Two capillary approach for a multifunctional nanoflow sheath liquid interface for capillary electrophoresis-mass spectrometry.

CE hyphenated to ESI-MS (CE-ESI-MS) is a well-established technique to analyze charged analytes in complex samples. Although various interfaces for CE-MS coupling are commercially available, the development of alternatives which combine sensitivity, simplicity, and robustness remains a topic of research. In this work, a nanoflow sheath liquid CE-MS interface with two movable capillaries inside a glass emitter is described. The setup enables a separation mode and a conditioning mode to guide the separation capillary effluent either into the electrospray or to the waste, respectively. This enables to exclude parts of the analysis from MS detection and unwanted matrix components reaching the mass spectrometer, comparable to divert valves in LC-MS coupling. Also, this function improves the overall robustness of the system by reduction of particles blocking the emitter. Preconditioning with electrospray interfering substances and even the application of coating materials for every analysis is enabled, even while the separation capillary is built into the interface with running electrospray. The functionality is demonstrated by analyses of heavy matrix bioreactor samples. Overall, this innovation offers a more convenient installation of the interface, improved handling with an extended lifetime of the emitter tips and additional functions compared to previous approaches, while keeping the higher sensitivity of nanoflow CE-MS-coupling.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Heart-cut nano-LC-CZE-MS for the characterization of proteins on the intact level.

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed-phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well-suited geometries/dimensions. Here, a heart-cut nano-LC-CZE-MS setup was developed utilizing for the first time a mechanical 4-port valve as LC-CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart-cut transfer of individual LC peaks and subsequent CZE-MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower µg/mL range were determined, which are considerably lower compared to traditional CZE-MS. In addition, this study represents the first application of an LC-CE-MS system for intact protein analysis. The nano-LC-CZE-MS system is expected to be applicable to various other analytical challenges.