홍지영 (Kist 도핑콘트롤센터)

김기훈 (Kist 도핑콘트롤센터)

홍지영 (Kist 도핑콘트롤센터)

윤지현 (Kist 도핑콘트롤센터)

오한빈 (서강대학교)

김기훈 (Kist 도핑콘트롤센터)

In Gram-negative bacteria, 3-deoxy-D-manno-octulosonic acid (Kdo)₂-lipid A is a fundamental substructure of lipopolysaccharide and acts as a selective activator of TLR4 on immune cells, which makes it a promising target for therapeutic development. Characterizing its structure across bacterial species is essential for elucidating structural diversity and role in virulence. However, due to its inherent heterogeneity, comprising a hydrophilic glucosamine bearing phosphate groups and hydrophobic acyl chains, it presents significant analytical challenges such as poor elution in reverse-phase liquid chromatography.

Herein, we developed and optimized a liquid chromatography-mass spectrometry method for Kdo₂-lipid A and monophosphoryl lipid A species. Triethylamine (TEA), a known ion-pairing reagent, was adopted as a mobile phase modifier. TEA formed a neutral ion pair with the target anionic analyte, leading to sufficient retention on the non-polar column. Particularly, implementing the mobile phase with 0.2% TEA under basic conditions led to a significant improvement in chromatographic separation and sensitivity. Additionally, data-independent acquisition (DIA) using a combination of CEs enabled the simultaneous detection of superimposed fragmentation patterns produced at distinct CEs, offering a simpler analytical procedure without the need for CE optimization. This analytical approach is amenable to the analysis of lipid A derivatives and structurally related amphiphilic lipids.