A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

Abstract

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides.

Keywords: ANOVA simultaneous component analysis; dual polarity; lipidomics; liquid-liquid extraction; orbitrap; polarity switching.

Fig. 1.

Typical normal-phase LC-MS chromatograms of lipid extract from normolipidemic LDL in positive (+) (top) and negative (−) ion mode (bottom). LDL was extracted using solvent system 3 (acidified Bligh and Dyer method). Dashed boxes represent the elution time windows for lipid classes eluting (in order of appearance): 1, TAGs+CEs; 2, PCs; 3, SMs; 4, lysoPCs; 5, CS; 6, LAAs; 7, Cer+HexCer; 8, FAs+LacCer; 9, PIs; 10, PEs. Inset in the LC-MS chromatogram in negative ion mode depicts zoomed region for the elution of CS, LAAs, Cer and FAs.

Fig. 2.

Average LC-MS mass spectra of 12 classes of the 19 individual classes identified in LDL population. LC-MS spectra of lipid extracts were acquired in a high-resolution instrument (orbitrap analyzer) equipped with simultaneous detection in both positive (+) and negative (−) ion modes. Lipids classes were extracted using solvent system 3 (acidified Bligh and Dyer) with TAGs, CEs, PCs, SMs, and lysoPC detected in positive ion mode and with Cer, STs, FAs, LacCer, PIs, PEs, and LAAs, detected in the negative ion mode.

Fig. 3.

Evaluation of extractability of total lipids in five different solvent systems using data set 1. Data set 1 consists of 169 lipid species identified in five different solvent systems following processing of LC-MS data through MzMatch R, as described in the Materials and Methods. Graph depicts intensity in counts per second (cps); bars represent standard deviation, ±SD.

Fig. 4.

Evaluation of extractability of individual lipid classes using data set 1. Data set 1 consists of 169 lipid species identified in five different solvent systems, as described in Materials and Methods, following processing of LC-MS data through MzMatch R. Graphs depict intensity in counts per second (cps) for individual classes in five solvent system; bars represent standard deviation, ±SD.

Fig. 5.

Principal component analysis plots for lipids in five solvent systems for data set 1 using ASCA model. A: Principal component analysis score plot of total lipids using ASCA model. B: Principal component analysis loading plot for the individual lipid classes in the five solvent systems.

Fig. 6.

Principal component analysis score plot for total lipids in five solvent systems for data set 2 using ASCA model.