Structural elucidation of triacylglycerol using online acetone Paternò-Büchi reaction coupled with reversed-phase liquid chromatography mass spectrometry

Triacylglycerol (TG) is a class of lipids that is responsible for energy storage and cell metabolism in biological systems; it is found in relatively high abundances in biological fluids such as human plasma. Due to structural complexity, analyzing TGs using shotgun lipidomic approaches is challenging because of the presence of multiple fatty acyl compositional isomers. In this work, reversed-phase liquid chromatography (RPLC) was used for separation of TG species due to the capability of separating lipids based on fatty acyl chain lengths and degrees of unsaturation. RPLC alone does not provide structurally informative information for the location of carbon-carbon double-bonds (C[double bond, length as m-dash]Cs) without using synthesized standards that correspond to each species analyzed. The Paternò-Büchi (PB) reaction was employed online to confidently characterize the location of C[double bond, length as m-dash]Cs within lipid species via photo-initiated modification of the alkene group with acetone, which was later subjected to electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) to form signature fragmentation peaks. This online RPLC-PB-MS/MS system was able to distinguish fatty acyl level and C[double bond, length as m-dash]C level isomeric species. The systems allowed for the identification of 46 TG molecular species in human plasma with confident C[double bond, length as m-dash]C location assignment in fatty acyls at a limit of identification of 50 nM.