Exploring the fatty acids of vernix caseosa in form of their methyl esters by off-line coupling of non-aqueous reversed phase high performance liquid chromatography and gas chromatography coupled to mass spectrometry

Vernix caseosa is a greasy biofilm formed on the skin of the human fetus in the last period of pregnancy. This matrix is known to contain a range of uncommon branched chain fatty acids. In this study, we studied the fatty acid composition of vernix caseosa by non-aqueous reversed phase high performance liquid chromatography (RP-HPLC) fractionation followed by gas chromatography–electron ionization mass spectrometry (GC/EI–MS) of the fractions. For this purpose the fatty acids from vernix caseosa were converted into the corresponding methyl esters. These were fractionated by non-aqueous RP-HPLC using three serially connected C18-columns and pure methanol as the eluent. Aliquots of the HPLC fractions were directly analyzed by GC/EI–MS in the selected ion monitoring mode. Data analysis and visualization were performed by the creation of a two dimensional (2D) contour plot, in which GC retention times were plotted against the HPLC fractions. Inspection of the plot resulted in the detection of 133 different fatty acids but only 16 of them contributed more than 1% to the total fatty acids detected. Identification was based on HPLC and GC retention data, GC/MS–SIM and full scan data, as well as plotting the logarithmic retention times against the longest straight carbon chain. In selected cases, aliquots of the HPLC fractions were hydrogenated or studied by means of the picolinyl esters. Using these techniques, the number of double bonds could be unequivocally assigned to all fatty acids. Moreover, the number of methyl branches, and in many cases the positions of methyl branches could be determined. The enantioselective analysis of chiral anteiso-fatty acids resulted in the dominance of the S-enantiomers. However, high proportions of R-a13:0, R-a15:0, and R-a17:1 were also detected while a17:0 was virtually S-enantiopure.