Comprehensive and Reproducible Untargeted Lipidomic Workflow Using LC-QTOF Validated for Human Plasma Analysis

The goal of this
work was to develop a label-free, comprehensive,
and reproducible high-resolution liquid chromatography–mass
spectrometry (LC-MS)-based untargeted lipidomic workflow using a single
instrument, which could be applied to biomarker discovery in both
basic and clinical studies. For this, we have (i) optimized lipid
extraction and elution to enhance coverage of polar and nonpolar lipids
as well as resolution of their isomers, (ii) ensured MS signal reproducibility
and linearity, and (iii) developed a bioinformatic pipeline to correct
remaining biases. Workflow validation is reported for 48 replicates
of a single human plasma sample: 1124 reproducible LC-MS signals were
extracted (median signal intensity RSD = 10%), 50% of which are redundant
due to adducts, dimers, in-source fragmentation, contaminations, or
positive and negative ion duplicates. From the resulting 578 unique
compounds, 428 lipids were identified by MS/MS, including acyl chain
composition, of which 394 had RSD < 30% inside their linear intensity
range, thereby enabling robust semiquantitation. MS signal intensity
spanned 4 orders of magnitude, covering 16 lipid subclasses. Finally,
the power of our workflow is illustrated by a proof-of-concept study
in which 100 samples from healthy human subjects were analyzed and
the data set was investigated using three different statistical testing
strategies in order to compare their capacity in identifying the impact
of sex and age on circulating lipids.