Characterization of complex, heterogeneous lipid A samples using HPLC–MS/MS technique II. Structural elucidation of non‐phosphorylated lipid A by negative‐ion mode tandem mass spectrometry

Non‐phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non‐phosphorylated lipid A species under low‐energy collision‐induced dissociation conditions of an electrospray ionization quadrupole time‐of‐flight instrument. Charge‐promoted consecutive and competitive eliminations of the acyl chains and cross‐ring cleavages of the sugar residues were observed. The A‐type fragment ion series and the complementary X‐type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non‐reducing and the reducing ends, respectively, of the non‐phosphorylated lipid A backbone. Reversed‐phase liquid chromatography in combination with negative‐ion electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non‐phosphorylated lipid A. As a standard, the hexa‐acylated ion at m / z 1636 with the Escherichia coli ‐type acyl distribution (from E .  coli O111) was used. The method was tested and refined with the analysis of other non‐phosphorylated hexa‐ and several hepta‐, penta‐, and tetra‐acylated lipid A species detected in crude lipid A fractions from E .  coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.