Profiling of soil fatty acids using comprehensive two-dimensional gas chromatography with mass spectrometry detection

• Soil bacterial fatty acids esters were studied by using GC × GC methods.
• A series of different column sets showed a non-polar × polar set to be most suitable.
• Forest soil comprised saturated, unsaturated, branched, hydroxyl and oxygenated FAME.
• Accurate mass QTOFMS analysis with GC × GC permitted interpretation of all FAME.

Profiling of phospholipid fatty acids (PLFA) represents a challenging goal for distinguishing the diversity of microbial communities and biomass in the complex and heterogeneous soil ecosystem. Comprehensive two-dimensional gas chromatography (GC × GC) coupled with simultaneous flame ionisation and mass spectrometry detection was applied as a culture-independent method for PLFA profiling of microbial classification in forest soil. A number of column sets were evaluated for the GC × GC separation of fatty acid methyl esters (FAME). Due to better isomeric separation and compound patterns on the 2D contour plot, an apolar–polar column combination was selected for soil microbial PLFA characterisation. A comprehensive view of PLFA composition with carbon chain length varying from 12 to 20 was observed in forest soil samples, with the commonly reported bacterial FAME of iso-/anteiso-, methyl-branched-, cyclopropyl-, and hydroxyl-substituted FA identified by their mass spectral and retention time according to authentic standards. Notably, some uncommon oxygenated FAME were found in high abundance and were further characterised by GC × GC coupled with high resolution mass spectrometry. This tentatively revealed geometric pairs of methyl 9,10-epoxyoctadecanoate isomers.