Detection of Lipopolysaccharides in Phospholipids and Liposomes Using the Limulus Test

Abstract

AbstractLPS (lipopolysaccharides) represent a feared pyrogenic impurity in parenterals and raw materials used for their production. In liposome dispersions detection of LPS via the standard Limulus amoebocyte lysate (LAL) test was proved unreliable in presence of phospholipids (liposomes). Attempts were made either to eliminate or inactivate disturbances of the LAL test by phospholipid(s). Common methods to overcome inhibition of the test, such as dilution of the sample, removal of the inhibiting substance by centrifugation or its inactivation by addition of detergents, were found not successful when LPS was present in liposome membrane-bound form. Another means to remove inhibiting substances is ultrafiltration. Ultrafiltration of aqueous lipid dispersions cannot be performed due to clogging of the filter membrane. Ultrafiltration upon addition of organic solvents turned out to be difficult due to the very limited resistance of celluloseacetate filter membranes against these solvents. Nevertheless a test...