Abstract
Glycerophospholipids (GPLs) are structurally diverse biomolecules that play crucial roles in cellular membranes, signaling, and metabolism. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been widely used for GPL identification due to its high sensitivity and specificity. However, this method often falls short in distinguishing isomeric lipids, such as those differing in the positions of carbon-carbon double bonds. Additionally, the ion types naturally generated during ESI are not always optimal for lipid detection and identification. In this work, we introduce a novel bifunctional tag, nitrophenyl pyrazole (DNPZ), which reacts with double bonds in lipids to form N-doped ozonides. In-situ tandem MS analysis of these modified lipids enables simultaneous identification of double-bond positional isomers and charge switching, facilitating the acquisition of comprehensive structural information. Our findings demonstrate that this approach significantly improves ionization efficiency of GPLs in negative ion mode and provides detailed insights into fatty acyl chain compositions and double-bond positions in GPLs. We have demonstrated that this method allows for the characterization of various lipid classes, lipids with multiple double bonds as well as polar lipid extracts from complex biological samples without the need for authentic lipid reference standards.