Bacterial endotoxin test (revision 7)
Old revision·07:00, 26 Oct 2024·Chromatokid
| Bacterial endotoxin test | |
|---|---|
| Also called | LAL test, BET |
| Detects | Lipopolysaccharide from Gram-negative bacterial cell walls |
| Compendial chapter | USP General Chapter 85 |
| Units | Endotoxin units per mg or per mL (EU) |
| Analytical method infobox · conventions | |
The bacterial endotoxin test detects lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria that provokes a febrile response when introduced parenterally. The test exploits a clotting cascade in amoebocyte lysate derived from horseshoe crab blood, which is activated by picogram quantities of endotoxin.[1]
Endotoxin is not a living organism and is not removed by sterilisation. A preparation can be sterile and pyrogenic at the same time: autoclaving kills bacteria but leaves their lipopolysaccharide intact and biologically active. Endotoxin is the commonest but not the only pyrogen, and a separate rabbit pyrogen test exists for the wider class.[2] This is why endotoxin and sterility are separate determinations answering separate questions.[1]
Results are expressed in endotoxin units, normalised to mass for a drug substance or to volume for a solution. Compendial limits for parenteral products are calculated from the maximum dose, and a research-chemical certificate reporting an endotoxin figure without the basis of normalisation is difficult to interpret.[3]
Methods
[edit]Three variants are recognised. The gel-clot method observes whether a firm clot forms at a given dilution and gives a limit result rather than a number. The turbidimetric method follows the increase in turbidity as the cascade proceeds and is quantitative. The chromogenic method measures release of a coloured product from a synthetic substrate and is also quantitative.[1]
Recombinant factor C assays, which use a recombinant version of the initiating enzyme rather than crab-derived lysate, have been introduced and are increasingly accepted. They avoid the animal-derived reagent and are more specific, since they respond only to endotoxin and not to β-glucans.
Interference is the routine difficulty. Sample matrices can enhance or inhibit the cascade, so a validated method includes a positive product control demonstrating recovery of a known spike. A result reported without evidence that interference was assessed is not fully interpretable.[1]