Identification of conjugated isomers of linolenic acid and arachidonic acid in cheese
A method has been developed for the separation and identification of conjugated fatty acid isomers as distinct from conjugated linoleic acid (CLA). Cheese fat was extracted using n-hexane and converted into fatty acid methyl esters (FAMEs) with potassium methylate. Because of the low concentration of conjugated fatty acids isomers, a prefractionation was performed to enhance and to separate these isomers from CLA and other fatty acids common in cheese 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 nm, so all FAMEs were detectable, including non-conjugated ones. All prefractions were analysed on a high polarity GC column fitted with a split-splitless injector and a flame ionisation detector. In addition prefraction 2, which contained linolenic acid and arachidonic acid, as well as their isomers, was fractionated by silver ion (Ag + )-HPLC. Two columns were used in series with 0.1% acetonitrile in n-hexane as isocratic mobile phase. All conjugated 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 identification 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 isomers of arachidonic acid (CAA) and six conjugated isomers of linolenic acid (CLnA) could be identified with regard to the position of their double bonds.