Endotoxin Testing

Endotoxin testing quantifies lipopolysaccharide, the gram-negative outer-membrane component that provokes fever and an inflammatory cascade when it reaches the bloodstream. Results are expressed in endotoxin units referenced to an international standard preparation rather than to a mass, because potency varies between lipopolysaccharide structures. The compendial test is performed with horseshoe crab lysate in gel-clot, turbidimetric or kinetic chromogenic format, or with recombinant Factor C, the animal-free reagent now in the official chapters.

The limit is not a fixed number for a product; it is calculated. The permitted endotoxin dose for most parenteral routes is five endotoxin units per kilogram of body weight per hour, and for intrathecal administration it is far lower. Dividing that by the maximum dose given in an hour yields the specification in units per millilitre or per milligram, so the same material can be compliant at microgram doses and grossly out of specification at tens of milligrams.

The decision this supports is whether a product can be injected at all, independently of what the peptide itself does. Endotoxin survives ordinary autoclaving and is not removed by sterilising filtration, so it must be excluded upstream by controlling bioburden and depyrogenating containers rather than removed at the end. A product can be sterile and pyrogenic at once; these are two questions with two answers.

The common failures are interpretive. Material sold without endotoxin data is presumed clean because a filtration step was mentioned, which misunderstands what a filter does. A figure in units per milligram quoted with no reference to the intended dose cannot be judged against anything, and a low-looking number attached to a product used in quantity may be well over the calculated limit.