Lipidomics by ultrahigh performance liquid chromatography-high resolution mass spectrometry and its application to complex biological samples

Abstract

An improved approach for selective and sensitive identification and quantitation of lipid molecular species using reversed phase chromatography coupled to high resolution mass spectrometry was developed. The method is applicable to a wide variety of biological matrices using a simple liquid-liquid extraction procedure. Together, this approach combines multiple selectivity criteria: Reversed phase chromatography separates lipids according to their acyl chain length and degree of unsaturation and is capable of resolving positional isomers of lysophospholipids, as well as structural isomers of diacyl phospholipids and glycerolipids. Orbitrap mass spectrometry delivers the elemental composition of both positive and negative ions with high mass accuracy. Finally, automatically generated tandem mass spectra provide structural insight into numerous glycerolipids, phospholipids, and sphingolipids within a single run. Calibration showed linearity ranges of more than four orders of magnitude, good values for accuracy and precision at biologically relevant concentration levels, and limits of quantitation of a few femtomoles on column. Hundreds of lipid molecular species were detected and quantified in three different biological matrices, which cover well the wide variety and complexity of various model organisms in lipidomic research. Together with a software package, this method is a prime choice for global lipidomic analysis of even the most complex biological samples.

Keywords: High performance liquid chromatography; High-resolution mass spectrometry; Lipid structural isomers; Quantitative lipidomics.

Figure 1

Construction of homologous series to aid in identification of lipid molecular species. Reversed phase retention behavior of phospholipids is based on number of carbon atoms and degree of unsaturation. Extracted ion chromatograms of protonated PE 37:4 shows numerous interferences from (a) isobaric PC 34:4, (b) the second isotope of PE 37:5, and (c) sodiated PE 35:1. Correct peaks identified by Lipid Data Analyzer are filled red, green background denotes that tandem mass spectra of the corresponding analytes are available. Note that most of the lipid molecular species depicted show multiple chromatographic peaks belonging to structural isomers differing in fatty acid composition.

Figure 2

Number of glycerolipids (GL), glycerophospholipids (GP), and sphingolipids (SP) identified in murine liver, A549 cells and C. elegans with a 1 % cutoff for each lipid class. Glycerolipids were not analysed in the A549 sample.

Figure 3

Separation of PE structural isomers from a C. elegans lipid extract. The chromatogram shows extracted ion chromatograms of deprotonated PE 34:2. Manual inspection of tandem mass spectra reveals three structural isomers, differing in fatty acyl composition separated by reversed phase chromatography. The positions of the fatty acyls can be determined by comparing the relative intensities of the carboxylate anions.