Improved separation and characterization of lipopolysaccharide related compounds by reverse phase ion pairing-HPLC/electrospray ionization-quadrupole-mass spectrometry (RPIP-HPLC/ESI-Q-MS)

A new approach for the separation and inline characterization of lipopolysaccharide (LPS) related compounds has been developed. The separation was based on the difference in the number of charged phosphate and ethanolamine groups, as non-stoichiometric substituents, on the polysaccharide backbone, and was achieved with reverse phase ion-pairing chromatography (RPIP-HPLC). Tributylamine was used as an ion-pair reagent. In the conditions used in this study, tributylammonium then binds to the LPS related compounds through the negatively charged phosphate groups. This changes the hydrophobicity of the analytes at different positions and allows for separation based on both the number and position of the substituents on the analyte. The RPIP-HPLC was found to be effective for the separation of the O,N-deacylated derivative (deON) and polysaccharide portion (PS) from the LPS of Escherichia coli C strain. Post-column fluorescence derivatization (FLD), using sodium periodate and taurine, was used to detect the separated LPS related species. On the other hand, the separated species were also detected by direct infusion into the ESI-Q-MS using a volatile ammonium acetate buffer rather than the more traditional potassium phosphate buffer. The signal to noise ratio (S/N ratio) was low for the total ion chromatogram, however, high S/N ratios as well as good resolution were attained by selected ion monitoring (SIM) using m/z numbers corresponding to species with different numbers of non-stoichiometric substituents. Five species for deON and ten species for PS were clearly identified on the SIM chromatogram on the RPIP-HPLC/ESI-Q-MS. Accordingly, the present method allows for the effective separation and inline identification of the species corresponding to the diverse non-stoichiometric substitutions in LPS related compounds.