What Is Endotoxins Test?

What Is Endotoxins Test?

Endotoxins are hydrophobic molecules that are part of the lipopolysaccharide complex that forms most of the outer membrane of Gram-negative bacteria. They are released when the bacteria die and their outer membranes disintegrate. Endotoxins are considered as the major contributors to the pyrogenic response. And parenteral products contaminated with pyrogens can lead to development of fever, induction of inflammatory response, shock, organ failure and death in humans.

Endotoxins test is the test to detect or quantify endotoxins from Gram-negative bacteria.

Rabbits are employed to detect and quantify endotoxins in pharmaceutical products at the first. According to USP, the RPT involves monitoring for an increase in temperature or a fever after intravenous injection of the pharmaceutical into rabbits. And 21 CFR 610.13(b) requires a rabbit pyrogen test for specified biological products.

In 1960s, Fredrick Bang and Jack Levin found that amebocytes of the horseshoe crab will clot in the presence of endotoxins. The Limulus Amebocyte Lysate (or Tachypleus Amebocyte Lysate) was developed accordingly to replace most RPT. On USP, LAL test is referred to as the bacterial endotoxin test (BET). And BET could be done by using 3 techniques: 1) the gel-clot technique; 2) the turbidimetric technique; 3)the chromogenic technique. Requirements for LAL test contains optimal pH, ionic strength, temperature, and time of incubation.

Compared with RPT, BET is rapid and efficient. However, BET could not replace RPT completely. Because LAL assay may be interfered by factors and it is unable to detect non-endotoxin pyrogens.