Endotoxin

Endotoxin is lipopolysaccharide from the outer membrane of Gram-negative bacteria, a heat-stable contaminant that laboratories measure with the Limulus amebocyte lysate (LAL) test or a recombinant equivalent.

Gram-negative bacteria carry lipopolysaccharide in their outer membrane and shed it as they grow and die. It survives the conditions that kill the bacteria themselves: autoclaving does not destroy it, and sterile filtration does not reliably remove it. A peptide can therefore be sterile and still contain endotoxin, which is why the two are tested separately.

The standard test uses a lysate of horseshoe crab blood cells, which clots in the presence of endotoxin. USP <85> describes gel-clot, turbidimetric and chromogenic versions, and USP <86> describes recombinant reagents made without horseshoe crabs. Amounts are reported in endotoxin units (EU), usually per milligram of peptide. Because some peptides inhibit or enhance the reaction, a valid test includes a spiked sample showing that a known amount of endotoxin can be recovered from the product.

Endotoxin matters in research because immune cells respond to it at very low concentrations, through the TLR4 receptor. In cell culture or animal studies, contamination can produce an inflammatory response that looks like an effect of the peptide itself. An HPLC purity figure does not reveal it, because endotoxin does not appear as a peptide peak.

Related terms

• Sterility testing
• Microbial limits testing
• Certificate of analysis

UPDATED OCTOBER 30, 2026 · REVIEWED BY DEL POTTER, PHD