Aqueous based extraction and GCMS

[Azimpatar]

1. I am in the middle to figure out to profiling the possible compounds from aqueous based extraction using GCMS. Does it possible? Please advise.

2. The source of extraction is from marine organism. Please someone help!

[Don_Hilton]

This depends a bit on what you are looking for. You see many methods that start with an aqueous extract or a biofluid (like urine) and the results go to GC/MS. There are many metabolomics papers showing this kind of work.

An aqueous extract typically has many polar moecules. One approach to take is to freeze-dry the extract, add methoxlylamine (to derivitize ketones and aldehydes) and then to add MSTFA/TMCS and pyridine. There is a publication describing this derivitization by Dr. Oliver Fiehn's group.

Edit: updated spelling of methoxylamine.

[Azimpatar]

Don_Hilton,

Thanks for the input, its very helpful. Since the GC/MS is analyzing volatile compounds (correct me if i wrong), do you think using an aqueous extract to GC/MS is practically right to do?
U mentioned about polar molecules from aqueous extracts.

I want to profile the possible bio-active molecules from this new species of marine organism. We are expecting the compounds are derives from aqueous extracts. The worst thing is we only have GCMS in our lab.

[Don_Hilton]

The whole point of derivitization with MSTFA is that you make volatile derivatives of non-volatile molecues. You can do GC/MS analysis of sugars once you make TMS derivatives. Look at what the metabolomics people examine with GC/MS and you will see a very wide variety of molecules, many of them found in body fluids or aqueous extracts.

[Peter Apps]

Can I suggest that you rethink your strategy. Any extract of a living organism, into any solvent contains thousands of compounds and "profiling" these is just a tedious excercise in generating long lists of questionable identities for compounds that are individually very unlikely to be the active component that you are looking for. You need to progressively fractionate (by whatever means) your original extract and see which fraction the activity is in, and then analyse that fraction.

Peter

[Azimpatar]

Dear Peter,

Thanks for invaluable comment. It is good suggestion though, it may add a couple of works to do with different solvents. At current moment, we are still interested to look up on aqueous compositions. This will strengthen the hypothesis of in vivo and in vitro studies that have been done in our lab. We probably may put into the consideration to fraction it in the future, thanks Peter.

Azim

[Azimpatar]

Dear Don Hilton,

Thanks for the enlightenment. Much appreciated. Thanks

Azim

[Peter Apps]

Hi Azim

It sounds as if you have demonstrated biological activity in the aqueous extracts, which is a good start. You are gambling at very long odds by using only GC-MS because only a small fraction of water soluble compounds will ever get through a GC column, even if they are derivatized - think of all the complex sugars, glycosylates, small peptides etc etc. Also - what is it that you are looking for as a result of the analysis - something that is already known to be a drug ?, something with a new structure similar to that of a known drug ?, a "no hits in the library" unknown which might be a new exciting compound, or might just be something irrelevant (to you) that is not in the library. Only in the first two cases do you have any chance at all of linking biological activity to a particular peak on the chromatogram.

Of course, if you have the genetics and biochemistry of the organism to back up your GC-MS you could challenge you marine organism with whatever triggers the genes to make the target molecule, and then search for a peak that gets bigger (beware though that this approach more often than not simply finds generalised responses to infection or stress).

Peter

[Don_Hilton]

Peter picks up a very important point. If you are looking to identify compounds which have activity in the mixture - do you want to profile the mixture or do you want to discover compounds which have activity?

If you want to profile a group of organisms based on active compounds - you need to know which compounds are indeed active. And if you have a list of active compounds already, you need to obtain authentic standards of each to confirm identities -- which also answers the question of whether the compounds are amenable to GC/MS.

Picking up on Peters recommendation of progressive fractionation: Look for separation techniques which will permit scale-up or preparation of what might appear to be "large quantities" of material so you can take multiple separation steps, testing fractions from each and still having enough material to continue on to other steps. Techniques which provide useful information about the compounds help as well.

Separation with size exclusion chromatography immediately gives you an idea about the size of the molecule -- and works nicely with aqueous extracts. (If the active molecule is of sufficient size, you will never get it down a GC column. And if you only have GC/MS to identify compounds which turn out to be large, you know that you have already reached a dead end. )

Separation with an SPE technique provides separation with a different separation mechanism - so you could try SPE on each fraction from the size exclusion chromatography with fractionation on an SPE cartridge.

And the process of separating away irrelevant matrix compounds continues with additional fractionation…