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

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.