Endotoxins are toxic substances that are released from the outer membrane of Gram-negative bacteria when they die or multiply. Endotoxins can cause fever, inflammation, shock, and even death if they enter the bloodstream of humans or animals.
Therefore, it is essential to measure the levels of endotoxins in pharmaceutical products, medical devices, and biological materials that are intended for injection.
One of the most widely used methods to detect and quantify endotoxins is the Limulus Amebocyte Lysate (LAL) test. The LAL test is based on the principle that the blood cells of horseshoe crabs, called amebocytes, clot in the presence of endotoxins.
The LAL Test Procedure
1. Preparation of the sample: The sample must be diluted to a suitable concentration that does not interfere with the test or exceed the endotoxin limit. The endotoxin limit is the maximum amount of endotoxin allowed per dose of the product, and it depends on the route of administration, the dose, and the threshold pyrogenic dose. The endotoxin limit can be calculated using the formula EL = K/M, where K is the threshold pyrogenic dose and M is the dose of the product per kg of body weight per hour. The maximum valid dilution (MVD) is the highest dilution factor of the sample that can be tested without violating the endotoxin limit. The MVD can be calculated using the formula MVD = EL x Potency / λ, where Potency is the activity of the product per mL, and λ is the sensitivity of the LAL reagent in EU/mL.
2. Addition of the LAL reagent: The LAL reagent is an extract from the amebocytes of horseshoe crabs that contains the enzymes and coagulation factors that react with endotoxins. The LAL reagent is added to the sample and incubated at a controlled temperature for a specific time.
3. LRW (LAL Reagent Water) used for dillution of sample.
4.CSE (Control Standard Endotoxin) used to make positive control.
5. Measurement of the reaction: Depending on the format of the LAL test, the reaction can be measured by observing the formation of a gel-clot.
The LAL test must be validated for each product to ensure its accuracy and reliability. The validation process involves testing the product for interference.
The LAL test is a simple, rapid, and sensitive method to detect endotoxins in pharmaceutical products. However, it also has some limitations, such as the variability of the LAL reagent, the potential for false positives or negatives, and the ethical concerns over the harvesting of horseshoe crabs.
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