Univerzita Pardubice Fakulta chemicko- technologická

Univerzita Pardubice

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Publikace detail

UHPSFC/MS as a New Approach in the Lipidomic Quantitation

Autoři: Holčapek Michal | Lísa Miroslav

Rok: 2015

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

Tituly:

Jazyk	Název	Abstrakt	Klíčová slova
eng	UHPSFC/MS as a New Approach in the Lipidomic Quantitation	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 [1]. Total lipid extracts from selected human tissues prepared by modified Folch procedure using chloroform – methanol – water extraction were used for the comprehensive lipidomic analysis. UHPSFC experiments were acquired on UPC2 instrument (Waters) using 1.7 μm UPC2 columns, separation temperature 60°C and gradient of methanol – water – ammonium acetate mixture as a modifier. Synapt HDMS G2Si instrument (Waters) with ESI ionization was used for MS experiments. Developed UHPSFC method is based on 1.7 μm particle bridged ethylene hybrid silica column using the gradient of methanol – water – ammonium acetate mixture as a modifier providing separation of total lipid extract into lipid classes. Individual parameters of UHPSFC analysis have been 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 minute 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 the extraction step. Ion mobility (IM) can be used as the third separation dimension, where ions are separated according to their collision cross-sections to increase the identification capacity. Developed UHPSFC/MS method is validated and applied for the comprehensive analysis of lipid composition in real biological samples, such as plasma or normal and cancer tissues. The comparison of obtained results with shotgun MS and LC/MS approaches will be shown as well.