CCC and HPLC enrichment and GC-MS databasing of more than 20 furan fatty acids (FuFAs) from two fish oil ethyl ester fractions

Abstract

Furan fatty acids (FuFAs) are an important family of fatty acids with health-promoting effects, which are especially present in marine sources but also found in other food categories, albeit only at low amounts. Hence, only six or fewer FuFAs were regularly measured in samples. However, some studies, carried out with great effort, demonstrated the existence of many more (uncommon) FuFAs (in total 42). To facilitate the GC-MS analysis of such rarely reported compounds, the present work focused on enriching and databasing as many FuFAs as possible. First, gram-amounts of two fish oil ethyl ester fractions, one rich in docosahexaenoic ethyl ester (DHA-EE oil) and one in eicosapentaenoic ethyl ester (EPA-EE oil), were hydrogenated, followed by urea complexation. The supernatant solutions rich in FuFA-EE were fractionated by countercurrent chromatography (CCC). Three CCC fractions with several (minor) FuFA-EE were selected, converted into methyl esters (ME), and further purified with HPLC. GC-MS scan analysis of the HPLC fractions enabled the detection of 20 FuFA-ME, including several rare (previously undetected) ones with even-numbered carboxyalkyl or alkyl chains. The data was used to develop a GC-MS retention time prediction tool for FuFA-ME. Based on only one measured FuFA-ME, the Excel-based method allows an accurate prediction of the GC retention times of 40 D-FuFA-ME with a carboxyalkyl chain of 7⎼14 and an alkyl chain length of 2⎼6 carbon atoms. In the CCC/HPLC fractions, several unsaturated FuFAs were also identified, along with the first proof of one with an isolated double bond in the carboxyalkyl chain.