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In this study, we developed and validated a CE-TOF-MS method for the quantification of glyphosate (N-(phosphonomethyl)glycine) and its major degradation product aminomethylphosphonic acid (AMPA) in different samples including beer, media from toxicological analysis with Daphnia magna, and sorption experiments. Using a background electrolyte (BGE) of very low pH, where glyphosate is still negatively charged but many matrix components become neutral or protonated, a very high separation selectivity was reached. The presence of inorganic salts in the sample was advantageous with regard to preconcentration via transient isotachophoresis. The advantages of our new method are the following: no derivatization is needed, high separation selectivity and thus matrix tolerance, speed of analysis, limits of detection suitable for many applications in food and environmental science, negligible disturbance by metal chelation. LODs for glyphosate were < 5 μg/L for both aqueous and beer samples, the linear range in aqueous samples was 5-3000 μg/L, for beer samples 10-3000 μg/L. For AMPA, LODs were 3.3 and 30.6 μg/L, and the linear range 10-3000 μg/L and 50-3000 μg/L, for aqueous and beer samples, respectively. Recoveries in beer samples for glyphosate were 94.3-110.7% and for AMPA 80.2-100.4%. We analyzed 12 German and 2 Danish beer samples. Quantification of glyphosate and AMPA was possible using isotopically labeled standards without enrichment, purification, or dilution, only degassing and filtration were required for sample preparation. Finally, we demonstrate the applicability of the method for other strong acids, relevant in food and environmental sciences such as N-acetyl glyphosate, N-acetyl AMPA (present in some glyphosate resistant crop), trifluoroacetic acid, 2-methyl-4-chlorophenoxyacetic acid, glufosinate and its degradation product 3-(methylphosphinico)propionic acid, oxamic acid, and others.

Statically adsorbed or covalently coupled capillary coatings are of crucial importance in capillary electrophoresis-mass spectrometry for the separation of peptides and proteins. So far, published coating strategies and commercially available coated capillaries have a limited pH-stability so that the analysis at strongly acidic pH is limited, or harsh rinsing procedures for biological sample analysis cannot be applied. We here present a capillary coating based on Si-C linkages to N-acryloylamido ethoxyethanol (AAEE) with a new synthetic strategy including LiAlH 4 surface reaction. We optimized the coating method with emphasis on stability and reproducibility applying harsh rinsing procedures (strong acid, strong base and organic solvent), using the electroosmotic mobility and separation efficiency of tryptic peptides as performance measure. Complete synthesis is performed in less than 2 days for up to 8 capillaries in parallel of more than 16 m total length. Intra- and inter-batch reproducibility were determined regarding electroosmotic mobility, separation efficiency and migration time precision in CE-MS separations of tryptically digested bovine serum albumin. Coating stability towards rinsing with strong acid (1 mol/L HCl), organic solvent (acetonitrile) and strong base (1 mol/L NaOH) was investigated. Outstanding performance was found for single capillaries. However, inter-capillary reproducibility is discussed critically. The new coating was successfully applied for reproducible CE-MS separation of large proteins in diluted serum, medium-sized peptides and small and highly charged polyamines in fish egg extracts using a very acidic background electrolyte containing 0.75 mol/L acetic acid and 0.25 mol/L formic acid (pH 2.2).

In this study, we characterized unexpected side-products in a commercially synthesized peptide with the sequence RPRTRLHTHRNR. This so-called peptide D3 was selected by mirror phage display against low molecular weight amyloid-β-peptide (Aβ) associated with Alzheimer's disease. Capillary electrophoresis (CE) was the method of choice for structure analysis because the extreme hydrophilicity of the peptide did not allow reversed-phase liquid chromatography (RPLC) and hydrophilic interaction stationary phases (HILIC). CE-MS analysis, applying a strongly acidic background electrolyte and different statically adsorbed capillary coatings, provided fast and efficient analysis and revealed that D3 unexpectedly showed strong ion-pairing with sulfuric acid. Moreover, covalent O-sulfonation at one or two threonine residues was identified as a result of a side reaction during peptide synthesis, and deamidation was found at either the asparagine residue or at the C-terminus. In total, more than 10 different species with different m/z values were observed. Tandem-MS analysis with collision induced dissociation (CID) using a CE-quadrupole-time-of-flight (QTOF) setup predominantly resulted in sulfate losses and did not yield any further characteristic fragment ions at high collision energies. Therefore, direct infusion Fourier transform ion cyclotron resonance (FT-ICR) MS was employed to identify the covalent modification and discriminate O-sulfonation from possible O-phosphorylation by using an accurate mass analysis. Electron transfer dissociation (ETD) was used for the identification of the threonine O-sulfation sites. In this work, it is shown that the combination of CE-MS and FT-ICR-MS with ETD fragmentation was essential for the full characterization of this extremely basic peptide with labile modifications.