A very old recipe : free fatty acids in boiling methanol, under reflux, say 30 min, with a drop of HCl to provide acidic conditions. This is enough to obtain fatty acid methyl esters. If you use ethanol, instead of methanol, you will obtain fatty acid ethyl esters. I used this recipe long time ago (45 years) to obtain the methyl esters from Greek tobacco leaves, and from organic matter in ancient sediments, before characterization by GC/MS. This a mild methylating method, and it will not permethylate, eg proteins, or produce ethers from primary alcohols, thus it will probably not methylate hydroxyl groups from oxylipins. Depending on the number of remaining free hydroxyl groups, the resulting FA methyl esters may be or not amenable to GC/MS. Note that you may obtain the methyl esters first, using the above method, then silylate the hydroxyl groups using BSTFA.

Other strong permethylating agents include: methyl fluorosulfonate (magic methyl) , but is probably as toxic to prepare and use as diazomethane. I have not used it. Permethylation of sugars and proteins used to be performed using methylsulfinyl carbanion, and methyl iodide. Methylsulfinyl carbanion is prepared from DMSO and sodium hydride and can be prepared in advance, then stored in sealed  ampules before use. It is somewhat cumbersome, but it produces a very strong methylating agent.

Dear Fabien,

There are several strategies of methylation and every reaction that involves methylating agents is carcinogenic.

In addition to diazomethane you can methylate samples using CH3I (i've used it for polisaccharides) or you can perform a transmethylation reaction of your FA with a methanolic solution of KOH (2M) to obtain FAME derivatives for GC-MS analysis. Identification is achieved through a database.

Hope this helps,

Ana

A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be 96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.

Thank you very much for all the answers. I am sorry that I haven`t been answering earlier, I`ve been traveling to Copenhagen.

I will definitely try the method using HCl and methanol and will give you a feedback soon.

I should have mentioned earlier the MeOH/BF3 solution, as a mild method for just converting the free carboxylic acid into methyl esters. Glassware don't like the solution; the reagent can be prepared in the lab and stored in glass ampules; it used to be commercially available.  Nevertheless, the MeOH/HCl method is simple and worth to be tested first.

ok, i will boil it in Methanol with a drop of HCl today, then silylate and have a look at the TIC/mass spectra compared to the same sample that was methylated with TMS-diazomethane. I think, there might be some column chromatography necessary because of the water and HCl in GC-MS detection. Thanks for all the suggestions.

If you add pyridine during the silylation step, it may take care of the remaining HCl from the previous methyl ester preparation and avoid a column purification.

Are you interested in analysing any conjugated dienes? If so, acid catalyzed methylation can lead to some artifact generation.

Thank you. I am mainly analyzing oxygenated fatty acids with one double bound.

http://onlinelibrary.wiley.com/doi/10.1002/ejlt.201400404/abstract

Hi Fabien!

Noch aktuell? Nimm mal statt HCl lieber Acetylchlorid (ca. 1% auf die Menge MeOH bezogen einsetzen). Vorsichtig reintröpfeln lassen, kann heftig reagieren, evtl. den Kolben mit Eis oder Wasser kühlen. Vorteil ist, dass Du kein Wasser im Ansatz hast, was ja den Ester wieder spalten kann.

Es gibt auch Protokolle mit TMSH im Liner des GC. Habe ich aber nicht mehr im Kopf, kann man aber Googlen.

Grüße!

Hi Konrad,

vielen Dank. Ich werde das mal mit unserem Team durchsprechen und mich dann nochmal bei dir melden. Hoffe es geht euch gut!

Grüße zurück!

Hi Fabien!

Danke, uns geht es gut! Langsam steigt die Spannung...

Viele Grüße!

trimethylsulfonium hydroxide (TMSH)...a few drops and inject...easy

Use BF3/methanol at waterbath with closed tubes for 3-4 hours. after that extract with n-hexane and evaporate it on mild heat. then use methanol as solvent and run on GC

Use methanhol and 10% sulfuric acid, silicic acid or sodium sulfate. Reflux for one hour for linear acids and for 4 hours for cyclic carboxylic acids, steranoic, hopanoic, etc. After that, add dichloromethane or hexane / acetate 20% and treat the reaction with 5% Bicarbonate or carbonate solution ...