Abstract (eng)
The central motif of this work is to face the challenge of validating an absolute quantification proce-dure based on a newly developed LC-MS method for simultaneous metabolomics and exposomics analysis in a way that approximates the validation needs for a clinical study. The presented method is a novel HPLC-HRMS approach employing simultaneous chromatography under complementing elu-tion conditions: Hydrophilic interaction liquid chromatography (HILIC) provides good separation con-ditions for the more hydrophilic metabolites, whereas reversed phase (RP) chromatography poses a better environment for analyzing compounds of higher lipophilicity, a property quite common for small molecule pharmaceuticals. To get a sense of a medicines authority’s regulatory expectations of analytical method validation, the U.S. FDA’s guideline Bioanalytical Method Validation – Guidance for Industry is sought for advice. The guideline conveniently suggests strategies how to evaluate the ca-pabilities of a chromatographic method. It serves as a starting point for approaching the specific is-sues of an HPLC-HRMS metabolomics method.
An already acquired data set is exploited to gain deeper insight into the capabilities and weak spots of the novel dual-chromatography HPLC-HRMS method and in deed suggests its versatility for com-bined metabolomics and exposomics research. With the exception of a few targeted metabolites with overlapping HILIC and RP peaks, the 2D chromatography concept saves time without considera-ble drawbacks for the quality of the analysis. This work especially maps out the benefits of internal standardization with a fully 13C-labelled metabolite extract from Pichia pastoris while, on the other hand, also discussing its possible pitfalls. Analyzing the data arises questions that will have to be ad-dressed by further research.
Linking basic university research with a highly regulated application environment is an ambitious endeavor, which, in the course of this work, turned out to be hardly feasible for the current state of method development. Nonetheless, the thesis collates general issues specific for a full validation of a metabolomics method according to medicines authorities’ provisions. Among them are the complexi-ty of matrix effect assessment and the problem that an approach via blank matrix, as suggested by the guideline, is not viable for metabolomics. Internal standardization with a fully 13C-labeled yeast cell extract is presented as a possible solution. Dedicated evaluation is needed but lies beyond the scope of this thesis.