Testing And Detection Of Bacterial Endotoxins

Introduction

Within the pharmaceutical and medical device industry, bacterial endo toxins are a major safety and quality concern, and thusly, detection of theses toxins is important. The main reason endotoxins are of concern, is that they are pyrogenic. Pyrogens are a group of compounds that have the ability to raise the core body temperature of the individual once introduced into the bloodstream. This can lead to rapid inflammation and shock and in some cases organ failure which leads to eventual death. Bacterial endotoxins are most likely to found in a manufacturing environment, therefore, testing usually focuses on the detection of the compounds. Bacterial endotoxins are synonymous with lipopolysaccharides (LPS), which is found as a major

This method was carried out by injecting rabbits with a test solution and then monitoring the rabbits for any rise in body temperature and/or symptoms of fever. The main disadvantages of the rabbit pyrogen test is that the test is both expensive to preform and time-consuming plus it cannot be made quantitative. Its use is also only limited to products which would not cause any adverse effects to the test animals. While the test is still an approved method of detecting endotoxins, it is now rarely used, as it has been mostly replaced by the LAL or Limulus Amoebocyte Lysate

The gel-clot method is the simplest and most basic form of detection. It is carried out by adding the reagent to a sample in a test tube and incubating said sample at 37°C for one hour. The tube is then inverted, if a clot is present then the result is positive. It is and endpoint method that can be made semi-quantitative by testing using serial dilutions. The results are expressed as endotoxin units (EU), which is a measure of endotoxin potency rather than the quantity. This reflects the variability in toxicity of naturally occurring LPS (rapidmicrobiology n.d.). The chromogenic method uses a synthetic substrate that brings about a colour change when it is cleaved by endotoxin-activated protease. The turbidimetric method on the other hand, relies on the a coagulin gel cot forming which will alter the turbidity of the sample. Both the turbidimetric and the chromogenic methods can be used as quantitative kinetic methods simply by plotting standard curves of time vs endotoxin concentration. Spectrophotometric instruments can be used to detect changes in colour and turbidity at much lower concentration than that need to form a visible gel-clot. Doing this makes the turbidimetric and chromogenic methods much more sensitive that the gel-clot method. The sensitivity in this case is determined by the lowest concentration that is on the standard