AimsThe bacterial endotoxins test (BET) is a sensitive assay for measuring endotoxin levels in solution and uses the limulus amebocyte lysate (LAL) coagulation reaction. We sought to identify the mechanisms through which certain substances interfere with the interaction between LAL and bacterial lipopolysaccharide (LPS). Methods and ResultsEndotoxin lipid A was inactivated by the addition of iron sulfate, which acted on endotoxin directly and strongly inhibited LAL coagulation activity. Size-exclusion, anion-exchange and reverse-phase liquid chromatography/mass spectrometry were used to examine changes in inactivated lipid A in terms of complex formation, negative charge status, hydrophobic interaction and structure. Furthermore, we verified the involvement of the lipid A phosphoryl group in the interference of iron sulfate with lipid A-factor C binding activity. Iron sulfate-inactivated lipid A was cleaved at its glycosidic bond, resulting in loss of hydrophobic interactions and disruption of lipid A complexes without alteration of negative charge status and lipid A-factor C interaction. ConclusionsLipid A cleavage was a direct result of interfering factors, including iron sulfate, which acted on endotoxin directly to disrupt lipid A complexes rather than interfering with LAL coagulation. Significance and Impact of the StudyOur data provide new insights into the mechanisms of lipid A activity.