What endotoxin is
Endotoxin is lipopolysaccharide (LPS), a molecule from the outer membrane of Gram-negative bacteria. It is released when bacteria divide or die, and it turns up wherever bacteria have been: water, glassware, reagents and hands.
It is also tough. Endotoxin survives autoclaving, and removing it from glassware takes dry heat at around 250 °C. A 0.22 µm sterile filter removes bacteria, but it does not reliably remove their endotoxin.
Why it matters in research
Immune cells detect endotoxin through the TLR4 receptor and respond to remarkably small amounts. In cell culture, that response can look like an effect of the peptide you are testing: cytokine release, macrophage activation, changes in cell growth. Without an endotoxin result, you can't rule it out.
Why HPLC and mass spec miss it
Endotoxin is present at trace levels by weight, it doesn't show up as a clean UV peak, and it is a family of molecules rather than one mass. The purity and identity tests on a report aren't built to find it. It needs an assay of its own.
How labs test for it
- LAL assays. Limulus amebocyte lysate, from horseshoe crab blood, clots or changes color in the presence of endotoxin. The main formats are gel-clot, turbidimetric and chromogenic. Standards: USP <85> and Ph. Eur. 2.6.14.
- Recombinant factor C (rFC). A lab-made version of the protein that sets off the LAL reaction, with no horseshoe crabs involved. Standard: Ph. Eur. 2.6.32.
Results are given in endotoxin units (EU). For a peptide, the useful figure is EU per mg.
What to look for on a report
- The method: gel-clot, kinetic turbidimetric, kinetic chromogenic or rFC.
- The result in EU/mg, and the limit it was judged against.
- A valid spike recovery. A peptide can boost or block the assay, so labs add a known amount of endotoxin to the sample and check that they get it back. For kinetic methods, USP <85> accepts a recovery of 50 to 200%.
Keeping it out at the bench
- Make stock solutions with endotoxin-free water and buffers.
- Use sterile single-use plastics, or glassware depyrogenated with dry heat.
- Keep tubes closed and wear gloves. Most endotoxin arrives from the room, not the vial.
- Aliquot a fresh stock once, so each tube is opened only once.