Hello all,
I just finished lipid extraction using Bligh Dyer method. I used 100ul of plasma samples for extraction. I got about 0.9-2.2 mg dry weight total lipid. I want to know how much organic solvent would be suitable to add to the dry lipid to shoot sample into mass spec. I have seen people using about 10ul to run through LC-MS. Someone suggested me to resuspend in 1ml organic solvent.
Could anyone of you please suggest me how much solvent do I need to add to the total dry lipid so that I can get enough signal in MS analysis.
Thank you.
The answer is... as much as it takes to fully dissolve it in solution. No matter which lipid you have, it needs to be fully dissolved before diluting with mobile phase for introduction to the mass spec's source. Failure to fully dissolve the sample may result in contamination of the MS source ($$$$$) and/or precipitation of the sample. Be sure and use HPLC grade solvent(s) and which specific solvent(s) to use will also depend on the mobile phase which will be used by the LC/MS system (try and add your dissolved sample to a vial with mobile phase so the solution is weaker, if possible, then the mobile phase for loading on the column. Note: If you are using flow injection mode only, where no HPLC column is used, then ignore this warning, but the sample still needs to be fully dissolved). Your final solution should have a concentration of less than 0.5 ug/ml to start with. You can adjust the final concentration once you determine the signal to noise ratio of the peak.
Examples (which may or may not be appropriate to use in your case): DCM (aka: methylene chloride), chloroform, Hexane/IPA and others. *Remember, the sample needs to be fully dissolved in solution, then filtered through an appropriate 0.45u filter before placing in the HPLC vial.
If you have no internal standard you'll have to be careful to dissolve your samples in the same volume and avoid evaporation of the small solvent volume you'll use . Hopefully your samples are dried in some kind of small conical tube such as an eppendorf tube. Try dissolving each in 100 microlitres then submit a portion (10 microlitres ?) for LS-MS. If it's not enough you can inject more or dry them down and concentrate them. Your signal depends on what you are looking for in the samples.
Thank you respected people. I really appreciate your answers. This has been a great way to learn from the experts especially the beginners like me. Suggestions are very clear and helpful.
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