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

초록

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 Kdo2-lipid A acts as an endotoxin, its derivative, Kdo2-monophosphoryl lipid A (Kdo2-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 Kdo2 sugar units–causes 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 Kdo2-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 Kdo2 sugar units 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 Kdo2-lipid A species with great selectivity and sensitivity. This approach provides detailed structural information as an identification tool for engineered Kdo2-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. © 2026 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. http://creativecommons.org/licenses/by-nc-nd/4.0/

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