Identification of conjugated isomers of linolenic acid and arachidonic acid in cheese

A method has been developed for the separation and identification of conjug ated fatty acid isomers as distinct from conjugated linoleic acid (CLA). Ch eese fat was extracted using n-hexane and converted into fatty acid methyl esters (FAMEs) with potassium methylate. Because of the low concentration o f conjugated fatty acids isomers, a prefractionation was performed to enhan ce and to separate these isomers from CLA and other fatty acids common in c heese fat. The separation was carried out by preparative reversed phase (RP )-HPLC. The FAMEs were detected with a UV detector at a wavelength of 208 n m, so all FAMEs were detectable, including non-conjugated ones. All prefrac tions were analysed on a high polarity GC column fitted with a split-splitl ess injector and a flame ionisation detector. In addition prefraction 2, wh ich contained linolenic acid and arachidonic acid, as well as their isomers , was fractionated by silver ion (Ag+)-HPLC. Two columns were used in serie s with 0.1 % acetonitrile in n-hexane as isocratic mobile phase. All conjug ated compounds were detected with a photodiode array detector at 234 nm and the spectrum from 200 to 400 nm was recorded. Thus characteristic spectra of conjugated dienes and conjugated trienes could be distinguished. For ide ntification of the position of double bonds, each fraction collected after Ag -HPLC was converted into 4,4-dimethyloxazoline (DMOX) derivatives. These derivatives were analysed by gas chromatography-electron impact ionization mass spectrometry (GC-EI-MS). With the proposed method, 13 conjugated isom ers of arachidonic acid (CAA) and six conjugated isomers of linolenic acid (CLnA) could be identified with regard to the position of their double bond s.