Development of an Ion-pairing Reagent-assisted LC–MS/MS Method Employing Fragmentation with Stepped Collision Energy for monophosphoryl Lipid A containing 3-deoxy-D-manno-octulosonic acid and its Derivatives

• Triethylamine as a mobile phase additive for Kdo 2 -lipid A analysis by RP-LC-MS/MS. • Eight engineered Kdo 2 -monophosphoryl lipid A species were simultaneously separated in 13 min. • Fragmentations of multiple adducts were characterized to support structural interpretation. • The method can be used as an identification tool for engineering Kdo 2 -lipid A species. As interest in lipopolysaccharide (LPS)-based vaccine adjuvants continues to grow, clarification of the 3-deoxy-D-manno-octulosonic acid (Kdo) 2 -lipid A structure, a key component of LPS that maintains outer membrane integrity and ensures bacterial viability, is essential. Separately, LPS functions as an immune stimulant by activating Toll-like receptor 4 and caspase-4 in human cells. While Kdo 2 -lipid A acts as an endotoxin, its derivative, Kdo 2 -monophosphoryl lipid A (Kdo 2 -MPLA), shows reduced toxicity and has been explored as a therapeutic agent, including a vaccine adjuvant. Structural variations in their acyl chains and phosphate groups determine both biological activity and safety, emphasizing the need for accurate molecular characterization. However, its inherent heterogeneity and amphiphilic structure–arising from phosphate groups, lipid chains, and the Kdo 2 sugar moieties–cause significant analytical challenges in reverse-phase liquid chromatography. Herein, we developed a liquid chromatography mass spectrometry method using triethylamine as an ion-pairing reagent. The complex Kdo 2 -MPLA mixtures, extracted from Escherichia coli , were successfully separated and detected. Furthermore, the use of stepped collision energy enabled the acquisition of mass spectra with wider fragment coverage, providing detailed structural information such as Kdo 2 sugar as well as the specific locations and lengths of O -acyl chains. By incorporating an ion-pairing reagent, we successfully established a reliable LC-MS/MS method for Kdo 2 -lipid A species with great selectivity and sensitivity. This approach provides detailed structural information as an identification tool for engineered Kdo 2 -lipid A species, with potential to elucidate biosynthetic variations and establish a foundation for engineering lipid A analogs as next-generation LPS-based vaccine adjuvants.