Two-dimensional liquid chromatography-mass spectrometry for lipidomics using off-line coupling of hydrophilic interaction liquid chromatography with 50 cm long reversed phase capillary columns

Comprehensive characterization of the lipidome remains a challenge requiring development of new ana-lytical approaches to expand lipid coverage in complex samples. In this work, offline two-dimensional liq-uid chromatography-mass spectrometry was investigated for lipidomics from human plasma. Hydrophilic interaction liquid chromatography was implemented in the first dimension to fractionate lipid classes. Nine fractions were collected and subjected to a second-dimension separation utilizing 50 cm capillary columns packed with 1.7 mu m C18 particles operated on custom-built instrumentation at 35 kpsi. On-line coupling with time-of-flight mass spectrometry allowed putative lipid identification from precursor -mass based library searching. The method had good orthogonality (fractional coverage of similar to 40%), achieved a peak capacity of approximately 1900 in 600 min, and detected over 1000 lipids from a 5 mu L in-jection of a human plasma extract while consuming less than 3 mL of solvent. The results demon-strate the expected gains in peak capacity when employing long columns and two-dimensional sep-arations and illustrate practical approaches for improving lipidome coverage from complex biological samples.(c) 2022 Elsevier B.V. All rights reserved.