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, Nacetyl 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.
Polyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines.
Capillary coatings are crucial for high-quality separation performance in capillary electrophoresis analysis of proteins or peptides as they prevent analyte adsorption at the capillary wall. These coating materials have to fulfill many requirements such as a good separation performance and ensuring a good repeatability. The number of commercially available coating materials is still limited, especially with regard to the charge density on the coating material and the induced electroosmotic flow (EOF) velocity. In this work, we compare the separation performance of the novel self-made cationic capillary coating OHNOON and two commercially available coating materials, the acrylamide based, neutral LN® and the cationic hexadimethrine bromide (Polybrene), using the same coating procedure for all three coating materials. The coatings are investigated regarding the separation efficiency, analyte resolution, coating stability, and migration time stability in tryptic peptide analysis. Good separation performance was confirmed for all three coating materials: all coatings provided high plate numbers of up to 400,000-500,000 and a repeatability of the EOF and the analyte migration times in the range of 1% relative standard deviation or below. Our results reveal a moderate EOF velocity for the novel OHNOON coating in comparison to the Polybrene coating. We present a detailed discussion of the impact of this reduced EOF velocity and the separation performance. The results presented here will help to define the necessary properties of coating materials to achieve the best compromise between speed of analysis and resolution for the respective application. We show that our novel OHNOON coating is especially valuable for the analysis of low mobility analytes and for samples with a broad range of analyte mobilities.
A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis-mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3-12 and 9-29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields.
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