Nanoflow Interfacing for High Sensitivity CE-MS Coupling: Technical Development for Trace Analysis and Non-Targeted Metabolomics

Capillary electrophoresis coupled with mass spectrometry (CE-MS) is a powerful technique in many fields of analytical chemistry, especially for the separation of charged molecules. However, the widespread standard sheath liquid interface lacks sensitivity which is why many studies and inventions have attempted improvement. Based upon two novel principles, a porous tip interface and nanoflow sheath liquid interface were developed to systematically study their performance compared to the standard sheath liquid interface. Both nanoflow interfaces showed similar improvements in sensitivity of 13 to 114 times over the standard interface for organic acids, peptides, and monoclonal antibodies. In these experiments, the nanoflow sheath liquid interface demonstrated higher versatility and easier manufacturing compared to the sheathless porous tip approach. Therefore, it was chosen for further technical improvement and application for trace analysis of drinking water and non-targeted metabolomics analysis. The characterization of its electrical current flows and resistances disproved the misconception that electroosmosis is the main driving force for the sheath liquid flow, as found in the literature. Improvements in handling and robustness were achieved by introducing a second capillary into the emitter for sheath liquid supply with a switching function for both capillaries. The resulting valve mechanism enabled a conditioning mode and separation mode. By this means, not only emitter lifetime and usability were strongly improved but also unique functions added, such as a divert to waste function for exclusion of MS interfering matrix components, online preconditioning with MS incompatible cleaning/coating agents, and the potential of online capillary isoelectric focusing with MS detection (CIEF-MS). This two-capillary nanoflow sheath liquid interface prototype was then applied for enrichment-free trace analysis and quantification of anionic micropollutants in drinking water. A CE-Orbitrap method with nanoflow interfacing was developed to enable the quantification of halogenated acetic acids down to the ng/L range, required to meet the strict WHO Guidelines for Drinking Water Quality. Seven drinking water samples from various production plants in Germany were analyzed and the quantitative results (0.1 to 6.2 µg/L) verified by a validated liquid chromatography-MS (LC-MS) method, which emphasizes the strength of CE-MS as an alternative technique for the analysis of highly polar and ionic compounds in water. A subsequent suspect screening indicated the presence of halogenated methanesulfonic acids, which were identified and quantified by standard compounds (0.2 to 2.6 µg/L). Reviewing the data, a screening revealed around 20 additional anionic suspects such as sweeteners, organic sulfonates, and sulfates as well as inorganic ions, which shows the perspective for non-targeted analysis and discovery of contaminants. Non-targeted screening can be used as an exploratory tool in many fields of application. In this context, of a workflow for the discovery of metabolites by LC- and CE-MS in bioreactors was implemented. Substrate samples of biogas plants for methane production were analyzed to uncover the differences in metabolite concentration in varying process conditions. This work strongly focused on evaluating the influence of different separation techniques, mass spectrometer instruments and data processing software on the outcome of a non-targeted analysis. The samples were separated by both capillary zone electrophoresis (CZE) and reversed phase LC (RPLC) coupled to time-of-flight (TOF) and Orbitrap mass spectrometers. The non-targeted data were processed with mzMine and XCMS and the resulting features prioritized by partial least square regression (PLSR), which enabled the distinction between high and low gas yield reactor conditions with ranking by Variable Importance in Projection (VIP). As expected, RPLC-MS and CZE-MS delivered complementary information but also good correlation between 10% of the commonly detected features regarding fold change and importance for the PLSR model. Unexpectedly, strong differences between data sets were observed between TOF and Orbitrap (<50% common features), which suggest a major influence on the discovery of biomarkers if different MS instrumentation is used. Between MZmine and XCMS, significant differences in the total number of features were found (<57% common features), despite having a good overlap for features which were prioritized by the VIP ranking (up to 95% common features). This indicates a comparable performance for finding true positive features, but also highlights the drawbacks of automated data processing tools which generate high numbers of false positive features. This work underlined the high complexity of non-targeted workflows, and it was clearly shown that different approaches and slight variations can have a strong impact on the final outcome.

Overall, next generation nanoflow electrospray interfaces for CE-MS coupling offers a strong potential to establish new fields of application, by combining its strength for the separation of ionic compounds with high sensitivity detection. Yet, automation, affordable instrument parts and broad instrument compatibility are key to stimulate a broader use of CE-MS in the future. Hopefully, this thesis supports CE-MS as a technique on its way to become a more widely accepted technique, next to chromatography.


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