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=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=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=C level isomeric species. The systems allowed for the identification of 46 TG molecular species in human plasma with confident C=C location assignment in fatty acyls at a limit of identification of 50 nM.

• This study establishes a reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) platform coupling with online acetone Paternò-Büchi (PB) reaction for triacylglycerol (TG) structural analysis.
• The platform allows for confident identification of TG isomers at both fatty acyl and C=C location levels without requiring synthesized standards, resulting in 46 TG molecular species with defined C=C positions from human plasma.