BACKGROUND:

Endotoxins are bacterial structural components that are released when such a cell is lysed. These components are toxic if administered to humans and/or animals, causing a pyrogenic response (rise in body temperature). For this reason it is important that drugs and medical devices which are either injected or implanted must be tested for their endotoxin content.  There are several methods available for conducting the endotoxin  test, which includes the in vivo rabbit pyrogen test and several in vitro alternatives that utilize the Limulus Amebocyte Lysate (LAL) system.  These have become the method of choice and include gel clot, chromogenic, kinetic chromogenic or turbidimetric assays.  Medical devices usually require extraction with Pyrogen-Free Water (PFW) which is then treated as the test sample.

PRINCIPLE:

Kinetic Chromogenic method and Turbidimetric method

Ams Laboratories are able to perform both the kinetic chromogenic and turbidimetric assays.  The chromogenic method involves an enzymatic reaction between the endotoxin and lysate which results in the production of a yellow colour in the presence of endotoxin. The intensity of the colour production is directly linked to the quantity of endotoxin present in the sample. With the kinetic variation of the assay, the time of onset of the colour reaction is measured. Therefore, with the use of endotoxin standards we are able to calculate the value of endotoxin present in or on the product.  Some products are of a colour that would interfere with this form of testing, and so the turbidimetric method can be used to avoid any such interference. In this case a different lysate is used and the reaction with endotoxin results in the solution becoming turbid, thus allowing quantitation of endotoxin content without relying on the colour present. Both methods are equally effective in obtaining the endotoxin content in a product, but often, one is more suitable than the other. Both methods use objective measurements to determine endotoxin content and are quantitative in nature.  These tests can be carried out relatively quickly, and results can be available within 2-3 days of sample receipt.

Gel Clot Assay method

The gel clot assay was the original LAL method and relies upon the operator to distinguish the formation of the gel clot in the reaction tubes.  It is a qualitative or semi-quantitative test that is used to screen for the presence of endotoxins. A clot formation is interpreted as a positive result for the presence of endotoxin and if no clot forms, this is interpreted as the sample being endotoxin free.  The results are from the subjective interpretation of the clot formation.

SAMPLE REQUIREMENTS AND TURNAROUND TIMES:

Generally one container is sufficient for method qualification purposes.  For  validation of a new product, the pharmacopoeias require samples from three separate batches to be tested to ensure any batch-to-batch variation is taken into account.

Routine test results can be completed in 2 – 3 working days.  Depending on work loads in the laboratory, validations can be completed in 4 – 5 working days.