A method of coupling the Paternò–Büchi reaction with direct infusion ESI-MS/MS for locating the CC bond in glycerophospholipids

Abstract

Tandem mass spectrometry (MS/MS) coupled with soft ionization is established as an essential platform for lipid analysis; however, determining high order structural information, such as the carbon–carbon double bond (CC) location, remains challenging. Recently, our group demonstrated a method for sensitive and confident lipid CC location determination by coupling online the Paternò–Büchi (PB) reaction with nanoelectrospray ionization (nanoESI) and MS/MS. Herein, we aimed to expand the scope of the PB reaction for lipid analysis by enabling the reaction with infusion ESI-MS/MS at much higher flow rates than demonstrated in the nanoESI setup (∼20 nL min⁻¹). In the new design, the PB reaction was effected in a fused silica capillary solution transfer line, which also served as a microflow UV reactor, prior to ESI. This setup allowed PB reaction optimization and kinetics studies. Under optimized conditions, a maximum of 50% PB reaction yield could be achieved for a standard glycerophosphocholine (PC) within 6 s of UV exposure over a wide flow rate range (0.1–10 μL min⁻¹). A solvent composition of 7 : 3 acetone : H₂O (with 1% acid or base modifier) allowed the highest PB yields and good lipid ionization, while lower yields were obtained with an addition of a variety of organic solvents. Radical induced lipid peroxidation was identified to induce undesirable side reactions, which could be effectively suppressed by eliminating trace oxygen in the solution via N₂ purge. Finally, the utility of coupling the PB reaction with infusion ESI-MS/MS was demonstrated by analyzing a yeast polar lipid extract where CC bond locations were revealed for 35 glycerophospholipids (GPs).