Creation and evaluation of a two-dimensional contour plot of fatty acid methyl esters after off-line coupling of reversed-phase HPLC and GC/EI-MS.

Fatty acid methyl esters obtained from a fish oil sample were fractionated by non-aqueous reversed-phase high-performance liquid chromatography (RP-HPLC) using three serially connected C(18)-columns and pure methanol as the eluent. The HPLC fractions were analyzed by gas chromatography-electron ionization mass spectrometry in the selected ion monitoring mode. Data analysis and visualization was performed by the creation of a two-dimensional (2D) contour plot, in which GC retention times were plotted against the HPLC fractions. The 2D contour plot resulted in a full resolution of more than 120 fatty acids. The fatty acids were arranged on predictable lines and curves in dependence of the number of carbons and double bonds. The 2D contour plot enabled both the recognition of unknown fatty acids (which were found off the lines and curves) and the prediction of the coordinates of known fatty acids. Finally, selected HPLC fractions were subjected to further experiments (hydrogenation, silver ion fractionation, specific GC/MS measurements) in order to verify the structural assignments predicted from the 2D contour plot. All in all, the structures of over 100 FAs could be assigned to the peaks detected in the 2D contour plots.