Determining double-bond positions in monoenoic 2-hydroxy fatty acids of glucosylceramides by gas chromatography-mass spectrometry

We applied a gas chromatography-mass spectrometry (GC-MS) method using dimethyl disulfide (DMDS) adducts and were able to determine the double-bond positions in monounsaturated 2-hydroxy fatty acids (2-HFA). 2-HFA methyl esters, prepared from the hydrolysate ofArabidopsis thaliana leaf glucosylceramides, were acetylated and methylthiolated. GC-MS analysis of the resulting DMDS adducts showed simple mass spectra with recognizable molecular ions and a series of key fragment ions indicating the original double-bond positions in the aliphatic chain. Based on this GC-MS elucidation, we confirmed thatArabidopsis leaf glucosylceramides have C₂₂, C₂₃, C₂₄, C₂₅, and C₂₆ chain length 2-HFA with monounsaturation, and all their double bonds are placed at the n−9 position. This procedure is simple, time efficient, and highly sensitive.