I am working on human urine and fecal water.I need to choose an extraction solvent for GC-MS sample preparation
The solvent choice depends a lot on which substance you intent to extract.
These two solvents you mention are miscible with water so how do you plan to extract anything from the mostly water samples?
Although, both ACN and Methanol are equally good enough to use, however, there are few differences. Thus, I would suggest to have a look on the type of molecules you targeting prior to selection of extraction solvent.
1. When mixed with water in same ratio, ACN will give you higher elution strength. One can achieve same retention time using ACN with half the ratio of methanol.
There is a difference in the separation selectivity of both chemicals. ACN is aprotic whereas methanol is protic in nature. So, (if you want to go for untargeted approach) if ACN does not offer you passable separation selectivity, you can try methanol.
Dear Amit,
Thank you very much. I have come across few literature using ACN or methanol or both in 3:1 or 1:3 ratio for untargeted metabolomics of Human biofluids such as urine,fecal,saliva,plasma.
I also have used ACN:MeoH(3:1) followed by MeOH:Water(8:1) for human fecal samples and managed to extract around 120 metabolites including amino acids,carbohydrates,phenolics and indoles.But I need a proper technical justification of why this combination has produced good results?I am attaching a paper for your reference .
Dear Prof Andersen,
Thank you very much for your response. I have come across few literature using ACN or methanol or both in 3:1 or 1:3 ratio for untargeted metabolomics of Human biofluids such as urine,fecal,saliva,plasma.
I also have used ACN:MeoH(3:1) followed by MeOH:Water(8:1) for human fecal samples and managed to extract around 120 metabolites including amino acids,carbohydrates,phenolics and indoles.But I am not very sure what could be the proper explanation ? some studies explained that ACN has better protein precipitation capacity.Please see the attached paper.Thanks
Dear Dr Jain,
I agree that the reason for using acetonitrile is that it precipitates protein and some salts. Often procedures are designed so you get about 1:1 acetonitrile to water in the sample. In my methods I always used methanol under the assumption that it is suitable to get smaller molecules out of the solids.
The second step with just methanol is likely to get a more complete extraction and as proteins already denatured and precipitated in the first step they cannot get into solution.
My first comment was to that "extracting" samples with mostly water using methanol and acetonitrile. You will have the water from the sample mixed with the two solvents. This is fine for LC-MS but not so suitable for injecting on a GC. Don't you have a step there you seperate the target compounds from the water in the sample and possibly change to a more suitable solvent for injection into a GC.
Dear prof Anderson,
It is really helpful.
After adding solvents ,the supertnatant (aqueous phase) was air dried using a heat block at 37 degree.Then Trimethylsilylation derivatization was performed before injection.Thanks
Seems quite similar to some of my procedures. It is likely that the removal of solvent at 37 °C takes a long time. There is a risk that some of the products you are measuring partially degrade by the heat and reaction with oxygen during this procedure. Try once to protect the extract and accelerate the evaporation by applying a stream of nitrogen during the evaporation. Ideally adjust the end of a glass pipe with the hose supplying nitrogen so it makes waves on the surface of the solvent-water mixture but doesn't make it splash.
Another method for larger extracts is to remove the liquid using a rotavapor.
For GC-MS methanol has one advantage - it is more volatile than ACN, thus on the chromatogram it will be present just after the dead time. The ACN can "hide" some important peaks.
regards,
G
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