detection of benzodiazepines and their respective metabolites and target analytes in urine by LC-MS/MS is described. This overview shows substantial differences in the approach to detection using this technique including optional use of ß-glucuronidase to hydrolyze conjugates present in urine. There are also significant variations in the extraction method employed from the use of direct injection, liquid-liquid extraction to solid-phase extraction options, with little apparent difference in limits of detection. Chromatography was largely based on the use of C18-bonded columns; however both C8- and phenyl-bonded columns were used to affect separation. Modern-day tandem mass spectrometers are capable of exceptional sensitivity enabling detection of sub-nanogram per milliliter amounts in urine, which provide for longer detection times in the urine of suspected drug-facilitated assaults. A method employed in the laboratory of the authors is provided by way of an example for readers wishing to establish a method in their own laboratory.

The analysis of 132 novel psychoactive substances in human hair using a single step extraction by tandem LC/MS.

A rapid LC-MS/MS method for the targeted screening of 132 NPS in hair is described. Drugs include cathinones and synthetic cannabinoids, as well as amphetamine-type stimulants, piperazines and other hallucinogenic compounds. This method utilizes hair pulverization in acidified methanol followed by analysis using liquid chromatography coupled with tandem MS. The limit of detection varied from 0.001 to 0.1ng/mg hair among the various analytes. The method was validated in terms of sensitivity, selectivity, repeatability and stability. The limit of reporting was set at 0.1ng/mg of hair. The method was successfully applied to 23 medico-legal cases where NPS were detected in blood or where NPS use was suspected. The identified NPS included acetyl fentanyl, 25C-NBOMe, MDPV, PB-22 and AB-FUBINACA, allowing hair to be used where historical or retrospective information on use of NPS is sought. This technique has proven to be efficient for the one step extraction from hair of different classes of NPS in routine toxicological investigations; from unstable and volatile compounds, such as most of the cathinones, to hydrophobic compounds such as synthetic cannabinoids.

A Cluster of Fentanyl-Laced Heroin Deaths in 2015 in Melbourne, Australia.

The prevalence of opioid use in therapeutic and recreational settings has steadily increased throughout the western world. The addition of fentanyl into heroin products can produce potentially dangerous consequences, even to opioid tolerant individuals who may be unaware of such additions. Following an observed spike of heroin-fentanyl related deaths in Melbourne, Australia, a study was undertaken to determine the prevalence of these cases. All reportable deaths occurring in Victoria during 2015 and submitted to the toxicology laboratory were analysed using LC-MS-MS to confirm the combination of the heroin marker 6-acetylmorphine and/or morphine, and fentanyl. Over 4,000 coronial cases in 2015 underwent toxicological analysis for these drugs, there were nine cases identified that involved fentanyl-laced heroin. There was no specific mention of fentanyl use in any of these cases. All occurred within 2 months and in two distinct locations. The first four deaths occurred within 3 days of each other, in neighboring suburbs. The ages ranged from 25 to 57 years with an average of 40 and median of 37 years, and consisted of eight males and one female. The average and median femoral blood concentration of fentanyl was 18 and 20 ng/mL (range: <1-45 ng/mL), and morphine 140 and 80 ng/mL (range: 20-400 ng/mL), respectively. All nine cases had 6-acetylmorphine detectable in blood. Urine analysis was also performed where available. A syringe, powder and spoon found at the scene of one case were also analysed and found to be positive for both heroin and fentanyl, which supported the likelihood of fentanyl-laced heroin. This is the first reported case series of fatalities involving heroin and fentanyl outside of North America in published literature. These findings may help inform public health and prevention strategies serving to decrease the potential for such fatalities in the future.

Ultra-rapid targeted analysis of 40 drugs of abuse in oral fluid by LC-MS/MS using carbon-13 isotopes of methamphetamine and MDMA to reduce detector saturation.

The number of oral fluid samples collected by the road policing authority in Victoria, Australia, requiring confirmatory laboratory analysis for drugs proscribed under Victorian legislation (methamphetamine, MDMA and Δ9-tetrahydrocannabinol) has greatly increased in recent years, driving the need for improved analysis techniques to enable expedient results. The aim of this study was to develop an LC-MS/MS-based targeted oral fluid screening technique that covers a broad range of basic and neutral drugs of abuse that can satisfy increased caseload while monitoring other compounds of interest for epidemiological purposes. By combining small sample volume, simple extraction procedure, rapid LC-MS/MS analysis and automated data processing, 40 drugs of abuse including amphetamines, benzodiazepines, cocaine and major metabolites, opioids, cannabinoids and some designer stimulants were separated over 5 min (with an additional 0.5 min re-equilibration time). The analytes were detected using a Sciex® API 4500 Q-Trap LC-MS/MS system with positive ESI in MRM mode monitoring three transitions per analyte. The method was fully validated in accordance with international guidelines and also monitored carbon-13 isotopes of MDMA and MA to reduce detector saturation effects, allowing for confirmation of large concentrations of these compounds without the need for dilution or re-analysis. The described assay has been successfully used for analysis of oral fluid collected as part of law enforcement procedures at the roadside in Victoria, providing forensic results as well as epidemiological prevalence in the population tested. The fast and reliable detection of a broad range of drugs and subsequent automated data processing gives the opportunity for high throughput and fast turnaround times for forensic toxicology.

Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS.

The aim of this study was to develop an LC-MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100µL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5µm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex(®) API 2000 LC-MS/MS system (ESI+ and -, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC-MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology.