University of PardubiceFaculty of Chemical Technology

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Supercritical fluid chromatography – mass spectrometry: novel approach for high-throughput lipidomics

Authors: Holčapek Michal | Wolrab Denise | Peterka Ondřej | Jirásko Robert

Year: 2018

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Supercritical fluid chromatography – mass spectrometry: novel approach for high-throughput lipidomics	Numerous lipid molecules and present in eukaryotic cells, and they fulfill various physiological functions. The dysregulation of lipidomic pathways is often related to serious human diseases, such as cancer, cardiovascular diseases, diabetes, etc. The complexity of lipids results in the fact that multiple analytical methods are needed to cover a broader range of lipidome. Accurate, comprehensive and high-throughput methods are required for the analysis of large sample sets in the clinical research, which is challenging for analytical techniques due to the enormous complexity of lipid structures. Two well-established approaches are commonly used in lipidomics, such as shotgun and LC/MS, both having some advantages and limitations. Now we have developed a new high-throughput and comprehensive approach for analysis of lipids in biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) followed by ESI-MS identification and quantitation. Total lipid extracts from selected human tissues and body fluids (plasma and urine) are prepared by chloroform – methanol – water extraction. UHPSFC experiments were performed on UPC2 instrument (Waters) using 1.7 μm particle bridged ethylene hybrid silica column, separation temperature 60°C and gradient of methanol – water – ammonium acetate mixture as a modifier coupled with Synapt HDMS G2Si instrument (Waters) using electrospray ionization. Individual parameters of UHPSFC analysis are carefully optimized to achieve a maximum number of separated lipid classes. Final UHPSFC method enables a fast separation up to 28 nonpolar and polar lipid classes within 6 min analysis including the partial separation of species inside individual classes. Mass spectra with high mass accuracy and high resolving power are acquired using ESI in both positive- and negative-ion modes for the identification of individual species. The quantitative analysis is performed using internal standards for each lipid class added during	Supercritical fluid chromatography; mass spectrometry; lipidomics