Encyclopedia of Lipidomics

Living Edition
| Editors: Markus R. Wenk

Liquid Extraction: Single-Phase Butanol/Methanol Extraction

  • Zahir H. Alshehry
  • Peter J. MeikleEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-94-007-7864-1_90-1



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

The extraction method consists of the following steps:
  1. 1.

    Transfer 10 μL of plasma sample into a 1.5 mL eppendorf tube.

  2. 2.

    Transfer 100 μL of 1-butanol/methanol (1:1, v/v) with 5 mM ammonium formate containing internal standards (ISTDs, Table 1)

  3. 3.

    Vortex the sample for 10 s

  4. 4.

    Sonicate the sample for 60 min in a sonic water bath at 18–22 °C

  5. 5.

    Centrifuge for 10 min at (16,000 × g, 20 °C)

  6. 6.

    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

Lipid recovery for the single phase butanol/methanol lipid extraction method was assessed by performing extractions (five replicates) in which the ISTDs were added prior to or after the extraction step. The signal intensity of each ISTD added prior to the extraction relative to the signal intensity of the same ISTD added after extraction was then used to calculate recovery. The ISTDs recoveries were above 95 % for all ISTDs except for cholesteryl ester (CE(18:0) (d6)) and lysophosphatidylethanolamine (LPE(14:0)) which were approximately 90 % (Fig. 1).
Fig. 1

Recovery of ISTD lipids with the single phase butanol/methanol extraction method. ISTD lipids were added to plasma samples (n = 5) either before or after extraction by the single phase butanol/methanol method. Signal intensities were used to calculate the percent recovery of the ISTDs (green bars). The whiskers represent the standard error or recovery

Recovery of endogenous plasma lipids was assessed by sequential extraction. Following an initial extraction of a control plasma (n = 3 replicates), the pellets were re-extracted. Both the initial extract and the secondary extract were analyzed by LC ESI-MS/MS. The recovered lipids (second extraction) were expressed as a percentage of the total lipid in both extractions combined. The percentage of lipids recovered in the second extraction were below 18 % of the total lipid for all lipid species analyzed. Of the 293 lipid species analyzed, 217 (74 %) had less than 10 % in the second extraction (greater than 90 % in the initial extraction). Those lipids that showed the lowest recovery in the initial extraction were typically low-abundant and neutral, non-polar lipids (Fig. 2).
Fig. 2

Recovery of endogenous plasma lipids following sequential lipid extractions. A pooled plasma sample (n = 3 replicates) was sequentially extracted. Each lipid species recovered in the second extraction was expressed as a percentage of the total of that lipid species recovered in the first and second extractions combined. The x-axis represents the percent of lipid recovered in the second extraction (% of unextracted lipid). The y-axis shows the number of lipids within each extraction range

Reproducibility of Lipid Measurements Following Single Phase Butanol/Methanol Extraction

To assess the reproducibility of the lipid measurements following the single phase butanol/methanol extraction, a pooled plasma sample (n = 10) was extracted as a single batch and analyzed by LC ESI-MS/MS. The intra-assay CV% was less than 20 % for 276 out of 293 lipid species. Low abundance lipids typically gave the highest CV% results (Fig. 3). Inter-assay variation was evaluated by the extraction of seven batches (n = 7 pooled plasma samples, in each batch) over a 7-week period. The total of 49 extracted samples were then randomized and analyzed in a single LC ESI-MS/MS run. The inter-assay CV% showed a similar profile to the intra-assay CV% although fewer lipids showed a CV% less than 5 % (Fig. 3).
Fig. 3

Within-batch and between-batch coefficient of variations resulting from the single phase butanol/methanol extraction. Pooled plasma samples (n = 10 replicates) were extracted in a single batch and analyzed by LC ESI-MS/MS. The within-batch CV% was calculated for each lipid species and is shown as the number of lipid species within each CV% range (orange bars). The same pooled plasma sample was also analyzed (n = 7 replicates) on 7 separate days over a 7-week period. All samples were then analyzed in a single run by LC ESI-MS/MS. The between-batch CV% was calculated for each lipid species and is shown as the number of lipid species within each CV% range (purple bars)


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.



  1. Alshehry ZH, Barlow CK, Weir JM, Zhou Y, McConville MJ, Meikle PJ. An efficient single phase method for the extraction of plasma lipids. Metabolites. 2015;5(2):389–403.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Folch J, Lees M, Sloane-Stanley G. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226(1):497–509.PubMedGoogle Scholar
  3. Löfgren L, Ståhlman M, Forsberg G-B, Saarinen S, Nilsson R, Hansson GI. The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma. J Lipid Res. 2012;53(8):1690–700.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res. 2008;49(5):1137–46.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Meikle PJ, Wong G, Tsorotes D, Barlow CK, Weir JM, Christopher MJ, et al. Plasma lipidomic analysis of stable and unstable coronary artery disease. Arterioscler Thromb Vasc Biol. 2011;31(11):2723–32.CrossRefPubMedGoogle Scholar
  6. Weir JM, Wong G, Barlow CK, Greeve MA, Kowalczyk A, Almasy L, et al. Plasma lipid profiling in a large population-based cohort. J Lipid Res. 2013;54(10):2898–908.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Department of MetabolomicsBaker IDI Heart and Diabetes InstituteMelbourneAustralia
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of MelbourneMelbourneAustralia
  3. 3.King Fahad Medical CityRiyadhKingdom of Saudi Arabia