1. Imon Rahman

2. Introduction During the early days of the pharmaceutical industry it was noticed that some solutions when injected into the bloodstream induced fevers. Investigations found that almost all of these fevers were associated with a group of contaminants termed pyrogens ('heat or fever generating'). These were classified as either exogenous or endogenous pyrogens. Exogenous pyrogens are fever causing materials found in the environment, of these - endotoxins are the most researched and are lipopolysacchrides (LPS), found in the outer membrane of the cell wall of Gram -ve microorganisms. They are heat stable and can cause severe patient reactions when present in parenterals or medical devices.

3. Endotoxin toxicity is not dependent upon a living cell. Heat sterilization or other chemical/physical processes are ineffective control measures as killing the cells actually releases 'free' endotoxin from the cell wall. As all mammals can be affected by endotoxins (although sensitivity levels vary) one of the first tests used to determine if endotoxins were present, was the Rabbit Pyrogen Test (RPT). A rabbit was inoculated with the test substance and then monitored it to see if a fever was induced. This test however does not give a quantitative result, is time consuming and is not suitable for products that may in themselves adversely affect the animal. The most commonly used approach now is a Limulus Amoebocyte Lysate or LAL test. The LAL test focuses in particular on 2-keto-3- deoxyoctonoic acid - and it is this which is used as an indicator in the majority of endotoxin assays.

4. Detection Techniques LAL is a reagent derived from the blood cells of the horseshoe crab (Limulus polyphemus). Unlike a mammal the crab does not have a developed immune system, however the LAL component in its blood will bind to and inactivate endotoxins, in the crab the resulting clot also forms a protective barrier against bacterial infection. The test is known as Limulus Amoebocyte Lysate because it uses the cellular Lysate from Limulus Amoebocyte as the main reagent. The Lysate reagent contains a protein that binds to a portion of LPS molecule, reacts with it and coagulates it. At its simplest the LAL test consists of adding LAL reagent to the sample in a test tube, incubating at 37°C for 1 hour. The tube is then gently inverted - if a gel or clot has formed then a positive result is recorded.

5. A variety of LAL assay options are available - gel-clot; turbidimetric; and chromogenic measurement. The latter 2 can be measured either as an endpoint or kinetic reaction. The primary requirement for all methods is that the test reagents, containers, pipettes etc. are pyrogen free. A. The Gel-clot is the simplest and most widely used. \The method can be qualitative or semi-quantitative when comparing a sample against a dilution series of an endotoxin standard. Can be used to provide a pass/fail for a certain limit and is best used with low sample numbers. The method may be performed manually with little or no requirement for instrumentation.

6. Turbidimetric systems depend upon an increasing concentration of insoluble coagulin released as the test progresses; this changes the turbidity of the sample solution. The change in turbidity can be measured either as transmission or absorbance requiring at least a spectrophotometer. Chromogenic assays depend upon a chromogen which changes color if endotoxin is present in the sample. The higher the concentration of endotoxin then the more chromgen is released. Optical reading device operating at the chromgen wavelength is required. Both the turbidimetric and chromogenic assays can be performed either as endpoint or kinetic assays. Endpoint assays involve the measurement of the turbidity or color after incubation at a fixed temperature and time period.

7. Kinetic assays measure the rate of change in turbidity or color during the assay. Instrumentation is used to incubate at a fixed temperature, take measurements, compare those measurements and generate the results. Kinetic assays can provide a greater sensitivity over a wider range than endpoint assays but more sophisticated instrumentation is usually required. Because the potency of an endotoxin to cause pyrogenic reaction will vary according to the nature of the toxin, the FDA developed Endotoxin Units (EU) for result comparisons.

8. Choice of LAL technique may be prescribed by the pharmacopeias but may also be affected by other factors such as the inherent turbidity or color of the sample as well as required sensitivities. Electronics technology has also advanced to the stage that portable endotoxin systems with results available in 15 minutes are now on the market, which are more suited for lower sample numbers with the advantage of little operator training being required. If higher sample numbers are to be processed more sophisticated instrumentation in the form of dedicated systems or incubated microplate readers with appropriate software programs can be used, automated robotic systems are also available. Recently there have been developments intended to decrease the reliance on horseshoe crabs. Examples of these Alternative methods include: - genetically engineered recombinant factor C - the initial element of the LAL clotting cascade is linked to fluorogenic substrates; - cell lines sensitive to LPS linked to color changes in the test medium;