Quantitative determination of conjugated linoleic acid isomers by silver ion HPLC in ewe milk fat

The aim of this research was to validate a method for the quantification of conjugated linoleic acid (CLA) isomers in the fat of ewe's milk. The isomers in the form of methyl ester (FAME) are separated by silver ion high performance liquid chromatography (Ag+HPLC) and the quantification was based on the measurement of the integrated area under the 231 nm peaks, using external CLA reference standards. The response linearity is maintained in a wide range of concentrations and the quantification limit estimated was of 0.005 μg/injection for the cis-9,trans-11 and trans-10,cis-12 isomers. Referred to the samples this limit would be 0.008 mg/g of fat in the usual working conditions. In the precision assay an RSDr of 2.14% for the isomer cis-9,trans-11 was calculated and for the rest of the CLA isomers varied between 2.08 and 7.5%. The recovery assays were performed adding CLA cis-9,trans-11 triglyceride and the mean recovery was 96%. The method proposed allows the determination of the individual content of different CLA isomers using one single technique, HPLC and supposes saving time and resources.