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Anal Chem. 2018 Jun 5;90(11):6494-6501. doi: 10.1021/acs.analchem.7b05382. Epub 2018 May 10.

A Novel Lipidomics Workflow for Improved Human Plasma Identification and Quantification Using RPLC-MSn Methods and Isotope Dilution Strategies.

Author information

1
Department of Analytical Chemistry, Faculty of Chemistry , University of Vienna , Währingerstrasse 38 , 1090 Vienna , Austria.
2
Vienna Metabolomics Center (VIME) , University of Vienna , Althanstraße 14 , 1090 Vienna , Austria.
3
Chemistry Meets Microbiology , Althanstraße 14 , 1090 Vienna , Austria.
4
Thermo Fisher Scientific (Bremen GmbH) , Hanna-Kunath-Str. 11 , 28199 Bremen , Germany.
5
Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy , Universität Leipzig , Leipzig , Germany.
6
ISOtopic solutions , Währingerstrasse 38 , 1090 Vienna , Austria.
7
Thermo Fisher Scientific , 1 Boundary Park , Hemel Hempstead HP2 7GE , United Kingdom.
8
Thermo Fisher Scientific , 355 River Oaks Parkway , 95134 San Jose , California United States.

Abstract

Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples. Moreover, we used lipidome isotope-labeling of yeast (LILY)-a fast and efficient in vivo labeling strategy in Pichia pastoris-to produce (nonradioactive) isotopically labeled eukaryotic lipid standards in yeast. We integrated the 13C lipids in the LC-MS workflow to enable relative and absolute compound-specific quantification in yeast and human plasma samples by isotope dilution. Label-free and compound-specific quantification was validated by comparison against a recent international interlaboratory study on human plasma SRM 1950. In this way, we were able to prove that LILY enabled quantification leads to accurate results, even in complex matrices. Excellent analytical figures of merit with enhanced trueness, precision and linearity over 4-5 orders of magnitude were observed applying compound-specific quantification with 13C-labeled lipids. We strongly believe that lipidomics studies will benefit from incorporating isotope dilution and LC-MSn strategies.

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