The Bacterial Endotoxins test, or LAL test, per USP <85>, is a test that is used for the detection and quantification of bacterial endotoxins. Bacterial Endotoxins are a type of pyrogen, which are fever-inducing substances that can be harmful if present in high concentrations. The primary application for the USP <85> Bacterial Endotoxin test is the testing of parenteral (injectable) pharmaceuticals and medical devices that contact blood or cerebrospinal fluid.

There are three basic LAL test methodologies: gel-clot, turbidimetric, and chromogenic. Nucro-Technics bacterial endotoxin testing lab performs the gel-clot method. This test is most commonly conducted as a release test in alignment with Good Manufacturing Practice (GMP) Regulations.

What is the Bacterial Endotoxin Gel-Clot Method and How is it Performed?

The gel-clot method is one of the techniques outlined in USP <85> for detecting and quantifying endotoxin contamination in pharmaceutical products, medical devices, and other materials. This method is based on the clotting reaction that occurs when Limulus Amebocyte Lysate (LAL) comes into contact with endotoxins.

The principles of the bacterial endotoxin gel-clot test are straightforward. Sample is diluted and mixed with LAL reagent. If endotoxins are present, they will make the mixture form a solid gel clot. If multiple endotoxin concentrations are tested, the test can be used in a semi-quantitative way. It can not only determine the presence or absence of endotoxins, but also provide an estimate of the endotoxin concentration.

Inhibition / Enhancement Testing: Validating the USP <85> Bacterial Endotoxins Method

As with most tests performed in Nucro-Technics’ microbiology testing lab, for a test compliant with regulatory requirements, Inhibition / Enhancement testing needs to be performed to demonstrate the sensitivity of the LAL reagents as well as to identify any interfering factors in the sample. To do this, known amounts of endotoxin are spiked into the diluted sample (sample dilution based on the endotoxin limit and on preliminary testing). The recovery is compared to the control. A successful suitability test will provide evidence that there is no inhibition caused by the sample that is preventing endotoxin (if present) from being detected.

If interfering factors are found, they can generally be mitigated through the use of dispersion reagents, pH adjustment, heat treatment, or other techniques that may otherwise have been specified within the USP <85> general chapter.

Alternative Approaches for Endotoxin Testing

There are other ways to test for endotoxins. The USP <151> Pyrogen Test is a most common and accepted approach that may work in scenarios where performing USP <85> is not an option. In more recent years, the Monocyte Activation Test has also gained popularity as a promising alternative and has been accepted by the European Pharmacopoeia for the testing of pyrogenicity in drugs. Nucro-Technics also routinely performs both of these alternative tests.

For clients looking for a bacterial endotoxin testing lab, Nucro-Technics is an excellent choice. Our organization has run thousands of endotoxin tests across multiple methodologies and can help meet the specific needs of the pharmaceutical industry.