Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis

Abstract

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.

Keywords: Accucore C30 column; Monolithic column; Off-line two-dimensional liquid chromatography; Rat liver lipidome; Rat plasma lipidome; Untargeted lipidomics.

Figure 1

Mixed mode LC-ELSD chromatogram of nonpolar and polar lipids obtained from a standard mixture solution, rat plasma and rat liver. 1: cholesterol ester; 2: triacylglycerol; 3: cholesterol; 4: 1,3- diacylglycerol; 5: 1,2- diacylglycerol; 6: 1-monoacylglycerol; 7: ceramide; 8: free fatty acids; 9: phosphatidylglycerol; 10: cardiolipin; 11: phosphatidylethanolamine; 12: phosphatidylethanolamine plasmalogen; 13: lysophosphatidylglycerol; 14: phosphatidylinositol; 15: phosphatidylserine; 16: lysophosphatidylethanolamine; 17: phosphatidylcholine; 18: phosphatidylcholine plasmalogen; 19: phosphatidic acid; 20: sphingomyelin; 21: sn2-lysophosphatidylcholine; 22: sn1-lysophosphatidylcholine.

Figure 2

Lipid molecular species identified in rat liver and rat plasma by one-dimensional RPLC-MS/MS (1D-RPLC) of total lipid extract [3] and by off-line two-dimensional mixed mode LC-RPLC-MS/MS (2D-mixed mode LC-RPLC).

Figure 3

Full MS spectrum acquired in positive ion mode at retention time 10.79 min for total lipids from rat liver (A) and mixed mode LC-purified phosphatidylcholine lipid fraction from rat liver (B).

Figure 4

Product-ion spectrum of m/z 766.5 in positive mode from isobaric species PE(18:1,20:4) and PC(18:1/17:4) obtained using one-dimensional RPLC-MS/MS where both species co-elute (A) and off-line two-dimensional mixed mode LC-RPLC-MS/MS in PE (B) and PC (C) fractions of rat plasma lipid extract.