Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis
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Abstract
<p class="first" id="P1">The confident identification and in-depth profiling of molecular
lipid species remain
to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional
mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field
quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids
from complex biological samples. In the first dimension, 22 different lipid classes
were separated on a monolithic silica column with elution order from neutral to polar
lipids. A total of 13 fractions were collected and run on a RPLC C30 column in the
second dimension for further separation of the lipid molecular species based on their
hydrophobicity, with the elution order being determined by both the length and degree
of unsaturation in the fatty-acyl chain. The method was applied to analyze lipids
extracted from rat plasma and rat liver. Fatty acid methyl ester analysis by gas chromatography-mass
spectrometry was used to identify the fatty acyls from total lipid extracts, which
provided a more confident identification of the lipid species present in these samples.
More than 800 lipids were identified in each sample and their molecular structures
were confidentially confirmed using tandem mass spectrometry (MS/MS). The number of
lipid molecular species identified in both rat plasma and rat liver by this off-line
two-dimensional method is approximately twice of that by one-dimensional RPLC-MS/MS
employing a C30 column. This off-line two-dimensional mixed-mode LC-RPLC-MS/MS method
is a promising technique for comprehensive lipid profiling in complex biological matrices.
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