An Efficient Single Phase Method for the Extraction of Plasma Lipids

Abstract

Lipidomic approaches are now widely used to investigate the relationship between lipid metabolism, health and disease. Large-scale lipidomics studies typically aim to quantify hundreds to thousands of lipid molecular species in a large number of samples. Consequently, high throughput methodology that can efficiently extract a wide range of lipids from biological samples is required. Current methods often rely on extraction in chloroform:methanol with or without two phase partitioning or other solvents, which are often incompatible with liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS). Here, we present a fast, simple extraction method that is suitable for high throughput LC ESI-MS/MS. Plasma (10 μL) was mixed with 100 μL 1-butanol:methanol (1:1 v/v) containing internal standards resulting in efficient extraction of all major lipid classes (including sterols, glycerolipids, glycerophospholipids and sphingolipids). Lipids were quantified using positive-ion mode LC ESI-MS/MS. The method showed high recovery (>90%) and reproducibility (%CV < 20%). It showed a strong correlation of all lipid measures with an established chloroform:methanol extraction method (R2 = 0.976). This method uses non-halogenated solvents, requires no drying or reconstitution steps and is suitable for large-scale LC ESI-MS/MS-based lipidomic analyses in research and clinical laboratories.

Keywords: 1-butanol/methanol extraction; lipidomics; mass spectrometry.

Figure 1

Recovery of lipids with the different extraction methods. The recovery calculated the percent of recovery of the method (n = 5) against its spiked equivalent method (n = 5). The black bars represent the recovery of chloroform/methanol against spiked chloroform/methanol, the gray bars represent the recovery of 1-butanol/methanol (3:1 v/v) against spiked 1-butanol/methanol (3:1 v/v) and the white bars represent the recovery of 1-butanol/methanol (1:1 v/v) against spiked 1-butanol/methanol (1:1 v/v).

Figure 2

Percentage of endogenous lipids not extracted. Pellets remaining after the initial extraction (n = 3) were re-extracted and the lipids quantified. The re-extracted lipids were expressed as a percentage of the total lipids extracted in the first and second extractions combined.

Figure 3

Correlation of plasma lipid measurements following different extraction procedures. Plasma (10 µL, n=10) was extracted via the 1-butanol/methanol (1:1 v/v) or chloroform/methanol methods and analyzed for 293 lipid species via liquid chromatography electrospray ionization-tandem mass spectrometry. The concentration of each lipid was calculated by comparing the area under the chromatogram with the corresponding internal standard. The concentration of each lipid determined via the 1-butanol/methanol (1:1 v/v) method was plotted against the concentration of the same lipid as determined via the chloroform/methanol method. The line of best fit was y = 1.0278x (R² = 0.976).

Figure 4

Within-batch coefficient of variation of the different extraction methods. The CV% were calculated for chloroform/methanol, 1-butanol/methanol (3:1 v/v) and 1-butanol/methanol (1:1 v/v) methods (n = 10). Out of 293 lipid species, 271, 252 and 275 lipid species extracted via chloroform/methanol, 1-butanol/methanol (3:1 v/v) and 1-butanol/methanol (1:1), respectively, had CV% less than 20%.

Figure 5

Coefficient of variation of the batch-to-batch extraction. The CV% was calculated for (n = 49) samples (n = 7/day) over two months. Out of 293 lipid species, 274 lipid species showed a CV% less than 20%.