I usually isolate lipids from foodstuffs and biological samples using Folch´s method substituting Chloroform with Dichloromethane with excellent results. However recently some publications point out the possibility of using MTBE with similar results as that of Matyash et al. (see attachment).
I would like to know the lab experience of other researchers using this solvent in lipidomics. Thank you!.
Article Lipid Extraction by Methyl-Tert-Butyl Ether for High-Through...
Dear Luis,
Generally, all solvent systems are appropriate for the extraction of predominant lipid classes with high recoveries. Matyash compared MTBE with the Folch method, others have compared the Folch and Bligh and Dyer methods (Iverson et al., Lipids, 2001, 36, 1283), and methanol based method with Folch method (Lofgren et al., J Lipid Res, 2012, 53, 1690) in the extraction of lipophilic compounds.
In my own experience, the challenge lies in the extraction of minor and less abundant lipid classes, and in this case each solvent system will be more suitable depending on the class of lipids you're interested in isolating. For instance, hexane based system will be particularly suitable for the extraction of FA, TAG, Cer and CE; using MTBE system will efficiently extract sphingolipids (SM, Cer, LacCer); whereas using the acidified Bligh and Dyer will extract polar lipid classes (LysoPC, LAA, CS and LacCer).
In case you're aiming to an exploratory analysis you can find some guidelines in (http://www.ncbi.nlm.nih.gov/pubmed/23670529) before implementing a routine analysis.
Hope this helps,
Dear Luis,
Generally, all solvent systems are appropriate for the extraction of predominant lipid classes with high recoveries. Matyash compared MTBE with the Folch method, others have compared the Folch and Bligh and Dyer methods (Iverson et al., Lipids, 2001, 36, 1283), and methanol based method with Folch method (Lofgren et al., J Lipid Res, 2012, 53, 1690) in the extraction of lipophilic compounds.
In my own experience, the challenge lies in the extraction of minor and less abundant lipid classes, and in this case each solvent system will be more suitable depending on the class of lipids you're interested in isolating. For instance, hexane based system will be particularly suitable for the extraction of FA, TAG, Cer and CE; using MTBE system will efficiently extract sphingolipids (SM, Cer, LacCer); whereas using the acidified Bligh and Dyer will extract polar lipid classes (LysoPC, LAA, CS and LacCer).
In case you're aiming to an exploratory analysis you can find some guidelines in (http://www.ncbi.nlm.nih.gov/pubmed/23670529) before implementing a routine analysis.
Hope this helps,
Dear Luis,
The advantage of MTBE is that you can take the upper phase. If you have a lot samples to extract, it can be very useful. In my experience, I recovered correctly most of phospholipids, sphingolipids and sterols. However, you can lose sphingoid bases because they are at the limit of the upper and lower phase and it's not easy to pipet there. As Ana says, the challenge is really " in the extraction of minor and less abundant lipid classes".
Best wishes,
Dear Ana and Charlotte,
thank you very much for your contributions.I competely agree with you about the key are minor compounds. I currently use a variation of the Iverson et al. method with Dichloromethane. We have also compared Radin, Iverson, Bligh and Dyer in both foodstuffs and biological samples. We are particullarly interested in phospholipids but isolating whole lipids. In my experience only Folch-like methods are able to isolate PLs. Have you found diffenreces in the fatty acid analysis?.
In the last year I am finding many reviews and research papers convering MTB for lipidomics. One of the points is that organic phase is the upper.
I will take a look to your link Ana.
Thank you two very much. More comments?.
I never used MTBE ... I think that comments/suggestions of Ana and Charlotte are extremely useful
Thank you Cristina for your feedback. I usually evaporate extraction solvent. I rather use dichloromethane.
We have skipped all chlorinated solvents for environmental issues. we are generally using MTBE instead and are quiete happy. However, if you do work with acid (e.g. for clean-up purposes) You will experience that MTBE is collected back into the acidified aqueous layer, if strong acids are used.
Hi everyone,
I usually use Folch method for extracting the lipid fraction from cells, but by using MTBE for extracting the lipids I appreciated a precipitate (yellow and quite abundant) in the vials, so I could not inject the samples. Changing the extraction solvent was the only change I did to the protocol. Has anyone had this problem before? Do you think this solvent may had extracted other biomolecules as well?
Thanks in advanced
Dear Anna,
first thing coming to my mind is are washing you cell pellets?. I have no experienced problems using the MTBE/MeOH method with bacteria. Maybe that precipitate is coming from the growth medium.
In my experience one main source of problems when isolating lipids is water. You need to separate perfectly well your phases and also having a weak interlayer. You can achieve separation by adding water but you have to know the existing water content in the sample to adjust the added volume.
When I found undesired residues in my samples, a common solution is filtration. However you can be eliminating part of your sample.
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