Dear Mohsen,

I have attached a paper from Nature protocols that deals directly with NMR-based metabolomic analysis of plants.

All the best,

Niko

Dear Niko,

Thank you for attached paper. I have read this article. They used methanol as solvent, since I am beginner in plant metabolomics, I am wondering if  it is possible to change that protocol and use chloroform+methanol instead of methanol.

Dear Mohsen,

I suggest you to try to find answers in

http://www.bio-protocol.org/searchdb.aspx?q=plant+metabolites&sid=20150824041252731

Methanol is the most used solvent for metabolite extraction by plant leaves. Using this solvent you can avoid to extract a large number of pigments, which must be eliminated before analysis especially for LC/MS or NMR determinations. Chloroform is used to improve extraction, but needs a long preparation of the analytical sample. The best extraction method depends also by the technique of separation you would like to use after extraction (TLC, GC, LC?).

Best wishes

Sabino

http://bio-protocol.org/e1562

Dear Followers,

I know that you know role of maleate but  author of  protocol which I want to use said: Maleate was used for absolute quantification of metabolites v.s. FW (Fresh Weight) of samples. I decided to use lower amount of leaf for metabolite extraction. 

Thank you all for your advices and your papers.

Best regards,

Mohsen

I would go with Sabino and Thamere...for a NMR profiling approach it is only necessary to extract compounds in a reporducible way....methanol (or in combination with the toxix chlorofom) is the best compromise, when do you not looking for a pecific analyte....here, it is to get then rid of the methanol and re-dissolve in an appropriate NMR solvent....I do not see the need for using malate in the extraction solvent....never heard of that before.....I have seen a study where they used it as internal standard (?).....

Dear Sascha,

Thank you for your helpful response. Yes, Malate was used as internal control for absolute quantification  of metabolite on that study and I do not need to use it, the author of that article said. 

All the  best.

You may try with microwave assisted extraction (MAE). Chloroform is microwave transparent, methanol is not. A mixture of both is likely to give high extraction efficiency.

See the attachments.

Regards.

Dear Vijay Kothari,

Thank you for attached paper. They are some helpful.

All the best.

Dear Mohsen,

I don't know if it is still current, but maybe it'll help.

1. take fresh sample of leaf

2. as soon as possible, freeze the sample to avoid metabolism and enzymatic degradation of intracellular components

3. lyophilisation of frozen sample

4. weighing your sample for extraction

5. if you add chloroform, your extraction will have higher yield, but only in case of non polar lipophilic metabolites and pigments. if you use pure methanol, you also take some pigments and smaller amount of nonpolar metabolites. It depends what kind of metabolites you are focusing. you also should tray 60 - 95% aq. methanol, your extraction take almost nothing of nonpolar compounds, but very nice yield of polar and weak polar metabolites. next problem usually by oxidation followed to degradation of you extracted compounds, there are some possibilities, you can add to your ex. solvent some acid to decrease pH, somebody adds base on the other hand to increase pH, or you can add some few small grains of antioxidant [like BHA, vit. E, etc..].it doesn't matter what you add to ex.solvent before/after extraction, it always give you different extraction results.

6. the step of extraction, is up to your lab. possibilities, but ultrasonic bath is good way

7. centrifugation and filtration and your sample is done

8. do not use to big ration of sample:ex. solvent. Maximal 1:10. but also it is up to you, if you will extracted some sample several times with smaller volumes of liquid, or once with bigger volume followed to evaporation.

9. on the network there are many other strategies, e.g. SPE, destillation etc. but always it depend what kind of metabolites you focuse in.

Dear Martin Sák

Thank you for your helpful advises