Liquid Extraction: Single-Phase Butanol/Methanol Extraction
Liquid extraction consists of transferring solutes (lipids) into a liquid phase. In the case of single-phase liquid extraction, the extract consists of a single phase with the feed solution (or solid) being mixed in a single homogeneous phase with the extraction solvent.
Population-based lipidomic studies have shown the potential to identify the association of lipid species with disease risk and progression. However, the collinearity between lipids and many conventional risk factors may obscure the independent associations, requiring adjustment of regression models for multiple covariates. Further, the nature of lipidomic analyses involving hundreds of lipid species requires statistical correction for multiple comparisons. Thus, to obtain statistical power to identify independent associations, such studies require cohorts with hundreds to thousands of samples.
To facilitate such studies, a simple, robust, and high-throughput lipidomic m ethodology is required. Several methods have been developed for plasma lipid extraction. The gold standard is the “Folch” method (Folch et al. 1957), see chapter “Liquid Extraction: Folch” which is based on two-phase chloroform:methanol (2:1, v:v) extraction, where most lipids partition into the lower organic phase. Collection of lipids from the lower phase is challenging, which limits throughput and reproducibility. Matyash et al. developed methyl-tert-butyl ether (MTBE) extraction method in which the lipids partition into the upper organic phase (Matyash et al. 2008), see chapter “Methyl-Tert-Butyl Ether extraction” to overcome the limitations of the Folch method. Recently, an automated, two phase 1-butanol/methanol method (BUME method) was developed for shotgun “direct infusion” mass spectrometry analyses in which the lipids also partition into the upper organic phase (Löfgren et al. 2012), see chapter “Liquid Extraction: BUME” However, avoiding the interface between the two phases in both methods is still required and both suffer from incomplete recovery of more polar lipids.
A single-phase chloroform:methanol (2:1, v:v) lipid extraction method has been developed in our laboratory (Meikle et al. 2011; Weir et al. 2013). Our method is suitable for reverse phase liquid chromatography electrospray ionization tandem mass spectrometry (LC ESI-MS/MS) analyses. A limitation of this method was the requirement for removal of the extraction solvent and reconstitution in 1-butanol/methanol (1:1, v/v) to facilitate reverse phase liquid chromatography.
We subsequently developed a single-phase butanol/methanol lipid extraction, utilizing 1-butanol/methanol (1:1, v/v) that does not require a change of solvents prior to liquid chromatography (Alshehry et al. 2015). The method is simple, rapid, provides high recoveries of a wide range of both polar and non-polar lipids, and shows a high level of reproducibility.
Methodology: Single Phase Butanol/Methanol Lipid Extraction
Transfer 10 μL of plasma sample into a 1.5 mL eppendorf tube.
Transfer 100 μL of 1-butanol/methanol (1:1, v/v) with 5 mM ammonium formate containing internal standards (ISTDs, Table 1)
Vortex the sample for 10 s
Sonicate the sample for 60 min in a sonic water bath at 18–22 °C
Centrifuge for 10 min at (16,000 × g, 20 °C)
Transfer 80–90 μL of the supernatant to a 0.2 mL micro-insert in 32 × 11.6 mm glass vials with Teflon insert cap for analysis by LC ESI-MS/MS
Recovery of Lipids
Reproducibility of Lipid Measurements Following Single Phase Butanol/Methanol Extraction
The single phase butanol/methanol extraction method does not efficiently remove salts and polar metabolites. Therefore, the resulting extracts are not appropriate for direct infusion (shotgun) lipidomics. However, liquid chromatography can overcome this limitation by eluting salts and polar metabolites to waste prior to the elution of the less polar lipids. We have recently completed a practical demonstration of high throughput lipidomics using this approach where we were able to perform 1000 injections (1 μL) before cleaning the mass spectrometer and 4000 injections before replacing the LC column.
The single phase butanol/methanol extraction represents a simple, fast, and high recovery lipid extraction methodology suitable for high throughput lipidomics. It avoids the use of halogenated solvents and so minimizes the risk associated with those solvents. We expect this method to be amenable to automation which would further increase the potential throughput.