## 1 Introduction  
The emergence of new psychoactive substances (NPSs) poses a growing challenge to public health and forensic sciences. These substances are designed to mimic the effects of controlled drugs while evading existing drug laws by altering their chemical structures [1]. Among these, flualprazolam and isotonitazone have gained prominence due to their high potency and associated risks, necessitating the development of advanced analytical techniques for their detection and quantification in biological matrices [2; 3]. Flualprazolam, a fluorinated analog of alprazolam, is a designer benzodiazepine known for its sedative and anxiolytic effects. Structurally, the addition of a fluorine atom at the ortho position of the phenyl ring enhances its potency and duration of action compared to alprazolam [4]. Studies have shown that flualprazol has a higher receptor affinity, potentially increasing the risk of abuse, dependence, and overdose [4; 5; 6]. Despite its rising prevalence, limited pharmacokinetic and pharmacodynamic data are available, creating a need for sensitive analytical methods to detect and quantify this compound in biological samples. Flualprazolam use has been increasingly reported in forensic cases globally. In Ontario, Canada, it was identified in 395 death investigations and 108 impaired driving cases between July and December 2021. The concentrations in death investigations ranged from 3 to 59 ng/mL, with a median of 6.8 ng/mL [7]. Similarly, in the United States, flualprazolam has been confirmed in multiple states, including Pennsylvania and Texas, with concentrations in impaired driving cases reaching up to 68 ng/mL [8]. These findings underscore the substance's significant impact on public safety and its potential for causing severe impairment, even at low concentrations. The adverse effects of flualprazolam align with those observed in other benzodiazepines, including sedation, drowsiness, dizziness, impaired coordination, cognitive disturbances, and respiratory depression [4; 9]. Severe respiratory depression is particularly concerning when flualprazolam is combined with opioids or alcohol, contributing to its role in polysubstance-related fatalities. In a reported case series involving adolescents, the ingestion of counterfeit alprazolam containing flualprazolam led to symptoms such as lethargy, confusion, and mild respiratory depression. Most patients recovered within hours, suggesting a relatively short duration of acute toxicity [8]. The growing detection of flualprazolam in clinical and forensic settings, combined with its significant health risks, underscores the urgent need for validated analytical techniques. Isotonitazene is a synthetic opioid belonging to the benzimidazole class, being structurally related to to tointazene. It acts as a \(\upmu\)-opioid receptor agonist and exhibits significantly higher potency compared to morphine and fentanyl, making it highly toxic even at low doses [2]. This high potency raises concerns about its misuse and fatal overdoses, especially as it is increasingly detected in illicit drug markets. The substance's growing prevalence in forensic cases underscores the importance of developing reliable detection methods to identify and monitor its use in clinical and toxicological settings [10]. Since its identification in 2019, isotonitazene has emerged as a significant concern for public health and law enforcement. Data from the Drug Enforcement Administration (DEA) indicates that isotonitazene has been implicated in over 826 forensic cases, with detections commonly linked to counterfeit opioids and other illicit substances [11]. These findings highlight its widespread distribution in the illegal drug market and its role in drug-related fatalities. In Europe, the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) reported multiple fatalities attributed to isotonitazene, with many cases involving evidence of intravenous use. Autopsy findings frequently reveal features of opioid toxicity, such as pulmonary and cerebral edemas.