The endotoxin contamination of parenteral therapeutic products can lead to severe consequences, including, fever, sepsis, and septic shock. Therefore, endotoxin testing has been established as a routine and critical component of the manufacturing and quality control of parenteral pharmaceutical products and medical devices.
The Limulus amebocyte lysate (LAL) assay is the best established test for endotoxin assessment.1 It relies on the incubation of an amoebocyte extract from the horseshoe crab with a sample examined for endotoxin contamination. If endotoxin is present in the analyzed sample, a clot is formed. The most traditional LAL assay technique is the qualitative gel-clot assay. Subsequently, quantitative versions of the LAL assay were developed, including the chromogenic and turbidimetric LAL assays.
The accuracy and reliability of the LAL assay have been confirmed. However, a variety of factors related to the tested products, assay mixture components, environment, and even containers may cause testing challenges and misleading results, including sample interference or endotoxin masking.2–4 Factors that may interfere with the appropriate performance of the LAL assay include chemical inhibitors (such as ethylenediaminetetraacetic acid, EDTA); denaturing agents (such as concentrated acids, bases, inorganic salts, or organic solvents); base alcohol molecules; a pH value outside of the optimal range; endotoxin masking (by substances such as excipients, surfactants, chelators, or N-dimethylamine oxide); or absorption of the endotoxin to container surfaces.1 For medical devices, anticoagulant coating, heavy metal content, or the presence of particulates may interfere with the test results.2 For products assessed with the chromogenic method, assay mixture components that turn a specific color when they come in contact with the LAL reagent could also interference with the test results.1 For samples examined with the turbidimetric method, turbid or suspended products may also lead to sample interference.1
Various strategies have been developed to mitigate the influence of factors causing sample interference or endotoxin masking: