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Optimization of UHPSFC/ESI-MS Determination of Polar and Nonpolar Lipids in Biological Samples
Authors: Peterka Ondřej | Wolrab Denise | Holčapek Michal
Year: 2018
Type of publication: ostatní - přednáška nebo poster
Page from-to: nestránkováno
Titles:
Language Name Abstract Keywords
eng Optimization of UHPSFC/ESI-MS Determination of Polar and Nonpolar Lipids in Biological Samples Supercritical fluid chromatography (SFC) uses supercritical CO2 as the main component of the mobile phase. The addition of organic modifiers enhances the elution strength of the mobile phase and allows also the analysis of polar analytes. The hyphenation with mass spectrometry (MS) broadens the application range of SFC and makes the method suitable for metabolomics and lipidomics. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) has higher separation efficiency and faster analysis time due to the use of sub-2 µm particles as stationary phase. Nowadays, the hyphenation of UHPSFC with mass spectrometry (UHPSFC/MS) is commonly done by splitting the flow before the backpressure regulator and employing a make-up solvent before connecting to the MS. We used UHPSFC Acquity UPC2 (Waters, Milford, MA, USA) connected with the hybrid quadrupole time-of-flight mass spectrometer Synapt G2Si (Waters). For the separation of lipid (sub)classes, Acquity BEH UPC2 (100 mm × 3 mm, 1,7 µm, Waters) column was employed, the column temperature was set to 60 °C, the ABPR was 1800 psi, the flow rate 1.9 mL/min, and the injection volume 1 µL. The injector needle was washed with a mixture of hexane-2-propanol-water (2:2:1, v/v/v) after each injection. The effect of various modified on the signal was tested, typically mixtures of methanol – water with ammonium acetate. All parameters affecting the sensitivity of nonpolar and polar lipid classes are systematically investigated with the goal to achieve the highest possible number of identified lipids in short analysis times lower than 10 minutes. Then, identified lipids are quantified using exogenous internal standards (IS) for particular lipid (sub)classes. The mixture of IS or pooled sample are used for the method optimization. The optimization of the make-up parameters for enhancing the sensitivity for the quantitation of lipid (sub)classes by employing various additives, solvents, and flow rates were aimed. The influence of m UHPSFC, Polar Lipids, Nonpolar Lipids, Biological Samples

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