Recent findings and advancements in the detection of designer benzodiazepines: a brief review

Adinazolam is a triazolobenzodiazepine of lower cardiac toxicity than other antidepressants like amitriptyline, as it does not significantly affect intraventricular conduction or cause arrhythmia, as observed in canine studies of post-myocardial infarction (18). Neuropharmacologically, adinazolam reduces norepinephrine and serotonin release in the hippocampal CA1 region, which may contribute to its anxiolytic and antidepressant properties (19). In elderly populations, the drug and its metabolite show reduced clearance, which, in turn, may lead to poorer psychomotor performance and cognitive impairment (20). Its profile is similar to atypical antidepressive and anxiolytic drugs that induce “sleep slow wave” EEG patterns at moderate and high doses (21). Adinazolam's antidepressant activity may be mediated by heightened serotonin neurotransmission in rat hippocampal pyramidal neurons (22). Its sedative and psychomotor effects are owed to its metabolite, N-demethyladinazolam (NDMAD), rather than the parent drug (23).

Being a designer drug, adinazolam is associated with growing public health concerns, especially in fatal and drug-related cases (24). At a dose of 30 mg, adinazolam can impair psychomotor and cognitive performance, and this effect is exacerbated when combined with ethanol, although a synergistic effect is not strongly supported (25). One study (26) reported that both adinazolam and its metabolite effectively induced sedation, psychomotor impairment, and memory impairment in healthy participants who were receiving 10 mg doses of adinazolam.

Clonazolam, the triazolo-analogue of clonazepam, was first synthesised in the 1970s and emerged as the most potent benzodiazepine with hypnotic, anxiolytic, sedative, muscle relaxant, and anticonvulsant properties (27). Prescribed quantities span from 0.5 mg (for non-addicted individuals) to 4 mg (for habitual benzodiazepine users). Sommerfeld-Klatta et al. (28) documented a case of a 26-year-old woman deliberately ingesting 10 mg of clonazolam powder. Despite the substantial dose and toxic blood levels, the patient's response was confined to an extended state of unconsciousness, reduced muscular tonicity, and impaired reflexes. Respiratory failure and cardiovascular complications, typically associated with benzodiazepine overdoses, were notably absent. The authors conclude that despite its high potency clonazolam is relatively safer in cases of acute overdose than other benzodiazepines.

Deschloroetizolam, classified as a short-acting thienotriazolodiazepine due to the absence of a chlorine substitution on its benzene ring, is a less potent analogue of etizolam. It was first reported by the UK Focal Point after the analysis of a seized blue tablet in September 2014 (29).

In a case reported by Balkhi et al. (30), the corpse of a 31-year-old male drug abuser was found with injection materials and small plastic bags containing various DBZs, including deschloroetizolam. Autopsy revealed multi-organ congestion, likely of toxic origin, with no evident natural diseases or traumatic lesions related to death. The time between death and discovery was estimated to be less than 48 hours. Analysis of femoral blood and urine samples, collected during autopsy, revealed deschloroetizolam at a concentration of 11 μg/L in the femoral blood and a concentration below the limit of quantification in urine.

Another study involved a researcher who self-experimented with 6 mg of deschloroetizolam (31). To monitor the drug's presence, he used the NeoSal device to gather saliva samples over 18 hours. The drug reached the highest concentration at 30 minutes, followed by a rapid decrease, but traces remained for the entire 18 hours. This decrease correlated with the quick onset and subsequent decline in the volunteer's physical symptoms, which included tiredness, dizziness, difficulty speaking, and problems with concentration.

Diclazepam's initial documentation dates back to a German report in August 2013 (32). Three patients were received by the emergency department over poisoning with diclazepam, in two cases combined with stimulants and dissociatives. Patients manifested agitation, disorientation, CNS depression, and withdrawal symptoms. A recent investigation (33) used a combination of nuclear magnetic resonance (NMR), LC-MS/MS, GC-MS, and self-experimentation to delve into the pharmacokinetics and metabolism of diclazepam within the human body. Diclazepam exhibited an approximate elimination half-life of 42 hours and underwent metabolic conversion into active benzodiazepines such as delorazepam, lorazepam, and lormetazepam. These metabolites could be detected in urine for 6–19 days post-ingestion, while unmetabolised diclazepam could be identified in blood serum for up to 99 hours. The study also showed that standard immunochemical drug tests were not sensitive enough to identify low doses of diclazepam. In fact, all serum samples tested negative for diclazepam metabolites (delorazepam, lorazepam, and lormetazepam), despite the assay having a sufficient cross-reactivity of 72 %. LC-MS/MS, in turn, could detect its metabolites for nearly three weeks post-ingestion. This comprehensive study underscores diclazepam's long activity and challenges in detection with conventional techniques.

Etizolam, a thienodiazepine akin to benzodiazepines in its effects, has been a prescription drug in a number of countries for over 35 years. In therapeutic dosages ranging from 0.5 to 3 mg per day, etizolam elicits pharmacological responses and exhibits a safety profile parallel to those of conventional benzodiazepines such as diazepam (34). Reports of harmful effects are mostly related to its non-medical application, particularly the ingestion of illegal pills containing varying doses of etizolam and other unidentified substances (35). In such settings, etizolam is often combined with opioids, alcohol, and other CNS depressants, which significantly heightens susceptibility to strong adverse reactions, including respiratory depression. Instances of lethal overdoses almost invariably involve a complex interplay of polydrug toxicity, with etizolam seldom standing as the sole culprit (36).

Flualprazolam is an analogue of alprazolam with a fluorene moiety, which was first reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) by Swedish police in January 2018 (37). A more recent report from California about traffic accidents related to driving under the influence of flualprazolam between 2018 and 2020 (38) shows that motorists with relatively low flualprazolam blood concentrations, averaging 12 ng/mL and ranging between 5.5 and 26 ng/mL, exhibited CNS depression consistent with benzodiazepine usage and were weaving between lanes. This account warns that flualprazolam can considerably impair driving ability even at concentrations below 10 ng/mL, which is much lower than anticipated for other benzodiazepines. Kriikku et al. (39) reported median blood flualprazolam concentration of 18.0 ng/g in 33 post-mortem cases and observed that Swedish victims were significantly older than the Finnish. Polydrug use, especially of flualprazolam and opioids was common, and flualprazolam was determined as the cause of death in 13 cases.

Flubromazepam originates from the 1960s, but became widespread as recreational drug only in 2012. It was retailed by online vendors to be used in formulations of psychoactive blends. Its primary metabolic pathway is hydroxylation, most likely at position 3 of the molecule, leading to 3-hydroxy-flubromazepam, apparently catalysed by CYP450 enzymes (40). In terms of pharmacokinetics, flubromazepam has a prolonged action and elimination half-life of 106 hours, similar to other long-acting benzodiazepines like diazepam and nordazepam. Moosmann et al. (40) report that a 4 mg dose reached its highest serum concentration of approximately 78 ng/mL six hours after intake and that its monohydroxylated metabolites remained detectable in serum for up to 23 days and in urine for up to 28 days. Cases admitted to emergency departments were described as experiencing severe agitation, delirium, and CNS depression. Fatalities linked directly to flubromazepam are scarce, and more common are those attributed to other co-administered substances (41).

Flubromazolam stands out for its pronounced potency. A mere 0.5 mg dose, resulting in serum concentrations peaking between 8 and 10 ng/mL (42) can cause sedation. This compound can be found in urine for up to 6.5 days, while its hydroxylated metabolite remains traceable for up to 8 days following a single administration (43). Its potency, long life, and easy accessibility make flubromazolam particularly alluring to abusers. Abdul et al. (44) report that flubromazolam in post-mortem cases ranged from 1 to 70 mg/L, which is noteworthy, as no range has been established that elicits specific clinical manifestations. All samples, the authors also noted, showed several other sedatives, including heroin and other benzodiazepines. They emphasised the importance of including flubromazolam in post-mortem screens due to its potency and potential contribution to fatalities, especially given the growing prevalence of new psychoactive substances in toxicology.

Phenazepam was designed in the Soviet Union in the 1970s as an anxiolytic and anticonvulsant but has become a popular recreational drug in Western countries in recent times. In comparison to benzodiazepines, phenazepam is more potent, with effects persisting for up to three weeks (45). Recreational doses range from 2 to 10 mg, and blood levels between 200 and 400 μg/L have been associated with functional impairments and death (46, 47). For instance, the first UK death directly involving phenazepam was reported in July 2011, followed by a second in November 2011 (48). These incidents underscored the potential for severe adverse health consequences of the recreational use of the drug.

Pyrazolam is a benzodiazepine derivative originally developed in the 1970s (49) and first reported to the EMCDDA in August 2012 (50). Moosmann et al. (51) profiled pyrazolam using NMR spectroscopy and LC-MS/MS. One of the authors ingested a 1 mg dose of and gave serum and urine samples across several days to establish its pharmacokinetic behaviour. The analysis showed an elimination half-life of about 17 h, as opposed to 6 h claimed by the online vendor. The parent compound remained detectable in the serum for over 50 h, and in urine for up to 6 days. The absence of metabolites, furthermore, suggests that pyrazolam does not biotransform in the body as much as other analogous benzodiazepines. Lehmann et al. (52) detected it in various body fluids and tissues, which indicates widespread distribution in the body after ingestion. Its concentration varied from 11 μg/L in pericardial fluid to as high as 500 μg/L in urine.

Recent findings provide initial insights into the clinical effects of DBZs and related toxicological risks. All DBZs exhibit sedative-hypnotic properties, impair the psychomotor function, and cause CNS depression, but their potency and duration of effects vary substantially. What is clear, though, is that their co-administration with other CNS depressants significantly elevates the risk of serious adverse events.

However, their pharmacokinetic properties and metabolism in humans have yet to be elucidated. Preliminary data indicate metabolic pathways analogous to traditional benzodiazepines, but we still need to identify specific enzymes involved in their biotransformation. In this respect, knowing more about the contribution of genetic polymorphisms and drug interactions could inform clinical treatment choices.

Another avenue of progress concerns advances in DBZ detection and measurement. There we see much more progress, but further refinements are needed to expand the scope of detectable DBZs and their metabolites.

Further toxicological research should also characterise the risks of chronic and polydrug abuse to facilitate clinical, forensic, and regulatory responses to this growing issue.