Bifunctional Tagging Through N-doped Ozonide for Charge Switch-ing and Isomeric Characterization of Glycerophospholipids Using Tandem Mass Spectrometry

Glycerophospholipids (GPLs) are structurally diverse and play essential roles in cellular structure, signaling, and metabolism. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been an effective approach for identifying GPLs. How-ever, this approach often fails to differentiate between isomeric lipids. In this work, we have introduced a novel bifunctional tag, nitrophenyl pyrazole, that can react with the double bonds in lipids to simultaneously achieve charge switching and dou-ble bond positional identification. Our results indicate that this approach not only enhances the ionization efficiency of GPLs in the negative mode but also effectively identifies them by providing detailed insights into the fatty acyl chain compositions and position of carbon-carbon bonds in tandem MS. We have applied the method to characterization of polar lipid extracts from complex biological samples without the requirement of authentic lipid reference standards.