Isobaric 6-plex and tosyl dual tagging for the determination of positional isomers and quantitation of monounsaturated fatty acids using rapid UHPLC-MS/MS

Isobaric labelling of fatty acids is complicated by chromatographic co-elution of double bond isomers. This produces contaminated spectra which can mask important biological changes. Here two derivatization strategies are combined to improve throughput and produce MS 2 reporters which change mass depending on double bond position. A 6-plex isobaric tag is attached to the acid group, followed by the tosylation of the double bond using chloramine-T. These two derivatizations allowed for the chromatographic resolution of nearly all investigated isomers using a 3.5 minute ultrafast method. Further isomer differentiation is achieved upon fragmentation as reporter masses scale with the double bond location. This occurs by a dual-fragmentation route which reveals the isobaric labelling and fragments along the double bond of each analyte. These unique fragments allowed for accurate quantitation of co-isolated double bond isomers where traditional isobaric tags would experience ratio distortion. Saturated and monounsaturated fatty acids were characterized by this rapid 6-plex method and produced an average signal RSD of 9.3% and R 2 of 0.99. The method was then used to characterize fatty acid dysregulation upon inhibition of stearoyl CoA desaturase with CAY10566. Dual tagging and fragmentation of fatty acids produced isotope encoded reporters which change in mass depending on double bond position. This allowed for ultrafast analysis without reporter contamination from co-isolated double bond isomers.