Pseudotargeted Method Based on Parallel Column Two-Dimensional Liquid Chromatography-Mass Spectrometry for Broad Coverage of Metabolome and Lipidome

Metabolite
and lipid profilings usually need two liquid chromatography-mass
spectrometry (LC-MS) methods because of a great polarity difference.
A pseudotargeted metabolomics method as a novel emerging approach
can integrate the advantages of nontargeted and targeted methods.
Here, we aim to establish a comprehensive method for metabolome and
lipidome by using a parallel column-based two-dimensional LC (PC-2DLC)-MS
and pseudotargeted approach. To simultaneously extract as many polar
metabolites and nonpolar lipids as possible, we systematically optimized
the sample pretreatment process, and isopropanol/methanol (3:1, v/v)
and isopropanol/water (7:3, v/v) were selected as the extraction and
reconstitution solvents, respectively. The detected triglycerides
significantly increased after the sample pretreatment optimization.
Then PC-2DLC coupled with Triple TOF MS was applied to analyze a mixed
sample from serum, urine, and liver tissue matrixes. The multiple
reaction monitoring (MRM) transitions of the metabolome and lipidome
were defined according to the “MRM-Ion Pair Finder”
software and lipidomics MRM-transition database, respectively. After
verification by QTRAP MS in the scheduled MRM mode, 1609 potential
metabolites and lipids corresponding to 1294 MRM transitions, and
847 potential metabolites and lipids corresponding to 687 MRM transitions
were detected in positive and negative ion modes, respectively. They
range at about 30 orders of magnitude in octanol/water partition coefficient.
The pseudotargeted 2DLC-MS method was validated to have good analytical
characteristics. As a proof of applicability, sera from type 2 diabetic
patients were investigated by the established method. The results
indicated that the pseudotargeted 2DLC-MS method is reliable and repeatable
and can be used in a metabolomics study.