Metformin impacts cecal bile acid profiles in mice.

Bile acids (BAs) are major components of bile synthesized from cholesterol and take part in the digestion of dietary lipids, as well as having signaling functions. They undergo extensive microbial metabolism inside the gastrointestinal tract. Here, we present a method of ultra-high pressure liquid chromatography coupled to ion trap mass spectrometry for quantification of 45 BAs in mouse cecum. The system was validated in regard to sensitivity with limits of detection and quantification (0.6-24.9 nM), interday accuracy (102.4%), interday precision (15.2%), recovery rate (74.7%), matrix effect (98.2%) and carry-over effect (<1.1%). Afterwards, we applied our method to investigate the effect of metformin on BA profiles. Diabetic mice were treated with metformin for 1 day or 14 days. One day of treatment resulted in a significant increase of total BA concentration (2.7-fold increase; db/db metformin 5.32 µmol/g, db/db control mice 1.95 µmol/g), most notable in levels of 7-oxodeoxycholic, 3-dehydrocholic and cholic acid. We observed only minor impact on BA metabolism after 14 days of metformin treatment, compared to the single treatment. Furthermore, healthy wild type mice had elevated concentrations of allocholic and ω-muricholic acid compared to diabetic mice. Our method proved the applicability of profiling BAs in cecum to investigate intestinal BA metabolism in diabetes and pharmacological applications.

Trends on Analytical Characterization of Polysorbates and Their Degradation Products in Biopharmaceutical Formulations.

Among many other applications, polysorbates (PSs) are used as the most common surfactants in biopharmaceutical products in particular to protect proteins against interfacial stress. Structural heterogeneity, presence of degradants and other impurities, and tendency for degradation are interrelated features found in commercial PSs with a direct impact on their functional properties in biopharmaceutical products. These pose a challenge for the analytical characterization of PSs at different stages of product development. This review article focuses on methods and strategies reported in the recent years for the analytical characterization of PSs, their degradants and other impurities within neat PS (i.e., PS raw materials), diluted PS solutions, as well as in biopharmaceutical formulations. The use of versatile and complementary methods applied in a systematic approach is crucial to understand the impact of the concentration, composition, and degradation of PSs on the quality of biopharmaceutical products.

Computational analysis and ratiometric comparison approaches aimed to assist column selection in hydrophilic interaction liquid chromatography-tandem mass spectrometry targeted metabolomics.

In the present work two different approaches, a semi-quantitative and a Derringer function approach, were developed to assist column selection for method development in targeted metabolomics. These approaches were applied in the performance assessment of three HILIC columns with different chemistries (an amide, a diol and a zwitterionic phase). This was the first step for the development of a HILIC UPLC-MS/MS method that should be capable to analyze a large number of polar metabolites. Two gradient elution profiles and two mobile phase pH values were tested for the analysis of multi-analyte mixtures. Acquired chromatographic data were firstly treated by a ratiometric, "semi-quantitative" approach which quantifies various overall analysis parameters (e.g. the percent of detected compounds, retentivity and resolved critical pairs). These parameters were used to assess chromatographic performance in a rather conventional/traditional and cumbersome/labor-intensive way. Secondly, a comprehensive and automated comparison of the three columns was performed by monitoring several well-known chromatographic parameters (peak width, resolution, tailing factor, etc.) using a lab-built programming script which calculates overall desirability utilizing Derringer functions. Derringer functions exhibit the advantage that column performance is ultimately expressed in an objective single and quantitative value which can be easily interpreted. In summary, results show that each column exhibits unique strengths in metabolic profiling of polar compounds. The applied methodology proved useful for the selection of the most effective chromatographic system during method development for LC-MS/MS targeted metabolomics, while it could further assist in the selection of chromatographic conditions for the development of multi-analyte methods.

Sensitivity improvement in hydrophilic interaction chromatography negative mode electrospray ionization mass spectrometry using 2-(2-methoxyethoxy)ethanol as a post-column modifier for non-targeted metabolomics.

The application of ammonia acetate buffered liquid chromatography (LC) eluents is known to concomitantly lead to ion suppression when electrospray ionization mass spectrometry (ESI-MS) detection is used. In negative ESI mode, post column infusion of 2-(2-methoxyethoxy)ethanol (2-MEE) was shown in the literature to help to compensate this adverse effect occurring in reversed phase liquid chromatography mass spectrometry (RP-LC-MS) analyses. Here a setup of direct infusion and hydrophilic interaction chromatography (HILIC) post-column infusion experiments was established in order to investigate systematically the beneficial effects of 2-MEE. We demonstrate that, 2-MEE can help to improve ESI-MS sensitivity in HILIC too and reveal analyte structure specific behaviors. Our study indicates that 2-MEE especially improves ESI response for small and polar molecules. The ESI response of stable isotope labeled amino acids spiked into biological matrices increases up to 50-fold (i.e. D5-l-glutamic acid) when post column infusion of 2-MEE is applied. A non-targeted analysis of a pooled urine sample via HILIC-ESI-QTOF-MS supports this hypothesis. In direct infusion, the combined application of an ammonia acetate buffered solution together with 2-MEE results in an improved ESI response compared to a non-buffered solution. We observed up to 60-fold increased ESI response of l-lysine. We propose this effect is putatively caused by the formation of smaller ESI droplets and stripping of positive charge from ESI droplets due to evaporation of acetic acid anions. In summary, post-column infusion of 2-MEE especially enhances ESI response of small and polar molecules. Therefore it can be regarded as a valuable add-on in targeted or non-targeted metabolomic HILIC-MS studies since this method sets a focus on this molecule category.