Within the last two decades liquid chromatography hyphenated to mass spectrometry has developed into an indispensable tool for the qualitative and quantitative analysis of organic compounds. The breakthrough of this analytical technique came with the increasing availability of atmospheric pressure ionisation techniques such as electrospray ionisation in the 1990s. Today, LC-MS represents a key platform in analytical science and has provided new insights into pharmaceutical, biomedical and environmental issues. However, the potential of this technology is still vast and far from exhausted. The presented thesis focused on the further improvement of LC-MS based analytical processes and the exploration of novel applications in the fields of forensic toxicology and metabolomics. The research work resulted in three new LC-MS/MS workflows applied in the field of forensic toxicology and three LC-MS/MS based methods and applications for targeted metabolomics. The dissertation is complemented by two review articles dealing with the role of liquid chromatography hyphenated to mass spectrometry in forensic toxicology.
In a first study, SWATH, a modern data-independent acquisition technique for the automatic generation of fragment ion spectra was applied for systematic toxicological analysis. SWATH enabled a more sensitive detection of compounds compared to traditional data-dependent workflows. The bottleneck of SWATH analysis remains data deconvolution, since wide precursor ion isolation windows create “chimeric” spectra, making a proper identification of compounds without appropriate spectra deconvolution impossible. Nevertheless, it could be demonstrated, that SWATH has the potential to revolutionize the way untargeted screening in forensic toxicology is performed.
In addition, a combined qualitative and quantitative LC-MS/MS assay using SWATH acquisition was developed and implemented for systematic toxicological analysis. The established SWATH workflow enabled simultaneous general unknown screening with automated library search and quantitation of a broad range of pharmaceuticals and illicit drugs in human plasma samples. Furthermore, the SWATH workflow allowed for the retrospective identification of compounds at a later time without the need to reanalyze samples.
In a second study, an established workflow for general unknown screening of forensic casework samples was successfully adapted to the analysis of wastewater samples. After modification of the sample preparation steps and optimization of storage conditions, it could be shown, that the employed LC-MS/MS assay with subsequent automatic library search is transferable to water analysis and thereby could prove its universality.
Another major part of the PhD thesis focused on the development and application of targeted metabolomics workflows. In this regard, a high resolution tandem mass spectrometry method combined with hydrophilic interaction liquid chromatography for the quantitation of tryptophan metabolites in human plasma was developed. The presented analytical method was completely validated. High resolution multiple reaction monitoring proved to be beneficial for specific and robust detection of the targets and allowed the use of a fast and simple sample preparation technique.
Furthermore, a commercially available kit for the LC-MS/MS based analysis of bile acids was adapted on a less sensitive analytical platform. After modifications in the sample preparation workflow and reducing complexity of mass spectrometric detection without loss of specificity, the kit could be run on our system. A successful participation in the first international ring trial for bile acids analysis was enabled.
As part of the service provided by the core facility metabolomics, the use of the AbsoluteIDQ p150 kit from Biocrates Life Sciences, an LC-MS/MS based kit for targeted metabolomics, in our laboratory was enabled. The challenge was to establish the assay on a QTRAP 3200 system. The manufacturer recommends to use the kit with a QTRAP 4000 system or higher, which is considered ten times more sensitive. After optimization, sensitivity of the QTRAP 3200 system was sufficient to detect endogenous levels of the respective analytes.
The PhD thesis is completed by two review articles evaluating the role of untargeted LC-MS/MS approaches and the status of compound identification in forensic toxicology.