High-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies... Show moreHigh-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies. This study aimed to investigate the possibilities and limitations of high-throughput Quan/Qual analysis by ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS), to develop a widely applicable Quan/Qual UHPLC-HRMS method and to provide recommendations for Quan/Qual method development. A widely applicable 4.25-min UHPLC method for small-molecules was used to investigate and optimize mass spectrometric parameters of a Synapt G2S for Quan/Qual analysis. The method was applied on a rat metabolomics study investigating the effect of the fasting state and administration of a dosing vehicle on the rat plasma metabolic profile. scan mode with a short scan time of 30 ms. This 4.25-min Quan/Qual analysis method enabled quantification with accuracy and precision values ≤ 20% at the lowest quality control (QC) level and ≤15% at higher QC levels for 16 out of 19 tested analytes. It provided both parent m/z values and fragmentation spectra for compound identification with limited loss of chromatographic resolution and it revealed biologically relevant metabolites in its application to the metabolomics study. Quan/Qual method development requires balancing between the amount of qualitative data, the quality of the quantitative data and the analysis time. Recommendations are provided for MS resolution, scan mode, scan rate, smoothing and peak integration in Quan/Qual method development and analysis. BACKGROUND METHODS RESULTS CONCLUSION Show less
A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer... Show moreA novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10(-9) m(2) V(-1) s(-1)) when compared with unmodified fused silica (5.9 ± 0.1 10(-8) m(2) V(-1) s(-1)). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1-1.8% coefficient-of-variation (CV) within a day) and 2-3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use. Show less
