University of PardubiceFaculty of Chemical Technology

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HILIC-UHPLC/MS for quantitative lipidomics: An optimized and validated method for the analysis of body fluids.

Authors: Chocholoušková Michaela | Wolrab Denise | Jirásko Robert | Vrána David | Melichar Bohuslav | Holčapek Michal

Year: 2019

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	HILIC-UHPLC/MS for quantitative lipidomics: An optimized and validated method for the analysis of body fluids.	The aim of this project is the development of a robust and accurate method for the determination of lipid profiles in human body fluids and the application of those for the analysis of blood samples of healthy volunteers and patients suffering from kidney cancer. A systematic study with regard to differences in the lipid profile depending on the blood collection tube has been performed. Generally, EDTA plasma is recommended for the bioanalysis of small molecules in the literature, therefore we evaluate the lipid profiles of heparin-lithium and serum in comparison to EDTA plasma. A special focus is given to the optimization of the sample preparation protocol for the analysis of lipids in biological samples, the choice of internal standards (structure and concentration), the extraction solvents for liquid-liquid extraction (Folch extraction, methyl-tert-butyl ether extraction, butanol/methanol extraction, etc.), the influence of pH during the extraction (ammonium carbonate, ammonium acetate, etc.), and the influence of different solvent mixtures for dissolving the extracts are investigated. All experiments are performed using hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry (HILIC-UHPLC/MS), and the optimized protocol is fully validated. The best results with regard to the recovery are obtained by employing a slightly modified double Folch extraction using basic conditions and the reconstitution of the extracts in a mixture of CHCl3/MeOH (1:1). The lipid concentration results of the optimized method applied for the analysis of the SRM 1950 reference plasma are in agreement with established values from the literature [1, 2]. All experiments are also performed using ultra-high performance supercritical fluid chromatography coupled to high-resolution mass spectrometry (UHPSFC/MS), and results are compared to each other using validation parameters and statistical analysis.