During the last decade, the number of compounds of interest in forensic toxicology has expanded considerably mainly due to the emergence of new psychoactive substances in addition to medicinal and illicit drugs, inhalants, metals, and toxins. Continuous development of efficient and reliable strategies is required to unequivocally detect this large number of xenobiotics. The concept of systematic toxicological analysis (STA) is widely applied in forensic toxicology laboratories to accomplish the analysis of unknown compounds by both screening and confirmatory methods. With the development of instrumental techniques, hyphenated mass spectrometry (MS) represents the most efficient analytical principle for most compounds. More recently, high-resolution mass spectrometry (HRMS) provides a comprehensive screening tool with its accurate mass determination capability and data independent acquisition mode. HRMS enables discovery of new analytes through retrospective data analysis (RDA) of previously analyzed samples without additional injections.The improvement of MS-based techniques for STA, primarily employing liquid chromatography (LC–HRMS), is the main topic of this thesis. To enhance STA, the massive amount of data recorded from the LC–HRMS screening was used for RDA to detect new targets. LC–HRMS screening is normally conducted in positive electrospray ionization mode (ESI+) to detect most compounds
involved in STA. Complementary methods are needed for acidic/neutral substances that are not covered by a LC–ESI+–HRMS screening. We conducted an RDA of post-mortem whole blood samples to find targets of salicylic acid and ibuprofen that can be indirectly employed in ESI+. Potential targets were observed with the metabolites and adducts such as sodium, potassium, calcium, and formic acid. In addition, an RDA workflow is employed to identify designer benzodiazepines in cases of driving under the influence of drugs. The workflow was assessed using previously quantified samples of typical benzodiazepines. Etizolam, phenazepam, lorazepam, and flualprazolam were the most frequently tentatively positive benzodiazepines that we discovered out of a total of sixteen designer and uncommon benzodiazepines. Finally, we presented a mini review of STA in forensic toxicology. An established workflow in our laboratory was also described to provide an example of a modern forensic toxicological analysis with focus on the coverage of the overall procedure.