Benzodiazepines are a class of psychoactive drugs that act on the central nervous system (CNS) by enhancing the activity of gamma-aminobutyric acid, the major inhibitory neurotransmitter in the brain (1). Since their introduction in the 1950s, benzodiazepines have been widely prescribed for treating anxiety, insomnia, seizures, and other disorders (2). Some common benzodiazepines include diazepam, alprazolam, clonazepam, and lorazepam.
Detection of benzodiazepines and their metabolites is important in therapeutic drug monitoring, forensic toxicology, and sports anti-doping testing (3). Traditionally, this had been done with immunochemical techniques like enzyme-linked immunosorbent assays (ELISA) (4), but immunoassays lack specificity and are open to interferences. Over the past two decades, they have been replaced by chromatography coupled with mass spectrometry (MS) (5), most notably gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (6, 7), as they are highly sensitive and specific enough to distinguish individual benzodiazepines and their metabolites.
The metabolism of benzodiazepines is complex and follows several pathways involving several enzymes. It starts with the phase I metabolism, which mainly involves oxidative reactions catalysed by cytochrome P450 (CYP) enzymes (8), most notably CYP3A4, CYP2C19, and CYP2B6 (9, 10). Follow the phase II conjugative reactions like glucuronidation mediated by uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes (11). High variability in benzodiazepine metabolites poses certain challenges to their detection and therapeutic monitoring, but recent advances in the understanding of benzodiazepine metabolism pathways have paved the way for developing more sensitive and selective detection methods, such as radiolabelling and high-resolution mass spectrometry (12), which have discovered benzodiazepine metabolism by intestinal microbiota and previously undetected circulating and excreted metabolites (13). Further discoveries of differences in benzodiazepine biotransformation between humans and preclinical animal models (14) have shed new light on the extrapolation of pharmacokinetic data from animal studies to humans, whereas new
Designer benzodiazepines (DBZs) are compounds derived from traditional benzodiazepines, featuring slight chemical modifications to achieve specific pharmacological effects or circumvent existing drug regulations. Unlike their well-studied counterparts, these designer variants have often escaped comprehensive clinical testing, so their safety profiles and potential risks are unclear. While traditional benzodiazepines are controlled and prescribed, DBZs may fall outside strict legal regulation, raising concerns about their misuse and safety. The aim of this review is to take a look at the toxicology of DBZs and the challenges of their detection, considering the recent advances and new detection techniques that can improve clinical practice, forensic toxicology, and drug safety monitoring of this widely used class of psychoactive drugs.
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.
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.
LC-MS/MS has become the gold standard for benzodiazepine and metabolite analysis in biological samples. Recent advances have improved its sensitivity, selectivity, and throughput. Bergstrand et al. (53) developed a method for urine analysis of 11 DBZs that has only a 3.1 min run time. The method is sensitive (with the detection limit of 1–10 ng/mL), accurate, precise, and selective. Mastrovito et al. (54), in turn, developed an LC-MS/MS method preceded by liquid-liquid extraction, able to detect and measure 12 DBZs and their metabolites in blood. It has good validation results and has detected DBZs in 70 % of samples that were confirmed negative in the second-step screening.
In addition, advanced methods improve DBZ separation from sample matrices and have faster runs. Behnoush et al. (55) compared conventional high and ultra-high performance liquid chromatography (UHPLC). UHPLC had 25 min shorter run time, lower solvent consumption, 5–10 times better detection limits, better resolution, sensitivity, and efficiency. Vårdal et al. (56) developed a method using parallel artificial liquid membrane extraction coupled with UHPLCMS/MS. Using less organic solvent, this method had higher throughput, better sensitivity, and reproducibility on small blood volumes.
On-line SPE-LC-MS/MS workflows integrate solid-phase extraction directly with LC, providing automated sample cleanup. Turbulent flow chromatography is also applied for rapid online extraction prior to LC-MS (57). Such approaches minimise sample handling while maximising detection sensitivity and accuracy. Improving analytical sensitivity can help detect metabolites at low concentrations, which can be achieved by optimising chromatographic conditions, selecting appropriate internal standards, and using selected (multiple) reaction monitoring. To that end, Racamonde et al. (58) have developed an online SPE-LC-MS/MS method for determining 23 DBZs and their metabolites in 100–200 mL wastewater samples using automated extraction with Oasis MCX cartridges and LC-MS/MS analysis with multiple reaction monitoring. The reported limits of quantification range from 0.1 to 18 ng/L and the method shows good recoveries and precision.
Nissilä et al. (59) report improved efficiency of electrospray ionisation – mass spectrometry (ESI-MS) if a new nanoelectrospray ionisation is used. Mass spectrometer sensitivity can be boosted with optimised ion optics, ion transfer, and vacuum systems. Coupling to the high-field asymmetric waveform ion mobility spectrometry (FAIMS) improves the selectivity of MS by separating ions based on mobility differences (60). Mass spectrometry can also run faster for high-volume benzodiazepine testing if the RapidFire technology is used, which enables quick online SPE (61, 62). In addition, multiple analytes can be determined in a single injection with multiplexing based on different isotope labelling or mass defect separation (63, 64). Multiplexing improves throughput while minimising analytical variability.
While LC-MS/MS remains the gold standard (65), immunoassays can find niche applications owing to their simplicity of use and cost-effectiveness. Recent advances with novel antigens and formats seem to improve their specificity. For example, hybridoma technology can produce monoclonal antibodies targeting specific benzodiazepine metabolites like oxazepam glucuronide and temazepam, which renders them superior to polyclonal antibodies used in traditional immunoassays (66).
As for sensitivity, Darragh et al. (67) reported that the hydrolysis-enhanced CEDIATM immunoassay was superior to traditional immunoassays but still missed 22 % of samples found positive by LC-MS/MS. It did not detect lorazepam and other benzodiazepines primarily excreted as glucuronides.
While some innovative immunoassays, such as enzyme-multiplied immunoassay technique (EMIT) relying on luciferase, greatly improve sensitivity (reaching the pg/mL range), Bertol et al. (68) report issues with EMIT benzodiazepine detection in urine owed to high cross-reactivity for some compounds, which leads to greatly overestimated concentrations and risk of false positives.
One avenue of improving DBZ detection and measurement is by simplifying blood collection. Dried blood spot (DBS) microsampling requires smaller volumes of capillary blood (20–50 μL) collected onto filter paper than standard blood draws and can be used for sensitive LC-MS/MS, as reported by Moretti et al. (69), who found good sensitivity, accuracy, and precision in a LCMS/MS using DBS for 27 benzodiazepines and their metabolites. Most analytes remained stable in DBS for three months.
Volumetric absorptive microsampling (VAMS), in turn, is a technique for collecting small, precise volumes of blood or other biological fluids using an absorbent tip allowing easier and more convenient sampling, storage, and transport for analysis. Mestad et al. (70) validated several common classes of drugs of abuse, including benzodiazepines using VAMS. All benzodiazepines achieved extraction recoveries above 70 %.
New high-resolution accurate-mass spectrometers like quadrupole time-of-flight (QToF) and Orbitrap make it possible to identify metabolites and their isomers in very low concentrations. Further support to identifying biotransformation pathways is provided by
Because DBZs can be surreptitiously added to alcoholic beverages like beer to incapacitate a victim, making them more vulnerable to assault or theft, Yao et al. (72) developed a polymer monolithic microextraction method using poly(
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.