Fatty Acid and C18 column extraction

Ketil Thorstensen ketilt at dmf.unit.no
Thu Aug 24 15:26:58 EST 1995

Looking for method to extract free fatty acids from culture medium using 
disposable C18 bonded columns.  

I have been adding EPA and C17:0 to RPMI culture medium with 2% albumin.  This 
sits for at least 4 hours at 4 degrees C.  The sample is then acidified with 
2M citric acid to pH 3.  The columns are activated with 6ml methanol followed 
by 6ml H2O acidified to pH 3 with citric acid.  The sample is added to the 
columns and when dry washed with 2ml hexane.  The sample is then eluted with 
2ml ethyl acetate which is then evaporated and reconstituted with small 
amounts of methanol.  I have been unable to detect fatty acids using HPLC, 
however, I am able to detect appropriate radioactivity when I add 14C linoleic 
acid to the sample.  This method has worked for eicosanoid extraction but so 
far, no luck with free fatty acids.  Any suggestions, experience to relate, 
hints on biochemical errors would be greatly appriciated.  Thanks.

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