Dear Dr Rania,

Thank you for your question. Please find the answer in the attached and published files.

Good luck

Aly R Abdel-Moemin

Article Healthy cookies from cooked fish bones

Are you using ethyl acetate as an extraction solvent? Transesterification to the ethyl esters can easily happen if you have any residual acid in your sample and are using ethyl acetate as an extraction solvent. Try another solvent like chloroform, dichloromethane, diethyl ether, diisopropyl ether, MTBE, hexane, cyclohexane, or even toluene. If you are able to do your experiment using the ethyl esters then you could prepare them using ethanol and acid (like HCl) and continue using the ethyl acetate for extraction. The ethyl esters will take longer to generate than the methyl esters; I suggest using at least 2 times the amount of time allowed for the methyl ester formation under similar conditions.

The library mass spectra can also be similar as the fragmentation patterns for the unsaturated fatty acids can be similar, particularly if the mass ion is lost. Check the fragmentation patterns - if there is a MacLafferty ion at m/z = 74, that's the telltale sign for a methyl ester and means the library is matching it wrong. If there's one at m/z=88, that corresponds for an ethyl ester and means that you've genuinely made them. If that happens, as Bruce said, check if you've used a solvent with an ethyl moiety somewhere - particularly ethanol or ethyl acetate.

for the cuantitation of FAMES or FAEES use the molecular mass for cuantification. Depending in the chromatographic conditions you could have a co-elution of the same FAME with the FAME and with very few differences in the retention time with  the free acid. That could be a problem of mass identification.

Another problem is the formation of an  artifac because of the presence of ethanol in the sample or in the extraction solvent.

Dear Rania,

Reading Tom's answer about the possibility that the compounds ara mis-identified, I have a question. How did you identify the fatty acid esters, did you only do a MS library search?

A MS library search is not enough to identify compounds! Don't forget you are not using a MS but a GCMS You ALWAYS have to use the GC retention data in combination with the mass-spectrum. If you only did a MS library search, then analyse certified methyl and ethyl esters of your fatty acids and use the retention data to confirm your identification.

Regards, Alex