Lipid A isolated from several bacteria ( Escherichia coli , Pseudomonas aeruginosa , Salmonella enterica , and various strains of Yersinia ) showed abundant formation of pyrophosphate anions upon ion dissociation. Pyrophosphate [H ₃ P ₂ O ₇ ] ⁻ and/or [HP ₂ O ₆ ] ⁻ anions were observed as dominant fragments from diphosphorylated lipid A anions regardless of the ionization mode (matrix-assisted laser desorption ionization or electrospray ionization), excitation mode (collisional activation or infrared photoexcitation), or mass analyzer (time-of-flight/time-of-flight, tandem quadrupole, Fourier transform–ion cyclotron resonance mass spectrometry). Dissociations of anions from model lipid phosphate, pyrophosphate, and hexose diphosphates confirmed that pyrophosphate fragments were formed abundantly only in the presence of an intact pyrophosphate group in the analyte molecule and were not due to intramolecular rearrangement upon ionization, ion-molecule reactions, or rearrangement following activation. This indicated that pyrophosphate groups are present in diphosphorylated lipid A from a variety of Gram-negative bacteria.