NACE–ESI‐MS/MS method for separation and characterization of phosphorylation and acylation isomers of lipid A

Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram‐negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE–ESI‐MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4’‐ and C1‐monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative‐ and positive‐ionization modes. The uniqueness of the NACE–ESI‐MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4’) and acylation pattern of native lipid A species or those designed for therapeutic applications.