UTILIZATION OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AS AN ENRICHMENT STEP FOR THE DETERMINATION OF CYCLIC FATTY-ACID MONOMERS IN HEATED FATS AND BIOLOGICAL SAMPLES

A method was developed to determine traces of cyclic fatty acid monomers (CFAM) in oils and animal tissues. This method is a combination of some techniques developed earlier but with the enrichment step being achieved by high-performance liquid chromatography (HPLC) instead of urea inclusion. After transformation of the lipids into methyl eaters, the latter were hydrogenated after addition of an internal standard (methyl heptadecanoate or ethyl hexadecanoate). The mixture was enriched in CFAM by HPLC on a semi-preparative C-18 reversed-phase column using acetonitrile-acetone (90:10, v/v) at 4 ml/min. The enriched fraction containing the CFAM and the internal standard was then analysed by gas chromatography on a polar column (cyanosilicone phase). This method was developed using known mixtures of CFAM isolated from both heated sunflower and Iinseed oils. Small amounts of CFAM (50 mu g/g of sample) were determined with good reproducibility without any loss during the HPLC enrichment step and with no modification of the relative proportions of the CFAM in the mixture. This method can be applied to either heated fats and oils or biological samples (heart cell culture) that contain only traces of CFAM. Ethyl hexadecanoate (16:0 ethyl ester) can be used as an internal standard for samples containing small amounts of 17:0.