Initially limited to academic environments, liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) has become a key tool for the detection and the identification of toxic substances. The advent of hybrid systems such as Q-TOF or Q-Orbitrap has revolutionised the way laboratories approach compound screening. HRMS offers sensitivity close to low-resolution mass spectrometry (LRMS), allowing the detection of a wide range of compounds with low detection limits. LC-HRMS allows specific identifications based on monoisotopic mass, retention time, isotopic patterns and fragmentation spectra. In this way, the laboratories contribute to the growth of shared libraries with crucial information for chemical identification. Large spectral libraries such as HighResNPS, MzCloud™, and NIST assist in the identification of new psychoactive substances (NPS). LC-HRMS is used for screening, quantitation, metabolism studies, and biomarker identification. Finally, LC-HRMS also enables retrospective analysis. Despite its strengths, LC-HRMS has limitations, particularly in the case of low concentration compounds or low abundance ion fragments. This makes identification challenging, often relying on indicators such as exact mass and isotopic patterns without sufficient fragmentation for a complete identification. To mitigate these problems, laboratories use inclusion/exclusion lists to select precursor ions and optimise the analysis. Another limitation is the increasing use of complex software to manage the large data sets generated, imposing high requirements for data storage and processing. Three case studies are presented to illustrate real-world analytical challenges.

Case 1 During a proficiency test, a synthetic cathinone in a urine sample could not be identified, despite its presence in the spectral library. The problem was due to co-elution and co-fragmentation with a natural compound present in the sample. Solutions include adjusting the sample preparation, chromatographic conditions or adding the spectrum including interfering ions to the library.

Case 2 A 23-year-old man suffered a severe serotonin syndrome after ingesting a powder identified as 25I-NBOH. Using untargeted LC-HRMS screening, 25H-NBOH was identified and quantified with CRM. A metabolism study was then carried out in collaboration with the University of Rennes, which revealed the presence of 25E-NBOH as the major NBOH derivative in the biological samples, with 25H-NBOH being a minor metabolite or an impurity. This case highlights the importance of bioinformatics tools to address the complexity of NPS cases, and the opportunity offered to develop national or international collaborations.

Case 3 In a forensic case, a prisoner died in custody with toxic levels of flecainide in his blood. A random error in mass measurement was identified, resulting in truncated chromatographic peaks at the calibration points, leading to an overestimation of concentrations. After correction, a true overdose was confirmed. This case illustrates that even advanced instruments such as LC-HRMS can be subject to errors that require careful analytical adjustments.

LC-HRMS is a powerful and essential tool in clinical and forensic toxicology. However, it is not without technical limitations, underlining the need for a cautious and methodical approach to effectively overcome identification challenges.